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Sample records for coat protein promoter

  1. Identification of a seed coat-specific promoter fragment from the Arabidopsis MUCILAGE-MODIFIED4 gene.

    Science.gov (United States)

    Dean, Gillian H; Jin, Zhaoqing; Shi, Lin; Esfandiari, Elahe; McGee, Robert; Nabata, Kylie; Lee, Tiffany; Kunst, Ljerka; Western, Tamara L; Haughn, George W

    2017-09-01

    The Arabidopsis seed coat-specific promoter fragment described is an important tool for basic and applied research in Brassicaceae species. During differentiation, the epidermal cells of the Arabidopsis seed coat produce and secrete large quantities of mucilage. On hydration of mature seeds, this mucilage becomes easily accessible as it is extruded to form a tightly attached halo at the seed surface. Mucilage is composed mainly of pectin, and also contains the key cell wall components cellulose, hemicellulose, and proteins, making it a valuable model for studying numerous aspects of cell wall biology. Seed coat-specific promoters are an important tool that can be used to assess the effects of expressing biosynthetic enzymes and diverse cell wall-modifying proteins on mucilage structure and function. Additionally, they can be used for production of easily accessible recombinant proteins of commercial interest. The MUCILAGE-MODIFIED4 (MUM4) gene is expressed in a wide variety of plant tissues and is strongly up-regulated in the seed coat during mucilage synthesis, implying the presence of a seed coat-specific region in its promoter. Promoter deletion analysis facilitated isolation of a 308 base pair sequence (MUM4 0.3Pro ) that directs reporter gene expression in the seed coat cells of both Arabidopsis and Camelina sativa, and is regulated by the same transcription factor cascade as endogenous MUM4. Therefore, MUM4 0.3Pro is a promoter fragment that serves as a new tool for seed coat biology research.

  2. Functional analysis of bipartite begomovirus coat protein promoter sequences

    International Nuclear Information System (INIS)

    Lacatus, Gabriela; Sunter, Garry

    2008-01-01

    We demonstrate that the AL2 gene of Cabbage leaf curl virus (CaLCuV) activates the CP promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for Tomato golden mosaic virus (TGMV). Binding studies indicate that sequences mediating repression and activation of the TGMV and CaLCuV CP promoter specifically bind different nuclear factors common to Nicotiana benthamiana, spinach and tomato. However, chromatin immunoprecipitation demonstrates that TGMV AL2 can interact with both sequences independently. Binding of nuclear protein(s) from different crop species to viral sequences conserved in both bipartite and monopartite begomoviruses, including TGMV, CaLCuV, Pepper golden mosaic virus and Tomato yellow leaf curl virus suggests that bipartite begomoviruses bind common host factors to regulate the CP promoter. This is consistent with a model in which AL2 interacts with different components of the cellular transcription machinery that bind viral sequences important for repression and activation of begomovirus CP promoters

  3. RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

    Science.gov (United States)

    Manfrini, Nicola; Trovesi, Camilla; Wery, Maxime; Martina, Marina; Cesena, Daniele; Descrimes, Marc; Morillon, Antonin; d'Adda di Fagagna, Fabrizio; Longhese, Maria Pia

    2015-02-01

    Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. © 2014 The Authors.

  4. Polyglycerol coatings of glass vials for protein resistance.

    Science.gov (United States)

    Höger, Kerstin; Becherer, Tobias; Qiang, Wei; Haag, Rainer; Friess, Wolfgang; Küchler, Sarah

    2013-11-01

    Proteins are surface active molecules which undergo non-specific adsorption when getting in contact with surfaces such as the primary packaging material. This process is critical as it may cause a loss of protein content or protein aggregation. To prevent unspecific adsorption, protein repellent coatings are of high interest. We describe the coating of industrial relevant borosilicate glass vials with linear methoxylated polyglycerol, hyperbranched polyglycerol, and hyperbranched methoxylated polyglycerol. All coatings provide excellent protein repellent effects. The hyperbranched, non-methoxylated coating performed best. The protein repellent properties were maintained also after applying industrial relevant sterilization methods (≥200 °C). Marginal differences in antibody stability between formulations stored in bare glass vials and coated vials were detected after 3 months storage; the protein repellent effect remained largely stable. Here, we describe a new material suitable for the coating of primary packaging material of proteins which significantly reduces the protein adsorption and thus could present an interesting new possibility for biomedical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Evidence for lysine acetylation in the coat protein of a polerovirus.

    Science.gov (United States)

    Cilia, Michelle; Johnson, Richard; Sweeney, Michelle; DeBlasio, Stacy L; Bruce, James E; MacCoss, Michael J; Gray, Stewart M

    2014-10-01

    Virions of the RPV strain of Cereal yellow dwarf virus-RPV were purified from infected oat tissue and analysed by MS. Two conserved residues, K147 and K181, in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional data are available for K147, K181 lies within an interfacial region critical for virion assembly and stability. The signature immonium ion at m/z 126.0919 demonstrated the presence of N-acetyllysine, and the sequence fragment ions enabled an unambiguous assignment of the epsilon-N-acetyl modification on K181. We hypothesize that selection favours acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.

  6. Roles for the coat protein telokin-like domain and the scaffolding protein amino-terminus

    Science.gov (United States)

    Suhanovsky, Margaret M.; Teschke, Carolyn M.

    2011-01-01

    Assembly of icosahedral capsids of proper size and symmetry is not understood. Residue F170 in bacteriophage P22 coat protein is critical for conformational switching during assembly. Substitutions at this site cause assembly of tubes of hexamerically arranged coat protein. Intragenic suppressors of the ts phenotype of F170A and F170K coat protein mutants were isolated. Suppressors were repeatedly found in the coat protein telokin-like domain at position 285, which caused coat protein to assemble into petite procapsids and capsids. Petite capsid assembly strongly correlated to the side chain volume of the substituted amino acid. We hypothesize that larger side chains at position 285 torque the telokin-like domain, changing flexibility of the subunit and intercapsomer contacts. Thus, a single amino acid substitution in coat protein is sufficient to change capsid size. In addition, the products of assembly of the variant coat proteins were affected by the size of the internal scaffolding protein. PMID:21784500

  7. Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

    Science.gov (United States)

    Munoz, L; Sadaie, Y; Doi, R H

    1978-10-10

    The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.

  8. Engineered Protein Coatings to Improve the Osseointegration of Dental and Orthopaedic Implants

    Science.gov (United States)

    Raphel, Jordan; Karlsson, Johan; Galli, Silvia; Wennerberg, Ann; Lindsay, Christopher; Haugh, Matthew; Pajarinen, Jukka; Goodman, Stuart B.; Jimbo, Ryo; Andersson, Martin; Heilshorn, Sarah C.

    2016-01-01

    Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 hours, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure. PMID:26790146

  9. Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

    Science.gov (United States)

    Yu, Xiaohua; Biedrzycki, Adam H; Khalil, Andrew S; Hess, Dalton; Umhoefer, Jennifer M; Markel, Mark D; Murphy, William L

    2017-09-01

    Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Hydroxyapatite coating on the titanium substrate modulated by a recombinant collagen-like protein

    International Nuclear Information System (INIS)

    Pan Mingli; Kong Xiangdong; Cai Yurong; Yao Juming

    2011-01-01

    Research highlights: → Hydroxyapatite was deposited on alkali-heat treated Ti substrate by immersing in 1.5 x SBF solution containing the recombinant collagen-like protein. → The recombinant collagen-like protein accelerated the preferential nucleation and growth of hydroxyapatite along c axis on the Ti substrate. → Hydroxyapatite-collagen composite on the Ti substrate promoted the attachment, subsequently proliferation and differentiation of MG-63 cells. - Abstract: Plenty of techniques have been developed to modify the surface character of titanium (Ti) and its alloys in order to realize their biological bond to natural bone. In this work, a biomimetic process was employed to form a hydroxyapatite (HAp) coating on the alkali-heat treated Ti substrate in 1.5 times simulated body fluid (1.5 x SBF) with the addition of a recombinant collagen-like protein. The coating was characterized using SEM-EDX, FESEM, and XRD. Results showed that the recombinant collagen-like protein could accelerate the preferential nucleation and directional growth along c axis of HAp on the pretreated Ti substrates. The investigation of in vitro cell cultivation showed that the existence of recombinant collagen-like protein in coating could improve the initial cell adhesion, proliferation and differentiation of MG-63 cells, which implied the materials possessed excellent biocompatibility and had a wide potential in biomedical application.

  11. Hydroxyapatite coating on the titanium substrate modulated by a recombinant collagen-like protein

    Energy Technology Data Exchange (ETDEWEB)

    Pan Mingli [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Kong Xiangdong [College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Cai Yurong [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Yao Juming, E-mail: yaoj@zstu.edu.cn [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China)

    2011-04-15

    Research highlights: {yields} Hydroxyapatite was deposited on alkali-heat treated Ti substrate by immersing in 1.5 x SBF solution containing the recombinant collagen-like protein. {yields} The recombinant collagen-like protein accelerated the preferential nucleation and growth of hydroxyapatite along c axis on the Ti substrate. {yields} Hydroxyapatite-collagen composite on the Ti substrate promoted the attachment, subsequently proliferation and differentiation of MG-63 cells. - Abstract: Plenty of techniques have been developed to modify the surface character of titanium (Ti) and its alloys in order to realize their biological bond to natural bone. In this work, a biomimetic process was employed to form a hydroxyapatite (HAp) coating on the alkali-heat treated Ti substrate in 1.5 times simulated body fluid (1.5 x SBF) with the addition of a recombinant collagen-like protein. The coating was characterized using SEM-EDX, FESEM, and XRD. Results showed that the recombinant collagen-like protein could accelerate the preferential nucleation and directional growth along c axis of HAp on the pretreated Ti substrates. The investigation of in vitro cell cultivation showed that the existence of recombinant collagen-like protein in coating could improve the initial cell adhesion, proliferation and differentiation of MG-63 cells, which implied the materials possessed excellent biocompatibility and had a wide potential in biomedical application.

  12. TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER-Golgi intermediate compartments.

    Science.gov (United States)

    Hanna, Michael G; Block, Samuel; Frankel, E B; Hou, Feng; Johnson, Adam; Yuan, Lin; Knight, Gavin; Moresco, James J; Yates, John R; Ashton, Randolph; Schekman, Randy; Tong, Yufeng; Audhya, Anjon

    2017-09-12

    The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER-Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

  13. Detection of spore coat protein of Bacillus subtilis by immunological method

    International Nuclear Information System (INIS)

    Uchida, Aritsune; Kadota, Hajime

    1976-01-01

    The spore coat protein of Bacillus subtilis was separated, and the qualitative assay for the spore coat protein was made by use of the immunological technique. The immunological method was found to be useful for judging the maturation of spore coat in the course of sporulation. The spore coat protein antigen appeared at t 2 stage of sporulation. The addition of rifampicin at the earlier stages of sporulation inhibited the increase in content of the spore coat antigen. (auth.)

  14. Enhanced protein adsorption and patterning on nanostructured latex-coated paper.

    Science.gov (United States)

    Juvonen, Helka; Määttänen, Anni; Ihalainen, Petri; Viitala, Tapani; Sarfraz, Jawad; Peltonen, Jouko

    2014-06-01

    Specific interactions of extracellular matrix proteins with cells and their adhesion to the substrate are important for cell growth. A nanopatterned latex-coated paper substrate previously shown to be an excellent substrate for cell adhesion and 2D growth was studied for directed immobilization of proteins. The nanostructured latex surface was formed by short-wavelength IR irradiation of a two-component latex coating consisting of a hydrophilic film-forming styrene butadiene acrylonitrile copolymer and hydrophobic polystyrene particles. The hydrophobic regions of the IR-treated latex coating showed strong adhesion of bovine serum albumin (cell repelling protein), fibronectin (cell adhesive protein) and streptavidin. Opposite to the IR-treated surface, fibronectin and streptavidin had a poor affinity toward the untreated pristine latex coating. Detailed characterization of the physicochemical surface properties of the latex-coated substrates revealed that the observed differences in protein affinity were mainly due to the presence or absence of the protein repelling polar and charged surface groups. The protein adsorption was assisted by hydrophobic (dehydration) interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Prediction of protein-protein interactions in dengue virus coat proteins guided by low resolution cryoEM structures

    Directory of Open Access Journals (Sweden)

    Srinivasan Narayanaswamy

    2010-06-01

    Full Text Available Abstract Background Dengue virus along with the other members of the flaviviridae family has reemerged as deadly human pathogens. Understanding the mechanistic details of these infections can be highly rewarding in developing effective antivirals. During maturation of the virus inside the host cell, the coat proteins E and M undergo conformational changes, altering the morphology of the viral coat. However, due to low resolution nature of the available 3-D structures of viral assemblies, the atomic details of these changes are still elusive. Results In the present analysis, starting from Cα positions of low resolution cryo electron microscopic structures the residue level details of protein-protein interaction interfaces of dengue virus coat proteins have been predicted. By comparing the preexisting structures of virus in different phases of life cycle, the changes taking place in these predicted protein-protein interaction interfaces were followed as a function of maturation process of the virus. Besides changing the current notion about the presence of only homodimers in the mature viral coat, the present analysis indicated presence of a proline-rich motif at the protein-protein interaction interface of the coat protein. Investigating the conservation status of these seemingly functionally crucial residues across other members of flaviviridae family enabled dissecting common mechanisms used for infections by these viruses. Conclusions Thus, using computational approach the present analysis has provided better insights into the preexisting low resolution structures of virus assemblies, the findings of which can be made use of in designing effective antivirals against these deadly human pathogens.

  16. Expression of tomato yellow leaf curl virus coat protein using baculovirus expression system and evaluation of its utility as a viral antigen.

    Science.gov (United States)

    Elgaied, Lamiaa; Salem, Reda; Elmenofy, Wael

    2017-08-01

    DNA encoding the coat protein (CP) of an Egyptian isolate of tomato yellow leaf curl virus (TYLCV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of polyhedrin promoter. The generated recombinant baculovirus construct harboring the coat protein gene was characterized using PCR analysis. The recombinant coat protein expressed in infected insect cells was used as a coating antigen in an indirect Enzyme-linked immunosorbent assay (ELISA) and dot blot to test its utility for the detection of antibody generated against TYLCV virus particles. The results of ELISA and dot blot showed that the TYLCV-antibodies reacted positively with extracts of infected cells using the recombinant virus as a coating antigen with strong signals as well as the TYLCV infected tomato and beat plant extracts as positive samples. Scanning electron microscope examination showed that the expressed TYLCV coat protein was self-assembled into virus-like particles (VLPs) similar in size and morphology to TYLCV virus particles. These results concluded that, the expressed coat protein of TYLCV using baculovirus vector system is a reliable candidate for generation of anti-CP antibody for inexpensive detection of TYLCV-infected plants using indirect CP-ELISA or dot blot with high specificity.

  17. Exploring the interaction network of the Bacillus subtilis outer coat and crust proteins.

    Science.gov (United States)

    Krajčíková, Daniela; Forgáč, Vladimír; Szabo, Adam; Barák, Imrich

    2017-11-01

    Bacillus subtilis spores, representatives of an exceptionally resistant dormant cell type, are encircled by a thick proteinaceous layer called the spore coat. More than 80 proteins assemble into four distinct coat layers: a basement layer, an inner coat, an outer coat and a crust. As the spore develops inside the mother cell, spore coat proteins synthesized in the cytoplasm are gradually deposited onto the prespore surface. A small set of morphogenetic proteins necessary for spore coat morphogenesis are thought to form a scaffold to which the rest of the coat proteins are attached. Extensive localization and proteomic studies using wild type and mutant spores have revealed the arrangement of individual proteins within the spore coat layers. In this study we examined the interactions between the proteins localized to the outer coat and crust using a bacterial two hybrid system. These two layers are composed of at least 25 components. Self-interactions were observed for most proteins and numerous novel interactions were identified. The most interesting contacts are those made with the morphogenetic proteins CotE, CotY and CotZ; these could serve as a basis for understanding the specific roles of particular proteins in spore coat morphogenesis. Copyright © 2017 Elsevier GmbH. All rights reserved.

  18. Structure-function relationship of viral coat proteins : a site-directed spectroscopic study of M13 coat protein

    NARCIS (Netherlands)

    Stopar, D.

    1997-01-01

    This thesis describes the results of a spectroscopic study of the major coat protein of bacteriophage M13. During the infection process this protein is incorporated into the cytoplasmic membrane of Escherichia coli host cells. To specifically monitor the local structural changes

  19. Simple Coatings to Render Polystyrene Protein Resistant

    Directory of Open Access Journals (Sweden)

    Marcelle Hecker

    2018-02-01

    Full Text Available Non-specific protein adsorption is detrimental to the performance of many biomedical devices. Polystyrene is a commonly used material in devices and thin films. Simple reliable surface modification of polystyrene to render it protein resistant is desired in particular for device fabrication and orthogonal functionalisation schemes. This report details modifications carried out on a polystyrene surface to prevent protein adsorption. The trialed surfaces included Pluronic F127 and PLL-g-PEG, adsorbed on polystyrene, using a polydopamine-assisted approach. Quartz crystal microbalance with dissipation (QCM-D results showed only short-term anti-fouling success of the polystyrene surface modified with F127, and the subsequent failure of the polydopamine intermediary layer in improving its stability. In stark contrast, QCM-D analysis proved the success of the polydopamine assisted PLL-g-PEG coating in preventing bovine serum albumin adsorption. This modified surface is equally as protein-rejecting after 24 h in buffer, and thus a promising simple coating for long term protein rejection of polystyrene.

  20. Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

    Science.gov (United States)

    Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H

    1978-09-01

    Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

  1. Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation

    International Nuclear Information System (INIS)

    Zhang, Rongyue; Li, Qiang; Li, Juan; Zhou, Weiqing; Ye, Peili; Gao, Yang; Ma, Guanghui; Su, Zhiguo

    2012-01-01

    Protein denaturation and nonspecific adsorption on polymer media as a chromatographic support have been a problem which needs to be overcome. Macroporous poly(glycidyl methacrylate–divinylbezene) (PGMA–DVB) microspheres prepared in this study were firstly covalently coated with dextran through a three-step method. The dextran was firstly adsorbed onto the microspheres and then covalently bound to the PGMA–DVB microsphere through ether bonds which were formed by hydroxyl group reacting with epoxy group at the presence of 4-(Dimethylamino) pyridine. Finally, the coating dextran layer was crosslinked by ethylene glycol diglycidyl ether to form the continuous network coating. The coated microspheres were characterized by Fourier transform infrared spectra, scanning electron microscope, mercury porosimetry measurements, laser scanning confocal microscope, and protein adsorption experiments. Results showed that PGMA–DVB microspheres coated with dextran successfully maintained the macroporous structure and high permeability. The backpressure was only 1.69 MPa at a high flow rate of 2891 cm/h. Consequently, the hydrophilicity and biocompatibility of modified microspheres were greatly improved, and the contact angle decreased from 184° to 13°, and nonspecific adsorption of proteins was decreased to little or none. The clad dextran coating with large amounts of hydroxyl group was easily derived to be various functional groups. The derived media have great potential applications in rapid protein chromatography. - Highlights: ► Macroporous PGMA–DVB microspheres were covalently coated with dextran. ► The hydrophilicity of the coated microspheres was significantly improved. ► The irreversible adsorption of proteins was reduced to zero. ► The coated microspheres can maintain the macropore structure. ► The coated microspheres were applied to rapid protein separation.

  2. Bioactive Coatings for Orthopaedic Implants—Recent Trends in Development of Implant Coatings

    Directory of Open Access Journals (Sweden)

    Bill G. X. Zhang

    2014-07-01

    Full Text Available Joint replacement is a major orthopaedic procedure used to treat joint osteoarthritis. Aseptic loosening and infection are the two most significant causes of prosthetic implant failure. The ideal implant should be able to promote osteointegration, deter bacterial adhesion and minimize prosthetic infection. Recent developments in material science and cell biology have seen the development of new orthopaedic implant coatings to address these issues. Coatings consisting of bioceramics, extracellular matrix proteins, biological peptides or growth factors impart bioactivity and biocompatibility to the metallic surface of conventional orthopaedic prosthesis that promote bone ingrowth and differentiation of stem cells into osteoblasts leading to enhanced osteointegration of the implant. Furthermore, coatings such as silver, nitric oxide, antibiotics, antiseptics and antimicrobial peptides with anti-microbial properties have also been developed, which show promise in reducing bacterial adhesion and prosthetic infections. This review summarizes some of the recent developments in coatings for orthopaedic implants.

  3. Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Rongyue; Li, Qiang; Li, Juan; Zhou, Weiqing; Ye, Peili; Gao, Yang; Ma, Guanghui, E-mail: ghma@home.ipe.ac.cn; Su, Zhiguo

    2012-12-01

    Protein denaturation and nonspecific adsorption on polymer media as a chromatographic support have been a problem which needs to be overcome. Macroporous poly(glycidyl methacrylate-divinylbezene) (PGMA-DVB) microspheres prepared in this study were firstly covalently coated with dextran through a three-step method. The dextran was firstly adsorbed onto the microspheres and then covalently bound to the PGMA-DVB microsphere through ether bonds which were formed by hydroxyl group reacting with epoxy group at the presence of 4-(Dimethylamino) pyridine. Finally, the coating dextran layer was crosslinked by ethylene glycol diglycidyl ether to form the continuous network coating. The coated microspheres were characterized by Fourier transform infrared spectra, scanning electron microscope, mercury porosimetry measurements, laser scanning confocal microscope, and protein adsorption experiments. Results showed that PGMA-DVB microspheres coated with dextran successfully maintained the macroporous structure and high permeability. The backpressure was only 1.69 MPa at a high flow rate of 2891 cm/h. Consequently, the hydrophilicity and biocompatibility of modified microspheres were greatly improved, and the contact angle decreased from 184 Degree-Sign to 13 Degree-Sign , and nonspecific adsorption of proteins was decreased to little or none. The clad dextran coating with large amounts of hydroxyl group was easily derived to be various functional groups. The derived media have great potential applications in rapid protein chromatography. - Highlights: Black-Right-Pointing-Pointer Macroporous PGMA-DVB microspheres were covalently coated with dextran. Black-Right-Pointing-Pointer The hydrophilicity of the coated microspheres was significantly improved. Black-Right-Pointing-Pointer The irreversible adsorption of proteins was reduced to zero. Black-Right-Pointing-Pointer The coated microspheres can maintain the macropore structure. Black-Right-Pointing-Pointer The coated microspheres

  4. Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation.

    Science.gov (United States)

    Abhyankar, Wishwas; Pandey, Rachna; Ter Beek, Alexander; Brul, Stanley; de Koning, Leo J; de Koster, Chris G

    2015-02-01

    Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically (15)N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Protein-resistant polymer coatings obtained by matrix assisted pulsed laser evaporation

    Energy Technology Data Exchange (ETDEWEB)

    Rusen, L. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Mustaciosu, C. [Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH, Magurele, Bucharest (Romania); Mitu, B.; Filipescu, M.; Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Dinca, V., E-mail: dinali@nipne.ro [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania)

    2013-08-01

    Adsorption of proteins and polysaccharides is known to facilitate microbial attachment and subsequent formation of biofilm on surfaces that ultimately results in its biofouling. Therefore, protein repellent modified surfaces are necessary to block the irreversible attachment of microorganisms. Within this context, the feasibility of using the Poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether (PEG-block-PCL Me) copolymer as potential protein-resistant coating was explored in this work. The films were deposited using Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique that allows good control of composition, thickness and homogeneity. The chemical and morphological characteristics of the films were examined using Fourier Transform Infrared Spectroscopy (FTIR), contact angle measurements and Atomic Force Microscopy (AFM). The FTIR data demonstrates that the functional groups in the MAPLE-deposited films remain intact, especially for fluences below 0.5 J cm{sup −2}. Optical Microscopy and AFM images show that the homogeneity and the roughness of the coatings are related to both laser parameters (fluence, number of pulses) and target composition. Protein adsorption tests were performed on the PEG-block-PCL Me copolymer coated glass and on bare glass surface as a control. The results show that the presence of copolymer as coating significantly reduces the adsorption of proteins.

  6. Highly specific salt bridges govern bacteriophage P22 icosahedral capsid assembly: identification of the site in coat protein responsible for interaction with scaffolding protein.

    Science.gov (United States)

    Cortines, Juliana R; Motwani, Tina; Vyas, Aashay A; Teschke, Carolyn M

    2014-05-01

    Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. Bacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving

  7. Chitosan-Coated Collagen Membranes Promote Chondrocyte Adhesion, Growth, and Interleukin-6 Secretion

    Directory of Open Access Journals (Sweden)

    Nabila Mighri

    2015-11-01

    Full Text Available Designing scaffolds made from natural polymers may be highly attractive for tissue engineering strategies. We sought to produce and characterize chitosan-coated collagen membranes and to assess their efficacy in promoting chondrocyte adhesion, growth, and cytokine secretion. Porous collagen membranes were placed in chitosan solutions then crosslinked with glutaraldehyde vapor. Fourier transform infrared (FTIR analyses showed elevated absorption at 1655 cm-1 of the carbon–nitrogen (N=C bonds formed by the reaction between the (NH2 of the chitosan and the (C=O of the glutaraldehyde. A significant peak in the amide II region revealed a significant deacetylation of the chitosan. Scanning electron microscopy (SEM images of the chitosan-coated membranes exhibited surface variations, with pore size ranging from 20 to 50 µm. X-ray photoelectron spectroscopy (XPS revealed a decreased C–C groups and an increased C–N/C–O groups due to the reaction between the carbon from the collagen and the NH2 from the chitosan. Increased rigidity of these membranes was also observed when comparing the chitosan-coated and uncoated membranes at dried conditions. However, under wet conditions, the chitosan coated collagen membranes showed lower rigidity as compared to dried conditions. Of great interest, the glutaraldehyde-crosslinked chitosan-coated collagen membranes promoted chondrocyte adhesion, growth, and interleukin (IL-6 secretion. Overall results confirm the feasibility of using designed chitosan-coated collagen membranes in future applications, such as cartilage repair.

  8. Polydopamine-coated open tubular column for the separation of proteins by capillary electrochromatography.

    Science.gov (United States)

    Xiao, Xing; Wang, Wentao; Chen, Jia; Jia, Li

    2015-08-01

    The separation and determination of proteins in food is an important aspect in food industry. Inspired by the self-polymerization of dopamine under alkaline conditions and the natural adhesive properties of polydopamine, in this paper, a simple and economical method was developed for the preparation of polydopamine-coated open tubular column, in which ammonium persulfate was used as the source of oxygen to induce and facilitate the polymerization of dopamine to form polydopamine. In comparison with a naked fused-silica capillary, the direction and magnitude of the electro-osmotic flow of the as-prepared polydopamine-coated open tubular column could be manipulated by varying the pH values of background solutions due to the existence of amine and phenolic hydroxyl groups on polydopamine coating. The surface morphology of the polydopamine-coated open tubular column was studied by scanning electron microscopy, and the thickness of polydopamine coating was 106 nm. The performance of the polydopamine-coated open tubular column was validated by analysis of proteins. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.5%. In addition, the feasibility of the polydopamine-coated open tubular column for real samples was verified by the separation of proteins in chicken egg white and pure milk. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The movement protein and coat protein of alfalfa mosaic virus accumulate in structurally modified plasmodesmata

    NARCIS (Netherlands)

    van der Wel, N. N.; Goldbach, R. W.; van Lent, J. W.

    1998-01-01

    In systemically infected tissues of Nicotiana benthamiana, alfalfa mosaic virus (AMV) coat protein (CP) and movement protein (MP) are detected in plasmodesmata in a layer of three to four cells at the progressing front of infection. Besides the presence of these viral proteins, the plasmodesmata are

  10. Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

    Science.gov (United States)

    Masuda, Hiro-taka; Ishihara, Seiichiro; Harada, Ichiro; Mizutani, Takeomi; Ishikawa, Masayori; Kawabata, Kazushige; Haga, Hisashi

    2014-01-01

    We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

  11. Optically and biologically active mussel protein-coated double-walled carbon nanotubes.

    Science.gov (United States)

    Jung, Yong Chae; Muramatsu, Hiroyuki; Fujisawa, Kazunori; Kim, Jin Hee; Hayashi, Takuya; Kim, Yoong Ahm; Endo, Morinobu; Terrones, Mauricio; Dresselhaus, Mildred S

    2011-12-02

    A method of dispersing strongly bundled double-walled carbon nanotubes (DWNTs) via a homogeneous coating of mussel protein in an aqueous solution is presented. Optical activity, mechanical strength, as well as electrical conductivity coming from the nanotubes and the versatile biological activity from the mussel protein make mussel-coated DWNTs promising as a multifunctional scaffold and for anti-fouling materials. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. An Intramolecular Chaperone Inserted in Bacteriophage P22 Coat Protein Mediates Its Chaperonin-independent Folding*

    Science.gov (United States)

    Suhanovsky, Margaret M.; Teschke, Carolyn M.

    2013-01-01

    The bacteriophage P22 coat protein has the common HK97-like fold but with a genetically inserted domain (I-domain). The role of the I-domain, positioned at the outermost surface of the capsid, is unknown. We hypothesize that the I-domain may act as an intramolecular chaperone because the coat protein folds independently, and many folding mutants are localized to the I-domain. The function of the I-domain was investigated by generating the coat protein core without its I-domain and the isolated I-domain. The core coat protein shows a pronounced folding defect. The isolated I-domain folds autonomously and has a high thermodynamic stability and fast folding kinetics in the presence of a peptidyl prolyl isomerase. Thus, the I-domain provides thermodynamic stability to the full-length coat protein so that it can fold reasonably efficiently while still allowing the HK97-like core to retain the flexibility required for conformational switching during procapsid assembly and maturation. PMID:24126914

  13. The interaction of M13 coat protein with lipid bilayers : a spectroscopic study

    NARCIS (Netherlands)

    Sanders, J.C.

    1992-01-01

    In this thesis a small part of the reproductive cycle of the M13 bacteriophage is studied in more detail, namely the interaction of the major coat protein (MW 5240) with lipid bilayers. During the infection process is the major coat protein of M13 bacteriophage stored in the cytoplasm

  14. Development of bioactive coatings based on γ-irradiated proteins to preserve strawberries

    International Nuclear Information System (INIS)

    Vu, K.D.; Hollingsworth, R.G.; Salmieri, S.; Takala, P.N.; Lacroix, M.

    2012-01-01

    Gamma irradiation was applied for creating cross-linked proteins to enhance the physicochemical properties of edible films made of calcium caseinate, whey protein isolate and glycerol. The characteristics of γ irradiated cross-linked proteins were analyzed by Fourier Transform Infrared spectroscopy. A second derivative spectra exhibited changes in band intensities that were correlated to an increase of β-sheet structure and a decrease of α-helix and unordered fractions of γ irradiated-cross-linked proteins as compared to the control without irradiation. Furthermore, on addition of methylcellulose to the irradiated protein matrix it was found that it has potential in enhancing the puncture strength and has no detrimental effect on water vapor permeability of protein based films. Finally, these film formulations were used as bioactive edible coatings containing natural antimicrobial agents (limonene and peppermint) to preserve the shelf life of fresh strawberries during storage. The bioactive coatings containing peppermint was found to be more efficient as preserving coatings than the formulations containing limonene. Irradiated proteins/methylcellulose/peppermint formulation had only 40% of decay at day 8 while it was 65% for the control. - Highlights: ► Crosslinked proteins and antimicrobials agents was able to preserve strawberries. ► Crosslinked protein structure was more ordered. ► Films based on crosslinked proteins and methylcellulose enhanced puncture strength.

  15. Sensing of heavy metal ions by intrinsic TMV coat protein fluorescence

    Science.gov (United States)

    Bayram, Serene S.; Green, Philippe; Blum, Amy Szuchmacher

    2018-04-01

    We propose the use of a cysteine mutant of TMV coat protein as a signal transducer for the selective sensing and quantification of the heavy metal ions, Cd2+, Pb2+, Zn2+ and Ni2+ based on intrinsic tryptophan quenching. TMV coat protein is inexpensive, can be mass-produced since it is expressed and extracted from E-coli. It also displays several different functional groups, enabling a wide repertoire of bioconjugation chemistries; thus it can be easily integrated into functional devices. In addition, TMV-ion interactions have been widely reported and utilized for metallization to generate organic-inorganic hybrid composite novel materials. Building on these previous observations, we herein determine, for the first time, the TMV-ion binding constants assuming the static fluorescence quenching model. We also show that by comparing TMV-ion interactions between native and denatured coat protein, we can distinguish between chemically similar heavy metal ions such as cadmium and zinc ions.

  16. 40 CFR 174.512 - Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Potato Virus Y...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.512 Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance. Residues of Coat Protein of Potato Virus Y are exempt from...

  17. Isolation of nuclear proteins from flax (Linum usitatissimum L. seed coats for gene expression regulation studies

    Directory of Open Access Journals (Sweden)

    Renouard Sullivan

    2012-01-01

    Full Text Available Abstract Background While seed biology is well characterized and numerous studies have focused on this subject over the past years, the regulation of seed coat development and metabolism is for the most part still non-elucidated. It is well known that the seed coat has an essential role in seed development and its features are associated with important agronomical traits. It also constitutes a rich source of valuable compounds such as pharmaceuticals. Most of the cell genetic material is contained in the nucleus; therefore nuclear proteins constitute a major actor for gene expression regulation. Isolation of nuclear proteins responsible for specific seed coat expression is an important prerequisite for understanding seed coat metabolism and development. The extraction of nuclear proteins may be problematic due to the presence of specific components that can interfere with the extraction process. The seed coat is a rich source of mucilage and phenolics, which are good examples of these hindering compounds. Findings In the present study, we propose an optimized nuclear protein extraction protocol able to provide nuclear proteins from flax seed coat without contaminants and sufficient yield and quality for their use in transcriptional gene expression regulation by gel shift experiments. Conclusions Routinely, around 250 μg of nuclear proteins per gram of fresh weight were extracted from immature flax seed coats. The isolation protocol described hereafter may serve as an effective tool for gene expression regulation and seed coat-focused proteomics studies.

  18. 40 CFR 174.531 - Coat protein of plum pox virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of plum pox virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.531 Coat protein of plum pox virus; exemption from the requirement of a tolerance. Residues of the coat protein of plum pox virus in or on the...

  19. Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations

    Energy Technology Data Exchange (ETDEWEB)

    Moreno-Gordaliza, Estefanía, E-mail: emorenog@ucm.es [Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden (Netherlands); Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040, Madrid (Spain); Stigter, Edwin C.A. [Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden (Netherlands); Department of Molecular Cancer Research, Universitair Medisch Centrum Utrecht, Wilhelmina Kinder Ziekenhuis, Lundlaan 6, 3584, EA Utrecht (Netherlands); Lindenburg, Petrus W.; Hankemeier, Thomas [Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden (Netherlands)

    2016-06-07

    A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10{sup −9} m{sup 2} V{sup −1} s{sup −1}) when compared with unmodified fused silica (5.9 ± 0.1 10{sup −8} m{sup 2} V{sup −1} s{sup −1}). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1–1.8% coefficient-of-variation (CV) within a day) and 2–3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use. - Highlights: • New coating using recrystallized surface-layer proteins on

  20. A simple coated-tube assay for alpha-foeto protein for clinical use

    International Nuclear Information System (INIS)

    Dakubu, S.; Ahene, I.S.; Foli, A.K.

    1977-01-01

    A standard method for coating plastic tubes with antiserum has been applied to coat tubes with rabbit antiserum to human alpha-foeto protein. The coated plastic tubes have been used to set up a radioimmunoassay system which is sensitive and convenient for use on the occasional clinical sample. For a successful coated-tube assay, it was found necessary to modify the final incubation mixture from what was suitable in a standard double antibody assay system. (orig.) [de

  1. Protein-adsorption and Ca-phosphate formation on chitosan-bioactive glass composite coatings

    Science.gov (United States)

    Wagener, V.; Boccaccini, A. R.; Virtanen, S.

    2017-09-01

    In the last years, chitosan-bioactive glass (BG) composites have been developed and investigated as bioactive coatings for orthopedic applications. The increase of bioactivity occurs due to the stimulation of calcium-phosphate/hydroxyapatite formation on the surface while the coating is degrading. In the present work, protein adsorption and its influence on calcium-phosphate precipitation was studied for the first time on such composite coatings. The experiments involved coating of 316L stainless steel substrates with chitosan (Ch) and chitosan-bioactive glass (Ch-BG) and immersion of the coated samples in two different bovine serum albumin (BSA) containing solutions, namely DI H2O (with pH adjusted to about 7.2 with diluted NaOH) and simulated body fluid (SBF). In order to investigate the influence of protein adsorption on calcium-phosphate precipitation, samples were also immersed in DI H2O and in SBF without BSA. Samples were analyzed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Surface analysis revealed that adsorption of BSA takes place on all studied samples and that protein adsorption is influenced by the presence of Ca2+ and PO43- ions. Bioactivity in the form of hydroxyapatite pre-stage formation is significantly increased on Ch-BG composite coating as compared with bare stainless steel surface. However, calcium-phosphate precipitation in SBF is reduced by the presence of BSA.

  2. Facile Photoimmobilization of Proteins onto Low-Binding PEG-Coated Polymer Surfaces

    DEFF Research Database (Denmark)

    Larsen, Esben Kjær Unmack; Mikkelsen, Morten Bo Lindholm; Larsen, Niels Bent

    2014-01-01

    was verified for both enzymes and antibodies, and their presence on the surface was confirmed by X-ray photoelectron spectroscopy (XPS) and confocal fluorescence microscopy. Conjugation of capture antibody onto the PEG coating was employed for a simplified ELISA protocol without the need for blocking uncoated...... surface areas, showing ng/mL sensitivity to a cytokine antigen target. Moreover, spatially patterned attachment of fluorescently labeled protein onto the low-binding PEG-coated surface was achieved with a projection lithography system that enabled the creation of micrometer-sized protein features....

  3. Molecular interactions of mussel protective coating protein, mcfp-1, from Mytilus californianus.

    Science.gov (United States)

    Lu, Qingye; Hwang, Dong Soo; Liu, Yang; Zeng, Hongbo

    2012-02-01

    Protective coating of the byssus of mussels (Mytilus sp.) has been suggested as a new paradigm of medical coating due to its high extensibility and hardness co-existence without their mutual detriment. The only known biomacromolecule in the extensible and tough coating on the byssus is mussel foot protein-1 (mfp-1), which is made up with positively charged residues (~20 mol%) and lack of negatively charged residues. Here, adhesion and molecular interaction mechanisms of Mytilus californianus foot protein-1 (mcfp-1) from California blue mussel were investigated using a surface forces apparatus (SFA) in buffer solutions of different ionic concentrations (0.2-0.7 M) and pHs (3.0-5.5). Strong and reversible cohesion between opposed positively charged mcfp-1 films was measured in 0.1 M sodium acetate buffer with 0.1 M KNO(3). Cohesion of mcfp-1 was gradually reduced with increasing the ionic strength, but was not changed with pH variations. Oxidation of 3,4-dihydroxyphenylalanine (DOPA) residues of mcfp-1, a key residue for adhesive and coating proteins of mussel, didn't change the cohesion strength of mcfp-1 films, but the addition of chemicals with aromatic groups (i.e., aspirin and 4-methylcatechol) increased the cohesion. These results suggest that the cohesion of mcfp-1 films is mainly mediated by cation-π interactions between the positively charged residues and benzene rings of DOPA and other aromatic amino acids (~20 mol% of total amino acids of mcfp-1), and π-π interactions between the phenyl groups in mcfp-1. The adhesion mechanism obtained for the mcfp-1 proteins provides important insight into the design and development of functional biomaterials and coatings mimicking the extensible and robust mussel cuticle coating. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Anisotropic biodegradable lipid coated particles for spatially dynamic protein presentation.

    Science.gov (United States)

    Meyer, Randall A; Mathew, Mohit P; Ben-Akiva, Elana; Sunshine, Joel C; Shmueli, Ron B; Ren, Qiuyin; Yarema, Kevin J; Green, Jordan J

    2018-05-01

    There has been growing interest in the use of particles coated with lipids for applications ranging from drug delivery, gene delivery, and diagnostic imaging to immunoengineering. To date, almost all particles with lipid coatings have been spherical despite emerging evidence that non-spherical shapes can provide important advantages including reduced non-specific elimination and increased target-specific binding. We combine control of core particle geometry with control of particle surface functionality by developing anisotropic, biodegradable ellipsoidal particles with lipid coatings. We demonstrate that these lipid coated ellipsoidal particles maintain advantageous properties of lipid polymer hybrid particles, such as the ability for modular protein conjugation to the particle surface using versatile bioorthogonal ligation reactions. In addition, they exhibit biomimetic membrane fluidity and demonstrate lateral diffusive properties characteristic of natural membrane proteins. These ellipsoidal particles simultaneously provide benefits of non-spherical particles in terms of stability and resistance to non-specific phagocytosis by macrophages as well as enhanced targeted binding. These biomaterials provide a novel and flexible platform for numerous biomedical applications. The research reported here documents the ability of non-spherical polymeric particles to be coated with lipids to form anisotropic biomimetic particles. In addition, we demonstrate that these lipid-coated biodegradable polymeric particles can be conjugated to a wide variety of biological molecules in a "click-like" fashion. This is of interest due to the multiple types of cellular mimicry enabled by this biomaterial based technology. These features include mimicry of the highly anisotropic shape exhibited by cells, surface presentation of membrane bound protein mimetics, and lateral diffusivity of membrane bound substrates comparable to that of a plasma membrane. This platform is demonstrated to

  5. Screening of Potential Inhibitor against Coat Protein of Apple Chlorotic Leaf Spot Virus.

    Science.gov (United States)

    Purohit, Rituraj; Kumar, Sachin; Hallan, Vipin

    2018-06-01

    In this study, we analyzed Coat protein (CP) of Apple chlorotic leaf spot virus (ACLSV), an important latent virus on Apple. Incidence of the virus is upto 60% in various apple cultivars, affecting yield losses of the order of 10-40% (depending upon the cultivar). CP plays an important role as the sole building block of the viral capsid. Homology approach was used to model 193 amino acid sequence of the coat protein. We used various servers such as ConSurf, TargetS, OSML, COACH, COFACTOR for the prediction of active site residues in coat protein. Virtual screening strategy was employed to search potential inhibitors for CP. Top twenty screened molecules considered for drugability, and toxicity analysis and one potential molecule was further analyzed by docking analysis. Here, we reported a potent molecule which could inhibit the formation of viron assembly by targeting the CP protein of virus.

  6. Promoters and proteins from Clostridium thermocellum and uses thereof

    Science.gov (United States)

    Wu, J. H. David; Newcomb, Michael

    2012-11-13

    The present invention relates to an inducible and a high expression nucleic acid promoter isolated from Clostridium thermocellum. These promoters are useful for directing expression of a protein or polypeptide encoded by a nucleic acid molecule operably associated with the nucleic acid promoters. The present invention also relates to nucleic acid constructs including the C. thermocellum promoters, and expression vectors and hosts containing such nucleic acid constructs. The present invention also relates to protein isolated from Clostridium thermocellum, including a repressor protein. The present invention also provides methods of using the isolated promoters and proteins from Clostridium thermocellum, including methods for directing inducible in vitro and in vivo expression of a protein or polypeptide in a host, and methods of producing ethanol from a cellulosic biomass.

  7. Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries.

    Science.gov (United States)

    Liu, Shaorong; Gao, Lin; Pu, Qiaosheng; Lu, Joann J; Wang, Xingjia

    2006-02-01

    We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.

  8. Crystal structure of the bacteriophage Qβ coat protein in complex with the RNA operator of the replicase gene.

    Science.gov (United States)

    Rumnieks, Janis; Tars, Kaspars

    2014-03-06

    The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein-RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA. © 2013.

  9. Membrane-bound conformation of M13 major coat protein : a structure validation through FRET-derived constraints

    NARCIS (Netherlands)

    Vos, W.L.; Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

    2005-01-01

    M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein

  10. Contrasting evolutionary patterns of spore coat proteins in two Bacillus species groups are linked to a difference in cellular structure

    Science.gov (United States)

    2013-01-01

    Background The Bacillus subtilis-group and the Bacillus cereus-group are two well-studied groups of species in the genus Bacillus. Bacteria in this genus can produce a highly resistant cell type, the spore, which is encased in a complex protective protein shell called the coat. Spores in the B. cereus-group contain an additional outer layer, the exosporium, which encircles the coat. The coat in B. subtilis spores possesses inner and outer layers. The aim of this study is to investigate whether differences in the spore structures influenced the divergence of the coat protein genes during the evolution of these two Bacillus species groups. Results We designed and implemented a computational framework to compare the evolutionary histories of coat proteins. We curated a list of B. subtilis coat proteins and identified their orthologs in 11 Bacillus species based on phylogenetic congruence. Phylogenetic profiles of these coat proteins show that they can be divided into conserved and labile ones. Coat proteins comprising the B. subtilis inner coat are significantly more conserved than those comprising the outer coat. We then performed genome-wide comparisons of the nonsynonymous/synonymous substitution rate ratio, dN/dS, and found contrasting patterns: Coat proteins have significantly higher dN/dS in the B. subtilis-group genomes, but not in the B. cereus-group genomes. We further corroborated this contrast by examining changes of dN/dS within gene trees, and found that some coat protein gene trees have significantly different dN/dS between the B subtilis-clade and the B. cereus-clade. Conclusions Coat proteins in the B. subtilis- and B. cereus-group species are under contrasting selective pressures. We speculate that the absence of the exosporium in the B. subtilis spore coat effectively lifted a structural constraint that has led to relaxed negative selection pressure on the outer coat. PMID:24283940

  11. EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy.

    Directory of Open Access Journals (Sweden)

    Catarina Oliveira Silva

    Full Text Available The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100-200 nm showed a plasmon absorption band located within the near-infrared range (650-900 nm, optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0-25%. Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue.

  12. EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy

    Science.gov (United States)

    Silva, Catarina Oliveira; Petersen, Steffen B.; Reis, Catarina Pinto; Rijo, Patrícia; Molpeceres, Jesús; Fernandes, Ana Sofia; Gonçalves, Odete; Gomes, Andreia C.; Correia, Isabel; Vorum, Henrik; Neves-Petersen, Maria Teresa

    2016-01-01

    The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100–200 nm) showed a plasmon absorption band located within the near-infrared range (650–900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0–25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue. PMID:27788212

  13. The Influence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores.

    Science.gov (United States)

    Abhyankar, Wishwas R; Kamphorst, Kiki; Swarge, Bhagyashree N; van Veen, Henk; van der Wel, Nicole N; Brul, Stanley; de Koster, Chris G; de Koning, Leo J

    2016-01-01

    Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14 N spores prepared on solid Schaeffer's-glucose (SG) agar plates and 15 N metabolically labeled spores prepared in shake flasks containing 3-( N -morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14 N: 15 N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the

  14. The Influence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores

    Science.gov (United States)

    Abhyankar, Wishwas R.; Kamphorst, Kiki; Swarge, Bhagyashree N.; van Veen, Henk; van der Wel, Nicole N.; Brul, Stanley; de Koster, Chris G.; de Koning, Leo J.

    2016-01-01

    Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14N spores prepared on solid Schaeffer’s-glucose (SG) agar plates and 15N metabolically labeled spores prepared in shake flasks containing 3-(N-morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14N:15N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the

  15. The influence of sporulation conditions on the spore coat protein composition of Bacillus subtilis spores.

    Directory of Open Access Journals (Sweden)

    Wishwas R. Abhyankar

    2016-10-01

    Full Text Available Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14N spores prepared on solid SG agar plates and 15N metabolically labelled spores prepared in shake flasks containing MOPS buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14N: 15N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and

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

    Science.gov (United States)

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

    2012-10-07

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

  17. Albizia lebbeck Seed Coat Proteins Bind to Chitin and Act as a Defense against Cowpea Weevil Callosobruchus maculatus.

    Science.gov (United States)

    Silva, Nadia C M; De Sá, Leonardo F R; Oliveira, Eduardo A G; Costa, Monique N; Ferreira, Andre T S; Perales, Jonas; Fernandes, Kátia V S; Xavier-Filho, Jose; Oliveira, Antonia E A

    2016-05-11

    The seed coat is an external tissue that participates in defense against insects. In some nonhost seeds, including Albizia lebbeck, the insect Callosobruchus maculatus dies during seed coat penetration. We investigated the toxicity of A. lebbeck seed coat proteins to C. maculatus. A chitin-binding protein fraction was isolated from seed coat, and mass spectrometry showed similarity to a C1 cysteine protease. By ELM program an N-glycosylation interaction motif was identified in this protein, and by molecular docking the potential to interact with N-acetylglucosamine (NAG) was shown. The chitin-binding protein fraction was toxic to C. maculatus and was present in larval midgut and feces but not able to hydrolyze larval gut proteins. It did not interfere, though, with the intestinal cell permeability. These results indicate that the toxicity mechanism of this seed coat fraction may be related to its binding to chitin, present in the larvae gut, disturbing nutrient absorption.

  18. Dual Function of Novel Pollen Coat (Surface) Proteins: IgE-binding Capacity and Proteolytic Activity Disrupting the Airway Epithelial Barrier

    Science.gov (United States)

    Bashir, Mohamed Elfatih H.; Ward, Jason M.; Cummings, Matthew; Karrar, Eltayeb E.; Root, Michael; Mohamed, Abu Bekr A.; Naclerio, Robert M.; Preuss, Daphne

    2013-01-01

    Background The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., “de-fatted”), and, as a result, their involvement in allergy has not been explored. Methodology/Principal Findings Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. Conclusions/Significance Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is

  19. Dual function of novel pollen coat (surface proteins: IgE-binding capacity and proteolytic activity disrupting the airway epithelial barrier.

    Directory of Open Access Journals (Sweden)

    Mohamed Elfatih H Bashir

    Full Text Available BACKGROUND: The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., "de-fatted", and, as a result, their involvement in allergy has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass pollen (BGP by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP and endoxylanase (EXY. The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. CONCLUSIONS/SIGNIFICANCE: Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic

  20. Promotion of tribological and hydrophobic properties of a coating on TPE substrates by atmospheric plasma-polymerization

    Science.gov (United States)

    Sainz-García, Elisa; Alba-Elías, Fernando; Múgica-Vidal, Rodolfo; Pantoja-Ruiz, Mariola

    2016-05-01

    Thermoplastic elastomers (TPE) are used in the automotive sealing industry with the objective of producing anti-friction and hydrophobic components. At present, the anti-friction property is achieved by the electrostatic flocking, which sometimes produces an irregular coating. Therefore, this paper's objective is the promotion of adhesion of an anti-friction (based on the silane aminopropyltriethoxysilane-APTES-) and hydrophobic (based on the fluorinated precursor 1-perfluorohexene-PFH-) coating by the adhesion promoter, APTES. Different mixtures of APTES and PFH have been applied to a TPE substrate by an Atmospheric Pressure Plasma Jet (APPJ) system with Dielectric Barrier Discharge (DBD) in order to determine the optimal mixture of precursors. The main difficulty in this work lies in the hydrophilic character of APTES and the low adhesion of the fluorinated coatings. The sample coated with a mixture of 50% APTES and 50% PFH (A50P50) was found to be the best one to satisfy both properties at the same time, despite not having the highest dynamic water contact angle (WCA) or the lowest friction coefficient.

  1. The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation.

    Science.gov (United States)

    Ribis, John W; Ravichandran, Priyanka; Putnam, Emily E; Pishdadian, Keyan; Shen, Aimee

    2017-01-01

    The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis , only two of these morphogenetic proteins have homologs in the Clostridia : SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis . Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis , C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia , but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and

  2. The regulated synthesis of a Bacillus anthracis spore coat protein that affects spore surface properties.

    Science.gov (United States)

    Aronson, A; Goodman, B; Smith, Z

    2014-05-01

    Examine the regulation of a spore coat protein and the effects on spore properties. A c. 23 kDa band in coat/exosporial extracts of Bacillus anthracis Sterne spores varied in amount depending upon the conditions of sporulation. It was identified by MALDI as a likely orthologue of ExsB of Bacillus cereus. Little if any was present in an exosporial preparation with a location to the inner coat/cortex region established by spore fractionation and immunogold labelling of electron micrograph sections. Because of its predominant location in the inner coat, it has been renamed Cotγ. It was relatively deficient in spores produced at 37°C and when acidic fermentation products were produced a difference attributable to transcriptional regulation. The deficiency or absence of Cotγ resulted in a less robust exosporium positioned more closely to the coat. These spores were less hydrophobic and germinated somewhat more rapidly. Hydrophobicity and appearance were rescued in the deletion strain by introduction of the cotγ gene. The deficiency or lack of a protein largely found in the inner coat altered spore hydrophobicity and surface appearance. The regulated synthesis of Cotγ may be a paradigm for other spore coat proteins with unknown functions that modulate spore properties in response to environmental conditions. © 2014 The Society for Applied Microbiology.

  3. The architecture of mammalian ribosomal protein promoters

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    Perry Robert P

    2005-02-01

    Full Text Available Abstract Background Mammalian ribosomes contain 79 different proteins encoded by widely scattered single copy genes. Coordinate expression of these genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. To date, detailed studies of only a very few ribosomal protein (rp promoters have been made. To elucidate the general features of rp promoter architecture, I made a detailed sequence comparison of the promoter regions of the entire set of orthologous human and mouse rp genes. Results A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y2C+1TY(T2(Y3. At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point. Conclusions This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes.

  4. Unraveling the Role of the C-terminal Helix Turn Helix of the Coat-binding Domain of Bacteriophage P22 Scaffolding Protein*

    Science.gov (United States)

    Padilla-Meier, G. Pauline; Gilcrease, Eddie B.; Weigele, Peter R.; Cortines, Juliana R.; Siegel, Molly; Leavitt, Justin C.; Teschke, Carolyn M.; Casjens, Sherwood R.

    2012-01-01

    Many viruses encode scaffolding and coat proteins that co-assemble to form procapsids, which are transient precursor structures leading to progeny virions. In bacteriophage P22, the association of scaffolding and coat proteins is mediated mainly by ionic interactions. The coat protein-binding domain of scaffolding protein is a helix turn helix structure near the C terminus with a high number of charged surface residues. Residues Arg-293 and Lys-296 are particularly important for coat protein binding. The two helices contact each other through hydrophobic side chains. In this study, substitution of the residues of the interface between the helices, and the residues in the β-turn, by aspartic acid was used examine the importance of the conformation of the domain in coat binding. These replacements strongly affected the ability of the scaffolding protein to interact with coat protein. The severity of the defect in the association of scaffolding protein to coat protein was dependent on location, with substitutions at residues in the turn and helix 2 causing the most significant effects. Substituting aspartic acid for hydrophobic interface residues dramatically perturbs the stability of the structure, but similar substitutions in the turn had much less effect on the integrity of this domain, as determined by circular dichroism. We propose that the binding of scaffolding protein to coat protein is dependent on angle of the β-turn and the orientation of the charged surface on helix 2. Surprisingly, formation of the highly complex procapsid structure depends on a relatively simple interaction. PMID:22879595

  5. Albumen foam stability and s-ovalbumin contents in eggs coated with whey protein concentrate

    Directory of Open Access Journals (Sweden)

    ACC Alleoni

    2004-06-01

    Full Text Available Food products such as breads, cakes, crackers, meringues, ice creams and several bakery items depend on air incorporation to maintain their texture and structure during or after processing. Proteins are utilized in the food industry since they improve texture attributes through their ability to encapsulate and retain air. The objectives of this work were to quantify s-ovalbumin contents in albumen and to determine alterations in egg white foam stability in fresh eggs, and in eggs coated and non-coated with a whey protein-based concentrate film (WPC, stored at 25°C for 28 days. The volume of drained liquid was higher in non-coated eggs than in coated eggs stored at 25°C at all storage periods. The difference on the third day of storage was in the order of 59% between coated and non-coated eggs, while on the twenty-eighth day it was 202%. During the storage period, an increase in pH and drainage volume was observed for non-coated eggs. After three days, the non-coated eggs showed a s-ovalbumin content 33% higher than coated eggs; this increase jumped to 205% at 28 days of storage. There was a positive correlation between s-ovalbumin content and the volume of drained liquid for coated and non-coated eggs; in other words, when the s-ovalbumin content increased, there was an increase in the volume of drained liquid and a decrease in foam stability. WPC coating maintain egg quality, since it is an effective barrier against the loss of CO2, avoiding changes in the pH of egg white.

  6. Protein Hydrolysates as Promoters of Non-Haem Iron Absorption

    Science.gov (United States)

    Li, Yanan; Jiang, Han; Huang, Guangrong

    2017-01-01

    Iron (Fe) is an essential micronutrient for human growth and health. Organic iron is an excellent iron supplement due to its bioavailability. Both amino acids and peptides improve iron bioavailability and absorption and are therefore valuable components of iron supplements. This review focuses on protein hydrolysates as potential promoters of iron absorption. The ability of protein hydrolysates to chelate iron is thought to be a key attribute for the promotion of iron absorption. Iron-chelatable protein hydrolysates are categorized by their absorption forms: amino acids, di- and tri-peptides and polypeptides. Their structural characteristics, including their size and amino acid sequence, as well as the presence of special amino acids, influence their iron chelation abilities and bioavailabilities. Protein hydrolysates promote iron absorption by keeping iron soluble, reducing ferric iron to ferrous iron, and promoting transport across cell membranes into the gut. We also discuss the use and relative merits of protein hydrolysates as iron supplements. PMID:28617327

  7. Protein Hydrolysates as Promoters of Non-Haem Iron Absorption

    Directory of Open Access Journals (Sweden)

    Yanan Li

    2017-06-01

    Full Text Available Iron (Fe is an essential micronutrient for human growth and health. Organic iron is an excellent iron supplement due to its bioavailability. Both amino acids and peptides improve iron bioavailability and absorption and are therefore valuable components of iron supplements. This review focuses on protein hydrolysates as potential promoters of iron absorption. The ability of protein hydrolysates to chelate iron is thought to be a key attribute for the promotion of iron absorption. Iron-chelatable protein hydrolysates are categorized by their absorption forms: amino acids, di- and tri-peptides and polypeptides. Their structural characteristics, including their size and amino acid sequence, as well as the presence of special amino acids, influence their iron chelation abilities and bioavailabilities. Protein hydrolysates promote iron absorption by keeping iron soluble, reducing ferric iron to ferrous iron, and promoting transport across cell membranes into the gut. We also discuss the use and relative merits of protein hydrolysates as iron supplements.

  8. Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels

    Science.gov (United States)

    Scott, Evan A.; Nichols, Michael D.; Cordova, Lee H.; George, Brandon J.; Jun, Young-Shin; Elbert, Donald L.

    2008-01-01

    Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically-active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating. PMID:18771802

  9. The promotion of osseointegration of titanium surfaces by coating with silk protein sericin.

    Science.gov (United States)

    Nayak, Sunita; Dey, Tuli; Naskar, Deboki; Kundu, Subhas C

    2013-04-01

    A promising strategy to influence the osseointegration process around orthopaedic titanium implants is the immobilization of bioactive molecules. This recruits appropriate interaction between the surface and the tissue by directing cells adhesion, proliferation, differentiation and active matrix remodelling. In this study, we aimed to investigate the functionalization of metallic implant titanium with silk protein sericin. Titanium surface was immobilized with non-mulberry Antheraea mylitta sericin using glutaraldehyde as crosslinker. To analyse combinatorial effects the sericin immobilized titanium was further conjugated with integrin binding peptide sequence Arg-Gly-Asp (RGD) using ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling agents. The surface of sericin immobilized titanium was characterized biophysically. Osteoblast-like cells were cultured on sericin and sericin/RGD functionalized titanium and found to be more viable than those on pristine titanium. The enhanced adhesion, proliferation, and differentiation of osteoblast cells were observed. RT-PCR analysis showed that mRNA expressions of bone sialoprotein, osteocalcin and alkaline phosphatase were upregulated in osteoblast cells cultured on sericin and sericin/RGD immobilized titanium substrates. Additionally, no significant amount of pro-inflammatory cytokines TNF-α, IL-1β and nitric oxide production were recorded when macrophages cells and osteoblast-macrophages co culture cells were grown on sericin immobilized titanium. The findings demonstrate that the sericin immobilized titanium surfaces are potentially useful bioactive coated materials for titanium-based medical implants. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Natural supramolecular building blocks: from virus coat proteins to viral nanoparticles.

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    Liu, Zhi; Qiao, Jing; Niu, Zhongwei; Wang, Qian

    2012-09-21

    Viruses belong to a fascinating class of natural supramolecular structures, composed of multiple copies of coat proteins (CPs) that assemble into different shapes with a variety of sizes from tens to hundreds of nanometres. Because of their advantages including simple/economic production, well-defined structural features, unique shapes and sizes, genetic programmability and robust chemistries, recently viruses and virus-like nanoparticles (VLPs) have been used widely in biomedical applications and materials synthesis. In this critical review, we highlight recent advances in the use of virus coat proteins (VCPs) and viral nanoparticles (VNPs) as building blocks in self-assembly studies and materials development. We first discuss the self-assembly of VCPs into VLPs, which can efficiently incorporate a variety of different materials as cores inside the viral protein shells. Then, the self-assembly of VNPs at surfaces or interfaces is summarized. Finally, we discuss the co-assembly of VNPs with different functional materials (178 references).

  11. Intravascular local gene transfer mediated by protein-coated metallic stent.

    Science.gov (United States)

    Yuan, J; Gao, R; Shi, R; Song, L; Tang, J; Li, Y; Tang, C; Meng, L; Yuan, W; Chen, Z

    2001-10-01

    To assess the feasibility, efficiency and selectivity of adenovirus-mediated gene transfer to local arterial wall by protein-coated metallic stent. A replication-defective recombinant adenovirus carrying the Lac Z reporter gene for nuclear-specific beta-galactosidase (Ad-beta gal) was used in this study. The coating for metallic stent was made by immersing it in a gelatin solution containing crosslinker. The coated stents were mounted on a 4.0 or 3.0 mm percutaneous transluminal coronary angioplasty (PTCA) balloon and submersed into a high-titer Ad-beta gal viral stock (2 x 10(10) pfu/ml) for 3 min, and then implanted into the carotid arteries in 4 mini-swines and into the left anterior descending branch of the coronary artery in 2 mini-swines via 8F large lumen guiding catheters. The animals were sacrificed 7 (n = 4), 14 (n = 1) and 21 (n = 1) days after implantation, respectively. The beta-galactosidase expression was assessed by X-gal staining. The results showed that the expression of transgene was detected in all animal. In 1 of carotid artery with an intact intima, the beta-gal expression was limited to endothelial cells. In vessels with denuded endothelium, gene expression was found in the sub-intima, media and adventitia. The transfection efficiency of medial smooth muscle cells was 38.6%. In 2 animals sacrificed 7 days after transfection, a microscopic examination of X-gal-stained samples did not show evidence of transfection in remote organs and arterial segments adjacent to the treated arterial site. Adenovirus-mediated arterial gene transfer to endothelial, smooth muscle cells and adventitia by protein-coated metallic stent is feasible. The transfection efficiency is higher. The coated stent may act as a good carrier of adenovirus-mediated gene transfer and have a potential to prevent restenosis following PTCA.

  12. Thickness and morphology of polyelectrolyte coatings on silica surfaces before and after protein exposure studied by atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Haselberg, Rob, E-mail: r.haselberg@vu.nl [Biomolecular Analysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht (Netherlands); AIMMS Division of BioMolecular Analysis, VU University Amsterdam, de Boelelaan 1083, 1081 HV Amsterdam (Netherlands); Flesch, Frits M. [Biomolecular Analysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht (Netherlands); Boerke, Arjan [Department of Biochemistry and Cell Biology, Utrecht University, Yalelaan 2, 3508 TD Utrecht (Netherlands); Somsen, Govert W. [Biomolecular Analysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht (Netherlands); AIMMS Division of BioMolecular Analysis, VU University Amsterdam, de Boelelaan 1083, 1081 HV Amsterdam (Netherlands)

    2013-05-24

    Graphical abstract: -- Highlights: •Atomic force microscopy is used to characterize polyelectrolyte coatings. •Coating procedure leads to nm-thick layers on a silica surface. •Polyelectrolyte coatings effectively prevent protein adsorption. •AFM provides the high resolution to investigate these thin films. •AFM results support earlier findings obtained with capillary electrophoresis. -- Abstract: Analyte–wall interaction is a significant problem in capillary electrophoresis (CE) as it may compromise separation efficiencies and migration time repeatability. In CE, self-assembled polyelectrolyte multilayer films of Polybrene (PB) and dextran sulfate (DS) or poly(vinylsulfonic acid) (PVS) have been used to coat the capillary inner wall and thereby prevent analyte adsorption. In this study, atomic force microscopy (AFM) was employed to investigate the layer thickness and surface morphology of monolayer (PB), bilayer, (PB-DS and PB-PVS), and trilayer (PB-DS-PB and PB-PVS-PB) coatings on glass surfaces. AFM nanoshaving experiments providing height distributions demonstrated that the coating procedures led to average layer thicknesses between 1 nm (PB) and 5 nm (PB-DS-PB), suggesting the individual polyelectrolytes adhere flat on the silica surface. Investigation of the surface morphology of the different coatings by AFM revealed that the PB coating does not completely cover the silica surface, whereas full coverage was observed for the trilayer coatings. The DS-containing coatings appeared on average 1 nm thicker than the corresponding PVS-containing coatings, which could be attributed to the molecular structure of the anionic polymers applied. Upon exposure to the basic protein cytochrome c, AFM measurements showed an increase of the layer thickness for bare (3.1 nm) and PB-DS-coated (4.6 nm) silica, indicating substantial protein adsorption. In contrast, a very small or no increase of the layer thickness was observed for the PB and PB-DS-PB coatings

  13. Coat protein-mediated resistance against an Indian isolate of the ...

    Indian Academy of Sciences (India)

    Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were ...

  14. The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein.

    Science.gov (United States)

    Saunders, Keith; Lomonossoff, George P

    2015-01-01

    Turnip crinkle virus (TCV), a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T = 3 structure) but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T = 1 structures that are free of RNA. The possibility of utilizing TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein's exterior P domain plays a key role in particle formation.

  15. Nanocomposited coatings produced by laser-assisted process to prevent silicone hydogels from protein fouling and bacterial contamination

    International Nuclear Information System (INIS)

    Huang, Guobang; Chen, Yi; Zhang, Jin

    2016-01-01

    Graphical abstract: Nanocomposited-coating was deposited on silicone hydrogel by using the matrix-assisted pulsed laser evaporation (MAPLE) process. The ZnO–PEG nanocomposited coating reduces over 50% protein absorption on silicone hydrogel, and can inhibit the bacterial growth efficiently. - Highlights: • We developed a nanocomposited coating to prevent silicone hydrogel from biofouling. • Matrix-assisted pulsed laser evaporation can deposit inorganic–organic nanomaterials. • The designed nanocomposited coating reduces protein absorption by over 50%. • The designed nanocomposited coating shows significant antimicrobial efficiency. - Abstract: Zinc oxide (ZnO) nanoparticles incorporating with polyethylene glycol (PEG) were deposited together on the surface of silicone hydrogel through matrix-assisted pulsed laser evaporation (MAPLE). In this process, frozen nanocomposites (ZnO–PEG) in isopropanol were irradiated under a pulsed Nd:YAG laser at 532 nm for 1 h. Our results indicate that the MAPLE process is able to maintain the chemical backbone of polymer and prevent the nanocomposite coating from contamination. The ZnO–PEG nanocomposited coating reduces over 50% protein absorption on silicone hydrogel. The cytotoxicity study shows that the ZnO–PEG nanocomposites deposited on silicone hydrogels do not impose the toxic effect on mouse NIH/3T3 cells. In addition, MAPLE-deposited ZnO–PEG nanocomposites can inhibit the bacterial growth significantly.

  16. Recyclability of PET/WPI/PE Multilayer Films by Removal of Whey Protein Isolate-Based Coatings with Enzymatic Detergents

    Directory of Open Access Journals (Sweden)

    Patrizia Cinelli

    2016-06-01

    Full Text Available Multilayer plastic films provide a range of properties, which cannot be obtained from monolayer films but, at present, their recyclability is an open issue and should be improved. Research to date has shown the possibility of using whey protein as a layer material with the property of acting as an excellent barrier against oxygen and moisture, replacing petrochemical non-recyclable materials. The innovative approach of the present research was to achieve the recyclability of the substrate films by separating them, with a simple process compatible with industrial procedures, in order to promote recycling processes leading to obtain high value products that will beneficially impact the packaging and food industries. Hence, polyethyleneterephthalate (PET/polyethylene (PE multi-layer film was prepared based on PET coated with a whey protein layer, and then the previous structure was laminated with PE. Whey proteins, constituting the coating, can be degraded by enzymes so that the coating films can be washed off from the plastic substrate layer. Enzyme types, dosage, time, and temperature optima, which are compatible with procedures adopted in industrial waste recycling, were determined for a highly-efficient process. The washing of samples based on PET/whey and PET/whey/PE were efficient when performed with enzymatic detergent containing protease enzymes, as an alternative to conventional detergents used in recycling facilities. Different types of enzymatic detergents tested presented positive results in removing the protein layer from the PET substrate and from the PET/whey/PE multilayer films at room temperature. These results attested to the possibility of organizing the pre-treatment of the whey-based multilayer film by washing with different available commercial enzymatic detergents in order to separate PET and PE, thus allowing a better recycling of the two different polymers. Mechanical properties of the plastic substrate, such as stress at

  17. Recyclability of PET/WPI/PE Multilayer Films by Removal of Whey Protein Isolate-Based Coatings with Enzymatic Detergents

    Science.gov (United States)

    Cinelli, Patrizia; Schmid, Markus; Bugnicourt, Elodie; Coltelli, Maria Beatrice; Lazzeri, Andrea

    2016-01-01

    Multilayer plastic films provide a range of properties, which cannot be obtained from monolayer films but, at present, their recyclability is an open issue and should be improved. Research to date has shown the possibility of using whey protein as a layer material with the property of acting as an excellent barrier against oxygen and moisture, replacing petrochemical non-recyclable materials. The innovative approach of the present research was to achieve the recyclability of the substrate films by separating them, with a simple process compatible with industrial procedures, in order to promote recycling processes leading to obtain high value products that will beneficially impact the packaging and food industries. Hence, polyethyleneterephthalate (PET)/polyethylene (PE) multi-layer film was prepared based on PET coated with a whey protein layer, and then the previous structure was laminated with PE. Whey proteins, constituting the coating, can be degraded by enzymes so that the coating films can be washed off from the plastic substrate layer. Enzyme types, dosage, time, and temperature optima, which are compatible with procedures adopted in industrial waste recycling, were determined for a highly-efficient process. The washing of samples based on PET/whey and PET/whey/PE were efficient when performed with enzymatic detergent containing protease enzymes, as an alternative to conventional detergents used in recycling facilities. Different types of enzymatic detergents tested presented positive results in removing the protein layer from the PET substrate and from the PET/whey/PE multilayer films at room temperature. These results attested to the possibility of organizing the pre-treatment of the whey-based multilayer film by washing with different available commercial enzymatic detergents in order to separate PET and PE, thus allowing a better recycling of the two different polymers. Mechanical properties of the plastic substrate, such as stress at yield, stress and

  18. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    International Nuclear Information System (INIS)

    Nielsen, Anders Lade

    2009-01-01

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  19. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, Anders Lade, E-mail: aln@humgen.au.dk [Department of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C (Denmark)

    2009-10-23

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  20. Formation of the outer layer of the Dictyostelium spore coat depends on the inner-layer protein SP85/PsB.

    Science.gov (United States)

    Metcalf, Talibah; Kelley, Karen; Erdos, Gregory W; Kaplan, Lee; West, Christopher M

    2003-02-01

    The Dictyostelium spore is surrounded by a 220 microm thick trilaminar coat that consists of inner and outer electron-dense layers surrounding a central region of cellulose microfibrils. In previous studies, a mutant strain (TL56) lacking three proteins associated with the outer layer exhibited increased permeability to macromolecular tracers, suggesting that this layer contributes to the coat permeability barrier. Electron microscopy now shows that the outer layer is incomplete in the coats of this mutant and consists of a residual regular array of punctate electron densities. The outer layer is also incomplete in a mutant lacking a cellulose-binding protein associated with the inner layer, and these coats are deficient in an outer-layer protein and another coat protein. To examine the mechanism by which this inner-layer protein, SP85, contributes to outer-layer formation, various domain fragments were overexpressed in forming spores. Most of these exert dominant negative effects similar to the deletion of outer-layer proteins, but one construct, consisting of a fusion of the N-terminal and Cys-rich C1 domain, induces a dense mat of novel filaments at the surface of the outer layer. Biochemical studies show that the C1 domain binds cellulose, and a combination of site-directed mutations that inhibits its cellulose-binding activity suppresses outer-layer filament induction. The results suggest that, in addition to a previously described early role in regulating cellulose synthesis, SP85 subsequently contributes a cross-bridging function between cellulose and other coat proteins to organize previously unrecognized structural elements in the outer layer of the coat.

  1. Biomimetic surface coatings from modular amphiphilic proteins

    Science.gov (United States)

    Harden, James; Wan, Fan; Fischer, Stephen; Dick, Scott

    2010-03-01

    Recombinant DNA methods have been used to develop a library of diblock protein polymers for creating designer biofunctional interfaces. These proteins are composed of a surface-active, amphiphilic block joined to a disordered, water soluble block with an end terminal bioactive domain. The amphiphilic block has a strong affinity for many synthetic polymer surfaces, providing a facile means of imparting biological functionality to otherwise bio-neutral materials through physical self-assembly. We have incorporated a series of bioactive end domains into this diblock motif, including sequences that encode specific cell binding and signaling functions of extracellular matrix constituents (e.g. RGD and YIGSR). In this talk, we show that these diblock constructs self-assemble into biofunctional surface coatings on several model synthetic polymer materials. We demonstrate that surface adsorption of the proteins has minimal impacts on the presentation of the bioactive domains in the soluble block, and through the use of microscopic and cell proliferation assays, we show that the resulting biofunctional interfaces are capable of inducing appropriate cellular responses in a variety of human cell types.

  2. Poly(norepinephrine)-coated open tubular column for the separation of proteins and recombination human erythropoietin by capillary electrochromatography.

    Science.gov (United States)

    Xiao, Xue; Zhang, Yamin; Wu, Jia; Jia, Li

    2017-12-01

    Recombinant human erythropoietin is an important therapeutic protein with high economic interest due to the benefits provided by its clinical use for the treatment of anemias associated with chronic renal failure and chemotherapy. In this work, a poly(norepinephrine)-coated open tubular column was successfully prepared based on the self-polymerization of norepinephrine under mild alkaline condition, the favorable film forming and easy adhesive properties of poly(norepinephrine). The poly(norepinephrine) coating was characterized by scanning electron microscopy and measurement of the electro-osmotic flow. The thickness of the coating was about 431 nm. The electrochromatographic performance of the poly(norepinephrine)-coated open tubular column was evaluated by separation of proteins. Some basic and acidic proteins including two variants of bovine serum albumin and two variants of β-lactoglobulin achieved separation in the poly(norepinephrine)-coated open tubular column. More importantly, the column demonstrated separation ability for the glycoforms of recombinant human erythropoietin. In addition, the column demonstrated good repeatability with the run-to-run, day-to-day, and column-to-column relative standard deviations of migration times of proteins less than 3.40%. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Regulation of expression of a select group of Bacillus anthracis spore coat proteins.

    Science.gov (United States)

    Aronson, Arthur

    2018-04-01

    The spore coat of Bacilli is a relatively complex structure comprised of about 70 species of proteins in 2 or 3 layers. While some are involved in assembly or protection, the regulation of many are not well defined so lacZ transcriptional fusions were constructed to six Bacillus anthracis spore coat genes in order to gain insight into their possible functions. The genes were selected on the basis of the location of the encoded proteins within the coat and distribution among spore forming species. Conditions tested were temperature and media either as solid or liquid. The most extensive differences were for the relatively well expressed fusions to the cotH and cotM genes, which were greatest at 30°C on plates of a nutrient rich medium. The cotJ operon was moderately expressed under all conditions although somewhat higher on enriched plates at 30°C. Cot S was low under all conditions except for a substantial increase in biofilm medium. Cot∝ and cotF were essentially invariant with a somewhat greater expression in the more enriched medium. The capacity of a subset of coat genes to respond to various conditions reflects a flexibility in spore coat structure that may be necessary for adaptation to environmental challenges. This could account, at least in part, for the complexity of this structure.

  4. Binding constants of Southern rice black-streaked dwarf virus Coat Protein with ferulic acid derivatives

    Directory of Open Access Journals (Sweden)

    Longlu Ran

    2018-04-01

    Full Text Available The data present binding constants between ferulic acid derivatives and the Coat Protein (P10 by fluorescence titration in this article, which is hosted in the research article entitled “Interaction Research on an Antiviral Molecule that Targets the Coat Protein of Southern rice black-streaked dwarf virus’’ (Ran et al., 2017 [1]. The data include fluorescence quenching spectrum, Stern–Volmer quenching constants, and binding parameters. In this article, a more comprehensive data interpretation and analysis is explained.

  5. Health issues of whey proteins: 3. gut health promotion

    NARCIS (Netherlands)

    Gertjan Schaafsma

    2007-01-01

    This paper reviews the potential of whey protein to promote gut health. The high digestibility and specific amino acid composition of whey protein, as present in whey powder, whey protein concentrate and whey protein isolate, explain why ingestion of whey protein will exert this beneficial effect.

  6. Formulating food protein-stabilized indomethacin nanosuspensions into pellets by fluid-bed coating technology: physical characterization, redispersibility, and dissolution.

    Science.gov (United States)

    He, Wei; Lu, Yi; Qi, Jianping; Chen, Lingyun; Yin, Lifang; Wu, Wei

    2013-01-01

    Drug nanosuspensions are very promising for enhancing the dissolution and bioavailability of drugs that are poorly soluble in water. However, the poor stability of nanosuspensions, reflected in particle growth, aggregation/agglomeration, and change in crystallinity state greatly limits their applications. Solidification of nanosuspensions is an ideal strategy for addressing this problem. Hence, the present work aimed to convert drug nanosuspensions into pellets using fluid-bed coating technology. Indomethacin nanosuspensions were prepared by the precipitation-ultrasonication method using food proteins (soybean protein isolate, whey protein isolate, β-lactoglobulin) as stabilizers. Dried nanosuspensions were prepared by coating the nanosuspensions onto pellets. The redispersibility, drug dissolution, solid-state forms, and morphology of the dried nanosuspensions were evaluated. The mean particle size for the nanosuspensions stabilized using soybean protein isolate, whey protein isolate, and β-lactoglobulin was 588 nm, 320 nm, and 243 nm, respectively. The nanosuspensions could be successfully layered onto pellets with high coating efficiency. Both the dried nanosuspensions and nanosuspensions in their original amorphous state and not influenced by the fluid-bed coating drying process could be redispersed in water, maintaining their original particle size and size distribution. Both the dried nanosuspensions and the original drug nanosuspensions showed similar dissolution profiles, which were both much faster than that of the raw crystals. Fluid-bed coating technology has potential for use in the solidification of drug nanosuspensions.

  7. TALE factors poise promoters for activation by Hox proteins.

    Science.gov (United States)

    Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G

    2014-01-27

    Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

    Science.gov (United States)

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

  9. Sulfonate-terminated carbosilane dendron-coated nanotubes: a greener point of view in protein sample preparation.

    Science.gov (United States)

    González-García, Estefanía; Gutiérrez Ulloa, Carlos E; de la Mata, Francisco Javier; Marina, María Luisa; García, María Concepción

    2017-09-01

    Reduction or removal of solvents and reagents in protein sample preparation is a requirement. Dendrimers can strongly interact with proteins and have great potential as a greener alternative to conventional methods used in protein sample preparation. This work proposes the use of single-walled carbon nanotubes (SWCNTs) functionalized with carbosilane dendrons with sulfonate groups for protein sample preparation and shows the successful application of the proposed methodology to extract proteins from a complex matrix. SEM images of nanotubes and mixtures of nanotubes and proteins were taken. Moreover, intrinsic fluorescence intensity of proteins was monitored to observe the most significant interactions at increasing dendron generations under neutral and basic pHs. Different conditions for the disruption of interactions between proteins and nanotubes after protein extraction and different concentrations of the disrupting reagent and the nanotube were also tried. Compatibility of extraction and disrupting conditions with the enzymatic digestion of proteins for obtaining bioactive peptides was also studied. Finally, sulfonate-terminated carbosilane dendron-coated SWCNTs enabled the extraction of proteins from a complex sample without using non-environmentally friendly solvents that were required so far. Graphical Abstract Green protein extraction from a complex sample employing carbosilane dendron coated nanotubes.

  10. Dissolution, agglomerate morphology, and stability limits of protein-coated silver nanoparticles.

    Science.gov (United States)

    Martin, Matthew N; Allen, Andrew J; MacCuspie, Robert I; Hackley, Vincent A

    2014-09-30

    Little is understood regarding the impact that molecular coatings have on nanoparticle dissolution kinetics and agglomerate formation in a dilute nanoparticle dispersion. Dissolution and agglomeration processes compete in removing isolated nanoparticles from the dispersion, making quantitative time-dependent measurements of the mechanisms of nanoparticle loss particularly challenging. In this article, we present in situ ultra-small-angle X-ray scattering (USAXS) results, simultaneously quantifying dissolution, agglomeration, and stability limits of silver nanoparticles (AgNPs) coated with bovine serum albumin (BSA) protein. When the BSA corona is disrupted, we find that the loss of silver from the nanoparticle core is well matched by a second-order kinetic rate reaction, arising from the oxidative dissolution of silver. Dissolution and agglomeration are quantified, and morphological transitions throughout the process are qualified. By probing the BSA-AgNP suspension around its stability limits, we provide insight into the destabilization mechanism by which individual particles rapidly dissolve as a whole rather than undergo slow dissolution from the aqueous interface inward, once the BSA layer is breached. Because USAXS rapidly measures over the entire nanometer to micrometer size range during the dissolution process, many insights are also gained into the stabilization of NPs by protein and its ability to protect the labile metal core from the solution environment by prohibiting the diffusion of reactive species. This approach can be extended to a wide variety of coating molecules and reactive metal nanoparticle systems to carefully survey their stability limits, revealing the likely mechanisms of coating breakdown and ensuing reactions.

  11. The fluoride coated AZ31B magnesium alloy improves corrosion resistance and stimulates bone formation in rabbit model

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Wei; Zhang, Guangdao [Department of Prosthodontics, School of Stomatology, China Medical University, Shenyang 110001 (China); Tan, Lili; Yang, Ke [Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Ai, Hongjun, E-mail: aihongjuna@sina.com [Department of Prosthodontics, School of Stomatology, China Medical University, Shenyang 110001 (China)

    2016-06-01

    This study aimed to evaluate the effect of fluorine coated Mg alloy and clarify its mechanism in bone formation. We implanted the fluorine coated AZ31B Mg alloy screw (group F) in rabbit mandibular and femur in vivo. Untreated AZ31B Mg alloy screw (group A) and titanium screw (group T) were used as control. Then, scanning electron microscopy, the spectral energy distribution analysis, hard and decalcified bone tissues staining were performed. Immunohistochemistry was employed to examine the protein expressions of bone morphogenetic protein 2 (BMP-2) and collagen type I in the vicinity of the implant. Compared with the group A, the degradation of the alloy was reduced, the rates of Mg corrosion and Mg ion release were slowed down, and the depositions of calcium and phosphate increased in the group F in the early stage of implantation. Histological results showed that fluorine coated Mg alloy had well osteogenic activity and biocompatibility. Moreover, fluoride coating obviously up-regulated the expressions of collagen type I and BMP-2. This study confirmed that the fluorine coating might improve the corrosion resistance of AZ31B Mg alloy and promote bone formation by up-regulated the expressions of collagen type I and BMP-2. - Highlights: • Fluoride coating inhibited the degradation of the alloy in the early implantation. • Fluorine coating could slow down the rate of Mg corrosion and Mg ion release. • Fluorine coating could promote the deposition of Ca and P in vivo. • Fluorine coated Mg alloy had well osteogenic activity and biocompatibility. • Fluorine coating up-regulated the expression of BMP-2 and collagen type I protein.

  12. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    International Nuclear Information System (INIS)

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

    2013-01-01

    A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection

  13. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    Energy Technology Data Exchange (ETDEWEB)

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi, E-mail: kumasaka@spring8.or.jp [Japan Synchrotron Radiation Research Institute (JASRI/SPring-8), 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan)

    2013-09-01

    A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection.

  14. Role of Charge Regulation and Size Polydispersity in Nanoparticle Encapsulation by Viral Coat Proteins

    NARCIS (Netherlands)

    Kusters, Remy; Lin, Hsiang-Ku; Zandi, Roya; Tsvetkova, Irina; Dragnea, Bogdan; van der Schoot, Paul

    2015-01-01

    Nanoparticles can be encapsulated by virus coat proteins if their surfaces are functionalized to acquire a sufficiently large negative charge. A minimal surface charge is required to overcome (i) repulsive interactions between the positively charged RNA-binding domains on the proteins and (ii) the

  15. Health issues of whey proteins: 3. Gut health promotion

    NARCIS (Netherlands)

    Schaafsma, G.

    2007-01-01

    This paper reviews the potential of whey protein to promote gut health. The high digestibility and specific amino acid composition of whey protei, as present in whey powder, whey protein concentrate and whey protein isolate, explain why ingestion of whey protein will exert this beneficial effect.

  16. Effect of Protein-Based Edible Coating from Red Snapper (Lutjanus sp.) Surimi on Cooked Shrimp

    Science.gov (United States)

    Rostini, I.; Ibrahim, B.; Trilaksani, W.

    2018-02-01

    Surimi can be used as a raw material for making protein based edible coating to protect cooked shrimp color. The purpose of this study was to determine consumers preference level on cooked shrimp which coated by surimi edible coating from red snapper and to know the microscopic visualization of edible coating layer on cooked shrimp. The treatments for surimi edible coating were without and added by sappan wood (Caesalpinia sappan Linn) extract. Application of surimi edible coating on cooked shrimp was comprised methods (1) boiled then coated and (2) coated then boiled. Edible coating made from surimi with various concentrations which were 2, 6, 10 and 14% of distillated water. The analysis were done using hedonic test and microscopic observation with microscope photographs. Effect of surimi edible coating on cooked shrimp based on the hedonic and colour test results showed that the 14% surimi concentration, added by sappan wood (Caesalpinia sappan Linn) extract on edible coating was the most preferable by panellist and giving the highest shrimp colour. The edible coating surimi application on cooked shrimp which gave the best result was processed by boiling followed by coating.

  17. Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway.

    Science.gov (United States)

    Blackburn, Brody J; Li, Shuaizhi; Roznowski, Aaron P; Perez, Alexis R; Villarreal, Rodrigo H; Johnson, Curtis J; Hardy, Margaret; Tuckerman, Edward C; Burch, April D; Fane, Bentley A

    2017-12-15

    Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability. IMPORTANCE Genetic analyses have been

  18. Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.

    Science.gov (United States)

    Rizzo, Alessandro A; Suhanovsky, Margaret M; Baker, Matthew L; Fraser, LaTasha C R; Jones, Lisa M; Rempel, Don L; Gross, Michael L; Chiu, Wah; Alexandrescu, Andrei T; Teschke, Carolyn M

    2014-06-10

    Some capsid proteins built on the ubiquitous HK97-fold have accessory domains imparting specific functions. Bacteriophage P22 coat protein has a unique insertion domain (I-domain). Two prior I-domain models from subnanometer cryoelectron microscopy (cryoEM) reconstructions differed substantially. Therefore, the I-domain's nuclear magnetic resonance structure was determined and also used to improve cryoEM models of coat protein. The I-domain has an antiparallel six-stranded β-barrel fold, not previously observed in HK97-fold accessory domains. The D-loop, which is dynamic in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. The S-loop is important for capsid size determination, likely through intrasubunit interactions. Ten of 18 coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Characterization of a Lactococcus lactis promoter for heterologous protein production

    Directory of Open Access Journals (Sweden)

    Christian E. Ogaugwu

    2018-03-01

    Full Text Available Constitutively active promoter elements for heterologous protein production in Lactococcus lactis are scarce. Here, the promoter of the PTS-IIC gene cluster from L. lactis NZ3900 is described. This promoter was cloned upstream of an enhanced green fluorescent protein, GFPmut3a, and transformed into L. lactis. Transformants produced up to 13.5 μg of GFPmut3a per milliliter of log phase cells. Addition of cellobiose further increased the production of GFPmut3a by up to two-fold when compared to glucose. Analysis of mutations at two specific positions in the PTS-IIC promoter showed that a ‘T’ to ‘G’ mutation within the −35 element resulted in constitutive expression in glucose, while a ‘C’ at nucleotide 7 in the putative cre site enhanced promoter activity in cellobiose. Finally, this PTS-IIC promoter is capable of mediating protein expression in Bacillus subtilis and Escherichia coli Nissle 1917, suggesting the potential for future biotechnological applications of this element and its derivatives.

  20. Small proteins link coat and cortex assembly during sporulation in Bacillus subtilis

    Science.gov (United States)

    Ebmeier, Sarah E.; Tan, Irene S.; Clapham, Katie Rose; Ramamurthi, Kumaran S.

    2015-01-01

    Summary Mature spores of the bacterium Bacillus subtilis are encased by two concentric shells: an inner shell (the ‘cortex’), made of peptidoglycan; and an outer proteinaceous shell (the ‘coat’), whose basement layer is anchored to the surface of the developing spore via a 26-amino-acid-long protein called SpoVM. During sporulation, initiation of cortex assembly depends on the successful initiation of coat assembly, but the mechanisms that co-ordinate the morphogenesis of both structures are largely unknown. Here, we describe a sporulation pathway involving SpoVM and a 37-amino-acid-long protein named ‘CmpA’ that is encoded by a previously un-annotated gene and is expressed under control of two sporulation-specific transcription factors (σE and SpoIIID). CmpA localized to the surface of the developing spore and deletion of cmpA resulted in cells progressing through the sporulation programme more quickly. Overproduction of CmpA did not affect normal growth or cell division, but delayed entry into sporulation and abrogated cortex assembly. In those cells that had successfully initiated coat assembly, CmpA was removed by a posttranslational mechanism, presumably in order to overcome the sporulation inhibition it imposed. We propose a model in which CmpA participates in a developmental checkpoint that ensures the proper orchestration of coat and cortex morphogenesis by repressing cortex assembly until coat assembly successfully initiates. PMID:22463703

  1. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    Science.gov (United States)

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Surface properties of nanocrystalline TiO2 coatings in relation to the in vitro plasma protein adsorption

    International Nuclear Information System (INIS)

    Lorenzetti, M; Kobe, S; Novak, S; Bernardini, G; Santucci, A; Luxbacher, T

    2015-01-01

    This study reports on the selective adsorption of whole plasma proteins on hydrothermally (HT) grown TiO 2 -anatase coatings and its dependence on the three main surface properties: surface charge, wettability and roughness. The influence of the photo-activation of TiO 2 by UV irradiation was also evaluated. Even though the protein adhesion onto Ti-based substrates was only moderate, better adsorption of any protein (at pH = 7.4) occurred for the most negatively charged and hydrophobic substrate (Ti non-treated) and for the most nanorough and hydrophilic surface (HT Ti3), indicating that the mutual action of the surface characteristics is responsible for the attraction and adhesion of the proteins. The HT coatings showed a higher adsorption of certain proteins (albumin ‘passivation’ layer, apolipoproteins, vitamin D-binding protein, ceruloplasmin, α-2-HS-glycoprotein) and higher ratios of albumin to fibrinogen and albumin to immunoglobulin γ-chains. The UV pre-irradiation affected the surface properties and strongly reduced the adsorption of the proteins. These results provide in-depth knowledge about the characterization of nanocrystalline TiO 2 coatings for body implants and provide a basis for future studies on the hemocompatibility and biocompatibility of such surfaces. (paper)

  3. Impact of surface coating and food-mimicking media on nanosilver-protein interaction

    Energy Technology Data Exchange (ETDEWEB)

    Burcza, Anna, E-mail: anna.burcza@mri.bund.de; Gräf, Volker; Walz, Elke; Greiner, Ralf [Max Rubner-Institute, Department of Food Technology and Bioprocess Engineering (Germany)

    2015-11-15

    The application of silver nanoparticles (AgNPs) in food contact materials has recently become a subject of dispute due to the possible migration of silver in nanoform into foods and beverages. Therefore, the analysis of the interaction of AgNPs with food components, especially proteins, is of high importance in order to increase our knowledge of the behavior of nanoparticles in food matrices. AgPURE™ W10 (20 nm), an industrially applied nanomaterial, was compared with AgNPs of similar size frequently investigated for scientific purposes differing in the surface capping agent (spherical AgNP coated with either PVP or citrate). The interactions of the AgNPs with whey proteins (BSA, α-lactalbumin and β-lactoglobulin) at different pH values (4.2, 7 or 7.4) were investigated using surface plasmon resonance, SDS-PAGE, and asymmetric flow field-flow fractionation. The data obtained by the three different methods correlated well. Besides the nature of the protein and the nanoparticle coating, the environment was shown to affect the interaction significantly. The strongest interaction was obtained with BSA and AgNPs in an acidic environment. Neutral and slightly alkaline conditions however, seemed to prevent the AgNP-protein interaction almost completely. Furthermore, the interaction of whey proteins with AgPURE™ W10 was found to be weaker compared to the interaction with the other two AgNPs under all conditions investigated.

  4. Impact of surface coating and food-mimicking media on nanosilver-protein interaction

    International Nuclear Information System (INIS)

    Burcza, Anna; Gräf, Volker; Walz, Elke; Greiner, Ralf

    2015-01-01

    The application of silver nanoparticles (AgNPs) in food contact materials has recently become a subject of dispute due to the possible migration of silver in nanoform into foods and beverages. Therefore, the analysis of the interaction of AgNPs with food components, especially proteins, is of high importance in order to increase our knowledge of the behavior of nanoparticles in food matrices. AgPURE™ W10 (20 nm), an industrially applied nanomaterial, was compared with AgNPs of similar size frequently investigated for scientific purposes differing in the surface capping agent (spherical AgNP coated with either PVP or citrate). The interactions of the AgNPs with whey proteins (BSA, α-lactalbumin and β-lactoglobulin) at different pH values (4.2, 7 or 7.4) were investigated using surface plasmon resonance, SDS-PAGE, and asymmetric flow field-flow fractionation. The data obtained by the three different methods correlated well. Besides the nature of the protein and the nanoparticle coating, the environment was shown to affect the interaction significantly. The strongest interaction was obtained with BSA and AgNPs in an acidic environment. Neutral and slightly alkaline conditions however, seemed to prevent the AgNP-protein interaction almost completely. Furthermore, the interaction of whey proteins with AgPURE™ W10 was found to be weaker compared to the interaction with the other two AgNPs under all conditions investigated

  5. Poly(oligoethylene glycol methacrylate) dip-coating: turning cellulose paper into a protein-repellent platform for biosensors.

    Science.gov (United States)

    Deng, Xudong; Smeets, Niels M B; Sicard, Clémence; Wang, Jingyun; Brennan, John D; Filipe, Carlos D M; Hoare, Todd

    2014-09-17

    The passivation of nonspecific protein adsorption to paper is a major barrier to the use of paper as a platform for microfluidic bioassays. Herein we describe a simple, scalable protocol based on adsorption and cross-linking of poly(oligoethylene glycol methacrylate) (POEGMA) derivatives that reduces nonspecific adsorption of a range of proteins to filter paper by at least 1 order of magnitude without significantly changing the fiber morphology or paper macroporosity. A lateral-flow test strip coated with POEGMA facilitates effective protein transport while also confining the colorimetric reporting signal for easier detection, giving improved performance relative to bovine serum albumin (BSA)-blocked paper. Enzyme-linked immunosorbent assays based on POEGMA-coated paper also achieve lower blank values, higher sensitivities, and lower detection limits relative to ones based on paper blocked with BSA or skim milk. We anticipate that POEGMA-coated paper can function as a platform for the design of portable, disposable, and low-cost paper-based biosensors.

  6. Blocking of bacterial biofilm formation by a fish protein coating

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk; Klemm, Per

    2008-01-01

    Bacterial biofilm formation on inert surfaces is a significant health and economic problem in a wide range of environmental, industrial, and medical areas. Bacterial adhesion is generally a prerequisite for this colonization process and, thus, represents an attractive target for the development......, this proteinaceous coating is characterized with regards to its biofilm-reducing properties by using a range of urinary tract infectious isolates with various pathogenic and adhesive properties. The antiadhesive coating significantly reduced or delayed biofilm formation by all these isolates under every condition...... examined. The biofilm-reducing activity did, however, vary depending on the substratum physicochemical characteristics and the environmental conditions studied. These data illustrate the importance of protein conditioning layers with respect to bacterial biofilm formation and suggest that antiadhesive...

  7. Polyclonal Antibodies to a Recombinant Coat Protein of Potato Virus A

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Velemínský, Jiří

    2002-01-01

    Roč. 46, - (2002), s. 147-151 ISSN 0001-723X R&D Projects: GA ČR GA310/00/0381 Institutional research plan: CEZ:AV0Z5038910 Keywords : Potato virus A * recombinant coat protein * Escherichia coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.660, year: 2002

  8. Impact of whey protein coating incorporated with Bifidobacterium and Lactobacillus on sliced ham properties.

    Science.gov (United States)

    Odila Pereira, Joana; Soares, José; J P Monteiro, Maria; Gomes, Ana; Pintado, Manuela

    2018-05-01

    Edible coatings/films with functional ingredients may be a solution to consumers' demands for high-quality food products and an extended shelf-life. The aim of this work was to evaluate the antimicrobial efficiency of edible coatings incorporated with probiotics on sliced ham preservation. Coatings was developed based on whey protein isolates with incorporation of Bifidobacterium animalis Bb-12® or Lactobacillus casei-01. The physicochemical analyses showed that coating decreased water and weight loss on the ham. Furthermore, color analysis showed that coated sliced ham, exhibited no color change, comparatively to uncoated slices. The edible coatings incorporating the probiotic strains inhibited detectable growth of Staphylococcus spp., Pseudomonas spp., Enterobacteriaceae and yeasts/molds, at least, for 45days of storage at 4°C. The sensory evaluation demonstrated that there was a preference for the sliced coated ham. Probiotic bacteria viable cell numbers were maintained at ca. 10 8 CFU/g throughout storage time, enabling the slice of ham to act as a suitable carrier for the beneficial bacteria. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non-structural Triple Gene Block Protein 1 and Coat Protein

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Plchová, Helena; Hoffmeisterová, Hana; Dědič, P.

    2012-01-01

    Roč. 160, č. 5 (2012), s. 251-254 ISSN 0931-1785 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato virus M * recombinant protein * coat protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.000, year: 2012

  10. Interplay between chaperones and protein disorder promotes the evolution of protein networks.

    Directory of Open Access Journals (Sweden)

    Sebastian Pechmann

    2014-06-01

    Full Text Available Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the

  11. On-Chip Manipulation of Protein-Coated Magnetic Beads via Domain-Wall Conduits

    DEFF Research Database (Denmark)

    Donolato, Marco; Vavassori, Paolo; Gobbi, Marco

    2010-01-01

    Geometrically constrained magnetic domain walls (DWs) in magnetic nanowires can be manipulated at the nanometer scale. The inhomogeneous magnetic stray field generated by a DW can capture a magnetic nanoparticle in solution. On-chip nanomanipulation of individual magnetic beads coated with proteins...

  12. Expression and purification of coat protein of citrus tristeza virus ...

    African Journals Online (AJOL)

    CTV coat protein gene (CTV-cp) cloned in pQE30 vector and transformed to DH5α containing 666bp long from Thailand MK-50 isolate was amplified with a forward primer CTV-CP1 (5' CAC CGA CGA AAC AAA GAA ATT GAA GAA CA 3') and a reverse primer CTVCP2 (5' TCA ACG TGT GTT AAA TTT CCC AAG C 3') and ...

  13. Adaptive growth factor delivery from a polyelectrolyte coating promotes synergistic bone tissue repair and reconstruction

    Science.gov (United States)

    Shah, Nisarg J.; Hyder, Md. Nasim; Quadir, Mohiuddin A.; Dorval Courchesne, Noémie-Manuelle; Seeherman, Howard J.; Nevins, Myron; Spector, Myron; Hammond, Paula T.

    2014-01-01

    Traumatic wounds and congenital defects that require large-scale bone tissue repair have few successful clinical therapies, particularly for craniomaxillofacial defects. Although bioactive materials have demonstrated alternative approaches to tissue repair, an optimized materials system for reproducible, safe, and targeted repair remains elusive. We hypothesized that controlled, rapid bone formation in large, critical-size defects could be induced by simultaneously delivering multiple biological growth factors to the site of the wound. Here, we report an approach for bone repair using a polyelectrolye multilayer coating carrying as little as 200 ng of bone morphogenetic protein-2 and platelet-derived growth factor-BB that were eluted over readily adapted time scales to induce rapid bone repair. Based on electrostatic interactions between the polymer multilayers and growth factors alone, we sustained mitogenic and osteogenic signals with these growth factors in an easily tunable and controlled manner to direct endogenous cell function. To prove the role of this adaptive release system, we applied the polyelectrolyte coating on a well-studied biodegradable poly(lactic-co-glycolic acid) support membrane. The released growth factors directed cellular processes to induce bone repair in a critical-size rat calvaria model. The released growth factors promoted local bone formation that bridged a critical-size defect in the calvaria as early as 2 wk after implantation. Mature, mechanically competent bone regenerated the native calvaria form. Such an approach could be clinically useful and has significant benefits as a synthetic, off-the-shelf, cell-free option for bone tissue repair and restoration. PMID:25136093

  14. High genetic diversity in the coat protein and 3' untranslated regions

    Indian Academy of Sciences (India)

    The 3′ terminal region consisting of the coat protein (CP) coding sequence and 3′ untranslated region (3′UTR) was cloned and sequenced from seven isolates. Sequence comparisons revealed considerable genetic diversity among the isolates in their CP and 3′UTR, making CdMV one of the highly variable members ...

  15. Functional improvement of antibody fragments using a novel phage coat protein III fusion system

    DEFF Research Database (Denmark)

    Jensen, Kim Bak; Larsen, Martin; Pedersen, Jesper Søndergaard

    2002-01-01

    Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of het......(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity....

  16. Mammalian amyloidogenic proteins promote prion nucleation in yeast.

    Science.gov (United States)

    Chandramowlishwaran, Pavithra; Sun, Meng; Casey, Kristin L; Romanyuk, Andrey V; Grizel, Anastasiya V; Sopova, Julia V; Rubel, Aleksandr A; Nussbaum-Krammer, Carmen; Vorberg, Ina M; Chernoff, Yury O

    2018-03-02

    Fibrous cross-β aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-β (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    Science.gov (United States)

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

    2013-01-01

    Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection. PMID:23999307

  18. Phosphorylation of stress protein pp80 is related to promotion of transformation

    International Nuclear Information System (INIS)

    Smith, B.M.; Gindhart, T.D.; Hirano, K.; Colburn, N.H.

    1986-01-01

    The JB6 mouse epidermal cell system is an in vitro model of late stage promotion, and includes cell lines sensitive (P+) or resistant (P-) to phorbol ester-induced anchorage independent transformation, and transformed (T/sub x/) lines. Certain promoter-induced changes in phosphoproteins, identified by gel electrophoresis, are unique to cells of one phenotype, and occur only with specific promoters. An 80Kd protein is inversely correlated with phenotype: P- cells have a constitutively higher level (p 35 S-methionine. pp80 shares properties with the 80Kd heat stress protein: molecular weight relative abundance, and isoelectric point (4.5). Pharmacological analogs of calcium, the lanthanides, promote transformation of JB6 cells, but have no effect on phosphorylation of the 80Kd protein. If pp80 is on the promotion pathway, it is limited to a specific subset of transformation promoters

  19. Disease specific protein corona

    Science.gov (United States)

    Rahman, M.; Mahmoudi, M.

    2015-03-01

    It is now well accepted that upon their entrance into the biological environments, the surface of nanomaterials would be covered by various biomacromolecules (e.g., proteins and lipids). The absorption of these biomolecules, so called `protein corona', onto the surface of (nano)biomaterials confers them a new `biological identity'. Although the formation of protein coronas on the surface of nanoparticles has been widely investigated, there are few reports on the effect of various diseases on the biological identity of nanoparticles. As the type of diseases may tremendously changes the composition of the protein source (e.g., human plasma/serum), one can expect that amount and composition of associated proteins in the corona composition may be varied, in disease type manner. Here, we show that corona coated silica and polystyrene nanoparticles (after interaction with in the plasma of the healthy individuals) could induce unfolding of fibrinogen, which promotes release of the inflammatory cytokines. However, no considerable releases of inflammatory cytokines were observed for corona coated graphene sheets. In contrast, the obtained corona coated silica and polystyrene nanoparticles from the hypofibrinogenemia patients could not induce inflammatory cytokine release where graphene sheets do. Therefore, one can expect that disease-specific protein coronas can provide a novel approach for applying nanomedicine to personalized medicine, improving diagnosis and treatment of different diseases tailored to the specific conditions and circumstances.

  20. Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins

    International Nuclear Information System (INIS)

    Wnęk, M; Stockley, P G; Górzny, M Ł; Evans, S D; Ward, M B; Brydson, R; Wälti, C; Davies, A G

    2013-01-01

    The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating. (paper)

  1. Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins

    Science.gov (United States)

    Wnęk, M.; Górzny, M. Ł.; Ward, M. B.; Wälti, C.; Davies, A. G.; Brydson, R.; Evans, S. D.; Stockley, P. G.

    2013-01-01

    The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating.

  2. Preparation of recombinant coat protein of Prunus necrotic ringspot virus.

    Science.gov (United States)

    Petrzik, K; Mráz, I; Kubelková, D

    2001-02-01

    The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

  3. Transfer in SDS of biotinylated proteins from acrylamide gels to an avidin-coated membrane filter.

    Science.gov (United States)

    Karlin, Arthur; Wang, Chaojian; Li, Jing; Xu, Qiang

    2004-06-01

    Avidin was covalently linked to aldehyde-derivatized polyethersulfone membrane filters. These filters were used in Western blot analysis of proteins reacted with biotinylation reagents and electrophoresed in sodium dodecyl sulfate (SDS) on polyacrylamide gels. Electrophoretic transfer from the gels to these filters was in 0.1% SDS, in which the covalently bound avidin retained its biotin-binding capacity. We compared Western blots on avidin-coated membrane filters of biotinylated and nonbiotinylated forms of mouse immunoglobulin G (IgG), mouse IgG heavy chain, muscle-type acetylcholine receptor alpha subunit, and fused alpha and beta subunits of receptor. Biotinylated proteins were captured with high specificity compared to their nonbiotinylated counterparts and sensitively detected on the avidin-coated membranes.

  4. Glial cell adhesion and protein adsorption on SAM coated semiconductor and glass surfaces of a microfluidic structure

    Science.gov (United States)

    Sasaki, Darryl Y.; Cox, Jimmy D.; Follstaedt, Susan C.; Curry, Mark S.; Skirboll, Steven K.; Gourley, Paul L.

    2001-05-01

    The development of microsystems that merge biological materials with microfabricated structures is highly dependent on the successful interfacial interactions between these innately incompatible materials. Surface passivation of semiconductor and glass surfaces with thin organic films can attenuate the adhesion of proteins and cells that lead to biofilm formation and biofouling of fluidic structures. We have examined the adhesion of glial cells and serum albumin proteins to microfabricated glass and semiconductor surfaces coated with self-assembled monolayers of octadecyltrimethoxysilane and N-(triethoxysilylpropyl)-O- polyethylene oxide urethane, to evaluate the biocompatibility and surface passivation those coatings provide.

  5. Preparation of non-aggregated fluorescent nanodiamonds (FNDs) by non-covalent coating with a block copolymer and proteins for enhancement of intracellular uptake.

    Science.gov (United States)

    Lee, Jong Woo; Lee, Seonju; Jang, Sangmok; Han, Kyu Young; Kim, Younggyu; Hyun, Jaekyung; Kim, Seong Keun; Lee, Yan

    2013-05-01

    Fluorescent nanodiamonds (FNDs) are very promising fluorophores for use in biosystems due to their high biocompatibility and photostability. To overcome their tendency to aggregate in physiological solutions, which severely limits the biological applications of FNDs, we developed a new non-covalent coating method using a block copolymer, PEG-b-P(DMAEMA-co-BMA), or proteins such as BSA and HSA. By simple mixing of the block copolymer with FNDs, the cationic DMAEMA and hydrophobic BMA moieties can strongly interact with the anionic and hydrophobic moieties on the FND surface, while the PEG block can form a shell to prevent the direct contact between FNDs. The polymer-coated FNDs, along with BSA- and HSA-coated FNDs, showed non-aggregation characteristics and maintained their size at the physiological salt concentration. The well-dispersed, polymer- or protein-coated FNDs in physiological solutions showed enhanced intracellular uptake, which was confirmed by CLSM. In addition, the biocompatibility of the coated FNDs was expressly supported by a cytotoxicity assay. Our simple non-covalent coating with the block copolymer, which can be easily modified by various chemical methods, projects a very promising outlook for future biomedical applications, especially in comparison with covalent coating or protein-based coating.

  6. Efficacy of various protein-based coating on enhancing the shelf life of fresh eggs during storage.

    Science.gov (United States)

    Caner, Cengiz; Yüceer, Muhammed

    2015-07-01

    The effectiveness of various coatings (whey protein isolate [WPI], whey protein concentrate [WPC], zein, and shellac) on functional properties, interior quality, and eggshell breaking strength of fresh eggs were evaluated during storage at 24 °: C for 6 weeks. Coatings and storage time had significant effects on Haugh unit, yolk index, albumen pH, dry matter (DMA), relative whipping capacity (RWC), and albumen viscosity. Uncoated eggs had higher albumen pH (9.56) and weight loss, and lower albumen viscosity (5.73), Haugh unit (HU), and yolk index (YI) during storage. Among the coated eggs, the shellac and zein coated eggs had the highest value of albumen viscosity (27.26 to 26.90), HU (74.10 to 73.61), and YI (44.84 to 44.63) after storage. Shellac (1.44%) was more effective in preventing weight loss than WPC (4.59%), WPI (4.60%), and zein (2.13%) coatings. Uncoated eggs had the higest value (6.71%) of weight lost. All coatings increased shell strength (5.18 to 5.73 for top and 3.58 to 4.71 for bottom) significantly (P eggs (4.70 for top and 3.15 for bottom). The functional properties such as albumen DMA (14.50 to 16.66 and 18.97 for uncoated) and albumen RWC (841 to 891 and 475 for uncoated) of fresh eggs can be preserved during storage when they are coated. The shellac and zein coatings were more effective for maintaining the internal quality of fresh eggs during storage. Fourier transform near infrared (FT-NIR) in the 800 to 2500 nm reflection spectra were used to quantify the contents of the fresh eggs at the end of storage. Eggs coated with shellac or zein displayed a higher absorbance at 970 and 1,197 nm respectively (OH vibration of water) compared with those coated with WPI or WPC and the uncoated group at the end of storage. The coatings improved functional properties and also shell strength and could be a viable alternative technology for maintaining the internal quality of eggs during long-term storage. This study highlights the promising use of

  7. Calcium carbonate hybrid coating promotes the formation of biomimetic hydroxyapatite on titanium surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Cruz, Marcos Antônio E.; Ruiz, Gilia C.M. [Departamento de Química-Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, SP (Brazil); Faria, Amanda N. [Departamento de Química-Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, SP (Brazil); Departamento de Bioquímica e Imunologia-Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Zancanela, Daniela C.; Pereira, Lourivaldo S.; Ciancaglini, Pietro [Departamento de Química-Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, SP (Brazil); Ramos, Ana P., E-mail: anapr@ffclrp.usp.br [Departamento de Química-Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, SP (Brazil)

    2016-05-01

    Graphical abstract: - Highlights: • CaCO{sub 3} continuous films were deposited on titanium discs using a biomimetic approach. • The coatings origin hydroxyapatite when immersed in simulated body fluid. • The wettability and the free energy of the surfaces were increased after the treatment. • The coated titanium discs are bioactive and non-toxic to osteoblasts. - Abstract: CaCO{sub 3} particles dispersed in liquid media have proven to be good inductors of hydroxyapatite (HAp) growth. However, the use of CaCO{sub 3} deposited as thin films for this propose is unknown. Here, we report the growth of CaCO{sub 3} continuous films on Langmuir–Blodgett (LB) modified titanium surfaces and its use as HAp growth inductor. The Ti surfaces were modified with two, four, and six layers of dihexadecylphosphate (DHP)-LB films containing Ca{sup 2+}, exposed to CO{sub 2} (g) for 12 h. The modified surfaces were immersed in simulated body fluid (SBF) at 37 °C for 36 h and submitted to bioactivity studies. This procedure originates bioactive coatings composed by non-stoichiometric HAp as evidenced by Fourier-Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), and X-ray Photoelectron Spectroscopy (XPS). The presence of the CaCO{sub 3} film as pre-coating diminished the time necessary to growth continuous and homogeneous HAp films using a biomimetic approach. The surface properties of the films regarding their roughness, composition, charge, wettability, and surface free energy (γ{sub s}) were accessed. The presence of HAp increased the wettability and γ{sub s} of the surfaces. The coatings are not toxic for osteoblasts as observed for cell viability assays obtained after 7 and 14 days of culture. Moreover, the CaCO{sub 3} thin films promote the recovery of the osteoblasts viability more than the Ti surfaces themselves.

  8. Calcium carbonate hybrid coating promotes the formation of biomimetic hydroxyapatite on titanium surfaces

    International Nuclear Information System (INIS)

    Cruz, Marcos Antônio E.; Ruiz, Gilia C.M.; Faria, Amanda N.; Zancanela, Daniela C.; Pereira, Lourivaldo S.; Ciancaglini, Pietro; Ramos, Ana P.

    2016-01-01

    Graphical abstract: - Highlights: • CaCO 3 continuous films were deposited on titanium discs using a biomimetic approach. • The coatings origin hydroxyapatite when immersed in simulated body fluid. • The wettability and the free energy of the surfaces were increased after the treatment. • The coated titanium discs are bioactive and non-toxic to osteoblasts. - Abstract: CaCO 3 particles dispersed in liquid media have proven to be good inductors of hydroxyapatite (HAp) growth. However, the use of CaCO 3 deposited as thin films for this propose is unknown. Here, we report the growth of CaCO 3 continuous films on Langmuir–Blodgett (LB) modified titanium surfaces and its use as HAp growth inductor. The Ti surfaces were modified with two, four, and six layers of dihexadecylphosphate (DHP)-LB films containing Ca 2+ , exposed to CO 2 (g) for 12 h. The modified surfaces were immersed in simulated body fluid (SBF) at 37 °C for 36 h and submitted to bioactivity studies. This procedure originates bioactive coatings composed by non-stoichiometric HAp as evidenced by Fourier-Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), and X-ray Photoelectron Spectroscopy (XPS). The presence of the CaCO 3 film as pre-coating diminished the time necessary to growth continuous and homogeneous HAp films using a biomimetic approach. The surface properties of the films regarding their roughness, composition, charge, wettability, and surface free energy (γ s ) were accessed. The presence of HAp increased the wettability and γ s of the surfaces. The coatings are not toxic for osteoblasts as observed for cell viability assays obtained after 7 and 14 days of culture. Moreover, the CaCO 3 thin films promote the recovery of the osteoblasts viability more than the Ti surfaces themselves.

  9. Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating.

    Science.gov (United States)

    Sorkio, Anni; Hongisto, Heidi; Kaarniranta, Kai; Uusitalo, Hannu; Juuti-Uusitalo, Kati; Skottman, Heli

    2014-02-01

    Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

  10. Chemistry and stability of thiol based polyethylene glycol surface coatings on colloidal gold and their relationship to protein adsorption and clearance in vivo

    Science.gov (United States)

    Carpinone, Paul

    Nanomaterials have presented a wide range of novel biomedical applications, with particular emphasis placed on advances in imaging and treatment delivery. Of the many particulate nanomaterials researched for biomedical applications, gold is one of the most widely used. Colloidal gold has been of great interest due to its chemical inertness and its ability to perform multiple functions, such as drug delivery, localized heating of tissues (hyperthermia), and imaging (as a contrast agent). It is also readily functionalized through the use of thiols, which spontaneously form sulfur to gold bonds with the surface. Polyethylene glycol (PEG) is the most widely used coating material for these particles as it provides both steric stability to the suspension and protein resistance. These properties extend the circulation time of the particles in blood, and consequently the efficacy of the treatment. Despite widespread use of PEG coated gold particles, the coating chemistry and stability of these particles are largely unknown. The goal of this work was to identify the mechanisms leading to degradation and stability of thiol based polyethylene glycol coatings on gold particles and to relate this behavior to protein adsorption and clearance in vivo. The results indicate that the protective PEG coating is susceptible to sources of oxidation (including dissolved oxygen) and competing adsorbates, among other factors. The quality of commercially available thiolated PEG reagents was also found to play a key role in the quality and protein resistance of the final PEG coating. Analysis of the stability of these coatings indicated that they rapidly degrade under physiological conditions, leading to the onset of protein adsorption when exposed to plasma or blood. Paralleling the protein adsorption behavior and onset of coating degradation observed in vitro, blood clearance of parenterally administered PEG coated particles in mice began after approximately 2h of circulation time. Taken

  11. PCP-B class pollen coat proteins are key regulators of the hydration checkpoint in Arabidopsis thaliana pollen-stigma interactions.

    Science.gov (United States)

    Wang, Ludi; Clarke, Lisa A; Eason, Russell J; Parker, Christopher C; Qi, Baoxiu; Scott, Rod J; Doughty, James

    2017-01-01

    The establishment of pollen-pistil compatibility is strictly regulated by factors derived from both male and female reproductive structures. Highly diverse small cysteine-rich proteins (CRPs) have been found to play multiple roles in plant reproduction, including the earliest stages of the pollen-stigma interaction. Secreted CRPs found in the pollen coat of members of the Brassicaceae, the pollen coat proteins (PCPs), are emerging as important signalling molecules that regulate the pollen-stigma interaction. Using a combination of protein characterization, expression and phylogenetic analyses we identified a novel class of Arabidopsis thaliana pollen-borne CRPs, the PCP-Bs (for pollen coat protein B-class) that are related to embryo surrounding factor (ESF1) developmental regulators. Single and multiple PCP-B mutant lines were utilized in bioassays to assess effects on pollen hydration, adhesion and pollen tube growth. Our results revealed that pollen hydration is severely impaired when multiple PCP-Bs are lost from the pollen coat. The hydration defect also resulted in reduced pollen adhesion and delayed pollen tube growth in all mutants studied. These results demonstrate that AtPCP-Bs are key regulators of the hydration 'checkpoint' in establishment of pollen-stigma compatibility. In addition, we propose that interspecies diversity of PCP-Bs may contribute to reproductive barriers in the Brassicaceae. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  12. Denatured protein-coated docetaxel nanoparticles: Alterable drug state and cytosolic delivery.

    Science.gov (United States)

    Zhang, Li; Xiao, Qingqing; Wang, Yiran; Zhang, Chenshuang; He, Wei; Yin, Lifang

    2017-05-15

    Many lead compounds have a low solubility in water, which substantially hinders their clinical application. Nanosuspensions have been considered a promising strategy for the delivery of water-insoluble drugs. Here, denatured soy protein isolate (SPI)-coated docetaxel nanosuspensions (DTX-NS) were developed using an anti-solvent precipitation-ultrasonication method to improve the water-solubility of DTX, thus improving its intracellular delivery. DTX-NS, with a diameter of 150-250nm and drug-loading up to 18.18%, were successfully prepared by coating drug particles with SPI. Interestingly, the drug state of DTX-NS was alterable. Amorphous drug nanoparticles were obtained at low drug-loading, whereas at a high drug-loading, the DTX-NS drug was mainly present in the crystalline state. Moreover, DTX-NS could be internalized at high levels by cancer cells and enter the cytosol by lysosomal escape, enhancing cell cytotoxicity and apoptosis compared with free DTX. Taken together, denatured SPI has a strong stabilization effect on nanosuspensions, and the drug state in SPI-coated nanosuspensions is alterable by changing the drug-loading. Moreover, DTX-NS could achieve cytosolic delivery, generating enhanced cell cytotoxicity against cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Apoptosis inhibitor of macrophage (AIM) diminishes lipid droplet-coating proteins leading to lipolysis in adipocytes

    International Nuclear Information System (INIS)

    Iwamura, Yoshihiro; Mori, Mayumi; Nakashima, Katsuhiko; Mikami, Toshiyuki; Murayama, Katsuhisa; Arai, Satoko; Miyazaki, Toru

    2012-01-01

    Highlights: ► AIM induces lipolysis in a distinct manner from that of hormone-dependent lipolysis. ► AIM ablates activity of peroxisome proliferator-activated receptor in adipocytes. ► AIM reduces mRNA levels of lipid-droplet coating proteins leading to lipolysis. -- Abstract: Under fasting conditions, triacylglycerol in adipose tissue undergoes lipolysis to supply fatty acids as energy substrates. Such lipolysis is regulated by hormones, which activate lipases via stimulation of specific signalling cascades. We previously showed that macrophage-derived soluble protein, AIM induces obesity-associated lipolysis, triggering chronic inflammation in fat tissue which causes insulin resistance. However, the mechanism of how AIM mediates lipolysis remains unknown. Here we show that AIM induces lipolysis in a manner distinct from that of hormone-dependent lipolysis, without activation or augmentation of lipases. In vivo and in vitro, AIM did not enhance phosphorylation of hormone-sensitive lipase (HSL) in adipocytes, a hallmark of hormone-dependent lipolysis activation. Similarly, adipose tissue from obese AIM-deficient and wild-type mice showed comparable HSL phosphorylation. Consistent with the suppressive effect of AIM on fatty acid synthase activity, the amount of saturated and unsaturated fatty acids was reduced in adipocytes treated with AIM. This response ablated transcriptional activity of peroxisome proliferator-activated receptor (PPARγ), leading to diminished gene expression of lipid-droplet coating proteins including fat-specific protein 27 (FSP27) and Perilipin, which are indispensable for triacylglycerol storage in adipocytes. Accordingly, the lipolytic effect of AIM was overcome by a PPARγ-agonist or forced expression of FSP27, while it was synergized by a PPARγ-antagonist. Overall, distinct modes of lipolysis appear to take place in different physiological situations; one is a supportive response against nutritional deprivation achieved by

  14. Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissues regeneration.

    Science.gov (United States)

    Kaufman, Gili; Whitescarver, Ryan; Nunes, Laiz; Palmer, Xavier-Lewis; Skrtic, Drago; Tutak, Wojtek

    2017-10-09

    Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by imageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within 3 days of incubation, fibroblast spheroids interacted with the fibers and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for

  15. Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissue regeneration.

    Science.gov (United States)

    Kaufman, Gili; Whitescarver, Ryan A; Nunes, Laiz; Palmer, Xavier-Lewis; Skrtic, Drago; Tutak, Wojtek

    2018-01-24

    Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by ImageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within three days of incubation, fibroblast spheroids interacted with the fibers, and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for

  16. 'Let the phage do the work': Using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants

    International Nuclear Information System (INIS)

    Teschke, Carolyn M.; Parent, Kristin N.

    2010-01-01

    The amino acid sequence of viral capsid proteins contains information about their folding, structure and self-assembly processes. While some viruses assemble from small preformed oligomers of coat proteins, other viruses such as phage P22 and herpesvirus assemble from monomeric proteins (Fuller and King, 1980). The subunit assembly process is strictly controlled through protein:protein interactions such that icosahedral structures are formed with specific symmetries, rather than aberrant structures. dsDNA viruses commonly assemble by first forming a precursor capsid that serves as a DNA packaging machine. DNA packaging is accompanied by a conformational transition of the small precursor procapsid into a larger capsid for isometric viruses. Here we highlight the pseudo-atomic structures of phage P22 coat protein and rationalize several decades of data about P22 coat protein folding, assembly and maturation generated from a combination of genetics and biochemistry.

  17. Improvement of food packaging related properties in whey protein isolate‑based nanocomposite films and coatings by addition of montmorillonite nanoplatelets

    Science.gov (United States)

    Schmid, Markus; Merzbacher, Sarah; Brzoska, Nicola; Müller, Kerstin; Jesdinszki, Marius

    2017-11-01

    In the present study the effects of the addition of montmorillonite (MMT) nanoplatelets on whey protein isolate (WPI)-based nanocomposite films and coatings were investigated. The main objective was the development of WPI-based MMT-nanocomposites with enhanced barrier and mechanical properties. WPI-based nanocomposite cast-films and coatings were prepared by dispersing 0 % (reference sample), 3 %, 6 %, 9 % (w/w protein) MMT, or, depending on the protein concentration, also 12 % and 15 % (w/w protein) MMT into native WPI-based dispersions, followed by subsequent denaturation during the drying and curing process. The natural MMT nanofillers could be randomly dispersed into film-forming WPI-based nanodispersions, displaying good compatibility with the hydrophilic biopolymer matrix. As a result, by addition of 15 % (w/w protein) MMT into 10 % (w/w dispersion) WPI-based cast-films or coatings, the oxygen permeability (OP) was reduced by 91 % for glycerol-plasticized and 84 % for sorbitol-plasticized coatings, water vapor transmission rate (WVTR) was reduced by 58 % for sorbitol-plasticized cast-films. Due to the addition of MMT- nanofillers the Young’s modulus and tensile strength improved by 315 % and 129 %, respectively, whereas elongation at break declined by 77 % for glycerol-plasticized cast-films. In addition, comparison of plasticizer type revealed that sorbitol-plasticized cast-films were generally stiffer and stronger, but less flexible compared glycerol-plasticized cast-films. Viscosity measurements demonstrated good processability and suitability for up-scaled industrial processes of native WPI-based nanocomposite dispersions, even at high nanofiller-loadings. These results suggest that the addition of natural MMT- nanofillers into native WPI-based matrices to form nanocomposite films and coatings holds great potential to replace well-established, fossil-based packaging materials for at least certain applications such as oxygen barriers as part of

  18. Antifouling coatings: recent developments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Indrani; Pangule, Ravindra C.; Kane, Ravi S. [Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Ricketts Building, Troy, NY 12180 (United States)

    2011-02-08

    The major strategies for designing surfaces that prevent fouling due to proteins, bacteria, and marine organisms are reviewed. Biofouling is of great concern in numerous applications ranging from biosensors to biomedical implants and devices, and from food packaging to industrial and marine equipment. The two major approaches to combat surface fouling are based on either preventing biofoulants from attaching or degrading them. One of the key strategies for imparting adhesion resistance involves the functionalization of surfaces with poly(ethylene glycol) (PEG) or oligo(ethylene glycol). Several alternatives to PEG-based coatings have also been designed over the past decade. While protein-resistant coatings may also resist bacterial attachment and subsequent biofilm formation, in order to overcome the fouling-mediated risk of bacterial infection it is highly desirable to design coatings that are bactericidal. Traditional techniques involve the design of coatings that release biocidal agents, including antibiotics, quaternary ammonium salts (QAS), and silver, into the surrounding aqueous environment. However, the emergence of antibiotic- and silver-resistant pathogenic strains has necessitated the development of alternative strategies. Therefore, other techniques based on the use of polycations, enzymes, nanomaterials, and photoactive agents are being investigated. With regard to marine antifouling coatings, restrictions on the use of biocide-releasing coatings have made the generation of nontoxic antifouling surfaces more important. While considerable progress has been made in the design of antifouling coatings, ongoing research in this area should result in the development of even better antifouling materials in the future. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  19. Adaptive covariation between the coat and movement proteins of prunus necrotic ringspot virus.

    Science.gov (United States)

    Codoñer, Francisco M; Fares, Mario A; Elena, Santiago F

    2006-06-01

    The relative functional and/or structural importance of different amino acid sites in a protein can be assessed by evaluating the selective constraints to which they have been subjected during the course of evolution. Here we explore such constraints at the linear and three-dimensional levels for the movement protein (MP) and coat protein (CP) encoded by RNA 3 of prunus necrotic ringspot ilarvirus (PNRSV). By a maximum-parsimony approach, the nucleotide sequences from 46 isolates of PNRSV varying in symptomatology, host tree, and geographic origin have been analyzed and sites under different selective pressures have been identified in both proteins. We have also performed covariation analyses to explore whether changes in certain amino acid sites condition subsequent variation in other sites of the same protein or the other protein. These covariation analyses shed light on which particular amino acids should be involved in the physical and functional interaction between MP and CP. Finally, we discuss these findings in the light of what is already known about the implication of certain sites and domains in structure and protein-protein and RNA-protein interactions.

  20. Soy protein isolate inhibits hepatic tumor promotion in mice fed a high-fat liquid diet.

    Science.gov (United States)

    Mercer, Kelly E; Pulliam, Casey F; Pedersen, Kim B; Hennings, Leah; Ronis, Martin Jj

    2017-03-01

    Alcoholic and nonalcoholic fatty liver diseases are risk factors for development of hepatocellular carcinoma, but the underlying mechanisms are poorly understood. On the other hand, ingestion of soy-containing diets may oppose the development of certain cancers. We previously reported that replacing casein with a soy protein isolate reduced tumor promotion in the livers of mice with alcoholic liver disease after feeding a high fat ethanol liquid diet following initiation with diethylnitrosamine. Feeding soy protein isolate inhibited processes that may contribute to tumor promotion including inflammation, sphingolipid signaling, and Wnt/β-catenin signaling. We have extended these studies to characterize liver tumor promotion in a model of nonalcoholic fatty liver disease produced by chronic feeding of high-fat liquid diets in the absence of ethanol. Mice treated with diethylnitrosamine on postnatal day 14 were fed a high-fat liquid diet made with casein or SPI as the sole protein source for 16 weeks in adulthood. Relative to mice fed normal chow, a high fat/casein diet led to increased tumor promotion, hepatocyte proliferation, steatosis, and inflammation. Replacing casein with soy protein isolate counteracted these effects. The high fat diets also resulted in a general increase in transcripts for Wnt/β-catenin pathway components, which may be an important mechanism, whereby hepatic tumorigenesis is promoted. However, soy protein isolate did not block Wnt signaling in this nonalcoholic fatty liver disease model. We conclude that replacing casein with soy protein isolate blocks development of steatosis, inflammation, and tumor promotion in diethylnitrosamine-treated mice fed high fat diets. Impact statement The impact of dietary components on cancer is a topic of great interest for both the general public and the scientific community. Liver cancer is currently the second leading form of cancer deaths worldwide. Our study has addressed the effect of the protein

  1. UV-activated 7-dehydrocholesterol-coated titanium implants promote differentiation of human umbilical cord mesenchymal stem cells into osteoblasts.

    Science.gov (United States)

    Satué, María; Ramis, Joana M; Monjo, Marta

    2016-01-01

    Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23℃. Further, we investigated in vitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants. © The Author(s) 2015.

  2. Interaction of an IHF-like protein with the Rhizobium etli nifA promoter.

    Science.gov (United States)

    Benhassine, Traki; Fauvart, Maarten; Vanderleyden, Jos; Michiels, Jan

    2007-06-01

    The nifA gene fulfills an essential role in the regulation of nitrogen fixation genes in Rhizobium etli. Transcription analysis of the nifA gene, assessed using promoter deletions, indicated an oxygen-independent expression, threefold higher during symbiosis as compared with free-living conditions. Electrophoretic mobility shift assays using those nifA promoter deletion fragments, which were actively transcribed, demonstrated the specific interaction with R. etli cellular protein(s) resulting in the formation of two DNA-protein complexes. An interacting protein was purified by liquid chromatography on Heparin Sepharose and Mono S columns. The purified 12 kDa R. etli protein cross-reacted with antibodies directed against Escherichia coli integration host factor (IHF). Furthermore, purified E. coli IHF was able to specifically bind to the R. etli nifA promoter region. These results point to an as yet undisclosed function of IHF in the regulation of R. etli nifA expression.

  3. Raising the shields: PCR in the presence of metallic surfaces protected by tailor-made coatings.

    Science.gov (United States)

    Scherag, Frank D; Brandstetter, Thomas; Rühe, Jürgen

    2014-10-01

    The implementation of PCR reactions in the presence of metallic surfaces is interesting for the generation of novel bioanalytical devices, because metals exhibit high mechanical stability, good thermal conductivity, and flexibility during deformation. However, metallic substrates are usually non-compatible with enzymatic reactions such as PCR due to poisoning of the active center of the enzyme or nonspecific adsorption of the enzymeto the metal surface, which could result in protein denaturation. We present a method for the generation of polymer coatings on metallic surfaces which are designed to minimize protein adsorption and also prevent the release of metal ions. These coatings consist of three layers covalently linked to each other; a self-assembled monolayer to promote adhesion, a photochemically generated barrier layer and a photochemically generated hydrogel. The coatings can be deposited onto aluminum, stainless steel, gold and copper surfaces. We compare PCR efficiencies in the presence of bare metallic surfaces with those of surfaces treated with the novel coating system. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. All-atom normal-mode analysis reveals an RNA-induced allostery in a bacteriophage coat protein.

    Science.gov (United States)

    Dykeman, Eric C; Twarock, Reidun

    2010-03-01

    Assembly of the T=3 bacteriophage MS2 is initiated by the binding of a 19 nucleotide RNA stem loop from within the phage genome to a symmetric coat protein dimer. This binding event effects a folding of the FG loop in one of the protein subunits of the dimer and results in the formation of an asymmetric dimer. Since both the symmetric and asymmetric forms of the dimer are needed for the assembly of the protein container, this allosteric switch plays an important role in the life cycle of the phage. We provide here details of an all-atom normal-mode analysis of this allosteric effect. The results suggest that asymmetric contacts between the A -duplex RNA phosphodiester backbone of the stem loop with the EF loop in one coat protein subunit results in an increased dynamic behavior of its FG loop. The four lowest-frequency modes, which encompass motions predominantly on the FG loops, account for over 90% of the increased dynamic behavior due to a localization of the vibrational pattern on a single FG loop. Finally, we show that an analysis of the allosteric effect using an elastic network model fails to predict this localization effect, highlighting the importance of using an all-atom full force field method for this problem.

  5. Decreased Bacterial Attachment and Protein Adsorption to Coatings Produced by Low Enegy Plasma Polymerization

    DEFF Research Database (Denmark)

    Andersen, T.E.; Kingshott, Peter; Benter, M.

    .figure) .and E. coli grown on uncoated silicone compared to PP-PVP coated silicone (right figure). Results from the flow chamber analysis shows PP-PVP to be very good at preventing E. coli colonization during prolonged growth in flow chamber. At this point other surfaces and bacteria remains to be tested...... adsorption and bacteria attachment/colonization. This is emphasized by the fact that long dwelling urinary catheters, which is a typical silicone medical device, causes 5% per day incidence of urinary tract infection [1,2]. A demand therefore exists for surface modifications providing the silicone material......-coated crystals were then treated with one of the plasma polymerized coatings. Adsorption of fibrinogen, human serum albumin or immunoglobulin G was measured using a QCM-D instrument [5] (model E4, Q-Sense AB, Vastra Frolunda, Sweden) using a solution of 50llg/1 protein in PBS buffer. Results and Discussion: Our...

  6. Enhanced healing of rabbit segmental radius defects with surface-coated calcium phosphate cement/bone morphogenetic protein-2 scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yi; Hou, Juan; Yin, ManLi [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Wang, Jing, E-mail: biomatwj@163.com [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Liu, ChangSheng, E-mail: csliu@sh163.net [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China)

    2014-11-01

    Large osseous defects remain a difficult clinical problem in orthopedic surgery owing to the limited effective therapeutic options, and bone morphogenetic protein-2 (BMP-2) is useful for its potent osteoinductive properties in bone regeneration. Here we build a strategy to achieve prolonged duration time and help inducting new bone formation by using water-soluble polymers as a protective film. In this study, calcium phosphate cement (CPC) scaffolds were prepared as the matrix and combined with sodium carboxymethyl cellulose (CMC-Na), hydroxypropylmethyl cellulose (HPMC), and polyvinyl alcohol (PVA) respectively to protect from the digestion of rhBMP-2. After being implanted in the mouse thigh muscles, the surface-modified composite scaffolds evidently induced ectopic bone formation. In addition, we further evaluated the in vivo effects of surface-modified scaffolds in a rabbit radius critical defect by radiography, three dimensional micro-computed tomographic (μCT) imaging, synchrotron radiation-based micro-computed tomographic (SRμCT) imaging, histological analysis, and biomechanical measurement. The HPMC-modified CPC scaffold was regarded as the best combination for segmental bone regeneration in rabbit radius. - Highlights: • A simple surface-coating method was used to fabricate composite scaffolds. • Growth factor was protected from rapid depletion via superficial coating. • Significant promotion of bone regeneration was achieved. • HPMC-modification displayed optimal effect of bone regeneration.

  7. Apoptosis inhibitor of macrophage (AIM) diminishes lipid droplet-coating proteins leading to lipolysis in adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Iwamura, Yoshihiro; Mori, Mayumi; Nakashima, Katsuhiko [Laboratory of Molecular Biomedicine for Pathogenesis, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Mikami, Toshiyuki; Murayama, Katsuhisa [Genomic Science Laboratories, Dainippon Sumitomo Pharma Co. Ltd., 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-0022 (Japan); Arai, Satoko [Laboratory of Molecular Biomedicine for Pathogenesis, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Miyazaki, Toru, E-mail: tm@m.u-tokyo.ac.jp [Laboratory of Molecular Biomedicine for Pathogenesis, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer AIM induces lipolysis in a distinct manner from that of hormone-dependent lipolysis. Black-Right-Pointing-Pointer AIM ablates activity of peroxisome proliferator-activated receptor in adipocytes. Black-Right-Pointing-Pointer AIM reduces mRNA levels of lipid-droplet coating proteins leading to lipolysis. -- Abstract: Under fasting conditions, triacylglycerol in adipose tissue undergoes lipolysis to supply fatty acids as energy substrates. Such lipolysis is regulated by hormones, which activate lipases via stimulation of specific signalling cascades. We previously showed that macrophage-derived soluble protein, AIM induces obesity-associated lipolysis, triggering chronic inflammation in fat tissue which causes insulin resistance. However, the mechanism of how AIM mediates lipolysis remains unknown. Here we show that AIM induces lipolysis in a manner distinct from that of hormone-dependent lipolysis, without activation or augmentation of lipases. In vivo and in vitro, AIM did not enhance phosphorylation of hormone-sensitive lipase (HSL) in adipocytes, a hallmark of hormone-dependent lipolysis activation. Similarly, adipose tissue from obese AIM-deficient and wild-type mice showed comparable HSL phosphorylation. Consistent with the suppressive effect of AIM on fatty acid synthase activity, the amount of saturated and unsaturated fatty acids was reduced in adipocytes treated with AIM. This response ablated transcriptional activity of peroxisome proliferator-activated receptor (PPAR{gamma}), leading to diminished gene expression of lipid-droplet coating proteins including fat-specific protein 27 (FSP27) and Perilipin, which are indispensable for triacylglycerol storage in adipocytes. Accordingly, the lipolytic effect of AIM was overcome by a PPAR{gamma}-agonist or forced expression of FSP27, while it was synergized by a PPAR{gamma}-antagonist. Overall, distinct modes of lipolysis appear to take place in different physiological

  8. Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

    International Nuclear Information System (INIS)

    Henry, G.D.; Weiner, J.H.; Sykes, B.D.

    1987-01-01

    Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13 C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D 2 O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H 2 O solutions; in 1:1 H 2 O/D 2 O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13 C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects, the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule. A model of the detergent-solubilized coat protein is constructed from these H-exchange data which is consistent with circular dichroism and other NMR results

  9. Dynamics of Spore Coat Morphogenesis in Bacillus subtilis

    Science.gov (United States)

    McKenney, Peter T.; Eichenberger, Patrick

    2011-01-01

    SUMMARY Spores of Bacillus subtilis are encased in a protective coat made up of at least 70 proteins. The structure of the spore coat has been examined using a variety of genetic, imaging and biochemical techniques, however, the majority of these studies have focused on mature spores. In this study we use a library of 41 spore coat proteins fused to the Green Fluorescent Protein (GFP) to examine spore coat morphogenesis over the time-course of sporulation. We found considerable diversity in the localization dynamics of coat proteins and were able to establish 6 classes based on localization kinetics. Localization dynamics correlate well with the known transcriptional regulators of coat gene expression. Previously, we described the existence of multiple layers in the mature spore coat. Here, we find that the spore coat initially assembles a scaffold that is organized into multiple layers on one pole of the spore. The coat then encases the spore in multiple coordinated waves. Encasement is driven, at least partially, by transcription of coat genes and deletion of sporulation transcription factors arrests encasement. We also identify the trans-compartment SpoIIIAH-SpoIIQ channel as necessary for encasement. This is the first demonstration of a forespore contribution to spore coat morphogenesis. PMID:22171814

  10. Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Rosecká, Pavla; Dědič, P.; Filigarová, Marie

    2003-01-01

    Roč. 151, č. 4 (2003), s. 195-200 ISSN 0931-1785 R&D Projects: GA ČR GA522/01/1121 Institutional research plan: CEZ:AV0Z5038910 Keywords : potato mop-top virus * recombinant coat protein * Escherichia Coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.557, year: 2003

  11. Hydroxyapatite coatings for biomedical applications

    CERN Document Server

    Zhang, Sam

    2013-01-01

    Hydroxyapatite coatings are of great importance in the biological and biomedical coatings fields, especially in the current era of nanotechnology and bioapplications. With a bonelike structure that promotes osseointegration, hydroxyapatite coating can be applied to otherwise bioinactive implants to make their surface bioactive, thus achieving faster healing and recovery. In addition to applications in orthopedic and dental implants, this coating can also be used in drug delivery. Hydroxyapatite Coatings for Biomedical Applications explores developments in the processing and property characteri

  12. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

    Directory of Open Access Journals (Sweden)

    Sophie A Comyn

    2016-07-01

    Full Text Available Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

  13. Zinc phosphate conversion coatings

    Science.gov (United States)

    Sugama, Toshifumi

    1997-01-01

    Zinc phosphate conversion coatings for producing metals which exhibit enhanced corrosion prevention characteristics are prepared by the addition of a transition-metal-compound promoter comprising a manganese, iron, cobalt, nickel, or copper compound and an electrolyte such as polyacrylic acid, polymethacrylic acid, polyitaconic acid and poly-L-glutamic acid to a phosphating solution. These coatings are further improved by the incorporation of Fe ions. Thermal treatment of zinc phosphate coatings to generate .alpha.-phase anhydrous zinc phosphate improves the corrosion prevention qualities of the resulting coated metal.

  14. The influence of the N- and C- terminal modifications of Potato virus X coat protein on virus properties

    Czech Academy of Sciences Publication Activity Database

    Hoffmeisterová, Hana; Moravec, Tomáš; Plchová, Helena; Folwarczna, Jitka; Čeřovská, Noemi

    2012-01-01

    Roč. 56, č. 4 (2012), s. 775-779 ISSN 0006-3134 R&D Projects: GA ČR GA521/09/1525 Institutional research plan: CEZ:AV0Z50380511 Keywords : chimeric coat protein * expression of recombinant protein * Nicotiana benthamiana Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.692, year: 2012

  15. Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria.

    Science.gov (United States)

    Zhou, Jie; Zhang, Haifeng; Meng, Hengkai; Zhu, Yan; Bao, Guanhui; Zhang, Yanping; Li, Yin; Ma, Yanhe

    2014-03-28

    Cyanobacteria are oxygenic photosynthetic prokaryotes that play important roles in the global carbon cycle. Recently, engineered cyanobacteria capable of producing various small molecules from CO2 have been developed. However, cyanobacteria are seldom considered as factories for producing proteins, mainly because of the lack of efficient strong promoters. Here, we report the discovery and verification of a super-strong promoter P(cpc560), which contains two predicted promoters and 14 predicted transcription factor binding sites (TFBSs). Using P(cpc560), functional proteins were produced at a level of up to 15% of total soluble protein in the cyanobacterium Synechocystis sp. 6803, a level comparable to that produced in Escherichia coli. We demonstrated that the presence of multiple TFBSs in P(cpc560) is crucial for its promoter strength. Genetically transformable cyanobacteria neither have endotoxins nor form inclusion bodies; therefore, P(cpc560) opens the possibility to use cyanobacteria as alternative hosts for producing heterogeneous proteins from CO2 and inorganic nutrients.

  16. How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

    Directory of Open Access Journals (Sweden)

    Angela Schwede

    2015-12-01

    Full Text Available Variations on the statement "the variant surface glycoprotein (VSG coat that covers the external face of the mammalian bloodstream form of Trypanosoma brucei acts a physical barrier" appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.

  17. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling.

    Science.gov (United States)

    Kandasamy, Saveetha; Loganathan, Karthiba; Muthuraj, Raveendran; Duraisamy, Saravanakumar; Seetharaman, Suresh; Thiruvengadam, Raguchander; Ponnusamy, Balasubramanian; Ramasamy, Samiyappan

    2009-12-24

    Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

  18. Dependence of M13 Major Coat Protein Oligomerization and Lateral Segregation of Bilayer Composition

    NARCIS (Netherlands)

    Fernandes, F.; Loura, L.M.S.; Prieto, M.; Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

    2003-01-01

    M13 major coat protein was derivatized with BODIPY (n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide), and its aggregation was studied in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or

  19. Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes

    International Nuclear Information System (INIS)

    Nalcacioglu, Remziye; Marks, Hendrik; Vlak, Just M.; Demirbag, Zihni; Oers, Monique M. van

    2003-01-01

    The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an immediate-early gene and confirmed that the major capsid protein (MCP) is a late gene. Transcription of DNApol initiated 35 nt upstream and that of MCP 14 nt upstream of the translational start site. In a luciferase reporter gene assay both promoters were active only when cells were infected with CIV. For DNApol sequences between position -27 and -6, relative to the transcriptional start site, were essential for promoter activity. Furthermore, mutation of a G within the sequence TTGTTTT located just upstream of the DNApol transcription initiation site reduced the promoter activity by 25%. Sequences crucial for MCP promoter activity are located between positions -53 and -29

  20. Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain*

    Science.gov (United States)

    Harprecht, Christina; Okifo, Oghenefejiro; Robbins, Kevin J.; Motwani, Tina; Alexandrescu, Andrei T.; Teschke, Carolyn M.

    2016-01-01

    The I-domain is a genetic insertion in the phage P22 coat protein that chaperones its folding and stability. Of 11 acidic residues in the I-domain, seven participate in stabilizing electrostatic interactions with basic residues across elements of secondary structure, fastening the β-barrel fold. A hydrogen-bonded salt bridge between Asp-302 and His-305 is particularly interesting as Asp-302 is the site of a temperature-sensitive-folding mutation. The pKa of His-305 is raised to 9.0, indicating the salt bridge stabilizes the I-domain by ∼4 kcal/mol. Consistently, urea denaturation experiments indicate the stability of the WT I-domain decreases by 4 kcal/mol between neutral and basic pH. The mutants D302A and H305A remove the pH dependence of stability. The D302A substitution destabilizes the I-domain by 4 kcal/mol, whereas H305A had smaller effects, on the order of 1–2 kcal/mol. The destabilizing effects of D302A are perpetuated in the full-length coat protein as shown by a higher sensitivity to protease digestion, decreased procapsid assembly rates, and impaired phage production in vivo. By contrast, the mutants have only minor effects on capsid expansion or stability in vitro. The effects of the Asp-302–His-305 salt bridge are thus complex and context-dependent. Substitutions that abolish the salt bridge destabilize coat protein monomers and impair capsid self-assembly, but once capsids are formed the effects of the substitutions are overcome by new quaternary interactions between subunits. PMID:27006399

  1. The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module

    DEFF Research Database (Denmark)

    Rosenthal, J A; Chen, H; Slepnev, V I

    1999-01-01

    Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. E...... fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.......Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries...

  2. A study of strontium doped calcium phosphate coatings on AZ31

    International Nuclear Information System (INIS)

    Singh, Satish S.; Roy, Abhijit; Lee, Boeun E.; Ohodnicki, John; Loghmanian, Autrine; Banerjee, Ipsita; Kumta, Prashant N.

    2014-01-01

    Calcium phosphate (CaP) coatings have been studied to tailor the uncontrolled non-uniform corrosion of Mg based alloys while simultaneously enhancing bioactivity. The use of immersion techniques to deposit CaP coatings is attractive due to the ability of the approach to coat complex structures. In the current study, AZ31 substrates were subjected to various pretreatment conditions prior to depositing Sr 2+ doped and undoped CaP coatings. It was hypothesized that the bioactivity and corrosion protection of CaP coatings could be improved by doping with Sr 2+ . Heat treatment to elevated temperatures resulted in the diffusion of alloying elements, Mg and Zn, into the pretreated layer. Sr 2+ doped and undoped CaP coatings formed on the pretreated substrates consisted of biphasic mixtures of β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA). Electrochemical corrosion experiments indicated that the extent of Sr 2+ doping and pretreatment both influenced the corrosion protection. Cytotoxicity was evaluated with MC3T3-E1 mouse preosteoblasts and human mesenchymal stem cells (hMSCs). For both cell types, proliferation decreased upon increasing the Sr 2+ concentration. However, both osteogenic gene and protein expression significantly increased upon increasing Sr 2+ concentration. These results suggest that Sr 2+ doped coatings are capable of promoting osteogenic differentiation on degradable Mg alloys, while also enhancing corrosion protection, in comparison to undoped CaP coatings

  3. Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses.

    Science.gov (United States)

    Klaassen, V A; Boeshore, M; Dolja, V V; Falk, B W

    1994-07-01

    Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000. LIYV-infected plants contained multiple dsRNAs. The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2. To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned. Northern blot hybridization analysis showed no detectable sequence homology between these RNAs. A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone. The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli. Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses. The LIYV coat protein appears to be most closely related to the coat proteins of two closteroviruses, beet yellows virus and citrus tristeza virus.

  4. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

    Directory of Open Access Journals (Sweden)

    Thiruvengadam Raguchander

    2009-12-01

    Full Text Available Abstract Background Plant Growth Promoting Rhizobacteria (PGPR, Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

  5. Knotted vs. unknotted proteins: evidence of knot-promoting loops.

    Directory of Open Access Journals (Sweden)

    Raffaello Potestio

    Full Text Available Knotted proteins, because of their ability to fold reversibly in the same topologically entangled conformation, are the object of an increasing number of experimental and theoretical studies. The aim of the present investigation is to assess, on the basis of presently available structural data, the extent to which knotted proteins are isolated instances in sequence or structure space, and to use comparative schemes to understand whether specific protein segments can be associated to the occurrence of a knot in the native state. A significant sequence homology is found among a sizeable group of knotted and unknotted proteins. In this family, knotted members occupy a primary sub-branch of the phylogenetic tree and differ from unknotted ones only by additional loop segments. These "knot-promoting" loops, whose virtual bridging eliminates the knot, are found in various types of knotted proteins. Valuable insight into how knots form, or are encoded, in proteins could be obtained by targeting these regions in future computational studies or excision experiments.

  6. RNA2 of grapevine fanleaf virus: sequence analysis and coat protein cistron location.

    Science.gov (United States)

    Serghini, M A; Fuchs, M; Pinck, M; Reinbolt, J; Walter, B; Pinck, L

    1990-07-01

    The nucleotide sequence of the genomic RNA2 (3774 nucleotides) of grapevine fanleaf virus strain F13 was determined from overlapping cDNA clones and its genetic organization was deduced. Two rapid and efficient methods were used for cDNA cloning of the 5' region of RNA2. The complete sequence contained only one long open reading frame of 3555 nucleotides (1184 codons, 131K product). The analysis of the N-terminal sequence of purified coat protein (CP) and identification of its C-terminal residue have allowed the CP cistron to be precisely positioned within the polyprotein. The CP produced by proteolytic cleavage at the Arg/Gly site between residues 680 and 681 contains 504 amino acids (Mr 56019) and has hydrophobic properties. The Arg/Gly cleavage site deduced by N-terminal amino acid sequence analysis is the first for a nepovirus coat protein and for plant viruses expressing their genomic RNAs by polyprotein synthesis. Comparison of GFLV RNA2 with M RNA of cowpea mosaic comovirus and with RNA2 of two closely related nepoviruses, tomato black ring virus and Hungarian grapevine chrome mosaic virus, showed strong similarities among the 3' non-coding regions but less similarity among the 5' end non-coding sequences than reported among other nepovirus RNAs.

  7. Neural regeneration protein is a novel chemoattractive and neuronal survival-promoting factor

    International Nuclear Information System (INIS)

    Gorba, Thorsten; Bradoo, Privahini; Antonic, Ana; Marvin, Keith; Liu, Dong-Xu; Lobie, Peter E.; Reymann, Klaus G.; Gluckman, Peter D.; Sieg, Frank

    2006-01-01

    Neurogenesis and neuronal migration are the prerequisites for the development of the central nervous system. We have identified a novel rodent gene encoding for a neural regeneration protein (NRP) with an activity spectrum similar to the chemokine stromal-derived factor (SDF)-1, but with much greater potency. The Nrp gene is encoded as a forward frameshift to the hypothetical alkylated DNA repair protein AlkB. The predicted protein sequence of NRP contains domains with homology to survival-promoting peptide (SPP) and the trefoil protein TFF-1. The Nrp gene is first expressed in neural stem cells and expression continues in glial lineages. Recombinant NRP and NRP-derived peptides possess biological activities including induction of neural migration and proliferation, promotion of neuronal survival, enhancement of neurite outgrowth and promotion of neuronal differentiation from neural stem cells. NRP exerts its effect on neuronal survival by phosphorylation of the ERK1/2 and Akt kinases, whereas NRP stimulation of neural migration depends solely on p44/42 MAP kinase activity. Taken together, the expression profile of Nrp, the existence in its predicted protein structure of domains with similarities to known neuroprotective and migration-inducing factors and the high potency of NRP-derived synthetic peptides acting in femtomolar concentrations suggest it to be a novel gene of relevance in cellular and developmental neurobiology

  8. The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition.

    Science.gov (United States)

    Moll, Lorna; Ben-Gedalya, Tziona; Reuveni, Hadas; Cohen, Ehud

    2016-04-01

    The discovery that the alteration of aging by reducing the activity of the insulin/IGF-1 signaling (IIS) cascade protects nematodes and mice from neurodegeneration-linked, toxic protein aggregation (proteotoxicity) raises the prospect that IIS inhibitors bear therapeutic potential to counter neurodegenerative diseases. Recently, we reported that NT219, a highly efficient IGF-1 signaling inhibitor, protects model worms from the aggregation of amyloid β peptide and polyglutamine peptides that are linked to the manifestation of Alzheimer's and Huntington's diseases, respectively. Here, we employed cultured cell systems to investigate whether NT219 promotes protein homeostasis (proteostasis) in mammalian cells and to explore its underlying mechanisms. We found that NT219 enhances the aggregation of misfolded prion protein and promotes its deposition in quality control compartments known as "aggresomes." NT219 also elevates the levels of certain molecular chaperones but, surprisingly, reduces proteasome activity and impairs autophagy. Our findings show that IGF-1 signaling inhibitors in general and NT219 in particular can promote proteostasis in mammalian cells by hyperaggregating hazardous proteins, thereby bearing the potential to postpone the onset and slow the progression of neurodegenerative illnesses in the elderly.-Moll, L., Ben-Gedalya, T., Reuveni, H., Cohen, E. The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition. © FASEB.

  9. Tobacco arabinogalactan protein NtEPc can promote banana (Musa AAA) somatic embryogenesis.

    Science.gov (United States)

    Shu, H; Xu, L; Li, Z; Li, J; Jin, Z; Chang, S

    2014-12-01

    Banana is an important tropical fruit worldwide. Parthenocarpy and female sterility made it impossible to improve banana varieties through common hybridization. Genetic transformation for banana improvement is imperative. But the low rate that banana embryogenic callus was induced made the transformation cannot be performed in many laboratories. Finding ways to promote banana somatic embryogenesis is critical for banana genetic transformation. After tobacco arabinogalactan protein gene NtEPc was transformed into Escherichia coli (DE3), the recombinant protein was purified and filter-sterilized. A series of the sterilized protein was added into tissue culture medium. It was found that the number of banana immature male flowers developing embryogenic calli increased significantly in the presence of NtEPc protein compared with the effect of the control medium. Among the treatments, explants cultured on medium containing 10 mg/l of NtEPc protein had the highest chance to develop embryogenic calli. The percentage of lines that developed embryogenic calli on this medium was about 12.5 %. These demonstrated that NtEPc protein can be used to promote banana embryogenesis. This is the first paper that reported that foreign arabinogalactan protein (AGP) could be used to improve banana somatic embryogenesis.

  10. Hydrogen exchange kinetics in a membrane protein determined by 15N NMR spectroscopy: Use of the INEPT [insensitive nucleus enhancement by polarization transfer] experiment to follow individual amides in detergent-solubilized M13 coat protein

    International Nuclear Information System (INIS)

    Henry, G.D.; Sykes, B.D.

    1990-01-01

    The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous 1 H nuclear magnetic resonance (NMR) study, multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow kinetic sets containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10 5 -fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein the authors use 15 N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiments can be used to transfer magnetization to the 15 N nucleus from a coupled proton; when 15 N-labeled protonated protein is dissolved in 2 H 2 O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H + and OH - ions. The time-dependent exchange-out experiment is suitable for slow exchange rates (k ex ). The INEPT experiment was also adapted to measure some of the more rapidly exchanging amides in the coat protein using either saturation transfer from water or exchange effects on the polarization transfer step itself. The results of all of these experiments are consistent with previous models of the coat protein in which a stable segment extends from the hydrophobic membrane-spanning region through to the C-terminus, whereas the N-terminal region is undergoing more extensive dynamic fluctuations

  11. Rapid capillary coating by epoxy-poly-(dimethylacrylamide): Performance in capillary zone electrophoresis of protein and polystyrene carboxylate

    Czech Academy of Sciences Publication Activity Database

    Chiari, M.; Cretich, M.; Šťastná, Miroslava; Radko, S. P.; Chrambach, A.

    2001-01-01

    Roč. 22, č. 4 (2001), s. 656-659 ISSN 0173-0835 Institutional research plan: CEZ:AV0Z4031919 Keywords : capillary coating * capillary zone electrophoresis * proteins Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.282, year: 2001

  12. Changing folding and binding stability in a viral coat protein: a comparison between substitutions accessible through mutation and those fixed by natural selection.

    Science.gov (United States)

    Miller, Craig R; Lee, Kuo Hao; Wichman, Holly A; Ytreberg, F Marty

    2014-01-01

    Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy). No analogous studies have been carried out for protein-protein binding. Here we use a structure-based model of the major coat protein in a simple virus, bacteriophage φX174, to estimate the free energy of folding of a single coat protein and binding of five coat proteins within a pentameric unit. We confirm and extend previous work in finding that most accessible substitutions destabilize both protein folding and protein-protein binding. We compare the pool of accessible substitutions with those observed among the φX174-like wild phage and in experimental evolution with φX174. We find that observed substitutions have smaller effects on stability than expected by chance. An analysis of adaptations at high temperatures suggests that selection favors either substitutions with no effect on stability or those that simultaneously stabilize protein folding and slightly destabilize protein binding. We speculate that these mutations might involve adjusting the rate of capsid assembly. At normal laboratory temperature there is little evidence of directional selection. Finally, we show that cumulative changes in stability are highly variable; sometimes they are well beyond the bounds of single substitution changes and sometimes they are not. The variation leads us to conclude that phenotype selection acts on more than just stability. Instances of larger cumulative stability change (never via a single substitution despite their availability) lead us to conclude that selection views stability at a local, not a global, level.

  13. Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS

    DEFF Research Database (Denmark)

    Boyd, A. R.; Burke, G. A.; Duffy, H.

    2011-01-01

    Protein adsorption onto calcium phosphate (Ca–P) bioceramics utilised in hard tissue implant applications has been highlighted as one of the key events that influences the subsequent biological response, in vivo. This work reports on the use of surface-matrix assisted laser desorption ionisation...... to a combination of growth factors and lipoproteins present in serum. From the data obtained here it is evident that surface-MALDI-MS has significant utility as a tool for studying the dynamic nature of protein adsorption onto the surfaces of bioceramic coatings, which most likely plays a significant role...

  14. Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells

    DEFF Research Database (Denmark)

    Reuter, Lauri J.; Shahbazi, Mohammad-Ali; Makila, Ermei M.

    2017-01-01

    can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration...... to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of concept for the functionalization...

  15. Electrospun silk fibroin scaffolds coated with reduced graphene promote neurite outgrowth of PC-12 cells under electrical stimulation.

    Science.gov (United States)

    Aznar-Cervantes, Salvador; Pagán, Ana; Martínez, Jose G; Bernabeu-Esclapez, Antonia; Otero, Toribio F; Meseguer-Olmo, Luis; Paredes, Juan I; Cenis, Jose L

    2017-10-01

    Novel approaches to neural research require biocompatible materials capable to act as electrode structures or scaffolds for tissue engineering in order to stimulate or restore the functionality of damaged tissues. This work offers promising results that indicate the potential use of electrospun silk fibroin (SF) scaffolds coated with reduced graphene oxide (rGO) in this sense. The coated material becomes conductor and electroactive. A complete characterisation of SF/rGO scaffolds is provided in terms of electrochemistry, mechanical behaviour and chemical conformation of fibroin. The excellent biocompatibility of this novel material is proved with cultures of PC-12 cells. The coating with rGO improved the adhesion of cells in comparison with cells growing onto the surface of pure SF scaffolds. Also, the use of SF/rGO scaffolds combined with electrical stimulation promoted the differentiation into neural phenotypes reaching comparable or even superior levels to those obtained by means of the traditional treatment with neural growth factor (NGF). Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Diverse supramolecular structures formed by self‐assembling proteins of the B acillus subtilis spore coat

    Science.gov (United States)

    Jiang, Shuo; Wan, Qiang; Krajcikova, Daniela; Tang, Jilin; Tzokov, Svetomir B.; Barak, Imrich

    2015-01-01

    Summary Bacterial spores (endospores), such as those of the pathogens C lostridium difficile and B acillus anthracis, are uniquely stable cell forms, highly resistant to harsh environmental insults. B acillus subtilis is the best studied spore‐former and we have used it to address the question of how the spore coat is assembled from multiple components to form a robust, protective superstructure. B . subtilis coat proteins (CotY, CotE, CotV and CotW) expressed in E scherichia coli can arrange intracellularly into highly stable macro‐structures through processes of self‐assembly. Using electron microscopy, we demonstrate the capacity of these proteins to generate ordered one‐dimensional fibres, two‐dimensional sheets and three‐dimensional stacks. In one case (CotY), the high degree of order favours strong, cooperative intracellular disulfide cross‐linking. Assemblies of this kind could form exquisitely adapted building blocks for higher‐order assembly across all spore‐formers. These physically robust arrayed units could also have novel applications in nano‐biotechnology processes. PMID:25872412

  17. Identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells.

    Science.gov (United States)

    Ho, Steven C L; Yang, Yuansheng

    2014-08-01

    Promoters are essential on plasmid vectors to initiate transcription of the transgenes when generating therapeutic recombinant proteins expressing mammalian cell lines. High and sustained levels of gene expression are desired during therapeutic protein production while gene expression is useful for cell engineering. As many finely controlled promoters exhibit cell and product specificity, new promoters need to be identified, optimized and carefully evaluated before use. Suitable promoters can be identified using techniques ranging from simple molecular biology methods to modern high-throughput omics screenings. Promoter engineering is often required after identification to either obtain high and sustained expression or to provide a wider range of gene expression. This review discusses some of the available methods to identify and engineer promoters for therapeutic recombinant protein expression in mammalian cells.

  18. Silica-coated Gd(DOTA)-loaded protein nanoparticles enable magnetic resonance imaging of macrophages

    Science.gov (United States)

    Bruckman, Michael A.; Randolph, Lauren N.; Gulati, Neetu M.; Stewart, Phoebe L.; Steinmetz, Nicole F.

    2015-01-01

    The molecular imaging of in vivo targets allows non-invasive disease diagnosis. Nanoparticles offer a promising platform for molecular imaging because they can deliver large payloads of imaging reagents to the site of disease. Magnetic resonance imaging (MRI) is often preferred for clinical diagnosis because it uses non-ionizing radiation and offers both high spatial resolution and excellent penetration. We have explored the use of plant viruses as the basis of for MRI contrast reagents, specifically Tobacco mosaic virus (TMV), which can assemble to form either stiff rods or spheres. We loaded TMV particles with paramagnetic Gd ions, increasing the ionic relaxivity compared to free Gd ions. The loaded TMV particles were then coated with silica maintaining high relaxivities. Interestingly, we found that when Gd(DOTA) was loaded into the interior channel of TMV and the exterior was coated with silica, the T1 relaxivities increased by three-fold from 10.9 mM−1 s−1 to 29.7 mM−1s−1 at 60 MHz compared to uncoated Gd-loaded TMV. To test the performance of the contrast agents in a biological setting, we focused on interactions with macrophages because the active or passive targeting of immune cells is a popular strategy to investigate the cellular components involved in disease progression associated with inflammation. In vitro assays and phantom MRI experiments indicate efficient targeting and imaging of macrophages, enhanced contrast-to-noise ratio was observed by shape-engineering (SNP > TMV) and silica-coating (Si-TMV/SNP > TMV/SNP). Because plant viruses are in the food chain, antibodies may be prevalent in the population. Therefore we investigated whether the silica-coating could prevent antibody recognition; indeed our data indicate that mineralization can be used as a stealth coating option to reduce clearance. Therefore, we conclude that the silica-coated protein-based contrast agent may provide an interesting candidate material for further investigation

  19. TiO2 coating promotes human mesenchymal stem cell proliferation without the loss of their capacity for chondrogenic differentiation

    International Nuclear Information System (INIS)

    Kaitainen, Salla; Lappalainen, Reijo; Mähönen, Anssi J; J Lammi, Mikko; Qu, Chengjuan; Kröger, Heikki

    2013-01-01

    Human mesenchymal stem cells (hMSCs) are used in applications, which may require a large amount of cells; therefore, efficient expansion of the cells is desired. We studied whether TiO 2 coating on plastic cell culture dishes could promote proliferation of hMSCs without adverse effects in chondrogenic differentiation. TiO 2 -films were deposited on polystyrene dishes and glass coverslips using an ultrashort pulsed laser deposition technique. Human MSCs from three donors were expanded on them until 95% confluence, and the cells were evaluated by morphology, immunocytochemistry and quantitative RT-PCR (qRT-PCR). The chondrogenic differentiation in pellets was performed after cultivation on TiO 2 -coated dishes. Chondrogenesis was evaluated by histological staining of proteoglycans and type II collagen, and qRT-PCR. Human MSC-associated markers STRO-1, CD44, CD90 and CD146 did not change after expansion on TiO 2 -coated coverslips. However, the cell number after a 48h-culture period was significantly higher on TiO 2 -coated culture dishes. Importantly, TiO 2 coating caused no significant differences in the proteoglycan and type II collagen staining of the pellets, or the expression of chondrocyte-specific genes in the chondrogenesis assay. Thus, the proliferation of hMSCs could be significantly increased when cultured on TiO 2 -coated dishes without weakening their chondrogenic differentiation capacity. The transparency of TiO 2 -films allows easy monitoring of the cell growth and morphology under a phase-contrast microscope. (paper)

  20. Nanostructured Polyaniline Coating on ITO Glass Promotes the Neurite Outgrowth of PC 12 Cells by Electrical Stimulation.

    Science.gov (United States)

    Wang, Liping; Huang, Qianwei; Wang, Jin-Ye

    2015-11-10

    A conducting polymer polyaniline (PANI) with nanostructure was synthesized on indium tin oxide (ITO) glass. The effect of electrical stimulation on the proliferation and the length of neurites of PC 12 cells was investigated. The dynamic protein adsorption on PANI and ITO surfaces in a cell culture medium was also compared with and without electrical stimulation. The adsorbed proteins were characterized using SDS-PAGE. A PANI coating on ITO surface was shown with 30-50 nm spherical nanostructure. The number of PC 12 cells was significantly greater on the PANI/ITO surface than on ITO and plate surfaces after cell seeding for 24 and 36 h. This result confirmed that the PANI coating is nontoxic to PC 12 cells. The electrical stimulation for 1, 2, and 4 h significantly enhanced the cell numbers for both PANI and ITO conducting surfaces. Moreover, the application of electrical stimulation also improved the neurite outgrowth of PC 12 cells, and the number of PC 12 cells with longer neurite lengths increased obviously under electrical stimulation for the PANI surface. From the mechanism, the adsorption of DMEM proteins was found to be enhanced by electrical stimulation for both PANI/ITO and ITO surfaces. A new band 2 (around 37 kDa) was observed from the collected adsorbed proteins when PC 12 cells were cultured on these surfaces, and culturing PC 12 cells also seemed to increase the amount of band 1 (around 90 kDa). When immersing PANI/ITO and ITO surfaces in a DMEM medium without a cell culture, the number of band 3 (around 70 kDa) and band 4 (around 45 kDa) proteins decreased compared to that of PC 12 cell cultured surfaces. These results are valuable for the design and improvement of the material performance for neural regeneration.

  1. High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.

    Science.gov (United States)

    Zhang, Kang; Su, Lingqia; Duan, Xuguo; Liu, Lina; Wu, Jing

    2017-02-20

    We recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, P amyQ from Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536. Using the enzyme β-cyclodextrin glycosyltransferase (β-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the P amyQ' promoter produced the greatest extracellular β-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter P HpaII -P amyQ' produced the highest extracellular β-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter P HpaII -P amyQ' also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of β-CGTase using the dual-promoter P HpaII -P amyQ' system was investigated in a 3-L fermenter. Extracellular expression of β-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications. The dual-promoter P HpaII -P amyQ' system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.

  2. Bone Morphogenetic Protein Coating on Titanium Implant Surface: a Systematic Review

    Directory of Open Access Journals (Sweden)

    Haim Haimov

    2017-06-01

    Full Text Available Objectives: The purpose of the study is to systematically review the osseointegration process improvement by bone morphogenetic protein coating on titanium implant surface. Material and Methods: An electronic literature search was conducted through the MEDLINE (PubMed and EMBASE databases. The search was restricted for articles published during the last 10 years from October 2006 to September 2016 and articles were limited to English language. Results: A total of 41 articles were reviewed, and 8 of the most relevant articles that are suitable to the criteria were selected. Articles were analysed regarding concentration of bone morphogenetic protein (BMP, delivery systems, adverse reactions and the influence of the BMP on the bone and peri-implant surface in vivo. Finally, the present data included 340 implants and 236 models. Conclusions: It’s clearly shown from most of the examined studies that bone morphogenetic protein increases bone regeneration. Further studies should be done in order to induce and sustain bone formation activity. Osteogenic agent should be gradually liberated and not rapidly released with priority to three-dimension reservoir (incorporated titanium implant surface in order to avoid following severe side effects: inflammation, bleeding, haematoma, oedema, erythema, and graft failure.

  3. Coat protein deletion mutants elicit more severe symptoms than wild-type virus in multiple cereal hosts

    Science.gov (United States)

    The coat protein (CP) of Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. This study demonstrates that deletion of CP amino acids 58 to 84, but not 36 to 57, from WSMV genome induced severe ...

  4. TPA-inducible proteins may account for sensitivity to promotion of transformation

    International Nuclear Information System (INIS)

    Hirano, K.; Smith, B.; Colburn, N.H.

    1986-01-01

    The preneoplastic JB6 mouse epidermal cell system includes cell lines sensitive (P + ) or resistant (P - ) to tumor promoter induced neoplastic transformation. The authors investigated whether a difference in TPA-inducible proteins may explain this differential sensitivity. The synthesis of a 39 Kd cytoplasmic protein (Major Excreted Protein) was TPA-inducible, but to a similar extent in both P + and P - cells. TPA stimulated phosphorylation but not synthesis of the previously described stress protein pp80 in both P + and P - cells from 1 to 5 hr after treatment. Pulse labelling of P + and P - cells with 35 S-methionine revealed a TPA dependent P + specific transient increase in the synthesis of 58Kd protein. Induction was observed at 1 hr, and returned to basal levels by 4 hr and 20 hr, in nuclear and cytoplasmic fractions, respectively. This protein is not phosphorylated in response to TPA treatment. P + cells differ from P - cells in one or more genes that specify sensitivity to promotion of transformation, designated pro genes. Antibodies to three peptides representing the pro-1 open reading frame were used in immunoprecipitation and Western blotting to isolate the pro-1 gene product. A 43 Kd protein was immunologically responsive to the pro-1 peptide antibodies, and showed an increased signal 40 min after TPA treatment. Since the predicted molecular weight of a pro-1 gene product is only 7 Kd, the possibility of a modification of the protein by poly(ADP-ribosylation) or glycosylation is being investigated

  5. Multi-component adsorption model for pellicle formation: the influence of salivary proteins and non-salivary phospho proteins on the binding of histatin 5 onto hydroxyapatite.

    Science.gov (United States)

    Yin, A; Margolis, H C; Yao, Y; Grogan, J; Oppenheim, F G

    2006-02-01

    The acquired enamel pellicle formed by selective adsorption of proteins in whole saliva is a protective integument on the tooth surface. The purpose of the present study was to investigate the formation of human acquired enamel pellicle using an in vitro hydroxyapatite (HA) model and 3H-histatin 5 to allow accurate measurement of histatin 5 binding in a multi-component experimental system. A binary system was employed by mixing 3H-histatin 5 with one unlabeled protein prior to incubation with HA or by first incubating 3H-histatin 5 with the HA which had been pre-coated with one of a panel of unlabeled proteins (human albumin, salivary amylase, lysozyme, acidic PIFs, statherin, the N-terminal fragment of statherin, and egg yolk phosvitin). A ternary system was employed by mixing 3H-histatin 5 with HA sequentially pre-coated with two different unlabeled proteins, including recombinant histatin 1. The results showed that only salivary statherin and egg yolk phosvitin promote histatin 5 adsorption significantly. The amount of histatin 5 adsorbed was also found to increase as a function of the amount of phosvitin and statherin used to pre-coat HA up to a maximum level that was two- to four-fold greater than that observed on untreated HA. These data suggest that specific protein-protein interactions may play important roles in pellicle formation in vivo.

  6. Growth-promoting effect on iron-sulfur proteins on axenic cultures of Entamoeba dispar

    Directory of Open Access Journals (Sweden)

    Khalifa S.A.M.

    2006-03-01

    Full Text Available A growth-promoting factor (GPF that promotes the growth of Entamoeba dispar under axenic culture conditions was found in fractions of mitochondria (Mt, hydrogenosomes (Hg and chloroplasts (Cp obtained from cells of six different protozoan, mammalian and plant species. We were able to extract the GPF from the Cp-rich leaf cells of a plant (spiderwort: Commelina communis L. in an acetone-soluble fraction as a complex of chlorophyll with low molecular weight proteins (molecular weight [MW] approximately 4,600. We also found that on treatment with 0.6 % complexes of 2-mercapthoethanol (2ME, complexes of chlorophyll-a with iron-sulphur (Fe-S proteins (e.g., ferredoxins [Fd] from spinach and Clostridium pasteurianum and noncomplex rubredoxin (Rd from C. pasteurianum have a growth-promoting effect on E. dispar. These findings suggest that E. dispar may lack a sufficient quantity of some essential components of Fe-S proteins, such as Fe-S center.

  7. Neocellularization and neovascularization of nanosized bioactive glass-coated decellularized trabecular bone scaffolds

    KAUST Repository

    Gerhardt, Lutz Christian

    2012-09-11

    In this study, the in vivo recellularization and neovascularization of nanosized bioactive glass (n-BG)-coated decellu-larized trabecular bone scaffolds were studied in a rat model and quantified using stereological analyses. Based on the highest amount of vascular endothelial growth factor (VEGF) secreted by human fibroblasts grown on n-BG coatings (0-1.245 mg/cm 2), decellularized trabecular bone samples (porosity: 43-81%) were coated with n-BG particles. Grown on n-BG particles at a coating density of 0.263 mg/cm2, human fibroblasts produced 4.3 times more VEGF than on uncoated controls. After 8 weeks of implantation in Sprague-Dawley rats, both uncoated and n-BG-coated samples were well infiltrated with newly formed tissue (47-48%) and blood vessels (3-4%). No significant differences were found in cellularization and vascularization between uncoated bone scaffolds and n-BG-coated scaffolds. This finding indicates that the decellularized bone itself may exhibit growth-promoting properties induced by the highly interconnected pore microarchitecture and/or proteins left behind on decellularized scaffolds. Even if we did not find proangiogenic effects in n-BG-coated bone scaffolds, a bioactive coating is considered to be beneficial to impart osteoinductive and osteoconductive properties to decellularized bone. n-BG-coated bone grafts have thus high clinical potential for the regeneration of complex tissue defects given their ability for recellularization and neovascularization. © 2012 Wiley Periodicals, Inc.

  8. Evaluation of silk biomaterials in combination with extracellular matrix coatings for bladder tissue engineering with primary and pluripotent cells.

    Science.gov (United States)

    Franck, Debra; Gil, Eun Seok; Adam, Rosalyn M; Kaplan, David L; Chung, Yeun Goo; Estrada, Carlos R; Mauney, Joshua R

    2013-01-01

    Silk-based biomaterials in combination with extracellular matrix (ECM) coatings were assessed as templates for cell-seeded bladder tissue engineering approaches. Two structurally diverse groups of silk scaffolds were produced by a gel spinning process and consisted of either smooth, compact multi-laminates (Group 1) or rough, porous lamellar-like sheets (Group 2). Scaffolds alone or coated with collagen types I or IV or fibronectin were assessed independently for their ability to support attachment, proliferation, and differentiation of primary cell lines including human bladder smooth muscle cells (SMC) and urothelial cells as well as pluripotent cell populations, such as murine embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. AlamarBlue evaluations revealed that fibronectin-coated Group 2 scaffolds promoted the highest degree of primary SMC and urothelial cell attachment in comparison to uncoated Group 2 controls and all Group 1 scaffold variants. Real time RT-PCR and immunohistochemical (IHC) analyses demonstrated that both fibronectin-coated silk groups were permissive for SMC contractile differentiation as determined by significant upregulation of α-actin and SM22α mRNA and protein expression levels following TGFβ1 stimulation. Prominent expression of epithelial differentiation markers, cytokeratins, was observed in urothelial cells cultured on both control and fibronectin-coated groups following IHC analysis. Evaluation of silk matrices for ESC and iPS cell attachment by alamarBlue showed that fibronectin-coated Group 2 scaffolds promoted the highest levels in comparison to all other scaffold formulations. In addition, real time RT-PCR and IHC analyses showed that fibronectin-coated Group 2 scaffolds facilitated ESC and iPS cell differentiation toward both urothelial and smooth muscle lineages in response to all trans retinoic acid as assessed by induction of uroplakin and contractile gene and protein expression. These results

  9. Protective Coatings

    Science.gov (United States)

    1980-01-01

    General Magnaplate Corporation's pharmaceutical machine is used in the industry for high speed pressing of pills and capsules. Machine is automatic system for molding glycerine suppositories. These machines are typical of many types of drug production and packaging equipment whose metal parts are treated with space spinoff coatings that promote general machine efficiency and contribute to compliance with stringent federal sanitation codes for pharmaceutical manufacture. Collectively known as "synergistic" coatings, these dry lubricants are bonded to a variety of metals to form an extremely hard slippery surface with long lasting self lubrication. The coatings offer multiple advantages; they cannot chip, peel or be rubbed off. They protect machine parts from corrosion and wear longer, lowering maintenance cost and reduce undesired heat caused by power-robbing friction.

  10. Mycobacterium tuberculosis UvrD1 and UvrA proteins suppress DNA strand exchange promoted by cognate and noncognate RecA proteins.

    Science.gov (United States)

    Singh, Pawan; Patil, K Neelakanteshwar; Khanduja, Jasbeer Singh; Kumar, P Sanjay; Williams, Alan; Rossi, Franca; Rizzi, Menico; Davis, Elaine O; Muniyappa, K

    2010-06-15

    DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coli RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.

  11. Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins

    Science.gov (United States)

    Yu, Siming; Perálvarez-Marín, Alex; Minelli, Caterina; Faraudo, Jordi; Roig, Anna; Laromaine, Anna

    2016-07-01

    The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the outside of the SPIONs and their binding strength to the SPIONs is about 3.5 × 10-4 M, ten times higher than the adsorption of fetal bovine serum (FBS) on the same SPIONs. We elucidate a strong electrostatic interaction between BSA and the SPIONs, although the secondary structure of the protein is not affected. We present data that supports the strong binding of the BSA monolayer on SPIONs and the properties of the BSA layer as a protein-resistant coating. We believe that a complete understanding of the behavior and morphology of BSA-SPIONs and how the protein interacts with SPIONs is crucial for improving NP surface design and expanding the potential applications of SPIONs in nanomedicine.The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the

  12. Fabrication and biological evaluation of uniform extracellular matrix coatings on discontinuous photolithography generated micropallet arrays.

    Science.gov (United States)

    Gunn, Nicholas M; Bachman, Mark; Li, Guann-Pyng; Nelson, Edward L

    2010-11-01

    The recent identification of rare cell populations within tissues that are associated with specific biological behaviors, for example, progenitor cells, has illuminated a limitation of current technologies to study such adherent cells directly from primary tissues. The micropallet array is a recently developed technology designed to address this limitation by virtue of its capacity to isolate and recover single adherent cells on individual micropallets. The capacity to apply this technology to primary tissues and cells with restricted growth characteristics, particularly adhesion requirements, is critically dependent on the capacity to generate functional extracellular matrix (ECM) coatings. The discontinuous nature of the micropallet array surface provides specific constraints on the processes for generating the desired ECM coatings that are necessary to achieve the full functional capacity of the micropallet array. We have developed strategies, reported herein, to generate functional coatings with various ECM protein components: fibronectin, EHS tumor basement membrane extract, collagen, and laminin-5; confirmed by evaluation for rapid cellular adherence of four dissimilar cell types: fibroblast, breast epithelial, pancreatic epithelial, and myeloma. These findings are important for the dissemination and expanded use of micropallet arrays and similar microtechnologies requiring the integrated use of ECM protein coatings to promote cellular adherence.

  13. Highly Stable Trypsin-Aggregate Coatings on Polymer Nanofibers for Repeated Protein Digestion

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Byoung Chan; Lopez-Ferrer, Daniel; Lee, Sang-mok; Ahn, Hye-kyung; Nair, Sujith; Kim, Seong H.; Kim, Beom S.; Petritis, Konstantinos; Camp, David G.; Grate, Jay W.; Smith, Richard D.; Koo, Yoon-mo; Gu, Man Bock; Kim, Jungbae

    2009-04-01

    A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This new process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was also resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with enzyme aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

  14. Structure of clathrin-coated vesicles from small-angle scattering experiments

    DEFF Research Database (Denmark)

    Pedersen, J.S.

    1993-01-01

    Previously published small-angle neutron and X-ray scattering data from coated vesicles, reassembled coats, and stripped vesicles have been analyzed in terms of one common model. The neutron data sets include contrast variation measurements at three different D2O solvent concentrations. The model...... used for interpreting the data has spherical symmetry and explicitly takes into account polydispersity, which is described by a Gaussian distribution. A constant thickness of the clathrin coats is assumed. The fitting of the model shows that the coated vesicles consist of a low-density outer protein....... Thus, the membrane and the high-density protein shell overlap in space, which shows that the lipid membrane contains protein. The molecular mass of the average particle is 27 x 10(6) Da. The coated vesicles consist, on average, of approximately 85% protein and 15% lipids. About 40% of the protein mass...

  15. Analysis of agouti signaling protein (ASIP) gene polymorphisms and association with coat color in Tibetan sheep (Ovis aries).

    Science.gov (United States)

    Han, J L; Yang, M; Yue, Y J; Guo, T T; Liu, J B; Niu, C E; Yang, B H

    2015-02-06

    Tibetan sheep, an indigenous breed, have a wide variety of phenotypes and a colorful coat, which make this breed an interesting model for evaluating the effects of coat-color gene mutations on this phenotypic trait. The agouti signaling protein (ASIP) gene is a positional candidate gene, as was inferred based on previous study. In our research, ASIP gene copy numbers in genomic DNA were detected using a novel approach, and the exon 2 g.100-104 mutation and copy number variation (CNV) of ASIP were associated with coat color in 256 sheep collected from eight populations with different coat colors by high-resolution melting curve assay. We found that the relative copy numbers of ASIP ranged from one to eight in Tibetan sheep. All of the g.100-104 genotypes in the populations were in Hardy-Weinberg equilibrium, and there was no relationship between the g.100-104 genotype and coat color (P > 0.05). The single ASIP CNV allele was found to be almost entirely associated with solid-black coat color; however, not all solid-black sheep displayed the putative single ASIP CNV genotype. From our study, we speculate that the ASIP CNV is under great selective pressure and the single ASIP CNV allows selection for black coat color in Tibetan sheep, but this does not explain all black phenotypes in Tibetan sheep.

  16. Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization

    DEFF Research Database (Denmark)

    Coda, L; Salcini, A E; Confalonieri, S

    1998-01-01

    in NIH-3T3 cells overexpressing the receptor, even at low levels of receptor occupancy, thus behaving as physiological substrates. A role for eps15R in clathrin-mediated endocytosis is suggested by its localization in plasma membrane-coated pits and in vivo association to the coated pits' adapter protein...... AP-2. Finally, we demonstrate that a sizable fraction of eps15R exists in the cell as a complex with eps15 and that its EH domains exhibit binding specificities that are partially distinct from those of eps15. We propose that eps15 and eps15R are multifunctional binding proteins that serve...

  17. Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Hye-Kyung; Kim, Byoung Chan; Jun, Seung-Hyun; Chang, Mun Seock; Lopez-Ferrer, Daniel; Smith, Richard D.; Gu, Man Bock; Lee, Sang-Won; Kim, Beom S.; Kim, Jungbae

    2010-12-15

    An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently-attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 °C and 50 °C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 0.24 and 163.20 hours at 40 ºC, respectively. The improved stability of EC-TR can be explained by covalent-linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40 °C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of one week. In the same condition, CA-TR showed poor performance from the beginning, and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis, but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.

  18. Studies on soft centered coated snacks.

    Science.gov (United States)

    Pavithra, A S; Chetana, Ramakrishna; Babylatha, R; Archana, S N; Bhat, K K

    2013-04-01

    Roasted groundnut seeds, amaranth and dates pulp formed the center filling which was coated with sugar, breadings, desiccated coconut and roasted Bengalgram flour (BGF) to get 4 coated snacks. Physicochemical characteristics, microbiological profile, sorption behaviour and sensory quality of 4 coated snacks were determined. Centre filling to coating ratio of the products were in the range of 3:2-7:1, the product having BGF coating had the thinnest coating. Center filling had soft texture and the moisture content was 10.2-16.2% coating had lower moisture content (4.4-8.6%) except for Bengal gram coating, which had 11.1% moisture. Sugar coated snack has lowest fat (11.6%) and protein (7.2%) contents. Desiccated coconut coated snack has highest fat (25.4%) and Bengal gram flour coated snack had highest protein content (15.4%). Sorption studies showed that the coated snack had critical moisture content of 11.2-13.5%. The products were moisture sensitive and hence require packaging in films having higher moisture barrier property. In freshly prepared snacks coliforms, yeast and mold were absent. Mesophillic aerobes count did not show significant change during 90 days of storage at 27 °C and 37 °C. Sensory analysis showed that products had a unique texture due to combined effect of fairly hard coating and soft center. Flavour and overall quality of all the products were rated as very good.

  19. Wheat streak mosaic virus coat protein is a determinant for vector transmission by the wheat curl mite

    Science.gov (United States)

    Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae), is transmitted by the wheat curl mite (Aceria tosichella Keifer). The requirement of coat protein (CP) for WSMV transmission by the wheat curl mite was examined using a series of viable deletion and point mutations. Mite trans...

  20. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    Science.gov (United States)

    Fauteux, François; Strömvik, Martina V

    2009-01-01

    Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP) gene promoters from three plant families, namely Brassicaceae (mustards), Fabaceae (legumes) and Poaceae (grasses) using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L.) Heynh.), soybean (Glycine max (L.) Merr.) and rice (Oryza sativa L.) respectively. We have identified three conserved motifs (two RY-like and one ACGT-like) in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs

  1. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    Directory of Open Access Journals (Sweden)

    Fauteux François

    2009-10-01

    Full Text Available Abstract Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP gene promoters from three plant families, namely Brassicaceae (mustards, Fabaceae (legumes and Poaceae (grasses using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L. Heynh., soybean (Glycine max (L. Merr. and rice (Oryza sativa L. respectively. We have identified three conserved motifs (two RY-like and one ACGT-like in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination

  2. Evaluation of an edible coating based whey protein and beeswax on the physical and chemical quality of gooseberry (Physalis peruviana L.

    Directory of Open Access Journals (Sweden)

    Oswaldo Osorio Mora

    2016-10-01

    Full Text Available It was developed and optimized an edible coating based whey protein concentrate and beeswax. An experimental design 32 was used, this was evaluated by response surface methodology, it was obtained that the optimal formulation with a concentration of 15% beeswax and 10% whey protein, reduced by 35.49% weight loss of the fruit with respect to weight loss of control treatments. The optimal treatment was characterized and evaluated on the physicochemical properties of the gooseberry (Physalis peruviana L. in two storage conditions: environment (17- 2 ºC y HR: 69% and cooling (4 -2 ºC y HR: 66%. The results for the 15th day evaluation indicated a decrease in the percentage of weight loss, in storage environment and cooling (36.20% and 41.50% respectively. Control treatments decreased acidity in storage environment and cooling 3.88% and 4.92% respectively, compared to coating treatments. pH have not significantly change with any treatment. The coating prevent reduction of soluble solids 3.76% to environmental conditions and 2.27% to cooling conditions. For maturity index were not any significant changes between control treatments and coating treatments, in both conditions. The firmness remained without any significant changes except treatment environment uncoated, this presented a loss of firmness of 12.04% compared to the treatment environment coated. The respiration rate indicates a climacteric peak at day 8th for environment and at day 10th for cooling. For some properties, the cooling treatment uncoated and environment treatment coated have not any significant changes whereby the coating application can be an alternative to the cooling.

  3. TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine.

    Directory of Open Access Journals (Sweden)

    Stephanie Jemielity

    2013-03-01

    Full Text Available Human T-cell Immunoglobulin and Mucin-domain containing proteins (TIM1, 3, and 4 specifically bind phosphatidylserine (PS. TIM1 has been proposed to serve as a cellular receptor for hepatitis A virus and Ebola virus and as an entry factor for dengue virus. Here we show that TIM1 promotes infection of retroviruses and virus-like particles (VLPs pseudotyped with a range of viral entry proteins, in particular those from the filovirus, flavivirus, New World arenavirus and alphavirus families. TIM1 also robustly enhanced the infection of replication-competent viruses from the same families, including dengue, Tacaribe, Sindbis and Ross River viruses. All interactions between TIM1 and pseudoviruses or VLPs were PS-mediated, as demonstrated with liposome blocking and TIM1 mutagenesis experiments. In addition, other PS-binding proteins, such as Axl and TIM4, promoted infection similarly to TIM1. Finally, the blocking of PS receptors on macrophages inhibited the entry of Ebola VLPs, suggesting that PS receptors can contribute to infection in physiologically relevant cells. Notably, infection mediated by the entry proteins of Lassa fever virus, influenza A virus and SARS coronavirus was largely unaffected by TIM1 expression. Taken together our data show that TIM1 and related PS-binding proteins promote infection of diverse families of enveloped viruses, and may therefore be useful targets for broad-spectrum antiviral therapies.

  4. SEC16 in COPII coat dynamics at ER exit sites

    NARCIS (Netherlands)

    Sprangers, Joep; Rabouille, Catherine

    Protein export from the endoplasmic reticulum (ER), the first step in protein transport through the secretory pathway, is mediated by coatomer protein II (COPII)-coated vesicles at ER exit sites. COPII coat assembly on the ER is well understood and the conserved large hydrophilic protein Sec16

  5. Multi-protein delivery by nanodiamonds promotes bone formation.

    Science.gov (United States)

    Moore, L; Gatica, M; Kim, H; Osawa, E; Ho, D

    2013-11-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE(®) for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation.

  6. Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector

    Directory of Open Access Journals (Sweden)

    Juliette Doumayrou

    2016-11-01

    Full Text Available Understanding the molecular mechanisms involved in plant virus–vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus, Luteoviridae and Pea enation mosaic virus 2 (PEMV2, Umbravirus, Tombusviridae are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum, and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum. Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

  7. Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector.

    Science.gov (United States)

    Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C; Miller, W Allen

    2016-11-18

    Understanding the molecular mechanisms involved in plant virus-vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus , Luteoviridae ) and Pea enation mosaic virus 2 (PEMV2, Umbravirus , Tombusviridae ) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum , and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum . Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

  8. Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.

    Science.gov (United States)

    Wanka, Franziska; Arentshorst, Mark; Cairns, Timothy C; Jørgensen, Thomas; Ram, Arthur F J; Meyer, Vera

    2016-08-20

    The filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency. In this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein. Both hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.

  9. Combination of the Endogenous lhcsr1 Promoter and Codon Usage Optimization Boosts Protein Expression in the Moss Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Manuel Hiss

    2017-10-01

    Full Text Available The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.

  10. 40 CFR 174.516 - Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. 174.516 Section 174.516 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance...

  11. 40 CFR 174.515 - Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. 174.515 Section 174.515 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance...

  12. Ablation behavior of rare earth La-modified ZrC coating for SiC-coated carbon/carbon composites under an oxyacetylene torch

    International Nuclear Information System (INIS)

    Jia, Yujun; Li, Hejun; Feng, Lei; Sun, Jiajia; Li, Kezhi; Fu, Qiangang

    2016-01-01

    Highlights: • La-modified ZrC coating was prepared by supersonic atmosphere plasma spraying. • The oxyacetylene ablation behavior of La-modified ZrC/SiC coating was evaluated. • The coating shows a good ablation resistance under heat flux of 2.4 MW/m"2. • La promotes the liquid phase sintering of ZrO_2 and the formation of a compact scale. • The protection of the scale results in retaining elemental C in its inner layer. - Abstract: To improve the ablation resistance of carbon/carbon (C/C) composites at ultra-high temperature, La-modified ZrC coating was prepared on SiC-coated C/C composites by supersonic atmosphere plasma spraying. The coating shows a significant improvement on the ablation resistance compared with ZrC coating and could protect C/C composites for more than 120 s under heat flux of 2.4 MW/m"2. La acted as a role in promoting the liquid phase sintering of ZrO_2 and forming a compact scale with high thermal stability, improving the ablation resistance of C/C composites.

  13. Tailoring odorant-binding protein coatings characteristics for surface acoustic wave biosensor development

    Energy Technology Data Exchange (ETDEWEB)

    Di Pietrantonio, F., E-mail: fabio.dp@idasc.cnr.it [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Benetti, M. [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Dinca, V. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125 Magurele (Romania); Cannatà, D. [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Verona, E. [Institute for Photonics and Nanotechnologies, National Research Council of Italy, Via del Cineto Romano 42, 00156 Rome (Italy); D’Auria, S. [Institute of Protein Biochemistry, National Research Council of Italy, Via Pietro Castellino 111, 80131 Naples (Italy); Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125 Magurele (Romania)

    2014-05-01

    In this study, wild type bovine odorant-binding proteins (wtbOBPs) were deposited by matrix-assisted pulsed laser evaporation (MAPLE) and utilized as active material on surface acoustic wave (SAW) biosensors. Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM) were used to determine the chemical, morphological characteristics of the protein thin films. The FTIR data demonstrates that the functional groups of wtbOBPs do not suffer significant changes in the MAPLE-deposited films when compared to the reference one. The topographical studies show that the homogeneity, density and the roughness of the coatings are related mainly to the laser parameters (fluence and number of pulses). SAW biosensor responses to different concentrations of R-(–)-1-octen-3-ol (octenol) and R-(–)-carvone (carvone) were evaluated. The obtained sensitivities, achieved through the optimization of deposition parameters, demonstrated that MAPLE is a promising deposition technique for SAW biosensor implementation.

  14. Maize seed coatings and seedling sprayings with chitosan and hydrogen peroxide:their influence on some phenological and biochemical behaviors

    Institute of Scientific and Technical Information of China (English)

    Eva-Guadalupe LIZ(A)RRAGA-PAUL(I)N; Susana-Patricia MIRANDA-CASTRO; Ernesto MORENO-MART(I)NEZ; Alma-Virginia LARA-SAGAH(O)N; Irineo TORRES-PACHECO

    2013-01-01

    Objective:To evaluate the effect of chitosan(CH)and hydrogen peroxide(H2O2)seed coatings and seedling sprinklings on two different maize varieties by measuring their phenology,the H2O2 presence,the catalase (CAT)activity,and the protein quantity.Methods:Seven groups of ten seeds for each maize variety were treated with CH(2%(20 g/L)and 0.2%(2 g/L))or H2O2(8 mmol/L)by coating,sprinkling,or both.Germination and seedling growth were measured.One month after germination,the presence of H2O2 in seedlings in the coated seed treatments was evaluated.Protein content and CAT activity were determined under all treatments.Results:H2O2 seed coating enhanced the germination rate and increased seedling and stem length in the quality protein maize(QPM)variety.Seedlings had a higher emergence velocity under this treatment in both varieties.CH and H2O2 sprinklings did not have an effect on seedling phenology.Exogenous application of H2O2 promoted an increase of endogenous H2O2.CH and H2O2 seedling sprinkling increased the protein content in both maize varieties,while there was no significant effect on the CAT activity of treated seeds and seedlings.Conclusions:CH and H2O2 enhance some phenological and biochemical features of maize depending on their method of application.

  15. A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly

    Science.gov (United States)

    D'Lima, Nadia G.

    2015-01-01

    ABSTRACT Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as “molecular staples” at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging. IMPORTANCE Many dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by intercapsomer cross-linking, but most virus capsids are stabilized by alternate means. Here we show that the I domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I domain allows for stabilizing interactions across the capsid 2-fold axis of symmetry via a D-loop. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products, including tubes, are formed instead

  16. Interaction of phosphorylcholine with fibronectin coatings: Surface characterization and biological performances

    Energy Technology Data Exchange (ETDEWEB)

    Montaño-Machado, Vanessa, E-mail: vanessa.montano-machado.1@ulaval.ca [Laboratory for Biomaterials and Bioengineering, Dept. of Min-Met-Materials Eng., & University Hospital Research Center, Laval University, University Campus, PLT-1745G, Québec, Québec, G1 V 0A6 (Canada); ERRMECe, University of Cergy-Pontoise, Site Saint-Martin, 2 Avenue Adolphe Chauvin, 95302 Cergy-Pontoise Cedex (France); Noël, Céline, E-mail: celine.noel@unamur.be [Research Centre in Physics of Matter and Radiation (PMR), Université de Namur, 61 rue de Bruxelles, B-5000 Namur (Belgium); Chevallier, Pascale, E-mail: pascale.chevallier@crchudequebec.ulaval.ca [Laboratory for Biomaterials and Bioengineering, Dept. of Min-Met-Materials Eng., & University Hospital Research Center, Laval University, University Campus, PLT-1745G, Québec, Québec, G1 V 0A6 (Canada); Turgeon, Stéphane, E-mail: stephane.turgeon@crchudequebec.ulaval.ca [Laboratory for Biomaterials and Bioengineering, Dept. of Min-Met-Materials Eng., & University Hospital Research Center, Laval University, University Campus, PLT-1745G, Québec, Québec, G1 V 0A6 (Canada); Houssiau, Laurent, E-mail: laurent.houssiau@unamur.be [Research Centre in Physics of Matter and Radiation (PMR), Université de Namur, 61 rue de Bruxelles, B-5000 Namur (Belgium); Pauthe, Emmanuel, E-mail: emmanuel.pauthe@u-cergy.fr [ERRMECe, University of Cergy-Pontoise, Site Saint-Martin, 2 Avenue Adolphe Chauvin, 95302 Cergy-Pontoise Cedex (France); and others

    2017-02-28

    Highlights: • Fibronectin/phosphorylcholine coatings on plasma deposited fluorocarbon films were created. • The effect of several coating techniques on the surface biological performances was evaluated. • XPS, DWCA, immunostaining and ToF-SIMS (imaging and depth profiling) techniques were applied. • Potential for cardiovascular applications was showed by endothelial cell and blood interactions. - Abstract: Coating medical devices with several bioactive molecules is an interesting approach to achieve specific biological targets upon the interaction of the biomaterial with the living environment. In this work, a fluorocarbon polymer (CF{sub x}) was first deposited by plasma treatment on stainless steel (SS) substrate and thereafter, coatings containing fibronectin (FN) and phosphorylcholine (PRC) were created for cardiovascular applications. These two biomolecules were chosen to promote endothelialization and to avoid thrombus formation, respectively. Adsorption and grafting techniques were applied – and combined – to accomplish 4 different coatings containing both molecules. However, big challenge was found to characterize a small molecule (PRC: 184 g/mol) interacting with a protein (FN: 450 kD). For the first time XPS, dynamic water contact angle, immunostaining and ToF-SIMS (imaging and depth profiling) analyses were combined to accomplish the characterization of such a coating. The most encouraging biological performances were obtained for samples where FN was grafted to the CF{sub x} film followed by the adsorption of PRC: proliferation of endothelial cells and hemocompatibility properties were observed. Promising coatings for cardiovascular applications were developed. The relevance of characterizing the coatings with high sensitive techniques and the further correlation with their biological performances were evidenced.

  17. The influence of Sr content in calcium phosphate coatings

    International Nuclear Information System (INIS)

    Lindahl, Carl; Pujari-Palmer, Shiuli; Hoess, Andreas; Ott, Marjam; Engqvist, Håkan; Xia, Wei

    2015-01-01

    In this study calcium phosphate coatings with different amounts of strontium (Sr) were prepared using a biomineralization method. The incorporation of Sr changed the composition and morphology of coatings from plate-like to sphere-like morphology. Dissolution testing indicated that the solubility of the coatings increased with increased Sr concentration. Evaluation of extracts (with Sr concentrations ranging from 0 to 2.37 μg/mL) from the HA, 0.06Sr, 0.6Sr, and 1.2Sr coatings during in vitro cell cultures showed that Sr incorporation into coatings significantly enhanced the ALP activity in comparison to cells treated with control and HA eluted media. These findings show that calcium phosphate coatings could promote osteogenic differentiation even in a low amount of strontium. - Highlights: • Calcium phosphate coating doping with low Sr contents was prepared via a biomineralization process. • The solubility of the coatings increased with increased Sr concentration. • Present findings show the potential that Sr has on promoting osteogenic differentiation even in a low amount

  18. LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor

    Directory of Open Access Journals (Sweden)

    Rian de Laat

    2015-03-01

    Full Text Available Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP by β-secretase and γ-secretase generate amyloid β (Aβ peptides, which are thought to contribute to Alzheimer's disease (AD. Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD.

  19. Properties of Whey-Protein-Coated Films and Laminates as Novel Recyclable Food Packaging Materials with Excellent Barrier Properties

    Directory of Open Access Journals (Sweden)

    Markus Schmid

    2012-01-01

    Full Text Available In case of food packaging applications, high oxygen and water vapour barriers are the prerequisite conditions for preserving the quality of the products throughout their whole lifecycle. Currently available polymers and/or biopolymer films are mostly used in combination with barrier materials derived from oil based plastics or aluminium to enhance their low barrier properties. In order to replace these non-renewable materials, current research efforts are focused on the development of sustainable coatings, while maintaining the functional properties of the resulting packaging materials. This article provides an introduction to food packaging requirements, highlights prior art on the use of whey-based coatings for their barriers properties, and describes the key properties of an innovative packaging multilayer material that includes a whey-based layer. The developed whey protein formulations had excellent barrier properties almost comparable to the ethylene vinyl alcohol copolymers (EVOH barrier layer conventionally used in food packaging composites, with an oxygen barrier (OTR of <2 [cm³(STP/(m²d bar] when normalized to a thickness of 100 μm. Further requirements of the barrier layer are good adhesion to the substrate and sufficient flexibility to withstand mechanical load while preventing delamination and/or brittle fracture. Whey-protein-based coatings have successfully met these functional and mechanical requirements.

  20. Chemical stability and osteogenic activity of plasma-sprayed boron-modified calcium silicate-based coatings.

    Science.gov (United States)

    Lu, Xiang; Li, Kai; Xie, Youtao; Huang, Liping; Zheng, Xuebin

    2016-11-01

    In recent years, CaSiO 3 bio-ceramic coatings have attracted great attention because of their good bioactivity. However, their high degradation rates in physiological environment restrict their practical applications. In this work, boron-modified CaSiO 3 ceramic (Ca 11 Si 4 B 2 O 22 , B-CS) coating was developed on Ti substrates by plasma-spraying technique attempting to obtain enhanced chemical stability and osteogenic activity. The B-CS coating possessed significantly increased chemical stability due to the introduction of boron and consequently the modified crystal structure, while maintaining good bioactivity. Scanning electron microscope and immunofluorescence studies showed that better cellular adhesion and extinctive filopodia-like processes were observed on the B-CS coating. Compared with the pure CaSiO 3 (CS) coating, the B-CS coating promoted MC3T3-E1 cells attachment and proliferation. In addition, enhanced collagen I (COL-I) secretion, alkaline phosphatase activity, and extracellular matrix mineralization levels were detected from the B-CS coating. According to RT-PCR results, notable up-regulation expressions of mineralized tissue-related genes, such as runt-related transcription factor 2 (Runx2), bone sialoprotein and osteocalcin, and bone morphogenetic protein 7 (BMP-7) were observed on the B-CS coating compared with the CS coating. The above results suggested that Ca 11 Si 4 B 2 O 22 coatings possess excellent osteogenic activity and might be a promising candidate for orthopedic applications.

  1. Dominant Red Coat Color in Holstein Cattle Is Associated with a Missense Mutation in the Coatomer Protein Complex, Subunit Alpha (COPA Gene.

    Directory of Open Access Journals (Sweden)

    Ben Dorshorst

    Full Text Available Coat color in Holstein dairy cattle is primarily controlled by the melanocortin 1 receptor (MC1R gene, a central determinant of black (eumelanin vs. red/brown pheomelanin synthesis across animal species. The major MC1R alleles in Holsteins are Dominant Black (MC1RD and Recessive Red (MC1Re. A novel form of dominant red coat color was first observed in an animal born in 1980. The mutation underlying this phenotype was named Dominant Red and is epistatic to the constitutively activated MC1RD. Here we show that a missense mutation in the coatomer protein complex, subunit alpha (COPA, a gene with previously no known role in pigmentation synthesis, is completely associated with Dominant Red in Holstein dairy cattle. The mutation results in an arginine to cysteine substitution at an amino acid residue completely conserved across eukaryotes. Despite this high level of conservation we show that both heterozygotes and homozygotes are healthy and viable. Analysis of hair pigment composition shows that the Dominant Red phenotype is similar to the MC1R Recessive Red phenotype, although less effective at reducing eumelanin synthesis. RNA-seq data similarly show that Dominant Red animals achieve predominantly pheomelanin synthesis by downregulating genes normally required for eumelanin synthesis. COPA is a component of the coat protein I seven subunit complex that is involved with retrograde and cis-Golgi intracellular coated vesicle transport of both protein and RNA cargo. This suggests that Dominant Red may be caused by aberrant MC1R protein or mRNA trafficking within the highly compartmentalized melanocyte, mimicking the effect of the Recessive Red loss of function MC1R allele.

  2. Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

    Science.gov (United States)

    Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

    2012-03-01

    Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

  3. Synthesis of protein-coated biocompatible methotrexate-loaded PLA-PEG-PLA nanoparticles for breast cancer treatment

    Directory of Open Access Journals (Sweden)

    Salam Massadeh

    2016-06-01

    Full Text Available Background: PLA-PEG-PLA triblock polymer nanoparticles are promising tools for targeted dug delivery. The main aim in designing polymeric nanoparticles for drug delivery is achieving a controlled and targeted release of a specific drug at the therapeutically optimal rate and choosing a suitable preparation method to encapsulate the drug efficiently, which depends mainly on the nature of the drug (hydrophilic or hydrophobic. In this study, methotrexate (MTX-loaded nanoparticles were prepared by the double emulsion method. Method: Biodegradable polymer polyethylene glycol-polylactide acid tri-block was used with poly(vinyl alcohol as emulsifier. The resulting methotrexate polymer nanoparticles were coated with bovine serum albumin in order to improve their biocompatibility. This study focused on particle size distribution, zeta potential, encapsulation efficiency, loading capacity, and in vitro drug release at various concentrations of PVA (0.5%, 1%, 2%, and 3%. Results: Reduced particle size of methotrexate-loaded nanoparticles was obtained using lower PVA concentrations. Enhanced encapsulation efficiency and loading capacity was obtained using 1% PVA. FT-IR characterization was conducted for the void polymer nanoparticles and for drug-loaded nanoparticles with methotrexate, and the protein-coated nanoparticles in solid state showed the structure of the plain PEG-PLA and the drug-loaded nanoparticles with methotrexate. The methotrexate-loaded PLA-PEG-PLA nanoparticles have been studied in vitro; the drug release, drug loading, and yield are reported. Conclusion: The drug release profile was monitored over a period of 168 hours, and was free of burst effect before the protein coating. The results obtained from this work are promising; this work can be taken further to develop MTX based therapies.

  4. Synthesis of protein-coated biocompatible methotrexate-loaded PLA-PEG-PLA nanoparticles for breast cancer treatment

    Science.gov (United States)

    Massadeh, Salam; Alaamery, Manal; Al-Qatanani, Shatha; Alarifi, Saqer; Bawazeer, Shahad; Alyafee, Yusra

    2016-01-01

    Background PLA-PEG-PLA triblock polymer nanoparticles are promising tools for targeted dug delivery. The main aim in designing polymeric nanoparticles for drug delivery is achieving a controlled and targeted release of a specific drug at the therapeutically optimal rate and choosing a suitable preparation method to encapsulate the drug efficiently, which depends mainly on the nature of the drug (hydrophilic or hydrophobic). In this study, methotrexate (MTX)-loaded nanoparticles were prepared by the double emulsion method. Method Biodegradable polymer polyethylene glycol-polylactide acid tri-block was used with poly(vinyl alcohol) as emulsifier. The resulting methotrexate polymer nanoparticles were coated with bovine serum albumin in order to improve their biocompatibility. This study focused on particle size distribution, zeta potential, encapsulation efficiency, loading capacity, and in vitro drug release at various concentrations of PVA (0.5%, 1%, 2%, and 3%). Results Reduced particle size of methotrexate-loaded nanoparticles was obtained using lower PVA concentrations. Enhanced encapsulation efficiency and loading capacity was obtained using 1% PVA. FT-IR characterization was conducted for the void polymer nanoparticles and for drug-loaded nanoparticles with methotrexate, and the protein-coated nanoparticles in solid state showed the structure of the plain PEG-PLA and the drug-loaded nanoparticles with methotrexate. The methotrexate-loaded PLA-PEG-PLA nanoparticles have been studied in vitro; the drug release, drug loading, and yield are reported. Conclusion The drug release profile was monitored over a period of 168 hours, and was free of burst effect before the protein coating. The results obtained from this work are promising; this work can be taken further to develop MTX based therapies.

  5. Application of edible coating with essential oil in food preservation.

    Science.gov (United States)

    Ju, Jian; Xie, Yunfei; Guo, Yahui; Cheng, Yuliang; Qian, He; Yao, Weirong

    2018-03-26

    Compared with other types of packaging, edible coatings are becoming more and more popular because of their more environmentally friendly properties and active ingredients carrying ability. The edible coating can reduce the influence of essential oils (EOs) on the flavor of the product and also can prolong the action time of EOs through the slow-release effect, which effectively promote the application of EOs in food. Understanding the different combinations of edible coatings and EOs as well as their antimicrobial effects on different microorganisms will be more powerful and targeted to promote the application of EOs in real food systems. The review focus on the contribution of the combination of EOs and edible coatings (EO-edible coatings) to prolong the shelf life of food products, (1) specifically addressing the main materials used in the preparation of EO-edible coatings and the application of EO-edible coatings in the product, (2) systematically summarizing the main production method of EO-edible coatings, (3) discussing the antiseptic activity of EO-edible coatings on different microorganisms in food.

  6. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Science.gov (United States)

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  7. Molecular characterization and intermolecular interaction of coat protein of Prunus necrotic ringspot virus: implications for virus assembly.

    Science.gov (United States)

    Kulshrestha, Saurabh; Hallan, Vipin; Sharma, Anshul; Seth, Chandrika Attri; Chauhan, Anjali; Zaidi, Aijaz Asghar

    2013-09-01

    Coat protein (CP) and RNA3 from Prunus necrotic ringspot virus (PNRSV-rose), the most prevalent virus infecting rose in India, were characterized and regions in the coat protein important for self-interaction, during dimer formation were identified. The sequence analysis of CP and partial RNA 3 revealed that the rose isolate of PNRSV in India belongs to PV-32 group of PNRSV isolates. Apart from the already established group specific features of PV-32 group member's additional group-specific and host specific features were also identified. Presence of methionine at position 90 in the amino acid sequence alignment of PNRSV CP gene (belonging to PV-32 group) was identified as the specific conserved feature for the rose isolates of PNRSV. As protein-protein interaction plays a vital role in the infection process, an attempt was made to identify the portions of PNRSV CP responsible for self-interaction using yeast two-hybrid system. It was found (after analysis of the deletion clones) that the C-terminal region of PNRSV CP (amino acids 153-226) plays a vital role in this interaction during dimer formation. N-terminal of PNRSV CP is previously known to be involved in CP-RNA interactions, but our results also suggested that N-terminal of PNRSV CP represented by amino acids 1-77 also interacts with C-terminal (amino acids 153-226) in yeast two-hybrid system, suggesting its probable involvement in the CP-CP interaction.

  8. Coating thickness measuring device

    International Nuclear Information System (INIS)

    Joffe, B.B.; Sawyer, B.E.; Spongr, J.J.

    1984-01-01

    A device especially adapted for measuring the thickness of coatings on small, complexly-shaped parts, such as, for example, electronic connectors, electronic contacts, or the like. The device includes a source of beta radiation and a radiation detector whereby backscatter of the radiation from the coated part can be detected and the thickness of the coating ascertained. The radiation source and detector are positioned in overlying relationship to the coated part and a microscope is provided to accurately position the device with respect to the part. Means are provided to control the rate of descent of the radiation source and radiation detector from its suspended position to its operating position and the resulting impact it makes with the coated part to thereby promote uniformity of readings from operator to operator, and also to avoid excessive impact with the part, thereby improving accuracy of measurement and eliminating damage to the parts

  9. The structure of the COPII transport-vesicle coat assembled on membranes.

    Science.gov (United States)

    Zanetti, Giulia; Prinz, Simone; Daum, Sebastian; Meister, Annette; Schekman, Randy; Bacia, Kirsten; Briggs, John A G

    2013-09-17

    Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available. We have used cryo-electron tomography and subtomogram averaging to determine the structure of the complete, membrane-assembled COPII coat. We describe a novel arrangement of the outer coat and find that the inner coat can assemble into regular lattices. The data reveal how coat subunits interact with one another and with the membrane, suggesting how coordinated assembly of inner and outer coats can mediate and regulate packaging of vesicles ranging from small spheres to large tubular carriers. DOI:http://dx.doi.org/10.7554/eLife.00951.001.

  10. Sustained release of neurotrophin-3 via calcium phosphate-coated sutures promotes axonal regeneration after spinal cord injury.

    Science.gov (United States)

    Hanna, Amgad; Thompson, Daniel L; Hellenbrand, Daniel J; Lee, Jae-Sung; Madura, Casey J; Wesley, Meredith G; Dillon, Natalie J; Sharma, Tapan; Enright, Connor J; Murphy, William L

    2016-07-01

    Because of the dynamics of spinal cord injury (SCI), the optimal treatment will almost certainly be a combination approach to control the environment and promote axonal growth. This study uses peripheral nerve grafts (PNGs) as scaffolds for axonal growth while delivering neurotrophin-3 (NT-3) via calcium phosphate (CaP) coatings on surgical sutures. CaP coating was grown on sutures, and NT-3 binding and release were characterized in vitro. Then, the NT-3-loaded sutures were tested in a complete SCI model. Rats were analyzed for functional improvement and axonal growth into the grafts. The CaP-coated sutures exhibited a burst release of NT-3, followed by a sustained release for at least 20 days. Functionally, the rats with PNGs + NT-3-loaded sutures and the rats treated with PNGs scored significantly higher than controls on day 56 postoperatively. However, functional scores in rats treated with PNGs + NT-3-loaded suture were not significantly different from those of rats treated with PNGs alone. Cholera toxin subunit B (CTB) labeling rostral to the graft was not observed in any controls, but CTB labeling rostral to the graft was observed in almost all rats that had had a PNG. Neurofilament labeling on transverse sections of the graft revealed that the rats treated with the NT-3-loaded sutures had significantly more axons per graft than rats treated with an NT-3 injection and rats without NT-3. These data demonstrate that PNGs serve as scaffolds for axonal growth after SCI and that CaP-coated sutures can efficiently release NT-3 to increase axonal regeneration. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

    Directory of Open Access Journals (Sweden)

    Vidal M.S.

    2002-01-01

    Full Text Available Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22. Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

  12. Ceramic protective coatings applied by sol-gel or electrophoresis

    International Nuclear Information System (INIS)

    Stoch, A.

    1993-01-01

    Sol-gel and electrophoresis are the complementary techniques which may be used for obtaining the ceramic coatings. The composition of such a coatings depends on the composition of electrophoresis bath or sol solution. Thermal treatment is used for densifying the coating and promoting the adherence of coating to the substrate. In presented work silica, silica-alumina or alumina coatings are applied by sol-gel dip coating procedure on steel, aluminium or ceramic substrates. Electrophoresis is employed for obtaining zirconia, alumina or hydroxyapatite coatings on stainless steel. (author). 7 refs

  13. Characterization and functional analyses of the human G protein-coupled receptor kinase 4 gene promoter.

    Science.gov (United States)

    Hasenkamp, Sandra; Telgmann, Ralph; Staessen, Jan A; Hagedorn, Claudia; Dördelmann, Corinna; Bek, Martin; Brand-Herrmann, Stefan-Martin; Brand, Eva

    2008-10-01

    The G protein-coupled receptor kinase 4 is involved in renal sodium handling and blood pressure regulation. Missense variants have already been tested functionally and are associated with hypertension, but no data on promoter analyses are yet available. We scanned 94 hypertensive white subjects for genetic variation and performed promoter reporter gene analyses in HEK293T, COS7, and SaOs-2 cells. Transient transfections with various full lengths and wild-type deletion constructs revealed that 1851 bp of the flanking region and 275 bp of the 5'-untranslated region were sufficient for transcriptional activities and composed a powerful cis-active element in the distal 293 bp. The -1702T and +2T alleles resulted in drastic general reductions of promoter function, whereas an activity increasing effect of +268C was cell type specific. Electrophoretic mobility-shift assay, supershift, and cotransfection analyses of transcription factor binding sites predicted in silico (Alibaba2.1/Transfac7) resulted in allele-specific binding patterns of nuclear proteins and identified the participation of CCAAT/enhancer-binding protein transcription factor family members. The G protein-coupled receptor kinase 4 core promoter resides in the first 1851 bp upstream of its transcription start site. The 4 identified genetic variants within this region exert allele-specific impact on both cell type- and stimulation-dependent transcription and may affect the expression balance of renal G protein-coupled receptor kinase 4.

  14. Rapid and Efficient Protein Digestion using Trypsin Coated Magnetic Nanoparticles under Pressure Cycles

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Byoungsoo; Lopez-Ferrer, Daniel; Kim, Byoung Chan; Na, Hyon Bin; Park, Yong Il; Weitz, Karl K.; Warner, Marvin G.; Hyeon, Taeghwan; Lee, Sang-Won; Smith, Richard D.; Kim, Jungbae

    2011-01-01

    Trypsin-coated magnetic nanoparticles (EC-TR/NPs), prepared via a simple crosslinking of the enzyme to magnetic nanoparticles, were highly stable and could be easily captured using a magnet after the digestion was complete. EC-TR/NPs showed a negligible loss of trypsin activity after multiple uses and continuous shaking, while a control sample of covalently-attached trypsin on NPs resulted in a rapid inactivation under the same conditions due to the denaturation and autolysis of trypsin. Digestions were carried out on a single model protein, a five protein mixture, and a whole mouse brain proteome, and also compared for digestion at atmospheric pressure and 37 ºC for 12 h, and in combination with pressure cycling technology (PCT) at room temperature for 1 min. In all cases, the EC-TR/NPs performed equally as well or better than free trypsin in terms of the number of peptide/protein identifications and reproducibility across technical replicates. However, the concomitant use of EC-TR/NPs and PCT resulted in very fast (~1 min) and more reproducible digestions.

  15. Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.

    Science.gov (United States)

    Bando, Hiroki; Hisada, Hiromoto; Ishida, Hiroki; Hata, Yoji; Katakura, Yoshio; Kondo, Akihiko

    2011-11-01

    A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.

  16. Activation of mitochondrial promoter PH-binding protein in a radio-resistant Chinese hamster cell strain associated with Bcl-2

    International Nuclear Information System (INIS)

    Roychoudhury, Paromita; Ghosh, Utpal; Bhattacharyya, Nitai P.; Chaudhuri, Keya

    2006-01-01

    The cellular response to ionizing radiation is mediated by a complex interaction of number of proteins involving different pathways. Previously, we have shown that up regulation of mitochondrial genes ND1, ND4, and COX1 transcribed from the heavy strand promoter (P H ) has been increased in a radio-resistant cell strain designated as M5 in comparison with the parental Chinese hamster V79 cells. These genes are also up regulated in Chinese hamster V79 cells VB13 that express exogenous human Bcl2. In the present study, the expression of the gene ND6 that is expressed from the light strand promoter (P L ) was found to be similar in both the cell lines, as determined by RT-PCR. To test the possibility that this differential expression of mitochondrial genes under these two promoters was mediated by differences in proteins' affinity to interact with these promoters, we have carried out electrophoretic mobility shift assay (EMSA) using mitochondrial cell extracts from these two cell lines. Our result of these experiments revealed that two different proteins formed complex with the synthetic promoters and higher amount of protein from M5 cell extracts interacted with the P H promoter in comparison to that observed with cell extracts from Chinese hamster V79 cells. The promoter-specific differential binding of proteins was also observed in VB13. These results showed that differential mitochondrial gene expression observed earlier in the radio-resistant M5 cells was due to enhanced interaction proteins with the promoters P H and mediated by the expression of Bcl2

  17. Nucleotide sequence of the coat protein gene of Lettuce big-vein virus.

    Science.gov (United States)

    Sasaya, T; Ishikawa, K; Koganezawa, H

    2001-06-01

    A sequence of 1425 nt was established that included the complete coat protein (CP) gene of Lettuce big-vein virus (LBVV). The LBVV CP gene encodes a 397 amino acid protein with a predicted M(r) of 44486. Antisera raised against synthetic peptides corresponding to N-terminal or C-terminal parts of the LBVV CP reacted in Western blot analysis with a protein with an M(r) of about 48000. RNA extracted from purified particles of LBVV by using proteinase K, SDS and phenol migrated in gels as two single-stranded RNA species of approximately 7.3 kb (ss-1) and 6.6 kb (ss-2). After denaturation by heat and annealing at room temperature, the RNA migrated as four species, ss-1, ss-2 and two additional double-stranded RNAs (ds-1 and ds-2). The Northern blot hybridization analysis using riboprobes from a full-length clone of the LBVV CP gene indicated that ss-2 has a negative-sense nature and contains the LBVV CP gene. Moreover, ds-2 is a double-stranded form of ss-2. Database searches showed that the LBVV CP most resembled the nucleocapsid proteins of rhabdoviruses. These results indicate that it would be appropriate to classify LBVV as a negative-sense single-stranded RNA virus rather than as a double-stranded RNA virus.

  18. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

    DEFF Research Database (Denmark)

    Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M

    2003-01-01

    Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also found...... in the proteasome subunit SpRpn2 and the anaphase-promoting complex/cyclosome (APC/C) subunit Cut4. The putative role of Pus1 as a factor involved in allocation of ubiquitinylated substrates for the proteasome is discussed....

  19. Nanostructured titanium–silver coatings with good antibacterial activity and cytocompatibility fabricated by one-step magnetron sputtering

    Energy Technology Data Exchange (ETDEWEB)

    Bai, Long [Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan (China); Hang, Ruiqiang, E-mail: hangruiqiang@tyut.edu.cn [Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan (China); Gao, Ang [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China); Zhang, Xiangyu; Huang, Xiaobo; Wang, Yueyue; Tang, Bin [Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan (China); Zhao, Lingzhou, E-mail: zhaolingzhou1983@hotmail.com [State Key Laboratory of Military Stomatology, Department of Periodontology, School of Stomatology, The Fourth Military Medical University, Xi’an (China); Chu, Paul K. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China)

    2015-11-15

    Graphical abstract: - Highlights: • We fabricate Ti–Ag coatings with different Ag contents and surface morphologies. • The Ti–Ag coatings possess long-term antibacterial ability. • Increased Ag contents in the coatings leads to enhanced osteoblast functions. - Abstract: Bacterial infection and loosing are serious complications for biomedical implants in the orthopedic, dental, and other biomedical fields and the ideal implants should combine good antibacterial ability and bioactivity. In this study, nanostructured titanium–silver (Ti–Ag) coatings with different Ag contents (1.2 to 21.6 at%) are prepared on Ti substrates by magnetron sputtering. As the Ag concentration is increased, the coatings change from having dense columnar crystals to sparse ones and eventually no columnar structure. The Ti–Ag coatings can effectively kill Staphylococcus aureus during the first few days and remain moderately antibacterial after immersion for 75 days. Compared to pure Ti, the Ti–Ag coatings show good cytocompatibility as indicated by good osteoblast adhesion, proliferation, intracellular total protein synthesis, and alkaline phosphatase (ALP) activity. In addition, cell spreading, collagen secretion, and extracellular matrix mineralization are promoted on the coatings with the proper Ag contents due to the nanostructured morphological features. Our results indicate that favorable antibacterial activity and osseointegration ability can be simultaneously achieved by regulating the Ag contents in Ti–Ag coatings.

  20. Delivery of bone morphogenetic protein-2 and substance P using graphene oxide for bone regeneration

    Directory of Open Access Journals (Sweden)

    La WG

    2014-05-01

    Full Text Available Wan-Geun La,1 Min Jin,1 Saibom Park,1,2 Hee-Hun Yoon,1 Gun-Jae Jeong,1 Suk Ho Bhang,1 Hoyoung Park,1,2 Kookheon Char,1,2 Byung-Soo Kim1,31School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea; 2The National Creative Research Initiative Center for Intelligent Hybrids, Seoul National University, Seoul, Republic of Korea; 3Institute of Bioengineering, Institute of Chemical Processes, Engineering Research Institute, Seoul National University, Seoul, Republic of KoreaAbstract: In this study, we demonstrate that graphene oxide (GO can be used for the delivery of bone morphogenetic protein-2 (BMP-2 and substance P (SP, and that this delivery promotes bone formation on titanium (Ti implants that are coated with GO. GO coating on Ti substrate enabled a sustained release of BMP-2. BMP-2 delivery using GO-coated Ti exhibited a higher alkaline phosphatase activity in bone-forming cells in vitro compared with bare Ti. SP, which is known to recruit mesenchymal stem cells (MSCs, was co-delivered using Ti or GO-coated Ti to further promote bone formation. SP induced the migration of MSCs in vitro. The dual delivery of BMP-2 and SP using GO-coated Ti showed the greatest new bone formation on Ti implanted in the mouse calvaria compared with other groups. This approach may be useful to improve osteointegration of Ti in dental or orthopedic implants.Keywords: bone morphogenetic protein-2, bone regeneration, graphene oxides, stem cell recruitment, substance P

  1. Cationic osteogenic peptide P15-CSP coatings promote 3-D osteogenesis in poly(epsilon-caprolactone) scaffolds of distinct pore size.

    Science.gov (United States)

    Li, Xian; Ghavidel Mehr, Nima; Guzmán-Morales, Jessica; Favis, Basil D; De Crescenzo, Gregory; Yakandawala, Nandadeva; Hoemann, Caroline D

    2017-08-01

    P15-CSP is a biomimetic cationic fusion peptide that stimulates osteogenesis and inhibits bacterial biofilm formation when coated on 2-D surfaces. This study tested the hypothesis that P15-CSP coatings enhance 3-D osteogenesis in a porous but otherwise hydrophobic poly-(ɛ-caprolactone) (PCL) scaffold. Scaffolds of 84 µm and 141 µm average pore size were coated or not with Layer-by-Layer polyelectrolytes followed by P15-CSP, seeded with adult primary human mesenchymal stem cells (MSCs), and cultured 10 days in proliferation medium, then 21 days in osteogenic medium. Atomic analyses showed that P15-CSP was successfully captured by LbL. After 2 days of culture, MSCs adhered and spread more on P15-CSP coated pores than PCL-only. At day 10, all constructs contained nonmineralized tissue. At day 31, all constructs became enveloped in a "skin" of tissue that, like 2-D cultures, underwent sporadic mineralization in areas of high cell density that extended into some 141 µm edge pores. By quantitative histomorphometry, 2.5-fold more tissue and biomineral accumulated in edge pores versus inner pores. P15-CSP specifically promoted tissue-scaffold integration, fourfold higher overall biomineralization, and more mineral deposits in the outer 84 µm and inner 141 µm pores than PCL-only (p pore surfaces with 3-D topography. Biomineralization deeper than 150 µm from the scaffold edge was optimally attained with the larger 141 µm peptide-coated pores. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2171-2181, 2017. © 2017 Wiley Periodicals, Inc.

  2. Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture.

    Science.gov (United States)

    DeQuach, Jessica A; Mezzano, Valeria; Miglani, Amar; Lange, Stephan; Keller, Gordon M; Sheikh, Farah; Christman, Karen L

    2010-09-27

    The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu. We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells. This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell culture to more closely mimic the mature in vivo ECM microenvironment.

  3. The Timing of cotE Expression Affects Bacillus subtilis Spore Coat Morphology but Not Lysozyme Resistance▿

    Science.gov (United States)

    Costa, Teresa; Serrano, Mónica; Steil, Leif; Völker, Uwe; Moran, Charles P.; Henriques, Adriano O.

    2007-01-01

    The synthesis of structural components and morphogenetic factors required for the assembly of the Bacillus subtilis spore coat is governed by a mother cell-specific transcriptional cascade. The first two temporal classes of gene expression, which involve RNA polymerase sigma σE factor and the ancillary regulators GerR and SpoIIID, are deployed prior to engulfment of the prespore by the mother cell. The two last classes rely on σK, whose activation follows engulfment completion, and GerE. The cotE gene codes for a morphogenetic protein essential for the assembly of the outer coat layer and spore resistance to lysozyme. cotE is expressed first from a σE-dependent promoter and, in a second stage, from a promoter that additionally requires SpoIIID and that remains active under σK control. CotE localizes prior to engulfment completion close to the surface of the developing spore, but formation of the outer coat is a late, σK-controlled event. We have transplanted cotE to progressively later classes of mother cell gene expression. This created an early class of mutants in which cotE is expressed prior to engulfment completion and a late class in which expression of cotE follows the complete engulfment of the prespore. Mutants of the early class assemble a nearly normal outer coat structure, whereas mutants of the late class do not. Hence, the early expression of CotE is essential for outer coat assembly. Surprisingly, however, all mutants were fully resistant to lysozyme. The results suggest that CotE has genetically separable functions in spore resistance to lysozyme and spore outer coat assembly. PMID:17172339

  4. Shelf-life of fresh blueberries coated with quinoa protein/chitosan/sunflower oil edible film.

    Science.gov (United States)

    Abugoch, Lilian; Tapia, Cristián; Plasencia, Dora; Pastor, Ana; Castro-Mandujano, Olivio; López, Luis; Escalona, Victor H

    2016-01-30

    The aim of this study was to evaluate quinoa protein (Q), chitosan (CH) and sunflower oil (SO) as edible film material as well as the influence of this coating in extending the shelf-life of fresh blueberries stored at 4 °C and 75% relative humidity. These conditions were used to simulate the storage conditions in supermarkets and represent adverse conditions for testing the effects of the coating. The mechanical, barrier, and structural properties of the film were measured. The effectiveness of the coating in fresh blueberries (CB) was evaluated by changes in weight loss, firmness, color, molds and yeast count, pH, titratable acidity, and soluble solids content. The tensile strength and elongation at break of the edible film were 0.45 ± 0.29 MPa and 117.2% ± 7%, respectively. The water vapor permeability was 3.3 × 10(-12) ± 4.0 × 10(-13) g s(-1) m(-1) Pa(-1). In all of the color parameters CB presented significant differences. CB had slight delayed fruit ripening as evidenced by higher titratable acidity (0.3-0.5 g citric acid 100 g(-1)) and lower pH (3.4-3.6) than control during storage; however, it showed reduced firmness (up to 38%). The use of Q/CH/SO as a coating in fresh blueberries was able to control the growth of molds and yeasts during 32 days of storage, whereas the control showed an increasing of molds and yeast, between 1.8 and 3.1 log cycles (between 20 and 35 days). © 2015 Society of Chemical Industry.

  5. Cellular Handling of Protein Aggregates by Disaggregation Machines.

    Science.gov (United States)

    Mogk, Axel; Bukau, Bernd; Kampinga, Harm H

    2018-01-18

    Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Growth hormone-promoted tyrosyl phosphorylation of SHC proteins and SHC association with Grb2

    DEFF Research Database (Denmark)

    VanderKuur, J; Allevato, G; Billestrup, Nils

    1995-01-01

    . To gain insight into pathways coupling GH receptor (GHR) to MAP kinase activation and signaling molecules that might interact with GHR and its associated tyrosine kinase JAK2, we examined whether SHC and Grb2 proteins serve as signaling molecules for GH. Human GH was shown to promote the rapid tyrosyl...... phosphorylation of 66-, 52-, and 46-kDa SHC proteins in 3T3-F442A fibroblasts. GH also promoted binding of GHR and JAK2 to the SH2 domain of 46/52-kDa SHC protein fused to glutathione S-transferase (GST). Constitutively phosphorylated JAK2, from COS-7 cells transiently transfected with murine JAK2 cDNA, bound......-638 and GHR1-638(Y333,338F), GH stimulated phosphorylation of all 3 SHC proteins whereas GH stimulated phosphorylation of only the 66- and 52-kDa SHC proteins in cells expressing GHR1-454. GH had no effect on SHC phosphorylation in cells expressing GHR1-294 or GHR delta P, the latter lacking amino acids 297...

  7. Interfaces in graded coatings on titanium-based implants.

    Science.gov (United States)

    Lopez-Esteban, S; Gutierrez-Gonzalez, C F; Gremillard, L; Saiz, E; Tomsia, A P

    2009-03-15

    Graded bilayered glass-ceramic composite coatings on Ti6Al4V substrates were fabricated using an enameling technique. The layers consisted of a mixture of glasses in the CaO-MgO-Na(2)O-K(2)O-P(2)O(5) system with different amounts of calcium phosphates (CPs). Optimum firing conditions have been determined for the fabrication of coatings having good adhesion to the metal, while avoiding deleterious reactions between the glass and the ceramic particles. The final coatings do not crack or delaminate. The use of high-silica layers (>60 wt % SiO(2)) in contact with the alloy promotes long-term stability of the coating; glass-metal adhesion is achieved through the formation of a nanostructured Ti(5)Si(3) layer. A surface layer containing a mixture of a low-silica glass ( approximately 53 wt % SiO(2)) and synthetic hydroxyapatite particles promotes the precipitation of new apatite during tests in vitro. The in vitro behavior of the coatings in simulated body fluid depends both on the composition of the glass matrix and the CP particles, and is strongly affected by the coating design and the firing conditions.

  8. Effects of orthopedic implants with a polycaprolactone polymer coating containing bone morphogenetic protein-2 on osseointegration in bones of sheep.

    Science.gov (United States)

    Niehaus, Andrew J; Anderson, David E; Samii, Valerie F; Weisbrode, Steven E; Johnson, Jed K; Noon, Mike S; Tomasko, David L; Lannutti, John J

    2009-11-01

    To determine elution characteristics of bone morphogenetic protein (BMP)-2 from a polycaprolactone coating applied to orthopedic implants and determine effects of this coating on osseointegration. 6 sheep. An in vitro study was conducted to determine BMP-2 elution from polycaprolactone-coated implants. An in vivo study was conducted to determine the effects on osseointegration when the polycaprolactone with BMP-2 coating was applied to bone screws. Osseointegration was assessed via radiography, measurement of peak removal torque and bone mineral density, and histomorphometric analysis. Physiologic response was assessed by measuring serum bone-specific alkaline phosphatase activity and uptake of bone markers. Mean +/- SD elution on day 1 of the in vitro study was 263 +/- 152 pg/d, which then maintained a plateau at 59.8 +/- 29.1 pg/d. Mean peak removal torque for screws coated with polycalprolactone and BMP-2 (0.91 +/- 0.65 dN x m) and screws coated with polycaprolactone alone (0.97 +/- 1.30 dN.m) did not differ significantly from that for the control screws (2.34 +/- 1.62 dN x m). Mean bone mineral densities were 0.535 +/- 0.060 g/cm(2), 0.596 +/- 0.093 g/cm(2), and 0.524 +/- 0.142 g/cm(2) for the polycaprolactone-BMP-2-coated, polycaprolactone-coated, and control screws, respectively, and did not differ significantly among groups. Histologically, bone was in closer apposition to the implant with the control screws than with either of the coated screws. BMP-2 within the polycaprolactone coating did not stimulate osteogenesis. The polycaprolactone coating appeared to cause a barrier effect that prevented formation of new bone. A longer period or use of another carrier polymer may result in increased osseointegration.

  9. Architecture and assembly of the Bacillus subtilis spore coat.

    Science.gov (United States)

    Plomp, Marco; Carroll, Alicia Monroe; Setlow, Peter; Malkin, Alexander J

    2014-01-01

    Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of "nanodot" particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization

  10. Architecture and Assembly of the Bacillus subtilis Spore Coat

    Science.gov (United States)

    Plomp, Marco; Carroll, Alicia Monroe; Setlow, Peter; Malkin, Alexander J.

    2014-01-01

    Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of “nanodot” particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization

  11. Accelerated differentiation of osteoblast cells on polycaprolactone scaffolds driven by a combined effect of protein coating and plasma modification

    Energy Technology Data Exchange (ETDEWEB)

    Yildirim, Eda D; Gueceri, Selcuk; Sun, Wei [Department of Mechanical Engineering and Mechanics, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Besunder, Robyn; Allen, Fred [Drexel University, School of Biomedical Engineering Science and Health System, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Pappas, Daphne, E-mail: edy22@drexel.ed [Army Research Laboratory, Aberdeen Proving Ground, MD 21005 (United States)

    2010-03-15

    A combined effect of protein coating and plasma modification on the quality of the osteoblast-scaffold interaction was investigated. Three-dimensional polycaprolactone (PCL) scaffolds were manufactured by the precision extrusion deposition (PED) system. The structural, physical, chemical and biological cues were introduced to the surface through providing 3D structure, coating with adhesive protein fibronectin and modifying the surface with oxygen-based plasma. The changes in the surface properties of PCL after those modifications were examined by contact angle goniometry, surface energy calculation, surface chemistry analysis (XPS) and surface topography measurements (AFM). The effects of modification techniques on osteoblast short-term and long-term functions were examined by cell adhesion, proliferation assays and differentiation markers, namely alkaline phosphatase activity (ALP) and osteocalcin secretion. The results suggested that the physical and chemical cues introduced by plasma modification might be sufficient for improved cell adhesion, but for accelerated osteoblast differentiation the synergetic effects of structural, physical, chemical and biological cues should be introduced to the PCL surface.

  12. The Influence of Coating Structure on Micromachine Stiction

    Energy Technology Data Exchange (ETDEWEB)

    Kushmerick, J.G.; Hankins, M.G.; De Boer, M.P.; Clews, P.J.; Carpick, R.W.; Bunker, B.C.

    2000-10-03

    We have clearly shown that the film morphology dictates the anti-stiction properties of FDTS coatings. Release stiction is not observed when ideal monolayer films are present but can be extensive when thicker aggregate structures are present. This finding is significant because it indicates that agglomerate formation during processing is a major source of irreproducible behavior when FDTS coatings are used to release micromachined parts. The results could also help explain why coatings that are aged at high. humidity start to stick to each other. (AFM results show that humid environments promote the formation of aggregates from monolayer films.) The reason why aggregate structures promote stiction is currently unknown. However, it appears that aggregates interfere with the ability of FDTS to form dense, well-ordered coatings under microstructures, leading to surfaces that are sufficiently hydrophilic to allow for release stiction via an attractive Laplace force during drying.

  13. Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes

    KAUST Repository

    Alam, Tanvir

    2014-10-02

    Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future.

  14. Zinc-decorated silica-coated magnetic nanoparticles for protein binding and controlled release.

    Science.gov (United States)

    Bele, Marjan; Hribar, Gorazd; Campelj, Stanislav; Makovec, Darko; Gaberc-Porekar, Vladka; Zorko, Milena; Gaberscek, Miran; Jamnik, Janko; Venturini, Peter

    2008-05-01

    The aim of this study was to be able to reversibly bind histidine-rich proteins to the surface of maghemite magnetic nanoparticles via coordinative bonding using Zn ions as the anchoring points. We showed that in order to adsorb Zn ions on the maghemite, the surface of the latter needs to be modified. As silica is known to strongly adsorb zinc ions, we chose to modify the maghemite nanoparticles with a nanometre-thick silica layer. This layer appeared to be thin enough for the maghemite nanoparticles to preserve their superparamagnetic nature. As a model the histidine-rich protein bovine serum albumin (BSA) was used. The release of the BSA bound to Zn-decorated silica-coated maghemite nanoparticles was analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrated that the bonding of the BSA to such modified magnetic nanoparticles is highly reversible and can be controlled by an appropriate change of the external conditions, such as a pH decrease or the presence/supply of other chelating compounds.

  15. A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

    Directory of Open Access Journals (Sweden)

    Patrizia Marinelli

    2018-04-01

    Full Text Available Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states. Keywords: Protein oxidation, Protein misfolding, Protein aggregation, Oxidative stress, Post-translational modification

  16. Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-Terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis

    NARCIS (Netherlands)

    Houwing, C.J.; Huis in 't Veld, M.; Zuidema, D.; Graaff, de M.; Jaspars, E.M.J.

    2001-01-01

    Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural

  17. Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes

    NARCIS (Netherlands)

    Nalcacioglu, R.; Marks, H.; Vlak, J.M.; Demirbag, Z.; Oers, van M.M.

    2003-01-01

    The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an

  18. Method of manufacturing a graphite coated fuel can

    International Nuclear Information System (INIS)

    Saito, Koichi; Uchida, Shunsuke.

    1984-01-01

    Purpose: To improve the close bondability and homogeneity of a graphite coating formed at the inner surface of a fuel can. Method: A coating containing graphite dispersed in a volatile organic solvent is used and a graphite coating is formed to the inner surface of a fuel can by way of a plunger method. After applying graphite coating, an inert gas is caused to flow at a certain flow rate to the inside of the fuel can horizontally rotaged so that gassification and evaporation of the volatile organic solvent contained in the graphite coating may be promoted. Since drying of the graphite coating coated to the inner surface of the fuel can thus be controlled, a graphite coating with satisfactory close bondability and homogeneity can be formed. (Kawakami, Y.)

  19. Molecular Composition and Ultrastructure of the Caveolar Coat Complex

    Science.gov (United States)

    Ludwig, Alexander; Howard, Gillian; Mendoza-Topaz, Carolina; Deerinck, Thomas; Mackey, Mason; Sandin, Sara; Ellisman, Mark H.; Nichols, Benjamin J.

    2013-01-01

    Caveolae are an abundant feature of the plasma membrane of many mammalian cell types, and have key roles in mechano-transduction, metabolic regulation, and vascular permeability. Caveolin and cavin proteins, as well as EHD2 and pacsin 2, are all present in caveolae. How these proteins assemble to form a protein interaction network for caveolar morphogenesis is not known. Using in vivo crosslinking, velocity gradient centrifugation, immuno-isolation, and tandem mass spectrometry, we determine that cavins and caveolins assemble into a homogenous 80S complex, which we term the caveolar coat complex. There are no further abundant components within this complex, and the complex excludes EHD2 and pacsin 2. Cavin 1 forms trimers and interacts with caveolin 1 with a molar ratio of about 1∶4. Cavins 2 and 3 compete for binding sites within the overall coat complex, and form distinct subcomplexes with cavin 1. The core interactions between caveolin 1 and cavin 1 are independent of cavin 2, cavin 3, and EHD2 expression, and the cavins themselves can still interact in the absence of caveolin 1. Using immuno-electron microscopy as well as a recently developed protein tag for electron microscopy (MiniSOG), we demonstrate that caveolar coat complexes form a distinct coat all around the caveolar bulb. In contrast, and consistent with our biochemical data, EHD2 defines a different domain at the caveolar neck. 3D electron tomograms of the caveolar coat, labeled using cavin-MiniSOG, show that the caveolar coat is composed of repeating units of a unitary caveolar coat complex. PMID:24013648

  20. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    International Nuclear Information System (INIS)

    Parent, Kristin N.; Tang, Jinghua; Cardone, Giovanni; Gilcrease, Eddie B.; Janssen, Mandy E.; Olson, Norman H.; Casjens, Sherwood R.; Baker, Timothy S.

    2014-01-01

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer

  1. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Parent, Kristin N., E-mail: kparent@msu.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Tang, Jinghua; Cardone, Giovanni [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Gilcrease, Eddie B. [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Janssen, Mandy E.; Olson, Norman H. [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Casjens, Sherwood R., E-mail: sherwood.casjens@path.utah.edu [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Baker, Timothy S., E-mail: tsb@ucsd.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); University of California, San Diego, Division of Biological Sciences, La Jolla, CA, 92093 (United States)

    2014-09-15

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.

  2. Axon initial segment cytoskeleton comprises a multiprotein submembranous coat containing sparse actin filaments

    Science.gov (United States)

    Jones, Steven L.; Korobova, Farida

    2014-01-01

    The axon initial segment (AIS) of differentiated neurons regulates action potential initiation and axon–dendritic polarity. The latter function depends on actin dynamics, but actin structure and functions at the AIS remain unclear. Using platinum replica electron microscopy (PREM), we have characterized the architecture of the AIS cytoskeleton in mature and developing hippocampal neurons. The AIS cytoskeleton assembly begins with bundling of microtubules and culminates in formation of a dense, fibrillar–globular coat over microtubule bundles. Immunogold PREM revealed that the coat contains a network of known AIS proteins, including ankyrin G, spectrin βIV, neurofascin, neuronal cell adhesion molecule, voltage-gated sodium channels, and actin filaments. Contrary to existing models, we find neither polarized actin arrays, nor dense actin meshworks in the AIS. Instead, the AIS contains two populations of sparse actin filaments: short, stable filaments and slightly longer dynamic filaments. We propose that stable actin filaments play a structural role for formation of the AIS diffusion barrier, whereas dynamic actin may promote AIS coat remodeling. PMID:24711503

  3. The Coat Protein and NIa Protease of Two Potyviridae Family Members Independently Confer Superinfection Exclusion

    Science.gov (United States)

    French, Roy

    2016-01-01

    ABSTRACT Superinfection exclusion (SIE) is an antagonistic virus-virus interaction whereby initial infection by one virus prevents subsequent infection by closely related viruses. Although SIE has been described in diverse viruses infecting plants, humans, and animals, its mechanisms, including involvement of specific viral determinants, are just beginning to be elucidated. In this study, SIE determinants encoded by two economically important wheat viruses, Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) and Triticum mosaic virus (TriMV; genus Poacevirus, family Potyviridae), were identified in gain-of-function experiments that used heterologous viruses to express individual virus-encoded proteins in wheat. Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, or NIb cistrons permitted efficient superinfection by WSMV expressing green fluorescent protein (WSMV-GFP). In contrast, wheat infected with TriMV expressing WSMV NIa-Pro or coat protein (CP) substantially excluded superinfection by WSMV-GFP, suggesting that both of these cistrons are SIE effectors encoded by WSMV. Importantly, SIE is due to functional WSMV NIa-Pro or CP rather than their encoding RNAs, as altering the coded protein products by minimally changing RNA sequences led to abolishment of SIE. Deletion mutagenesis further revealed that elicitation of SIE by NIa-Pro requires the entire protein while CP requires only a 200-amino-acid (aa) middle fragment (aa 101 to 300) of the 349 aa. Strikingly, reciprocal experiments with WSMV-mediated expression of TriMV proteins showed that TriMV CP, and TriMV NIa-Pro to a lesser extent, likewise excluded superinfection by TriMV-GFP. Collectively, these data demonstrate that WSMV- and TriMV-encoded CP and NIa-Pro proteins are effectors of SIE and that these two proteins trigger SIE independently of each other. IMPORTANCE Superinfection exclusion (SIE) is an antagonistic virus-virus interaction that

  4. 40 CFR 174.514 - Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. 174.514 Section 174.514 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED...

  5. Super magnetic nanoparticles NiFe2O4, coated with aluminum-nickel oxide sol-gel lattices to safe, sensitive and selective purification of his-tagged proteins.

    Science.gov (United States)

    Mirahmadi-Zare, Seyede Zohreh; Allafchian, Alireza; Aboutalebi, Fatemeh; Shojaei, Pendar; Khazaie, Yahya; Dormiani, Kianoush; Lachinani, Liana; Nasr-Esfahani, Mohammad-Hossein

    2016-05-01

    Super magnetic nanoparticle NiFe2O4 with high magnetization, physical and chemical stability was introduced as a core particle which exhibits high thermal stability (>97%) during the harsh coating process. Instead of multi-stage process for coating, the magnetic nanoparticles was mineralized via one step coating by a cheap, safe, stable and recyclable alumina sol-gel lattice (from bohemite source) saturated by nickel ions. The TEM, SEM, VSM and XRD imaging and BET analysis confirmed the structural potential of NiFe2O4@NiAl2O4 core-shell magnetic nanoparticles for selective and sensitive purification of His-tagged protein, in one step. The functionality and validity of the nickel magnetic nanoparticles were attested by purification of three different bioactive His-tagged recombinant fusion proteins including hIGF-1, GM-CSF and bFGF. The bonding capacity of the nickel magnetics nanoparticles was studied by Bradford assay and was equal to 250 ± 84 μg Protein/mg MNP base on protein size. Since the metal ion leakage is the most toxicity source for purification by nickel magnetic nanoparticles, therefor the nickel leakage in purified final protein was determined by atomic absorption spectroscopy and biological activity of final purified protein was confirmed in comparison with reference. Also, in vitro cytotoxicity of nickel magnetic nanoparticles and trace metal ions were investigated by MTS assay analysis. The results confirmed that the synthesized nickel magnetic nanoparticles did not show metal ion toxicity and not affected on protein folding. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Cyclic adenosine 3',5'-monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter.

    Science.gov (United States)

    Clem, Brian F; Hudson, Elizabeth A; Clark, Barbara J

    2005-03-01

    Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5'-TGACGTCA-3') within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.

  7. NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

    2011-07-01

    Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

  8. Optimizing the coating process for double-coated, wood-containing papers. Double toko chushitsushi no toko process no saitekika

    Energy Technology Data Exchange (ETDEWEB)

    Mori, T [Sumitomo Jukikai Valmet Ltd., Tokyo (Japan)

    1991-09-01

    Medium weight coat (MWC) paper is judged to be optimum for the heating setting offsetting rotary press and appropriate also for the offset printing. Representative printed matters to use MWC paper comprise, among others, sales promotion and high grade propagation purpose documents, and specialized magazines. Judging from the viewpoint of comparison between the one-layer coating and two-layer coating, effect of different coating methods, optimization in drying, etc., the two-layer coating is more advantageous than the one-layer coating in quality to keep the homogeneity also even after the printing. There are cases that the two-layer coating lowers the total cost for necessary constituent elements for the coating. The drying method of precoating, if made by an applicator roll, hardly influences the brightness and smoothness. For the precoating, the use of roll applicator gives a better brightness than that of short dwell. It was also known that the use of roll applicator solves problems raised by the increase in coated quantity due to the use of short dwell. 8 figs., 3 tabs.

  9. The influence of sporulation conditions on the spore coat protein composition of Bacillus subtilis spores.

    OpenAIRE

    Wishwas R. Abhyankar; Wishwas R. Abhyankar; Kiki Kamphorst; Bhagyashree N. Swarge; Bhagyashree N. Swarge; Henk van Veen; Nicole N. van der Wel; Stanley Brul; Chris G. de Koster; Leo J. de Koning

    2016-01-01

    Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for t...

  10. The Influence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores

    OpenAIRE

    Abhyankar, Wishwas R.; Kamphorst, Kiki; Swarge, Bhagyashree N.; van Veen, Henk; van der Wel, Nicole N.; Brul, Stanley; de Koster, Chris G.; de Koning, Leo J.

    2016-01-01

    Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for t...

  11. A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G

    Science.gov (United States)

    A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite...

  12. Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum.

    Science.gov (United States)

    Heiss, Silvia; Hörmann, Angelika; Tauer, Christopher; Sonnleitner, Margot; Egger, Esther; Grabherr, Reingard; Heinl, Stefan

    2016-03-10

    Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector(®) micro-fermentation system. Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector(®) micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a

  13. The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA.

    Science.gov (United States)

    Aparicio, Frederic; Vilar, Marçal; Perez-Payá, Enrique; Pallás, Vicente

    2003-08-15

    Binding of coat protein (CP) to the 3' nontranslated region (3'-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo- and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3'-NTR of its genomic RNA using purified E. coli- expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3'-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3'-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg(2+), lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3'-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3'-NTR level in Alfamo- and Ilarvirus genera.

  14. The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA

    International Nuclear Information System (INIS)

    Aparicio, Frederic; Vilar, Marcal; Perez-Paya, Enrique; Pallas, Vicente

    2003-01-01

    Binding of coat protein (CP) to the 3' nontranslated region (3'-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo- and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3'-NTR of its genomic RNA using purified E. coli- expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3'-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3'-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg 2+ , lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3'-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3'-NTR level in Alfamo- and Ilarvirus genera

  15. Human FAN1 promotes strand incision in 5'-flapped DNA complexed with RPA.

    Science.gov (United States)

    Takahashi, Daisuke; Sato, Koichi; Hirayama, Emiko; Takata, Minoru; Kurumizaka, Hitoshi

    2015-09-01

    Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endonuclease that is considered to be involved in DNA incision at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been determined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 exhibited robust endonuclease activity with 5'-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 5'-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 5'-flapped DNA covered by RPA. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  16. The role of seed coat phenolics on water uptake and early protein synthesis during germination of dimorphic seeds of halopyrum mucronatum (L.) staph

    International Nuclear Information System (INIS)

    Siddiqui, Z. S.; Khan, M.A.

    2010-01-01

    Role of seed coat phenolics on water uptake and early protein synthesis of Halopyrum mucronatum dimorphic seeds during germination were tested. Scanning electron micrographs (SEM) showed seed texture with differential deposition of secondary metabolites in both morphs. Ability of both seed morphs to retain secondary deposition was dependent on exposure to either saline or non-saline conditions. More phenols leached from the brown seed during the initial hours of soaking when compared to black seeds. Water uptake pattern was slightly different in both seed type particularly during initial hours when imbibition in black seeds showed little water uptake while in brown seeds absorption was quick in the first hour under both saline and non saline condition. Change in total protein was somewhat similar in both seeds morphs showing early increase (4 and 8 h), reaching to the maximum (12 h) and decreasing (24 and 48 h) afterward. The results are discussed in relation to seed coat phenolics, water uptake and early protein synthesis during germination. (author)

  17. TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

    Science.gov (United States)

    Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

    2014-08-03

    Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

  18. A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Nan; Deng, Ling; Mei, Yuxia

    2012-01-01

    Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression...... to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high...... levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started...

  19. Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation.

    Science.gov (United States)

    Kim, K H; Hemenway, C

    1997-05-26

    The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation of Nicotiana tabacum NT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.

  20. BSA Nanoparticles for siRNA Delivery: Coating Effects on Nanoparticle Properties, Plasma Protein Adsorption, and In Vitro siRNA Delivery

    Directory of Open Access Journals (Sweden)

    Haran Yogasundaram

    2012-01-01

    Full Text Available Developing vehicles for the delivery of therapeutic molecules, like siRNA, is an area of active research. Nanoparticles composed of bovine serum albumin, stabilized via the adsorption of poly-L-lysine (PLL, have been shown to be potentially inert drug-delivery vehicles. With the primary goal of reducing nonspecific protein adsorption, the effect of using comb-type structures of poly(ethylene glycol (1 kDa, PEG units conjugated to PLL (4.2 and 24 kDa on BSA-NP properties, apparent siRNA release rate, cell viability, and cell uptake were evaluated. PEGylated PLL coatings resulted in NPs with ζ-potentials close to neutral. Incubation with platelet-poor plasma showed the composition of the adsorbed proteome was similar for all systems. siRNA was effectively encapsulated and released in a sustained manner from all NPs. With 4.2 kDa PLL, cellular uptake was not affected by the presence of PEG, but PEG coating inhibited uptake with 24 kDa PLL NPs. Moreover, 24 kDa PLL systems were cytotoxic and this cytotoxicity was diminished upon PEG incorporation. The overall results identified a BSA-NP coating structure that provided effective siRNA encapsulation while reducing ζ-potential, protein adsorption, and cytotoxicity, necessary attributes for in vivo application of drug-delivery vehicles.

  1. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen

    2015-01-01

    a less leaky Cu2+-inducible promoter based on CUP1. The basal expression level of the new promoter was approx. 61% below the wild-type CUP1 promoter, thus expanding the absolute range of Cu2+-based gene control. The stability of 3vGFP towards direct-repeat recombination was assayed in S. cerevisiae......Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP...... functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different...

  2. Centrosomal protein 55 activates NF-?B signalling and promotes pancreatic cancer cells aggressiveness

    OpenAIRE

    Peng, Tao; Zhou, Wei; Guo, Feng; Wu, He-shui; Wang, Chun-you; Wang, Li; Yang, Zhi-yong

    2017-01-01

    Centrosomal protein 55 (CEP55) is a microtubule-bundling protein that participants in cell mitosis. It is overexpressed in several solid tumours and promotes the growth and invasion of cancer cells. However, the role of CEP55 in pancreatic cancer (PANC) remains unclear. Herein, upregulated expression of CEP55 (associated with poor prognosis) was detected in PANC using quantitative real-time reverse transcription PCR, western blotting, and immunohistochemistry. Cell migration, colony formation...

  3. Protein-coated pH-responsive gold nanoparticles: Microwave-assisted synthesis and surface charge-dependent anticancer activity

    Directory of Open Access Journals (Sweden)

    Dickson Joseph

    2014-09-01

    Full Text Available The biocompatibility and ease of functionalization of gold nanoparticles underlie significant potential in biotechnology and biomedicine. Eight different proteins were examined in the preparation of gold nanoparticles (AuNPs in aqueous medium under microwave irradiation. Six of the proteins resulted in the formation of AuNPs. The intrinsic pH of the proteins played an important role in AuNPs with strong surface plasmon bands. The hydrodynamic size of the nanoparticles was larger than the values observed by TEM and ImageJ. The formation of a protein layer on the AuNPs accounts for this difference. The AuNPs exhibited sensitivity towards varying pH conditions, which was confirmed by determining the difference in the isoelectric points studied by using pH-dependent zeta potential titration. Cytotoxicity studies revealed anticancerous effects of the AuNPs at a certain micromolar concentration by constraining the growth of cancer cells with different efficacies due to the use of different proteins as capping agents. The positively charged AuNPs are internalized by the cells to a greater level than the negatively charged AuNPs. These AuNPs synthesized with protein coating holds promise as anticancer agents and would help in providing a new paradigm in area of nanoparticles.

  4. The Highly Conserved COPII Coat Complex Sorts Cargo from the Endoplasmic Reticulum and Targets It to the Golgi

    OpenAIRE

    Lord, Christopher; Ferro-Novick, Susan; Miller, Elizabeth A.

    2013-01-01

    Protein egress from the endoplasmic reticulum (ER) is driven by a conserved cytoplasmic coat complex called the COPII coat. The COPII coat complex contains an inner shell (Sec23/Sec24) that sorts cargo into ER-derived vesicles and an outer cage (Sec13/Sec31) that leads to coat polymerization. Once released from the ER, vesicles must tether to and fuse with the target membrane to deliver their protein and lipid contents. This delivery step also depends on the COPII coat, with coat proteins bin...

  5. Hibiscus chlorotic ringspot virus coat protein upregulates sulfur metabolism genes for enhanced pathogen defense.

    Science.gov (United States)

    Gao, Ruimin; Ng, Florence Kai Lin; Liu, Peng; Wong, Sek-Man

    2012-12-01

    In both Hibiscus chlorotic ringspot virus (HCRSV)-infected and HCRSV coat protein (CP) agroinfiltrated plant leaves, we showed that sulfur metabolism pathway related genes-namely, sulfite oxidase (SO), sulfite reductase, and adenosine 5'-phosphosulfate kinase-were upregulated. It led us to examine a plausible relationship between sulfur-enhanced resistance (SED) and HCRSV infection. We broadened an established method to include different concentrations of sulfur (0S, 1S, 2S, and 3S) to correlate them to symptom development of HCRSV-infected plants. We treated plants with glutathione and its inhibitor to verify the SED effect. Disease resistance was induced through elevated glutathione contents during HCRSV infection. The upregulation of SO was related to suppression of symptom development induced by sulfur treatment. In this study, we established that HCRSV-CP interacts with SO which, in turn, triggers SED and leads to enhanced plant resistance. Thus, we have discovered a new function of SO in the SED pathway. This is the first report to demonstrate that the interaction of a viral protein and host protein trigger SED in plants. It will be interesting if such interaction applies generally to other host-pathogen interactions that will lead to enhanced pathogen defense.

  6. p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma.

    Science.gov (United States)

    Di Vinci, Angela; Perdelli, Luisa; Banelli, Barbara; Salvi, Sandra; Casciano, Ida; Gelvi, Ilaria; Allemanni, Giorgio; Margallo, Edoardo; Gatteschi, Beatrice; Romani, Massimo

    2005-04-10

    The potential role of p16(INK4a) methylation in breast cancer is controversial whereas there are no data on fibroadenoma. To assess if inactivation of p16(INK4a) by promoter hypermethylation occurs in this hyperproliferative benign breast lesion or, on the contrary, it is strictly related to the carcinogenic process, we have tested the different histological components of 15 cases of fibroadenoma and the intraductal and infiltrating components of 15 cases of carcinoma and their adjacent non-tumoral epithelium. All samples were obtained by laser-assisted microdissection. The relationship between promoter methylation status, immunohistochemical protein expression and ki67 proliferative activity was evaluated for each lesion. Our data demonstrate that hypermethylation of p16(INK4a) promoter is a common event occurring at similar frequency in all the different histological areas of the benign and malignant breast lesions taken into exam. Conversely, protein p16 expression, although heterogeneously distributed within the section, is considerably higher in breast carcinoma as compared to fibroadenoma in both tumoral and non-tumoral epithelia and stroma. The protein localization was almost exclusively nuclear in fibroadenoma and non-tumoral epithelia whereas, in carcinoma, the staining was both nuclear and cytoplasmic or cytoplasmic alone. Furthermore, in a subset of fibroadenoma with higher proliferative activity, p16 protein expression was substantially decreased as compared to those showing lower proliferation. We did not observe this association in carcinomas. Our data demonstrate that the hypermethylation of the p16(INK4a) promoter is not specifically associated with malignancy and that, on the contrary, the overexpression of p16 and its cytoplasmic sequestration is a feature of breast carcinoma. (c) 2004 Wiley-Liss, Inc.

  7. The specific transmission of Grapevine fanleaf virus by its nematode vector Xiphinema index is solely determined by the viral coat protein

    International Nuclear Information System (INIS)

    Andret-Link, Peggy; Schmitt-Keichinger, Corinne; Demangeat, Gerard; Komar, Veronique; Fuchs, Marc

    2004-01-01

    The viral determinants involved in the specific transmission of Grapevine fanleaf virus (GFLV) by its nematode vector Xiphinema index are located within the 513 C-terminal residues of the RNA2-encoded polyprotein, that is, the 9 C-terminal amino acids of the movement protein (2B MP ) and contiguous 504 amino acids of the coat protein (2C CP ) [Virology 291 (2001) 161]. To further delineate the viral determinants responsible for the specific spread, the four amino acids that are different within the 9 C-terminal 2B MP residues between GFLV and Arabis mosaic virus (ArMV), another nepovirus which is transmitted by Xiphinema diversicaudatum but not by X. index, were subjected to mutational analysis. Of the recombinant viruses derived from transcripts of GFLV RNA1 and RNA2 mutants that systemically infected herbaceous host plants, all with the 2C CP of GFLV were transmitted by X. index unlike none with the 2C CP of ArMV, regardless of the mutations within the 2B MP C-terminus. These results demonstrate that the coat protein is the sole viral determinant for the specific spread of GFLV by X. index

  8. Silica-Coated Liposomes for Insulin Delivery

    Directory of Open Access Journals (Sweden)

    Neelam Dwivedi

    2010-01-01

    Full Text Available Liposomes coated with silica were explored as protein delivery vehicles for their enhanced stability and improved encapsulation efficiency. Insulin was encapsulated within the fluidic phosphatidylcholine lipid vesicles by thin film hydration at pH 2.5, and layer of silica was formed above lipid bilayer by acid catalysis. The presence of silica coating and encapsulated insulin was identified using confocal and electron microscopy. The native state of insulin present in the formulation was evident from Confocal Micro-Raman spectroscopy. Silica coat enhances the stability of insulin-loaded delivery vehicles. In vivo study shows that these silica coated formulations were biologically active in reducing glucose levels.

  9. Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

    Science.gov (United States)

    Oka, Yasuyoshi; Bekker-Jensen, Simon; Mailand, Niels

    2015-05-12

    Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway. © 2015 The Authors.

  10. Tailoring Escherichia coli for the L-rhamnose PBAD promoter-based production of membrane and secretory proteins

    NARCIS (Netherlands)

    Hjelm, Anna; Karyolaimos, Alexandros; Zhang, Zhe; Rujas, Edurne; Vikström, David; Slotboom, Dirk Jan; de Gier, Jan-Willem

    Membrane and secretory protein production in Escherichia coli requires precisely controlled production rates to avoid the deleterious saturation of their biogenesis pathways. Based on this requirement, the E. coli L-rhamnose PBAD promoter (PrhaBAD) is often used for membrane and secretory protein

  11. Role of alfalfa mosaic virus coat protein in regulation of the balance between viral plus and minus strand RNA synthesis

    NARCIS (Netherlands)

    van der Kuyl, A. C.; Neeleman, L.; Bol, J. F.

    1991-01-01

    Replication of wild type RNA 3 of alfalfa mosaic virus (AIMV) and mutants with frameshifts in the P3 or coat protein (CP) genes was studied in protoplasts from tobacco plants transformed with DNA copies of AIMV RNAs 1 and 2. Accumulation of viral plus and minus strand RNAs was monitored with

  12. Ursodeoxycholic acid reduces protein levels and nucleation-promoting activity in human gallbladder bile

    NARCIS (Netherlands)

    van Erpecum, K. J.; Portincasa, P.; Eckhardt, E.; Go, P. M.; vanBerge-Henegouwen, G. P.; Groen, A. K.

    1996-01-01

    Background & Aims: Ursodeoxycholic acid prevents gallstone formation in selected patients. The aim of this study was to examine whether decreased concentration and nucleation-promoting activity of various proteins contribute to this beneficial effect. Methods: Gallbladder bile of 13 patients with

  13. High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

    Directory of Open Access Journals (Sweden)

    D’Urzo Nunzia

    2013-02-01

    Full Text Available Abstract Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA. The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio and B. megaterium (from Mobitec, we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

  14. Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

    Directory of Open Access Journals (Sweden)

    Zainul A Khan

    Full Text Available Cotton leaf curl Burewala virus (CLCuBuV, belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV were fused with β-glucuronidase (GUS and green fluorescent protein (GFP reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

  15. IN-MACA-MCC: Integrated Multiple Attractor Cellular Automata with Modified Clonal Classifier for Human Protein Coding and Promoter Prediction

    Directory of Open Access Journals (Sweden)

    Kiran Sree Pokkuluri

    2014-01-01

    Full Text Available Protein coding and promoter region predictions are very important challenges of bioinformatics (Attwood and Teresa, 2000. The identification of these regions plays a crucial role in understanding the genes. Many novel computational and mathematical methods are introduced as well as existing methods that are getting refined for predicting both of the regions separately; still there is a scope for improvement. We propose a classifier that is built with MACA (multiple attractor cellular automata and MCC (modified clonal classifier to predict both regions with a single classifier. The proposed classifier is trained and tested with Fickett and Tung (1992 datasets for protein coding region prediction for DNA sequences of lengths 54, 108, and 162. This classifier is trained and tested with MMCRI datasets for protein coding region prediction for DNA sequences of lengths 252 and 354. The proposed classifier is trained and tested with promoter sequences from DBTSS (Yamashita et al., 2006 dataset and nonpromoters from EID (Saxonov et al., 2000 and UTRdb (Pesole et al., 2002 datasets. The proposed model can predict both regions with an average accuracy of 90.5% for promoter and 89.6% for protein coding region predictions. The specificity and sensitivity values of promoter and protein coding region predictions are 0.89 and 0.92, respectively.

  16. Electrodeposition, characterization, and antibacterial activity of zinc/silver particle composite coatings

    Energy Technology Data Exchange (ETDEWEB)

    Reyes-Vidal, Y.; Suarez-Rojas, R.; Ruiz, C.; Torres, J. [Center of Research and Technological Development in Electrochemistry (CIDETEQ), Parque Tecnológico Sanfandila, Pedro Escobedo, Querétaro, A.P.064, C.P.76703, Querétaro (Mexico); Ţălu, Ştefan [Technical University of Cluj-Napoca, Faculty of Mechanical Engineering, Department of AET, Discipline of Descriptive Geometry and Engineering Graphics, 103-105 B-dul Muncii St., Cluj-Napoca 400641 Cluj (Romania); Méndez, Alia [Centro de Química-ICUAP Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria Puebla, 72530 Puebla (Mexico); Trejo, G., E-mail: gtrejo@cideteq.mx [Center of Research and Technological Development in Electrochemistry (CIDETEQ), Parque Tecnológico Sanfandila, Pedro Escobedo, Querétaro, A.P.064, C.P.76703, Querétaro (Mexico)

    2015-07-01

    Highlights: • Zn/AgPs composites coatings were formed for electrodeposition. • CTAB promotes occlusion of silver particles in the coating. • Zn/AgPs coatings present very good antibacterial activity. - Abstract: Composite coatings consisting of zinc and silver particles (Zn/AgPs) with antibacterial activity were prepared using an electrodeposition technique. The morphology, composition, and structure of the Zn/AgPs composite coatings were analyzed using scanning electron microscopy (SEM) coupled with energy-dispersive spectroscopy (EDS), inductively coupled plasma (ICP) spectrometry, and X-ray diffraction (XRD). The antibacterial properties of the coatings against the microorganisms Escherichia coli as a model Gram-negative bacterium and Staphylococcus aureus as a model Gram-positive bacterium were studied quantitatively and qualitatively. The results revealed that the dispersant cetyltrimethylammonium bromide (CTAB) assisted in the formation of a stable suspension of Ag particles in the electrolytic bath for 24 h. Likewise, a high concentration of CTAB in the electrolytic bath promoted an increase in the number of Ag particles occluded in the Zn/AgPs coatings. The Zn/AgPs coatings that were obtained were compact, smooth, and shiny materials. Antimicrobial tests performed on the Zn/AgPs coatings revealed that the inhibition of bacterial growth after 30 min of contact time was between 91% and 98% when the AgPs content ranged from 4.3 to 14.0 mg cm{sup −3}.

  17. Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Dong; Neoh, Koon Gee, E-mail: chenkg@nus.edu.sg; Kang, En-Tang

    2016-01-01

    Graphical abstract: - Highlights: • Alkaline phosphatase was immobilized on carboxymethyl chitosan coating on Ti. • The coating is bifunctional; resists bacterial adhesion and enhances cell functions. • Osteogenic differentiation of osteoblasts and stem cells is enhanced on the coating. • The coating remains stable and functional after ethanol treatment and autoclaving. - Abstract: In this work, alkaline phosphatase (ALP) was covalently immobilized on carboxymethyl chitosan (CMCS)-coated polydopamine (PDA)-functionalized Ti to achieve a bifunctional surface. Our results showed ∼89% reduction in Staphylococcus epidermidis adhesion on this surface compared to that on pristine Ti. The ALP-modified Ti supported cell proliferation, and significantly enhanced cellular ALP activity and calcium deposition of osteoblasts, human mesenchymal stem cells (hMSCs) and human adipose-derived stem cells (hADSCs). The extent of enhancement in the functions of these cells is dependent on the surface density of immobilized ALP. The substrate prepared using an ALP solution of 50 μg/cm{sup 2} resulted in 44%, 54% and 129% increase in calcium deposited by osteoblasts, hMSCs and hADSCs, respectively, compared to those cultured on pristine Ti. The ALP-modified substrates also promoted the osteogenic differentiation of hMSCs and hADSCs by up-regulating gene expressions of runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteocalcin (OC) in the two types of stem cells. The surface-immobilized ALP was stable after being subjected to 1 h immersion in 70% ethanol and autoclaving at 121 °C for 20 min. However, the enzymatic bioactivity of the surface-immobilized ALP was reduced by about 50% after these substrates were immersed in phosphate buffered saline (PBS) or PBS containing lysozyme for 14 days.

  18. Nuclear proteins interacting with the promoter region of the human granulocyte/macrophage colony-stimulating factor gene

    International Nuclear Information System (INIS)

    Shannon, M.F.; Gamble, J.R.; Vadas, M.A.

    1988-01-01

    The gene for human granulocyte/macrophage colony-stimulating factor (GM-CSF) is expressed in a tissue-specific as well as an activation-dependent manner. The interaction of nuclear proteins with the promoter region of the GM-CSF gene that is likely to be responsible for this pattern of GM-CSF expression was investigated. The authors show that nuclear proteins interact with DNA fragments from the GM-CSF promoter in a cell-specific manner. A region spanning two cytokine-specific sequences, cytokine 1 (CK-1, 5', GAGATTCCAC 3') and cytokine 2 (CK-2, 5' TCAGGTA 3') bound two nuclear proteins from GM-CSF-expressing cells in gel retardation assays. NF-GMb was inducible with phorbol 12-myristate 13-acetate and accompanied induction of GM-CSF message. NF-GMb was absent in cell lines not producing GM-CSF, some of which had other distinct binding proteins. NF-GMa and NF-GMb eluted from a heparin-Sepharose column at 0.3 and 0.6 M KCl, respectively. They hypothesize that the sequences CK-1 and CK-2 bind specific proteins and regulate GM-CSF transcription

  19. Cellulose acetate propionate coated titanium: characterization and biotechnological application

    Directory of Open Access Journals (Sweden)

    Guilherme da Silva Gomes

    2007-12-01

    Full Text Available Surfaces of pure titanium and Ti coated with cellulose acetate propionate (CAP have been characterized by means of scanning electron microscopy X ray coupled with elemental microanalysis (SEM-EDS, ellipsometry, atomic force microscopy (AFM and contact angle measurements. Coating Ti surfaces with CAP ultrathin films reduced original surface roughness. Surface energy and wettability of CAP covered Ti surfaces pure Ti surfaces were similar. The adsorption of lysozyme (LYZ, an antibacterial protein, onto Ti and CAP-coated Ti surfaces has been studied by means of ellipsometry and atomic force microscopy (AFM. The adsorption of LYZ was mainly driven by hydrophobic interaction between protein hydrophobic residues and CAP propyl groups. Pure Ti and CAP coated Ti surfaces presented no cytotoxicity effect and proved to be adequate substrates for cell adhesion. The biocompatibility of CAP coated Ti surfaces was attributed to the surface enrichment in glucopyranosyl residues and short alkyl side groups.

  20. Hepatitis B X-interacting protein promotes cisplatin resistance and regulates CD147 via Sp1 in ovarian cancer.

    Science.gov (United States)

    Zou, Wei; Ma, Xiangdong; Yang, Hong; Hua, Wei; Chen, Biliang; Cai, Guoqing

    2017-03-01

    Ovarian cancer is the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure in malignant tumors. Hepatitis B X-interacting protein acts as an oncoprotein, regulates cell proliferation, and migration in breast cancer. We aimed to investigate the effects and mechanisms of hepatitis B X-interacting protein on resistance to cisplatin in human ovarian cancer cell lines. The mRNA and protein levels of hepatitis B X-interacting protein were detected using RT-PCR and Western blotting in cisplatin-resistant and cisplatin-sensitive tissues, cisplatin-resistant cell lines A2780/CP and SKOV3/CP, and cisplatin-sensitive cell lines A2780 and SKOV3. Cell viability and apoptosis were measured to evaluate cellular sensitivity to cisplatin in A2780/CP cells. Luciferase reporter gene assay was used to determine the relationship between hepatitis B X-interacting protein and CD147. The in vivo function of hepatitis B X-interacting protein on tumor burden was assessed in cisplatin-resistant xenograft models. The results showed that hepatitis B X-interacting protein was highly expressed in ovarian cancer of cisplatin-resistant tissues and cells. Notably, knockdown of hepatitis B X-interacting protein significantly reduced cell viability in A2780/CP compared with cisplatin treatment alone. Hepatitis B X-interacting protein and cisplatin cooperated to induce apoptosis and increase the expression of c-caspase 3 as well as the Bax/Bcl-2 ratio. We confirmed that hepatitis B X-interacting protein up-regulated CD147 at the protein expression and transcriptional levels. Moreover, we found that hepatitis B X-interacting protein was able to activate the CD147 promoter through Sp1. In vivo, depletion of hepatitis B X-interacting protein decreased the tumor volume and weight induced by cisplatin. Taken together, these results indicate that hepatitis B X-interacting protein promotes cisplatin resistance and regulated CD147 via Sp1 in

  1. Promoting resuscitation of viable but nonculturable cells of Vibrio harveyi by a resuscitation-promoting factor-like protein YeaZ.

    Science.gov (United States)

    Li, Y; Chen, J; Zhao, M; Yang, Z; Yue, L; Zhang, X

    2017-02-01

    To demonstrate the resuscitation-promoting activities of recombinant YeaZ from Vibrio harveyi SF-1. The gene of resuscitation-promoting factor YeaZ was cloned from genomic DNA of V. harveyi SF-1. The gene was expressed in Escherichia coli, and the expressed protein was purified by Ni 2+ -affinity chromatography. A yeaZ mutant was constructed by using the suicide plasmid pNQ705 with homologous recombination. Disruption of yeaZ did not affect cell growth significantly in 2216 E broth at 28°C. The wild-type and mutant viable but nonculturable (VBNC) cells could be resuscitated by temperature upshift method. In addition, the recombinant YeaZ increased the culturable counts from 1·27 × 10 4  CFU per ml and 1·99 × 10 4 CFU per ml to 2·88 × 10 5  CFU per ml and 4·59 × 10 5 CFU per ml, respectively. After the VBNC cells of wild-type and mutant cells were maintained at 4°C for 120 days, no resuscitation was obtained by temperature upshift method, but addition of the recombinant YeaZ promoted the resuscitation of the wild-type and mutant cells, with the culturable cell counts of 1·13 × 10 3 and 1·44 × 10 3 CFU per ml, respectively. Disruption of yeaZ decreased the virulence of V. harveyi in zebrafish. The lethal dose 50% of the yeaZ null mutant was more than 10-fold higher than that of the wild-type cells. The recombinant YeaZ could efficiently promote resuscitation of the wild-type and mutant cells of V. harveyi from VBNC to culturable state. The protein also promoted resuscitation of the VBNC wild-type and mutant cells, which were maintained at 4°C for 120 days and not recovered by temperature upshift method. Disruption of yeaZ decreased the virulence of V. harveyi in zebrafish. Here, we show clear evidence of a resuscitation-promoting factor YeaZ of V. harveyi and the roles in resuscitation of the VBNC cells and its pathogenicity. © 2016 The Society for Applied Microbiology.

  2. A poly(glycerol sebacate)-coated mesoporous bioactive glass scaffold with adjustable mechanical strength, degradation rate, controlled-release and cell behavior for bone tissue engineering.

    Science.gov (United States)

    Lin, Dan; Yang, Kai; Tang, Wei; Liu, Yutong; Yuan, Yuan; Liu, Changsheng

    2015-07-01

    Various requirements in the field of tissue engineering have motivated the development of three-dimensional scaffold with adjustable physicochemical properties and biological functions. A series of multiparameter-adjustable mesoporous bioactive glass (MBG) scaffolds with uncrosslinked poly(glycerol sebacate) (PGS) coating was prepared in this article. MBG scaffold was prepared by a modified F127/PU co-templating process and then PGS was coated by a simple adsorption and lyophilization process. Through controlling macropore parameters and PGS coating amount, the mechanical strength, degradation rate, controlled-release and cell behavior of the composite scaffold could be modulated in a wide range. PGS coating successfully endowed MBG scaffold with improved toughness and adjustable mechanical strength covering the bearing range of trabecular bone (2-12MPa). Multilevel degradation rate of the scaffold and controlled-release rate of protein from mesopore could be achieved, with little impact on the protein activity owing to an "ultralow-solvent" coating and "nano-cavity entrapment" immobilization method. In vitro studies indicated that PGS coating promoted cell attachment and proliferation in a dose-dependent manner, without affecting the osteogenic induction capacity of MBG substrate. These results first provide strong evidence that uncrosslinked PGS might also yield extraordinary achievements in traditional MBG scaffold. With the multiparameter adjustability, the composite MBG/PGS scaffolds would have a hopeful prospect in bone tissue engineering. The design considerations and coating method of this study can also be extended to other ceramic-based artificial scaffolds and are expected to provide new thoughts on development of future tissue engineering materials. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Electrophoretic deposition of composite hydroxyapatite-chitosan coatings

    International Nuclear Information System (INIS)

    Pang Xin; Zhitomirsky, Igor

    2007-01-01

    Cathodic electrophoretic deposition has been utilized for the fabrication of composite hydroxyapatite-chitosan coatings on 316L stainless steel substrates. The addition of chitosan to the hydroxyapatite suspensions promoted the electrophoretic deposition of the hydroxyapatite nanoparticles and resulted in the formation of composite coatings. The obtained coatings were investigated by X-ray diffraction, thermogravimetric and differential thermal analysis, scanning and transmission electron microscopy, potentiodynamic polarization measurements, and electrochemical impedance spectroscopy. It was shown that the deposit composition can be changed by a variation of the chitosan or hydroxyapatite concentration in the solutions. Experimental conditions were developed for the fabrication of hydroxyapatite-chitosan nanocomposites containing 40.9-89.8 wt.% hydroxyapatite. The method enabled the formation of adherent and uniform coatings of thicknesses up to 60 μm. X-ray studies revealed that the preferred orientation of the hydroxyapatite nanoparticles in the chitosan matrix increases with decreasing hydroxyapatite content in the composite coatings. The obtained coatings provided the corrosion protection for the 316L stainless steel substrates

  4. An essential GT motif in the lamin A promoter mediates activation by CREB-binding protein

    International Nuclear Information System (INIS)

    Janaki Ramaiah, M.; Parnaik, Veena K.

    2006-01-01

    Lamin A is an important component of nuclear architecture in mammalian cells. Mutations in the human lamin A gene lead to highly degenerative disorders that affect specific tissues. In studies directed towards understanding the mode of regulation of the lamin A promoter, we have identified an essential GT motif at -55 position by reporter gene assays and mutational analysis. Binding of this sequence to Sp transcription factors has been observed in electrophoretic mobility shift assays and by chromatin immunoprecipitation studies. Further functional analysis by co-expression of recombinant proteins and ChIP assays has shown an important regulatory role for CREB-binding protein in promoter activation, which is mediated by the GT motif

  5. Controlled protein adsorption on PMOXA/PAA based coatings by thermally induced immobilization

    Science.gov (United States)

    Mumtaz, Fatima; Chen, Chaoshi; Zhu, Haikun; Pan, Chao; Wang, Yanmei

    2018-05-01

    In this work, poly(2-methyl-2-oxazoline-random-glycidyl methacrylate) (PMOXA-r-GMA) and poly(acrylic acid)-block-poly(glycidyl methacrylate) (PAA-b-PGMA) copolymers were synthesized via cationic ring-opening polymerization (CROP) of 2-methyl-2-oxazoline (MOXA) and reversible addition-fragmentation chain transfer (RAFT) polymerization of acrylic acid (AA) followed by their random and block copolymerization with glycidyl methacrylate (GMA), respectively, and then characterized carefully. PMOXA/PAA based coatings were then prepared by simply spin coating the mixture of PMOXA-r-GMA and PAA-b-PGMA copolymer solutions onto silicon/glass substrates followed by annealing at 110 °C. The coatings were rigorously characterized by using X-ray photoelectron spectroscopy (XPS), the static water contact angle (WCA) test, ellipsometry and atomic force microscopy (AFM). The results demonstrated that the coating based mixed PMOXA/PAA brushes with desired surface composition could be attained by simply maintaining their percentage in the mixture of PMOXA-r-GMA and PAA-b-PGMA copolymer solutions. Finally, the switchable behavior of PMOXA/PAA based coatings toward bovine serum albumin (BSA) adsorption was investigated by fluorescein isothiocyanate-labelled BSA (FITC-BSA) assay and quartz crystal microbalance with dissipation monitoring (QCM-D), which indicated that the coating based mixed PMOXA/PAA brushes could control BSA adsorption/desorption from very low to high amount (>90% desorption) through adjusting the composition of PMOXA-r-GMA and PAA-b-PGMA solution used in preparing PMOXA/PAA based coatings upon pH and ionic strength change. Furthermore, PMOXA/PAA based coatings displayed efficient repeatability of reversible BSA adsorption/desorption cycles.

  6. Exogenous fatty acid binding protein 4 promotes human prostate cancer cell progression.

    Science.gov (United States)

    Uehara, Hisanori; Takahashi, Tetsuyuki; Oha, Mina; Ogawa, Hirohisa; Izumi, Keisuke

    2014-12-01

    Epidemiologic studies have found that obesity is associated with malignant grade and mortality in prostate cancer. Several adipokines have been implicated as putative mediating factors between obesity and prostate cancer. Fatty acid binding protein 4 (FABP4), a member of the cytoplasmic fatty acid binding protein multigene family, was recently identified as a novel adipokine. Although FABP4 is released from adipocytes and mean circulating concentrations of FABP4 are linked with obesity, effects of exogenous FABP4 on prostate cancer progression are unclear. In this study, we examined the effects of exogenous FABP4 on human prostate cancer cell progression. FABP4 treatment promoted serum-induced prostate cancer cell invasion in vitro. Furthermore, oleic acid promoted prostate cancer cell invasion only if FABP4 was present in the medium. These promoting effects were reduced by FABP4 inhibitor, which inhibits FABP4 binding to fatty acids. Immunostaining for FABP4 showed that exogenous FABP4 was taken up into DU145 cells in three-dimensional culture. In mice, treatment with FABP4 inhibitor reduced the subcutaneous growth and lung metastasis of prostate cancer cells. Immunohistochemical analysis showed that the number of apoptotic cells, positive for cleaved caspase-3 and cleaved PARP, was increased in subcutaneous tumors of FABP4 inhibitor-treated mice, as compared with control mice. These results suggest that exogenous FABP4 might promote human prostate cancer cell progression by binding with fatty acids. Additionally, exogenous FABP4 activated the PI3K/Akt pathway, independently of binding to fatty acids. Thus, FABP4 might be a key molecule to understand the mechanisms underlying the obesity-prostate cancer progression link. © 2014 UICC.

  7. Flow accelerated organic coating degradation

    Science.gov (United States)

    Zhou, Qixin

    well as promotes the migration of coating materials from the coating into the working fluid, where coatings experience more severe deterioration in their barrier property under flowing conditions. Pure water has shown to be a much more aggressive working fluid than electrolyte solutions. The flowing fluid over the coating surface could be used as an effective acceleration method.

  8. The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

    Science.gov (United States)

    2012-01-01

    Background Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter. Conclusions The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available

  9. NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62▿

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

    2011-01-01

    Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus. PMID:21543505

  10. Early Involvement of Death-Associated Protein Kinase Promoter Hypermethylation in the Carcinogenesis of Barrett's Esophageal Adenocarcinoma and Its Association with Clinical Progression

    Directory of Open Access Journals (Sweden)

    Doerthe Kuester

    2007-03-01

    Full Text Available Esophageal Barrett's adenocarcinoma (BA develops through a multistage process, which is associated with the transcriptional silencing of tumor-suppressor genes by promoter CpG island hypermethylation. In this study, we explored the promoter hypermethylation and protein expression of proapoptotic deathassociated protein kinase (DAPK during the multistep Barrett's carcinogenesis cascade. Early BA and paired samples of premalignant lesions of 61 patients were analyzed by methylation-specific polymerase chain reaction and immunohistochemistry. For the association of clinicopathological markers and protein expression, an immunohistochemical tissue microarray analysis of 66 additional BAs of advanced tumor stages was performed. Hypermethylation of DAPK promoter was detected in 20% of normal mucosa, 50% of Barrett's metaplasia, 53% of dysplasia, and 60% of adenocarcinomas, and resulted in a marked decrease in DAPK protein expression (P < .01. The loss of DAPK protein was significantly associated with advanced depth of tumor invasion and advanced tumor stages (P < .001. Moreover, the severity of reflux esophagitis correlated significantly with the hypermethylation rate of the DAPK promoter (P < .003. Thus, we consider DAPK inactivation by promoter hypermethylation as an early event in Barrett's carcinogenesis and suggest that a decreased protein expression of DAPK likely plays a role in the development and progression of BA.

  11. Microstructure of oxides in thermal barrier coatings grown under dry/humid atmosphere

    International Nuclear Information System (INIS)

    Zhou Zhaohui; Guo Hongbo; Wang Juan; Abbas, Musharaf; Gong Shengkai

    2011-01-01

    Graphical abstract: The presence of water vapor promoted the formation of spinels in the TBC. Highlights: → Thermal barrier coatings are produced by electron beam physical vapour deposition. → Oxidation behaviour of the coatings at 1100 deg. C has been investigated in dry/humid O 2 . → Thermally grown oxides formed in the coatings are characterized. → The presence of water vapour promotes the formation of spinel in the TBCs. - Abstract: The microstructure of thermally grown oxide (TGO) in thermal barrier coatings (TBCs) oxidized under dry/humid atmosphere at 1100 deg. C has been characterized by transmission electron microscopy. A thin and continuous oxide layer is formed in the as-deposited TBCs produced by electron beam physical vapor deposition. The TGO formed in dry atmosphere consists of an outer layer of fine α-alumina, zirconia grains and an inner layer of columnar α-alumina grains. However, a small amount of spinel is observed in the TGO under humid atmosphere. The presence of water vapour promotes the formation of spinel.

  12. Sr-doped nanowire modification of Ca-Si-based coatings for improved osteogenic activities and reduced inflammatory reactions

    Science.gov (United States)

    Li, Kai; Hu, Dandan; Xie, Youtao; Huang, Liping; Zheng, Xuebin

    2018-02-01

    Biomedical coatings for orthopedic implants should facilitate osseointegration and mitigate implant-induced inflammatory reactions. In our study, Ca-Si coatings with Sr-containing nanowire-like structures (NW-Sr-CS) were achieved via hydrothermal treatment. In order to identify the effect of nanowire-like topography and Sr dopant on the biological properties of Ca-Si-based coatings, the original Ca-Si coating, Ca-Si coatings modified with nanoplate (NP-CS) and similar nanowire-like structure (NW-CS) were fabricated as the control. Surface morphology, phase composition, surface area, zeta potential and ion release of these coatings were characterized. The in vitro osteogenic activities and immunomodulatory properties were evaluated with bone marrow stromal cells (BMSCs) and RAW 264.7 cells, a mouse macrophage cell line. Compared with the CS and NP-CS coatings, the NW-CS coating possessed a larger surface area and pore volume, beneficial protein adsorption, up-regulated the expression levels of integrin β1, Vinculin and focal adhesion kinase and promoted cell spreading. Furthermore, the NW-CS coating significantly enhanced the osteogenic differentiation and mineralization as indicated by the up-regulation of ALP activity, mineralized nodule formation and osteoblastogenesis-related gene expression. With the introduction of Sr, the NW-Sr-CS coatings exerted a greater effect on the BMSC proliferation rate, calcium sensitive receptor gene expression as well as PKC and ERK1/2 phosphorylation. In addition, the Sr-doped coatings significantly up-regulated the ratio of OPG/RANKL in the BMSCs. The NW-Sr-CS coatings could modulate the polarization of macrophages towards the wound-healing M2 phenotype, reduce the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and enhance anti-inflammatory cytokines (IL-1ra, IL-10). The Sr-doped nanowire modification may be a valuable approach to enhance osteogenic activities and reduce inflammatory reactions.

  13. A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus.

    Science.gov (United States)

    May, Jared; Johnson, Philip; Saleem, Huma; Simon, Anne E

    2017-04-15

    To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both in vitro and in vivo An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES region in vitro by use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA in vivo Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation. IMPORTANCE Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary

  14. In vitro haematic proteins adsorption and cytocompatibility study on acrylic copolymer to realise coatings for drug-eluting stents

    International Nuclear Information System (INIS)

    Gagliardi, Mariacristina

    2012-01-01

    In the present paper, a preliminary in vitro analysis of biocompatibility of newly-synthesised acrylic copolymers is reported. In particular, with the aim to obtain coatings for drug-eluting stents, blood protein absorption and cytocompatibility were studied. For protein absorption tests, bovine serum albumin and bovine plasma fibrinogen were considered. Cytocompatibility was tested using C2C12 cell line as model, analysing the behaviour of polymeric matrices and of drug-eluting systems, obtained loading polymeric matrices with paclitaxel, an anti-mitotic drug, in order to evaluate the efficacy of a pharmacological treatment locally administered from these materials. Results showed that the amount of albumin absorbed was greater than the amount of fibrinogen (comprised in the range of 70%–85% and 10%–22% respectively) and it is a good behaviour in terms of haemocompatibility. Cell culture tests showed good adhesion properties and a relative poor proliferation. In addition, a strong effect related to drug elution and a correlation with the macromolecular composition were detected. In this preliminary analysis, tested materials showed good characteristics and can be considered possible candidates to obtain coatings for drug-eluting stents. Highlights: ► Preliminary evaluation of haemo- and cytocompatibility of newly-synthesised acrylic copolymers ► Materials adsorb higher amounts of albumin and with a faster rate than fibrinogen. ► Protein adsorption depended on the macromolecular composition and surface properties. ► Cell viability on pure samples and efficacy of paclitaxel release were verified in C2C12 cultures.

  15. In vitro haematic proteins adsorption and cytocompatibility study on acrylic copolymer to realise coatings for drug-eluting stents

    Energy Technology Data Exchange (ETDEWEB)

    Gagliardi, Mariacristina, E-mail: mariacristina.gagliardi@iit.it

    2012-12-01

    In the present paper, a preliminary in vitro analysis of biocompatibility of newly-synthesised acrylic copolymers is reported. In particular, with the aim to obtain coatings for drug-eluting stents, blood protein absorption and cytocompatibility were studied. For protein absorption tests, bovine serum albumin and bovine plasma fibrinogen were considered. Cytocompatibility was tested using C2C12 cell line as model, analysing the behaviour of polymeric matrices and of drug-eluting systems, obtained loading polymeric matrices with paclitaxel, an anti-mitotic drug, in order to evaluate the efficacy of a pharmacological treatment locally administered from these materials. Results showed that the amount of albumin absorbed was greater than the amount of fibrinogen (comprised in the range of 70%-85% and 10%-22% respectively) and it is a good behaviour in terms of haemocompatibility. Cell culture tests showed good adhesion properties and a relative poor proliferation. In addition, a strong effect related to drug elution and a correlation with the macromolecular composition were detected. In this preliminary analysis, tested materials showed good characteristics and can be considered possible candidates to obtain coatings for drug-eluting stents. Highlights: Black-Right-Pointing-Pointer Preliminary evaluation of haemo- and cytocompatibility of newly-synthesised acrylic copolymers Black-Right-Pointing-Pointer Materials adsorb higher amounts of albumin and with a faster rate than fibrinogen. Black-Right-Pointing-Pointer Protein adsorption depended on the macromolecular composition and surface properties. Black-Right-Pointing-Pointer Cell viability on pure samples and efficacy of paclitaxel release were verified in C2C12 cultures.

  16. Control of autogenous activation of Herbaspirillum seropedicae nifA promoter by the IHF protein.

    Science.gov (United States)

    Wassem, Roseli; Pedrosa, Fábio O; Yates, Marshall G; Rego, Fabiane G M; Chubatsu, Leda S; Rigo, Liu U; Souza, Emanuel M

    2002-07-02

    Analysis of the expression of the Herbaspirillum seropedicae nifA promoter in Escherichia coli and Herbaspirillum seropedicae, showed that nifA expression is primarily dependent on NtrC but also required NifA for maximal expression under nitrogen-fixing conditions. Deletion of the IHF (integration host factor)-binding site produced a promoter with two-fold higher activity than the native promoter in the H. seropedicae wild-type strain but not in a nifA strain, indicating that IHF controls NifA auto-activation. IHF is apparently required to prevent overexpression of the NifA protein via auto-activation under nitrogen-fixing conditions in H. seropedicae.

  17. The Functionalization of Nanostructures and Their Potential Applications in Edible Coatings

    Directory of Open Access Journals (Sweden)

    Ricardo M. González-Reza

    2018-04-01

    Full Text Available Nowadays, edible coatings incorporated with nanostructures as systems of controlled release of flavors, colorants and/or antioxidants and antimicrobial substances, also used for thermal and environmental protection of active compounds, represent a gap of opportunity to increase the shelf life of food highly perishable, as well as for the development of new products. These functionalized nanostructures have the benefit of incorporating natural substances obtained from the food industry that are rich in polyphenols, dietary fibers, and antimicrobial substances. In addition, the polymers employed on its preparation, such as polysaccharides, solid lipids and proteins that are low cost and developed through sustainable processes, are friendly to the environment. The objective of this review is to present the materials commonly used in the preparation of nanostructures, the main ingredients with which they can be functionalized and used in the preparation of edible coatings, as well as the advances that these structures have represented when used as controlled release systems, increasing the shelf life and promoting the development of new products that meet the characteristics of functionality for fresh foods ready to eat.

  18. An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence.

    Science.gov (United States)

    Rodriguez, Patricia A; Escudero-Martinez, Carmen; Bos, Jorunn I B

    2017-03-01

    Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M persicae -host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

  19. Datasets of mung bean proteins and metabolites from four different cultivars

    Directory of Open Access Journals (Sweden)

    Akiko Hashiguchi

    2017-08-01

    Full Text Available Plants produce a wide array of nutrients that exert synergistic interaction among whole combinations of nutrients. Therefore comprehensive nutrient profiling is required to evaluate their nutritional/nutraceutical value and health promoting effect. In order to obtain such datasets for mung bean, which is known as a medicinal plant with heat alleviating effect, proteomic and metabolomic analyses were performed using four cultivars from China, Thailand, and Myanmar. In total, 449 proteins and 210 metabolic compounds were identified in seed coat; whereas 480 proteins and 217 metabolic compounds were detected in seed flesh, establishing the first comprehensive dataset of mung bean for nutraceutical evaluation.

  20. Polyethylenimine-coated Fe3O4 nanoparticles effectively quench fluorescent DNA, which can be developed as a novel platform for protein detection.

    Science.gov (United States)

    Ma, Long; Sun, Nana; Zhang, Jinyan; Tu, Chunhao; Cao, Xiuqi; Duan, Demin; Diao, Aipo; Man, Shuli

    2017-11-23

    We report a novel assembly of polyethyleneimine (PEI)-coated Fe 3 O 4 nanoparticles (NPs) with single-stranded DNA (ssDNA), and the fluorescence of the dye labeled in the DNA is remarkably quenched. In the presence of a target protein, the protein-DNA aptamer mutual interaction releases the ssDNA from this assembly and hence restores the fluorescence. This feature could be adopted to develop an aptasensor for protein detection. As a proof-of-concept, for the first time, we have used this proposed sensing strategy to detect thrombin selectively and sensitively. Furthermore, simultaneous multiple detection of thrombin and lysozyme in a complex protein mixture has been proven to be possible.

  1. Implication of the C terminus of the Prunus necrotic ringspot virus movement protein in cell-to-cell transport and in its interaction with the coat protein.

    Science.gov (United States)

    Aparicio, Frederic; Pallás, Vicente; Sánchez-Navarro, Jesús

    2010-07-01

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for viral transport. Previous analysis with MPs of other members of the family Bromoviridae has shown that the C-terminal part of these MPs plays a critical role in the interaction with the cognate coat protein (CP) and in cell-to-cell transport. Bimolecular fluorescence complementation and overlay analysis confirm an interaction between the C-terminal 38 aa of PNRSV MP and its cognate CP. Mutational analysis of the C-terminal region of the PNRSV MP revealed that its C-terminal 38 aa are dispensable for virus transport, however, the 4 aa preceding the dispensable C terminus are necessary to target the MP to the plasmodesmata and for the functionality of the protein. The capacity of the PNRSV MP to use either a CP-dependent or a CP-independent cell-to-cell transport is discussed.

  2. Microscopy analysis of reconstituted COPII coat polymerization and Sec16 dynamics.

    Science.gov (United States)

    Iwasaki, Hirohiko; Yorimitsu, Tomohiro; Sato, Ken

    2017-09-01

    The COPII coat and the small GTPase Sar1 mediate protein export from the endoplasmic reticulum (ER) via specialized domains known as the ER exit sites. The peripheral ER protein Sec16 has been proposed to organize ER exit sites. However, it remains unclear how these molecules drive COPII coat polymerization. Here, we characterized the spatiotemporal relationships between the Saccharomyces cerevisiae COPII components during their polymerization by performing fluorescence microscopy of an artificial planar membrane. We demonstrated that Sar1 dissociates from the membrane shortly after the COPII coat recruitment, and Sar1 is then no longer required for the COPII coat to bind to the membrane. Furthermore, we found that Sec16 is incorporated within the COPII-cargo clusters, and that this is dependent on the Sar1 GTPase cycle. These data show how Sar1 drives the polymerization of COPII coat and how Sec16 is spatially distributed during COPII coat polymerization. © 2017. Published by The Company of Biologists Ltd.

  3. Biocompatibility of chitosan-coated iron oxide nanoparticles with osteoblast cells

    Directory of Open Access Journals (Sweden)

    Shi S

    2012-10-01

    Full Text Available Si-Feng Shi,1 Jing-Fu Jia,2 Xiao-Kui Guo,3 Ya-Ping Zhao,2 De-Sheng Chen,1 Yong-Yuan Guo,1 Tao Cheng,1 Xian-Long Zhang11Department of Orthopedic Surgery, Shanghai Sixth People’s Hospital, School of Medicine, 2School of Chemistry and Chemical Technology, 3Department of Medical Microbiology and Parasitology, School of Medicine, Shanghai Jiao Tong University Shanghai, ChinaBackground: Bone disorders (including osteoporosis, loosening of a prosthesis, and bone infections are of great concern to the medical community and are difficult to cure. Therapies are available to treat such diseases, but all have drawbacks and are not specifically targeted to the site of disease. Chitosan is widely used in the biomedical community, including for orthopedic applications. The aim of the present study was to coat chitosan onto iron oxide nanoparticles and to determine its effect on the proliferation and differentiation of osteoblasts.Methods: Nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, x-ray diffraction, zeta potential, and vibrating sample magnetometry. Uptake of nanoparticles by osteoblasts was studied by transmission electron microscopy and Prussian blue staining. Viability and proliferation of osteoblasts were measured in the presence of uncoated iron oxide magnetic nanoparticles or those coated with chitosan. Lactate dehydrogenase, alkaline phosphatase, total protein synthesis, and extracellular calcium deposition was studied in the presence of the nanoparticles.Results: Chitosan-coated iron oxide nanoparticles enhanced osteoblast proliferation, decreased cell membrane damage, and promoted cell differentiation, as indicated by an increase in alkaline phosphatase and extracellular calcium deposition. Chitosan-coated iron oxide nanoparticles showed good compatibility with osteoblasts.Conclusion: Further research is necessary to optimize magnetic nanoparticles for the treatment of bone disease

  4. Enzyme-driven Bacillus spore coat degradation leading to spore killing.

    Science.gov (United States)

    Mundra, Ruchir V; Mehta, Krunal K; Wu, Xia; Paskaleva, Elena E; Kane, Ravi S; Dordick, Jonathan S

    2014-04-01

    The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable. © 2013 Wiley Periodicals, Inc.

  5. The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus Infectivity

    Directory of Open Access Journals (Sweden)

    Zhenwei Zhang

    2017-12-01

    Full Text Available Cucumber green mottle mosaic virus (CGMMV is one of the most devastating viruses infecting members of the family Cucurbitaceae. The assembly initiation site of CGMMV is located in the coding region of the coat protein, which is not only involved in virion assembly but is also a key factor determining the long-distance movement of the virus. To understand the effect of assembly initiation site and the adjacent region on CGMMV infectivity, we created a GTT deletion mutation in the GAGGTTG assembly initiation site of the infectious clone of CGMMV, which we termed V97 (deletion mutation at residue 97 of coat protein, followed by the construction of the V94A and T104A mutants. We observed that these three mutations caused mosaic after Agrobacterium-mediated transformation in Nicotiana benthamiana, albeit with a significant delay compared to the wild type clone. The mutants also had a common spontaneous E96K mutation in the coat protein. These results indicated that the initial assembly site and the sequence of the adjacent region affected the infectivity of the virus and that E96 might play an essential role in this process. We constructed two single point mutants—E96A and E96K—and three double mutants—V94A-E96K, V97-E96K and T104A-E96K—to further understand the role of E96 in CGMMV pathogenesis. After inoculation in N. benthamiana, E96A showed delayed systemic symptoms, but the E96K and three double mutants exhibited typical symptoms of mosaic at seven days post-infection. Then, sap from CGMMV-infected N. benthamiana leaves was mechanically inoculated on watermelon plants. We confirmed that E96 affected CGMMV infection using double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA, reverse transcription-polymerase chain reaction (RT-PCR, and sequencing, which further confirmed the successful infection of the related mutants, and that E96K can compensate the effect of the V94, V97, and T104 mutations on virus infectivity. In

  6. ZP Domain Proteins in the Abalone Egg Coat Include a Paralog of VERL under Positive Selection That Binds Lysin and 18-kDa Sperm Proteins

    Science.gov (United States)

    Aagaard, Jan E.; Vacquier, Victor D.; MacCoss, Michael J.; Swanson, Willie J.

    2010-01-01

    Identifying fertilization molecules is key to our understanding of reproductive biology, yet only a few examples of interacting sperm and egg proteins are known. One of the best characterized comes from the invertebrate archeogastropod abalone (Haliotis spp.), where sperm lysin mediates passage through the protective egg vitelline envelope (VE) by binding to the VE protein vitelline envelope receptor for lysin (VERL). Rapid adaptive divergence of abalone lysin and VERL are an example of positive selection on interacting fertilization proteins contributing to reproductive isolation. Previously, we characterized a subset of the abalone VE proteins that share a structural feature, the zona pellucida (ZP) domain, which is common to VERL and the egg envelopes of vertebrates. Here, we use additional expressed sequence tag sequencing and shotgun proteomics to characterize this family of proteins in the abalone egg VE. We expand 3-fold the number of known ZP domain proteins present within the VE (now 30 in total) and identify a paralog of VERL (vitelline envelope zona pellucida domain protein [VEZP] 14) that contains a putative lysin-binding motif. We find that, like VERL, the divergence of VEZP14 among abalone species is driven by positive selection on the lysin-binding motif alone and that these paralogous egg VE proteins bind a similar set of sperm proteins including a rapidly evolving 18-kDa paralog of lysin, which may mediate sperm–egg fusion. This work identifies an egg coat paralog of VERL under positive selection and the candidate sperm proteins with which it may interact during abalone fertilization. PMID:19767347

  7. Exosome purification based on PEG-coated Fe3O4 nanoparticles.

    Science.gov (United States)

    Chang, Ming; Chang, Yaw-Jen; Chao, Pei Yu; Yu, Qing

    2018-01-01

    Cancer cells secrete many exosomes, which facilitate metastasis and the later growth of cancer. For early cancer diagnosis, the detection of exosomes is a crucial step. Exosomes exist in biological fluid, such as blood, which contains various proteins. It is necessary to remove the proteins in the biological fluid to avoid test interference. This paper presented a novel method for exosome isolation using Fe3O4 magnetic nanoparticles (MNPs), which were synthesized using the chemical co-precipitation method and then coated with polyethylene glycol (PEG). The experimental results showed that the diameter of the PEG-coated Fe3O4 nanoparticles was about 20 nm, while an agglomerate of MNPs reached hundreds of nanometers in size. In the protein removal experiments, fetal bovine serum (FBS) was adopted as the analyte for bioassays of exosome purification. PEG-coated Fe3O4 MNPs reduced the protein concentration in FBS to 39.89% of the original solution. By observing a particle size distribution of 30~200 nm (the size range of various exosomes), the exosome concentrations were kept the same before and after purification. In the gel electrophoresis experiments, the bands of CD63 (~53 kDa) and CD9 (~22 kDa) revealed that exosomes existed in FBS as well as in the purified solution. However, the bands of the serum albumins (~66 kDa) and the various immunoglobulins (around 160 ~ 188 kDa) in the purified solution's lane explained that most proteins in FBS were removed by PEG-coated Fe3O4 MNPs. When purifying exosomes from serum, protein removal is critical for further exosome investigation. The proposed technique provides a simple and effective method to remove proteins in the serum using the PEG-coated Fe3O4 MNPs.

  8. Epigenetic repression of regulator of G-protein signaling 2 promotes androgen-independent prostate cancer cell growth.

    Science.gov (United States)

    Wolff, Dennis W; Xie, Yan; Deng, Caishu; Gatalica, Zoran; Yang, Mingjie; Wang, Bo; Wang, Jincheng; Lin, Ming-Fong; Abel, Peter W; Tu, Yaping

    2012-04-01

    G-protein-coupled receptor (GPCR)-stimulated androgen-independent activation of androgen receptor (AR) contributes to acquisition of a hormone-refractory phenotype by prostate cancer. We previously reported that regulator of G-protein signaling (RGS) 2, an inhibitor of GPCRs, inhibits androgen-independent AR activation (Cao et al., Oncogene 2006;25:3719-34). Here, we show reduced RGS2 protein expression in human prostate cancer specimens compared to adjacent normal or hyperplastic tissue. Methylation-specific PCR analysis and bisulfite sequencing indicated that methylation of the CpG island in the RGS2 gene promoter correlated with RGS2 downregulation in prostate cancer. In vitro methylation of this promoter suppressed reporter gene expression in transient transfection studies, whereas reversal of this promoter methylation with 5-aza-2'-deoxycytidine (5-Aza-dC) induced RGS2 reexpression in androgen-independent prostate cancer cells and inhibited their growth under androgen-deficient conditions. Interestingly, the inhibitory effect of 5-Aza-dC was significantly reduced by an RGS2-targeted short hairpin RNA, indicating that reexpressed RGS2 contributed to this growth inhibition. Restoration of RGS2 levels by ectopic expression in androgen-independent prostate cancer cells suppressed growth of xenografts in castrated mice. Thus, RGS2 promoter hypermethylation represses its expression and unmasks a latent pathway for AR transactivation in prostate cancer cells. Targeting this reversible process may provide a new strategy for suppressing prostate cancer progression by reestablishing its androgen sensitivity. Copyright © 2011 UICC.

  9. Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection.

    Science.gov (United States)

    DeBlasio, Stacy L; Johnson, Richard; Sweeney, Michelle M; Karasev, Alexander; Gray, Stewart M; MacCoss, Michael J; Cilia, Michelle

    2015-06-01

    Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a nonincorporated protein in concert with numerous insect and plant proteins to regulate virus movement/transmission and tissue tropism. Affinity purification coupled to quantitative MS was used to generate protein interaction networks for a PLRV mutant that is unable to produce the read through domain (RTD) and compared to the known wild-type PLRV protein interaction network. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and nonstructural viral proteins interacting with assembled coat protein (category I); plant proteins in complex with both coat protein and RTD (category II); plant proteins in complex with the RTD (category III); and plant proteins that had higher affinity for virions lacking the RTD (category IV). Proteins identified as interacting with the RTD are potential candidates for regulating viral processes that are mediated by the RTP such as phloem retention and systemic movement and can potentially be useful targets for the development of strategies to prevent infection and/or viral transmission of Luteoviridae species that infect important crop species. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells.

    Science.gov (United States)

    Behjati, M; Kazemi, M; Hashemi, M; Zarkesh-Esfahanai, S H; Bahrami, E; Hashemi-Beni, B; Ahmadi, R

    2013-08-20

    Enhanced attraction of selective vascular reparative cells is of great importance in order to increase vascular patency after endovascular treatments. We aimed to evaluate efficient attachment of endothelial cells and their progenitors on surfaces coated with mixture of specific antibodies, L-selectin and VE-cadherin, with prohibited platelet attachment. The most efficient conditions for coating of L-selectin-Fc chimera and VE-cadherin-Fc chimera proteins were first determined by protein coating on ELISA plates. The whole processes were repeated on titanium substrates, which are commonly used to coat stents. Endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry. Cell attachment, growth, proliferation, viability and surface cytotoxicity were evaluated using nuclear staining and MTT assay. Platelet and cell attachment were evaluated using scanning electron microscopy. Optimal concentration of each protein for surface coating was 50 ng/ml. The efficacy of protein coating was both heat and pH independent. Calcium ions had significant impact on simultaneous dual-protein coating (P<0.05). Coating stability data revealed more than one year stability for these coated proteins at 4°C. L-selectin and VE-cadherin (ratio of 50:50) coated surface showed highest EPC and HUVEC attachment, viability and proliferation compared to single protein coated and non-coated titanium surfaces (P<0.05). This double coated surface did not show any cytotoxic effect. Surfaces coated with L-selectin and VE-cadherin are friendly surface for EPC and endothelial cell attachment with less platelet attachment. These desirable factors make the L-selectin and VE-cadherin coated surfaces perfect candidate endovascular device. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  11. Polyclonal antibodies against the recombinantly expressed coat protein of the Citrus psorosis virus

    Directory of Open Access Journals (Sweden)

    Reda Salem

    2018-05-01

    Full Text Available Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV, the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in E. coli using recombinant DNA approach. The full length CP gene fragment was amplified by RT-PCR using total RNA extracted from CPsV infected citrus leaves and CP specific primers. The obtained product (1320bp was cloned, sequenced and sub-cloned into pET-30(+ expression vector. Expression was induced and screened in different bacterial clones by the presence of the expressed protein (48kDa and optimized in one clone. Expressed CP was purified using batch chromatography under denaturing conditions. Specificity of expressed protein was demonstrated by ELISA before used as antigen for raising PAbs in mice. Specificity of the raised PAbs to CPsV was verified by ELISA and western blotting. The raised PAbs were showed highly effectiveness in screening by ELISA comparing with the commercial antibodies purchased from Agritest, Valanzano, Italy.The expression of CPsV CP gene in E. coli, production of PAbs using recombinant protein as an antigen, the suitability of these antibodies for use in immunodiagnostics against the CPsV Egyptian isolate have been accomplished in this work. Keywords: CPsV, CP, PAbs, RT-PCR, ELISA, Western blotting

  12. The characterization of edible coating from tilapia surimi as a biodegradable packaging

    Science.gov (United States)

    Saputra, E.; Alamsjah, A.; Abdillah, A. A.

    2018-04-01

    One of the problems that often arise in the fisheries sector is maintaining the quality. In the room temperature, the fish more quickly enter the phase of rigor mortis and lasted shorter. The retention of fresh fish can be extended by adding antibacterial compounds in the form of synthetic chemicals or natural ingredients. One of the safe natural ingredients used to extend the freshness of the fish is the edible coating. Edible coatings may be composed of hydrocolloid, lipids and composites. In the food industry surimi can be used as an ingredient to make edible packaging or better known in the form of edible film and protein-based edible coating. Edible film and potential coatings are used as packaging materials as they may affect food quality, food safety, and shelf life. Protein-based edible film have superior inhibitory and mechanical properties compared to polysaccharide-based ones. This is because protein contains 20 different amino acids and has most special characteristics that produce functional characteristics when compared with polysaccharides used as an ingredient in edible film and coating making most homopolymers.

  13. Binding of mouse immunoglobulin G to polylysine-coated glass substrate for immunodiagnosis

    Science.gov (United States)

    Vashist, Sandeep Kumar; Tewari, Rupinder; Bajpai, Ram Prakash; Bharadwaj, Lalit Mohan; Raiteri, Roberto

    2006-12-01

    We report a method for immobilizing mouse immunoglobulin G (IgG) on polylysine-coated glass substrate for immunodiagnostic applications. Mouse IgG molecules were immobilized on polylysine-coated glass substrate employing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and protein A. The amino groups of the polylysine-coated glass slide were cross linked to the carboxyl groups of protein A employing EDC crosslinker. Protein A was employed as it binds to the constant Fc region of antibodies keeping their antigen binding sites on the variable F ab region free to bind to antigens. The qualitative analysis of surface immobilized mouse IgG was done by fluorescent microscopy employing fluorescein isothiocyanate (FITC) labeled mouse IgG molecules. The immobilization densities of protein A and mouse IgG were determined by 3, 3', 4, 4'-tetramethyl benzidine (TMB) substrate assay employing horse radish peroxidise labelled molecules and were found to be 130 +/- 17 ng/cm2 and 596 +/- 31 ng/cm2 respectively. The biomolecular coatings analyzed by atomic force microscopy (AFM) were found to be uniform.

  14. Polyurethane/poly(vinyl alcohol hydrogel coating improves the cytocompatibility of neural electrodes

    Directory of Open Access Journals (Sweden)

    Mei Li

    2015-01-01

    Full Text Available Neural electrodes, the core component of neural prostheses, are usually encapsulated in polydimethylsiloxane (PDMS. However, PDMS can generate a tissue response after implantation. Based on the physicochemical properties and excellent biocompatibility of polyurethane (PU and poly(vinyl alcohol (PVA when used as coating materials, we synthesized PU/PVA hydrogel coatings and coated the surface of PDMS using plasma treatment, and the cytocompatibility to rat pheochromocytoma (PC12 cells was assessed. Protein adsorption tests indicated that the amount of protein adsorption onto the PDMS substrate was reduced by 92% after coating with the hydrogel. Moreover, the PC12 cells on the PU/PVA-coated PDMS showed higher cell density and longer and more numerous neurites than those on the uncoated PDMS. These results indicate that the PU/PVA hydrogel is cytocompatible and a promising coating material for neural electrodes to improve their biocompatibility.

  15. pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion.

    Science.gov (United States)

    Batishchev, O V; Shilova, L A; Kachala, M V; Tashkin, V Y; Sokolov, V S; Fedorova, N V; Baratova, L A; Knyazev, D G; Zimmerberg, J; Chizmadzhev, Y A

    2016-01-01

    Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ~6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ~5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In

  16. Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding

    Science.gov (United States)

    Newcomer, Rebecca L.; Fraser, LaTasha C.R.; Teschke, Carolyn M.; Alexandrescu, Andrei T.

    2015-01-01

    The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining 3JNC’ couplings transmitted through H-bonds, the temperature and urea-concentration dependence of 1HN and 15N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and 3JNC’ H-bond couplings, are identified with an accuracy of 90% by 1HN temperature coefficients. The accuracy is improved to 95% when 15N temperature coefficients are also included. In contrast, the urea dependence of 1HN and 15N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. PMID:26682823

  17. Poly(dopamine) coating of 3D printed poly(lactic acid) scaffolds for bone tissue engineering

    International Nuclear Information System (INIS)

    Kao, Chia-Tze; Lin, Chi-Chang; Chen, Yi-Wen; Yeh, Chia-Hung; Fang, Hsin-Yuan; Shie, Ming-You

    2015-01-01

    3D printing is a versatile technique to generate large quantities of a wide variety of shapes and sizes of polymer. The aim of this study is to develop functionalized 3D printed poly(lactic acid) (PLA) scaffolds and use a mussel-inspired surface coating to regulate cell adhesion, proliferation and differentiation of human adipose-derived stem cells (hADSCs). We prepared PLA 3D scaffolds coated with polydopamine (PDA). The chemical composition and surface properties of PDA/PLA were characterized by XPS. PDA/PLA modulated hADSCs' responses in several ways. Firstly, adhesion and proliferation, and cell cycle of hADSCs cultured on PDA/PLA were significantly enhanced relative to those on PLA. In addition, the collagen I secreted from cells was increased and promoted cell attachment and cell cycle progression were depended on the PDA content. In osteogenesis assay, the ALP activity and osteocalcin of hADSCs cultured on PDA/PLA were significantly higher than seen in those cultured on pure PLA scaffolds. Moreover, hADSCs cultured on PDA/PLA showed up-regulation of the ang-1 and vWF proteins associated with angiogenic differentiation. Our results demonstrate that the bio-inspired coating synthetic PLA polymer can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to direct the specific responses of hADSCs. - Highlights: • A simple method of 3D printed poly(lactic acid) scaffold coated with PDA • Promoted proliferation of hADSCs on PDA/PLA scaffolds • Increased collagen I, cell cycle, and cell adhesion with a high PDA content • Up-regulation of angiogenic and osteogenic of hADSCs • A promising method for bioinspired surface modification on PLA using PDA

  18. Poly(dopamine) coating of 3D printed poly(lactic acid) scaffolds for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Kao, Chia-Tze [School of Dentistry, Chung Shan Medical University, Taichung City, Taiwan (China); Department of Stomatology, Chung Shan Medical University Hospital, Taichung City, Taiwan (China); Lin, Chi-Chang [Department of Chemical and Materials Engineering, Tunghai University, Taichung City, Taiwan (China); Chen, Yi-Wen; Yeh, Chia-Hung [3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan (China); Fang, Hsin-Yuan [3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan (China); Department of Thoracic Surgery, China Medical University Hospital, Taichung City, Taiwan (China); School of Medicine, College of Medicine, College of Public Health, Taichung City, Taiwan (China); Shie, Ming-You, E-mail: eviltacasi@gmail.com [3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan (China)

    2015-11-01

    3D printing is a versatile technique to generate large quantities of a wide variety of shapes and sizes of polymer. The aim of this study is to develop functionalized 3D printed poly(lactic acid) (PLA) scaffolds and use a mussel-inspired surface coating to regulate cell adhesion, proliferation and differentiation of human adipose-derived stem cells (hADSCs). We prepared PLA 3D scaffolds coated with polydopamine (PDA). The chemical composition and surface properties of PDA/PLA were characterized by XPS. PDA/PLA modulated hADSCs' responses in several ways. Firstly, adhesion and proliferation, and cell cycle of hADSCs cultured on PDA/PLA were significantly enhanced relative to those on PLA. In addition, the collagen I secreted from cells was increased and promoted cell attachment and cell cycle progression were depended on the PDA content. In osteogenesis assay, the ALP activity and osteocalcin of hADSCs cultured on PDA/PLA were significantly higher than seen in those cultured on pure PLA scaffolds. Moreover, hADSCs cultured on PDA/PLA showed up-regulation of the ang-1 and vWF proteins associated with angiogenic differentiation. Our results demonstrate that the bio-inspired coating synthetic PLA polymer can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to direct the specific responses of hADSCs. - Highlights: • A simple method of 3D printed poly(lactic acid) scaffold coated with PDA • Promoted proliferation of hADSCs on PDA/PLA scaffolds • Increased collagen I, cell cycle, and cell adhesion with a high PDA content • Up-regulation of angiogenic and osteogenic of hADSCs • A promising method for bioinspired surface modification on PLA using PDA.

  19. PHO1 Exports Phosphate from the Chalazal Seed Coat to the Embryo in Developing Arabidopsis Seeds.

    Science.gov (United States)

    Vogiatzaki, Evangelia; Baroux, Célia; Jung, Ji-Yul; Poirier, Yves

    2017-10-09

    Seed production requires the transfer of nutrients from the maternal seed coat to the filial endosperm and embryo. Because seed coat and filial tissues are symplasmically isolated, nutrients arriving in the seed coat via the phloem must be exported to the apoplast before reaching the embryo. Proteins implicated in the transfer of inorganic phosphate (Pi) from the seed coat to the embryo are unknown despite seed P content being an important agronomic trait. Here we show that the Arabidopsis Pi exporters PHO1 and PHOH1 are expressed in the chalazal seed coat (CZSC) of developing seeds. PHO1 is additionally expressed in developing ovules. Phosphorus (P) content and Pi flux between the seed coat and embryo were analyzed in seeds from grafts between WT roots and scions from either pho1, phoh1, or the pho1 phoh1 double mutant. Whereas P content and distribution between the seed coat and embryo in fully mature dry seeds of these mutants are similar to the WT, at the mature green stage of seed development the seed coat of the pho1 and pho1 phoh1 mutants, but not of the phoh1 mutant, retains approximately 2-fold more P than its WT control. Expression of PHO1 under a CZSC-specific promoter complemented the seed P distribution phenotype of the pho1 phoh1 double mutant. CZSC-specific down-expression of PHO1 also recapitulated the seed P distribution phenotype of pho1. Together, these experiments show that PHO1 expression in the CZSC is important for the transfer of P from the seed coat to the embryo in developing seeds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Performance of HVOF carbide coatings under erosion/corrosion

    International Nuclear Information System (INIS)

    Simard, S.; Arsenault, B.; Legoux, J.G.; Hawthorne, H.M.

    1999-01-01

    Cermet based materials are known to have an excellent performance under several wear conditions. High velocity oxy-fuel (HVOF) technology allows the deposition of such hard materials in the form of protective coatings onto different surfaces. Under slurry erosion, the performance of the coatings is influenced by the occurrence of corrosion reactions on the metallic matrix. Indeed, wet conditions promote the dissolution of metallic binder resulting in a potential synergic effect between the corrosion and wear mechanisms. The composition of the metallic matrix plays a key role on the stability of the coatings and their degradation rate. In this work, four coatings based on tungsten carbide embedded in different metallic binders were evaluated with regard to corrosion and wear. (author)

  1. Polymer Brush-Functionalized Chitosan Hydrogels as Antifouling Implant Coatings.

    Science.gov (United States)

    Buzzacchera, Irene; Vorobii, Mariia; Kostina, Nina Yu; de Los Santos Pereira, Andres; Riedel, Tomáš; Bruns, Michael; Ogieglo, Wojciech; Möller, Martin; Wilson, Christopher J; Rodriguez-Emmenegger, Cesar

    2017-06-12

    Implantable sensor devices require coatings that efficiently interface with the tissue environment to mediate biochemical analysis. In this regard, bioinspired polymer hydrogels offer an attractive and abundant source of coating materials. However, upon implantation these materials generally elicit inflammation and the foreign body reaction as a consequence of protein fouling on their surface and concomitant poor hemocompatibility. In this report we investigate a strategy to endow chitosan hydrogel coatings with antifouling properties by the grafting of polymer brushes in a "grafting-from" approach. Chitosan coatings were functionalized with polymer brushes of oligo(ethylene glycol) methyl ether methacrylate and 2-hydroxyethyl methacrylate using photoinduced single electron transfer living radical polymerization and the surfaces were thoroughly characterized by XPS, AFM, water contact angle goniometry, and in situ ellipsometry. The antifouling properties of these new bioinspired hydrogel-brush coatings were investigated by surface plasmon resonance. The influence of the modifications to the chitosan on hemocompatibility was assessed by contacting the surfaces with platelets and leukocytes. The coatings were hydrophilic and reached a thickness of up to 180 nm within 30 min of polymerization. The functionalization of the surface with polymer brushes significantly reduced the protein fouling and eliminated platelet activation and leukocyte adhesion. This methodology offers a facile route to functionalizing implantable sensor systems with antifouling coatings that improve hemocompatibility and pave the way for enhanced device integration in tissue.

  2. Silica-Coated Liposomes for Insulin Delivery

    OpenAIRE

    Neelam Dwivedi; M. A. Arunagirinathan; Somesh Sharma; Jayesh Bellare

    2010-01-01

    Liposomes coated with silica were explored as protein delivery vehicles for their enhanced stability and improved encapsulation efficiency. Insulin was encapsulated within the fluidic phosphatidylcholine lipid vesicles by thin film hydration at pH 2.5, and layer of silica was formed above lipid bilayer by acid catalysis. The presence of silica coating and encapsulated insulin was identified using confocal and electron microscopy. The native state of insulin present in the formulation was evid...

  3. Promoter activity of polypyrimidine tract-binding protein genes of potato responds to environmental cues.

    Science.gov (United States)

    Butler, Nathaniel M; Hannapel, David J

    2012-12-01

    Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that target specific RNAs for post-transcriptional processing by binding cytosine/uracil motifs. PTBs have established functions in a range of RNA processes including splicing, translation, stability and long-distance transport. Six PTB-like genes identified in potato have been grouped into two clades based on homology to other known plant PTBs. StPTB1 and StPTB6 are closely related to a PTB protein discovered in pumpkin, designated CmRBP50, and contain four canonical RNA-recognition motifs. CmRBP50 is expressed in phloem tissues and functions as the core protein of a phloem-mobile RNA/protein complex. Sequence from the potato genome database was used to clone the upstream sequence of these two PTB genes and analyzed to identify conserved cis-elements. The promoter of StPTB6 was enriched for regulatory elements for light and sucrose induction and defense. Upstream sequence of both PTB genes was fused to β-glucuronidase and monitored in transgenic potato lines. In whole plants, the StPTB1 promoter was most active in leaf veins and petioles, whereas StPTB6 was most active in leaf mesophyll. Both genes are active in new tubers and tuber sprouts. StPTB6 expression was induced in stems and stolon sections in response to sucrose and in leaves or petioles in response to light, heat, drought and mechanical wounding. These results show that CmRBP50-like genes of potato exhibit distinct expression patterns and respond to both developmental and environmental cues.

  4. Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway

    DEFF Research Database (Denmark)

    Oka, Yasuyoshi; Bekker-Jensen, Simon; Mailand, Niels

    2015-01-01

    in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function...

  5. HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.

    Science.gov (United States)

    Guillen-Ahlers, Hector; Rao, Prahlad K; Levenstein, Mark E; Kennedy-Darling, Julia; Perumalla, Danu S; Jadhav, Avinash Y L; Glenn, Jeremy P; Ludwig-Kubinski, Amy; Drigalenko, Eugene; Montoya, Maria J; Göring, Harald H; Anderson, Corianna D; Scalf, Mark; Gildersleeve, Heidi I S; Cole, Regina; Greene, Alexandra M; Oduro, Akua K; Lazarova, Katarina; Cesnik, Anthony J; Barfknecht, Jared; Cirillo, Lisa A; Gasch, Audrey P; Shortreed, Michael R; Smith, Lloyd M; Olivier, Michael

    2016-06-01

    Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of Saccharomyces cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. A missense mutation in the agouti signaling protein gene (ASIP) is associated with the no light points coat phenotype in donkeys.

    Science.gov (United States)

    Abitbol, Marie; Legrand, Romain; Tiret, Laurent

    2015-04-08

    Seven donkey breeds are recognized by the French studbook and are characterized by a black, bay or grey coat colour including light cream-to-white points (LP). Occasionally, Normand bay donkeys give birth to dark foals that lack LP and display the no light points (NLP) pattern. This pattern is more frequent and officially recognized in American miniature donkeys. The LP (or pangare) phenotype resembles that of the light bellied agouti pattern in mouse, while the NLP pattern resembles that of the mammalian recessive black phenotype; both phenotypes are associated with the agouti signaling protein gene (ASIP). We used a panel of 127 donkeys to identify a recessive missense c.349 T > C variant in ASIP that was shown to be in complete association with the NLP phenotype. This variant results in a cysteine to arginine substitution at position 117 in the ASIP protein. This cysteine is highly-conserved among vertebrate ASIP proteins and was previously shown by mutagenesis experiments to lie within a functional site. Altogether, our results strongly support that the identified mutation is causative of the NLP phenotype. Thus, we propose to name the c.[349 T > C] allele in donkeys, the a(nlp) allele, which enlarges the panel of coat colour alleles in donkeys and ASIP recessive loss-of-function alleles in animals.

  7. Methods for promoting wound healing and muscle regeneration with the cell signaling protein nell1

    Energy Technology Data Exchange (ETDEWEB)

    Culiat, Cymbeline T.

    2018-03-20

    The present invention provides methods for promoting wound healing and treating muscle atrophy in a mammal in need. The method comprises administering to the mammal a Nell1 protein or a Nell1 nucleic acid molecule.

  8. Methods for promoting wound healing and muscle regeneration with the cell signaling protein Nell1

    Science.gov (United States)

    Culiat, Cymbeline T [Oak Ridge, TN

    2011-03-22

    The present invention provides methods for promoting wound healing and treating muscle atrophy in a mammal in need. The method comprises administering to the mammal a Nell1 protein or a Nell1 nucleic acid molecule.

  9. Low-thrombogenic fibrin-heparin coating promotes in vitro endothelialization

    Czech Academy of Sciences Publication Activity Database

    Kaplan, Ondřej; Hierlemann, T.; Krajewski, S.; Kurz, J.; Nevoralová, Martina; Houska, Milan; Riedel, Tomáš; Riedelová, Zuzana; Zárubová, Jana; Wendel, H. P.; Brynda, Eduard

    2017-01-01

    Roč. 105, č. 11 (2017), s. 2995-3005 ISSN 1549-3296 R&D Projects: GA MZd(CZ) NV15-29153A Institutional support: RVO:61389013 ; RVO:67985823 Keywords : fibrin-heparin coating * hemocompatibility * endothelialization Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery; FA - Cardiovascular Diseases incl. Cardiotharic Surgery (FGU-C) OBOR OECD: Cardiac and Cardiovascular systems; Cardiac and Cardiovascular systems (FGU-C) Impact factor: 3.076, year: 2016

  10. Substrates coated with silver nanoparticles as a neuronal regenerative material

    Directory of Open Access Journals (Sweden)

    Alon N

    2014-05-01

    Full Text Available Noa Alon,1,3,* Yana Miroshnikov,2,3,* Nina Perkas,2,3 Ifat Nissan,2,3 Aharon Gedanken,2,3 Orit Shefi1,31Faculty of Engineering, 2Department of Chemistry, 3Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan, Israel*These authors contributed equally to this workAbstract: Much effort has been devoted to the design of effective biomaterials for nerve regeneration. Here, we report the novel use of silver nanoparticles (AgNPs as regenerative agents to promote neuronal growth. We grew neuroblastoma cells on surfaces coated with AgNPs and studied the effect on the development of the neurites during the initiation and the elongation growth phases. We find that the AgNPs function as favorable anchoring sites, and the growth on the AgNP-coated substrates leads to a significantly enhanced neurite outgrowth. Cells grown on substrates coated with AgNPs have initiated three times more neurites than cells grown on uncoated substrates, and two times more than cells grown on substrates sputtered with a plain homogenous layer of silver. The growth of neurites on AgNPs in the elongation phase was enhanced as well. A comparison with substrates coated with gold nanoparticles (AuNPs and zinc oxide nanoparticles (ZnONPs demonstrated a clear silver material-driven promoting effect, in addition to the nanotopography. The growth on substrates coated with AgNPs has led to a significantly higher number of initiating neurites when compared to substrates coated with AuNPs or ZnONPs. All nanoparticle-coated substrates affected and promoted the elongation of neurites, with a significant positive maximal effect for the AgNPs. Our results, combined with the well-known antibacterial effect of AgNPs, suggest the use of AgNPs as an attractive nanomaterial – with dual activity – for neuronal repair studies.Keywords: nerve regeneration, nanotopography, antibacterial material, neuroblastoma, gold nanoparticles, zinc oxide nanoparticles

  11. In vitro evidence for RNA binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses.

    Science.gov (United States)

    Pallás, V; Sánchez-Navarro, J A; Díez, J

    1999-01-01

    The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding.

  12. Screening a yeast promoter library leads to the isolation of the RP29/L32 and SNR17B/RPL37A divergent promoters and the discovery of a gene encoding ribosomal protein L37.

    Science.gov (United States)

    Santangelo, G M; Tornow, J; McLaughlin, C S; Moldave, K

    1991-08-30

    Two promoters (A7 and A23), isolated at random from the Saccharomyces cerevisiae genome by virtue of their capacity to activate transcription, are identical to known intergenic bidirectional promoters. Sequence analysis of the genomic DNA adjacent to the A7 promoter identified a split gene encoding ribosomal (r) protein L37, which is homologous to the tRNA-binding r-proteins, L35a (from human and rat) and L32 (from frogs).

  13. Gelatin methacrylamide as coating material in cell culture.

    Science.gov (United States)

    Egger, Michael; Tovar, Günter E M; Hoch, Eva; Southan, Alexander

    2016-06-13

    Unmodified gelatin (uG) is widely used as a coating material in cell culture for improving surface properties. In this study, the authors investigated if gelatin methacrylamide (GM) with a medium degree of methacrylamide modification (GM1.5) and a high degree of methacrylamide modification (GM4) are equally suitable for this purpose. Therefore, gold surfaces were coated with uG, GM1.5, and GM4 by adsorption of the polymers on the surfaces. Coating success was confirmed by spectroscopic ellipsometry, contact angle measurements, surface plasmon resonance spectroscopy (SPRS), and atomic force microscopy (AFM). The authors found that upon adsorption of uG, GM1.5, a nd GM4 on gold, thin films with thicknesses of 2.95 nm, 2.50 nm, and 2.26 nm were formed. The coated surfaces showed advancing contact angles of 46° (uG and GM1.5) and 52° (GM4) without alteration of the surface roughness determined by AFM. Protein adsorption taking place on the coated surfaces was measured during contact of the surfaces with fetal calf serum by SPRS. Protein adsorption on the coated surfaces was reduced by the factor of 6.4 (uG), 5.4 (GM1.5), and 4.6 (GM4) compared to gold surfaces. Human fibroblasts cultured on the surfaces showed excellent viability shown by water soluble tetrazolium salt assay as well as live/dead staining with propidium iodide and fluorescein diacetate. No cytotoxic effects of the GM coated surfaces were observed, giving rise to the conclusion that GMs are suitable materials as coatings in cell culture.

  14. Synthetic cold-inducible promoter enhances recombinant protein accumulation during Agrobacterium-mediated transient expression in Nicotiana excelsior at chilling temperatures.

    Science.gov (United States)

    Gerasymenko, I M; Sheludko, Y V

    2017-07-01

    To exploit cold-inducible biochemical processes beneficial for foreign mRNA transcription, translation and storage, as well as protein product stability, during Agrobacterium-mediated transient expression. The efficiency of three different 5'-regulatory sequences to achieve transient expression of the GFP-based reporter gene under chilling conditions (6-8 °C since the 3rd day post inoculation) was compared. We studied the upstream sequences of a cold-inducible Arabidopsis thaliana cor15a gene, the core element of 35S CaMV promoter fused to the TMV omega 5'-UTR, and the synthetic promoter including the 35S core sequence and two binding sites for cold-inducible CBF transcription factors (P_DRE::35S). Cultivation of plants transiently expressing reporter gene under control of the synthetic P_DRE::35S promoter under chilling conditions since the 3rd dpi led to the reliably higher reporter accumulation as compared to the other tested regulatory sequences under chilling or greenhouse conditions. Reporter protein fluorescence under chilling conditions using P_DRE::35S reached 160% as compared to the transient expression in the greenhouse. Period of transient expression considerably extended if plants were cultivated at chilling temperature since the 3rd dpi: reporter protein fluorescence reached its maximum at the 20th dpi and was detected in leaves up to the 65th dpi. The enhanced protein accumulation at low temperature was accompanied by the prolonged period of corresponding mRNA accumulation. Transient expression under chilling conditions using synthetic cold-inducible promoter enhances target protein accumulation and may decrease greenhouse heating expenses.

  15. In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation.

    Science.gov (United States)

    Aparicio, Frederic; Sánchez-Navarro, Jesús A; Pallás, Vicente

    2006-06-01

    Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

  16. Investigation of calcium phosphate coatings for biomedical applications

    International Nuclear Information System (INIS)

    Yusof Abdullah; Idris Besar; Muhammad Jamal Md Isa; Mohamad Abd Razak; Hyzan Mohd Yusof

    1999-01-01

    Calcium phosphate is the main constituent of our bone and tooth minerals. The use of this bioactive material for coating implant such as artificial joint prosthesis, therefore, can promote biological fixation and enhance biocompatibility. Our initial work has been focused on the evaluation of experimental conditions of coating preparation and the effects of post-deposition calcium phosphate coatings on stainless steel substrates. The coating layers were produced by the precipitation technique and coatings were carried out in sol-gel by the dipping method. For comparison purposes a wet method was used to obtain a fine calcium phosphate ceramic powder for fabrication of microcrystal suspension used as a coating material. Scanning electron microscopy (SEM), energy dispersive microanalysis (EDS), energy dispersive x-ray fluorescence (EDXRF) and x-ray diffraction (XRD) were used to characterise the morphology, chemical composition and structure of the coatings. The results showed that the dip coating of stainless steel substrates using viscous solutions lead to the formation of porous calcium phosphate layers. These results suggested that fabrication of bioactive calcium phosphate coatings using this route offers significant advantages over the currently used methods due to considerably lower temperature process involved and may produce better result for substrates with complex shapes

  17. RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

    Science.gov (United States)

    Galloway, Alison; Saveliev, Alexander; Łukasiak, Sebastian; Hodson, Daniel J; Bolland, Daniel; Balmanno, Kathryn; Ahlfors, Helena; Monzón-Casanova, Elisa; Mannurita, Sara Ciullini; Bell, Lewis S; Andrews, Simon; Díaz-Muñoz, Manuel D; Cook, Simon J; Corcoran, Anne; Turner, Martin

    2016-04-22

    Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint. Copyright © 2016, American Association for the Advancement of Science.

  18. An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence1[OPEN

    Science.gov (United States)

    2017-01-01

    Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M. persicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. PMID:28100451

  19. Nucleotide sequence of the coat protein gene of the Skierniewice isolate of plum pox virus (PPV)

    International Nuclear Information System (INIS)

    Wypijewski, K.; Musial, W.; Augustyniak, J.; Malinowski, T.

    1994-01-01

    The coat protein (CP) gene of the Skierniewice isolate of plum pox virus (PPV-S) has been amplified using the reverse transcription - polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide sequence of the gene and the deduced amino-acid sequences of PPV-S CP were compared with those of other PPV strains. The nucleotide sequence showed very high homology to most of the published sequences. The motif: Asp-Ala-Gly (DAG), important for the aphid transmissibility, was present in the amino-acid sequence. Our isolate did not react in ELISA with monoclonal antibodies MAb06 supposed to be specific for PPV-D. (author). 32 refs, 1 fig., 2 tabs

  20. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

    Science.gov (United States)

    Dhar, Jayeeta; Barik, Sailen

    2016-12-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

  1. A liver stress-endocrine nexus promotes metabolic integrity during dietary protein dilution

    DEFF Research Database (Denmark)

    Maida, Adriano; Zota, Annika; Sjøberg, Kim Anker

    2016-01-01

    of impaired glucose homeostasis independently of obesity and food intake. DPD-mediated metabolic inefficiency and improvement of glucose homeostasis were independent of uncoupling protein 1 (UCP1), but required expression of liver-derived fibroblast growth factor 21 (FGF21) in both lean and obese mice. FGF21...... expression and secretion as well as the associated metabolic remodeling induced by DPD also required induction of liver-integrated stress response-driven nuclear protein 1 (NUPR1). Insufficiency of select nonessential amino acids (NEAAs) was necessary and adequate for NUPR1 and subsequent FGF21 induction...... and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency-induced liver NUPR1/FGF21 axis....

  2. Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore.

    Science.gov (United States)

    Tang, Jilin; Krajcikova, Daniela; Zhu, Rong; Ebner, Andreas; Cutting, Simon; Gruber, Hermann J; Barak, Imrich; Hinterdorfer, Peter

    2007-01-01

    Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores. Copyright (c) 2007 John Wiley & Sons, Ltd.

  3. Probing the ability of the coat and vertex protein of the membrane-containing bacteriophage PRD1 to display a meningococcal epitope

    International Nuclear Information System (INIS)

    Huiskonen, Juha T.; Laakkonen, Liisa; Toropainen, Maija; Sarvas, Matti; Bamford, Dennis H.; Bamford, Jaana K.H.

    2003-01-01

    Bacteriophage PRD1 is an icosahedral dsDNA virus with a diameter of 740 A and an outer protein shell composed of 720 copies of major coat protein P3. Spike complexes at the vertices are composed of a pentameric base (protein P31) and a spike structure (proteins P5 and P2) where the N-terminal region of the trimeric P5 is associated with the base and the C-terminal region of P5 is associated with receptor-binding protein P2. The functionality of proteins P3 and P5 was investigated using insertions and deletions. It was observed that P3 did not tolerate changes whereas P5 tolerated changes much more freely. These properties support the hypothesis that viruses have core structures and functions, which remain stable over time, as well as other elements, responsible for host interactions, which are evolutionally more fluid. The insertional probe used was the apex of exposed loop 4 of group B meningococcal outer membrane protein PorA, a medically important subunit vaccine candidate. It was demonstrated that the epitope could be displayed on the virus surface as part of spike protein P5

  4. Mechanical particle coating using polymethacrylate nanoparticle agglomerates for the preparation of controlled release fine particles: The relationship between coating performance and the characteristics of various polymethacrylates.

    Science.gov (United States)

    Kondo, Keita; Kato, Shinsuke; Niwa, Toshiyuki

    2017-10-30

    We aimed to understand the factors controlling mechanical particle coating using polymethacrylate. The relationship between coating performance and the characteristics of polymethacrylate powders was investigated. First, theophylline crystals were treated using a mechanical powder processor to obtain theophylline spheres (grindability of the agglomerates were attributed to differences in particle structure, resulting from consolidation between colloidal particles. High-grindability agglomerates exhibited higher pulverization as their glass transition temperature (T g ) increased and the further pulverization promoted coating. We therefore conclude that the minimization of polymethacrylate powder by pulverization is an important factor in mechanical particle coating using polymethacrylate with low deformability. Meanwhile, when product temperature during coating approaches T g of polymer, polymethacrylate was soften to show high coating performance by plastic deformation. The effective coating by this mechanism may be accomplished by adjusting the temperature in the processor to the T g . Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Targets of DNA-binding proteins in bacterial promoter regions present enhanced probabilities for spontaneous thermal openings

    International Nuclear Information System (INIS)

    Apostolaki, Angeliki; Kalosakas, George

    2011-01-01

    We mapped promoter regions of double-stranded DNA with respect to the probabilities of appearance of relatively large bubble openings exclusively due to thermal fluctuations at physiological temperatures. We analyzed five well-studied promoter regions of procaryotic type and found a spatial correlation between the binding sites of transcription factors and the position of peaks in the probability pattern of large thermal openings. Other distinct peaks of the calculated patterns correlate with potential binding sites of DNA-binding proteins. These results suggest that a DNA molecule would more frequently expose the bases that participate in contacts with proteins, which would probably enhance the probability of the latter to reach their targets. It also stands for using this method as a means to analyze DNA sequences based on their intrinsic thermal properties

  6. Effect of amorphous fluorinated coatings on photocatalytic properties of anodized titanium surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Persico, Federico [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, via Mancinelli 7, 20131, Milano (Italy); Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali, Via G. Giusti, 9, 50121 Firenze (Italy); Sansotera, Maurizio, E-mail: maurizio.sansotera@polimi.it [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, via Mancinelli 7, 20131, Milano (Italy); Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali, Via G. Giusti, 9, 50121 Firenze (Italy); Diamanti, Maria Vittoria [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, via Mancinelli 7, 20131, Milano (Italy); Magagnin, Luca; Venturini, Francesco; Navarrini, Walter [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, via Mancinelli 7, 20131, Milano (Italy); Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali, Via G. Giusti, 9, 50121 Firenze (Italy)

    2013-10-31

    The photocatalytic activity promoted by anodized titanium surfaces coated with different amorphous perfluoropolymers was evaluated. A copolymer between tetrafluoroethylene and perfluoro-4-trifluoromethoxy-1,3-dioxole and two perfluoropolyethers containing ammonium phosphate and triethoxysilane functionalities, respectively, were tested as coating materials. These coatings revealed good adhesion to the anodized titanium substrate and conferred to it both hydrophobicity and oleophobicity. The photocatalytic activity of the coating on anodized titanium was evaluated by monitoring the degradation of stearic acid via Infrared spectroscopy. The degradation rate of stearic acid was reduced but not set to zero by the presence of the fluorinated coatings, leading to the development of advanced functional coatings. The morphological variations of the coatings as a result of photocatalysis were also determined by atomic force microscopy. - Highlights: • Coated anodized titanium surfaces show a decreased wettability. • Evaluation of the stability of perfluorinated coatings towards photocatalysis. • Amorphous perfluorinated coatings do not hinder photocatalytic activity.

  7. High quality protein microarray using in situ protein purification

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D

    2009-08-01

    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  8. GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells

    Directory of Open Access Journals (Sweden)

    Dawid Krokowski

    2017-12-01

    Full Text Available Summary: GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome. : Here, Krokowski et al. show that GADD34/PP1 protects the microtubule network, prevents Golgi fragmentation, and preserves protein trafficking independent of its action in the integrated stress response (ISR. In osmoadaptation, GADD34 facilitates trans-Golgi-mediated processing of the endoplasmic reticulum (ER-synthesized amino acid transporter SNAT2, which in turn increases amino acid uptake. Keywords: SNAT2, GADD34, hyperosmotic stress, amino acid transport, Golgi fragmentation, ISR

  9. Increase in Bacterial Colony Formation from a Permafrost Ice Wedge Dosed with a Tomitella biformata Recombinant Resuscitation-Promoting Factor Protein.

    Science.gov (United States)

    Puspita, Indun Dewi; Kitagawa, Wataru; Kamagata, Yoichi; Tanaka, Michiko; Nakatsu, Cindy H

    2015-01-01

    Resuscitation-promoting factor (Rpf) is a protein that has been found in a number of different Actinobacteria species and has been shown to promote the growth of active cells and resuscitate dormant (non-dividing) cells. We previously reported the biological activity of an Rpf protein in Tomitella biformata AHU 1821(T), an Actinobacteria isolated from a permafrost ice wedge. This protein is excreted outside the cell; however, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. Therefore, the aim of the present study was to determine whether Rpf from T. biformata impacted the cultivation of other bacteria from the permafrost ice wedge from which it was originally isolated. All experiments used recombinant Rpf proteins produced using a Rhodococcus erythropolis expression system. Dilutions of melted surface sterilized ice wedge samples mixed with different doses of the purified recombinant Rpf (rRpf) protein indicated that the highest concentration tested, 1250 pM, had a significantly (p permafrost sediments. The results of the present study demonstrated that rRpf not only promoted the growth of T. biformata from which it was isolated, but also enhanced colony formation by another Actinobacteria in an environmental sample.

  10. The coat protein of Alfalfa mosaic virus interacts and interferes with the transcriptional activity of the bHLH transcription factor ILR3 promoting salicylic acid-dependent defence signalling response.

    Science.gov (United States)

    Aparicio, Frederic; Pallás, Vicente

    2017-02-01

    During virus infection, specific viral component-host factor interaction elicits the transcriptional reprogramming of diverse cellular pathways. Alfalfa mosaic virus (AMV) can establish a compatible interaction in tobacco and Arabidopsis hosts. We show that the coat protein (CP) of AMV interacts directly with transcription factor (TF) ILR3 of both species. ILR3 is a basic helix-loop-helix (bHLH) family member of TFs, previously proposed to participate in diverse metabolic pathways. ILR3 has been shown to regulate NEET in Arabidopsis, a critical protein in plant development, senescence, iron metabolism and reactive oxygen species (ROS) homeostasis. We show that the AMV CP-ILR3 interaction causes a fraction of this TF to relocate from the nucleus to the nucleolus. ROS, pathogenesis-related protein 1 (PR1) mRNAs, salicylic acid (SA) and jasmonic acid (JA) contents are increased in healthy Arabidopsis loss-of-function ILR3 mutant (ilr3.2) plants, which implicates ILR3 in the regulation of plant defence responses. In AMV-infected wild-type (wt) plants, NEET expression is reduced slightly, but is induced significantly in ilr3.2 mutant plants. Furthermore, the accumulation of SA and JA is induced in Arabidopsis wt-infected plants. AMV infection in ilr3.2 plants increases JA by over 10-fold, and SA is reduced significantly, indicating an antagonist crosstalk effect. The accumulation levels of viral RNAs are decreased significantly in ilr3.2 mutants, but the virus can still systemically invade the plant. The AMV CP-ILR3 interaction may down-regulate a host factor, NEET, leading to the activation of plant hormone responses to obtain a hormonal equilibrium state, where infection remains at a level that does not affect plant viability. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  11. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

    DEFF Research Database (Denmark)

    Søgaard-Andersen, L; Martinussen, J; Møllegaard, N E

    1990-01-01

    We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) Cyt...

  12. Tumor promoter induced membrane-bound protein kinase C - its influence on hematogenous metastasis

    International Nuclear Information System (INIS)

    Gopalakrishna, R.; Barsky, S.H.

    1987-01-01

    A correlation between the amount of membrane-bound detergent-extractable protein kinase C activity in various B16 melanoma sublines (F10, F1, BL6) and their lung metastasizing abilities following intravenous injection was found. The F10 subline which exhibits higher metastasizing ability was found to have higher membrane-bound protein kinase C compared to the lower metastasizing subline, F1. Treatment of F1 cells with 100 nM 12-0 tetradecanoylphorbol-13-acetate (TPA) for 1h resulted in 90% decrease in protein kinase C activity in the cytosol with a concommitent increase in membrane-bound activity. These TPA-treated cells when injected intravenously in C57BL/6 mice produced 6-fold increase in pulmonary metastases compared to untreated F1 cells. However, biologically inactive analogues 4 α-phorbol 12,13-didecanoate and phorbol 13-acetate had no effect on either membrane-bound protein kinase C activity or pulmonary metastases. Treating F1 cells with the second-stage tumor promoter, mezerin, resulted in increase in both membrane association of protein kinase C and also lung metastases. Thus, these results strongly suggests that membrane associated protein kinase C activity influences hematogenous metastasis of these melanoma cells

  13. Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes

    KAUST Repository

    Alam, Tanvir; Medvedeva, Yulia A.; Jia, Hui; Brown, James B.; Lipovich, Leonard; Bajic, Vladimir B.

    2014-01-01

    raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted

  14. Index of refraction enhancement of calcite particles coated with zinc carbonate

    Science.gov (United States)

    Lattaud, Kathleen; Vilminot, Serge; Hirlimann, Charles; Parant, Hubert; Schoelkopf, Joachim; Gane, Patrick

    2006-10-01

    ZnCO 3 coating on calcite particles has been developed in order to enhance the index of refraction of this mineral that is used as a charge in paper, paint and polymer industries. Chemical reaction between calcite particles in an aqueous suspension with zinc chloride promotes the formation of a ZnCO 3 coating consisting of two layers with different interactions with the calcite particle. The refraction index of the resulting composite particles increases with the Zn/Ca ratio. A model allows to evaluate the coating thickness. The value of the scattering S and diffusion K coefficients of sheets coated with the ZnCO 3 coated particles reveal a dependence on the preparation conditions with a 15% increase for the best samples.

  15. Adhesion of Porphyromonas gingivalis and Tannerella forsythia to dentin and titanium with sandblasted and acid etched surface coated with serum and serum proteins - An in vitro study.

    Science.gov (United States)

    Eick, Sigrun; Kindblom, Christian; Mizgalska, Danuta; Magdoń, Anna; Jurczyk, Karolina; Sculean, Anton; Stavropoulos, Andreas

    2017-03-01

    To evaluate the adhesion of selected bacterial strains incl. expression of important virulence factors at dentin and titanium SLA surfaces coated with layers of serum proteins. Dentin- and moderately rough SLA titanium-discs were coated overnight with human serum, or IgG, or human serum albumin (HSA). Thereafter, Porphyromonas gingivalis, Tannerella forsythia, or a six-species mixture were added for 4h and 24h. The number of adhered bacteria (colony forming units; CFU) was determined. Arg-gingipain activity of P. gingivalis and mRNA expressions of P. gingivalis and T. forsythia proteases and T. forsythia protease inhibitor were measured. Coating specimens never resulted in differences exceeding 1.1 log10 CFU, comparing to controls, irrespective the substrate. Counts of T. forsythia were statistically significantly higher at titanium than dentin, the difference was up to 3.7 log10 CFU after 24h (p=0.002). No statistically significant variation regarding adhesion of the mixed culture was detected between surfaces or among coatings. Arg-gingipain activity of P. gingivalis was associated with log10 CFU but not with the surface or the coating. Titanium negatively influenced mRNA expression of T. forsythia protease inhibitor at 24h (p=0.026 uncoated, p=0.009 with serum). The present findings indicate that: a) single bacterial species (T. forsythia) can adhere more readily to titanium SLA than to dentin, b) low expression of T. forsythia protease inhibitor may influence the virulence of the species on titanium SLA surfaces in comparison with teeth, and c) surface properties (e.g. material and/or protein layers) do not appear to significantly influence multi-species adhesion. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Yeast Interacting Proteins Database: YDL239C, YDR273W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available of a Don1p-containing structure at the leading edge of the prospore membrane via interaction with spindle p...it as prey (1) YDR273W DON1 Meiosis-specific component of the spindle pole body, part of the leading... edge protein (LEP) coat, forms a ring-like structure at the leading edge of the prospore...ption Protein required for spore wall formation, thought to mediate assembly of a Don1p-containing structure at the leading...description Meiosis-specific component of the spindle pole body, part of the leading edge protein (LEP) coat

  17. Co-delivery of Cbfa-1-targeting siRNA and SOX9 protein using PLGA nanoparticles to induce chondrogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Jeon, Su Yeon; Park, Ji Sun; Yang, Han Na; Lim, Hye Jin; Yi, Se Won; Park, Hansoo; Park, Keun-Hong

    2014-09-01

    During stem cell differentiation, various cellular responses occur that are mediated by transcription factors and proteins. This study evaluated the abilities of SOX9, a crucial protein during the early stage of chondrogenesis, and siRNA targeting Cbfa-1, a transcription factor that promotes osteogenesis, to stimulate chondrogenesis. Non-toxic poly-(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. Coomassie blue staining and circular dichroism revealed that the loaded SOX9 protein maintained its stability and bioactivity. These NPs easily entered human mesenchymal stem cells (hMSCs) in vitro and caused them to differentiate into chondrocytes. Markers that are typically expressed in mature chondrocytes were examined. These markers were highly expressed at the mRNA and protein levels in hMSCs treated with PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. By contrast, these cells did not express osteogenesis-related markers. hMSCs were injected into mice following internalization of PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. When the injection site was excised, markers of chondrogenesis were found to be highly expressed at the mRNA and protein levels, similar to the in vitro results. When hMSCs internalized these NPs and were then cultured in vitro or injected into mice, chondrogenesis-related extracellular matrix components were highly expressed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

    Directory of Open Access Journals (Sweden)

    Nannenga Brent L

    2011-12-01

    Full Text Available Abstract Background Membrane proteins (MPs populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. Results Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells, toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host. Conclusions Our system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

  19. Influence of twisted tape turbulence promoter on fouling reduction in microfiltration of milk proteins

    Directory of Open Access Journals (Sweden)

    Popović Svetlana S.

    2011-01-01

    Full Text Available Membrane filtration has become one of the major technologies in the food industry. It is widely applied in the dairy industry, and it is mostly used for the concentration and fractionation of milk proteins and for the whey processing. Of all pressure driven membrane processes, ultrafiltration is the most widely used. The major disadvantage of pressure driven membrane processes is severe fouling of membrane during filtration particularly when the fluids containing proteins are processed. Fouling with proteins is complex phenomenon because it occurs at the membrane surface as well as in the pores of membrane, and depends on the operating conditions and on the interactions of proteins and membrane material. In order to reduce fouling of the membrane different techniques have been developed, and one of them relies on the changing of the hydrodynamic conditions in the membrane or module. In this study, influence of twisted tape turbulence promoters on the fouling reduction in cross-flow microfiltration of skim milk was investigated. Twisted tapes with tree characteristic ratios of helix element length to the tape diameter (aspect ratio were studied. It was shown that twisted tapes with different aspect ratios reduce fouling of membrane by a factor of three or more. The presence of twisted tape induces changes in the flow patterns from straight to helicoidally thus producing turbulence flow at the lower cross-flow rates. Turbulence intensification prevents accumulation of proteins at membrane surface enabling reduction in reversible fouling what results in the reduction of overall membrane fouling. The best performance was achieved using a twisted tape with the lowest aspect ratio of 1.0. This promoter reduces fouling seven times at low transmembrane pressure and low cross-flow velocity. The twisted tape with aspect ratio 1.0 induces the most intensive turbulence, the longest helicoidal flow path, and appearance of vortices near the membrane surfaces

  20. Reactive polymer coatings: A robust platform towards sophisticated surface engineering for biotechnology

    Science.gov (United States)

    Chen, Hsien-Yeh

    Functionalized poly(p-xylylenes) or so-called reactive polymers can be synthesized via chemical vapor deposition (CVD) polymerization. The resulting ultra-thin coatings are pinhole-free and can be conformally deposited to a wide range of substrates and materials. More importantly, the equipped functional groups can served as anchoring sites for tailoring the surface properties, making these reactive coatings a robust platform that can deal with sophisticated challenges faced in biointerfaces. In this work presented herein, surface coatings presenting various functional groups were prepared by CVD process. Such surfaces include aldehyde-functionalized coating to precisely immobilize saccharide molecules onto well-defined areas and alkyne-functionalized coating to click azide-modified molecules via Huisgen 1,3-dipolar cycloaddition reaction. Moreover, CVD copolymerization has been conducted to prepare multifunctional coatings and their specific functions were demonstrated by the immobilization of biotin and NHS-ester molecules. By using a photodefinable coating, polyethylene oxides were immobilized onto a wide range of substrates through photo-immobilization. Spatially controlled protein resistant properties were characterized by selective adsorption of fibrinogen and bovine serum albumin as model systems. Alternatively, surface initiator coatings were used for polymer graftings of polyethylene glycol) methyl ether methacrylate, and the resultant protein- and cell- resistant properties were characterized by adsorption of kinesin motor proteins, fibrinogen, and murine fibroblasts (NIH3T3). Accessibility of reactive coatings within confined microgeometries was systematically studied, and the preparation of homogeneous polymer thin films within the inner surface of microchannels was demonstrated. Moreover, these advanced coatings were applied to develop a dry adhesion process for microfluidic devices. This process provides (i) excellent bonding strength, (ii) extended

  1. Isolation and characterization of an ubiquitin extension protein gene (JcUEP) promoter from Jatropha curcas.

    Science.gov (United States)

    Tao, Yan-Bin; He, Liang-Liang; Niu, Long-Jian; Xu, Zeng-Fu

    2015-04-01

    The JcUEP promoter is active constitutively in the bio-fuel plant Jatropha curcas , and is an alternative to the widely used CaMV35S promoter for driving constitutive overexpression of transgenes in Jatropha. Well-characterized promoters are required for transgenic breeding of Jatropha curcas, a biofuel feedstock with great potential for production of bio-diesel and bio-jet fuel. In this study, an ubiquitin extension protein gene from Jatropha, designated JcUEP, was identified to be ubiquitously expressed. Thus, we isolated a 1.2 kb fragment of the 5' flanking region of JcUEP and evaluated its activity as a constitutive promoter in Arabidopsis and Jatropha using the β-glucuronidase (GUS) reporter gene. As expected, histochemical GUS assay showed that the JcUEP promoter was active in all Arabidopsis and Jatropha tissues tested. We also compared the activity of the JcUEP promoter with that of the cauliflower mosaic virus 35S (CaMV35S) promoter, a well-characterized constitutive promoter conferring strong transgene expression in dicot species, in various tissues of Jatropha. In a fluorometric GUS assay, the two promoters showed similar activities in stems, mature leaves and female flowers; while the CaMV35S promoter was more effective than the JcUEP promoter in other tissues, especially young leaves and inflorescences. In addition, the JcUEP promoter retained its activity under stress conditions in low temperature, high salt, dehydration and exogenous ABA treatments. These results suggest that the plant-derived JcUEP promoter could be an alternative to the CaMV35S promoter for driving constitutive overexpression of transgenes in Jatropha and other plants.

  2. Parallel nano-assembling of a multifunctional GO/HapNP coating on ultrahigh-purity magnesium for biodegradable implants

    International Nuclear Information System (INIS)

    Santos, C.; Piedade, C.; Uggowitzer, P.J.; Montemor, M.F.; Carmezim, M.J.

    2015-01-01

    Graphical abstract: - Highlights: • A GO/HapNP thin coating was applied on XHP-Mg using a parallel nano-assembling route. • The coating surface wettability can be controlled by adding HapNP and/or GO. • Nanostructured GO/HapNP/phosphate coating promotes the apatite mineralization. • Critical properties, including degradation on SBF, of the coating were studied. - Abstract: This work reports the one-step fabrication of a novel coating on ultra high purity magnesium using a parallel nano assembling process. The multifunctional biodegradable surface was obtained by adding hydroxyapatite nanoparticles (HapNP) plus graphene oxide (GO). The coating was characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffractometer (XRD), micro-Raman spectroscopy. The thin phosphate coating (thickness of 1 μm) reveals a uniform coverage with cypress like structures. The incorporation of HapNP and GO promotes the hydrophilic behavior of the coating surface. The results revealed that the proposed coating can be used to tailor the surface properties such as wettability by adjusting the contents of HapNP and GO. The in vitro degradation rate of the coated magnesium suggests that the presence of HapNP and GO/HapNP in the phosphate coating decreased the current density compared to the single phosphate coating and uncoated magnesium. This study also reveals the HapNP/GO/phosphate coating induces apatite formation, showing suitable degradability that makes it a promising coating candidate for enhanced bone regeneration

  3. Parallel nano-assembling of a multifunctional GO/HapNP coating on ultrahigh-purity magnesium for biodegradable implants

    Energy Technology Data Exchange (ETDEWEB)

    Santos, C., E-mail: catarina.santos@estsetubal.ips.pt [EST Setúbal, DEM, Instituto Politécnico de Setúbal, Campus IPS, 2914-508 Setúbal (Portugal); CQE, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa (Portugal); Piedade, C. [EST Setúbal, DEM, Instituto Politécnico de Setúbal, Campus IPS, 2914-508 Setúbal (Portugal); Uggowitzer, P.J. [Laboratory of Metal Physics and Technology, ETH Zurich, 8093 Zurich (Switzerland); Montemor, M.F. [CQE, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa (Portugal); Carmezim, M.J. [EST Setúbal, DEM, Instituto Politécnico de Setúbal, Campus IPS, 2914-508 Setúbal (Portugal); CQE, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa (Portugal)

    2015-08-01

    Graphical abstract: - Highlights: • A GO/HapNP thin coating was applied on XHP-Mg using a parallel nano-assembling route. • The coating surface wettability can be controlled by adding HapNP and/or GO. • Nanostructured GO/HapNP/phosphate coating promotes the apatite mineralization. • Critical properties, including degradation on SBF, of the coating were studied. - Abstract: This work reports the one-step fabrication of a novel coating on ultra high purity magnesium using a parallel nano assembling process. The multifunctional biodegradable surface was obtained by adding hydroxyapatite nanoparticles (HapNP) plus graphene oxide (GO). The coating was characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffractometer (XRD), micro-Raman spectroscopy. The thin phosphate coating (thickness of 1 μm) reveals a uniform coverage with cypress like structures. The incorporation of HapNP and GO promotes the hydrophilic behavior of the coating surface. The results revealed that the proposed coating can be used to tailor the surface properties such as wettability by adjusting the contents of HapNP and GO. The in vitro degradation rate of the coated magnesium suggests that the presence of HapNP and GO/HapNP in the phosphate coating decreased the current density compared to the single phosphate coating and uncoated magnesium. This study also reveals the HapNP/GO/phosphate coating induces apatite formation, showing suitable degradability that makes it a promising coating candidate for enhanced bone regeneration.

  4. Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

    Science.gov (United States)

    de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

    2016-11-01

    Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p  0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

  5. Minor Coat and Heat Shock Proteins Are Involved in the Binding of Citrus Tristeza Virus to the Foregut of Its Aphid Vector, Toxoptera citricida.

    Science.gov (United States)

    Killiny, N; Harper, S J; Alfaress, S; El Mohtar, C; Dawson, W O

    2016-11-01

    Vector transmission is a critical stage in the viral life cycle, yet for most plant viruses how they interact with their vector is unknown or is explained by analogy with previously described relatives. Here we examined the mechanism underlying the transmission of citrus tristeza virus (CTV) by its aphid vector, Toxoptera citricida, with the objective of identifying what virus-encoded proteins it uses to interact with the vector. Using fluorescently labeled virions, we demonstrated that CTV binds specifically to the lining of the cibarium of the aphid. Through in vitro competitive binding assays between fluorescent virions and free viral proteins, we determined that the minor coat protein is involved in vector interaction. We also found that the presence of two heat shock-like proteins, p61 and p65, reduces virion binding in vitro Additionally, treating the dissected mouthparts with proteases did not affect the binding of CTV virions. In contrast, chitinase treatment reduced CTV binding to the foregut. Finally, competition with glucose, N-acetyl-β-d-glucosamine, chitobiose, and chitotriose reduced the binding. These findings together suggest that CTV binds to the sugar moieties of the cuticular surface of the aphid cibarium, and the binding involves the concerted activity of three virus-encoded proteins. Limited information is known about the specific interactions between citrus tristeza virus and its aphid vectors. These interactions are important for the process of successful transmission. In this study, we localized the CTV retention site as the cibarium of the aphid foregut. Moreover, we demonstrated that the nature of these interactions is protein-carbohydrate binding. The viral proteins, including the minor coat protein and two heat shock proteins, bind to sugar moieties on the surface of the foregut. These findings will help in understanding the transmission mechanism of CTV by the aphid vector and may help in developing control strategies which interfere

  6. Metal matrix coated fiber composites and the methods of manufacturing such composites

    Science.gov (United States)

    Weeks, J.K. Jr.; Gensse, C.

    1993-09-14

    A fiber coating which allows ceramic or metal fibers to be wetted by molten metals is disclosed. The coating inhibits degradation of the physical properties caused by chemical reaction between the fiber and the coating itself or between the fiber and the metal matrix. The fiber coating preferably includes at least a wetting layer, and in some applications, a wetting layer and a barrier layer between the fiber and the wetting layer. The wetting layer promotes fiber wetting by the metal matrix. The barrier layer inhibits fiber degradation. The fiber coating permits the fibers to be infiltrated with the metal matrix resulting in composites having unique properties not obtainable in pure materials. 8 figures.

  7. Localization of the N-terminal domain of cauliflower mosaic virus coat protein precursor

    International Nuclear Information System (INIS)

    Champagne, Julie; Benhamou, Nicole; Leclerc, Denis

    2004-01-01

    Cauliflower mosaic virus (CaMV) open reading frame (ORF) IV encodes a coat protein precursor (pre-CP) harboring an N-terminal extension that is cleaved off by the CaMV-encoded protease. In transfected cells, pre-CP is present in the cytoplasm, while the processed form (p44) of CP is targeted to the nucleus, suggesting that the N-terminal extension might be involved in keeping the pre-CP in the cytoplasm for viral assembly. This study reports for the first time the intracellular localization of the N-terminal extension during CaMV infection in Brassica rapa. Immunogold-labeling electron microscopy using polyclonal antibodies directed to the N-terminal extension of the pre-CP revealed that this region is closely associated with viral particles present in small aggregates, which we called small bodies, adjacent to the main inclusion bodies typical of CaMV infection. Based on these results, we propose a model for viral assembly of CaMV

  8. Investigation of interactions between dendrimer-coated magnetite nanoparticles and bovine serum albumin

    International Nuclear Information System (INIS)

    Pan Bifeng; Gao Feng; Ao Limei

    2005-01-01

    We investigated the interactions between dendrimer-coated magnetite nanoparticles (MNPs) and the protein serum albumin. The investigation was based on the fluorescence quenching of tryptophan residue of serum albumin after binding with the dendrimer-coated magnetite nanoparticles. The extent of the interactions between bovine serum albumin and dendrimer-coated MNPs strongly depends on their surface groups and pH value

  9. Improving the osteointegration of Ti6Al4V by zeolite MFI coating

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yong [Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Jiao, Yilai [Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning 110016 (China); Li, Xiaokang [Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Guo, Zheng, E-mail: guozheng@fmmu.edu.cn [Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

    2015-05-01

    Osteointegration is crucial for success in orthopedic implantation. In recent decades, there have been numerous studies aiming to modify titanium alloys, which are the most widely used materials in orthopedics. Zeolites are solid aluminosilicates whose application in the biomedical field has recently been explored. To this end, MFI zeolites have been developed as titanium alloy coatings and tested in vitro. Nevertheless, the effect of the MFI coating of biomaterials in vivo has not yet been addressed. The aim of the present work is to evaluate the effects of MFI-coated Ti6Al4V implants in vitro and in vivo. After surface modification, the surface was investigated using field emission scanning electron microscopy (FE-SEM) and energy dispersive spectroscopy (EDS). No difference was observed regarding the proliferation of MC3T3-E1 cells on the Ti6Al4V (Ti) and MFI-coated Ti6Al4V (M−Ti) (p > 0.05). However, the attachment of MC3T3-E1 cells was found to be better in the M−Ti group. Additionally, ALP staining and activity assays and quantitative real-time RT-PCR indicated that MC3T3-E1 cells grown on the M−Ti displayed high levels of osteogenic differentiation markers. Moreover, Van-Gieson staining of histological sections demonstrated that the MFI coating on Ti6Al4V scaffolds significantly enhanced osteointegration and promoted bone regeneration after implantation in rabbit femoral condylar defects at 4 and 12 weeks. Therefore, this study provides a method for modifying Ti6Al4V to achieve improved osteointegration and osteogenesis. - Highlights: • Osteointegration is a crucial factor for orthopedic implants. • We coated MFI zeolite on Ti6Al4V substrates and investigated the effects in vitro and in vivo. • The MFI coating displayed good biocompatibility and promoted osteogenic differentiation in vitro. • The MFI coating promoted osteointegration and osteogenesis peri-implant in vivo.

  10. Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73

    International Nuclear Information System (INIS)

    Zhang Heng; Wu Shengnan

    2011-01-01

    p73 has been identified as a structural and functional homolog of the tumor suppressor p53. However, mechanisms that regulate the localization of p73 have not been fully clarified. The Yes-associated protein (YAP) is a transcriptional coactivator. As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. p73 is a reported YAP target transcription factors and YAP has been shown to positively regulate p73 in promoting apoptosis. Previous studies show that p73 interacts with YAP through its PPPY motif, and increases p73 transactivation of apoptotic genes. In this study, we focused on YAP's regulation of the localization of p73. After transient transfection into Rat pheochromocytoma (PC12) cells and Human embryonic kidney 293T cells with GFP-YAP and/or YFP-p73, and incubated for 24 hours expression. p73 was fused to YFP to allow the examination of its subcellular localization. When expressed alone, YFP-p73 was distributed throughout the cell. When coexpressed with YAP, nuclear accumulation of YFP-p73 became evident. We quantitated the effect of YAP on the redistribution of YFP-p73 by counting cells with nuclear-only YFP signal. We found that YAP can influence the subcellular distribution of p73. Altogether, coexpression with YAP affected the subcellular distribution of the p73 protein. Our studies attribute a central role to YAP in regulating p73 accumulation and YAP, at least in part, might promote the nuclear import of p73.

  11. Bio-Based Coatings for Paper Applications

    Directory of Open Access Journals (Sweden)

    Vibhore Kumar Rastogi

    2015-11-01

    Full Text Available The barrier resistance and wettability of papers are commonly controlled by the application of petroleum-based derivatives such as polyethylene, waxes and/or fluor- derivatives as coating. While surface hydrophobicity is improved by employing these polymers, they have become disfavored due to limitations in fossil-oil resources, poor recyclability, and environmental concerns on generated waste with lack of biodegradation. Alternatively, biopolymers including polysaccharides, proteins, lipids and polyesters can be used to formulate new pathways for fully bio-based paper coatings. However, difficulties in processing of most biopolymers may arise due to hydrophilicity, crystallization behavior, brittleness or melt instabilities that hinder a full exploitation at industrial scale. Therefore, blending with other biopolymers, plasticizers and compatibilizers is advantageous to improve the coating performance. In this paper, an overview of barrier properties and processing of bio-based polymers and their composites as paper coating will be discussed. In particular, recent technical advances in nanotechnological routes for bio-based nano- composite coatings will be summarized, including the use of biopolymer nanoparticles, or nanofillers such as nanoclay and nanocellulose. The combination of biopolymers along with surface modification of nanofillers can be used to create hierarchical structures that enhance hydrophobicity, complete barrier protection and functionalities of coated papers.

  12. Activation of the ATR kinase by the RPA-binding protein ETAA1

    DEFF Research Database (Denmark)

    Haahr, Peter; Hoffmann, Saskia; Tollenaere, Maxim A X

    2016-01-01

    Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway...... in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically...

  13. A biodegradable AZ91 magnesium alloy coated with a thin nanostructured hydroxyapatite for improving the corrosion resistance

    Energy Technology Data Exchange (ETDEWEB)

    Mukhametkaliyev, T.M.; Surmeneva, M.A. [National Research Tomsk Polytechnic University, 634050, Lenin Avenue 43, Tomsk (Russian Federation); Vladescu, A. [National Research Tomsk Polytechnic University, 634050, Lenin Avenue 43, Tomsk (Russian Federation); National Institute for Optoelectronics, 409 Atomistilor St., RO77125 Magurele (Romania); Cotrut, C.M. [National Research Tomsk Polytechnic University, 634050, Lenin Avenue 43, Tomsk (Russian Federation); Politehnica University of Bucharest, 313 Spl. Independentei, Bucharest (Romania); Braic, M.; Dinu, M. [National Institute for Optoelectronics, 409 Atomistilor St., RO77125 Magurele (Romania); Vranceanu, M.D. [Politehnica University of Bucharest, 313 Spl. Independentei, Bucharest (Romania); Pana, I. [National Institute for Optoelectronics, 409 Atomistilor St., RO77125 Magurele (Romania); Faculty of Physics, Bucharest University, 405 Atomistilor St., RO77125 Magurele (Romania); Mueller, M. [Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, 70569 Stuttgart (Germany); Surmenev, R.A., E-mail: rsurmenev@gmail.com [National Research Tomsk Polytechnic University, 634050, Lenin Avenue 43, Tomsk (Russian Federation)

    2017-06-01

    The main aim of this study was to investigate the properties of an AZ91 alloy coated with nanostructured hydroxyapatite (HA) prepared by radio frequency (RF) magnetron sputtering. The bioactivity and biomineralization of the AZ91 magnesium alloy coated with HA were investigated in simulated body fluid (SBF) via an in vitro test. Scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD) analyses were performed. The samples were immersed in SBF to study the ability of the surface to promote the formation of an apatite layer as well as corrosion resistance and mass change of the HA-coated AZ91 alloy. Electrochemical tests were performed to estimate the corrosion behaviour of HA-coated and uncoated samples. The results revealed the capability of the HA coating to significantly improve the corrosion resistance of the uncoated AZ91 alloy. - Highlights: • The nanostructured HA layer allows to control the degradation rate of the AZ91 alloy. • The HA coating significantly reduces the corrosion current density. • The HA coating significantly improves the polarization resistance in vitro. • The RF magnetron deposited HA coating promotes calcium-phosphate precipitation in SBF.

  14. A biodegradable AZ91 magnesium alloy coated with a thin nanostructured hydroxyapatite for improving the corrosion resistance

    International Nuclear Information System (INIS)

    Mukhametkaliyev, T.M.; Surmeneva, M.A.; Vladescu, A.; Cotrut, C.M.; Braic, M.; Dinu, M.; Vranceanu, M.D.; Pana, I.; Mueller, M.; Surmenev, R.A.

    2017-01-01

    The main aim of this study was to investigate the properties of an AZ91 alloy coated with nanostructured hydroxyapatite (HA) prepared by radio frequency (RF) magnetron sputtering. The bioactivity and biomineralization of the AZ91 magnesium alloy coated with HA were investigated in simulated body fluid (SBF) via an in vitro test. Scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD) analyses were performed. The samples were immersed in SBF to study the ability of the surface to promote the formation of an apatite layer as well as corrosion resistance and mass change of the HA-coated AZ91 alloy. Electrochemical tests were performed to estimate the corrosion behaviour of HA-coated and uncoated samples. The results revealed the capability of the HA coating to significantly improve the corrosion resistance of the uncoated AZ91 alloy. - Highlights: • The nanostructured HA layer allows to control the degradation rate of the AZ91 alloy. • The HA coating significantly reduces the corrosion current density. • The HA coating significantly improves the polarization resistance in vitro. • The RF magnetron deposited HA coating promotes calcium-phosphate precipitation in SBF.

  15. Effect of H2O and Y(O on Oxidation Behavior of NiCoCrAl Coating Within Thermal Barrier Coating

    Directory of Open Access Journals (Sweden)

    WANG Yi-qun

    2017-04-01

    Full Text Available NiCoCrAl coatings containing Y and Y oxide were made using vacuum plasma deposition and high-velocity oxygen fuel respectively, high temperature oxidation dynamics and cross-section microstructures of NiCoCrAl+Y and NiCoCrAl+Y(O coatings in Ar-16.7%O2, Ar-3.3%H2O and Ar-0.2%H2-0.9%H2O at 1100℃ were investigated by differential thermal analysis (DTA and optical and electron microscope. The influencing mechanism of Y oxide on the oxidation of coatings at different atmosphere was compared by computation using First-Principles. The results show that Al2O3 layer on NiCoCrAl+Y coatings has more holes for internal oxidation on account of the element Y diffusion and enrichment on the interface. In addition, steam can promote the internal oxidation. While a thinner and uniform alumina form on NiCoCrAl+Y(O coatings because element Y is pinned by oxygen atoms during the preparation of coatings. Water vapor has less influence on protective alumina formation on the NiCoCrAl+Y(O coating. Therefore, oxidation behavior of NiCoCrAl coatings vary in composition and structure in different oxidizing atmosphere. Besides, Y and Y-enrichment oxides have key influences on the microstructure and the growth rate.

  16. Immobilization of bacterial S-layer proteins from Caulobacter crescentus on iron oxide-based nanocomposite: synthesis and spectroscopic characterization of zincite-coated Fe₂O₃ nanoparticles.

    Science.gov (United States)

    Habibi, Neda

    2014-05-05

    Zinc oxide was coated on Fe2O3 nanoparticles using sol-gel spin-coating. Caulobacter crescentus have a crystalline surface layer (S-layer), which consist of one protein or glycoprotein species. The immobilization of bacterial S-layers obtained from C. crescentus on zincite-coated nanoparticles of iron oxide was investigated. The SDS PAGE results of S-layers isolated from C. crescentus showed the weight of 50 KDa. Nanoparticles of the Fe2O3 and zinc oxide were synthesized by a sol-gel technique. Fe2O3 nanoparticles with an average size of 50 nm were successfully prepared by the proper deposition of zinc oxide onto iron oxide nanoparticles surface annealed at 450 °C. The samples were characterized by field-emission scanning electron microscope (FESEM), atomic force microscopy (AFM), powder X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FT-IR). Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Characterization and tribology of PEG-like coatings on UHMWPE for total hip replacements.

    Science.gov (United States)

    Kane, Sheryl R; Ashby, Paul D; Pruitt, Lisa A

    2010-03-15

    A crosslinked hydrogel coating similar to poly(ethylene glycol) (PEG) was covalently bonded to the surface of ultrahigh molecular weight polyethylene (UHMWPE) to improve the lubricity and wear resistance of the UHWMPE for use in total joint replacements. The chemistry, hydrophilicity, and protein adsorption resistance of the coatings were determined, and the wear behavior of the PEG-like coating was examined by two methods: pin-on-disk tribometry to evaluate macroscale behavior, and atomic force microscopy (AFM) to simulate asperity wear. As expected, the coating was found to be highly PEG-like, with approximately 83% ether content by x-ray photoelectron spectroscopy and more hydrophilic and resistant to protein adsorption than uncoated UHMWPE. Pin-on-disk testing showed that the PEG-like coating could survive 3 MPa of contact pressure, comparable to that experienced by total hip replacements. AFM nanoscratching experiments uncovered three damage mechanisms for the coatings: adhesion/microfracture, pure adhesion, and delamination. The latter two mechanisms appear to correlate well with wear patterns induced by pin-on-disk testing and evaluated by attenuated total reflection Fourier transform infrared spectroscopy mapping. Understanding the mechanisms by which the PEG-like coatings wear is critical for improving the behavior of subsequent generations of wear-resistant hydrogel coatings. (c) 2009 Wiley Periodicals, Inc.

  18. Translationally controlled tumor protein supplemented chitosan modified glass ionomer cement promotes osteoblast proliferation and function

    Energy Technology Data Exchange (ETDEWEB)

    Sangsuwan, Jiraporn [Department of Molecular Biology and Bioinformatics, Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112 (Thailand); Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla 90112 (Thailand); Wanichpakorn, Supreya; Kedjarune-Leggat, Ureporn [Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla 90112 (Thailand)

    2015-09-01

    The objective of this study was to evaluate the effect of translationally controlled tumor protein (TCTP) supplemented in a novel glass ionomer cement (BIO-GIC) on normal human osteoblasts (NHost cells). BIO-GIC was a glass ionomer cement (GIC) modified by adding chitosan and albumin to promote the release of TCTP. NHost cells were seeded on specimens of GIC, GIC + TCTP, BIO-GIC and BIO-GIC + TCTP. Cell proliferation was determined by BrdU assay. It was found that BIO-GIC + TCTP had significantly higher proliferation of cells than other specimens. Bone morphogenetic protein-2 (BMP-2) and osteopontin (OPN) gene expressions assessed by quantitative real time PCR and alkaline phosphatase (ALP) activity were used to determine cell differentiation. Bone cell function was investigated by calcium deposition using alizarin assay. Both BMP-2 and OPN gene expressions of cells cultured on specimens with added TCTP increased gradually up-regulation after day 1 and reached the highest on day 3 then down-regulation on day 7. The ALP activity of cells cultured on BIO-GIC + TCTP for 7 days and calcium content after 14 days were significantly higher than other groups. BIO-GIC + TCTP can promote osteoblast cells proliferation, differentiation and function. - Highlights: • Developed a new GIC by supplementing TCTP in BIO-GIC (GIC with chitosan and albumin) • BIO-GIC + TCTP released a higher amount of TCTP than GIC + TCTP. • BIO-GIC + TCTP promoted cell proliferation higher than other specimens and control. • BIO-GIC + TCTP promoted osteoblasts differentiation and function.

  19. Translationally controlled tumor protein supplemented chitosan modified glass ionomer cement promotes osteoblast proliferation and function

    International Nuclear Information System (INIS)

    Sangsuwan, Jiraporn; Wanichpakorn, Supreya; Kedjarune-Leggat, Ureporn

    2015-01-01

    The objective of this study was to evaluate the effect of translationally controlled tumor protein (TCTP) supplemented in a novel glass ionomer cement (BIO-GIC) on normal human osteoblasts (NHost cells). BIO-GIC was a glass ionomer cement (GIC) modified by adding chitosan and albumin to promote the release of TCTP. NHost cells were seeded on specimens of GIC, GIC + TCTP, BIO-GIC and BIO-GIC + TCTP. Cell proliferation was determined by BrdU assay. It was found that BIO-GIC + TCTP had significantly higher proliferation of cells than other specimens. Bone morphogenetic protein-2 (BMP-2) and osteopontin (OPN) gene expressions assessed by quantitative real time PCR and alkaline phosphatase (ALP) activity were used to determine cell differentiation. Bone cell function was investigated by calcium deposition using alizarin assay. Both BMP-2 and OPN gene expressions of cells cultured on specimens with added TCTP increased gradually up-regulation after day 1 and reached the highest on day 3 then down-regulation on day 7. The ALP activity of cells cultured on BIO-GIC + TCTP for 7 days and calcium content after 14 days were significantly higher than other groups. BIO-GIC + TCTP can promote osteoblast cells proliferation, differentiation and function. - Highlights: • Developed a new GIC by supplementing TCTP in BIO-GIC (GIC with chitosan and albumin) • BIO-GIC + TCTP released a higher amount of TCTP than GIC + TCTP. • BIO-GIC + TCTP promoted cell proliferation higher than other specimens and control. • BIO-GIC + TCTP promoted osteoblasts differentiation and function

  20. Antimicrobial edible films and coatings for fresh and minimally processed fruits and vegetables: a review.

    Science.gov (United States)

    Valencia-Chamorro, Silvia A; Palou, Lluís; Del Río, Miguel A; Pérez-Gago, María B

    2011-01-01

    The use of edible films and coatings is an environmentally friendly technology that offers substantial advantages for shelf-life increase of many food products including fruits and vegetables. The development of new natural edible films and coatings with the addition of antimicrobial compounds to preserve fresh and minimally processed fruits and vegetables is a technological challenge for the industry and a very active research field worldwide. Antimicrobial agents have been successfully added to edible composite films and coatings based on polysaccharides or proteins such as starch, cellulose derivatives, chitosan, alginate, fruit puree, whey protein isolated, soy protein, egg albumen, wheat gluten, or sodium caseinate. This paper reviews the development of edible films and coatings with antimicrobial activity, typically through the incorporation of antimicrobial food additives as ingredients, the effect of these edible films on the control of target microorganisms, the influence of antimicrobial agents on mechanical and barrier properties of stand-alone edible films, and the effect of the application of antimicrobial edible coatings on the quality of fresh and fresh-cut fruits and vegetables.

  1. Development of Bioactive Edible Coatings and Biodegradable Packaging Using Gamma Irradiation

    International Nuclear Information System (INIS)

    Lacroix, M.; Salmieri, S.

    2010-01-01

    Gamma irradiation was used to cross-link milk proteins in order to enhance the physico-chemical properties of edible films made of calcium caseinate, whey protein isolate and glycerol. Fourier Transform Infrared analysis was used to characterize the conformation of proteins adopted after irradiation. The molecular weight of cross-linked proteins was measured by Size-Exclusion Chromatography. Furthermore, the effect of the addition of methylcellulose to the irradiated protein matrix on the rheological properties (puncture strength, puncture deformation and water vapor permeability) of films was also studied. Moreover, cross-linking of polysaccharides under paste-like state was investigated and the cross-linking degree of the gel products was determined by gel fraction measurements and solubility percentage. In order to prepare bioactive coatings, several antifungal compounds were evaluated as bioactive compounds in order to select one of them to prepare an antimicrobial solution to spray onto strawberries or to encapsulate them in film formulations composed of milk proteins and methylcellulose based films. In addition, the bioactive coatings containing the antifungals were used to increase the radiosensitivity under air of moulds and total flora in strawberries and the relative sensitivity of selected formulations was calculated from their D10 value. The film formulation selected was used as a bioactive edible coating in order to determine their efficiency to increase the shelf life of fresh strawberries and to preserve their quality during storage. (author)

  2. Biomimetic apatite-coated porous PVA scaffolds promote the growth of breast cancer cells

    International Nuclear Information System (INIS)

    Ye, Mao; Mohanty, Pravansu; Ghosh, Gargi

    2014-01-01

    Recapitulating the native environment of bone tissue is essential to develop in vitro models of breast cancer bone metastasis. The bone is a composite material consisting of organic matrix and inorganic mineral phase, primarily hydroxyapatite. In this study, we report the mineralization of porous poly vinyl alcohol (PVA) scaffolds upon incubation in modified Hanks' Balanced Salt Solution (HBSS) for 14 days. Scanning electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction analysis revealed that the deposited minerals have composition similar to hydroxyapatite. The study demonstrated that the rate of nucleation and growth of minerals was faster on surfaces of less porous scaffolds. However, upon prolonged incubation, formation of mineral layer was observed on the surface of all the scaffolds. In addition, the study also demonstrated that 3D mineralization only occurred for scaffolds with highly interconnected porous networks. The mineralization of the scaffolds promoted the adsorption of serum proteins and consequently, the adhesion and proliferation of breast cancer cells. - Highlights: • Porous PVA scaffolds fabricated via mechanical agitation followed by freeze-drying. • Mineralization of the scaffold was carried out by utilizing biomimetic approach. • Mineralization resulted in increased protein adsorption on the scaffold. • Increased breast cancer cell growth was observed on mineralized scaffolds

  3. Biomimetic apatite-coated porous PVA scaffolds promote the growth of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Mao; Mohanty, Pravansu; Ghosh, Gargi, E-mail: gargi@umich.edu

    2014-11-01

    Recapitulating the native environment of bone tissue is essential to develop in vitro models of breast cancer bone metastasis. The bone is a composite material consisting of organic matrix and inorganic mineral phase, primarily hydroxyapatite. In this study, we report the mineralization of porous poly vinyl alcohol (PVA) scaffolds upon incubation in modified Hanks' Balanced Salt Solution (HBSS) for 14 days. Scanning electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction analysis revealed that the deposited minerals have composition similar to hydroxyapatite. The study demonstrated that the rate of nucleation and growth of minerals was faster on surfaces of less porous scaffolds. However, upon prolonged incubation, formation of mineral layer was observed on the surface of all the scaffolds. In addition, the study also demonstrated that 3D mineralization only occurred for scaffolds with highly interconnected porous networks. The mineralization of the scaffolds promoted the adsorption of serum proteins and consequently, the adhesion and proliferation of breast cancer cells. - Highlights: • Porous PVA scaffolds fabricated via mechanical agitation followed by freeze-drying. • Mineralization of the scaffold was carried out by utilizing biomimetic approach. • Mineralization resulted in increased protein adsorption on the scaffold. • Increased breast cancer cell growth was observed on mineralized scaffolds.

  4. Pectin-honey coating as novel dehydrating bioactive agent for cut fruit: Enhancement of the functional properties of coated dried fruits.

    Science.gov (United States)

    Santagata, Gabriella; Mallardo, Salvatore; Fasulo, Gabriella; Lavermicocca, Paola; Valerio, Francesca; Di Biase, Mariaelena; Di Stasio, Michele; Malinconico, Mario; Volpe, Maria Grazia

    2018-08-30

    In this paper, a novel and sustainable process for the fruit dehydration was described. Specifically, edible coatings based on pectin and honey were prepared and used as dehydrating and antimicrobial agents of cut fruit samples, in this way promoting the fruit preservation from irreversible deteriorative processes. Pectin-honey coating was tested on apple, cantaloupe melon, mango and pineapple. The analysis were performed also on uncoated dehydrated fruits (control). The coated fruit evidenced enhanced dehydration percentage, enriched polyphenol and vitamin C contents, improved antioxidant activity and volatile molecules profile. Moreover, the antimicrobial activity against Pseudomonas and Escherichia coli was assessed. Finally, morphological analysis performed on fruit fractured surface, highlighted the formation of a non-sticky and homogeneous thin layer. These outcomes suggested that the novel fruit dehydration process, performed by using pectin-honey coating, was able to both preserve the safety and quality of dehydrated fruits, and enhance their authenticity and naturalness. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

    Science.gov (United States)

    Fire, E; Zwart, D E; Roth, M G; Henis, Y I

    1991-12-01

    Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543

  6. A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution

    Directory of Open Access Journals (Sweden)

    Ramesh K. Jha

    2018-06-01

    Full Text Available Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators. Keywords: Whole cell biosensor, Aromatic catabolism, Transcription factor, PcaU, Shikimate

  7. Genetic diversity of the movement and coat protein genes of South American isolates of Prunus necrotic ringspot virus.

    Science.gov (United States)

    Fiore, Nicola; Fajardo, Thor V M; Prodan, Simona; Herranz, María Carmen; Aparicio, Frederic; Montealegre, Jaime; Elena, Santiago F; Pallás, Vicente; Sánchez-Navarro, Jesús

    2008-01-01

    Prunus necrotic ringspot virus (PNRSV) is distributed worldwide, but no molecular data have been previously reported from South American isolates. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of 23 isolates of PNRSV from Chile, Brazil, and Uruguay, and from different Prunus species, have been obtained. Phylogenetic analysis performed with full-length MP and CP sequences from all the PNRSV isolates confirmed the clustering of the isolates into the previously reported PV32-I, PV96-II and PE5-III phylogroups. No association was found between specific sequences and host, geographic origin or symptomatology. Comparative analysis showed that both MP and CP have phylogroup-specific amino acids and all of the motifs previously characterized for both proteins. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that most amino acid sites are under the effect of negative purifying selection.

  8. Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy.

    Science.gov (United States)

    Bengali, Aditya N; Tessier, Peter M

    2009-10-01

    "Reversible" protein interactions govern diverse biological behavior ranging from intracellular transport and toxic protein aggregation to protein crystallization and inactivation of protein therapeutics. Much less is known about weak protein interactions than their stronger counterparts since they are difficult to characterize, especially in a parallel format (in contrast to a sequential format) necessary for high-throughput screening. We have recently introduced a highly efficient approach of characterizing protein self-association, namely self-interaction nanoparticle spectroscopy (SINS; Tessier et al., 2008; J Am Chem Soc 130:3106-3112). This approach exploits the separation-dependent optical properties of gold nanoparticles to detect weak self-interactions between proteins immobilized on nanoparticles. A limitation of our previous work is that differences in the sequence and structure of proteins can lead to significant differences in their affinity to adsorb to nanoparticle surfaces, which complicates analysis of the corresponding protein self-association behavior. In this work we demonstrate a highly specific approach for coating nanoparticles with proteins using biotin-avidin interactions to generate protein-nanoparticle conjugates that report protein self-interactions through changes in their optical properties. Using lysozyme as a model protein that is refractory to characterization by conventional SINS, we demonstrate that surface Plasmon wavelengths for gold-avidin-lysozyme conjugates over a range of solution conditions (i.e., pH and ionic strength) are well correlated with lysozyme osmotic second virial coefficient measurements. Since SINS requires orders of magnitude less protein and time than conventional methods (e.g., static light scattering), we envision this approach will find application in large screens of protein self-association aimed at either preventing (e.g., protein aggregation) or promoting (e.g., protein crystallization) these

  9. Enhancing physicochemical properties of emulsions by heteroaggregation of oppositely charged lactoferrin coated lutein droplets and whey protein isolate coated DHA droplets.

    Science.gov (United States)

    Li, Xin; Wang, Xu; Xu, Duoxia; Cao, Yanping; Wang, Shaojia; Wang, Bei; Sun, Baoguo; Yuan, Fang; Gao, Yanxiang

    2018-01-15

    The formation and physicochemical stability of mixed functional components (lutein & DHA) emulsions through heteroaggregation were studied. It was formed by controlled heteroaggregation of oppositely charged lutein and DHA droplets coated by cationic lactoferrin (LF) and anionic whey protein isolate (WPI), respectively. Heteroaggregation was induced by mixing the oppositely charged LF-lutein and WPI-DHA emulsions together at pH 6.0. Droplet size, zeta-potential, transmission-physical stability, microrheological behavior and microstructure of the heteroaggregates formed were measured as a function of LF-lutein to WPI-DHA droplet ratio. Lutein degradation and DHA oxidation by measurement of lipid hydroperoxides and thiobarbituric acid reactive substances were determined. Upon mixing the two types of bioactive compounds droplets together, it was found that the largest aggregates and highest physical stability occurred at a droplet ratio of 40% LF-lutein droplets to 60% WPI-DHA droplets. Heteroaggregates formation altered the microrheological properties of the mixed emulsions mainly by the special network structure of the droplets. When LF-coated lutein droplets ratios were more than 30% and less than 60%, the mixed emulsions exhibited distinct decreases in the Mean Square Displacement, which indicated that their limited scope of Brownian motion and stable structure. Mixed emulsions with LF-lutein/WPI-DHA droplets ratio of 4:6 exhibited Macroscopic Viscosity Index with 13 times and Elasticity Index with 3 times of magnitudes higher than the individual emulsions from which they were prepared. Compared with the WPI-DHA emulsion or LF-lutein emulsion, the oxidative stability of the heteroaggregate of LF-lutein/WPI-DHA emulsions was improved. Heteroaggregates formed by oppositely charged bioactive compounds droplets may be useful for creating specific food structures that lead to desirable physicochemical properties, such as microrheological property, physical and chemical

  10. Micro patterning of cell and protein non-adhesive plasma polymerized coatings for biochip applications

    DEFF Research Database (Denmark)

    Bouaidat, Salim; Berendsen, C.; Thomsen, P.

    2004-01-01

    Micro scale patterning of bioactive surfaces is desirable for numerous biochip applications. Polyethyleneoxide-like (PEO-like) coating with non-fouling functionality has been deposited using low frequency AC plasma polymerization. The non-fouling properties of the coating were tested with human c...... and versatility of the plasma-polymerized coatings, make this technology highly suitable for bio-MEMS and biochip applications, where patterned high contrast non-fouling surfaces are needed....

  11. Direct inhibition of TNF-α promoter activity by Fanconi anemia protein FANCD2.

    Directory of Open Access Journals (Sweden)

    Nobuko Matsushita

    Full Text Available Fanconi anemia (FA, an inherited disease, is associated with progressive bone marrow failure, predisposition to cancer, and genomic instability. Genes corresponding to 15 identified FA complementation groups have been cloned, and each gene product functions in the response to DNA damage induced by cross-linking agents and/or in protection against genome instability. Interestingly, overproduction of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α and aberrant activation of NF-κB-dependent transcriptional activity have been observed in FA cells. Here we demonstrated that FANCD2 protein inhibits NF-κB activity in its monoubiquitination-dependent manner. Furthermore, we detected a specific association between FANCD2 and an NF-κB consensus element in the TNF-α promoter by electrophoretic mobility shift assays (EMSA and chromatin immunoprecipitation (ChIP assay. Therefore, we propose FANCD2 deficiency promotes transcriptional activity of the TNF-α promoter and induces overproduction of TNF-which then sustains prolonged inflammatory responses. These results also suggest that artificial modulation of TNFα production could be a promising therapeutic approach to FA.

  12. Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

    Science.gov (United States)

    Aagaard, Jan E.; Yi, Xianhua; MacCoss, Michael J.; Swanson, Willie J.

    2006-01-01

    Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins. PMID:17085584

  13. Microwave plasma deposition of diamond like carbon coatings

    Indian Academy of Sciences (India)

    Abstract. The promising applications of the microwave plasmas have been appearing in the fields of chemical processes and semiconductor manufacturing. Applications include surface deposition of all types including diamond/diamond like carbon (DLC) coatings, etching of semiconductors, promotion of organic reactions, ...

  14. Sensory Quality Preservation of Coated Walnuts.

    Science.gov (United States)

    Grosso, Antonella L; Asensio, Claudia M; Grosso, Nelson R; Nepote, Valeria

    2017-01-01

    The objective of this study was to evaluate the sensory stability of coated walnuts during storage. Four walnut samples were prepared: uncoated (NC), and samples coated with carboxymethyl cellulose (NCMC), methyl cellulose (NMC), or whey protein (NPS). The samples were stored at room temperature for 210 d and were periodically removed from storage to perform a sensory descriptive analysis. A consumer acceptance test was carried out on the fresh product (storage day 0) to evaluate flavor. All samples exhibited significant differences in their sensory attributes initially and after storage. Intensity ratings for oxidized and cardboard flavors increased during storage. NC showed the highest oxidized and cardboard intensity ratings (39 and 22, respectively) and NMC exhibited the lowest intensity ratings for these negative attributes (8 and 17, respectively) after 210 d of storage. Alternatively, the intensity ratings for sweetness and walnut flavors were decreased for all samples. NMC had the lowest decrease at the end of storage for these positive attributes (75.86 in walnut flavor and 12.09 in sweetness). The results of this study suggest a protective effect of the use of an edible coating to preserve sensory attributes during storage, especially for samples coated with MC. The results of the acceptance test showed that addition of the coating negatively affected the flavor acceptance for NMC and NCMC coated walnuts. Edible coatings help to preserve sensory attributes in walnuts, improving their shelf-life, however, these coatings may affect consumer acceptance in some cases. © 2016 Institute of Food Technologists®.

  15. The PsB glycoprotein complex is secreted as a preassembled precursor of the spore coat in Dictyostelium discoideum.

    Science.gov (United States)

    Watson, N; McGuire, V; Alexander, S

    1994-09-01

    The PsB glycoprotein in Dictyostelium discoideum is one of a diverse group of developmentally regulated, prespore-cell-specific proteins, that contain a common O-linked oligosaccharide. This post-translational modification is dependent on the wild-type modB allele. The PsB protein exists as part of a multiprotein complex of six different proteins, which have different post-translational modifications and are held together by both covalent and non-covalent interactions (Watson et al. (1993). J. Biol. Chem. 268, 22634-22641). In this study we have used microscopic and biochemical analyses to examine the cellular localization and function of the PsB complex during development. We found that the PsB complex first accumulates in prespore vesicles in slug cells and is secreted later during culmination and becomes localized to both the extracellular matrix of the apical spore mass of mature fruiting bodies and to the inner layer of the spore coat. The PsB associated with the spore coat is covalently bound by disulfide bridges. The PsB protein always exists in a multiprotein complex, but the composition of the PsB complex changes during secretion and spore maturation. Some of the PsB complex proteins have been identified as spore coat proteins. These data demonstrate that some of the proteins that form the spore coat exist as a preassembled precursor complex. The PsB complex is secreted in a developmentally regulated manner during the process of spore differentiation, at which time proteins of the complex, as well as additional spore coat proteins, become covalently associated in at least two forms of extracellular matrix: the interspore matrix and the spore coat. These and other studies show that proteins with modB dependent O-linked oligosaccharides are involved in a wide variety of processes underlying morphogenesis in this organism. These developmental processes are the direct result of cellular mechanisms regulating protein targeting, assembly and secretion, and the

  16. Investigation of hydrogen evolution activity for the nickel, nickel-molybdenum nickel-graphite composite and nickel-reduced graphene oxide composite coatings

    International Nuclear Information System (INIS)

    Jinlong, Lv; Tongxiang, Liang; Chen, Wang

    2016-01-01

    Graphical abstract: - Highlights: • Improved HER efficiency of Ni-Mo coatings was attributed to ‘cauliflower’ like microstructure. • RGO in nickel-RGO composite coating promoted refined grain and facilitated HER. • Synergistic effect between nickel and RGO facilitated HER due to large specific surface of RGO. - Abstract: The nickel, nickel-molybdenum alloy, nickel-graphite and nickel-reduced graphene oxide composite coatings were obtained by the electrodeposition technique from a nickel sulfate bath. Nanocrystalline molybdenum, graphite and reduced graphene oxide in nickel coatings promoted hydrogen evolution reaction in 0.5 M H_2SO_4 solution at room temperature. However, the nickel-reduced graphene oxide composite coating exhibited the highest electrocatalytic activity for the hydrogen evolution reaction in 0.5 M H_2SO_4 solution at room temperature. A large number of gaps between ‘cauliflower’ like grains could decrease effective area for hydrogen evolution reaction in slight amorphous nickel-molybdenum alloy. The synergistic effect between nickel and reduced graphene oxide promoted hydrogen evolution, moreover, refined grain in nickel-reduced graphene oxide composite coating and large specific surface of reduced graphene oxide also facilitated hydrogen evolution reaction.

  17. Investigation of hydrogen evolution activity for the nickel, nickel-molybdenum nickel-graphite composite and nickel-reduced graphene oxide composite coatings

    Energy Technology Data Exchange (ETDEWEB)

    Jinlong, Lv, E-mail: ljlbuaa@126.com [Beijing Key Laboratory of Fine Ceramics, Institute of Nuclear and New Energy Technology, Tsinghua University, Zhongguancun Street, Haidian District, Beijing 100084 (China); State Key Lab of New Ceramic and Fine Processing, Tsinghua University, Beijing 100084 (China); Tongxiang, Liang; Chen, Wang [Beijing Key Laboratory of Fine Ceramics, Institute of Nuclear and New Energy Technology, Tsinghua University, Zhongguancun Street, Haidian District, Beijing 100084 (China); State Key Lab of New Ceramic and Fine Processing, Tsinghua University, Beijing 100084 (China)

    2016-03-15

    Graphical abstract: - Highlights: • Improved HER efficiency of Ni-Mo coatings was attributed to ‘cauliflower’ like microstructure. • RGO in nickel-RGO composite coating promoted refined grain and facilitated HER. • Synergistic effect between nickel and RGO facilitated HER due to large specific surface of RGO. - Abstract: The nickel, nickel-molybdenum alloy, nickel-graphite and nickel-reduced graphene oxide composite coatings were obtained by the electrodeposition technique from a nickel sulfate bath. Nanocrystalline molybdenum, graphite and reduced graphene oxide in nickel coatings promoted hydrogen evolution reaction in 0.5 M H{sub 2}SO{sub 4} solution at room temperature. However, the nickel-reduced graphene oxide composite coating exhibited the highest electrocatalytic activity for the hydrogen evolution reaction in 0.5 M H{sub 2}SO{sub 4} solution at room temperature. A large number of gaps between ‘cauliflower’ like grains could decrease effective area for hydrogen evolution reaction in slight amorphous nickel-molybdenum alloy. The synergistic effect between nickel and reduced graphene oxide promoted hydrogen evolution, moreover, refined grain in nickel-reduced graphene oxide composite coating and large specific surface of reduced graphene oxide also facilitated hydrogen evolution reaction.

  18. Accelerated healing of cardiovascular textiles promoted by an RGD peptide.

    Science.gov (United States)

    Tweden, K S; Harasaki, H; Jones, M; Blevitt, J M; Craig, W S; Pierschbacher, M; Helmus, M N

    1995-07-01

    Polytetrafluoroethylene (PTFE) and polyethylene terephthalate (Dacron polyester) fabrics are used extensively in cardiovascular devices, e.g. heart valve sewing cuffs and vascular prostheses. While devices containing these fabrics are generally successful, it is recognized that fabrics cause complications prior to tissue ingrowth due to their thrombogenic nature. A surface active synthetic peptide, called PepTite Coating (PepTite), which was modeled after the cell attachment domain of human fibronectin has been marketed as a biocompatible coating. This peptide stimulates cell attachment through the arginine-glycine-aspartic acid (RGD) sequence. Modification of medical implants with PepTite has been shown to promote ingrowth of surrounding cells into the material leading to better tissue integration, reduced inflammation and reduced fibrotic encapsulation. In this study, polyester and PTFE textiles were modified with PepTite. The effectiveness of this coating in enhancing wound healing was investigated in a simple vascular and cardiac valve model. Our results indicate that the RGD-containing peptide, PepTite, promoted the formation of an endothelial-like cell layer on both polyester and PTFE vascular patches in the dog model. PepTite was also found to promote the formation of a significantly thinner neointima (pannus) on polyester as compared to that on its uncoated control. These results were corroborated in the cardiac valve model in which a greater amount of thin pannus and less thrombus were seen on coated polyester sewing cuffs than on control uncoated cuffs. This research shows the promising tissue response to RGD coated textiles and the potential role of this peptide in material passivation via accelerated healing.

  19. Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

    Science.gov (United States)

    Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

  20. Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

    Science.gov (United States)

    Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

    2002-08-01

    Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

  1. 9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

    Science.gov (United States)

    Dodonova, Svetlana O; Aderhold, Patrick; Kopp, Juergen; Ganeva, Iva; Röhling, Simone; Hagen, Wim J H; Sinning, Irmgard; Wieland, Felix; Briggs, John A G

    2017-06-16

    COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.

  2. Side-chain dynamics of a detergent-solubilized membrane protein: Measurement of tryptophan and glutamine hydrogen-exchange rates in M13 coat protein by 1H NMR spectroscopy

    International Nuclear Information System (INIS)

    O'Neil, J.D.J.; Sykes, B.D.

    1989-01-01

    M13 coat protein is a small (50 amino acids) lipid-soluble protein that becomes an integral membrane protein during the infection stage of the life cycle of the M13 phage and is therefore used as a model membrane protein. To study side-chain dynamics in the protein, the authors have measured individual hydrogen-exchange rates for a primary amide in the side chain of glutamine-15 and for the indole amine of tryptophan-26. The protein was solubilized with the use of perdeuteriated sodium dodecyl sulfate (SDS), and hydrogen-exchange rates were measured by using 1 H nuclear magnetic resonance spectroscopy. The glutamine-15 syn proton exchanged at a rate identical with that in glutamine model peptides except that the pH corresponding to minimum exchange was elevated by about 1.5 pH units. The tryptophan-26 indole amine proton exchange was biphasic, suggesting that two populations of tryptophan-26 exist. It is suggested that the two populations may reflect protein dimerization or aggregation in the SDS micelles. The pH values of minimum exchange for tryptophan-26 in both environments were also elevated by 1.3-1.9 pH units. This phenomenon is reproduced when small tryptophan- and glutamine-containing hydrophobic peptides are dissolved in the presence of SDS micelles. The electrostatic nature of this phenomenon is proven by showing that the minimum pH for exchange can be reduced by dissolving the hydrophobic peptides in the positively charged detergent micelle dodecyltrimethylammonium bromide

  3. Properties of DLC coatings deposited by dc and dc with superimposed pulsed vacuum arc

    International Nuclear Information System (INIS)

    Zavaleyev, V.; Walkowicz, J.; Aksyonov, D.S.; Luchaninov, A.A.; Reshetnyak, E.N.; Strel'nitskij, V.E.

    2014-01-01

    Comparative studies of the structure, mechanical and tribological properties of DLC coatings deposited in DC and DC with superimposed high current pulse modes of operation vacuum-arc plasma source with the graphite cathode are presented. Imposition the pulses of high current on DC vacuum-arc discharge allows both increase the deposition rate of DLC coating and reduce the residual compressive stress in the coatings what promotes substantial improvement the adhesion to the substrate. Effect of vacuum arc plasma filtration with Venetian blind filter on the deposition rate and tribological characteristics of the coatings analyzed.

  4. The effect of layer-by-layer chitosan-hyaluronic acid coating on graft-to-bone healing of a poly(ethylene terephthalate) artificial ligament.

    Science.gov (United States)

    Li, Hong; Ge, Yunsheng; Zhang, Pengyun; Wu, Lingxiang; Chen, Shiyi

    2012-01-01

    Surface coating with an organic layer-by-layer self-assembled template of chitosan and hyaluronic acid on a poly(ethylene terephthalate) (PET) artificial ligament was designed for the promotion and enhancement of graft-to-bone healing after artificial ligament implantation in a bone tunnel. The results of in vitro culturing of MC3T3-E1 mouse osteoblastic cells supported the hypothesis that the layer-by-layer coating of chitosan and hyaluronic acid could promote the cell compatibility of grafts and could promote osteoblast proliferation. A rabbit extra-articular tendon-to-bone healing model was used to evaluate the effect of this kind of surface-modified stainless artificial ligament in vivo. The final results proved that this organic compound coating could significantly promote and enhance new bone formation at the graft-bone interface histologically and, correspondingly, the experimental group with coating had significantly higher biomechanical properties compared with controls at 8 weeks (P < 0.05).

  5. Biological Response of Human Bone Marrow-Derived Mesenchymal Stem Cells to Commercial Tantalum Coatings with Microscale and Nanoscale Surface Topographies

    Science.gov (United States)

    Skoog, Shelby A.; Kumar, Girish; Goering, Peter L.; Williams, Brian; Stiglich, Jack; Narayan, Roger J.

    2016-06-01

    Tantalum is a promising orthopaedic implant coating material due to its robust mechanical properties, corrosion resistance, and excellent biocompatibility. Previous studies have demonstrated improved biocompatibility and tissue integration of surface-treated tantalum coatings compared to untreated tantalum. Surface modification of tantalum coatings with biologically inspired microscale and nanoscale features may be used to evoke optimal tissue responses. The goal of this study was to evaluate commercial tantalum coatings with nanoscale, sub-microscale, and microscale surface topographies for orthopaedic and dental applications using human bone marrow-derived mesenchymal stem cells (hBMSCs). Tantalum coatings with different microscale and nanoscale surface topographies were fabricated using a diffusion process or chemical vapor deposition. Biological evaluation of the tantalum coatings using hBMSCs showed that tantalum coatings promote cellular adhesion and growth. Furthermore, hBMSC adhesion to the tantalum coatings was dependent on surface feature characteristics, with enhanced cell adhesion on sub-micrometer- and micrometer-sized surface topographies compared to hybrid nano-/microstructures. Nanostructured and microstructured tantalum coatings should be further evaluated to optimize the surface coating features to promote osteogenesis and enhance osseointegration of tantalum-based orthopaedic implants.

  6. IQ-domain GTPase-activating protein 1 promotes the malignant phenotype of invasive ductal breast carcinoma via canonical Wnt pathway.

    Science.gov (United States)

    Zhao, Huan-Yu; Han, Yang; Wang, Jian; Yang, Lian-He; Zheng, Xiao-Ying; Du, Jiang; Wu, Guang-Ping; Wang, En-Hua

    2017-06-01

    IQ-domain GTPase-activating protein 1 is a scaffolding protein with multidomain which plays a role in modulating dishevelled (Dvl) nuclear translocation in canonical Wnt pathway. However, the biological function and mechanism of IQ-domain GTPase-activating protein 1 in invasive ductal carcinoma (IDC) remain unknown. In this study, we found that IQ-domain GTPase-activating protein 1 expression was elevated in invasive ductal carcinoma, which was positively correlated with tumor grade, lymphatic metastasis, and poor prognosis. Coexpression of IQ-domain GTPase-activating protein 1 and Dvl in the nucleus and cytoplasm of invasive ductal carcinoma was significantly correlated but not in the membrane. Postoperative survival in the patients with their coexpression in the nucleus and cytoplasm was obviously lower than that without coexpression. The positive expression rates of c-myc and cyclin D1 were significantly higher in the patients with nuclear coexpression of Dvl and IQ-domain GTPase-activating protein 1 than that with cytoplasmic coexpression, correlating with poor prognosis. IQ-domain GTPase-activating protein 1 significantly enhanced cell proliferation and invasion in invasive ductal carcinoma cell lines by interacting with Dvl in cytoplasm to promote Dvl nuclear translocation so as to upregulate the expression of c-myc and cyclin D1. Collectively, our data suggest that IQ-domain GTPase-activating protein 1 may promote the malignant phenotype of invasive ductal carcinoma via canonical Wnt signaling, and it could be used as a potential prognostic biomarker for breast cancer patients.

  7. Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry.

    Science.gov (United States)

    van Andel, Esther; de Bus, Ian; Tijhaar, Edwin J; Smulders, Maarten M J; Savelkoul, Huub F J; Zuilhof, Han

    2017-11-08

    Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.

  8. Invited review: Whey proteins as antioxidants and promoters of cellular antioxidant pathways.

    Science.gov (United States)

    Corrochano, Alberto R; Buckin, Vitaly; Kelly, Phil M; Giblin, Linda

    2018-03-28

    Oxidative stress contributes to cell injury and aggravates several chronic diseases. Dietary antioxidants help the body to fight against free radicals and, therefore, avoid or reduce oxidative stress. Recently, proteins from milk whey liquid have been described as antioxidants. This review summarizes the evidence that whey products exhibit radical scavenging activity and reducing power. It examines the processing and treatment attempts to increase the antioxidant bioactivity and identifies 1 enzyme, subtilisin, which consistently produces the most potent whey fractions. The review compares whey from different milk sources and puts whey proteins in the context of other known food antioxidants. However, for efficacy, the antioxidant activity of whey proteins must not only survive processing, but also upper gut transit and arrival in the bloodstream, if whey products are to promote antioxidant levels in target organs. Studies reveal that direct cell exposure to whey samples increases intracellular antioxidants such as glutathione. However, the physiological relevance of these in vitro assays is questionable, and evidence is conflicting from dietary intervention trials, with both rats and humans, that whey products can boost cellular antioxidant biomarkers. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  9. Microwave assisted coating of bioactive amorphous magnesium phosphate (AMP) on polyetheretherketone (PEEK).

    Science.gov (United States)

    Ren, Yufu; Sikder, Prabaha; Lin, Boren; Bhaduri, Sarit B

    2018-04-01

    Polyetheretherketone (PEEK) with great thermal and chemical stability, desirable mechanical properties and promising biocompatibility is being widely used as orthopedic and dental implant materials. However, the bioinert surface of PEEK can hinder direct osseointegration between the host tissue and PEEK based implants. The important signatures of this paper are as follows. First, we report for the formation of osseointegrable amorphous magnesium phosphate (AMP) coating on PEEK surface using microwave energy. Second, coatings consist of nano-sized AMP particles with a stacked thickness of 800nm. Third, coatings enhance bioactivity in-vitro and induce significantly high amount of bone-like apatite coating, when soaked in simulated body fluid (SBF). Fourth, the as-deposited AMP coatings present no cytotoxicity effects and are beneficial for cell adhesion at early stage. Finally, the high levels of expression of osteocalcin (OCN) in cells cultured on AMP coated PEEK samples indicate that AMP coatings can promote new bone formation and hence osseointegration. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Thermally joining and/or coating or thermally separating the workpieces having heat-sensitive coating, comprises restoring coating by thermally coating the coating material after thermally joining and/or coating or thermally separating

    OpenAIRE

    Riedel, Frank; Winkelmann, Ralf; Puschmann, Markus

    2011-01-01

    The method for thermally joining and/or coating or thermally separating the workpieces (1), which have a heat-sensitive coating (2), comprises restoring the coating by thermally coating a coating material (3) after thermally joining and/or coating or thermally separating the workpieces. A part of the thermal energy introduced in the workpiece for joining and/or coating or separating or in the workpieces is used for thermally coating the coating material. Two workpieces are welded or soldered ...

  11. Inhibition of Staphylococcus epidermidis Biofilm by Trimethylsilane Plasma Coating

    Science.gov (United States)

    Ma, Yibao; Jones, John E.; Ritts, Andrew C.; Yu, Qingsong

    2012-01-01

    Biofilm formation on implantable medical devices is a major impediment to the treatment of nosocomial infections and promotes local progressive tissue destruction. Staphylococcus epidermidis infections are the leading cause of biofilm formation on indwelling devices. Bacteria in biofilms are highly resistant to antibiotic treatment, which in combination with the increasing prevalence of antibiotic resistance among human pathogens further complicates treatment of biofilm-related device infections. We have developed a novel plasma coating technology. Trimethylsilane (TMS) was used as a monomer to coat the surfaces of 316L stainless steel and grade 5 titanium alloy, which are widely used in implantable medical devices. The results of biofilm assays demonstrated that this TMS coating markedly decreased S. epidermidis biofilm formation by inhibiting the attachment of bacterial cells to the TMS-coated surfaces during the early phase of biofilm development. We also discovered that bacterial cells on the TMS-coated surfaces were more susceptible to antibiotic treatment than their counterparts in biofilms on uncoated surfaces. These findings suggested that TMS coating could result in a surface that is resistant to biofilm development and also in a bacterial community that is more sensitive to antibiotic therapy than typical biofilms. PMID:22964248

  12. Glass frits coated with silver nanoparticles for silicon solar cells

    International Nuclear Information System (INIS)

    Li, Yingfen; Gan, Weiping; Zhou, Jian; Li, Biyuan

    2015-01-01

    Graphical abstract: - Highlights: • Silver-coated glass frits for solar cells were prepared by electroless plating. • Gum Arabic was used as the activating agent of glass frits. • Silver-coated glass frits can improve the photovoltaic performances of solar cells. - Abstract: Glass frits coated with silver nanoparticles were prepared by electroless plating. Gum Arabic (GA) was used as the activating agent of glass frits without the assistance of stannous chloride or palladium chloride. The silver-coated glass frits prepared with different GA dosages were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), and thermogravimetric analysis (TGA). The characterization results indicated that silver-coated glass frits had the structures of both glass and silver. Spherical silver nanoparticles were distributed on the glass frits evenly. The density and particle size of silver nanoparticles on the glass frits can be controlled by adjusting the GA dosage. The silver-coated glass frits were applied to silver pastes to act as both the densification promoter and silver crystallite formation aid in the silver electrodes. The prepared silver-coated glass frits can improve the photovoltaic performances of solar cells

  13. Glass frits coated with silver nanoparticles for silicon solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yingfen, E-mail: lyf350857423@163.com; Gan, Weiping; Zhou, Jian; Li, Biyuan

    2015-06-30

    Graphical abstract: - Highlights: • Silver-coated glass frits for solar cells were prepared by electroless plating. • Gum Arabic was used as the activating agent of glass frits. • Silver-coated glass frits can improve the photovoltaic performances of solar cells. - Abstract: Glass frits coated with silver nanoparticles were prepared by electroless plating. Gum Arabic (GA) was used as the activating agent of glass frits without the assistance of stannous chloride or palladium chloride. The silver-coated glass frits prepared with different GA dosages were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), and thermogravimetric analysis (TGA). The characterization results indicated that silver-coated glass frits had the structures of both glass and silver. Spherical silver nanoparticles were distributed on the glass frits evenly. The density and particle size of silver nanoparticles on the glass frits can be controlled by adjusting the GA dosage. The silver-coated glass frits were applied to silver pastes to act as both the densification promoter and silver crystallite formation aid in the silver electrodes. The prepared silver-coated glass frits can improve the photovoltaic performances of solar cells.

  14. Role of protein and amino acids in promoting lean mass accretion with resistance exercise and attenuating lean mass loss during energy deficit in humans.

    Science.gov (United States)

    Churchward-Venne, Tyler A; Murphy, Caoileann H; Longland, Thomas M; Phillips, Stuart M

    2013-08-01

    Amino acids are major nutrient regulators of muscle protein turnover. After protein ingestion, hyperaminoacidemia stimulates increased rates of skeletal muscle protein synthesis, suppresses muscle protein breakdown, and promotes net muscle protein accretion for several hours. These acute observations form the basis for strategized protein intake to promote lean mass accretion, or prevent lean mass loss over the long term. However, factors such as protein dose, protein source, and timing of intake are important in mediating the anabolic effects of amino acids on skeletal muscle and must be considered within the context of evaluating the reported efficacy of long-term studies investigating protein supplementation as part of a dietary strategy to promote lean mass accretion and/or prevent lean mass loss. Current research suggests that dietary protein supplementation can augment resistance exercise-mediated gains in skeletal muscle mass and strength and can preserve skeletal muscle mass during periods of diet-induced energy restriction. Perhaps less appreciated, protein supplementation can augment resistance training-mediated gains in skeletal muscle mass even in individuals habitually consuming 'adequate' (i.e., >0.8 g kg⁻¹ day⁻¹) protein. Additionally, overfeeding energy with moderate to high-protein intake (15-25 % protein or 1.8-3.0 g kg⁻¹ day⁻¹) is associated with lean, but not fat mass accretion, when compared to overfeeding energy with low protein intake (5 % protein or ~0.68 g kg⁻¹ day⁻¹). Amino acids represent primary nutrient regulators of skeletal muscle anabolism, capable of enhancing lean mass accretion with resistance exercise and attenuating the loss of lean mass during periods of energy deficit, although factors such as protein dose, protein source, and timing of intake are likely important in mediating these effects.

  15. Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA.

    Science.gov (United States)

    Neeleman, L; Olsthoorn, R C; Linthorst, H J; Bol, J F

    2001-12-04

    On entering a host cell, positive-strand RNA virus genomes have to serve as messenger for the translation of viral proteins. Efficient translation of cellular messengers requires interactions between initiation factors bound to the 5'-cap structure and the poly(A) binding protein bound to the 3'-poly(A) tail. Initiation of infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV) and viruses from the genus Ilarvirus requires binding of a few molecules of coat protein (CP) to the 3' end of the nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs can be initiated by addition of the subgenomic messenger for CP, RNA 4. We report here that extension of the AMV RNAs with a poly(A) tail of 40 to 80 A-residues permitted initiation of infection independently of CP or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1 relieved an apparent bottleneck in the translation of the viral RNAs. Translation of RNA 4 in plant protoplasts was autocatalytically stimulated by its encoded CP. Mutations that interfered with CP binding to the 3' end of viral RNAs reduced translation of RNA 4 to undetectable levels. Possibly, CP of AMV and ilarviruses stimulates translation of viral RNAs by acting as a functional analogue of poly(A) binding protein or other cellular proteins.

  16. Frictional characteristics of erythrocytes on coated glass plates subject to inclined centrifugal forces.

    Science.gov (United States)

    Kandori, Takashi; Hayase, Toshiyuki; Inoue, Kousuke; Funamoto, Kenichi; Takeno, Takanori; Ohta, Makoto; Takeda, Motohiro; Shirai, Atsushi

    2008-10-01

    In recent years a diamond-like carbon (DLC) film and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer have attracted attention as coating materials for implantable artificial organs or devices. When these materials are coated on vascular devices, compatibility to blood is an important problem. The present paper focuses on friction characteristics of erythrocytes to these coating materials in a medium. With an inclined centrifuge microscope developed by the authors, observation was made for erythrocytes moving on flat glass plates with and without coating in a medium of plasma or saline under the effect of inclined centrifugal force. Friction characteristics of erythrocytes with respect to these coating materials were then measured and compared to each other to characterize DLC and MPC as coating materials. The friction characteristics of erythrocytes in plasma using the DLC-coated and noncoated glass plates are similar, changing approximately proportional to the 0.5th power of the cell velocity. The cells stick to these plates in saline as well, implying the influence of plasma protein. The results using the MPC-coated plate in plasma are similar to those of the other plates for large cell velocities, but deviate from the other results with decreased cell velocity. The results change nearly proportional to the 0.75th power of the cell velocity in the range of small velocities. The results for the MPC-coated plate in saline are similar to that in plasma but somewhat smaller, implying that the friction characteristics for the MPC-coated plate are essentially independent of plasma protein.

  17. Plastic Deformation Induced by Nanoindentation Test Applied on ZrN/Si3N4 Multilayer Coatings

    Directory of Open Access Journals (Sweden)

    Zhengtao Wu

    2017-12-01

    Full Text Available ZrN/Si3N4 multilayer coating that alternates with either nanocrystalline ZrN or amorphous Si3N4 interlayers was fabricated by reactively magnetron sputtering in an Ar-N2 mixture atmosphere. The thicknesses of the nanocrystalline ZrN and the amorphous Si3N4 interlayers are ~12.5 and 2.5 nm, respectively. The ZrN/Si3N4 coating exhibits a promoted hardness of 28.6 ± 1.2 GPa when compared to the binary ZrN. Microstructure evolution just underneath the nanoindentation impression of the ZrN/Si3N4 multilayer coating has been investigated. The result indicates that both ZrN nanograin rotations and plastic flow of the Si3N4 interlayers contribute to the permanent deformation of the multilayer coating induced by the nanoindentation. In addition, the introduction of the a-Si3N4 interlayers hinders both the initiation and propagation of microcracks when the multilayer coating was applied to the scratch test. The propagation deflection of the microcracks was observed attributed to the heterogenous interface, which produces the hardness promotion of the multilayer coating eventually.

  18. Smad3 induces atrogin-1, inhibits mTOR and protein synthesis, and promotes muscle atrophy in vivo.

    Science.gov (United States)

    Goodman, Craig A; McNally, Rachel M; Hoffmann, F Michael; Hornberger, Troy A

    2013-11-01

    Myostatin, a member of the TGF superfamily, is sufficient to induce skeletal muscle atrophy. Myostatin-induced atrophy is associated with increases in E3-ligase atrogin-1 expression and protein degradation and decreases in Akt/mechanistic target of rapamycin (mTOR) signaling and protein synthesis. Myostatin signaling activates the transcription factor Smad3 (Small Mothers Against Decapentaplegic), which has been shown to be necessary for myostatin-induced atrogin-1 expression and atrophy; however, it is not known whether Smad3 is sufficient to induce these events or whether Smad3 simply plays a permissive role. Thus, the aim of this study was to address these questions with an in vivo model. To accomplish this goal, in vivo transfection of plasmid DNA was used to create transient transgenic mouse skeletal muscles, and our results show for the first time that Smad3 expression is sufficient to stimulate atrogin-1 promoter activity, inhibit Akt/mTOR signaling and protein synthesis, and induce muscle fiber atrophy. Moreover, we propose that Akt/mTOR signaling is inhibited by a Smad3-induced decrease in microRNA-29 (miR-29) expression and a subsequent increase in the translation of phosphatase and tensin homolog (PTEN) mRNA. Smad3 is also sufficient to inhibit peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) promoter activity and to increase FoxO (Forkhead Box Protein, Subclass O)-mediated signaling and the promoter activity of plasminogen activator inhibitor 1 (PAI-1). Combined, this study provides the first evidence that Smad3 is sufficient to regulate many of the events associated with myostatin-induced atrophy and therefore suggests that Smad3 signaling may be a viable target for therapies aimed at preventing myostatin-induced muscle atrophy.

  19. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    Science.gov (United States)

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. TMV nanorods with programmed longitudinal domains of differently addressable coat proteins

    Science.gov (United States)

    Geiger, Fania C.; Eber, Fabian J.; Eiben, Sabine; Mueller, Anna; Jeske, Holger; Spatz, Joachim P.; Wege, Christina

    2013-04-01

    The spacing of functional nanoscopic elements may play a fundamental role in nanotechnological and biomedical applications, but is so far rarely achieved on this scale. In this study we show that tobacco mosaic virus (TMV) and the RNA-guided self-assembly process of its coat protein (CP) can be used to establish new nanorod scaffolds that can be loaded not only with homogeneously distributed functionalities, but with distinct molecule species grouped and ordered along the longitudinal axis. The arrangement of the resulting domains and final carrier rod length both were governed by RNA-templated two-step in vitro assembly. Two selectively addressable TMV CP mutants carrying either thiol (TMVCys) or amino (TMVLys) groups on the exposed surface were engineered and shown to retain reactivity towards maleimides or NHS esters, respectively, after acetic acid-based purification and re-assembly to novel carrier rod types. Stepwise combination of CPCys and CPLys with RNA allowed fabrication of TMV-like nanorods with a controlled total length of 300 or 330 nm, respectively, consisting of adjacent longitudinal 100-to-200 nm domains of differently addressable CP species. This technology paves the way towards rod-shaped scaffolds with pre-defined, selectively reactive barcode patterns on the nanometer scale.The spacing of functional nanoscopic elements may play a fundamental role in nanotechnological and biomedical applications, but is so far rarely achieved on this scale. In this study we show that tobacco mosaic virus (TMV) and the RNA-guided self-assembly process of its coat protein (CP) can be used to establish new nanorod scaffolds that can be loaded not only with homogeneously distributed functionalities, but with distinct molecule species grouped and ordered along the longitudinal axis. The arrangement of the resulting domains and final carrier rod length both were governed by RNA-templated two-step in vitro assembly. Two selectively addressable TMV CP mutants carrying

  1. Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis

    NARCIS (Netherlands)

    Sha, H.; Yang, L.; Liu, M.; Xia, S.; Liu, Y.; Liu, F.; Kersten, A.H.; Qi, L.

    2014-01-01

    The physiological role of the spliced form of X-box–binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in

  2. Chitosan-coated doxorubicin nano-particles drug delivery system inhibits cell growth of liver cancer via p53/PRC1 pathway.

    Science.gov (United States)

    Ye, Bai-Liang; Zheng, Ru; Ruan, Xiao-Jiao; Zheng, Zhi-Hai; Cai, Hua-Jie

    2018-01-01

    Nano-particles have been widely used in target-specific drug delivery system and showed advantages in cancers treatment. This study aims to evaluate the effect of chitosan coated doxorubicin nano-particles drug delivery system in liver cancer. The chitosan nano-particles were prepared by using the ionic gelation method. The characterizations of the nano-particles were determined by transmission electron microscopy. The cytotoxicity was detected by MTT assay, and the endocytosis, cell apoptosis and cell cycle were examined by flow cytometry. The protein level was analyzed with western blot. The dual luciferase reporter assay was performed to assess the interaction between p53 and the promoter of PRC1, and chromatin immune-precipitation was used to verify the binding between them. The FA-CS-DOX nano-particles were irregular and spherical particles around 30-40 nm, with uniform size and no adhesion. No significant difference was noted in doxorubicin release rate between CS-DOX and FA-CS-DOX. FA-CS-DOX nano-particles showed stronger cytotoxicity than CS-DOX. FA-CS-DOX nano-particles promoted the apoptosis and arrested cell cycle at G2/M phase, and they up-regulated p53. FA-CS-DOX nano-particles inhibited cell survival through p53/PRC1 pathway. Chitosan-coated doxorubicin nano-particles drug delivery system inhibits cell growth of liver cancer by promoting apoptosis and arresting cell cycle at G2/M phase through p53/PRC1 pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. The Effect of Salts in Promoting Specific and Competitive Interactions between Zinc Finger Proteins and Metals

    Science.gov (United States)

    Li, Gongyu; Yuan, Siming; Zheng, Shihui; Chen, Yuting; Zheng, Zhen; Liu, Yangzhong; Huang, Guangming

    2017-12-01

    Specific protein-metal interactions (PMIs) fulfill essential functions in cells and organic bodies, and activation of these functions in vivo are mostly modulated by the complex environmental factors, including pH value, small biomolecules, and salts. Specifically, the role of salts in promoting specific PMIs and their competition among various metals has remained untapped mainly due to the difficulty to distinguish nonspecific PMIs from specific PMIs by classic spectroscopic techniques. Herein, we report Hofmeister salts differentially promote the specific PMIs by combining nanoelectrospray ionization mass spectrometry and spectroscopic techniques (fluorescence measurement and circular dichroism). Furthermore, to explore the influence of salts in competitive binding between metalloproteins and various metals, we designed a series of competitive experiments and applied to a well-defined model system, the competitive binding of zinc (II) and arsenic (III) to holo-promyelocytic leukemia protein (PML). These experiments not only provided new insights at the molecular scale as complementary to previous NMR and spectroscopic results, but also deduced the relative binding ability between zinc finger proteins and metals at the molecular scale, which avoids the mass spectrometric titration-based determination of binding constants that is frequently affected and often degraded by variable solution conditions including salt contents. [Figure not available: see fulltext.

  4. Modeling the self-assembly of clathrin coats

    NARCIS (Netherlands)

    Giani, Matteo

    2017-01-01

    The assembly of clathrin coats in the presence of adaptor proteins was studied through computer simulations using coarse-grained models and through statistical mechanics. Adopting a reductionist approach based on recent experimental results, we aimed at reproducing and studying the minimal

  5. Promoter polymorphisms in two overlapping 6p25 genes implicate mitochondrial proteins in cognitive deficit in schizophrenia.

    LENUS (Irish Health Repository)

    Jablensky, A

    2011-10-04

    In a previous study, we detected a 6p25-p24 region linked to schizophrenia in families with high composite cognitive deficit (CD) scores, a quantitative trait integrating multiple cognitive measures. Association mapping of a 10 Mb interval identified a 260 kb region with a cluster of single-nucleotide polymorphisms (SNPs) significantly associated with CD scores and memory performance. The region contains two colocalising genes, LYRM4 and FARS2, both encoding mitochondrial proteins. The two tagging SNPs with strongest evidence of association were located around the overlapping putative promoters, with rs2224391 predicted to alter a transcription factor binding site (TFBS). Sequencing the promoter region identified 22 SNPs, many predicted to affect TFBSs, in a tight linkage disequilibrium block. Luciferase reporter assays confirmed promoter activity in the predicted promoter region, and demonstrated marked downregulation of expression in the LYRM4 direction under the haplotype comprising the minor alleles of promoter SNPs, which however is not driven by rs2224391. Experimental evidence from LYRM4 expression in lymphoblasts, gel-shift assays and modelling of DNA breathing dynamics pointed to two adjacent promoter SNPs, rs7752203-rs4141761, as the functional variants affecting expression. Their C-G alleles were associated with higher transcriptional activity and preferential binding of nuclear proteins, whereas the G-A combination had opposite effects and was associated with poor memory and high CD scores. LYRM4 is a eukaryote-specific component of the mitochondrial biogenesis of Fe-S clusters, essential cofactors in multiple processes, including oxidative phosphorylation. LYRM4 downregulation may be one of the mechanisms involved in inefficient oxidative phosphorylation and oxidative stress, increasingly recognised as contributors to schizophrenia pathogenesis.Molecular Psychiatry advance online publication, 4 October 2011; doi:10.1038\\/mp.2011.129.

  6. Identification of second harmonic optical effects from vaccine coated gold microparticles

    International Nuclear Information System (INIS)

    Jumah, N A; Ameer-Beg, S M; White, N S; Prasad, K V R; Bellhouse, B J

    2004-01-01

    This study investigates the optical effects observed from uncoated and protein vaccine coated gold microparticles while imaging with two-photon excitation in the Mie scattering regime. When observed with time correlated single photon counting fluorescence lifetime microscopy, the emission from the gold microparticles appeared as an intense instrument-limited temporal response. The intensity of the emission showed a second-order dependence on the laser power and frequency doubling of the emitted light was observed for fundamental light between 890 and 970 nm. The optical effect was attributed to two-photon induced second harmonic generation. The vaccine coated gold microparticles had a much weaker second harmonic signal than the uncoated gold microparticles. Chemical analysis of the surface of the gold microparticles revealed that the vaccine coating decreases the surface charge thereby diminishing the observed second harmonic signal. These optical properties can be exploited to identify both the location of the protein vaccine coating as well as the gold microparticles in vitro and potentially to investigate the vaccine delivery kinetics in vivo

  7. Sol-Gel-Derived Hydroxyapatite-Carbon Nanotube/Titania Coatings on Titanium Substrates

    Directory of Open Access Journals (Sweden)

    Chuantong Liu

    2012-04-01

    Full Text Available In this paper, hydroxyapatite-carbon nanotube/titania (HA-CNT/TiO2 double layer coatings were successfully developed on titanium (Ti substrates intended for biomedical applications. A TiO2 coating was firstly developed by anodization to improve bonding between HA and Ti, and then the layer of HA and CNTs was coated on the surface by the sol-gel process to improve the biocompatibility and mechanical properties of Ti. The surfaces of double layer coatings were uniform and crack-free with a thickness of about 7 μm. The bonding strength of the HA-CNT/TiO2 coating was higher than that of the pure HA and HA-CNT coatings. Additionally, in vitro cell experiments showed that CNTs promoted the adhesion of preosteoblasts on the HA-CNT/TiO2 double layer coatings. These unique surfaces combined with the osteoconductive properties of HA exhibited the excellent mechanical properties of CNTs. Therefore, the developed HA-CNT/TiO2 coatings on Ti substrates might be a promising material for bone replacement.

  8. HFCVD Diamond-Coated Mechanical Seals

    Directory of Open Access Journals (Sweden)

    Raul Simões

    2018-05-01

    Full Text Available A mechanical seal promotes the connection between systems or mechanisms, preventing the escape of fluids to the exterior. Nonetheless, due to extreme working conditions, premature failure can occur. Diamond, due to its excellent properties, is heralded as an excellent choice to cover the surface of these devices and extend their lifetime. Therefore, the main objective of this work was to deposit diamond films over mechanical seals and test the coated seals on a water pump, under real working conditions. The coatings were created by hot filament chemical vapor deposition (HFCVD and two consecutive layers of micro- and nanocrystalline diamond were deposited. One of the main difficulties is the attainment of a good adhesion between the diamond films and the mechanical seal material (WC-Co. Nucleation, deposition conditions, and pre-treatments were studied to enhance the coating. Superficial wear or delamination of the film was investigated using SEM and Raman characterization techniques, in order to draw conclusions about the feasibility of these coatings in the WC-Co mechanical seals with the purpose of increasing their performance and life time. The results obtained gave a good indication about the feasibility of this process and the deposition conditions used, with the mechanical seals showing no wear and no film delamination after a real work environment test.

  9. Biocompatibility of a functionally graded bioceramic coating made by wide-band laser cladding.

    Science.gov (United States)

    Weidong, Zhu; Qibin, Liu; Min, Zheng; Xudong, Wang

    2008-11-01

    The application of plasma spray is the most popular method by which a metal-bioceramic surface composite can be prepared for the repair of biological hard-tissue, but this method has disadvantages. These disadvantages include poor coating-to-substrate adhesion, low mechanical strength, and brittleness of the coating. In the investigation described in this article, a gradient bioceramic coating was prepared on a Ti-6Al-4V titanium alloy surface using a gradient composite design and wide-band laser cladding techniques. Using a trilayer-structure composed of a substratum, an alloy and bioceramics, the coating was chemically and metallurgically bonded with the substratum. The coating, which contains beta-tricalcium phosphate and hydroxyapatite, showed favorable biocompatibility with the bone tissue and promoted in vivo osteogenesis.

  10. Failure behavior of nano-SiO2 fillers epoxy coating under hydrostatic pressure

    International Nuclear Information System (INIS)

    Liu Li; Cui Yu; Li Ying; Zhang Tao; Wang Fuhui

    2012-01-01

    The failure of organic coating (epoxy resin filled with 5 mass% nano-SiO 2 particles) on mild steel under high hydrostatic pressure (35 atm) has been studied compared with that under atmospheric pressure (1 atm), using impedance measurements, gravimetric testing, adhesion testing and scanning electron microscopy (SEM). The results show that high hydrostatic pressure accelerated the failure of the organic coating by promoting diffusion of water in the coating, which speeds up water spread and electrochemical reactions at the interface. The roughness of the coating and steel has been discussed from point of view of their respective fractal dimensions D fc and D fdl , as deduced from impedance measurements

  11. The putative Agrobacterium transcriptional activator-like virulence protein VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell nucleus.

    Science.gov (United States)

    Wang, Yafei; Peng, Wei; Zhou, Xu; Huang, Fei; Shao, Lingyun; Luo, Meizhong

    2014-09-01

    Agrobacterium exports at least five virulence proteins (VirE2, VirE3, VirF, VirD2, VirD5) into host cells and hijacks some host plant factors to facilitate its transformation process. Random DNA binding selection assays (RDSAs), electrophoretic mobility shift assays (EMSAs) and yeast one-hybrid systems were used to identify protein-bound DNA elements. Bimolecular fluorescence complementation, glutathione S-transferase pull-down and yeast two-hybrid assays were used to detect protein interactions. Protoplast transformation, coprecipitation, competitive binding and cell-free degradation assays were used to analyze the relationships among proteins. We found that Agrobacterium VirD5 exhibits transcriptional activation activity in yeast, is located in the plant cell nucleus, and forms homodimers. A specific VirD5-bound DNA element designated D5RE (VirD5 response element) was identified. VirD5 interacted directly with Arabidopsis VirE2 Interacting Protein 1 (AtVIP1). However, the ternary complex of VirD5-AtVIP1-VirE2 could be detected, whereas that of VirD5-AtVIP1-VBF (AtVIP1 Binding F-box protein) could not. We demonstrated that VirD5 competes with VBF for binding to AtVIP1 and stabilizes AtVIP1 and VirE2 in the cell-free degradation system. Our results indicated that VirD5 may act as both a transcriptional activator-like effector to regulate host gene expression and a protector preventing the coat proteins of the T-complex from being quickly degraded by the host's ubiquitin proteasome system (UPS). © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  12. Quantifying the influence of polymer coatings on the serum albumin corona formation around silver and gold nanoparticles

    International Nuclear Information System (INIS)

    Treuel, Lennart; Malissek, Marcelina; Grass, Stefan; Diendorf, Jörg; Mahl, Dirk; Meyer-Zaika, Wolfgang; Epple, Matthias

    2012-01-01

    When nanoparticles (NPs) come into contact with biological fluids, proteins, and other biomolecules interact with their surface. Upon exposure to biological fluids a layer of proteins adsorbs onto their surface, the so-called protein corona, and interactions of biological systems with NPs are therefore mediated by this corona. Here, interactions of serum albumin with silver and gold NPs were quantitatively investigated using circular dichroism spectroscopy. Moreover, surface enhanced Raman spectroscopy was used for further elucidation of protein binding to silver surfaces. The decisive role of poly(vinylpyrrolidone), coatings on the protein adsorption was quantitatively described for the first time and the influential role of the polymer coatings is discussed. Research in nanotoxicology may benefit from such molecular scale data as well as scientific approaches seeking to improve nanomedical applications by using a wide range of polymer surface coatings to optimize biological transport and medical action of NPs.

  13. Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8.

    Directory of Open Access Journals (Sweden)

    Eleanna Stamatakou

    Full Text Available Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1 and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.

  14. Improving electrical conductivity in polycarbonate nanocomposites using highly conductive PEDOT/PSS coated MWCNTs

    KAUST Repository

    Zhou, Jian

    2013-07-10

    We describe a strategy to design highly electrically conductive polycarbonate nanocomposites by using multiwalled carbon nanotubes (MWCNTs) coated with a thin layer of poly(3,4-ethylenedioxythiophene)/ poly(styrenesulfonate), a conductive polymer. We found that this coating method improves the electrical properties of the nanocomposites in two ways. First, the coating becomes the main electrical conductive path. Second, the coating promotes the formation of a percolation network at a low filler concentration (0.3 wt %). To tailor the electrical properties of the conductive polymer coating, we used a polar solvent ethylene glycol, and we can tune the final properties of the nanocomposite by controlling the concentrations of the elementary constituents or the intrinsic properties of the conductive polymer coating. This very flexible technique allows for tailoring the properties of the final product. © 2013 American Chemical Society.

  15. The membrane protein LasM Promotes the Culturability of Legionella pneumophila in Water

    Directory of Open Access Journals (Sweden)

    Laam Li

    2016-09-01

    Full Text Available The water-borne pathogen Legionella pneumophila (Lp strongly expresses the lpg1659 gene in water. This gene encodes a hypothetical protein predicted to be a membrane protein using in silico analysis. While no conserved domains were identified in Lpg1659, similar proteins are found in many Legionella species and other aquatic bacteria. RT-qPCR showed that lpg1659 is positively regulated by the alternative sigma factor RpoS, which is essential for Lp to survive in water. These observations suggest an important role of this novel protein in the survival of Lp in water. Deletion of lpg1659 did not affect cell morphology, membrane integrity or tolerance to high temperature. Moreover, lpg1659 was dispensable for growth of Lp in rich medium, and during infection of the amoeba Acanthamoeba castellanii and of THP-1 human macrophages. However, deletion of lpg1659 resulted in an early loss of culturability in water, while over-expression of this gene promoted the culturability of Lp. Therefore, these results suggest that lpg1659 is required for Lp to maintain culturability, and possibly long-term survival, in water. Since the loss of culturability observed in the absence of Lpg1659 was complemented by the addition of trace metals into water, this membrane protein is likely a transporter for acquiring essential trace metal for maintaining culturability in water and potentially in other metal-deprived conditions. Given its role in the survival of Lp in water, Lpg1659 was named LasM for Legionella aquatic survival membrane protein.

  16. Hybrid Thin Film Organosilica Sol-Gel Coatings To Support Neuronal Growth and Limit Astrocyte Growth.

    Science.gov (United States)

    Capeletti, Larissa Brentano; Cardoso, Mateus Borba; Dos Santos, João Henrique Zimnoch; He, Wei

    2016-10-07

    Thin films of silica prepared by a sol-gel process are becoming a feasible coating option for surface modification of implantable neural sensors without imposing adverse effects on the devices' electrical properties. In order to advance the application of such silica-based coatings in the context of neural interfacing, the characteristics of silica sol-gel are further tailored to gain active control of interactions between cells and the coating materials. By incorporating various readily available organotrialkoxysilanes carrying distinct organic functional groups during the sol-gel process, a library of hybrid organosilica coatings is developed and investigated. In vitro neural cultures using PC12 cells and primary cortical neurons both reveal that, among these different types of hybrid organosilica, the introduction of aminopropyl groups drastically transforms the silica into robust neural permissive substrate, supporting neuron adhesion and neurite outgrowth. Moreover, when this organosilica is cultured with astrocytes, a key type of glial cells responsible for glial scar response toward neural implants, such cell growth promoting effect is not observed. These findings highlight the potential of organo-group-bearing silica sol-gel to function as advanced coating materials to selectively modulate cell response and promote neural integration with implantable sensing devices.

  17. Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA, umuDC, or sulA Promoters

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2005-01-01

    Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N'-......Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N......-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies....

  18. Self-assembled hemocompatible coating on poly (vinyl chloride) surface

    Energy Technology Data Exchange (ETDEWEB)

    Zha Zhengbao; Ma Yan; Yue Xiuli; Liu Meng [Nanobiotechnology Division, State Key Laboratory of Urban Water Resources and Environment, School of Sciences, Harbin Institute of Technology, Harbin 150001 (China); Dai Zhifei, E-mail: zhifei.dai@hit.edu.cn [Nanobiotechnology Division, State Key Laboratory of Urban Water Resources and Environment, School of Sciences, Harbin Institute of Technology, Harbin 150001 (China)

    2009-11-15

    A stable hemocompatible coating was fabricated by consecutive alternating adsorption of iron (III) and two kinds of polysaccharides, heparin (Hep) and dextran sulfate (DS), onto poly (vinyl chloride) (PVC) surfaces via electrostatic interaction. The fluctuation of contact angles with the alternative deposition of iron (III) and polysaccharides verified the progressive buildup of the mulitilayer coating onto the PVC surface. Atomic force microscopy (AFM) analysis revealed that the PVC surfaces were completely masked by iron-polysaccharides multilayer coatings. The activated partial thromboplastin time (APTT) assay showed that both Hep/Fe{sup 3+}/Hep and DS/Fe{sup 3+}/Hep coated PVC were less thrombogenic than the uncoated one. Chromogenic assay for heparin activity proved definitively that the inhibition of locally produced thrombin was ascribed to the thromboresistance of the surface-bound heparin. Compared with the unmodified PVC surfaces, iron-polysaccharide multilayer coating presented a drastically reduced adhesion in vitro of platelets, polymorphonuclear neutrophil leukocytes (PMN) and peripheral blood mononuclear cells (PBMC). Interestingly, the DS/Fe{sup 3+}/Hep coating was found to exhibit higher hydrophilicity and stability, hence lower non-specific protein adsorption in comparison with Hep/Fe{sup 3+}/Hep coating due to the incorporation of dextran sulfate into the multilayer coating.

  19. Functional promoter variant in zinc finger protein 202 predicts severe atherosclerosis and ischemic heart disease

    DEFF Research Database (Denmark)

    Frikke-Schmidt, R.; Nordestgaard, Børge; Grande, Peer

    2008-01-01

    Objectives This study was designed to test the hypotheses that single nucleotide polymorphisms ( SNPs), in zinc finger protein 202 ( ZNF202), predict severe atherosclerosis and ischemic heart disease ( IHD). Background ZNF202 is a transcriptional repressor controlling promoter elements in genes...... involved in vascular maintenance and lipid metabolism. Methods We first determined genotype association for 9 ZNF202 SNPs with severe atherosclerosis ( ankle brachial index >0.7 vs. ...,998 controls. Finally, we determined whether g. -660A>G altered transcriptional activity of the ZNF202 promoter in vitro. Results Cross-sectionally, ZNF202 g. -660 GG versus AA homozygosity predicted an odds ratio for severe atherosclerosis of 2.01 ( 95% confidence interval [CI]: 1.34 to 3.01). Prospectively...

  20. Understanding cracking failures of coatings: A fracture mechanics approach

    Science.gov (United States)

    Kim, Sung-Ryong

    A fracture mechanics analysis of coating (paint) cracking was developed. A strain energy release rate (G(sub c)) expression due to the formation of a new crack in a coating was derived for bending and tension loadings in terms of the moduli, thicknesses, Poisson's ratios, load, residual strain, etc. Four-point bending and instrumented impact tests were used to determine the in-situ fracture toughness of coatings as functions of increasing baking (drying) time. The system used was a thin coating layer on a thick substrate layer. The substrates included steel, aluminum, polycarbonate, acrylonitrile-butadiene-styrene (ABS), and Noryl. The coatings included newly developed automotive paints. The four-point bending configuration promoted nice transversed multiple coating cracks on both steel and polymeric substrates. The crosslinked type automotive coatings on steel substrates showed big cracks without microcracks. When theoretical predictions for energy release rate were compared to experimental data for coating/steel substrate samples with multiple cracking, the agreement was good. Crosslinked type coatings on polymeric substrates showed more cracks than theory predicted and the G(sub c)'s were high. Solvent evaporation type coatings on polymeric substrates showed clean multiple cracking and the G(sub c)'s were higher than those obtained by tension analysis of tension experiments with the same substrates. All the polymeric samples showed surface embrittlement after long baking times using four-point bending tests. The most apparent surface embrittlement was observed in the acrylonitrile-butadiene-styrene (ABS) substrate system. The impact properties of coatings as a function of baking time were also investigated. These experiments were performed using an instrumented impact tester. There was a rapid decrease in G(sub c) at short baking times and convergence to a constant value at long baking times. The surface embrittlement conditions and an embrittlement toughness

  1. Analysis of the epitope structure of Plum pox virus coat protein.

    Science.gov (United States)

    Candresse, Thierry; Saenz, Pilar; García, Juan Antonio; Boscia, Donato; Navratil, Milan; Gorris, Maria Teresa; Cambra, Mariano

    2011-05-01

    Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

  2. P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

    Science.gov (United States)

    Loll-Krippleber, Raphael; Brown, Grant W

    2017-09-15

    mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.P-bodies form in response to stress and act as sites of mRNA storage and degradation. Here the authors identify the mRNA targets of P-bodies during DNA replication stress, and show that P-body proteins act to prevent toxic accumulation of these target transcripts.

  3. Identification of the promoter region required for human adiponectin gene transcription: Association with CCAAT/enhancer binding protein-β and tumor necrosis factor-α

    International Nuclear Information System (INIS)

    Kita, Atsushi; Yamasaki, Hironori; Kuwahara, Hironaga; Moriuchi, Akie; Fukushima, Keiko; Kobayashi, Masakazu; Fukushima, Tetsuya; Takahashi, Ryoko; Abiru, Norio; Uotani, Shigeo; Kawasaki, Eiji; Eguchi, Katsumi

    2005-01-01

    Adiponectin, an adipose tissue-specific plasma protein, is involved in insulin sensitizing and has anti-atherosclerotic properties. Plasma levels of adiponectin are decreased in obese individuals and patients with type 2 diabetes with insulin resistance. Tumor necrosis factor-α (TNF-α) decreases the expression of adiponectin in adipocytes. The aims of the present study were: (1) to identify the promoter region responsible for basal transcription of the human adiponectin gene, and (2) to investigate the mechanism by which adiponectin was regulated by TNF-α. The human adiponectin promoter (2.1 kb) was isolated and used for luciferase reporter analysis by transient transfection into 3T3-L1 adipocytes. Deletion analysis demonstrated that the promoter region from -676 to +41 was sufficient for basal transcriptional activity. Mutation analysis of putative response elements for sterol regulatory element binding protein (SREBP) (-431 to -423) and CCAAT/enhancer binding protein (C/EBP) (-230 to -224) showed that both elements were required for basal promoter activity. Adiponectin transcription was increased 3-fold in cells that over-expressed constitutively active C/EBP-β. Electrophoretic mobility shift assay, using nuclear extract from 3T3-L1 cells and the -258 to -199 region as a probe, demonstrated specific DNA-protein binding, which was abolished by TNF-α treatment. The present data indicate that the putative response elements for SREBP and C/EBP are required for human adiponectin promoter activity, and that suppression by TNF-α may, at least in part, be associated with inactivation of C/EBP-β

  4. A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation.

    Science.gov (United States)

    Kabeche, Lilian; Nguyen, Hai Dang; Buisson, Rémi; Zou, Lee

    2018-01-05

    The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability. Copyright © 2018, American Association for the Advancement of Science.

  5. Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors

    Directory of Open Access Journals (Sweden)

    Kawe Martin

    2009-01-01

    Full Text Available Abstract Background Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such "benign" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored. Results We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size – all antibiotic-resistant non-producers – was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented. Conclusion The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein.

  6. Increased protein-energy intake promotes anabolism in critically ill infants with viral bronchiolitis: a double‑blind randomised controlled trial

    Science.gov (United States)

    de Betue, Carlijn T; van Waardenburg, Dick A; Deutz, Nicolaas E; van Eijk, Hans M; van Goudoever, Johannes B; Luiking, Yvette C; Zimmermann, Luc J; Joosten, Koen F

    2011-01-01

    Objective The preservation of nutritional status and growth is an important aim in critically ill infants, but difficult to achieve due to the metabolic stress response and inadequate nutritional intake, leading to negative protein balance. This study investigated whether increasing protein and energy intakes can promote anabolism. The primary outcome was whole body protein balance, and the secondary outcome was first pass splanchnic phenylalanine extraction (SPEPhe). Design This was a double-blind randomised controlled trial. Infants (n=18) admitted to the paediatric intensive care unit with respiratory failure due to viral bronchiolitis were randomised to continuous enteral feeding with protein and energy enriched formula (PE-formula) (n=8; 3.1±0.3 g protein/kg/24 h, 119±25 kcal/kg/24 h) or standard formula (S-formula) (n=10; 1.7±0.2 g protein/kg/24 h, 84±15 kcal/kg/24 h; equivalent to recommended intakes for healthy infants <6 months). A combined intravenous-enteral phenylalanine stable isotope protocol was used on day 5 after admission to determine whole body protein metabolism and SPEPhe. Results Protein balance was significantly higher with PE-formula than with S-formula (PE-formula: 0.73±0.5 vs S-formula: 0.02±0.6 g/kg/24 h) resulting from significantly increased protein synthesis (PE-formula: 9.6±4.4, S-formula: 5.2±2.3 g/kg/24 h), despite significantly increased protein breakdown (PE-formula: 8.9±4.3, S-formula: 5.2±2.6 g/kg/24 h). SPEPhe was not statistically different between the two groups (PE-formula: 39.8±18.3%, S-formula: 52.4±13.6%). Conclusions Increasing protein and energy intakes promotes protein anabolism in critically ill infants in the first days after admission. Since this is an important target of nutritional support, increased protein and energy intakes should be preferred above standard intakes in these infants. Dutch Trial Register number: NTR 515. PMID:21673183

  7. Functional patterned coatings by thin polymer film dewetting.

    Science.gov (United States)

    Telford, Andrew M; Thickett, Stuart C; Neto, Chiara

    2017-12-01

    An approach for the fabrication of functional polymer surface coatings is introduced, where micro-scale structure and surface functionality are obtained by means of self-assembly mechanisms. We illustrate two main applications of micro-patterned polymer surfaces obtained through dewetting of bilayers of thin polymer films. By tuning the physical and chemical properties of the polymer bilayers, micro-patterned surface coatings could be produced that have applications both for the selective attachment and patterning of proteins and cells, with potential applications as biomaterials, and for the collection of water from the atmosphere. In all cases, the aim is to achieve functional coatings using approaches that are simple to realize, use low cost materials and are potentially scalable. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Material Science Smart Coatings

    Energy Technology Data Exchange (ETDEWEB)

    Rubinstein, A. I. [Univ. of Nebraska Medical Center, Omaha, NE (United States); Sabirianov, R. F. [Univ. of Nebraska Medical Center, Omaha, NE (United States); Namavar, Fereydoon [Univ. of Nebraska Medical Center, Omaha, NE (United States)

    2014-07-01

    The contribution of electrostatic interactions to the free energy of binding between model protein and a ceramic implant surface in the aqueous solvent, considered in the framework of the nonlocal electrostatic model, is calculated as a function of the implant low-frequency dielectric constant. We show that the existence of a dynamically ordered (low-dielectric) interfacial solvent layer at the protein-solvent and ceramic-solvent interface markedly increases charging energy of the protein and ceramic implant, and consequently makes the electrostatic contribution to the protein-ceramic binding energy more favorable (attractive). Our analysis shows that the corresponding electrostatic energy between protein and oxide ceramics depends nonmonotonically on the dielectric constant of ceramic, εC. Obtained results indicate that protein can attract electrostatically to the surface if ceramic material has a moderate εC below or about 35 (in particularly ZrO2 or Ta2O5). This is in contrast to classical (local) consideration of the solvent, which demonstrates an unfavorable electrostatic interaction of protein with typical metal oxide ceramic materials (εC>10). Thus, a solid implant coated by combining oxide ceramic with a reduced dielectric constant can be beneficial to strengthen the electrostatic binding of the protein-implant complex.

  9. Crosslinkable coatings from phosphorylcholine-based polymers.

    Science.gov (United States)

    Lewis, A L; Cumming, Z L; Goreish, H H; Kirkwood, L C; Tolhurst, L A; Stratford, P W

    2001-01-01

    2-Methacryloyloxyethyl phosphorylcholine (MPC) was synthesised and then used in the preparation of crosslinked polymer membranes with lauryl methacrylate, hydroxypropyl methacrylate and trimethoxysilylpropyl methacrylate (crosslinker) comonomers. Some physical aspects of the membrane properties were evaluated in order to establish the basis for the synthesis of a series of post-crosslinkable polymers. These materials were made by copolymerisation of the constituent monomers via a free radical method, and characterised using NMR, FT-IR, viscometry and elemental analysis. The optimum crosslink density and conditions required for curing coatings of these polymers were investigated using atomic force microscopy (AFM) and showed the inclusion of 5 mol% silyl crosslinking agent to be ideal. A nanoindentation technique was employed to determine if the coating developed elasticity upon crosslinking. The biological properties of the coatings were evaluated using a variety of protein adsorption assays and blood contacting experiments, and an enzyme immunoassay was developed to detect E. coli in order to assess the level of bacterial adhesion to these biomaterials. Polymers of this type were shown to be very useful as coating materials for improving the biocompatibility of, or reducing the levels of adherent bacteria to medical devices.

  10. Inorganic coatings on stainless steel for protection against crevice corrosion

    International Nuclear Information System (INIS)

    Henrikson, Sture

    1989-12-01

    In order to create protection against crevice corrosion stainless steel test specimens of type 316 steel with various inorganic coatings applied on crevice surfaces were tested for 3-50 months at 25 and 30 degree C in natural seawater containing 0.2-1.5 ppm free chlorine. Various metallic coatings, Ni base alloys with Cr and Mo, Ni with W, pure Ag and pure Mo, as well as ceramic coatings - Cr 2 O 3 , TiO 2 and Al 2 O 3 - were studied. All the coatings tested, except pure Molybdenum applied by plasma spraying in a max 0.1 mm thick layer were found to promote crevice corrosion of the stainless steel. A significant reduction of the crevice corrosion susceptibility was obtained with Molybdenum. The result is considered promising enough to justify full scale tests in seawater on flange joints of pipes, valves or pumps. (author)

  11. Flagellin based biomimetic coatings: From cell-repellent surfaces to highly adhesive coatings.

    Science.gov (United States)

    Kovacs, Boglarka; Patko, Daniel; Szekacs, Inna; Orgovan, Norbert; Kurunczi, Sandor; Sulyok, Attila; Khanh, Nguyen Quoc; Toth, Balazs; Vonderviszt, Ferenc; Horvath, Robert

    2016-09-15

    Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. As reference surfaces synthetic polymers were applied which have anti-adhesive (PLL-g-PEG poly(l-lysine)-graft-poly(ethylene glycol)) or adhesion inducing properties (RGD-functionalized PLL-g-PEG). Quantitative adhesion data was obtained by employing optical biochips and microscopy. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. The developed novel RGD-displaying flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell-adhesion-regulating biomimetic coatings. In the present work, we show for the first time that. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  12. Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus.

    Science.gov (United States)

    Zanek, María Cecilia; Reyes, Carina Andrea; Cervera, Magdalena; Peña, Eduardo José; Velázquez, Karelia; Costa, Norma; Plata, Maria Inés; Grau, Oscar; Peña, Leandro; García, María Laura

    2008-01-01

    Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found. Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants, but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism, although the transformant failed to protect against the viral load used. Possible causes for the failed protection against the CPsV are discussed.

  13. Characteristics of chalcone isomerase promoter in crabapple leaves ...

    African Journals Online (AJOL)

    Administrator

    2011-09-05

    Sep 5, 2011 ... in flavonoid biosynthetic pathway, can convert chalcone to (2S)-naringenin in the ... Construction of expression vectors with McCHI promoter fragment ... Each sample was bombarded three times with tungsten particles coated ...

  14. The Zn Finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis

    Science.gov (United States)

    Glazer, Andrew; Wilkinson, Alex; Backer, Chelsea B.; Lapan, Sylvain; Gutzman, Jennifer H.; Cheeseman, Iain M.; Reddien, Peter W.

    2009-01-01

    Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and that utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian S. mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates. PMID:19852954

  15. Quantitative proteomic analysis reveals effects of epidermal growth factor receptor (EGFR) on invasion-promoting proteins secreted by glioblastoma cells.

    Science.gov (United States)

    Sangar, Vineet; Funk, Cory C; Kusebauch, Ulrike; Campbell, David S; Moritz, Robert L; Price, Nathan D

    2014-10-01

    Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Transmission electron microscopy of coatings formed by plasma electrolytic oxidation of titanium.

    Science.gov (United States)

    Matykina, E; Arrabal, R; Skeldon, P; Thompson, G E

    2009-05-01

    Transmission electron microscopy and supporting film analyses are used to investigate the changes in composition, morphology and structure of coatings formed on titanium during DC plasma electrolytic oxidation in a calcium- and phosphorus-containing electrolyte. The coatings are of potential interest as bioactive surfaces. The initial barrier film, of mixed amorphous and nanocrystalline structure, formed below the sparking voltage of 180 V, incorporates small amounts of phosphorus and calcium species, with phosphorus confined to the outer approximately 63% of the coating thickness. On commencement of sparking, calcium- and phosphorus-rich amorphous material forms at the coating surface, with local heating promoting crystallization in underlying and adjacent anodic titania. The amorphous material thickens with increased treatment time, comprising almost the whole of the approximately 5.7-microm-thick coating formed at 340 V. At this stage, the coating is approximately 4.4 times thicker than the oxidized titanium, with a near-surface composition of about 12 at.% Ti, 58 at.% O, 19 at.% P and 11 at.% Ca. Further, the amount of titanium consumed in forming the coating is similar to that calculated from the anodizing charge, although there may be non-Faradaic contributions to the coating growth.

  17. Ectodomain shedding of Limbic System-Associated Membrane Protein (LSAMP) by ADAM Metallopeptidases promotes neurite outgrowth in DRG neurons.

    Science.gov (United States)

    Sanz, Ricardo L; Ferraro, Gino B; Girouard, Marie-Pier; Fournier, Alyson E

    2017-08-11

    IgLONs are members of the immunoglobulin superfamily of cell adhesion proteins implicated in the process of neuronal outgrowth, cell adhesion and subdomain target recognition. IgLONs form homophilic and heterophilic complexes on the cell surface that repress or promote growth depending on the neuronal population, the developmental stage and surface repertoire of IgLON family members. In the present study, we identified a metalloproteinase-dependent mechanism necessary to promote growth in embryonic dorsal root ganglion cells (DRGs). Treatment of embryonic DRG neurons with pan-metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-3, or an inhibitor of ADAM Metallopeptidase Domain 10 (ADAM10) reduces outgrowth from DRG neurons indicating that metalloproteinase activity is important for outgrowth. The IgLON family members Neurotrimin (NTM) and Limbic System-Associated Membrane Protein (LSAMP) were identified as ADAM10 substrates that are shed from the cell surface of DRG neurons. Overexpression of LSAMP and NTM suppresses outgrowth from DRG neurons. Furthermore, LSAMP loss of function decreases the outgrowth sensitivity to an ADAM10 inhibitor. Together our findings support a role for ADAM-dependent shedding of cell surface LSAMP in promoting outgrowth from DRG neurons.

  18. Electrophoretic properties of BSA-coated quantum dots.

    Science.gov (United States)

    Bücking, Wendelin; Massadeh, Salam; Merkulov, Alexei; Xu, Shu; Nann, Thomas

    2010-02-01

    Low toxic InP/ZnS quantum dots (QDs), ZnS:Mn(2+)/ZnS nanocrystals and CdSe/ZnS nanoparticles were rendered water-dispersible by different ligand-exchange methods. Eventually, they were coated with bovine serum albumin (BSA) as a model protein. All particles were characterised by isotachophoresis (ITP), laser Doppler velocimetry (LDV) and agarose gel electrophoresis. It was found that the electrophoretic mobility and colloidal stability of ZnS:Mn(2+)/ZnS and CdSe/ZnS nanoparticles, which bore short-chain surface ligands, was primarily governed by charges on the nanoparticles, whereas InP/ZnS nanocrystals were not charged per se. BSA-coated nanoparticles showed lower electrophoretic mobility, which was attributed to their larger size and smaller overall charge. However, these particles were colloidally stable. This stability was probably caused by steric stabilisation of the BSA coating.

  19. Molecular Characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL Gene Family in Betula luminifera

    Directory of Open Access Journals (Sweden)

    Xiu-Yun Li

    2018-05-01

    Full Text Available As a major family of plant-specific transcription factors, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL genes play vital regulatory roles in plant growth, development and stress responses. In this study, 18 SPL genes were identified and cloned from Betula luminifera. Two zinc finger-like structures and a nuclear location signal (NLS segments were existed in the SBP domains of all BlSPLs. Phylogenetic analysis showed that these genes were clustered into nine groups (group I-IX. The intron/exon structure and motif composition were highly conserved within the same group. 12 of the 18 BlSPLs were experimentally verified as the targets of miR156, and two cleavage sites were detected in these miR156-targeted BlSPL genes. Many putative cis-elements, associated with light, stresses and phytohormones response, were identified in the promoter regions of BlSPLs, suggesting that BlSPL genes are probably involved in important physiological processes and developmental events. Tissue-specific expression analysis showed that miR156-targeted BlSPLs exhibited a more differential expression pattern, while most miR156-nontargeted BlSPLs tended to be constitutively expressed, suggesting the distinct roles of miR156-targeted and nontargeted BlSPLs in development and growth of B. luminifera. Further expression analysis revealed that miR156-targeted BlSPLs were dramatically up-regulated with age, whereas mature BlmiR156 level was apparently declined with age, indicating that miR156/SPL module plays important roles in vegetative phase change of B. luminifera. Moreover, yeast two-hybrid assay indicated that several miR156-targeted and nontargeted BlSPLs could interact with two DELLA proteins (BlRGA and BlRGL, which suggests that certain BlSPLs take part in the GA regulated processes through protein interaction with DELLA proteins. All these results provide an important basis for further exploring the biological functions of BlSPLs in B. luminifera.

  20. Secreted Frizzled related protein-4 (sFRP4) promotes epidermal differentiation and apoptosis

    International Nuclear Information System (INIS)

    Maganga, Richard; Giles, Natalie; Adcroft, Katharine; Unni, Ambili; Keeney, Diane; Wood, Fiona; Fear, Mark; Dharmarajan, Arunasalam

    2008-01-01

    The skin provides vital protection from infection and dehydration. Maintenance of the skin is through a constant program of proliferation, differentiation and apoptosis of epidermal cells, whereby proliferating cells in the basal layer differentiating to form the keratinized, anucleated stratum corneum. The WNT signalling pathway is known to be important in the skin. WNT signalling has been shown to be important both in epidermal development and in the maintenance and cycling of hair follicles and epidermal stem cells. However, the precise role for this pathway in epidermal differentiation remains unknown. We investigated the role of the WNT signalling inhibitor sFRP4 in epidermal differentiation. sFRP4 is expressed in both normal skin and keratinocytes in culture. Expression of sFRP4 mRNA and protein increases with keratinocyte differentiation and apoptosis, whilst exposure of keratinocytes to exogenous sFRP4 promotes apoptosis and expression of the terminal differentiation marker Involucrin. These data suggest sFRP4 promotes epidermal differentiation.

  1. CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer

    DEFF Research Database (Denmark)

    Caldeira, José Roberto F; Prando, Erika C; Quevedo, Francisco C

    2006-01-01

    prognosis, and metastasis. Differential CpG island methylation in the promoter region of the CDH1 gene might be an alternative way for the loss of expression and function of E-cadherin, leading to loss of tissue integrity, an essential step in tumor progression. METHODS: The aim of our study was to assess...... not statistically significant, the levels of E-cadherin expression tended to diminish with the CDH1 promoter region methylation. In the group of 71 ductal cancinomas, most of the cases of showing CDH1 hypermethylation also presented reduced levels of expression of ER and PgR proteins, and a possible association......BACKGROUND: The E-cadherin gene (CDH1) maps, at chromosome 16q22.1, a region often associated with loss of heterozygosity (LOH) in human breast cancer. LOH at this site is thought to lead to loss of function of this tumor suppressor gene and was correlated with decreased disease-free survival, poor...

  2. Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment

    NARCIS (Netherlands)

    Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

    2008-01-01

    We present a new and simple method based on site-directed fluorescence labeling using the BADAN label that allows to examine protein-lipid interactions in great detail. We apply this approach to a membrane-embedded mainly -helical reference protein, the M13 major coat protein, of which in a

  3. Au nanocrystals grown on a better-defined one-dimensional tobacco mosaic virus coated protein template genetically modified by a hexahistidine tag

    International Nuclear Information System (INIS)

    Liu Nan; Zhang Wei; Luo Zhaopeng; Zhai Niu; Zhang Hongfei; Li Zhonghao; Jiang Xingyi; Tang Gangling; Hu Qingyuan; Wang Chong; Tian Dandan

    2012-01-01

    In this paper, tobacco mosaic virus (TMV) coated protein (CP) was genetically modified by introducing a hexahistidine tag into it for a well-defined one-dimensional template, on which Au nanocrystals (NCs) were grown. The results showed that genetic modification could not only ameliorate the one-dimensional structure of the template, but also improve the growth density of Au NCs on the template. This indicated that genetic modification could be an effective method to modulate the structure of the TMVCP template-based nanocomposites allowing for a broader application of them. (paper)

  4. Magnetically actuated mechanical stimuli on Fe3O4/mineralized collagen coatings to enhance osteogenic differentiation of the MC3T3-E1 cells.

    Science.gov (United States)

    Zhuang, Junjun; Lin, Suya; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

    2018-04-15

    Mechanical stimuli at the bone-implant interface are considered to activate the mechanotransduction pathway of the cell to improve the initial osseointegration establishment and to guarantee clinical success of the implant. However, control of the mechanical stimuli at the bone-implant interface still remains a challenge. In this study, we have designed a strategy of a magnetically responsive coating on which the mechanical stimuli is controlled because of coating deformation under static magnetic field (SMF). The iron oxide nanoparticle/mineralized collagen (IOP-MC) coatings were electrochemically codeposited on titanium substrates in different quantities of IOPs and distributions; the resulting coatings were verified to possess swelling behavior with flexibility same as that of hydrogel. The relative quantity of IOP to collagen and the IOP distribution in the coatings were demonstrated to play a critical role in mediating cell behavior. The cells present on the outer layer of the distributed IOP-MC (O-IOP-MC) coating with a mass ratio of 0.67 revealed the most distinct osteogenic differentiation activity being promoted, which could be attributed to the maximized mechanical stimuli with exposure to SMF. Furthermore, the enhanced osteogenic differentiation of the stimulated MC3T3-E1 cells originated from magnetically actuated mechanotransduction signaling pathway, embodying the upregulated expression of osteogenic-related and mechanotransduction-related genes. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface and thus to promote osseointegration. The magnetically actuated coating is designed to produce mechanical stimuli to cells for promoting osteogenic differentiation based on the coating deformation. Iron oxide nanoparticles (IOPs) were incorporated into the mineralized collagen coatings (MC) forming the composite coatings (IOP-MC) with spatially distributed IOPs

  5. European coatings conference - Marine coatings. Proceedings

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2008-07-01

    This volume contains 13 lectures (manuscripts or powerpoint foils) with the following topics: 1. Impact of containerization on polyurethane and polyurea in marine and protective coatings (Malte Homann); 2. The application of combinatorial/high-throughput methods to the development of marine coatings (Bret Chisholm); 3. Progress and perspectives in the AMBIO (advanced nanostructured surfaces for the control of biofouling) Project (James Callow); 4. Release behaviour due to shear and pull-off of silicone coatings with a thickness gradient (James G. Kohl); 5. New liquid rheology additives for high build marine coatings (Andreas Freytag); 6. Effective corrosion protection with polyaniline, polpyrrole and polythiophene as anticorrosice additives for marine paints (Carlos Aleman); 7. Potential applications of sol gel technology for marine applications (Robert Akid); 8: Performance of biocide-free Antifouling Coatings for leisure boats (Bernd Daehne); 9. Novel biocidefree nanostructured antifouling coatings - can nano do the job? (Corne Rentrop); 10. One component high solids, VOC compliant high durability finish technology (Adrian Andrews); 11. High solid coatings - the hybrid solution (Luca Prezzi); 12. Unique organofunctional silicone resins for environmentally friendly high-performance coatings (Dieter Heldmann); 13. Silicone-alkyd paints for marine applications: from battleship-grey to green (Thomas Easton).

  6. Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype.

    Science.gov (United States)

    Zhang, Yuanyuan; He, Yujiang; Bharadwaj, Shantaram; Hammam, Nevin; Carnagey, Kristen; Myers, Regina; Atala, Anthony; Van Dyke, Mark

    2009-08-01

    Recent studies have shown that extracellular matrix (ECM) substitutes can have a dramatic impact on cell growth, differentiation and function. However, these ECMs are often applied generically and have yet to be developed for specific cell types. In this study, we developed tissue-specific ECM-based coating substrates for skin, skeletal muscle and liver cell cultures. Cellular components were removed from adult skin, skeletal muscle, and liver tissues, and the resulting acellular matrices were homogenized and dissolved. The ECM solutions were used to coat culture dishes. Tissue matched and non-tissue matched cell types were grown on these coatings to assess adhesion, proliferation, maintenance of phenotype and cell function at several time points. Each cell type showed better proliferation and differentiation in cultures containing ECM from their tissue of origin. Although subtle compositional differences in the three ECM types were not investigated in this study, these results suggest that tissue-specific ECMs provide a culture microenvironment that is similar to the in vivo environment when used as coating substrates, and this new culture technique has the potential for use in drug development and the development of cell-based therapies.

  7. The role of nanocrystalline titania coating on nanostructured austenitic stainless steel in enhancing osteoblasts functions for regeneration of tissue

    Energy Technology Data Exchange (ETDEWEB)

    Shah, J.S.; Venkatsurya, P.K.C.; Thein-Han, W.W. [Biomaterials and Biomedical Engineering Research Laboratory, Center for Structural and Functional Materials, University of Louisiana at Lafayette, P.O. Box 44130, Lafayette, LA 70504 (United States); Misra, R.D.K., E-mail: dmisra@louisiana.edu [Biomaterials and Biomedical Engineering Research Laboratory, Center for Structural and Functional Materials, University of Louisiana at Lafayette, P.O. Box 44130, Lafayette, LA 70504 (United States); Pesacreta, T.C. [Department of Biology, University of Louisiana at Lafayette, P.O. Box 42451, Lafayette, LA 70504 (United States); Somani, M.C.; Karjalainen, L.P. [Department of Mechanical Engineering, University of Oulu, P.O. Box 4200, 90014 Oulu (Finland)

    2011-03-12

    In the context of osseointegration of metallic implants, while nanostructuring the surface favorably modulates cellular response, the disinfective attributes required during the healing process are not available. Thus, in the present study, we demonstrate that nanocrystalline titania provides cumulative benefit of enhancing osteoblasts functions to promote the efficacy of metal implants together with the disinfective attributes. To this end, the primary objective here is to examine the select functions of bone forming cells (osteoblasts) on electrocrystallized nanonodular titania-coated nanograined/ultrafine grained (NG/UFG) austenitic stainless steel. The accompanying objective is to study the disinfective/antimicrobial activity. To the best of our understanding this is the first study of nanophase titania on a non-titanium substrate. The osteoblasts functions were investigated in terms of cell attachment, proliferation, and quantitative analysis of proteins, actin and vinculin. In comparison to the bare NG/UFG substrate, the nanophase titania-coated substrate exhibited higher degree of cell attachment and proliferation which are regulated via cellular and molecular interactions with proteins, actin and vinculin. The enhanced functions of osteoblasts suggest that nanophase titania adsorbs extracellular matrix proteins, fibronectin and vitronectin from serum enhancing protein, with subsequent binding of integrins and osteoblasts precursor to titania. The antimicrobial attributes assessed in terms of degradation of methyl orange and effectiveness in killing E. coli supports the viewpoint that large surface area of titania would be instrumental in reducing the detrimental effect of biologically reactive oxygen species produced by macrophages in the vicinity of the metal bone/implant interface. In summary, the study provides some new insights concerning nanostructuring of metallic substrates with specific physical and surface properties for medical devices with

  8. The in vitro biomineralization and cytocompatibility of polydopamine coated carbon nanotubes

    International Nuclear Information System (INIS)

    Yan Penghua; Wang Jinqing; Wang Lin; Liu Bin; Lei Ziqiang; Yang Shengrong

    2011-01-01

    In this work, polydopamine coated carbon nanotubes were firstly prepared by a simple and feasible route. Then, for comparison, the in vitro bioactivity and cytocompatibility of the carbon nanotubes and the polydopamine coated carbon nanotubes were assessed by immersion study in simulated body fluids and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide test using osteoblast cells (MC3T3-E1), respectively. As a result, it has been demonstrated that the introduction of polydopamine coating can greatly enhance the bioactivity and promote cell proliferation of the carbon nanotubes. The improvement of bioactive behavior is attributed to the good combination of catecholamines structure of the polydopamine and the structural advantages of carbon nanotubes as a framework material. It is anticipated that the polydopamine coated carbon nanotubes would find potential applications in bone tissue engineering and other biomedical areas.

  9. The in vitro biomineralization and cytocompatibility of polydopamine coated carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Yan Penghua [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000 (China); Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China); Wang Jinqing, E-mail: jqwang@licp.cas.cn [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000 (China); Wang Lin; Liu Bin [School of Stomatology, Lanzhou University, Lanzhou 730000 (China); Lei Ziqiang [Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China); Yang Shengrong, E-mail: sryang@lzb.ac.cn [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000 (China); Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China)

    2011-03-15

    In this work, polydopamine coated carbon nanotubes were firstly prepared by a simple and feasible route. Then, for comparison, the in vitro bioactivity and cytocompatibility of the carbon nanotubes and the polydopamine coated carbon nanotubes were assessed by immersion study in simulated body fluids and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide test using osteoblast cells (MC3T3-E1), respectively. As a result, it has been demonstrated that the introduction of polydopamine coating can greatly enhance the bioactivity and promote cell proliferation of the carbon nanotubes. The improvement of bioactive behavior is attributed to the good combination of catecholamines structure of the polydopamine and the structural advantages of carbon nanotubes as a framework material. It is anticipated that the polydopamine coated carbon nanotubes would find potential applications in bone tissue engineering and other biomedical areas.

  10. A review paper on biomimetic calcium phosphate coatings

    NARCIS (Netherlands)

    Lin, X.; de Groot, K.; Wang, D.; Hu, Q.; Wismeijer, D.; Liu, Y.

    2015-01-01

    Biomimetic calcium phosphate coatings have been developed for bone regeneration and repair because of their biocompatibility, osteoconductivity, and easy preparation. They can be rendered osteoinductive by incorporating an osteogenic agent, such as bone morphogenetic protein 2 (BMP-2), into the

  11. The Murine Factor H-Related Protein FHR-B Promotes Complement Activation

    Directory of Open Access Journals (Sweden)

    Marcell Cserhalmi

    2017-09-01

    Full Text Available Factor H-related (FHR proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH. While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3, and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

  12. Selectivity in protein degradation during sporulation of Bacillus subtilis

    International Nuclear Information System (INIS)

    Mitani, Takahiko; Kadota, Hajime

    1976-01-01

    The breakdown of cellular protein was investigated in Bacillus subtilis ATCC 6051 labeled with glycine-2- 3 H or L-phenylalanine-U- 14 C at the different stages of vegetative growth and sporulation. The growth of the culture was determined by measuring optical density at 660 nm. The heat-resistant spores were scored by plating after heating at 80 deg C for 10 minutes. A question whether the turnover of glycine-labeled protein is similar to that of phenylalanine-labeled protein was experimentally studied. The patterns obtained with the glycine-labeled protein were different from those of phenylalanine-labeled protein. This was not multiple turnover. The cellular protein which was labeled with glycine at an early stage of sporulation showed rapid degradation, but the degradation of the protein labeled with glycine at later stages did not occur at all. Another question whether the labeled glycine incorporated into cells at the different stages of growth and sporulation was present in the spore coat fraction of matured spores was studied. Experiment demonstrated that the glycine incorporated into cells at the late sporulation stage was mainly utilized for the biosynthesis of the spore coat protein. These data suggest that the spore coat protein which contains relatively large amount of glycine is rarely subject to further degradation. (Iwakiri, K.)

  13. Design of Protein-Coated Carbon Nanotubes Loaded with Hydrophobic Drugs through Sacrificial Templating of Mesoporous Silica Shells.

    Science.gov (United States)

    Fiegel, Vincent; Harlepp, Sebastien; Begin-Colin, Sylvie; Begin, Dominique; Mertz, Damien

    2018-03-26

    One key challenge in the fields of nanomedicine and tissue engineering is the design of theranostic nanoplatforms able to monitor their therapeutic effect by imaging. Among current developed nano-objects, carbon nanotubes (CNTs) were found suitable to combine imaging, photothermal therapy, and to be loaded with hydrophobic drugs. However, a main problem is their resulting low hydrophilicity. To face this problem, an innovative method is developed here, which consists in loading the surface of carbon nanotubes (CNTs) with drugs followed by a protein coating around them. The originality of this method relies on first covering CNTs with a sacrificial template mesoporous silica (MS) shell grafted with isobutyramide (IBAM) binders on which a protein nanofilm is strongly adhered through IBAM-mediated physical cross-linking. This concept is first demonstrated without drugs, and is further improved with the suitable loading of hydrophobic drugs, curcumin (CUR) and camptothecin (CPT), which are retained between the CNTs and human serum albumin (HSA) layer. Such novel nanocomposites with favorable photothermal properties are very promising for theranostic systems, drug delivery, and phototherapy applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. PMEA polymer-coated PVC tubing maintains anti-thrombogenic properties during in vitro whole blood circulation

    NARCIS (Netherlands)

    Yoshizaki, T; Tabuchi, N; Van Oeveren, W; Shibamiya, A; Koyama, T; Sunamori, M

    Poly(2-methoxyethylacrylate) (PMEA) is a new coating material that appears to reduce protein and platelet adsorption. However, the exact performance of PMEA coated circuit remains to be revealed in well-controlled experiments. Therefore, we compared its hemocompatibility with covalent-bound

  15. Pea proteins oral supplementation promotes muscle thickness gains during resistance training: a double-blind, randomized, Placebo-controlled clinical trial vs. Whey protein.

    Science.gov (United States)

    Babault, Nicolas; Païzis, Christos; Deley, Gaëlle; Guérin-Deremaux, Laetitia; Saniez, Marie-Hélène; Lefranc-Millot, Catherine; Allaert, François A

    2015-01-01

    The effects of protein supplementation on muscle thickness and strength seem largely dependent on its composition. The current study aimed at comparing the impact of an oral supplementation with vegetable Pea protein (NUTRALYS®) vs. Whey protein and Placebo on biceps brachii muscle thickness and strength after a 12-week resistance training program. One hundred and sixty one males, aged 18 to 35 years were enrolled in the study and underwent 12 weeks of resistance training on upper limb muscles. According to randomization, they were included in the Pea protein (n = 53), Whey protein (n = 54) or Placebo (n = 54) group. All had to take 25 g of the proteins or placebo twice a day during the 12-week training period. Tests were performed on biceps muscles at inclusion (D0), mid (D42) and post training (D84). Muscle thickness was evaluated using ultrasonography, and strength was measured on an isokinetic dynamometer. Results showed a significant time effect for biceps brachii muscle thickness (P Pea, Whey and Placebo, respectively; P Pea group as compared to Placebo whereas there was no difference between Whey and the two other conditions. Muscle strength also increased with time with no statistical difference between groups. In addition to an appropriate training, the supplementation with pea protein promoted a greater increase of muscle thickness as compared to Placebo and especially for people starting or returning to a muscular strengthening. Since no difference was obtained between the two protein groups, vegetable pea proteins could be used as an alternative to Whey-based dietary products. The present trial has been registered at ClinicalTrials.gov (NCT02128516).

  16. Phorbol ester tumor promoter induced the synthesis of two major cytoplasmic proteins: identity with two proteins induced under heat-shocked and glucose-starved conditions

    International Nuclear Information System (INIS)

    Zhang, H.; Chen, K.Y.; Liu, A.Y.C.

    1987-01-01

    The regulation of specific protein synthesis by the phorbol ester tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was evaluated using the L-8 and C-2 myoblast and the 3T3-L1 fibroblast cell cultures. TPA increased, by 2-4 fold, the synthesis rates of two cytoplasmic proteins with apparent molecular weights of 89,000 and 74,000 as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. The concentration of TPA and the time of incubation needed to elicit this induction was determined to be 10 μg/ml and 20 hrs, respectively. Increasing the concentration of TPA to 100, 200, and 500 ng/ml did not result in a greater magnitude of induction. The possibility that these two TPA-induced proteins may be identical to proteins with similar molecular weights induced under heat-shocked or glucose-starved conditions was evaluated by 1-D and 2-D gel electrophoresis and autoradiography. Results provided evidence that the TPA-induced 89,000- and 74,000-dalton proteins were identical to hsp 89 and hsp 74, 2 out of a set of 8-9 proteins induced under heat shocked conditions. Furthermore, they are identical to two of the set of glucose-regulated proteins induced under a glucose-starved condition

  17. Enhanced ductility in thermally sprayed titania coating synthesized using a nanostructured feedstock

    International Nuclear Information System (INIS)

    Lima, R.S.; Marple, B.R.

    2005-01-01

    Nanostructured and conventional titania (TiO 2 ) feedstock powders were thermally sprayed via high velocity oxy-fuel (HVOF). The microstructure, porosity, Vickers hardness, crack propagation resistance, bond strength (ASTM C633), abrasion behavior (ASTM G65) and the wear scar characteristics of these two types of coatings were analyzed and compared. The coating made from the nanostructured feedstock exhibited a bimodal microstructure, with regions containing particles that were fully molten (conventional matrix) and regions with embedded particles that were semi-molten (nanostructured zones) during the thermal spraying process. The bimodal coating also exhibited higher bond strength and higher wear resistance when compared to the conventional coating. By comparing the wear scars of both coatings (via scanning electron microscopy and roughness measurements) it was observed that when the coatings were subjected to the same abrasive conditions the wear scar of the bimodal coating was smoother, with more plastically deformed regions than the conventional coating. It was concluded that this enhanced ductility of the bimodal coating was caused by its higher toughness. The results suggest that nanostructured zones randomly distributed in the microstructure of the bimodal coating act as crack arresters, thereby enhancing toughness and promoting higher critical depth of cut, which provides a broader plastic deformation range than that exhibited by the conventional coating. This work provides evidence that the enhanced ductility of the bimodal coating is a nanostructured-related property, not caused by any other microstructural artifact

  18. Wear resistance increase of the modified coatings, deposited in the beam of relativistic electrons

    International Nuclear Information System (INIS)

    Poletika, I.M.; Perovskaya, M.V.; Balushkina, M.A.

    2015-01-01

    The 1.5-3 mm thickness coatings have been obtained by vacuum - free electron beam cladding of tungsten carbide on low - carbon steel sub state. The coatings have an increased hardness but low wear resistance. Adding both nickel and titanium carbide to the tungsten carbide results in essentially improving the wear resistance of the coatings due to austenite-promoting effect of nickel and precipitation of fine Tic particles resulting in the formation of the final and nano grain structure. In the layer of weld one can find 30-100 nm grain - size structures. (authors)

  19. Improvement of interfacial interactions using natural polyphenol-inspired tannic acid-coated nanoclay enhancement of soy protein isolate biofilms

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhong; Kang, Haijiao; Zhang, Wei [MOE Key Laboratory of Wooden Material Science and Application, Beijing Forestry University, Beijing, 100083 (China); Beijing Key Laboratory of Wood Science and Engineering, Beijing Forestry University, Beijing, 100083 (China); Zhang, Shifeng, E-mail: shifeng.zhang@bjfu.edu.cn [MOE Key Laboratory of Wooden Material Science and Application, Beijing Forestry University, Beijing, 100083 (China); Beijing Key Laboratory of Wood Science and Engineering, Beijing Forestry University, Beijing, 100083 (China); Li, Jianzhang, E-mail: lijzh@bjfu.edu.cn [MOE Key Laboratory of Wooden Material Science and Application, Beijing Forestry University, Beijing, 100083 (China); Beijing Key Laboratory of Wood Science and Engineering, Beijing Forestry University, Beijing, 100083 (China)

    2017-04-15

    Highlights: • A novel interface of MMT was fabricated by natural polyphenol (TA)-inspired chemistry. • The resultant biomimetic surface exhibited good interface and surface compatibility. • TA can act as a bridge between MMT and SPI to enhance the interfacial interaction. • Surface-modified MMT gets the potential to be used in the modification of SPI biofilms for improving the mechanical properties and water resistance apparently. - Abstract: In this study, a novel and economic surface modification technique for montmorillonite (MMT) nanosheets, a biocompatible coupling cross-linking agent, was developed on an attempt at improving the interfacial adhesion with soy protein isolate (SPI) matrix. Inspired by natural polyphenol, the “green dip-coating” method using tannic acid (TA) to surface-modify MMT (TA@MMT). SPI nanocomposite films modified with MMT or TA@MMT, as well as the control ones, were prepared via the casting method. The TA layer was successfully coated on the MMT surface through the (Fe{sup III}) ions coordination chemistry and the synthetic samples were characterized by the Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), thermogravimetric analysis (TGA), atomic force microscopy (AFM), and transmission electron microscopy (TEM). The compatibility and interfacial interactions between modified MMT and SPI matrix were greatly enhanced by the TA-Fe{sup III} coating on the MMT surface. The mechanical properties, water resistance, and thermal stability of the resultant biofilm were increased accordingly. Compared with that of the unmodified SPI film, the tensile strength of the nanocomposite films modified by the green dip-coating was increased by 113.3%. These SPI-based nanocomposite films showed the favorable potential in terms of food packing applications due to their efficient barriers to water vapor and UV and/or visible light.

  20. Fish gelatin combined with chitosan coating inhibits myofibril degradation of golden pomfret (Trachinotus blochii) fillet during cold storage.

    Science.gov (United States)

    Feng, Xiao; Bansal, Nidhi; Yang, Hongshun

    2016-06-01

    Coating of gelatin and chitosan can improve fish fillet's quality, but the mechanism is not clear. Chitosan/gelatin coatings significantly prevented deterioration of golden pomfret fillet at 4 °C. Chitosan with 7.2% gelatin group showed the best effect on preserving the length of myofibril, which remained greater than 15 μm at day 17 of storage, while for control, chitosan and chitosan combined with 3.6% gelatin group, it was 5.03, 10.04 and 9.02 μm, respectively. The MALDI-TOF MS result revealed that the coatings slowed down the protein deterioration of fillet. On days 13 and 17, the myosin light chain and myoglobin in control group degraded, while the two proteins still existed in chitosan/gelatin coated groups. Overall, the chitosan with 7.2% gelatin coating had the best effect on preserving fillet's quality during storage. The coating may exert its protective effect via inhibiting myofibril degradation within fillet. Copyright © 2016 Elsevier Ltd. All rights reserved.