Complete nucleotide sequence of a novel Hibiscus-infecting Cilevirus from Florida and its relationship with closely associated Cileviruses

USDA-ARS?s Scientific Manuscript database

The complete nucleotide sequence of a recently discovered Florida (FL) isolate of Hibiscus infecting Cilevirus (HiCV) was determined by Sanger sequencing. The movement- and coat- protein gene sequences of the HiCV-FL isolate are more divergent than other genes of the previously sequenced HiCV-HA (Ha...

Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

PubMed Central

Deber, C M; Khan, A R; Li, Z; Joensson, C; Glibowicka, M; Wang, J

1993-01-01

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins. Images Fig. 2 Fig. 4 PMID:8265602

Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

PubMed

Deber, C M; Khan, A R; Li, Z; Joensson, C; Glibowicka, M; Wang, J

1993-12-15

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins.

Nucleotide sequence and phylogenetic analysis of Cucurbit yellow stunting disorder virus RNA 2.

PubMed

Livieratos, Ioannis C; Coutts, Robert H A

2002-06-01

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA 2, a whitefly (Bemisia tabaci)-transmitted closterovirus with a bi-partite genome, is reported. CYSDV RNA 2 is 7,281 nucleotides long and contains the closterovirus hallmark gene array with a similar arrangement to the prototype member of the genus Crinivirus, Lettuce infectious yellows virus (LIYV). CYSDV RNA 2 contains open reading frames (ORFs) potentially encoding in a 5' to 3' direction for proteins of 5 kDa (ORF 1; hydrophobic protein), 62 kDa (ORF 2; heat shock protein 70 homolog, HSP70h), 59 kDa (ORF 3; protein of unknown function), 9 kDa (ORF 4; protein of unknown function), 28.5 kDa (ORF 5; coat protein, CP), 53 kDa (ORF 6; coat protein minor, CPm), and 26.5 kDa (ORF 7; protein of unknown function). Pairwise comparisons of CYSDV RNA 2-encoded proteins (HSP70h, p59 and CPm) among the closteroviruses showed that CYSDV is closely related to LIYV. Phylogenetic analysis based on the amino acid sequence of the HSP70h, indicated that CYSDV clusters with other members of the genus Crinivirus, and it is related to Little cherry virus-1 (LChV-1), but is distinct from the aphid- or mealybug-transmitted closteroviruses.

Hairpin plum pox virus coat protein (hpPPV-CP) structure in 'HoneySweet' C5 plum provides PPV resistance when genetically engineered into plum (Prunus domestica) seedlings

USDA-ARS?s Scientific Manuscript database

The genetically engineered plum 'HoneySweet' (aka C5) has proven to be highly resistant to Plum pox virus (PPV) for over 10 years in field trials. The original vector used for transformation to develop 'HoneySweet' carried a single sense sequence of the full length PPV coat protein (ppv-cp) gene, y...

In Planta Synthesis of Designer-Length Tobacco Mosaic Virus-Based Nano-Rods That Can Be Used to Fabricate Nano-Wires.

PubMed

Saunders, Keith; Lomonossoff, George P

2017-01-01

We have utilized plant-based transient expression to produce tobacco mosaic virus (TMV)-based nano-rods of predetermined lengths. This is achieved by expressing RNAs containing the TMV origin of assembly sequence (OAS) and the sequence of the TMV coat protein either on the same RNA molecule or on two separate constructs. We show that the length of the resulting nano-rods is dependent upon the length of the RNA that possesses the OAS element. By expressing a version of the TMV coat protein that incorporates a metal-binding peptide at its C-terminus in the presence of RNA containing the OAS we have been able to produce nano-rods of predetermined length that are coated with cobalt-platinum. These nano-rods have the properties of defined-length nano-wires that make them ideal for many developing bionanotechnological processes.

In Planta Synthesis of Designer-Length Tobacco Mosaic Virus-Based Nano-Rods That Can Be Used to Fabricate Nano-Wires

PubMed Central

Saunders, Keith; Lomonossoff, George P.

2017-01-01

We have utilized plant-based transient expression to produce tobacco mosaic virus (TMV)-based nano-rods of predetermined lengths. This is achieved by expressing RNAs containing the TMV origin of assembly sequence (OAS) and the sequence of the TMV coat protein either on the same RNA molecule or on two separate constructs. We show that the length of the resulting nano-rods is dependent upon the length of the RNA that possesses the OAS element. By expressing a version of the TMV coat protein that incorporates a metal-binding peptide at its C-terminus in the presence of RNA containing the OAS we have been able to produce nano-rods of predetermined length that are coated with cobalt-platinum. These nano-rods have the properties of defined-length nano-wires that make them ideal for many developing bionanotechnological processes. PMID:28878782

Genetic diversity of the movement and coat protein genes of South American isolates of Prunus necrotic ringspot virus.

PubMed

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.

A Spore Coat Protein, CotS, of Bacillus subtilis Is Synthesized under the Regulation of ςK and GerE during Development and Is Located in the Inner Coat Layer of Spores

PubMed Central

Takamatsu, Hiromu; Chikahiro, Yukari; Kodama, Takeko; Koide, Hidekatsu; Kozuka, Satoshi; Tochikubo, Kunio; Watabe, Kazuhito

1998-01-01

The spore coat of Bacillus subtilis has a unique morphology and consists of polypeptides of different sizes, whose synthesis and assembly are precisely regulated by a cascade of transcription factors and regulatory proteins. We examined the factors that regulate cotS gene expression and CotS assembly into the coat layer of B. subtilis by Northern blot and Western blot analysis. Transcription of cotS mRNA was not detected in sporulating cells of ςK and gerE mutants by Northern blot analysis. By Western blot analysis using anti-CotS antibody, CotS was first detected in protein samples solubilized from wild-type cells at 5 h after the start of sporulation. CotS was not detected in the vegetative cells and spores of a gerE mutant or in the spores of mutants deficient in ςE, ςF, ςG, or ςK. CotS was detected in the sporangium but not in the spores of a cotE mutant. The sequence of the promoter region of cotS was similar to the consensus sequences for binding of ςK and GerE. These results demonstrate that ςK and GerE are required for cotS expression and that CotE is essential for the assembly of CotS in the coat. Immunoelectron microscopic observation using anti-CotS antibody revealed that CotS is located within the spore coat, in particular in the inner coats of dormant spores. PMID:9603889

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

PubMed

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.

Conformational states of mutant M13 coat proteins are regulated by transmembrane residues.

PubMed

Li, Z; Glibowicka, M; Joensson, C; Deber, C M

1993-03-05

Mutational and structural analysis of the 28 viable bacteriophage M13 mutants obtained by randomized mutagenesis of the effective transmembrane (TM) segment of the 50-residue major coat (gene VIII) protein (residues 21-39) demonstrated that M13 coat protein functionality, as reflected by phage viability, is incompatible with an increase in Gly + beta-branched residue content in its TM core. SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy performed in membrane environments on purified mutant coat proteins revealed that these proteins exist in a range of state(s), identified as helical monomers and dimers and polymeric (alpha-helical and/or beta-sheet) species, of which relative populations, and thermally induced conformational transitions, were dependent uniquely upon mutation type and locus. Mutations to relatively polar residues (e.g. G23D, Y24D, Y24H, A27E, I32T, and T36S) stabilized principally monomeric species, while mutants with decreased beta-branched content in the protein TM hydrophobic core (e.g. V29A, V30A, V31A, V31L, and V33A) displayed mainly dimeric species. Mutation of Ile37-->Thr within a "Sternberg-Gullick" consensus sequence of the coat protein TM segment led to a highly oligomerized/polymerized protein. The overall results suggest that TM residues in M13 coat protein are not universal components of a hydrophobic anchor segment per se, but are further selected (i) to impart conformational flexibility to the TM segment through helix destabilization and (ii) to retain the capacity to regulate protein-protein association and packing motifs within membranes.

A review of protein adsorption on bioceramics

PubMed Central

Wang, Kefeng; Zhou, Changchun; Hong, Youliang; Zhang, Xingdong

2012-01-01

Bioceramics, because of its excellent biocompatible and mechanical properties, has always been considered as the most promising materials for hard tissue repair. It is well know that an appropriate cellular response to bioceramics surfaces is essential for tissue regeneration and integration. As the in vivo implants, the implanted bioceramics are immediately coated with proteins from blood and body fluids, and it is through this coated layer that cells sense and respond to foreign implants. Hence, the adsorption of proteins is critical within the sequence of biological activities. However, the biological mechanisms of the interactions of bioceramics and proteins are still not well understood. In this review, we will recapitulate the recent studies on the bioceramic–protein interactions. PMID:23741605

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

PubMed Central

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

Analysis of the beak and feather disease viral genome indicates the existence of several genotypes which have a complex psittacine host specificity.

PubMed

de Kloet, E; de Kloet, S R

2004-12-01

A study was made of the phylogenetic relationships between fifteen complete nucleotide sequences as well as 43 nucleotide sequences of the putative coat protein gene of different strains belonging to the virus species Beak and feather disease virus obtained from 39 individuals of 16 psittacine species. The species included among others, cockatoos ( Cacatuini), African grey parrots ( Psittacus erithacus) and peach-faced lovebirds ( Agapornis roseicollis), which were infected at different geographical locations, within and outside Australia, the native origin of the virus. The derived amino acid sequences of the putative coat protein were highly diverse, with differences between some strains amounting to 50 of the 250 amino acids. Phylogenetic analysis demonstrated that the putative coat gene sequences form six clusters which show a varying degree of psittacine species specificity. Most, but not all strains infecting African grey parrots formed a single cluster as did the strains infecting the cockatoos. Strains infecting the lovebirds clustered with those infecting such Australasian species as Eclectus roratus, Psittacula kramerii and Psephotus haematogaster. Although individual birds included in this study were, where studied, often infected by closely related strains, infection by highly diverged trains was also detected. The possible relationship between BFD viral strains and clinical disease signs is discussed.

Deciphering the mechanisms for targeting and interaction of Arabidopsis Lipid Droplet Associated Protein (LDAP) to the lipid droplet surface

USDA-ARS?s Scientific Manuscript database

We recently identified a new class of lipid-droplet associated proteins (LDAPs) in plants that share extensive sequence similarity with abundant structural proteins that coat rubber particles in rubber-producing plants. A majority of higher plants, however, including those that do not produce rubber...

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs.

PubMed

Wang, C; Deber, C M

2000-05-26

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.

Species specific identification of spore-producing microbes using the gene sequence of small acid-soluble spore coat proteins for amplification based diagnostics

DOEpatents

McKinney, Nancy

2002-01-01

PCR (polymerase chain reaction) primers for the detection of certain Bacillus species, such as Bacillus anthracis. The primers specifically amplify only DNA found in the target species and can distinguish closely related species. Species-specific PCR primers for Bacillus anthracis, Bacillus globigii and Clostridium perfringens are disclosed. The primers are directed to unique sequences within sasp (small acid soluble protein) genes.

Self-assembling triblock proteins for biofunctional surface modification

NASA Astrophysics Data System (ADS)

Fischer, Stephen E.

Despite the tremendous promise of cell/tissue engineering, significant challenges remain in engineering functional scaffolds to precisely regulate the complex processes of tissue growth and development. As the point of contact between the cells and the scaffold, the scaffold surface plays a major role in mediating cellular behaviors. In this dissertation, the development and utility of self-assembling, artificial protein hydrogels as biofunctional surface modifiers is described. The design of these recombinant proteins is based on a telechelic triblock motif, in which a disordered polyelectrolyte central domain containing embedded bioactive ligands is flanked by two leucine zipper domains. Under moderate conditions of temperature and pH, the leucine zipper end domains form amphiphilic alpha-helices that reversibly associate into homo-trimeric aggregates, driving hydrogel formation. Moreover, the amphiphilic nature of these helical domains enables surface adsorption to a variety of scaffold materials to form biofunctional protein coatings. The nature and stability of these coatings in various solution conditions, and their interaction with mammalian cells is the primary focus of this dissertation. In particular, triblock protein coatings functionalized with cell recognition sequences are shown to produce well-defined surfaces with precise control over ligand density. The impact of this is demonstrated in multiple cell types through ligand density-dependent cell-substrate interactions. To improve the stability of these physically self-assembled coatings, two covalent crosslinking strategies are described---one in which a zero-length chemical crosslinker (EDC) is utilized and a second in which disulfide bonds are engineered into the recombinant proteins. These targeted crosslinking approaches are shown to increase the stability of surface adsorbed protein layers with minimal effect on the presentation of many bioactive ligands. Finally, to demonstrate the versatility of the triblock protein hydrogels, and the ease of introducing multiple functionalities to a substrate surface, a surface coating is tailored for neural stem cell culture in order to improve proliferation on the scaffold, while maintaining the stem cell phenotype. These studies demonstrate the unique advantages of genetic engineering over traditional techniques for surface modification. In addition to their unmatched sequence fidelity, recombinant proteins can easily be modified with bioactive ligands and their organization into coherent, supramolecular structures mimics natural self-assembly processes.

Biofunctionalization of silica-coated magnetic particles mediated by a peptide

NASA Astrophysics Data System (ADS)

Care, Andrew; Chi, Fei; Bergquist, Peter L.; Sunna, Anwar

2014-08-01

A linker peptide sequence with affinity to silica-containing materials was fused to Streptococcus protein G', an antibody-binding protein. This recombinant fusion protein, linker-protein G (LPG) was produced in E. coli and exhibited strong affinity to silica-coated magnetic particles and was able to bind to them at different pHs, indicating a true pH-independent binding. LPG was used as an anchorage point for the oriented immobilization of antibodies onto the surface of the particles. These particle-bound "LPG-Antibody complexes" mediated the binding and recovery of different cell types (e.g., human stem cells, Legionella, Cryptosporidium and Giardia), enabling their rapid and simple visualization and identification. This strategy was used also for the efficient capture of Cryptosporidium oocysts from water samples. These results demonstrate that LPG can mediate the direct biofunctionalization of silica-coated magnetic particles without the need for complex surface chemical modification.

Characterization of the yrbA Gene of Bacillus subtilis, Involved in Resistance and Germination of Spores

PubMed Central

Takamatsu, Hiromu; Kodama, Takeko; Nakayama, Tatsuo; Watabe, Kazuhito

1999-01-01

Insertional inactivation of the yrbA gene of Bacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with l-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in various sig mutants indicated that yrbA was transcribed by RNA polymerase with ςE beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the −35 (TCATAAC) and −10 (CATATGT) regions were similar to the consensus sequences of genes recognized by ςE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination of yrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination. PMID:10438771

Distribution, genetic diversity and recombination analysis of Citrus tristeza virus of India

USDA-ARS?s Scientific Manuscript database

Citrus tristeza virus (CTV) isolates representing all the citrus growing geographical zones of India were analyzed for sequence of the 5'ORF1a fragments of the partial LProI domain and for the coat protein (CP) gene. The sequences were compared with previously reported Indian and CTV genotypes from...

Armored RNA Technology for Production of Ribonuclease-Resistant Viral RNA Controls and Standards

PubMed Central

Pasloske, Brittan L.; Walkerpeach, Cindy R.; Obermoeller, R. Dawn; Winkler, Matthew; DuBois, Dwight B.

1998-01-01

The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. “Armored RNA” is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at −20°C or for 60 days at 4°C. Additionally, Armored RNA preparations are homogeneous and noninfectious. PMID:9817878

Evidence for the role of basic amino acids in the coat protein arm region of Cucumber necrosis virus in particle assembly and selective encapsidation of viral RNA.

PubMed

Alam, Syed Benazir; Reade, Ron; Theilmann, Jane; Rochon, D'Ann

2017-12-01

Cucumber necrosis virus (CNV) is a T = 3 icosahedral virus with a (+)ssRNA genome. The N-terminal CNV coat protein arm contains a conserved, highly basic sequence ("KGRKPR"), which we postulate is involved in RNA encapsidation during virion assembly. Seven mutants were constructed by altering the CNV "KGRKPR" sequence; the four basic residues were mutated to alanine individually, in pairs, or in total. Virion accumulation and vRNA encapsidation were significantly reduced in mutants containing two or four substitutions and virion morphology was also affected, where both T = 1 and intermediate-sized particles were produced. Mutants with two or four substitutions encapsidated significantly greater levels of truncated RNA than that of WT, suggesting that basic residues in the "KGRKPR" sequence are important for encapsidation of full-length CNV RNA. Interestingly, "KGRKPR" mutants also encapsidated relatively higher levels of host RNA, suggesting that the "KGRKPR" sequence also contributes to selective encapsidation of CNV RNA. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Human endomembrane H+ pump strongly resembles the ATP-synthetase of Archaebacteria.

PubMed Central

Südhof, T C; Fried, V A; Stone, D K; Johnston, P A; Xie, X S

1989-01-01

Preparations of mammalian H+ pumps that acidify intracellular vesicles contain eight or nine polypeptides, ranging in size from 116 to 17 kDa. Biochemical analysis indicates that the 70- and 58-kDa polypeptides are subunits critical for ATP hydrolysis. The amino acid sequences of the major catalytic subunits (58 and 70 kDa) of the endomembrane H+ pump are unknown from animal cells. We report here the complete sequence of the 58-kDa subunit derived from a human kidney cDNA clone and partial sequences of the 70- and 58-kDa subunits purified from clathrin-coated vesicles of bovine brain. The amino acid sequences of both proteins strongly resemble the sequences of the corresponding subunits of the vacuolar H+ pumps of Archaebacteria, plants, and fungi. The archaebacterial enzyme is believed to use a H+ gradient to synthesize ATP. Thus, a common ancestral protein has given rise to a H+ pump that synthesizes ATP in one organism and hydrolyzes it in another and is highly conserved from prokaryotes to humans. The same pump appears to mediate the acidification of intracellular organelles, including coated vesicles, lysosomes, and secretory granules, as well as extracellular fluids such as urine. PMID:2527371

Molecular and Cellular Mechanisms for the Interaction between Gold Nanoparticles and Neuroimmune Cells Based on Size, Shape, and Charge

DTIC Science & Technology

2014-04-25

IgG secretion. 2.3 Designing of Synthetic peptide The immunogenic peptides against the foot and mouth disease virus ( FMDV ) were designed and...synthesized based on viral protein 1 of type O FMDV . The amino acid sequence for pFMDV is NGSSKYGDTSTNNVRGDLQVLAQKAERTLC. An extra cysteine was added...peptides were synthesized based on the amino acid sequence of the VP1 coat protein of the FMDV (table 1). The peptide pFMDVD (19 amino acids in length

Genetic variations associated with six-white-point coat pigmentation in Diannan small-ear pigs

PubMed Central

Lü, Meng-Die; Han, Xu-Man; Ma, Yun-Fei; Irwin, David M.; Gao, Yun; Deng, Jia-Kun; Adeola, Adeniyi C.; Xie, Hai-Bing; Zhang, Ya-Ping

2016-01-01

A common phenotypic difference among domestic animals is variation in coat color. Six-white-point is a pigmentation pattern observed in varying pig breeds, which seems to have evolved through several different mechanistic pathways. Herein, we re-sequenced whole genomes of 31 Diannan small-ear pigs from China and found that the six-white-point coat color in Diannan small-ear pigs is likely regulated by polygenic loci, rather than by the MC1R locus. Strong associations were observed at three loci (EDNRB, CNTLN, and PINK1), which explain about 20 percent of the total coat color variance in the Diannan small-ear pigs. We found a mutation that is highly differentiated between six-white-point and black Diannan small-ear pigs, which is located in a conserved noncoding sequence upstream of the EDNRB gene and is a putative binding site of the CEBPB protein. This study advances our understanding of coat color evolution in Diannan small-ear pigs and expands our traditional knowledge of coat color being a monogenic trait. PMID:27270507

Differentiation of closely related but biologically distinct cherry isolates of Prunus necrotic ringspot virus by polymerase chain reaction.

PubMed

Hammond, R W; Crosslin, J M; Pasini, R; Howell, W E; Mink, G I

1999-07-01

Prunus necrotic ringspot ilarvirus (PNRSV) exists as a number of biologically distinct variants which differ in host specificity, serology, and pathology. Previous nucleotide sequence alignment and phylogenetic analysis of cloned reverse transcription-polymerase chain reaction (RT-PCR) products of several biologically distinct sweet cherry isolates revealed correlations between symptom type and the nucleotide and amino acid sequences of the 3a (putative movement protein) and 3b (coat protein) open reading frames. Based upon this analysis, RT-PCR assays have been developed that can identify isolates displaying different symptoms and serotypes. The incorporation of primers in a multiplex PCR protocol permits rapid detection and discrimination among the strains. The results of PCR amplification using type-specific primers that amplify a portion of the coat protein gene demonstrate that the primer-selection procedure developed for PNRSV constitutes a reliable method of viral strain discrimination in cherry for disease control and will also be useful for examining biological diversity within the PNRSV virus group.

Prunus necrotic ringspot ilarvirus: nucleotide sequence of RNA3 and the relationship to other ilarviruses based on coat protein comparison.

PubMed

Guo, D; Maiss, E; Adam, G; Casper, R

1995-05-01

The RNA3 of prunus necrotic ringspot ilarvirus (PNRSV) has been cloned and its entire sequence determined. The RNA3 consists of 1943 nucleotides (nt) and possesses two large open reading frames (ORFs) separated by an intergenic region of 74 nt. The 5' proximal ORF is 855 nt in length and codes for a protein of molecular mass 31.4 kDa which has homologies with the putative movement protein of other members of the Bromoviridae. The 3' proximal ORF of 675 nt is the cistron for the coat protein (CP) and has a predicted molecular mass of 24.9 kDa. The sequence of the 3' non-coding region (NCR) of PNRSV RNA3 showed a high degree of similarity with those of tobacco streak virus (TSV), prune dwarf virus (PDV), apple mosaic virus (ApMV) and also alfalfa mosaic virus (AIMV). In addition it contained potential stem-loop structures with interspersed AUGC motifs characteristic for ilar- and alfamoviruses. This conserved primary and secondary structure in all 3' NCRs may be responsible for the interaction with homologous and heterologous CPs and subsequent activation of genome replication. The CP gene of an ApMV isolate (ApMV-G) of 657 nt has also been cloned and sequenced. Although ApMV and PNRSV have a distant serological relationship, the deduced amino acid sequences of their CPs have an identity of only 51.8%. The N termini of PNRSV and ApMV CPs have in common a zinc-finger motif and the potential to form an amphipathic helix.

Expanding the versatility of phage display I: efficient display of peptide-tags on protein VII of the filamentous phage.

PubMed

Løset, Geir Åge; Bogen, Bjarne; Sandlie, Inger

2011-02-24

Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display. Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS(6) or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether. Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations.

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

PubMed

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.

The complete nucleotide sequence and genome organization of a novel betaflexivirus infecting Citrullus lanatus.

PubMed

Xin, Min; Zhang, Peipei; Liu, Wenwen; Ren, Yingdang; Cao, Mengji; Wang, Xifeng

2017-10-01

The complete nucleotide sequence of a novel positive single-stranded (+ss) RNA virus, tentatively named watermelon virus A (WVA), was determined using a combination of three methods: RNA sequencing, small RNA sequencing, and Sanger sequencing. The full genome of WVA is comprised of 8,372 nucleotides (nt), excluding the poly (A) tail, and contains four open reading frames (ORFs). The largest ORF, ORF1 encodes a putative replication-associated polyprotein (RP) with three conserved domains. ORF2 and ORF4 encode a movement protein (MP) and coat protein (CP), respectively. The putative product encoded by ORF3, of an estimated molecular mass of 25 kDa, has no significant similarity with other proteins. Identity and phylogenetic analysis indicate that WVA is a new virus, closely related to members of the family Betaflexiviridae. However, the final taxonomic allocation of WVA within the family is yet to be determined.

Biological Nanoplatforms for Self-Assembled Electronics

DTIC Science & Technology

2015-03-24

as M13 , a virus that infects Escherichia coli. Approximately one billion different amino acid sequences are displayed on different viruses in the...sequence when contained within a phage M13 coat protein sequence, not chemically linked to the surface of phage MS2 VLPs. Thus, binding properties may...gallium arsenide in a bacteriophage M13 phage display library, MS2 VLPs modified with the metal binding peptides do not display the same activity

Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Hip1R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo.

PubMed

Chen, Chih-Ying; Brodsky, Frances M

2005-02-18

Clathrin heavy and light chains form triskelia, which assemble into polyhedral coats of membrane vesicles that mediate transport for endocytosis and organelle biogenesis. Light chain subunits regulate clathrin assembly in vitro by suppressing spontaneous self-assembly of the heavy chains. The residues that play this regulatory role are at the N terminus of a conserved 22-amino acid sequence that is shared by all vertebrate light chains. Here we show that these regulatory residues and others in the conserved sequence mediate light chain interaction with Hip1 and Hip1R. These related proteins were previously found to be enriched in clathrin-coated vesicles and to promote clathrin assembly in vitro. We demonstrate Hip1R binding preference for light chains associated with clathrin heavy chain and show that Hip1R stimulation of clathrin assembly in vitro is blocked by mutations in the conserved sequence of light chains that abolish interaction with Hip1 and Hip1R. In vivo overexpression of a fragment of clathrin light chain comprising the Hip1R-binding region affected cellular actin distribution. Together these results suggest that the roles of Hip1 and Hip1R in affecting clathrin assembly and actin distribution are mediated by their interaction with the conserved sequence of clathrin light chains.

(Mechanisms of inhibition of viral replication in plants)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1991-01-01

During the last year we have made a number of important observations in the fields of virology and plant molecular biology. By directly sequencing Tomato Mosaic Virus (ToMV) movement genes, previously undetected sequence alterations common to specific viral strains were found. The difficulty in regenerating transgenic tomato plants containing the Tm-2 gene was overcome. Tobacco plants transformed with Cucumber Mosaic Virus (CMV) are being characterized. Analysis of transgenic tobacco plants expressing CMV coat protein have shown no correlation between coat protein expression and level of resistance. Specific amino acid changes have been found to correlate with CMV resistance breaking andmore » degree of pathogenicity. Satellite RNAs are shown to be too unstable for use as a biological control agent. The aphid transmission domain CMV has been localized to one (or more) of three amino acids; constructs have been made to determine the exact amino acids involved. 15 refs.« less

Putative recombination events and evolutionary history of five economically important viruses of fruit trees based on coat protein-encoding gene sequence analysis.

PubMed

Boulila, Moncef

2010-06-01

To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

Characterization of Hungarian isolates of zucchini yellow mosaic virus (ZYMV, potyvirus) transmitted by seeds of Cucurbita pepo var Styriaca.

PubMed

Tóbiás, István; Palkovics, László

2003-04-01

Zucchini yellow mosaic virus (ZYMV) has emerged as an important pathogen of cucurbits within the last few years in Hungary. The Hungarian isolates show a high biological variability, have specific nucleotide and amino acid sequences in the N-terminal region of coat protein and form a distinct branch in the phylogenetic tree. The virus is spread very efficiently in the field by several aphid species in a non-persistent manner. It can be transmitted by seed in holl-less seeded oil pumpkin (Cucurbita pepo (L) var Styriaca), although at a very low rate. Three isolates from seed transmission assay experiments were chosen and their nucleotide sequences of coat proteins have been compared with the available CP sequences of ZYMV. According to the sequence analysis, the Hungarian isolates belong to the Central European branch in the phylogenetic tree and, together with the ZYMV isolates from Austria and Slovenia, share specific amino acids at positions 16, 17, 27 and 37 which are characteristic only to these isolates. The phylogenetic tree suggests the common origin of distantly distributed isolates which can be attributed to widespread seed transmission.

Lima bean (Phaseolus lunatus) seed coat phaseolin is detrimental to the cowpea weevil (Callosobruchus maculatus).

PubMed

Moraes, R A; Sales, M P; Pinto, M S; Silva, L B; Oliveira, A E; Machado, O L; Fernandes, K V; Xavier-Filho, J

2000-02-01

The presence of phaseolin (a vicilin-like 7S storage globulin) peptides in the seed coat of the legume Phaseolus lunatus L. (lima bean) was demonstrated by N-terminal amino acid sequencing. Utilizing an artificial seed system assay we showed that phaseolin, isolated from both cotyledon and testa tissues of P. lunatus, is detrimental to the nonhost bruchid Callosobruchus maculatus (F) (cowpea weevil) with ED50 of 1.7 and 3.5%, respectively. The level of phaseolin in the seed coat (16.7%) was found to be sufficient to deter larval development of this bruchid. The expression of a C. maculatus-detrimental protein in the testa of nonhost seeds suggests that the protein may have played a significant role in the evolutionary adaptation of bruchids to legume seeds.

Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin.

PubMed

Scheele, Urte; Alves, Jurgen; Frank, Ronald; Duwel, Michael; Kalthoff, Christoph; Ungewickell, Ernst

2003-07-11

Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.

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

PubMed

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.

Bypassing bacterial infection in phage display by sequencing DNA released from phage particles.

PubMed

Villequey, Camille; Kong, Xu-Dong; Heinis, Christian

2017-11-01

Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type. A potential strategy for bypassing the bacterial infection step is to directly sequence DNA extracted from phage particles after a single round of phage panning using high-throughput sequencing. In this work, we have quantified the fraction of phage clones that can be identified by directly sequencing DNA from phage particles. The results show that the DNA of essentially all of the phage particles can be 'decoded', and that the sequence coverage for mutants equals that of amplified DNA extracted from cells infected with wild-type phage. This procedure is particularly attractive for selections with phage that have a compromised infection capacity, and it may allow phage display to be performed with particles that are not infective at all. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genetic variation of coat protein gene among the isolates of Rice tungro spherical virus from tungro-endemic states of the India.

PubMed

Mangrauthia, Satendra K; Malathi, P; Agarwal, Surekha; Ramkumar, G; Krishnaveni, D; Neeraja, C N; Madhav, M Sheshu; Ladhalakshmi, D; Balachandran, S M; Viraktamath, B C

2012-06-01

Rice tungro disease, one of the major constraints to rice production in South and Southeast Asia, is caused by a combination of two viruses: Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV). The present study was undertaken to determine the genetic variation of RTSV population present in tungro endemic states of Indian subcontinent. Phylogenetic analysis based on coat protein sequences showed distinct divergence of Indian RTSV isolates into two groups; one consisted isolates from Hyderabad (Andhra Pradesh), Cuttack (Orissa), and Puducherry and another from West Bengal, Coimbatore (Tamil Nadu), and Kanyakumari (Tamil Nadu). The results obtained from phylogenetic study were further supported with the SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. In addition, sequence difference count matrix revealed 2-68 nucleotides differences among all the Indian RTSV isolates taken in this study. However, at the protein level these differences were not significant as revealed by Ka/Ks ratio calculation. Sequence identity at nucleotide and amino acid level was 92-100% and 97-100%, respectively, among Indian isolates of RTSV. Understanding of the population structure of RTSV from tungro endemic regions of India would potentially provide insights into the molecular diversification of this virus.

Molecular characterization of two prunus necrotic ringspot virus isolates from Canada.

PubMed

Cui, Hongguang; Hong, Ni; Wang, Guoping; Wang, Aiming

2012-05-01

We determined the entire RNA1, 2 and 3 sequences of two prunus necrotic ringspot virus (PNRSV) isolates, Chr3 from cherry and Pch12 from peach, obtained from an orchard in the Niagara Fruit Belt, Canada. The RNA1, 2 and 3 of the two isolates share nucleotide sequence identities of 98.6%, 98.4% and 94.5%, respectively. Their RNA1- and 2-encoded amino acid sequences are about 98% identical to the corresponding sequences of a cherry isolate, CH57, the only other PNRSV isolate with complete RNA1 and 2 sequences available. Phylogenetic analysis of the coat protein and movement protein encoded by RNA3 of Pch12 and Chr3 and published PNRSV isolates indicated that Chr3 belongs to the PV96 group and Pch12 belongs to the PV32 group.

Two missense mutations in melanocortin 1 receptor (MC1R) are strongly associated with dark ventral coat color in reindeer (Rangifer tarandus).

PubMed

Våge, D I; Nieminen, M; Anderson, D G; Røed, K H

2014-10-01

The protein-coding region of melanocortin 1 receptor (MC1R) was sequenced to identify potential variation affecting coat color in reindeer (Rangifer tarandus). A T→C sequence variation at nucleotide position 218 (c.218T>C) causing an amino acid (aa) change from methionine to threonine at aa position 73 (p.Met73Thr) was identified. In addition, a T→G sequence variation was found at nucleotide position 839 (c.839T>G), causing phenylalanine to be exchanged by cysteine at aa position 280 (p.Phe280Cys). The two sequence variants (c.218C and c.839G) were found to be closely associated with a darker belly coat compared with animals not having any of these two variants. The aa acid change p.Met73Thr affects the same position as p.Met73Lys previously reported to give constitutive activation of MC1R in black sheep (Ovis aries), whereas p.Phe280Cys is identical to one of two variants previously reported to be associated with dark coat color in Arctic fox (Alopex lagopus), supporting that the two variants found in reindeer are functional. The complete absence of Thr73 and Cys280 among the 51 wild reindeer analyzed provides some evidence that these variants are more common in the domestic herds. © 2014 Stichting International Foundation for Animal Genetics.

A dual host vector for Fab phage display and expression of native IgG in mammalian cells.

PubMed

Tesar, Devin; Hötzel, Isidro

2013-10-01

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity.

Genetic diversity of rice tungro spherical virus in tungro-endemic provinces of the Philippines and Indonesia.

PubMed

Azzam, O; Yambao, M L; Muhsin, M; McNally, K L; Umadhay, K M

2000-01-01

The two adjacent genes of coat protein 1 and 2 of rice tungro spherical virus (RTSV) were amplified from total RNA extracts of serologically indistinguishable field isolates from the Philippines and Indonesia, using reverse transcriptase polymerase chain reaction (RT-PCR). Digestion with HindIII and BstYI restriction endonucleases differentiated the amplified DNA products into eight distinct coat protein genotypes. These genotypes were then used as indicators of virus diversity in the field. Inter- and intra-site diversities were determined over three cropping seasons. At each of the sites surveyed, one or two main genotypes prevailed together with other related minor or mixed genotypes that did not replace the main genotype over the sampling time. The cluster of genotypes found at the Philippines sites was significantly different from the one at the Indonesia sites, suggesting geographic isolation for virus populations. Phylogenetic studies based on the nucleotide sequences of 38 selected isolates confirm the spatial distribution of RTSV virus populations but show that gene flow may occur between populations. Under the present conditions, rice varieties do not seem to exert selective pressure on the virus populations. Based on the selective constraints in the coat protein amino acid sequences and the virus genetic composition per site, a negative selection model followed by random-sampling events due to vector transmissions is proposed to explain the inter-site diversity observed.

A Motif in the Clathrin Heavy Chain Required for the Hsc70/Auxilin Uncoating Reaction

PubMed Central

Rapoport, Iris; Boll, Werner; Yu, Anan; Böcking, Till

2008-01-01

The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent “disassembly enzyme” for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631–1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP- and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70–facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone. PMID:17978091

Variability and molecular typing of the woody-tree infecting prunus necrotic ringspot ilarvirus.

PubMed

Vasková, D; Petrzik, K; Karesová, R

2000-01-01

The 3'-part of the movement protein gene, the intergenic region and the complete coat protein gene of sixteen isolates of Prunus necrotic ringspot virus (PNRSV) from five different host species from the Czech Republic were sequenced in order to search for the bases of extensive variability of viroses caused by this pathogen. According to phylogenetic analyses all the 46 isolates sequenced to date split into three main groups, which correlated to a certain extend with their geographic origin. Modelled serological properties showed that all the new isolates belong to one serotype.

A case study to determine the geographical origin of unknown GM papaya in routine food sample analysis, followed by identification of papaya events 16-0-1 and 18-2-4.

PubMed

Prins, Theo W; Scholtens, Ingrid M J; Bak, Arno W; van Dijk, Jeroen P; Voorhuijzen, Marleen M; Laurensse, Emile J; Kok, Esther J

2016-12-15

During routine monitoring for GMOs in food in the Netherlands, papaya-containing food supplements were found positive for the genetically modified (GM) elements P-35S and T-nos. The goal of this study was to identify the unknown and EU unauthorised GM papaya event(s). A screening strategy was applied using additional GM screening elements including a newly developed PRSV coat protein PCR. The detected PRSV coat protein PCR product was sequenced and the nucleotide sequence showed identity to PRSV YK strains indigenous to China and Taiwan. The GM events 16-0-1 and 18-2-4 could be identified by amplifying and sequencing events-specific sequences. Further analyses showed that both papaya event 16-0-1 and event 18-2-4 were transformed with the same construct. For use in routine analysis, derived TaqMan qPCR methods for events 16-0-1 and 18-2-4 were developed. Event 16-0-1 was detected in all samples tested whereas event 18-2-4 was detected in one sample. This study presents a strategy for combining information from different sources (literature, patent databases) and novel sequence data to identify unknown GM papaya events. Copyright © 2016 Elsevier Ltd. All rights reserved.

Adaptive Covariation between the Coat and Movement Proteins of Prunus Necrotic Ringspot Virus

PubMed Central

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

2006-01-01

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. PMID:16731922

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

PubMed

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.

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

PubMed

Dai, Xinghong; Li, Zhihai; Lai, Mason; Shu, Sara; Du, Yushen; Zhou, Z Hong; Sun, Ren

2017-01-05

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification.

PubMed

Sinclair, Robert M; Ravantti, Janne J; Bamford, Dennis H

2017-04-15

Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids. Copyright © 2017 Sinclair et al.

Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification

PubMed Central

Sinclair, Robert M.; Ravantti, Janne J.

2017-01-01

ABSTRACT Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids. PMID:28122979

Characterization, genetic diversity, and evolutionary link of Cucumber mosaic virus strain New Delhi from India.

PubMed

Koundal, Vikas; Haq, Qazi Mohd Rizwanul; Praveen, Shelly

2011-02-01

The genome of Cucumber mosaic virus New Delhi strain (CMV-ND) from India, obtained from tomato, was completely sequenced and compared with full genome sequences of 14 known CMV strains from subgroups I and II, for their genetic diversity. Sequence analysis suggests CMV-ND shares maximum sequence identity at the nucleotide level with a CMV strain from Taiwan. Among all 15 strains of CMV, the encoded protein 2b is least conserved, whereas the coat protein (CP) is most conserved. Sequence identity values and phylogram results indicate that CMV-ND belongs to subgroup I. Based on the recombination detection program result, it appears that CMV is prone to recombination, and different RNA components of CMV-ND have evolved differently. Recombinational analysis of all 15 CMV strains detected maximum recombination breakpoints in RNA2; CP showed the least recombination sites.

Comprehensive mutational analysis of the M13 major coat protein: improved scaffolds for C-terminal phage display.

PubMed

Held, Heike A; Sidhu, Sachdev S

2004-07-09

A peptide was fused to the C terminus of the M13 bacteriophage major coat protein (P8), and libraries of P8 mutants were screened to select for variants that displayed the peptide with high efficiency. Over 600 variants were sequenced to compile a comprehensive database of P8 sequence diversity compatible with assembly into the wild-type phage coat. The database reveals that, while the alpha-helical P8 molecule was highly tolerant to mutations, certain functional epitopes were required for efficient incorporation. Three hydrophobic epitopes were located approximately equidistantly along the length of the alpha-helix. In addition, a positively charged epitope was required directly opposite the most C-terminal hydrophobic epitope and on the same side as the other two epitopes. Both ends of the protein were highly tolerant to mutations, consistent with the use of P8 as a scaffold for both N and C-terminal phage display. Further rounds of selection were used to enrich for P8 variants that supported higher levels of C-terminal peptide display. The largest improvements in display resulted from mutations around the junction between P8 and the C-terminal linker, and additional mutations in the N-terminal region were selected for further improvements in display. The best P8 variants improved C-terminal display more than 100-fold relative to the wild-type, and these variants could support the simultaneous display of N and C-terminal fusions. These finding provide information on the requirements for filamentous phage coat assembly, and provide improved scaffolds for phage display technology. Copyright 2004 Elsevier Ltd.

The complete genome sequence and genetic analysis of ΦCA82 a novel uncultured microphage from the turkey gastrointestinal system

PubMed Central

2011-01-01

The genomic DNA sequence of a novel enteric uncultured microphage, ΦCA82 from a turkey gastrointestinal system was determined utilizing metagenomics techniques. The entire circular, single-stranded nucleotide sequence of the genome was 5,514 nucleotides. The ΦCA82 genome is quite different from other microviruses as indicated by comparisons of nucleotide similarity, predicted protein similarity, and functional classifications. Only three genes showed significant similarity to microviral proteins as determined by local alignments using BLAST analysis. ORF1 encoded a predicted phage F capsid protein that was phylogenetically most similar to the Microviridae ΦMH2K member's major coat protein. The ΦCA82 genome also encoded a predicted minor capsid protein (ORF2) and putative replication initiation protein (ORF3) most similar to the microviral bacteriophage SpV4. The distant evolutionary relationship of ΦCA82 suggests that the divergence of this novel turkey microvirus from other microviruses may reflect unique evolutionary pressures encountered within the turkey gastrointestinal system. PMID:21714899

A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format.

PubMed

Beer, Meike V; Rech, Claudia; Diederichs, Sylvia; Hahn, Kathrin; Bruellhoff, Kristina; Möller, Martin; Elling, Lothar; Groll, Jürgen

2012-04-01

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a /sup 13/C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D/sub 2/O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H/sub 2/O solutions; in 1:1 H/sub 2/O/D/sub 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 /sup 13/C at the peptide carbonyls ofmore » 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.« less

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

PubMed

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.

ZP Domain Proteins in the Abalone Egg Coat Include a Paralog of VERL under Positive Selection That Binds Lysin and 18-kDa Sperm Proteins

PubMed Central

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

Involvement of Superoxide Dismutase in Spore Coat Assembly in Bacillus subtilis

PubMed Central

Henriques, Adriano O.; Melsen, Lawrence R.; Moran, Charles P.

1998-01-01

Endospores of Bacillus subtilis are enclosed in a proteinaceous coat which can be differentiated into a thick, striated outer layer and a thinner, lamellar inner layer. We found that the N-terminal sequence of a 25-kDa protein present in a preparation of spore coat proteins matched that of the Mn-dependent superoxide dismutase (SOD) encoded by the sodA locus. sodA is transcribed throughout the growth and sporulation of a wild-type strain and is responsible for the SOD activity detected in total cell extracts prepared from B. subtilis. Disruption of the sodA locus produced a mutant that lacked any detectable SOD activity during vegetative growth and sporulation. The sodA mutant was not impaired in the ability to form heat- or lysozyme-resistant spores. However, examination of the coat layers of sodA mutant spores revealed increased extractability of the tyrosine-rich outer coat protein CotG. We showed that this condition was not accompanied by augmented transcription of the cotG gene in sporulating cells of the sodA mutant. We conclude that SodA is required for the assembly of CotG into the insoluble matrix of the spore and suggest that CotG is covalently cross-linked into the insoluble matrix by an oxidative reaction dependent on SodA. Ultrastructural analysis revealed that the inner coat formed by a sodA mutant was incomplete. Moreover, the outer coat lacked the characteristic striated appearance of wild-type spores, a pattern that was accentuated in a cotG mutant. These observations suggest that the SodA-dependent formation of the insoluble matrix containing CotG is largely responsible for the striated appearance of this coat layer. PMID:9573176

[Preparation and activity validation of PP7 bacteriophage-like particles displaying PAP114-128 peptide].

PubMed

Sun, Yanli; Sun, Yanhua

2016-10-01

Objective To obtain the PP7 bacteriophage-like particles carrying the peptide of prostatic acid phosphatase PAP 114-128 , and prove that they retain the original biological activity. Methods First, the plasmid pETDuet-2PP7 was constructed as follows: the gene of PP7 coat protein dimer was amplified by gene mutation combined with overlapping PCR technology, and inserted into the vector pETDuet-1. Following that, the plasmid pETDuet-2PP7-PAP 114-128 was constructed as follows: the PP7 coat protein gene carrying the coding gene of PAP 114-128 peptide was amplified using PCR, and then inserted into the vector pETDuet-2PP7. Both pETDuet-2PP7 and pETDuet-2PP7-PAP 114-128 were transformed into E.coli and expressed. The expression product was verified by SDS-PAGE, double immunodiffusion assay and ELISA. Results The gene fragment of PP7 coat protein dimer was obtained by overlapping PCR using Ex Taq DNA polymerase, and the antigenicity of its expression product was the same as that of the coat protein of wild-type PP7 bacteriophage. Moreover, the PAP 114-128 peptide epitope that was displayed on the surface of PP7 bacteriophage was identical with the corresponding epitope of natural human PAP, and it was able to induce high levels of antibodies. Conclusion The gene of PP7 coat protein dimer with repeated sequences can be prepared by gene mutation combined with overlapping PCR. Based on this, PP7 bacteriophage-like particles carrying PAP peptide can be prepared, which not only solves the problem of the instability of the peptides, but also lays a foundation for the study on their delivery and function.

The effects of variations in the number and sequence of targeting signals on nuclear uptake

PubMed Central

1988-01-01

To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors. It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals. PMID:3170630

Occurrence of Cucumber mosaic virus on vanilla (Vanilla planifolia Andrews) in India.

PubMed

Madhubala, R; Bhadramurthy, V; Bhat, A I; Hareesh, P S; Retheesh, S T; Bhai, R S

2005-06-01

Cucumber mosaic virus (CMV) causing mosaic, leaf distortion and stunting of vanilla (Vanilla planifolia Andrews) in India was characterized on the basis of biological and coat protein (CP) nucleotide sequence properties. In mechanical inoculation tests, the virus was found to infect members of Chenopodiaceae, Cucurbitaceae, Fabaceae and Solanaceae. Nicotiana benthamiana was found to be a suitable host for the propagation of CMV. The virus was purified from inoculated N. benthamiana plants and negatively stained purified preparations contained isometric particles of about 28 nm in diameter. The molecular weight of the viral coat protein subunits was found to be 25.0 kDa. Polyclonal antiserum was produced in New Zealand white rabbit, immunoglobulin G (IgG) was purified and conjugated with alkaline phosphatase enzyme. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) method was standardized for the detection of CMV infection in vanilla plants. CP gene of the virus was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR), cloned and sequenced. Sequenced region contained a single open reading frame of 657 nucleotides potentially coding for 218 amino acids. Sequence analyses with other CMV isolates revealed the greatest identity with black pepper isolate of CMV (99%) and the phylogram clearly showed that CMV infecting vanilla belongs to subgroup IB. This is the first report of occurrence of CMV on V. planifolia from India.

Virulence and molecular polymorphism of Prunus necrotic ringspot virus isolates.

PubMed

Hammond, R W; Crosslin, J M

1998-07-01

Prunus necrotic ringspot virus (PNRSV) occurs as numerous strains or isolates that vary widely in their pathogenic, biophysical and serological properties. Prior attempts to distinguish pathotypes based upon physical properties have not been successful; our approach was to examine the molecular properties that may distinguish these isolates. The nucleic acid sequence was determined from 1.65 kbp RT-PCR products derived from RNA 3 of seven distinct isolates of PNRSV that differ serologically and in pathology on sweet cherry. Sequence comparisons of ORF 3a (putative movement protein) and ORF 3b (coat protein) revealed single nucleotide and amino acid differences with strong correlations to serology and symptom types (pathotypes). Sequence differences between serotypes and pathotypes were also reflected in the overall phylogenetic relationships between the isolates.

Particle sizer and DNA sequencer

DOEpatents

Olivares, Jose A.; Stark, Peter C.

2005-09-13

An electrophoretic device separates and detects particles such as DNA fragments, proteins, and the like. The device has a capillary which is coated with a coating with a low refractive index such as Teflon.RTM. AF. A sample of particles is fluorescently labeled and injected into the capillary. The capillary is filled with an electrolyte buffer solution. An electrical field is applied across the capillary causing the particles to migrate from a first end of the capillary to a second end of the capillary. A detector light beam is then scanned along the length of the capillary to detect the location of the separated particles. The device is amenable to a high throughput system by providing additional capillaries. The device can also be used to determine the actual size of the particles and for DNA sequencing.

Nucleotide sequence of a chickpea chlorotic stunt virus relative that infects pea and faba bean in China.

PubMed

Zhou, Cui-Ji; Xiang, Hai-Ying; Zhuo, Tao; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2012-07-01

We determined the genome sequence of a new polerovirus that infects field pea and faba bean in China. Its entire nucleotide sequence (6021 nt) was most closely related (83.3% identity) to that of an Ethiopian isolate of chickpea chlorotic stunt virus (CpCSV-Eth). With the exception of the coat protein (encoded by ORF3), amino acid sequence identities of all gene products of this virus to those of CpCSV-Eth and other poleroviruses were <90%. This suggests that it is a new member of the genus Polerovirus, and the name pea mild chlorosis virus is proposed.

Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina.

PubMed

Trucco, Verónica; de Breuil, Soledad; Bejerman, Nicolás; Lenardon, Sergio; Giolitti, Fabián

2014-06-01

The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.

Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F-108 tri-block copolymer and capillary electrophoresis.

PubMed

Martin, Lauren; Damaso, Natalie; Mills, DeEtta

2016-10-01

Molecular methods for the detection of mammalian coat color phenotypes have expanded greatly within the past decade. Many phenotypes are associated with a single nucleotide polymorphism mutation in the genetic sequence. Traditionally, these mutations are detected through sequencing, hybridization assays or mini-sequencing. However, these techniques can be expensive and tedious. Previously, CE-SSCP using the F-108 polymer was able to distinguish SNPs for the melanocortin-1 receptor (mc1r) coat color gene in horses (Equus caballus) that differed by one nucleotide substitution. The objective of this study was to expand the detection of coat color SNPs in horses. The genes for the solute carrier family member 2 (slc45a2/matp), type III receptor protein-tyrosine kinase (kit) and mc1r genes using CE-SSCP and F-108 polymer were compared to mini-sequencing with the SNaPshot TM kit. The F-108 polymer reproducibly resolved homozygous and heterozygous individuals for the mc1r and kit markers, but was unable to resolve heterozygous individuals for slc45a2 at 38ºC. The need for temperatures <15ºC, the SNP position being close to the 5'-end, and conformational structures/free energy with similar values resulted in the inability to resolve the secondary structures. Despite this limitation, the CE-SSCP method could be used to provide a rapid phenotypic description for equine forensic investigations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prevalence of Tobacco mosaic virus in Iran and Evolutionary Analyses of the Coat Protein Gene

PubMed Central

Alishiri, Athar; Rakhshandehroo, Farshad; Zamanizadeh, Hamid-Reza; Palukaitis, Peter

2013-01-01

The incidence and distribution of Tobacco mosaic virus (TMV) and related tobamoviruses was determined using an enzyme-linked immunosorbent assay on 1,926 symptomatic horticultural crops and 107 asymptomatic weed samples collected from 78 highly infected fields in the major horticultural crop-producing areas in 17 provinces throughout Iran. The results were confirmed by host range studies and reverse transcription-polymerase chain reaction. The overall incidence of infection by these viruses in symptomatic plants was 11.3%. The coat protein (CP) gene sequences of a number of isolates were determined and disclosed to be a high identity (up to 100%) among the Iranian isolates. Phylogenetic analysis of all known TMV CP genes showed three clades on the basis of nucleotide sequences with all Iranian isolates distinctly clustered in clade II. Analysis using the complete CP amino acid sequence showed one clade with two subgroups, IA and IB, with Iranian isolates in both subgroups. The nucleotide diversity within each sub-group was very low, but higher between the two clades. No correlation was found between genetic distance and geographical origin or host species of isolation. Statistical analyses suggested a negative selection and demonstrated the occurrence of gene flow from the isolates in other clades to the Iranian population. PMID:25288953

Synergistic effect of two cell recognition systems: glycosphingolipid-glycosphingolipid interaction and integrin receptor interaction with pericellular matrix protein.

PubMed

Kojima, N; Hakomori, S

1991-12-01

GM3-expressing cells adhere, spread and migrate on plastic plates coated with Gg3, LacCer and Gb4, but not with other glycosphingolipids (GSLs). Thus, cell adhesion, spreading and migration through GSL-GSL interaction occur in an analogous fashion to the interaction of cells with adhesive matrix proteins [AP, e.g. fibronectin (FN), laminin (LN)] through their integrin receptors. In this study, the adhesion of two GM3-expressing cell lines (B16 melanoma and HEL299 fibroblast) on plastic plates co-coated with GSL plus AP is compared with adhesion on plates coated with GSL (Gg3 or LacCer) alone, or coated with AP alone. Results show that: (i) cell adhesion on GSL-coated plates takes place earlier in the incubation period than that on AP-coated plates; (ii) cell adhesion, as well as spreading, was greatly enhanced (in terms of strength and rapidity) on plates co-coated with GSL plus AP; (iii) repulsion (negative adhesion) of cells was observed on plates co-coated with AP plus N-acetyl-GM3 (NAcGM3) and was presumably based on repulsive NAcGM3-NAcGM3 interaction; (iv) GM3-dependent cell adhesion on GSL-coated plates, as well as synergistic promotion of cell adhesion (based on the GSL-GSL and AP-integrin systems), was suppressed by incubation of cells with anti-GM3 monoclonal antibody DH2 or sialidase. Synergistic adhesion of cells on GSL/AP co-coated plates was less inhibited by incubation with peptide sequences RGDS or YIGSR than was adhesion on plates coated with AP alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

PubMed

Dröge, Melloney J; Boersma, Ykelien L; Braun, Peter G; Buining, Robbert Jan; Julsing, Mattijs K; Selles, Karin G A; van Dijl, Jan Maarten; Quax, Wim J

2006-07-01

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

Poly(Lactic Acid) Nanoparticles Targeting α5β1 Integrin as Vaccine Delivery Vehicle, a Prospective Study

PubMed Central

Gutjahr, Alice; Terrat, Céline; Exposito, Jean-Yves; Verrier, Bernard; Lethias, Claire

2016-01-01

Biodegradable polymeric nanoparticles are vehicles of choice for drug delivery and have the ability to encapsulate and present at their surface different molecules of interest. Among these bio-nanocarriers, poly(lactic acid) (PLA) nanoparticles have been used as adjuvant and vehicle for enhanced vaccine efficacy. In order to develop an approach to efficient vaccine delivery, we developed nanoparticles to target α5β1 positive cells. We first overproduced, in bacteria, human fibronectin FNIII9/10 recombinant proteins possessing an integrin α5β1 binding site, the RGDS sequence, or a mutated form of this site. After having confirmed the integrin binding properties of these recombinant proteins in cell culture assays, we were able to formulate PLA nanoparticles with these FNIII9/10 proteins at their surface. We then confirmed, by fluorescence and confocal microscopy, an enhanced cellular uptake by α5β1+ cells of RGDS-FNIII9/10 coated PLA nanoparticles, in comparison to KGES-FNIII9/10 coated or non-coated controls. As a first vaccination approach, we prepared PLA nanoparticles co-coated with p24 (an HIV antigen), and RGDS- or KGES-FNIII9/10 proteins, followed by subcutaneous vaccine administration, in mice. Although we did not detect improvements in the apparent humoral response to p24 antigen in the serum of RGDS/p24 nanoparticle-treated mice, the presence of the FNIII proteins increased significantly the avidity index of anti-p24 antibodies compared to p24-nanoparticle-injected control mice. Future developments of this innovative targeted vaccine are discussed. PMID:27973577

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parent, Kristin N., E-mail: kparent@msu.edu; Tang, Jinghua; Cardone, Giovanni

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 morphologymore » 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.« less

Genome analysis of the platypus reveals unique signatures of evolution.

PubMed

Warren, Wesley C; Hillier, LaDeana W; Marshall Graves, Jennifer A; Birney, Ewan; Ponting, Chris P; Grützner, Frank; Belov, Katherine; Miller, Webb; Clarke, Laura; Chinwalla, Asif T; Yang, Shiaw-Pyng; Heger, Andreas; Locke, Devin P; Miethke, Pat; Waters, Paul D; Veyrunes, Frédéric; Fulton, Lucinda; Fulton, Bob; Graves, Tina; Wallis, John; Puente, Xose S; López-Otín, Carlos; Ordóñez, Gonzalo R; Eichler, Evan E; Chen, Lin; Cheng, Ze; Deakin, Janine E; Alsop, Amber; Thompson, Katherine; Kirby, Patrick; Papenfuss, Anthony T; Wakefield, Matthew J; Olender, Tsviya; Lancet, Doron; Huttley, Gavin A; Smit, Arian F A; Pask, Andrew; Temple-Smith, Peter; Batzer, Mark A; Walker, Jerilyn A; Konkel, Miriam K; Harris, Robert S; Whittington, Camilla M; Wong, Emily S W; Gemmell, Neil J; Buschiazzo, Emmanuel; Vargas Jentzsch, Iris M; Merkel, Angelika; Schmitz, Juergen; Zemann, Anja; Churakov, Gennady; Kriegs, Jan Ole; Brosius, Juergen; Murchison, Elizabeth P; Sachidanandam, Ravi; Smith, Carly; Hannon, Gregory J; Tsend-Ayush, Enkhjargal; McMillan, Daniel; Attenborough, Rosalind; Rens, Willem; Ferguson-Smith, Malcolm; Lefèvre, Christophe M; Sharp, Julie A; Nicholas, Kevin R; Ray, David A; Kube, Michael; Reinhardt, Richard; Pringle, Thomas H; Taylor, James; Jones, Russell C; Nixon, Brett; Dacheux, Jean-Louis; Niwa, Hitoshi; Sekita, Yoko; Huang, Xiaoqiu; Stark, Alexander; Kheradpour, Pouya; Kellis, Manolis; Flicek, Paul; Chen, Yuan; Webber, Caleb; Hardison, Ross; Nelson, Joanne; Hallsworth-Pepin, Kym; Delehaunty, Kim; Markovic, Chris; Minx, Pat; Feng, Yucheng; Kremitzki, Colin; Mitreva, Makedonka; Glasscock, Jarret; Wylie, Todd; Wohldmann, Patricia; Thiru, Prathapan; Nhan, Michael N; Pohl, Craig S; Smith, Scott M; Hou, Shunfeng; Nefedov, Mikhail; de Jong, Pieter J; Renfree, Marilyn B; Mardis, Elaine R; Wilson, Richard K

2008-05-08

We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

Genome analysis of the platypus reveals unique signatures of evolution

PubMed Central

Warren, Wesley C.; Hillier, LaDeana W.; Marshall Graves, Jennifer A.; Birney, Ewan; Ponting, Chris P.; Grützner, Frank; Belov, Katherine; Miller, Webb; Clarke, Laura; Chinwalla, Asif T.; Yang, Shiaw-Pyng; Heger, Andreas; Locke, Devin P.; Miethke, Pat; Waters, Paul D.; Veyrunes, Frédéric; Fulton, Lucinda; Fulton, Bob; Graves, Tina; Wallis, John; Puente, Xose S.; López-Otín, Carlos; Ordóñez, Gonzalo R.; Eichler, Evan E.; Chen, Lin; Cheng, Ze; Deakin, Janine E.; Alsop, Amber; Thompson, Katherine; Kirby, Patrick; Papenfuss, Anthony T.; Wakefield, Matthew J.; Olender, Tsviya; Lancet, Doron; Huttley, Gavin A.; Smit, Arian F. A.; Pask, Andrew; Temple-Smith, Peter; Batzer, Mark A.; Walker, Jerilyn A.; Konkel, Miriam K.; Harris, Robert S.; Whittington, Camilla M.; Wong, Emily S. W.; Gemmell, Neil J.; Buschiazzo, Emmanuel; Vargas Jentzsch, Iris M.; Merkel, Angelika; Schmitz, Juergen; Zemann, Anja; Churakov, Gennady; Kriegs, Jan Ole; Brosius, Juergen; Murchison, Elizabeth P.; Sachidanandam, Ravi; Smith, Carly; Hannon, Gregory J.; Tsend-Ayush, Enkhjargal; McMillan, Daniel; Attenborough, Rosalind; Rens, Willem; Ferguson-Smith, Malcolm; Lefèvre, Christophe M.; Sharp, Julie A.; Nicholas, Kevin R.; Ray, David A.; Kube, Michael; Reinhardt, Richard; Pringle, Thomas H.; Taylor, James; Jones, Russell C.; Nixon, Brett; Dacheux, Jean-Louis; Niwa, Hitoshi; Sekita, Yoko; Huang, Xiaoqiu; Stark, Alexander; Kheradpour, Pouya; Kellis, Manolis; Flicek, Paul; Chen, Yuan; Webber, Caleb; Hardison, Ross; Nelson, Joanne; Hallsworth-Pepin, Kym; Delehaunty, Kim; Markovic, Chris; Minx, Pat; Feng, Yucheng; Kremitzki, Colin; Mitreva, Makedonka; Glasscock, Jarret; Wylie, Todd; Wohldmann, Patricia; Thiru, Prathapan; Nhan, Michael N.; Pohl, Craig S.; Smith, Scott M.; Hou, Shunfeng; Renfree, Marilyn B.; Mardis, Elaine R.; Wilson, Richard K.

2009-01-01

We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation. PMID:18464734

Structural Basis of Egg Coat-Sperm Recognition at Fertilization.

PubMed

Raj, Isha; Sadat Al Hosseini, Hamed; Dioguardi, Elisa; Nishimura, Kaoru; Han, Ling; Villa, Alessandra; de Sanctis, Daniele; Jovine, Luca

2017-06-15

Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus

PubMed Central

May, Jared; Johnson, Philip; Saleem, Huma

2017-01-01

ABSTRACT 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 structure has IRES activity and produces low levels of viral coat protein in vitro and in vivo. Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common. PMID:28179526

Quantitative analysis of RNA-protein interactions on a massively parallel array for mapping biophysical and evolutionary landscapes

PubMed Central

Buenrostro, Jason D.; Chircus, Lauren M.; Araya, Carlos L.; Layton, Curtis J.; Chang, Howard Y.; Snyder, Michael P.; Greenleaf, William J.

2015-01-01

RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of MS2 coat protein to >107 RNA targets generated on a flow-cell surface by in situ transcription and inter-molecular tethering of RNA to DNA. We decompose the binding energy contributions from primary and secondary RNA structure, finding that differences in affinity are often driven by sequence-specific changes in association rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis, and a long-hypothesized structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNAMaP) relationships across molecular variants. PMID:24727714

Molecular characterization of two genotypes of a new polerovirus infecting brassicas in China.

PubMed

Xiang, Hai-Ying; Dong, Shu-Wei; Shang, Qiao-Xia; Zhou, Cui-Ji; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2011-12-01

The genomic RNA sequences of two genotypes of a brassica-infecting polerovirus from China were determined. Sequence analysis revealed that the virus was closely related to but significantly different from turnip yellows virus (TuYV). This virus and other poleroviruses, including TuYV, had less than 90% amino acid sequence identity in all gene products except the coat protein. Based on the molecular criterion (>10% amino acid sequence difference) for species demarcation in the genus Polerovirus, the virus represents a distinct species for which the name Brassica yellows virus (BrYV) is proposed. Interestingly, there were two genotypes of BrYV, which mainly differed in the 5'-terminal half of the genome.

Kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab) coat protein and its mutants with ssDNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Priyadarshini, C.G. Poornima; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.i

Gemini viral assembly and transport of viral DNA into nucleus for replication, essentially involve DNA-coat protein interactions. The kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali recombinant coat protein (rCP) with DNA was studied by electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR). The rCP interacted with ssDNA with a K{sub A}, of 2.6 +- 0.29 x 10{sup 8} M{sup -1} in a sequence non-specific manner. The CP has a conserved C2H2 type zinc finger motif composed of residues C68, C72, H81 and H85. Mutation of these residues to alanine resulted in reduced binding to DNA probes.more » The H85A mutant rCP showed the least binding with approximately 756 fold loss in the association rate and a three order magnitude decrease in the binding affinity as compared to rCP. The CP-DNA interactions via the zinc finger motif could play a crucial role in virus assembly and in nuclear transport.« less

An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins.

PubMed Central

Gems, D; Maizels, R M

1996-01-01

Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode surface coat, which is shed in response to binding by host antibody molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein series. We have isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of TES-120. The mRNA is absent from T. canis adults but hyperabundant in larvae, making up approximately 10% of total mRNA, and is trans-spliced with the nematode 5' leader sequence SL1. It encodes a 15.8-kDa protein (after signal peptide removal) containing a typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized into an array of heptameric repeats, interspersed with proline residues. At the C-terminal end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a motif that can also be identified in several genes in Caenorhabditis elegans. Although TES-120 displays size and charge heterogeneity, there is a single copy gene and a homogeneous size of mRNA. The association of overexpression of some membrane-associated mucins with immunosuppression and tumor metastasis suggests a possible model for the role of the surface coat in immune evasion by parasitic nematodes. Images Fig. 1 Fig. 4 PMID:8643687

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

PubMed

Ferriol, I; Silva Junior, D M; Nigg, J C; Zamora-Macorra, E J; Falk, B W

2016-11-01

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Convergent Evolution of Slick Coat in Cattle through Truncation Mutations in the Prolactin Receptor.

PubMed

Porto-Neto, Laercio R; Bickhart, Derek M; Landaeta-Hernandez, Antonio J; Utsunomiya, Yuri T; Pagan, Melvin; Jimenez, Esbal; Hansen, Peter J; Dikmen, Serdal; Schroeder, Steven G; Kim, Eui-Soo; Sun, Jiajie; Crespo, Edward; Amati, Norman; Cole, John B; Null, Daniel J; Garcia, Jose F; Reverter, Antonio; Barendse, William; Sonstegard, Tad S

2018-01-01

Evolutionary adaptations are occasionally convergent solutions to the same problem. A mutation contributing to a heat tolerance adaptation in Senepol cattle, a New World breed of mostly European descent, results in the distinct phenotype known as slick, where an animal has shorter hair and lower follicle density across its coat than wild type animals. The causal variant, located in the 11 th exon of prolactin receptor , produces a frameshift that results in a truncated protein. However, this mutation does not explain all cases of slick coats found in criollo breeds. Here, we obtained genome sequences from slick cattle of a geographically distinct criollo breed, namely Limonero, whose ancestors were originally brought to the Americas by the Spanish. These data were used to identify new causal alleles in the 11 th exon of the prolactin receptor , two of which also encode shortened proteins that remove a highly conserved tyrosine residue. These new mutations explained almost 90% of investigated cases of animals that had slick coats, but which also did not carry the Senepol slick allele. These results demonstrate convergent evolution at the molecular level in a trait important to the adaptation of an animal to its environment.

Convergent Evolution of Slick Coat in Cattle through Truncation Mutations in the Prolactin Receptor

PubMed Central

Porto-Neto, Laercio R.; Bickhart, Derek M.; Landaeta-Hernandez, Antonio J.; Utsunomiya, Yuri T.; Pagan, Melvin; Jimenez, Esbal; Hansen, Peter J.; Dikmen, Serdal; Schroeder, Steven G.; Kim, Eui-Soo; Sun, Jiajie; Crespo, Edward; Amati, Norman; Cole, John B.; Null, Daniel J.; Garcia, Jose F.; Reverter, Antonio; Barendse, William; Sonstegard, Tad S.

2018-01-01

Evolutionary adaptations are occasionally convergent solutions to the same problem. A mutation contributing to a heat tolerance adaptation in Senepol cattle, a New World breed of mostly European descent, results in the distinct phenotype known as slick, where an animal has shorter hair and lower follicle density across its coat than wild type animals. The causal variant, located in the 11th exon of prolactin receptor, produces a frameshift that results in a truncated protein. However, this mutation does not explain all cases of slick coats found in criollo breeds. Here, we obtained genome sequences from slick cattle of a geographically distinct criollo breed, namely Limonero, whose ancestors were originally brought to the Americas by the Spanish. These data were used to identify new causal alleles in the 11th exon of the prolactin receptor, two of which also encode shortened proteins that remove a highly conserved tyrosine residue. These new mutations explained almost 90% of investigated cases of animals that had slick coats, but which also did not carry the Senepol slick allele. These results demonstrate convergent evolution at the molecular level in a trait important to the adaptation of an animal to its environment. PMID:29527221

Discovery and Development of Synthetic and Natural Biomaterials for Protein Therapeutics and Medical Device Applications

NASA Astrophysics Data System (ADS)

Keefe, Andrew J.

Controlling nonspecific protein interactions is important for applications from medical devices to protein therapeutics. The presented work is a compilation of efforts aimed at using zwitterionic (ionic yet charge neutral) polymers to modify and stabilize the surface of sensitive biomedical and biological materials. Traditionally, when modifying the surface of a material, the stability of the underlying substrate. The materials modified in this dissertation are unique due to their unconventional amorphous characteristics which provide additional challenges. These are poly(dimethyl siloxane) (PDMS) rubber, and proteins. These materials may seem dissimilar, but both have amorphous surfaces, that do not respond well to chemical modification. PDMS is a biomaterial extensively used in medical device manufacturing, but experiences unacceptably high levels of non-specific protein fouling when used with biological samples. To reduce protein fouling, surface modification is often needed. Unfortunately conventional surface modification methods, such as Poly(ethylene glycol) (PEG) coatings, do not work for PDMS due to its amorphous state. Herein, we demonstrate how a superhydrophilic zwitterionic material, poly(carboxybetaine methacrylate) (pCBMA), can provide a highly stable nonfouling coating with long term stability due to the sharp the contrast in hydrophobicity between pCBMA and PDMS. Biological materials, such as proteins, also require stabilization to improve shelf life, circulation time, and bioactivity. Conjugation of proteins with PEG is often used to increase protein stability, but has a detrimental effect on bioactivity. Here we have shown that pCBMA conjugation improves stability in a similar fashion to PEG, but also retains, or even improves, binding affinity due to enhanced protein-substrate hydrophobic interactions. Recognizing that pCBMA chemically resembles the combination of lysine (K) and glutamic acid (E) amino acids, we have shown how zwitterionic nonfouling peptides can be genetically engineered onto a protein to form recombinant protein-polymer conjugates. This technique avoids the need to post-modify proteins, that is often expensive and time consuming in protein manufacturing. Finally, we have developed two new peptide screening methods that were able to select for nonfouling peptide sequences. The selection for nonfouling sequences is not possible using traditional methods (phage display, yeast display, bacterial display and resin display) due to the presence of background interference. In our first nonfouling peptide screening method, we measured the fouling properties of peptides that were immobilized on the surface of solid glass beads. Peptides first needed to be rationally designed, and then subsequently evaluated for protein binding. Using this method, we were able to screen of 10's of sequences. Our second nonfouling peptide screening method is able to screen thousands of peptide sequences using a combinatorially generated peptide library. This was accomplished using controlled pore glass (CPG) beads as substrates to develop one-bead-one-compound (OBOC) peptide libraries. The choice of a porous substrate made it possible to synthesize enough peptide material to allow for peptide sequencing from a single bead using mass spectrometry techniques.

Two-Exon Skipping within MLPH Is Associated with Coat Color Dilution in Rabbits

PubMed Central

Lehner, Stefanie; Gähle, Marion; Dierks, Claudia; Stelter, Ricarda; Gerber, Jonathan; Brehm, Ralph; Distl, Ottmar

2013-01-01

Coat color dilution turns black coat color to blue and red color to cream and is a characteristic in many mammalian species. Matings among Netherland Dwarf, Loh, and Lionhead Dwarf rabbits over two generations gave evidence for a monogenic autosomal recessive inheritance of coat colour dilution. Histological analyses showed non-uniformly distributed, large, agglomerating melanin granules in the hair bulbs of coat color diluted rabbits. We sequenced the cDNA of MLPH in two dilute and one black rabbit for polymorphism detection. In both color diluted rabbits, skipping of exons 3 and 4 was present resulting in altered amino acids at p.QGL[37-39]QWA and a premature stop codon at p.K40*. Sequencing of genomic DNA revealed a c.111-5C>A splice acceptor mutation within the polypyrimidine tract of intron 2 within MLPH. This mutation presumably causes skipping of exons 3 and 4. In 14/15 dilute rabbits, the c.111-5C>A mutation was homozygous and in a further dilute rabbit, heterozygous and in combination with a homozygous frame shift mutation within exon 6 (c.585delG). In conclusion, our results demonstrated a colour dilution associated MLPH splice variant causing a strongly truncated protein (p.Q37QfsX4). An involvement of further MLPH-associated mutations needs further investigations. PMID:24376820

Coat Protein Regulation by CK2, CPIP, HSP70, and CHIP Is Required for Potato Virus A Replication and Coat Protein Accumulation

PubMed Central

Lõhmus, Andres; Hafrén, Anders

2016-01-01

ABSTRACT We demonstrate here that both coat protein (CP) phosphorylation by protein kinase CK2 and a chaperone system formed by two heat shock proteins, CP-interacting protein (CPIP) and heat shock protein 70 (HSP70), are essential for potato virus A (PVA; genus Potyvirus) replication and that all these host proteins have the capacity to contribute to the level of PVA CP accumulation. An E3 ubiquitin ligase called carboxyl terminus Hsc70-interacting protein (CHIP), which may participate in the CPIP-HSP70-mediated CP degradation, is also needed for robust PVA gene expression. Residue Thr243 within the CK2 consensus sequence of PVA CP was found to be essential for viral replication and to regulate CP protein stability. Substitution of Thr243 either with a phosphorylation-mimicking Asp (CPADA) or with a phosphorylation-deficient Ala (CPAAA) residue in CP expressed from viral RNA limited PVA gene expression to the level of nonreplicating PVA. We found that both the CPAAA mutant and CK2 silencing inhibited, whereas CPADA mutant and overexpression of CK2 increased, PVA translation. From our previous studies, we know that phosphorylation reduces the RNA binding capacity of PVA CP and an excess of CP fully blocks viral RNA translation. Together, these findings suggest that binding by nonphosphorylated PVA CP represses viral RNA translation, involving further CP phosphorylation and CPIP-HSP70 chaperone activities as prerequisites for PVA replication. We propose that this mechanism contributes to shifting potyvirus RNA from translation to replication. IMPORTANCE Host protein kinase CK2, two host chaperones, CPIP and HSP70, and viral coat protein (CP) phosphorylation at Thr243 are needed for potato virus A (PVA) replication. Our results show that nonphosphorylated CP blocks viral translation, likely via binding to viral RNA. We propose that this translational block is needed to allow time and space for the formation of potyviral replication complex around the 3′ end of viral RNA. Progression into replication involves CP regulation by both CK2 phosphorylation and chaperones CPIP and HSP70. PMID:27852853

Desmoglein 4 diversity and correlation analysis with coat color in goat.

PubMed

E, G X; Zhao, Y J; Ma, Y H; Cao, G L; He, J N; Na, R S; Zhao, Z Q; Jiang, C D; Zhang, J H; Arlvd, S; Chen, L P; Qiu, X Y; Hu, W; Huang, Y F

2016-03-04

Desmoglein 4 (DSG4) has an important role in the development of wool traits in domestic animals. The full-length DSG4 gene, which contains 3918 bp, a complete open-reading-frame, and encodes a 1040-amino acid protein, was amplified from Liaoning cashmere goat. The sequence was compared with that of DSG4 from other animals and the results show that the DSG4 coding region is consistent with interspecies conservation. Thirteen single-nucleotide polymorphisms (SNPs) were identified in a highly variable region of DSG4, and one SNP (M-1, G>T) was significantly correlated with white and black coat color in goat. Haplotype distribution of the highly variable region of DSG4 was assessed in 179 individuals from seven goat breeds to investigate its association with coat color and its differentiation among populations. However, the lack of a signature result indicates DGS4 haplotypes related with the color of goat coat.

Genetic analysis of a novel Alaska barley yellow dwarf virus in the family Luteoviridae.

PubMed

Robertson, N L; French, R

2007-02-01

A new plant virus belonging to the family Luteoviridae and isolated from diseased oat (Avena sativa L.) plants was discovered in Alaska in 2003. Even though plants with red/orange leaves were indicative of barley yellow dwarf disease, they were not reactive to specific antibodies corresponding to barley yellow dwarf virus (BYDV)-MAV, -PAV, -SGV, and cereal yellow dwarf virus-RPV from enzyme-linked immunosorbent assays (ELISA). An alternative RT-PCR assay that incorporated Shu-F/Yan-R primers for detection of BYDV-MAV, -PAS, -PAV, and SGV was effective in producing approximately 830-nt fragments that contained genomic sequences to the 3'-terminus of the polymerase gene (ORF 2), the intergenic region ( approximately 113 nt), the coat protein gene (ORF 3), and the putative movement gene (ORF 4). The Alaskan isolates were most similar to BYDV-MAV with only about 77 and 80% amino acid identity in the CP and ORF 4, respectively. The Alaska isolates coat protein gene sequences differed in several regions that otherwise are conserved among BYDV-MAV isolates, and may be important in serological variations, accounting for the negative ELISA results. Based upon sequence and serological differences, we concluded that the Alaskan BYDV-MAV-like isolates formed a novel species tentatively in the genus Luteovirus, and propose the name BYDV-ORV (oat red-leaf virus).

A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana.

PubMed

Jailani, A Abdul Kader; Solanki, Vikas; Roy, Anirban; Sivasudha, T; Mandal, Bikash

2017-04-02

A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits. Copyright © 2017 Elsevier B.V. All rights reserved.

The Bacillus subtilis yabG Gene Is Transcribed by SigK RNA Polymerase during Sporulation, and yabG Mutant Spores Have Altered Coat Protein Composition

PubMed Central

Takamatsu, Hiromu; Kodama, Takeko; Imamura, Atsuo; Asai, Kei; Kobayashi, Kazuo; Nakayama, Tatsuo; Ogasawara, Naotake; Watabe, Kazuhito

2000-01-01

The expression of six novel genes located in the region from abrB to spoVC of the Bacillus subtilis chromosome was analyzed, and one of the genes, yabG, had a predicted promoter sequence conserved among SigK-dependent genes. Northern blot analysis revealed that yabG mRNA was first detected from 4 h after the cessation of logarithmic growth (T4) in wild-type cells and in a gerE36 (GerE−) mutant but not in spoIIAC (SigF−), spoIIGAB (SigE−), spoIIIG (SigG−), and spoIVCB (SigK−) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase. Inactivation of the yabG gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yabG spores in l-alanine and in a mixture of l-asparagine, d-glucose, d-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from yabG spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that yabG encodes a sporulation-specific protein which is involved in coat protein composition in B. subtilis. PMID:10714992

Complete genome analysis of jasmine virus T from Jasminum sambac in China.

PubMed

Tang, Yajun; Gao, Fangluan; Yang, Zhen; Wu, Zujian; Yang, Liang

2016-07-01

The genome of a potyvirus (isolate JaVT_FZ) recovered from jasmine (Jasminum sambac L.) showing yellow ringspot symptoms in Fuzhou, China, was sequenced. JaVT_FZ is closely related to seven other potyviruses with completely sequenced genomes, with which it shares 66-70 % nucleotide and 52-56 % amino acid sequence identity. However, the coat protein (CP) gene shares 82-92 % nucleotide and 90-97 % amino acid sequence identity with those of two partially sequenced potyviruses, named jasmine potyvirus T (JaVT-jasmine) and jasmine yellow mosaic potyvirus (JaYMV-India), respectively. This suggests that JaVT_FZ, JaVT-jasmine and JaYMV-India should be regarded as members of a single potyvirus species, for which the name "Jasmine virus T" has priority.

Copy number variation and missense mutations of the agouti signaling protein (ASIP) gene in goat breeds with different coat colors.

PubMed

Fontanesi, L; Beretti, F; Riggio, V; Gómez González, E; Dall'Olio, S; Davoli, R; Russo, V; Portolano, B

2009-01-01

In goats, classical genetic studies reported a large number of alleles at the Agouti locus with effects on coat color and pattern distribution. From these early studies, the dominant A(Wt) (white/tan) allele was suggested to cause the white color of the Saanen breed. Here, we sequenced the coding region of the goat ASIP gene in 6 goat breeds (Girgentana, Maltese, Derivata di Siria, Murciano-Granadina, Camosciata delle Alpi, and Saanen), with different coat colors and patterns. Five single nucleotide polymorphisms (SNPs) were identified, 3 of which caused missense mutations in conserved positions of the cysteine-rich carboxy-terminal domain of the protein (p.Ala96Gly, p.Cys126Gly, and p.Val128Gly). Allele and genotype frequencies suggested that these mutations are not associated or not completely associated with coat color in the investigated goat breeds. Moreover, genotyping and sequencing results, deviation from Hardy-Weinberg equilibrium, as well as allele copy number evaluation from semiquantitative fluorescent multiplex PCR, indicated the presence of copy number variation (CNV) in all investigated breeds. To confirm the presence of CNV and evaluate its extension, we applied a bovine-goat cross-species array comparative genome hybridization (aCGH) experiment using a custom tiling array based on bovine chromosome 13. aCGH results obtained for 8 goat DNA samples confirmed the presence of CNV affecting a region of less that 100 kb including the ASIP and AHCY genes. In Girgentana and Saanen breeds, this CNV might cause the A(Wt) allele, as already suggested for a similar structural mutation in sheep affecting the ASIP and AHCY genes, providing evidence for a recurrent interspecies CNV. However, other mechanisms may also be involved in determining coat color in these 2 breeds. Copyright 2009 S. Karger AG, Basel.

Nucleotide Sequences and Comparison of Two Large Conjugative Plasmids from Different Campylobacter species

DTIC Science & Technology

2004-01-01

alleles have different predicted lengths, e.g. in pCC31, cpp46 starts with ATGATG whereas in pTet this gene starts with only one ATG; in ssb1 , cmgB7 and...homologues in plasmid pVT745 from Actinobacillus actinomycetemcomitans, and a single-stranded DNA-binding protein ssb1 that may coat the single-stranded

Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding

PubMed Central

Peng, Tao; Free, Paul; Fernig, David G.; Lim, Sierin; Tomczak, Nikodem

2016-01-01

Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently available approaches and provides a new route to design tailored and well-controlled hybrid nanoparticles. PMID:27622533

Biomimetic surface coatings from modular amphiphilic proteins

NASA Astrophysics Data System (ADS)

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.

Clathrin- and AP-2-binding sites in HIP1 uncover a general assembly role for endocytic accessory proteins.

PubMed

Mishra, S K; Agostinelli, N R; Brett, T J; Mizukami, I; Ross, T S; Traub, L M

2001-12-07

Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis.

Nucleotide sequence analysis of the 3' terminal region of a wasabi strain of crucifer tobamovirus genomic RNA: subgrouping of crucifer tobamoviruses.

PubMed

Shimamoto, I; Sonoda, S; Vazquez, P; Minaka, N; Nishiguchi, M

1998-01-01

The 3' terminal 2378 nucleotides of a wasabi strain of crucifer tobamovirus (CTMV-W) infectious to crucifer plants was determined. This includes the 3' non-coding region of 235 nucleotides, coat protein (CP) gene (468 nucleotides), movement protein (MP) gene (798 nucleotides) and C-terminal partial readthrough portion of 180 K protein gene (940 nucleotides). Comparison of the sequence with homologous regions of thirteen other tobamovirus genomes showed that it had much higher identity to those of four other crucifer tobamoviruses, 85.2% to cr-TMV and turnip vein-clearing virus (TVCV), 87.4% to oilseed rape mosaic virus (ORMV) and 87.1% to TMV-Cg, than to those of other tobamoviruses. Thus CTMV-W was most similar to ORMV and TMV-Cg in sequence, but only marginally so, whereas the location and size of its MP gene was the same as cr-TMV amd TVCV. These results, together with other analyses, show that CTMV-W is a new crucifer tobamovirus, that the five crucifer tobamoviruses can be classified into two subgroups based on MP gene organization, and that the rate of sequence change is not the same in all lineages.

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

PubMed

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.

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins.

PubMed

Rebière-Huët, J; Guérillon, J; Pimenta, A L; Di Martino, P; Orange, N; Hulen, C

2002-09-24

Bacterial adherence is a complex phenomenon involving specific interactions between receptors, including matricial fibronectin, and bacterial ligands. We show here that fibronectin and outer membrane proteins of Pseudomonas fluorescens were able to inhibit adherence of P. fluorescens to fibronectin-coated wells. We identified at least six fibronectin-binding proteins with molecular masses of 70, 55, 44, 37, 32 and 28 kDa. The presence of native (32 kDa) and heat-modified forms (37 kDa) of OprF was revealed by immuno-analysis and the 44-kDa band was composed of three proteins, their N-terminal sequences showing homologies with Pseudomonas aeruginosa porins (OprD, OprE1 and OprE3).

In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

PubMed Central

Beltzer, J P; Spiess, M

1991-01-01

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

Skin transcriptome profiles associated with coat color in sheep

PubMed Central

2013-01-01

Background Previous molecular genetic studies of physiology and pigmentation of sheep skin have focused primarily on a limited number of genes and proteins. To identify additional genes that may play important roles in coat color regulation, Illumina sequencing technology was used to catalog global gene expression profiles in skin of sheep with white versus black coat color. Results There were 90,006 and 74,533 unigenes assembled from the reads obtained from white and black sheep skin, respectively. Genes encoding for the ribosomal proteins and keratin associated proteins were most highly expressed. A total of 2,235 known genes were differentially expressed in black versus white sheep skin, with 479 genes up-regulated and 1,756 genes down-regulated. A total of 845 novel genes were differentially expressed in black versus white sheep skin, consisting of 107 genes which were up-regulated (including 2 highly expressed genes exclusively expressed in black sheep skin) and 738 genes that were down-regulated. There was also a total of 49 known coat color genes expressed in sheep skin, from which 13 genes showed higher expression in black sheep skin. Many of these up-regulated genes, such as DCT, MATP, TYR and TYRP1, are members of the components of melanosomes and their precursor ontology category. Conclusion The white and black sheep skin transcriptome profiles obtained provide a valuable resource for future research to understand the network of gene expression controlling skin physiology and melanogenesis in sheep. PMID:23758853

Variability Studies of Two Prunus-Infecting Fabaviruses with the Aid of High-Throughput Sequencing

PubMed Central

Sarkisova, Tatiana; Lenz, Ondřej; Přibylová, Jaroslava; Špak, Josef; Lotos, Leonidas; Beta, Christina; Katsiani, Asimina; Candresse, Thierry

2018-01-01

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species. PMID:29670059

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

PubMed Central

Gustafson, G; Armour, S L

1986-01-01

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs. Images PMID:3754962

Allexiviruses may have acquired inserted sequences between the CP and CRP genes to change the translation reinitiation strategy of CRP.

PubMed

Yoshida, Naoto; Shimura, Hanako; Masuta, Chikara

2018-06-01

Allexiviruses are economically important garlic viruses that are involved in garlic mosaic diseases. In this study, we characterized the allexivirus cysteine-rich protein (CRP) gene located just downstream of the coat protein (CP) gene in the viral genome. We determined the nucleotide sequences of the CP and CRP genes from numerous allexivirus isolates and performed a phylogenetic analysis. According to the resulting phylogenetic tree, we found that allexiviruses were clearly divided into two major groups (group I and group II) based on the sequences of the CP and CRP genes. In addition, the allexiviruses in group II had distinct sequences just before the CRP gene, while group I isolates did not. The inserted sequence between the CP and CRP genes was partially complementary to garlic 18S rRNA. Using a potato virus X vector, we showed that the CRPs affected viral accumulation and symptom induction in Nicotiana benthamiana, suggesting that the allexivirus CRP is a pathogenicity determinant. We assume that the inserted sequences before the CRP gene may have been generated during viral evolution to alter the termination-reinitiation mechanism for coupled translation of CP and CRP.

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

PubMed

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.

Phylogeny of geminivirus coat protein sequences and digital PCR aid in identifying Spissistilus festinus (Say) as a vector of Grapevine red blotch-associated virus

USDA-ARS?s Scientific Manuscript database

Grapevine red blotch-associated virus (GRBaV) is a newly identified virus of grapevines, and a putative member of a new genus within the family Geminiviridae. This virus is associated with red blotch disease that was first reported in California in 2008. It affects the profitability of vineyards by ...

Analysis of the complete genome of peach chlorotic mottle virus: identification of non-AUG start codons, in vitro coat protein expression, and elucidation of serological cross-reactions.

PubMed

James, D; Varga, A; Croft, H

2007-01-01

The entire genome of peach chlorotic mottle virus (PCMV), originally identified as Prunus persica cv. Agua virus (4N6), was sequenced and analysed. PCMV cross-reacts with antisera to diverse viruses, such as plum pox virus (PPV), genus Potyvirus, family Potyviridae; and apple stem pitting virus (ASPV), genus Foveavirus, family Flexiviridae. The PCMV genome consists of 9005 nucleotides (nts), excluding a poly(A) tail at the 3' end of the genome. Five open reading frames (ORFs) were identified with four untranslated regions (UTR) including a 5', a 3', and two intergenic UTRs. The genome organisation of PCMV is similar to that of ASPV and the two genomes share a nucleotide (nt) sequence identity of 58%. PCMV ORF1 encodes the replication-associated protein complex (Mr 241,503), ORF2-ORF4 code for the triple gene block proteins (TGBp; Mr 24,802, 12,370, and 7320, respectively), and ORF5 encodes the coat protein (CP) (Mr 42,505). Two non-AUG start codons participate in the initiation of translation: 35AUC and 7676AUA initiate translation of ORF1 and ORF5. In vitro expression with subsequent Western blot analysis confirmed ORF5 as the CP-encoding gene and confirmed that the codon AUA is able to initiate translation of the CP. Expression of a truncated CP fragment (Mr 39, 689) was demonstrated, and both proteins are expressed in vivo, since both were observed in Western blot analysis of PCMV-infected peach and Nicotiana occidentalis. The expressed proteins cross-reacted with an antiserum against ASPV. The amino acid sequences of the CPs of PCMV and ASPV CP share only 37% identity, but there are 11 shared peptides 4-8 aa residues long. These may constitute linear epitopes responsible for ASPV antiserum cross reactions. No significant common linear epitopes were associated with PPV. Extensive phylogenetic analysis indicates that PCMV is closely related to ASPV and is a new and distinct member of the genus Foveavirus.

Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection.

PubMed

Ghoshal, Kankana; Theilmann, Jane; Reade, Ron; Maghodia, Ajay; Rochon, D'Ann

2015-11-01

Next-generation sequence analysis of virus-like particles (VLPs) produced during agroinfiltration of cucumber necrosis virus (CNV) coat protein (CP) and of authentic CNV virions was conducted to assess if host RNAs can be encapsidated by CNV CP. VLPs containing host RNAs were found to be produced during agroinfiltration, accumulating to approximately 1/60 the level that CNV virions accumulated during infection. VLPs contained a variety of host RNA species, including the major rRNAs as well as cytoplasmic, chloroplast, and mitochondrial mRNAs. The most predominant host RNA species encapsidated in VLPs were chloroplast encoded, consistent with the efficient targeting of CNV CP to chloroplasts during agroinfiltration. Interestingly, droplet digital PCR analysis showed that the CNV CP mRNA expressed during agroinfiltration was the most efficiently encapsidated mRNA, suggesting that the CNV CP open reading frame may contain a high-affinity site or sites for CP binding and thus contribute to the specificity of CNV RNA encapsidation. Approximately 0.09% to 0.7% of the RNA derived from authentic CNV virions contained host RNA, with chloroplast RNA again being the most prominent species. This is consistent with our previous finding that a small proportion of CNV CP enters chloroplasts during the infection process and highlights the possibility that chloroplast targeting is a significant aspect of CNV infection. Remarkably, 6 to 8 of the top 10 most efficiently encapsidated nucleus-encoded RNAs in CNV virions correspond to retrotransposon or retrotransposon-like RNA sequences. Thus, CNV could potentially serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution. Viruses predominantly encapsidate their own virus-related RNA species due to the possession of specific sequences and/or structures on viral RNA which serve as high-affinity binding sites for the coat protein. In this study, we show, using next-generation sequence analysis, that CNV also encapsidates host RNA species, which account for ∼0.1% of the RNA packaged in CNV particles. The encapsidated host RNAs predominantly include chloroplast RNAs, reinforcing previous observations that CNV CP enters chloroplasts during infection. Remarkably, the most abundantly encapsidated cytoplasmic mRNAs consisted of retrotransposon-like RNA sequences, similar to findings recently reported for flock house virus (A. Routh, T. Domitrovic, and J. E. Johnson, Proc Natl Acad Sci U S A 109:1907-1912, 2012). Encapsidation of retrotransposon sequences may contribute to their horizontal transmission should CNV virions carrying retrotransposons infect a new host. Such an event could lead to large-scale genomic changes in a naive plant host, thus facilitating host evolutionary novelty. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase of coat protein subgroup II gene from Cucumber mosaic virus

USDA-ARS?s Scientific Manuscript database

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These...

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro

PubMed Central

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3′ untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication. PMID:15141033

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro.

PubMed

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.

Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter.

PubMed

Man, Michal; Epel, Bernard L

2004-06-01

A replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3'-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3' terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem-loop structures, which are followed by an enhancer region.

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

PubMed

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.

[The primary structure of a vaccine strain of tobacco mosaic virus V-69].

PubMed

Shiian, A N; Mil'shina, N V; Snegireva, P B; Pukhal'skiĭ, V A

1994-12-01

A random set of cDNA fragments were synthesized on genomic RNA of TMV vaccine strain V-69, using random primers and reverse transcriptase. Following synthesis of double-stranded cDNA, they were cloned into the pUC-19 plasmid; and 28 clones were sequenced (insert size 100-500 bp). High nucleotide sequence homology of V-69 (more than 95%) was shown only with tomato strain TMV-L [1]. Sequenced clones represent 54% of the genome (50% of the replicase gene, 98% of the transport protein gene, and 60% of the coat protein gene). In this genome region, 24 base substitutions were revealed, as compared to the wild-type TMV-L sequence. Six base substitutions resulted in changes in corresponding amino acid codons. No substitutions coincided with those discovered in the related TMV vaccine strain L11A [2], while two substitutions in the replicase gene were identical to those found in TMV strain Lta1 [3], which is capable of overcoming protection in tomatoes with the resistance gene Tm-1.

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

PubMed Central

Culver, J N; Lehto, K; Close, S M; Hilf, M E; Dawson, W O

1993-01-01

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes. Images Fig. 2 Fig. 3 Fig. 4 PMID:8446627

Poinsettia latent virus is not a cryptic virus, but a natural polerovirus-sobemovirus hybrid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siepen, Marc aus dem; Pohl, Jens O.; Koo, Bong-Jin

2005-06-05

The biochemical and genetic features of Poinsettia latent virus (PnLV, formerly named Poinsettia cryptic virus), which is spread worldwide in commercial cultivars of Euphorbia pulcherrima without inducing symptoms, have been determined using virus-purification, immunological techniques, electron microscopy, cloning, and sequencing. PnLV was found to be a chimeric virus with one 4652 bases, plus strand RNA showing a close relationship to poleroviruses within the first three quarters of its genome but to sobemoviruses in the last quarter. Thus, we propose to classify this virus as 'polemovirus'. Similarities of protein and nucleic acid sequences at the 5' and extreme 3' end ofmore » its RNA suggest a replication mode like that of poleroviruses, whereas the coat protein sequence is closely related to that of sobemoviruses. Consistent with these results, PnLV forms stable icosahedra of 34 nm in diameter. The consequences for the taxonomy of PnLV and for gardeners' practice are discussed.« less

Polyglycerol coatings of glass vials for protein resistance.

PubMed

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.

Single Molecule Spectroscopy of Amino Acids and Peptides by Recognition Tunneling

PubMed Central

Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, JongOne; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

2014-01-01

The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single molecule protein sequencing is a critical step in the search for protein biomarkers. Here we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules and measuring the electron tunneling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic ‘fingerprints’ associated with each binding motif. With this recognition tunneling technique, we are able to identify D, L enantiomers, a methylated amino acid, isobaric isomers, and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore. PMID:24705512

Widespread distribution and a new recombinant species of Brazilian virus associated with cotton blue disease

PubMed Central

Silva, TF; Corrêa, RL; Castilho, Y; Silvie, P; Bélot, J-L; Vaslin, MFS

2008-01-01

Background Cotton blue disease (CBD), an important global cotton crop pathology responsible for major economic losses, is prevalent in the major cotton-producing states of Brazil. Typical CBD symptoms include stunting due to internodal shortening, leaf rolling, intense green foliage, and yellowing veins. Atypical CBD symptoms, including reddish and withered leaves, were also observed in Brazilian cotton fields in 2007. Recently, a Polerovirus named Cotton leafroll dwarf virus (CLRDV) was shown to be associated with CBD. Results To understand the distribution and genetic diversity of CLRDV in Brazil, we analyzed 23 CBD-symptomatic plants from susceptible cotton varieties originating from five of the six most important cotton-growing states, from 2004–2007. Here, we report on CLRDV diversity in plants with typical or atypical CBD symptoms by comparing viral coat protein, RNA polymerase (RdRp), and intergenic region genomic sequences. Conclusion The virus had a widespread distribution with a low genetic diversity; however, three divergent isolates were associated with atypical CBD symptoms. These divergent isolates had a CLRDV-related coat protein but a distinct RdRp sequence, and probably arose from recombination events. Based on the taxonomic rules for the family Luteoviridae, we propose that these three isolates represent isolates of a new species in the genus Polerovirus. PMID:18937850

Widespread distribution and a new recombinant species of Brazilian virus associated with cotton blue disease.

PubMed

Silva, T F; Corrêa, R L; Castilho, Y; Silvie, P; Bélot, J-L; Vaslin, M F S

2008-10-20

Cotton blue disease (CBD), an important global cotton crop pathology responsible for major economic losses, is prevalent in the major cotton-producing states of Brazil. Typical CBD symptoms include stunting due to internodal shortening, leaf rolling, intense green foliage, and yellowing veins. Atypical CBD symptoms, including reddish and withered leaves, were also observed in Brazilian cotton fields in 2007. Recently, a Polerovirus named Cotton leafroll dwarf virus (CLRDV) was shown to be associated with CBD. To understand the distribution and genetic diversity of CLRDV in Brazil, we analyzed 23 CBD-symptomatic plants from susceptible cotton varieties originating from five of the six most important cotton-growing states, from 2004-2007. Here, we report on CLRDV diversity in plants with typical or atypical CBD symptoms by comparing viral coat protein, RNA polymerase (RdRp), and intergenic region genomic sequences. The virus had a widespread distribution with a low genetic diversity; however, three divergent isolates were associated with atypical CBD symptoms. These divergent isolates had a CLRDV-related coat protein but a distinct RdRp sequence, and probably arose from recombination events. Based on the taxonomic rules for the family Luteoviridae, we propose that these three isolates represent isolates of a new species in the genus Polerovirus.

New Insights into Asian Prunus Viruses in the Light of NGS-Based Full Genome Sequencing.

PubMed

Marais, Armelle; Faure, Chantal; Candresse, Thierry

2016-01-01

Double stranded RNAs were purified from five Prunus sources of Asian origin and submitted to 454 pyrosequencing after a random, whole genome amplification. Four complete genomes of Asian prunus virus 1 (APV1), APV2 and APV3 were reconstructed from the sequencing reads, as well as four additional, near-complete genome sequences. Phylogenetic analyses confirmed the close relationships of these three viruses and the taxonomical position previously proposed for APV1, the only APV so far completely sequenced. The genetic distances in the respective polymerase and coat protein genes as well as their gene products suggest that APV2 should be considered as a distinct viral species in the genus Foveavirus, even if the amino acid identity levels in the polymerase are very close to the species demarcation criteria for the family Betaflexiviridae. However, the situation is more complex for APV1 and APV3, for which opposite conclusions are obtained depending on the gene (polymerase or coat protein) analyzed. Phylogenetic and recombination analyses suggest that recombination events may have been involved in the evolution of APV. Moreover, genome comparisons show that the unusually long 3' non-coding region (3' NCR) is highly variable and a hot spot for indel polymorphisms. In particular, two APV3 variants differing only in their 3' NCR were identified in a single Prunus source, with 3' NCRs of 214-312 nt, a size similar to that observed in other foveaviruses, but 567-850 nt smaller than in other APV3 isolates. Overall, this study provides critical genome information of these viruses, frequently associated with Prunus materials, even though their precise role as pathogens remains to be elucidated.

New Insights into Asian Prunus Viruses in the Light of NGS-Based Full Genome Sequencing

PubMed Central

Marais, Armelle; Faure, Chantal; Candresse, Thierry

2016-01-01

Double stranded RNAs were purified from five Prunus sources of Asian origin and submitted to 454 pyrosequencing after a random, whole genome amplification. Four complete genomes of Asian prunus virus 1 (APV1), APV2 and APV3 were reconstructed from the sequencing reads, as well as four additional, near-complete genome sequences. Phylogenetic analyses confirmed the close relationships of these three viruses and the taxonomical position previously proposed for APV1, the only APV so far completely sequenced. The genetic distances in the respective polymerase and coat protein genes as well as their gene products suggest that APV2 should be considered as a distinct viral species in the genus Foveavirus, even if the amino acid identity levels in the polymerase are very close to the species demarcation criteria for the family Betaflexiviridae. However, the situation is more complex for APV1 and APV3, for which opposite conclusions are obtained depending on the gene (polymerase or coat protein) analyzed. Phylogenetic and recombination analyses suggest that recombination events may have been involved in the evolution of APV. Moreover, genome comparisons show that the unusually long 3’ non-coding region (3' NCR) is highly variable and a hot spot for indel polymorphisms. In particular, two APV3 variants differing only in their 3’ NCR were identified in a single Prunus source, with 3' NCRs of 214–312 nt, a size similar to that observed in other foveaviruses, but 567–850 nt smaller than in other APV3 isolates. Overall, this study provides critical genome information of these viruses, frequently associated with Prunus materials, even though their precise role as pathogens remains to be elucidated. PMID:26741704

Processing of the mother-cell sigma factor, sigma K, may depend on events occurring in the forespore during Bacillus subtilis development.

PubMed Central

Lu, S; Halberg, R; Kroos, L

1990-01-01

During sporulation of the Gram-positive bacterium Bacillus subtilis, transcription of genes encoding spore coat proteins in the mother-cell compartment of the sporangium is controlled by RNA polymerase containing the sigma subunit called sigma K. Based on comparison of the N-terminal amino acid sequence of sigma K with the nucleotide sequence of the gene encoding sigma K (sigK), the primary product of sigK was inferred to be a pro-protein (pro-sigma K) with 20 extra amino acids at the N terminus. Using antibodies generated against pro-sigma K, we have detected pro-sigma K beginning at the third hour of sporulation and sigma K beginning about 1 hr later. Even when pro-sigma K is expressed artificially during growth and throughout sporulation, sigma K appears at the normal time and expression of a sigma K-controlled gene occurs normally. These results suggest that pro-sigma K is an inactive precursor that is proteolytically processed to active sigma K in a developmentally regulated fashion. Mutations that block forespore gene expression block accumulation of sigma K but not accumulation of pro-sigma K, suggesting that pro-sigma K processing is a regulatory device that couples the programs of gene expression in the two compartments of the sporangium. We propose that this regulatory device ensures completion of forespore morphogenesis prior to the synthesis in the mother-cell of spore coat proteins that will encase the forespore. Images PMID:2124700

The complete nucleotide sequence of the barley yellow dwarf GPV isolate from China shows that it is a new member of the genus Polerovirus.

PubMed

Zhang, Wenwei; Cheng, Zhuomin; Xu, Lei; Wu, Maosen; Waterhouse, Peter; Zhou, Guanghe; Li, Shifang

2009-01-01

The complete nucleotide sequence of the ssRNA genome of a Chinese GPV isolate of barley yellow dwarf virus (BYDV) was determined. It comprised 5673 nucleotides, and the deduced genome organization resembled that of members of the genus Polerovirus. It was most closely related to cereal yellow dwarf virus-RPV (77% nt identity over the entire genome; coat protein amino acid identity 79%). The GPV isolate also differs in vector specificity from other BYDV strains. Biological properties, phylogenetic analyses and detailed sequence comparisons suggest that GPV should be considered a member of a new species within the genus, and the name Wheat yellow dwarf virus-GPV is proposed.

Phylogeny of isolates of Prunus necrotic ringspot virus from the Ilarvirus Ringtest and identification of group-specific features.

PubMed

Hammond, R W

2003-06-01

Isolates of Prunus necrotic ringspot virus (PNRSV) were examined to establish the level of naturally occurring sequence variation in the coat protein (CP) gene and to identify group-specific genome features that may prove valuable for the generation of diagnostic reagents. Phylogenetic analysis of a 452 bp sequence of 68 virus isolates, 20 obtained from the European Union Ilarvirus Ringtest held in October 1998, confirmed the clustering of the isolates into three distinct groups. Although no correlation was found between the sequence and host or geographic origin, there was a general trend for severe isolates to cluster into one group. Group-specific features have been identified for discrimination between virus strains.

A brief review of other notable protein blotting methods.

PubMed

Kurien, Biji T; Scofield, R Hal

2009-01-01

A plethora of methods have been used for transferring proteins from the gel to the membrane. These include centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low and high molecular weight proteins, blotting of Coomassie Brilliant Blue (CBB)-stained proteins from polyacrylamide gels to transparencies, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before MALDI tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

The nucleotide sequence and genome organization of Plasmopara halstedii virus.

PubMed

Heller-Dohmen, Marion; Göpfert, Jens C; Pfannstiel, Jens; Spring, Otmar

2011-03-17

Only very few viruses of Oomycetes have been studied in detail. Isometric virions were found in different isolates of the oomycete Plasmopara halstedii, the downy mildew pathogen of sunflower. However, complete nucleotide sequences and data on the genome organization were lacking. Viral RNA of different P. halstedii isolates was subjected to nucleotide sequencing and analysis of the viral genome. The N-terminal sequence of the viral coat protein was determined using Top-Down MALDI-TOF analysis. The complete nucleotide sequences of both single-stranded RNA segments (RNA1 and RNA2) were established. RNA1 consisted of 2793 nucleotides (nt) exclusive its 3' poly(A) tract and a single open-reading frame (ORF1) of 2745 nt. ORF1 was framed by a 5' untranslated region (5' UTR) of 18 nt and a 3' untranslated region (3' UTR) of 30 nt. ORF1 contained motifs of RNA-dependent RNA polymerases (RdRp) and showed similarities to RdRp of Scleropthora macrospora virus A (SmV A) and viruses within the Nodaviridae family. RNA2 consisted of 1526 nt exclusive its 3' poly(A) tract and a second ORF (ORF2) of 1128 nt. ORF2 coded for the single viral coat protein (CP) and was framed by a 5' UTR of 164 nt and a 3' UTR of 234 nt. The deduced amino acid sequence of ORF2 was verified by nano-LC-ESI-MS/MS experiments. Top-Down MALDI-TOF analysis revealed the N-terminal sequence of the CP. The N-terminal sequence represented a region within ORF2 suggesting a proteolytic processing of the CP in vivo. The CP showed similarities to CP of SmV A and viruses within the Tombusviridae family. Fragments of RNA1 (ca. 1.9 kb) and RNA2 (ca. 1.4 kb) were used to analyze the nucleotide sequence variation of virions in different P. halstedii isolates. Viral sequence variation was 0.3% or less regardless of their host's pathotypes, the geographical origin and the sensitivity towards the fungicide metalaxyl. The results showed the presence of a single and new virus type in different P. halstedii isolates. Insignificant viral sequence variation indicated that the virus did not account for differences in pathogenicity of the oomycete P. halstedii.

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis.

PubMed

Wang, Katherine H; Isidro, Anabela L; Domingues, Lia; Eskandarian, Haig A; McKenney, Peter T; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O; Eichenberger, Patrick

2009-11-01

Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.

Colorimetric and dynamic light scattering detection of DNA sequences by using positively charged gold nanospheres: a comparative study with gold nanorods

NASA Astrophysics Data System (ADS)

Pylaev, T. E.; Khanadeev, V. A.; Khlebtsov, B. N.; Dykman, L. A.; Bogatyrev, V. A.; Khlebtsov, N. G.

2011-07-01

We introduce a new genosensing approach employing CTAB (cetyltrimethylammonium bromide)-coated positively charged colloidal gold nanoparticles (GNPs) to detect target DNA sequences by using absorption spectroscopy and dynamic light scattering. The approach is compared with a previously reported method employing unmodified CTAB-coated gold nanorods (GNRs). Both approaches are based on the observation that whereas the addition of probe and target ssDNA to CTAB-coated particles results in particle aggregation, no aggregation is observed after addition of probe and nontarget DNA sequences. Our goal was to compare the feasibility and sensitivity of both methods. A 21-mer ssDNA from the human immunodeficiency virus type 1 HIV-1 U5 long terminal repeat (LTR) sequence and a 23-mer ssDNA from the Bacillus anthracis cryptic protein and protective antigen precursor (pagA) genes were used as ssDNA models. In the case of GNRs, unexpectedly, the colorimetric test failed with perfect cigar-like particles but could be performed with dumbbell and dog-bone rods. By contrast, our approach with cationic CTAB-coated GNPs is easy to implement and possesses excellent feasibility with retention of comparable sensitivity—a 0.1 nM concentration of target cDNA can be detected with the naked eye and 10 pM by dynamic light scattering (DLS) measurements. The specificity of our method is illustrated by successful DLS detection of one-three base mismatches in cDNA sequences for both DNA models. These results suggest that the cationic GNPs and DLS can be used for genosensing under optimal DNA hybridization conditions without any chemical modifications of the particle surface with ssDNA molecules and signal amplification. Finally, we discuss a more than two-three-order difference in the reported estimations of the detection sensitivity of colorimetric methods (0.1 to 10-100 pM) to show that the existing aggregation models are inconsistent with the detection limits of about 0.1-1 pM DNA and that other explanations should be developed.

Evolutionary interplay between structure, energy and epistasis in the coat protein of the ϕX174 phage family.

PubMed

Redondo, Rodrigo A F; de Vladar, Harold P; Włodarski, Tomasz; Bollback, Jonathan P

2017-01-01

Viral capsids are structurally constrained by interactions among the amino acids (AAs) of their constituent proteins. Therefore, epistasis is expected to evolve among physically interacting sites and to influence the rates of substitution. To study the evolution of epistasis, we focused on the major structural protein of the ϕX174 phage family by first reconstructing the ancestral protein sequences of 18 species using a Bayesian statistical framework. The inferred ancestral reconstruction differed at eight AAs, for a total of 256 possible ancestral haplotypes. For each ancestral haplotype and the extant species, we estimated, in silico, the distribution of free energies and epistasis of the capsid structure. We found that free energy has not significantly increased but epistasis has. We decomposed epistasis up to fifth order and found that higher-order epistasis sometimes compensates pairwise interactions making the free energy seem additive. The dN/dS ratio is low, suggesting strong purifying selection, and that structure is under stabilizing selection. We synthesized phages carrying ancestral haplotypes of the coat protein gene and measured their fitness experimentally. Our findings indicate that stabilizing mutations can have higher fitness, and that fitness optima do not necessarily coincide with energy minima. © 2017 The Authors.

Rational Design of a Transferrin-Binding Peptide Sequence Tailored to Targeted Nanoparticle Internalization.

PubMed

Santi, Melissa; Maccari, Giuseppe; Mereghetti, Paolo; Voliani, Valerio; Rocchiccioli, Silvia; Ucciferri, Nadia; Luin, Stefano; Signore, Giovanni

2017-02-15

The transferrin receptor (TfR) is a promising target in cancer therapy owing to its overexpression in most solid tumors and on the blood-brain barrier. Nanostructures chemically derivatized with transferrin are employed in TfR targeting but often lose their functionality upon injection in the bloodstream. As an alternative strategy, we rationally designed a peptide coating able to bind transferrin on suitable pockets not involved in binding to TfR or iron by using an iterative multiscale-modeling approach coupled with quantitative structure-activity and relationship (QSAR) analysis and evolutionary algorithms. We tested that selected sequences have low aspecific protein adsorption and high binding energy toward transferrin, and one of them is efficiently internalized in cells with a transferrin-dependent pathway. Furthermore, it promotes transferrin-mediated endocytosis of gold nanoparticles by modifying their protein corona and promoting oriented adsorption of transferrin. This strategy leads to highly effective nanostructures, potentially useful in diagnostic and therapeutic applications, which exploit (and do not suffer) the protein solvation for achieving a better targeting.

Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells.

PubMed

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.

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes.

PubMed

Crosslin, James M; Hamm, Philip B; Kirk, William W; Hammond, Rosemarie W

2010-04-01

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20-25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3' terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.

Other notable protein blotting methods: a brief review.

PubMed

Kurien, Biji T; Scofield, R Hal

2015-01-01

Proteins have been transferred from the gel to the membrane by a variety of methods. These include vacuum blotting, centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low- and high-molecular-weight proteins, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

The TRANSPARENT TESTA16 Locus Encodes the ARABIDOPSIS BSISTER MADS Domain Protein and Is Required for Proper Development and Pigmentation of the Seed Coat

PubMed Central

Nesi, Nathalie; Debeaujon, Isabelle; Jond, Clarisse; Stewart, Amanda J.; Jenkins, Gareth I.; Caboche, Michel; Lepiniec, Loïc

2002-01-01

Screening for seed pigmentation phenotypes in Arabidopsis led to the isolation of three allelic yellow-seeded mutants, which defined the novel TRANSPARENT TESTA16 (TT16) locus. Cloning of TT16 was performed by T-DNA tagging and confirmed by genetic complementation and sequencing of two mutant alleles. TT16 encodes the ARABIDOPSIS BSISTER (ABS) MADS domain protein. ABS belongs to the recently identified “B-sister” (BS) clade, which contains genes of unknown function that are expressed mainly in female organs. Phylogenetic analyses using a maximum parsimony approach confirmed that TT16/ABS and related proteins form a monophyletic group. TT16/ABS was expressed mainly in the ovule, as are the other members of the BS clade. TT16/ABS is necessary for BANYULS expression and proanthocyanidin accumulation in the endothelium of the seed coat, with the exception of the chalazal-micropylar area. In addition, mutant phenotype and ectopic expression analyses suggested that TT16/ABS also is involved in the specification of endothelial cells. Nevertheless, TT16/ABS apparently is not required for proper ovule function. We report the functional characterization of a member of the BS MADS box gene subfamily, demonstrating its involvement in endothelial cell specification as well as in the increasingly complex genetic control of flavonoid biosynthesis in the Arabidopsis seed coat. PMID:12368498

Complete genome sequence of a Chinese isolate of pepper vein yellows virus and evolutionary analysis based on the CP, MP and RdRp coding regions.

PubMed

Liu, Maoyan; Liu, Xiangning; Li, Xun; Zhang, Deyong; Dai, Liangyin; Tang, Qianjun

2016-03-01

The genome sequence of pepper vein yellows virus (PeVYV) (PeVYV-HN, accession number KP326573), isolated from pepper plants (Capsicum annuum L.) grown at the Hunan Vegetables Institute (Changsha, Hunan, China), was determined by deep sequencing of small RNAs. The PeVYV-HN genome consists of 6244 nucleotides, contains six open reading frames (ORFs), and is similar to that of an isolate (AB594828) from Japan. Its genomic organization is similar to that of members of the genus Polerovirus. Sequence analysis revealed that PeVYV-HN shared 92% sequence identity with the Japanese PeVYV genome at both the nucleotide and amino acid levels. Evolutionary analysis based on the coat protein (CP), movement protein (MP), and RNA-dependent RNA polymerase (RdRP) showed that PeVYV could be divided into two major lineages corresponding to their geographical origins. The Asian isolates have a higher population expansion frequency than the African isolates. Negative selection and genetic drift (founder effect) were found to be the potential drivers of the molecular evolution of PeVYV. Moreover, recombination was not the distinct cause of PeVYV evolution. This is the first report of a complete genomic sequence of PeVYV in China.

Identification of a novel vitivirus from grapevines in New Zealand.

PubMed

Blouin, Arnaud G; Keenan, Sandi; Napier, Kathryn R; Barrero, Roberto A; MacDiarmid, Robin M

2018-01-01

We report a sequence of a novel vitivirus from Vitis vinifera obtained using two high-throughput sequencing (HTS) strategies on RNA. The initial discovery from small-RNA sequencing was confirmed by HTS of the total RNA and Sanger sequencing. The new virus has a genome structure similar to the one reported for other vitiviruses, with five open reading frames (ORFs) coding for the conserved domains described for members of that genus. Phylogenetic analysis of the complete genome sequence confirmed its affiliation to the genus Vitivirus, with the closest described viruses being grapevine virus E (GVE) and Agave tequilana leaf virus (ATLV). However, the virus we report is distinct and shares only 51% amino acid sequence identity with GVE in the replicase polyprotein and 66.8% amino acid sequence identity with ATLV in the coat protein. This is well below the threshold determined by the ICTV for species demarcation, and we propose that this virus represents a new species. It is provisionally named "grapevine virus G".

Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages

PubMed Central

Vogt, Carmen; Pernemalm, Maria; Kohonen, Pekka; Laurent, Sophie; Hultenby, Kjell; Vahter, Marie; Lehtiö, Janne; Toprak, Muhammet S.; Fadeel, Bengt

2015-01-01

Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated versus dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs. PMID:26444829

Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages.

PubMed

Vogt, Carmen; Pernemalm, Maria; Kohonen, Pekka; Laurent, Sophie; Hultenby, Kjell; Vahter, Marie; Lehtiö, Janne; Toprak, Muhammet S; Fadeel, Bengt

2015-01-01

Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated versus dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs.

Spatially controlled coating of continuous liquid Interface production microneedles for transdermal protein delivery.

PubMed

Caudill, Cassie L; Perry, Jillian L; Tian, Shaomin; Luft, J Christopher; DeSimone, Joseph M

2018-06-09

Microneedle patches, arrays of micron-scale projections that penetrate skin in a minimally invasive manner, are a promising tool for transdermally delivering therapeutic proteins. However, current microneedle fabrication techniques are limited in their ability to fabricate microneedles rapidly and with a high degree of control over microneedle design parameters. We have previously demonstrated the ability to fabricate microneedle patches with a range of compositions and geometries using the novel additive manufacturing technique Continuous Liquid Interface Production (CLIP). Here, we establish a method for dip coating CLIP microneedles with protein cargo in a spatially controlled manner. Microneedle coating mask devices were fabricated with CLIP and utilized to coat polyethylene glycol-based CLIP microneedles with model proteins bovine serum albumin, ovalbumin, and lysozyme. The design of the coating mask device was used to control spatial deposition and loading of coated protein cargo on the microneedles. CLIP microneedles rapidly released coated protein cargo both in solution and upon insertion into porcine skin. The model enzyme lysozyme was shown to retain its activity throughout the CLIP microneedle coating process, and permeation of bovine serum albumin across full thickness porcine skin was observed after application with coated CLIP microneedles. Protein-coated CLIP microneedles were applied to live mice and showed sustained retention of protein cargo in the skin over 72 h. These results demonstrate the utility of a versatile coating platform for preparation of precisely coated microneedles for transdermal therapeutic delivery. Copyright © 2018. Published by Elsevier B.V.

A Bacillus subtilis Secreted Protein with a Role in Endospore Coat Assembly and Function

PubMed Central

Serrano, Mónica; Zilhão, Rita; Ricca, Ezio; Ozin, Amanda J.; Moran, Charles P.; Henriques, Adriano O.

1999-01-01

Bacterial endospores are encased in a complex protein coat, which confers protection against noxious chemicals and influences the germination response. In Bacillus subtilis, over 20 polypeptides are organized into an amorphous undercoat, a lamellar lightly staining inner structure, and an electron-dense outer coat. Here we report on the identification of a polypeptide of about 30 kDa required for proper coat assembly, which was extracted from spores of a gerE mutant. The N-terminal sequence of this polypeptide matched the deduced product of the tasA gene, after removal of a putative 27-residue signal peptide, and TasA was immunologically detected in material extracted from purified spores. Remarkably, deletion of tasA results in the production of asymmetric spores that accumulate misassembled material in one pole and have a greatly expanded undercoat and an altered outer coat structure. Moreover, we found that tasA and gerE mutations act synergistically to decrease the efficiency of spore germination. We show that tasA is the most distal member of a three-gene operon, which also encodes the type I signal peptidase SipW. Expression of the tasA operon is enhanced 2 h after the onset of sporulation, under the control of ςH. When tasA transcription is uncoupled from sipW expression, a presumptive TasA precursor accumulates, suggesting that its maturation depends on SipW. Mature TasA is found in supernatants of sporulating cultures and intracellularly from 2 h of sporulation onward. We suggest that, at an early stage of sporulation, TasA is secreted to the septal compartment. Later, after engulfment of the prespore by the mother cell, TasA acts from the septal-proximal pole of the spore membranes to nucleate the organization of the undercoat region. TasA is the first example of a polypeptide involved in coat assembly whose production is not mother cell specific but rather precedes its formation. Our results implicate secretion as a mechanism to target individual proteins to specific cellular locations during the assembly of the bacterial endospore coat. PMID:10368135

Evolutionary relationships in the ilarviruses: nucleotide sequence of prunus necrotic ringspot virus RNA 3.

PubMed

Sánchez-Navarro, J A; Pallás, V

1997-01-01

The complete nucleotide sequence of an isolate of prunus necrotic ringspot virus (PNRSV) RNA 3 has been determined. Elucidation of the amino acid sequence of the proteins encoded by the two large open reading frames (ORFs) allowed us to carry out comparative and phylogenetic studies on the movement (MP) and coat (CP) proteins in the ilarvirus group. Amino acid sequence comparison of the MP revealed a highly conserved basic sequence motif with an amphipathic alpha-helical structure preceding the conserved motif of the '30K superfamily' proposed by Mushegian and Koonin [26] for MP's. Within this '30K' motif a strictly conserved transmembrane domain is present in all ilarviruses sequenced so far. At the amino-terminal end, prune dwarf virus (PDV) has an extension not present in other ilarviruses but which is observed in all bromo- and cucumoviruses, suggesting a common ancestor or a recombinational event in the Bromoviridae family. Examination of the N-terminus of the CP's of all ilarviruses revealed a highly basic region, part of which resembles the Arg-rich motif that has been characterized in the RNA-binding protein family. This motif has also been found in the other members of the Bromoviridae family, suggesting its involvement in a structural function. Furthermore this region is required for infectivity in ilarviruses. The similarities found in this Arg-rich motif are discussed in terms of this process known as genome activation. Finally, phylogenetic analysis of both the MP and CP proteins revealed a higher relationship of A1MV to PNRSV, apple mosaic virus (ApMV) and PDV than any other member of the ilarvirus group. In that sense, A1MV should be considered as a true ilarvirus instead of forming a distinct group of viruses.

Packaging signals in two single-stranded RNA viruses imply a conserved assembly mechanism and geometry of the packaged genome.

PubMed

Dykeman, Eric C; Stockley, Peter G; Twarock, Reidun

2013-09-09

The current paradigm for assembly of single-stranded RNA viruses is based on a mechanism involving non-sequence-specific packaging of genomic RNA driven by electrostatic interactions. Recent experiments, however, provide compelling evidence for sequence specificity in this process both in vitro and in vivo. The existence of multiple RNA packaging signals (PSs) within viral genomes has been proposed, which facilitates assembly by binding coat proteins in such a way that they promote the protein-protein contacts needed to build the capsid. The binding energy from these interactions enables the confinement or compaction of the genomic RNAs. Identifying the nature of such PSs is crucial for a full understanding of assembly, which is an as yet untapped potential drug target for this important class of pathogens. Here, for two related bacterial viruses, we determine the sequences and locations of their PSs using Hamiltonian paths, a concept from graph theory, in combination with bioinformatics and structural studies. Their PSs have a common secondary structure motif but distinct consensus sequences and positions within the respective genomes. Despite these differences, the distributions of PSs in both viruses imply defined conformations for the packaged RNA genomes in contact with the protein shell in the capsid, consistent with a recent asymmetric structure determination of the MS2 virion. The PS distributions identified moreover imply a preferred, evolutionarily conserved assembly pathway with respect to the RNA sequence with potentially profound implications for other single-stranded RNA viruses known to have RNA PSs, including many animal and human pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

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 correctly shaped and sized procapsids and that the lack of these proper protein-protein interfaces leads to aberrant structures. The present work represents an important contribution supporting the hypothesis that virus capsid assembly is governed by seemingly simple interactions. The highly specific nature of the subunit interfaces suggests that these could be good targets for antivirals.

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis

PubMed Central

Wang, Katherine H.; Isidro, Anabela L.; Domingues, Lia; Eskandarian, Haig A.; McKenney, Peter T.; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N.; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O.; Eichenberger, Patrick

2009-01-01

SUMMARY Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to GFP and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homolog of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement. PMID:19775244

Genomic Approaches for Detection and Treatment of Breast Cancer

DTIC Science & Technology

2008-07-01

number display fused to the T7 coat protein, 10B. The library was then extensively characterized by sequencing several hundred individual phage clones at...FLAG-tagged T7 phage (1:1000) into a native phage population and diluting an anti-FLAG antibody (1:1000) into a non-specific isotype control antibody...plaque lift assay) by systematically varying the following parameters: T7 phage concentration, antibody concentration, time of immunoprecipitation, and

Using Genetics and Genomics for the Detection and Treatment of Breast Cancer

DTIC Science & Technology

2009-09-01

display fused to the 13 T7 coat protein, 10B. The library was then extensively characterized by sequencing several hundred individual phage clones at...tagged T7 phage (1:1000) into a native phage population and diluting an anti-FLAG antibody (1:1000) into a non-specific isotype control antibody, we...assay) by systematically varying the following parameters: T7 phage concentration, antibody concentration, time of immunoprecipitation, and number of

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

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

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

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

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

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

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

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

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

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

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

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

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

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

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

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

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

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt...

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

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are exempt...

Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

PubMed

Smirnova, Ekaterina; Firth, Andrew E; Miller, W Allen; Scheidecker, Danièle; Brault, Véronique; Reinbold, Catherine; Rakotondrafara, Aurélie M; Chung, Betty Y-W; Ziegler-Graff, Véronique

2015-05-01

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement

PubMed Central

Smirnova, Ekaterina; Firth, Andrew E.; Miller, W. Allen; Scheidecker, Danièle; Brault, Véronique; Reinbold, Catherine; Rakotondrafara, Aurélie M.; Chung, Betty Y.-W.; Ziegler-Graff, Véronique

2015-01-01

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement. PMID:25946037

Characterization of a Novel Polerovirus Infecting Maize in China

PubMed Central

Chen, Sha; Jiang, Guangzhuang; Wu, Jianxiang; Liu, Yong; Qian, Yajuan; Zhou, Xueping

2016-01-01

A novel virus, tentatively named Maize Yellow Mosaic Virus (MaYMV), was identified from the field-grown maize plants showing yellow mosaic symptoms on the leaves collected from the Yunnan Province of China by the deep sequencing of small RNAs. The complete 5642 nucleotide (nt)-long genome of the MaYMV shared the highest nucleotide sequence identity (73%) to Maize Yellow Dwarf Virus-RMV. Sequence comparisons and phylogenetic analyses suggested that MaYMV represents a new member of the genus Polerovirus in the family Luteoviridae. Furthermore, the P0 protein encoded by MaYMV was demonstrated to inhibit both local and systemic RNA silencing by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene, which further supported the identification of a new polerovirus. The biologically-active cDNA clone of MaYMV was generated by inserting the full-length cDNA of MaYMV into the binary vector pCB301. RT-PCR and Northern blot analyses showed that this clone was systemically infectious upon agro-inoculation into N. benthamiana. Subsequently, 13 different isolates of MaYMV from field-grown maize plants in different geographical locations of Yunnan and Guizhou provinces of China were sequenced. Analyses of their molecular variation indicate that the 3′ half of P3–P5 read-through protein coding region was the most variable, whereas the coat protein- (CP-) and movement protein- (MP-)coding regions were the most conserved. PMID:27136578

Characterization of a Novel Polerovirus Infecting Maize in China.

PubMed

Chen, Sha; Jiang, Guangzhuang; Wu, Jianxiang; Liu, Yong; Qian, Yajuan; Zhou, Xueping

2016-04-28

A novel virus, tentatively named Maize Yellow Mosaic Virus (MaYMV), was identified from the field-grown maize plants showing yellow mosaic symptoms on the leaves collected from the Yunnan Province of China by the deep sequencing of small RNAs. The complete 5642 nucleotide (nt)-long genome of the MaYMV shared the highest nucleotide sequence identity (73%) to Maize Yellow Dwarf Virus-RMV. Sequence comparisons and phylogenetic analyses suggested that MaYMV represents a new member of the genus Polerovirus in the family Luteoviridae. Furthermore, the P0 protein encoded by MaYMV was demonstrated to inhibit both local and systemic RNA silencing by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene, which further supported the identification of a new polerovirus. The biologically-active cDNA clone of MaYMV was generated by inserting the full-length cDNA of MaYMV into the binary vector pCB301. RT-PCR and Northern blot analyses showed that this clone was systemically infectious upon agro-inoculation into N. benthamiana. Subsequently, 13 different isolates of MaYMV from field-grown maize plants in different geographical locations of Yunnan and Guizhou provinces of China were sequenced. Analyses of their molecular variation indicate that the 3' half of P3-P5 read-through protein coding region was the most variable, whereas the coat protein- (CP-) and movement protein- (MP-)coding regions were the most conserved.

A Japanese Encephalitis Virus Peptide Present on Johnson Grass Mosaic Virus-Like Particles Induces Virus-Neutralizing Antibodies and Protects Mice against Lethal Challenge

PubMed Central

Saini, Manisha; Vrati, Sudhanshu

2003-01-01

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge. PMID:12610124

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

PubMed

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.

[Molecular cloning and characterization of a novel Clonorchis sinensis antigenic protein containing tandem repeat sequences].

PubMed

Liu, Qian; Xu, Xue-Nian; Zhou, Yan; Cheng, Na; Dong, Yu-Ting; Zheng, Hua-Jun; Zhu, Yong-Qiang; Zhu, Yong-Qiang

2013-08-01

To find and clone new antigen genes from the lambda-ZAP cDNA expression library of adult Clonorchis sinensis, and determine the immunological characteristics of the recombinant proteins. The cDNA expression library of adult C. sinensis was screened by pooled sera of clonorchiasis patients. The sequences of the positive phage clones were compared with the sequences in EST database, and the full-length sequence of the gene (Cs22 gene) was obtained by RT-PCR. cDNA fragments containing 2 and 3 times tandem repeat sequences were generated by jumping PCR. The sequence encoding the mature peptide or the tandem repeat sequence was respectively cloned into the prokaryotic expression vector pET28a (+), and then transformed into E. coli Rosetta DE3 cells for expression. The recombinant proteins (rCs22-2r, rCs22-3r, rCs22M-2r, and rCs22M-3r) were purified by His-bind-resin (Ni-NTA) affinity chromatography. The immunogenicity of rCs22-2r and rCs22-3r was identified by ELISA. To evaluate the immunological diagnostic value of rCs22-2r and rCs22-3r, serum samples from 35 clonorchiasis patients, 31 healthy individuals, 15 schistosomiasis patients, 15 paragonimiasis westermani patients and 13 cysticercosis patients were examined by ELISA. To locate antigenic determinants, the pooled sera of clonorchiasis patients and healthy persons were analyzed for specific antibodies by ELISA with recombinant protein rCs22M-2r and rCs22M-3r containing the tandem repeat sequences. The full-length sequence of Cs22 antigen gene of C. sinensis was obtained. It contained 13 times tandem repeat sequences of EQQDGDEEGMGGDGGRGKEKGKVEGEDGAGEQKEQA. Bioinformatics analysis indicated that the protein (Cs22) belonged to GPI-anchored proteins family. The recombinant proteins rCs22-2r and rCs22-3r showed a certain level of immunogenicity. The positive rate by ELISA coated with the purified PrCs22-2r and PrCs22-3r for sera of clonorchiasis patients both were 45.7% (16/35), and 3.2% (1/31) for those of healthy persons. There was no cross reaction with sera of schistosomiasis and cysticercosis patients. The cross reaction with sera of paragonimiasis westermani patients was 1/15. The recombinant proteins rCs22M-2r and rCs22M-3r which only contained tandem repeats were specifically recognized by pooled sera of clonorchiasis patients. The Cs22 antigen gene of Clonorchis sinensis is obtained, and the recombinant proteins have certain diagnostic value. The antigenic determinant is located in tandem repeat sequences.

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

Code of Federal Regulations, 2010 CFR

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...

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

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Watermelon Mosaic... Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. Residues of Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic...

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

Code of Federal Regulations, 2010 CFR

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...

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

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 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...

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

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 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...

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

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 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...

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

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 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...

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

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 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...

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

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 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...

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

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 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...

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

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 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...

Novel rod-shaped viruses isolated from garlic, Allium sativum, possessing a unique genome organization.

PubMed

Sumi, S; Tsuneyoshi, T; Furutani, H

1993-09-01

Rod-shaped flexuous viruses were partially purified from garlic plants (Allium sativum) showing typical mosaic symptoms. The genome was shown to be composed of RNA with a poly(A) tail of an estimated size of 10 kb as shown by denaturing agarose gel electrophoresis. We constructed cDNA libraries and screened four independent clones, which were designated GV-A, GV-B, GV-C and GV-D, using Northern and Southern blot hybridization. Nucleotide sequence determination of the cDNAs, two of which correspond to nearly one-third of the virus genomic RNA, shows that all of these viruses possess an identical genomic structure and that also at least four proteins are encoded in the viral cDNA, their M(r)s being estimated to be 15K, 27K, 40K and 11K. The 15K open reading frame (ORF) encodes the core-like sequence of a zinc finger protein preceded by a cluster of basic amino acid residues. The 27K ORF probably encodes the viral coat protein (CP), based on both the existence of some conserved sequences observed in many other rod-shaped or flexuous virus CPs and an overall amino acid sequence similarity to potexvirus and carlavirus CPs. The 11K ORF shows significant amino acid sequence similarities to the corresponding 12K proteins of the potexviruses and carlaviruses. On the other hand, the 40K ORF product does not resemble any other plant virus gene products reported so far. The genomic organization in the 3' region of the garlic viruses resembles, but clearly differs from, that of carlaviruses. Phylogenetic analysis based upon the amino acid sequence of the viral capsid protein also indicates that the garlic viruses have a unique and distinct domain different from those of the potexvirus and carlavirus groups. The results suggest that the garlic viruses described here belong to an unclassified and new virus group closely related to the carlaviruses.

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

PubMed

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.

A novel, highly divergent ssDNA virus identified in Brazil infecting apple, pear and grapevine.

PubMed

Basso, Marcos Fernando; da Silva, José Cleydson Ferreira; Fajardo, Thor Vinícius Martins; Fontes, Elizabeth Pacheco Batista; Zerbini, Francisco Murilo

2015-12-02

Fruit trees of temperate and tropical climates are of great economical importance worldwide and several viruses have been reported affecting their productivity and longevity. Fruit trees of different Brazilian regions displaying virus-like symptoms were evaluated for infection by circular DNA viruses. Seventy-four fruit trees were sampled and a novel, highly divergent, monopartite circular ssDNA virus was cloned from apple, pear and grapevine trees. Forty-five complete viral genomes were sequenced, with a size of approx. 3.4 kb and organized into five ORFs. Deduced amino acid sequences showed identities in the range of 38% with unclassified circular ssDNA viruses, nanoviruses and alphasatellites (putative Replication-associated protein, Rep), and begomo-, curto- and mastreviruses (putative coat protein, CP, and movement protein, MP). A large intergenic region contains a short palindromic sequence capable of forming a hairpin-like structure with the loop sequence TAGTATTAC, identical to the conserved nonanucleotide of circoviruses, nanoviruses and alphasatellites. Recombination events were not detected and phylogenetic analysis showed a relationship with circo-, nano- and geminiviruses. PCR confirmed the presence of this novel ssDNA virus in field plants. Infectivity tests using the cloned viral genome confirmed its ability to infect apple and pear tree seedlings, but not Nicotiana benthamiana. The name "Temperate fruit decay-associated virus" (TFDaV) is proposed for this novel virus. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression of a foot-and-mouth disease virus immunodominant epitope by a filamentous bacteriophage vector.

PubMed

Kim, Y J; Lebreton, F; Kaiser, C; Crucière, C; Rémond, M

2004-02-01

We described the construction of a recombinant filamentous phage displaying on its surface the immunodominant site of VP1 protein of foot-and-mouth disease virus (FMDV). The coding sequence was inserted at the amino-terminus of the major coat protein pVIII via a spacer. The hybrid phage proved to be antigenic as it was recognized by polyclonal and monoclonal anti FMDV sera. In two experiments involving immunisation of guinea-pigs with the recombinant phage, a low antibody response was generated. This suggests a possible role for phage displayed peptides in inducing anti FMDV immunity and the possibility of further development is discussed.

Structural evolution of the P22-like phages: Comparison of Sf6 and P22 procapsid and virion architectures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parent, Kristin N.; Gilcrease, Eddie B.; Casjens, Sherwood R., E-mail: sherwood.casjens@path.utah.edu

Coat proteins of tailed, dsDNA phages and in herpesviruses include a conserved core similar to the bacteriophage HK97 subunit. This core is often embellished with other domains such as the telokin Ig-like domain of phage P22. Eighty-six P22-like phages and prophages with sequenced genomes share a similar set of virion assembly genes and, based on comparisons of twelve viral assembly proteins (structural and assembly/packaging chaperones), these phages are classified into three groups (P22-like, Sf6-like, and CUS-3-like). We used cryo-electron microscopy and 3D image reconstruction to determine the structures of Sf6 procapsids and virions ({approx} 7 A resolution), and the structuremore » of the entire, asymmetric Sf6 virion (16-A resolution). The Sf6 coat protein is similar to that of P22 yet it has differences in the telokin domain and in its overall quaternary organization. Thermal stability and agarose gel experiments show that Sf6 virions are slightly less stable than those of P22. Finally, bacterial host outer membrane proteins A and C were identified in lipid vesicles that co-purify with Sf6 particles, but are not components of the capsid.« less

Protein Corona Composition of Superparamagnetic Iron Oxide Nanoparticles with Various Physico-Chemical Properties and Coatings

NASA Astrophysics Data System (ADS)

Sakulkhu, Usawadee; Mahmoudi, Morteza; Maurizi, Lionel; Salaklang, Jatuporn; Hofmann, Heinrich

2014-05-01

Because of their biocompatibility and unique magnetic properties, superparamagnetic iron oxide nanoparticles NPs (SPIONs) are recognized as some of the most prominent agents for theranostic applications. Thus, understanding the interaction of SPIONs with biological systems is important for their safe design and efficient applications. In this study, SPIONs were coated with 2 different polymers: polyvinyl alcohol polymer (PVA) and dextran. The obtained NPs with different surface charges (positive, neutral, and negative) were used as a model study of the effect of surface charges and surface polymer materials on protein adsorption using a magnetic separator. We found that the PVA-coated SPIONs with negative and neutral surface charge adsorbed more serum proteins than the dextran-coated SPIONs, which resulted in higher blood circulation time for PVA-coated NPs than the dextran-coated ones. Highly abundant proteins such as serum albumin, serotransferrin, prothrombin, alpha-fetoprotein, and kininogen-1 were commonly found on both PVA- and dextran-coated SPIONs. By increasing the ionic strength, soft- and hard-corona proteins were observed on 3 types of PVA-SPIONs. However, the tightly bound proteins were observed only on negatively charged PVA-coated SPIONs after the strong protein elution.

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

PubMed Central

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 warranted and could potentially lead to the development of improved diagnostic and therapeutic tools. PMID:23308195

Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles.

PubMed

Longkumer, Toshisangba; Kamireddy, Swetha; Muthyala, Venkateswar Reddy; Akbarpasha, Shaikh; Pitchika, Gopi Krishna; Kodetham, Gopinath; Ayaluru, Murali; Siddavattam, Dayananda

2013-01-01

While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002.

Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles

PubMed Central

Longkumer, Toshisangba; Kamireddy, Swetha; Muthyala, Venkateswar Reddy; Akbarpasha, Shaikh; Pitchika, Gopi Krishna; Kodetham, Gopinath; Ayaluru, Murali; Siddavattam, Dayananda

2013-01-01

While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002. PMID:23867905

A base substitution in the donor site of intron 12 of KIT gene is responsible for the dominant white coat colour of blue fox (Alopex lagopus).

PubMed

Yan, S Q; Hou, J N; Bai, C Y; Jiang, Y; Zhang, X J; Ren, H L; Sun, B X; Zhao, Z H; Sun, J H

2014-04-01

The dominant white coat colour of farmed blue fox is inherited as a monogenic autosomal dominant trait and is suggested to be embryonic lethal in the homozygous state. In this study, the transcripts of KIT were identified by RT-PCR for a dominant white fox and a normal blue fox. Sequence analysis showed that the KIT transcript in normal blue fox contained the full-length coding sequence of 2919 bp (GenBank Acc. No KF530833), but in the dominant white individual, a truncated isoform lacking the entire exon 12 specifically co-expressed with the normal transcript. Genomic DNA sequencing revealed that a single nucleotide polymorphism (c.1867+1G>T) in intron 12 appeared only in the dominant white individuals and a 1-bp ins/del polymorphism in the same intron showed in individuals representing two different coat colours. Genotyping results of the SNP with PCR-RFLP in 185 individuals showed all 90 normal blue foxes were homozygous for the G allele, and all dominant white individuals were heterozygous. Due to the truncated protein with a deletion of 35 amino acids and an amino acid replacement (p.Pro623Ala) located in the conserved ATP binding domain, we propose that the mutant receptor had absent tyrosine kinase activity. These findings reveal that the base substitution at the first nucleotide of intron 12 of KIT gene, resulting in skipping of exon 12, is a causative mutation responsible for the dominant white phenotype of blue fox. © 2013 Stichting International Foundation for Animal Genetics.

Nanoparticle formulation enhanced protective immunity provoked by PYGPI8p-transamidase related protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

PubMed

Cherif, Mahamoud Sama; Shuaibu, Mohammed Nasir; Kodama, Yukinobu; Kurosaki, Tomoaki; Helegbe, Gideon Kofi; Kikuchi, Mihoko; Ichinose, Akitoyo; Yanagi, Tetsuo; Sasaki, Hitoshi; Yui, Katsuyuki; Tien, Nguyen Huy; Karbwang, Juntra; Hirayama, Kenji

2014-04-07

We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamic acid (γ-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mouse malaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vaccine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positive genes picked up using web-based bioinformatics tools; though the observed protection was not complete. Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and γ-PGA complex as delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated empty plasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 and CD8T cells producing IFN-γ in the splenocytes and IL-4, IFN-γ, IL-12 and TNF-α levels in the sera and in the supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vaccine. These data indicates that NP augments PyTAM protective immune response, and this enhancement was associated with increased DC activation and concomitant IL-12 production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genome activation by raspberry bushy dwarf virus coat protein.

PubMed

Macfarlane, Stuart A; McGavin, Wendy J

2009-03-01

Two sets of infectious cDNA clones of raspberry bushy dwarf virus (RBDV) have been constructed, enabling either the synthesis of infectious RNA transcripts or the delivery of infectious binary plasmid DNA by infiltration of Agrobacterium tumefaciens. In whole plants and in protoplasts, inoculation of RBDV RNA1 and RNA2 transcripts led to a low level of infection, which was greatly increased by the addition of RNA3, a subgenomic RNA coding for the RBDV coat protein (CP). Agroinfiltration of RNA1 and RNA2 constructs did not produce a detectable infection but, again, inclusion of a construct encoding the CP led to high levels of infection. Thus, RBDV replication is greatly stimulated by the presence of the CP, a mechanism that also operates with ilarviruses and alfalfa mosaic virus, where it is referred to as genome activation. Mutation to remove amino acids from the N terminus of the CP showed that the first 15 RBDV CP residues are not required for genome activation. Other experiments, in which overlapping regions at the CP N terminus were fused to the monomeric red fluorescent protein, showed that sequences downstream of the first 48 aa are not absolutely required for genome activation.

Identification of the earliest collagen- and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel).

PubMed

Solazzo, Caroline; Courel, Blandine; Connan, Jacques; van Dongen, Bart E; Barden, Holly; Penkman, Kirsty; Taylor, Sheila; Demarchi, Beatrice; Adam, Pierre; Schaeffer, Philippe; Nissenbaum, Arie; Bar-Yosef, Ofer; Buckley, Michael

2016-08-09

Mortuary practices in human evolution record cognitive, social changes and technological innovations. The Neolithic Revolution in the Levant was a watershed in this domain that has long fascinated the archaeological community. Plaster modelled skulls are well known at Jericho and several other Neolithic sites, and in Nahal Hemar cave (Israel, ca. 8200 -7300 cal. BC) excavations yielded six unique human skulls covered with a black organic coating applied in a net pattern evoking a headdress. This small cave was used as storage for paraphernalia in the semi-arid area of the Judean desert and the dry conditions preserved other artefacts such as baskets coated with a similar dark substance. While previous analysis had revealed the presence of amino acids consistent with a collagen signature, in the present report, specific biomarkers were characterised using combined proteomic and lipid approaches. Basket samples yielded collagen and blood proteins of bovine origin (Bos genus) and a large sequence coverage of a plant protein charybdin (Charybdis genus). The skull residue samples were dominated by benzoate and cinnamate derivatives and triterpenes consistent with a styrax-type resin (Styrax officinalis), thus providing the earliest known evidence of an odoriferous plant resin used in combination with an animal product.

Identification of the earliest collagen- and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)

PubMed Central

Solazzo, Caroline; Courel, Blandine; Connan, Jacques; van Dongen, Bart E.; Barden, Holly; Penkman, Kirsty; Taylor, Sheila; Demarchi, Beatrice; Adam, Pierre; Schaeffer, Philippe; Nissenbaum, Arie; Bar-Yosef, Ofer; Buckley, Michael

2016-01-01

Mortuary practices in human evolution record cognitive, social changes and technological innovations. The Neolithic Revolution in the Levant was a watershed in this domain that has long fascinated the archaeological community. Plaster modelled skulls are well known at Jericho and several other Neolithic sites, and in Nahal Hemar cave (Israel, ca. 8200 −7300 cal. BC) excavations yielded six unique human skulls covered with a black organic coating applied in a net pattern evoking a headdress. This small cave was used as storage for paraphernalia in the semi-arid area of the Judean desert and the dry conditions preserved other artefacts such as baskets coated with a similar dark substance. While previous analysis had revealed the presence of amino acids consistent with a collagen signature, in the present report, specific biomarkers were characterised using combined proteomic and lipid approaches. Basket samples yielded collagen and blood proteins of bovine origin (Bos genus) and a large sequence coverage of a plant protein charybdin (Charybdis genus). The skull residue samples were dominated by benzoate and cinnamate derivatives and triterpenes consistent with a styrax-type resin (Styrax officinalis), thus providing the earliest known evidence of an odoriferous plant resin used in combination with an animal product. PMID:27503740

A two-stage mechanism of viral RNA compaction revealed by single molecule fluorescence

PubMed Central

Borodavka, Alexander; Tuma, Roman; Stockley, Peter G.

2013-01-01

Long RNAs often exist as multiple conformers in equilibrium. For the genomes of single-stranded RNA viruses, one of these conformers must include a compacted state allowing the RNA to be confined within the virion. We have used single molecule fluorescence correlation spectroscopy to monitor the conformations of viral genomes and sub-fragments in the absence and presence of coat proteins. Cognate RNA-coat protein interactions in two model viruses cause a rapid collapse in the hydrodynamic radii of their respective RNAs. This is caused by protein binding at multiple sites on the RNA that facilitate additional protein-protein contacts. The collapsed species recruit further coat proteins to complete capsid assembly with great efficiency and fidelity. The specificity in RNA-coat protein interactions seen at single-molecule concentrations reflects the packaging selectivity seen for such viruses in vivo. This contrasts with many in vitro reassembly measurements performed at much higher concentrations. RNA compaction by coat protein or polycation binding are distinct processes, implying that defined RNA-coat protein contacts are required for assembly. PMID:23422316

Embryonal Control of Yellow Seed Coat Locus ECY1 Is Related to Alanine and Phenylalanine Metabolism in the Seed Embryo of Brassica napus

PubMed Central

Wang, Fulin; He, Jiewang; Shi, Jianghua; Zheng, Tao; Xu, Fei; Wu, Guanting; Liu, Renhu; Liu, Shengyi

2016-01-01

Seed coat color is determined by the type of pigment deposited in the seed coat cells. It is related to important agronomic traits of seeds such as seed dormancy, longevity, oil content, protein content and fiber content. In Brassica napus, inheritance of seed coat color is related to maternal effects and pollen effects (xenia effects). In this research we isolated a mutation of yellow seeded B. napus controlled by a single Mendelian locus, which is named Embryonal Control of Yellow seed coat 1 (Ecy1). Microscopy of transverse sections of the mature seed show that pigment is deposited only in the outer layer of the seed coat. Using Illumina Hisequation 2000 sequencing technology, a total of 12 GB clean data, 116× coverage of coding sequences of B. napus, was achieved from seeds 26 d after pollination (DAP). It was assembled into 172,238 independent transcripts, and 55,637 unigenes. A total of 139 orthologous genes of Arabidopsis transparent testa (TT) genes were mapped in silico to 19 chromosomes of B. napus. Only 49 of the TT orthologous genes are transcribed in seeds. However transcription of all orthologs was independent of embryonal control of seed coat color. Only 55 genes were found to be differentially expressed between brown seeds and the yellow mutant. Of these 55, 50 were upregulated and five were downregulated in yellow seeds as compared to their brown counterparts. By KEGG classification, 14 metabolic pathways were significantly enriched. Of these, five pathways: phenylpropanoid biosynthesis, cyanoamino acid metabolism, plant hormone signal transduction, metabolic pathways, and biosynthesis of secondary metabolites, were related with seed coat pigmentation. Free amino acid quantification showed that Ala and Phe were present at higher levels in the embryos of yellow seeds as compared to those of brown seeds. This increase was not observed in the seed coat. Moreover, the excess amount of free Ala was exactly twice that of Phe in the embryo. The pigment substrate chalcone is synthesized from two molecules of Ala and one molecule of Phe. The correlation between accumulation of Ala and Phe, and disappearance of pigment in the yellow seeded mutant, suggests that embryonal control of seed coat color is related with Phe and Ala metabolism in the embryo of B. napus. PMID:26896439

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro.

PubMed

Cherwa, James E; Tyson, Joshua; Bedwell, Gregory J; Brooke, Dewey; Edwards, Ashton G; Dokland, Terje; Prevelige, Peter E; Fane, Bentley A

2017-01-01

During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity. Copyright © 2016 American Society for Microbiology.

Engineering of filamentous bacteriophage for protein sensing

NASA Astrophysics Data System (ADS)

Brasino, Michael

Methods of high throughput, sensitive and cost effective quantification of proteins enables personalized medicine by allowing healthcare professionals to better monitor patient condition and response to treatment. My doctoral research has attempted to advance these methods through the use of filamentous bacteriophage (phage). These bacterial viruses are particularly amenable to both genetic and chemical engineering and can be produced efficiently in large amounts. Here, I discuss several strategies for modifying phage for use in protein sensing assays. These include the expression of bio-orthogonal conjugation handles on the phage coat, the incorporation of specific recognition sequences within the phage genome, and the creation of antibody-phage conjugates via a photo-crosslinking non-canonical amino acid. The physical and chemical characterization of these engineered phage and the results of their use in modified protein sensing assays will be presented.

Complete nucleotide sequence and genome organization of a Chinese isolate of Tobacco vein distorting virus.

PubMed

Mo, Xiao-han; Chen, Zheng-bin; Chen, Jian-ping

2010-12-01

Tobacco bushy top disease is caused by tobacco bushy top virus (TBTV, a member of the genus Umbravirus) which is dependent on tobacco vein-distorting virus (TVDV) to act as a helper virus encapsidating TBTV and enabling its transmission by aphids. Isometric virions from diseased tobacco plants were purified and disease symptoms were reproduced after experimental aphid transmission. The complete genome of TVDV was determined from cloned RT-PCR products derived from viral RNA. It was 5,920 nucleotides (nts) long and had the six major open reading frames (ORFs) typical of a member of the genus Polerovirus. Sequence comparisons showed that it differed significantly from any of the other species in the genus and this was confirmed by phylogenetic analyses of the RdRp and coat protein. SDS-PAGE analysis of purified virions gave two protein bands of about 26 and 59 kDa both of which reacted strongly in Western blots with antiserum produced to prokaryotically expressed TVDV CP showing that the two forms of the TVDV CP were the only protein components of the capsid.

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

PubMed

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.

RNase-Resistant Virus-Like Particles Containing Long Chimeric RNA Sequences Produced by Two-Plasmid Coexpression System▿

PubMed Central

Wei, Yuxiang; Yang, Changmei; Wei, Baojun; Huang, Jie; Wang, Lunan; Meng, Shuang; Zhang, Rui; Li, Jinming

2008-01-01

RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments—hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)—was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 106 to 102 copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection. PMID:18305135

Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage.

PubMed

Løset, Geir Åge; Roos, Norbert; Bogen, Bjarne; Sandlie, Inger

2011-02-24

Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display. Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient. Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.

Structure and dynamics of a detergent-solubilized membrane protein: measurement of amide hydrogen exchange rates in M13 coat protein by /sub 1/H NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Neil, J.D.J.; Sykes, B.D.

The coat protein of bacteriophage M13 is inserted into the inner membrane of Escherichia coli where it exists as an integral membrane protein during the reproductive cycle of the phage. The protein sequence consists of a highly hydrophobic 19-residue central segment flanked by an acidic 20-residue N-terminus and a basic 11-residue C-terminus. The authors have measured backbone amide hydrogen exchange of the protein solubilized in perdeuteriated sodium dodecyl sulfate using /sup 1/H nuclear magnetic resonance (NMR) spectroscopy. Direct proton exchange-out measurements in D/sub 2/O at 24 /sup 0/C were used to follow the exchange of the slowest amides in themore » protein. Multiple exponential fitting of the exchange data showed that these amides exchanged in two kinetic sets with exchange rates that differed by more than 100-fold. Steady-state saturation-transfer techniques were also used to measure exchange. These methods showed that 15-20 amides in the protein are very stable at 55/sup 0/C and that bout 30 amides have exchange rates retarded by at least 10/sup 5/-fold at 24/sup 0/C. Saturation-transfer studies also showed that the pH dependence of exchange in the hydrophilic termini was unusual. Relaxation and solid-state NMR experiments have previously shown that the majority of the protein backbone is rigid on the picosecond to microsecond time scale, except for the extreme ends of the molecule which are mobile. The hydrogen exchange results, which are sensitive to a much longer time scale, suggest a stable core with a progressive increase in amplitude or frequency of motions as the ends of the protein are approached.« less

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Use of hydrophilic polymer coatings for control of electroosmosis and protein adsorption

NASA Technical Reports Server (NTRS)

Harris, J. Milton

1987-01-01

The purpose of this project was to examine the utility of polyethylene glycol (PEG) and dextran coatings for control of electroosmosis and protein adsorption; electroosmosis is an important, deleterious process affecting electrophoretic separations, and protein adsorption is a factor which needs to be controlled during protein crystal growth to avoid multiple nucleation sites. Performance of the project required use of X-ray photoelectron spectroscopy to refine previously developed synthetic methods. The results of this spectroscopic examination are reported. Measurements of electroosmotic mobility of charged particles in appropriately coated capillaries reveals that a new, one-step route to coating capillaries gives a surface in which electroosmosis is dramatically reduced. Similarly, both PEG and dextran coatings were shown by protein adsorption measurements to be highly effective at reducing protein adsorption on solid surfaces. These results should have impact on future low-g electrophoretic and protein crystal growth experiments.

Integrating next-generation sequencing and traditional tongue diagnosis to determine tongue coating microbiome

PubMed Central

Jiang, Bai; Liang, Xujun; Chen, Yang; Ma, Tao; Liu, Liyang; Li, Junfeng; Jiang, Rui; Chen, Ting; Zhang, Xuegong; Li, Shao

2012-01-01

Tongue diagnosis is a unique method in traditional Chinese medicine (TCM). This is the first investigation on the association between traditional tongue diagnosis and the tongue coating microbiome using next-generation sequencing. The study included 19 gastritis patients with a typical white-greasy or yellow-dense tongue coating corresponding to TCM Cold or Hot Syndrome respectively, as well as eight healthy volunteers. An Illumina paired-end, double-barcode 16S rRNA sequencing protocol was designed to profile the tongue-coating microbiome, from which approximately 3.7 million V6 tags for each sample were obtained. We identified 123 and 258 species-level OTUs that were enriched in patients with Cold/Hot Syndromes, respectively, representing "Cold Microbiota" and "Hot Microbiota". We further constructed the tongue microbiota-imbalanced networks associated with Cold/Hot Syndromes. The results reveal an important connection between the tongue-coating microbiome and traditional tongue diagnosis, and illustrate the potential of the tongue-coating microbiome as a novel holistic biomarker for characterizing patient subtypes. PMID:23226834

Donkey Orchid Symptomless Virus: A Viral ‘Platypus’ from Australian Terrestrial Orchids

PubMed Central

Wylie, Stephen J.; Li, Hua; Jones, Michael G. K.

2013-01-01

Complete and partial genome sequences of two isolates of an unusual new plant virus, designated Donkey orchid symptomless virus (DOSV) were identified using a high-throughput sequencing approach. The virus was identified from asymptomatic plants of Australian terrestrial orchid Diuris longifolia (Common donkey orchid) growing in a remnant forest patch near Perth, western Australia. DOSV was identified from two D. longifolia plants of 264 tested, and from at least one plant of 129 Caladenia latifolia (pink fairy orchid) plants tested. Phylogenetic analysis of the genome revealed open reading frames (ORF) encoding seven putative proteins of apparently disparate origins. A 69-kDa protein (ORF1) that overlapped the replicase shared low identity with MPs of plant tymoviruses (Tymoviridae). A 157-kDa replicase (ORF2) and 22-kDa coat protein (ORF4) shared 32% and 40% amino acid identity, respectively, with homologous proteins encoded by members of the plant virus family Alphaflexiviridae. A 44-kDa protein (ORF3) shared low identity with myosin and an autophagy protein from Squirrelpox virus. A 27-kDa protein (ORF5) shared no identity with described proteins. A 14-kDa protein (ORF6) shared limited sequence identity (26%) over a limited region of the envelope glycoprotein precursor of mammal-infecting Crimea-Congo hemorrhagic fever virus (Bunyaviridae). The putative 25-kDa movement protein (MP) (ORF7) shared limited (27%) identity with 3A-like MPs of members of the plant-infecting Tombusviridae and Virgaviridae. Transmissibility was shown when DOSV systemically infected Nicotiana benthamiana plants. Structure and organization of the domains within the putative replicase of DOSV suggests a common evolutionary origin with ‘potexvirus-like’ replicases of viruses within the Alphaflexiviridae and Tymoviridae, and the CP appears to be ancestral to CPs of allexiviruses (Alphaflexiviridae). The MP shares an evolutionary history with MPs of dianthoviruses, but the other putative proteins are distant from plant viruses. DOSV is not readily classified in current lower order virus taxa. PMID:24223974

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

PubMed

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.

Investigation of the effects of process sequence on the contact resistance characteristics of coated metallic bipolar plates for polymer electrolyte membrane fuel cells

NASA Astrophysics Data System (ADS)

Turan, Cabir; Cora, Ömer Necati; Koç, Muammer

2013-12-01

In this study, results of an investigation on the effects of manufacturing and coating process sequence on the contact resistance (ICR) of metallic bipolar plates (BPP) for polymer electrolyte membrane fuel cells (PEMFCs) are presented. Firstly, uncoated stainless steel 316L blanks were formed into BPP through hydroforming and stamping processes. Then, these formed BPP samples were coated with three different PVD coatings (CrN, TiN and ZrN) at three different thicknesses (0.1, 0.5 and 1 μm). Secondly, blanks of the same alloy were coated first with the same coatings, thickness and technique; then, they were formed into BPPs of the same shape and dimensions using the manufacturing methods as in the first group. Finally, these two groups of BPP samples were tested for their ICR to reveal the effect of process sequence. ICR tests were also conducted on the BPP plates both before and after exposure to corrosion to disclose the effect of corrosion on ICR. Coated-then-formed BPP samples exhibited similar or even better ICR performance than formed-then-coated BPP samples. Thus, manufacturing of coated blanks can be concluded to be more favorable and worth further investigation in quest of making cost effective BPPs for mass production of PEMFC.

Identification and sequence determination of a new chrysovirus infecting the entomopathogenic fungus Isaria javanica.

PubMed

Herrero, Noemi

2017-04-01

A new double-stranded RNA (dsRNA) mycovirus has been identified in the isolate NB IFR-19 of the entomopathogenic fungus Isaria javanica. Isaria javanica chrysovirus-1 (IjCV-1) constitutes a new member of the Chrysoviridae family, and its genome is made up of four dsRNA elements designated dsRNA1, 2, 3 and 4 from largest to smallest. dsRNA1 and dsRNA2 encode an RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. dsRNA3 and 4 encode hypothetical proteins of unknown function. IjCV-1 constitutes the first report of a chrysovirus infecting the entomopathogenic fungus Isaria javanica.

Disoxaril mutants of Coxsackievirus B1: phenotypic characteristics and analysis of the target VP1 gene.

PubMed

Nikolova, Ivanka; Galabov, Angel S; Petkova, Rumena; Chakarov, Stoyan; Atanasov, Boris

2011-01-01

Disoxaril inhibits enterovirus replication by binding to the hydrophobic pocket within the VP1 coat protein, thus stabilizing the virion and blocking its uncoating. Disoxaril-resistant (RES) mutants of the Coxsackievirus B1 (CVB1/RES) were derived from the wild disoxaril-sensitive (SOF) strain (CVB1/SOF) using a selection approach. A disoxaril-dependent (DEP) mutant (CVB1/DEP) was obtained following nine consecutive passages of the disoxaril-resistant mutant in the presence of disoxaril. Phenotypic characteristics of the disoxaril mutants were investigated. A timing-of-addition study of the CVB1/DEP replication demonstrated that in the absence of disoxaril the virus particle assembly stopped. VP1 RNA sequences of disoxaril mutants were compared with the existing Gen Bank CVB1 reference structure. The amino acid sequence of a large VP1 196-258 peptide (disoxaril-binding region) of CVB1/RES was significantly different from that of the CVB1/SOF. Crucially important changes in CVB1/RES were two point mutations, M213H and F237L, both in the ligand-binding pocket. The sequence analysis of the CVB1/DEP showed some reversion to CVB1/SOF. The amino acid sequences of the three VP1 proteins are presented.

Microbeads display of proteins using emulsion PCR and cell-free protein synthesis.

PubMed

Gan, Rui; Yamanaka, Yumiko; Kojima, Takaaki; Nakano, Hideo

2008-01-01

We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell-free protein synthesis in emulsion. A PCR mixture containing streptavidin-coated microbeads was compartmentalized by water-in-oil (w/o) emulsion with estimated 0.5 template molecules per droplet. The template molecules were amplified and immobilized on beads via bead-linked reverse primers and biotinylated forward primers. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti-glutathione S-transferase (GST) antibody. The pool of beads was then subjected to cell-free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We mixed two types of DNA templates of Histidine6 tag (His6)-fused and FLAG tag-fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. After incubation with fluorescein isothiocyanate (FITC)-labeled anti-His6 (C-term) antibody, the beads with the His6 gene were enriched 917-fold in a single-round screening by using flow cytometry. A library with a theoretical diversity of 10(6) was constructed by randomizing the middle four residues of the His6 tag. After a two-round screening, the randomized sequences were substantially converged to peptide-encoding sequences recognized by the anti-His6 antibody.

Mapping the subgenomic RNA promoter of the Citrus leaf blotch virus coat protein gene by Agrobacterium-mediated inoculation.

PubMed

Renovell, Agueda; Gago, Selma; Ruiz-Ruiz, Susana; Velázquez, Karelia; Navarro, Luis; Moreno, Pedro; Vives, Mari Carmen; Guerri, José

2010-10-25

Citrus leaf blotch virus has a single-stranded positive-sense genomic RNA (gRNA) of 8747 nt organized in three open reading frames (ORFs). The ORF1, encoding a polyprotein involved in replication, is translated directly from the gRNA, whereas ORFs encoding the movement (MP) and coat (CP) proteins are expressed via 3' coterminal subgenomic RNAs (sgRNAs). We characterized the minimal promoter region critical for the CP-sgRNA expression in infected cells by deletion analyses using Agrobacterium-mediated infection of Nicotiana benthamiana plants. The minimal CP-sgRNA promoter was mapped between nucleotides -67 and +50 nt around the transcription start site. Surprisingly, larger deletions in the region between the CP-sgRNA transcription start site and the CP translation initiation codon resulted in increased CP-sgRNA accumulation, suggesting that this sequence could modulate the CP-sgRNA transcription. Site-specific mutational analysis of the transcription start site revealed that the +1 guanylate and the +2 adenylate are important for CP-sgRNA synthesis. Copyright © 2010 Elsevier Inc. All rights reserved.

Yarrowia lipolytica vesicle-mediated protein transport pathways

PubMed Central

Swennen, Dominique; Beckerich, Jean-Marie

2007-01-01

Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y. lipolytica proteins are closer to animal ones, whereas they are only 13% in the case of S. cerevisiae. Conclusion These results provide further support for the idea, previously noted about the endoplasmic reticulum translocation pathway, that Y. lipolytica is more representative of vesicular secretion of animals and other fungi than is S. cerevisiae. PMID:17997821

The optimal SAM surface functional group for producing a biomimetic HA coating on Ti.

PubMed

Liu, D P; Majewski, P; O'Neill, B K; Ngothai, Y; Colby, C B

2006-06-15

Commercial interest is growing in biomimetic methods that employ self assembled mono-layers (SAMs) to produce biocompatible HA coatings on Ti-based orthopedic implants. Recently, separate studies have considered HA formation for various SAM surface functional groups. However, these have often neglected to verify crystallinity of the HA coating, which is essential for optimal bioactivity. Furthermore, differing experimental and analytical methods make performance comparisons difficult. This article investigates and evaluates HA formation for four of the most promising surface functional groups: --OH, --SO(3)H, --PO(4)H(2) and --COOH. All of them successfully formed a HA coating at Ca/P ratios between 1.49 and 1.62. However, only the --SO(3)H and --COOH end groups produced a predominantly crystalline HA. Furthermore, the --COOH end group yielded the thickest layer and possessed crystalline characteristics very similar to that of the human bone. The --COOH end group appears to provide the optimal SAM surface interface for nucleation and growth of biomimetic crystalline HA. Intriguingly, this finding may lend support to explanations elsewhere of why human bone sialoprotein is such a potent nucleator of HA and is attributed to the protein's glutamic acid-rich sequences.

RNA virus interference via CRISPR/Cas13a system in plants.

PubMed

Aman, Rashid; Ali, Zahir; Butt, Haroon; Mahas, Ahmed; Aljedaani, Fatimah; Khan, Muhammad Zuhaib; Ding, Shouwei; Mahfouz, Magdy

2018-01-04

CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

Recognition of cis-acting sequences in RNA 3 of Prunus necrotic ringspot virus by the replicase of Alfalfa mosaic virus.

PubMed

Aparicio, F; Sánchez-Navarro, J A; Olsthoorn, R C; Pallás, V; Bol, J F

2001-04-01

Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMOVIRUS: and ILARVIRUS:, respectively, of the family BROMOVIRIDAE: Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3' termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. CIS:-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV-PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.

Cymbidium chlorotic mosaic virus, a new sobemovirus isolated from a spring orchid (Cymbidium goeringii) in Japan.

PubMed

Kondo, Hideki; Takemoto, Shogo; Maruyama, Kazuyuki; Chiba, Sotaro; Andika, Ida Bagus; Suzuki, Nobuhiro

2015-08-01

Cymbidium chlorotic mosaic virus (CyCMV), isolated from a spring orchid (Cymbidium goeringii), was characterized molecularly. CyCMV isometric virions comprise a single, positive-strand RNA genome of 4,083 nucleotides and 30-kDa coat protein. The virus genome contains five overlapping open reading frames with a genomic organization similar to that of sobemoviruses. BLAST searches and phylogenetic analysis revealed that CyCMV is most closely related to papaya lethal yellowing virus, a proposed dicot-infecting sobemovirus (58.8 % nucleotide sequence identity), but has a relatively distant relationship to monocot-infecting sobemoviruses, with only modest sequence identities. This suggests that CyCMV is a new monocot-infecting member of the floating genus Sobemovirus.

Nodavirus infections in Israeli mariculture.

PubMed

Ucko, M; Colorni, A; Diamant, A

2004-08-01

Viral encephalopathy and retinopathy (VER) infections were diagnosed in five fish species: Epinephelus aeneus, Dicentrarchus labrax, Sciaenops ocellatus, Lates calcarifer and Mugil cephalus cultured on both the Red Sea and Mediterranean coasts of Israel during 1998-2002. Spongiform vacuolation of nervous tissue was observed in histological sections of all examined species. With transmission electron microscopy, paracrystalline arrays and pieces of membrane-associated non-enveloped virions measuring approximately 30 nm in diameter were observed in the brain and retina of all species. At the molecular level, the nodavirus was detected by using a primer set that amplified the T4 region of the coat protein gene. When the same set of primers was used to search for VER in an additional fish species, Sparus aurata, it was found to produce non-specific amplicons, giving rise to false-positive results. This problem was overcome by using a different primer set (F1/VR3), designed on a highly conserved region of the virus gene, which amplified a fragment of 254 bp, and confirmed that S. aurata was nodavirus-free. This set was validated on all five species of infected fish, as well as clinically healthy fish. Comparison of the coat protein genes from the Israeli isolated sequences indicated that more than one viral strain was involved. No strict host-specificity was evident. Red Sea and Mediterranean isolated sequences grouped in distinct clusters, together with several foreign isolates from the Mediterranean area and the Far East, as phylogenetically close to the Epinephelus akaara RGNNV type.

A direct biocombinatorial strategy toward next generation, mussel-glue inspired saltwater adhesives.

PubMed

Wilke, Patrick; Helfricht, Nicolas; Mark, Andreas; Papastavrou, Georg; Faivre, Damien; Börner, Hans G

2014-09-10

Biological materials exhibit remarkable, purpose-adapted properties that provide a source of inspiration for designing new materials to meet the requirements of future applications. For instance, marine mussels are able to attach to a broad spectrum of hard surfaces under hostile conditions. Controlling wet-adhesion of synthetic macromolecules by analogue processes promises to strongly impact materials sciences by offering advanced coatings, adhesives, and glues. The de novo design of macromolecules to mimic complex aspects of mussel adhesion still constitutes a challenge. Phage display allows material scientists to design specifically interacting molecules with tailored affinity to material surfaces. Here, we report on the integration of enzymatic processing steps into phage display biopanning to expand the biocombinatorial procedure and enable the direct selection of enzymatically activable peptide adhesion domains. Adsorption isotherms and single molecule force spectroscopy show that those de novo peptides mimic complex aspects of bioadhesion, such as enzymatic activation (by tyrosinase), the switchability from weak to strong binders, and adsorption under hostile saltwater conditions. Furthermore, peptide-poly(ethylene oxide) conjugates are synthesized to generate protective coatings, which possess anti-fouling properties and suppress irreversible interactions with blood-plasma protein cocktails. The extended phage display procedure provides a generic way to non-natural peptide adhesion domains, which not only mimic nature but also improve biological sequence sections extractable from mussel-glue proteins. The de novo peptides manage to combine several tasks in a minimal 12-mer sequence and thus pave the way to overcome major challenges of technical wet glues.

Temperature-responsive peptide-mimetic coating based on poly(N-methacryloyl-l-leucine): properties, protein adsorption and cell growth.

PubMed

Raczkowska, Joanna; Ohar, Mariya; Stetsyshyn, Yurij; Zemła, Joanna; Awsiuk, Kamil; Rysz, Jakub; Fornal, Katarzyna; Bernasik, Andrzej; Ohar, Halyna; Fedorova, Svitlana; Shtapenko, Oksana; Polovkovych, Svyatoslav; Novikov, Volodymyr; Budkowski, Andrzej

2014-06-01

Poly(N-methacryloyl-l-leucine) (PNML) coatings were successfully fabricated via polymerization from peroxide initiator grafted to premodified glass substrate. Chemical composition and thickness of PNML coatings were determined using time of flight-secondary ion mass spectrometry (TOF- SIMS) and ellipsometry, respectively. PNML coatings exhibit thermal response of the wettability, between 4 and 28°C, which indicates a transition between hydrated loose coils and hydrophobic collapsed chains. Morphology of the PNML coating was observed with the AFM, transforming with increasing temperature from initially relatively smooth surface to rough and more structured surface. Protein adsorption observed by fluorescence microscopy for model proteins (bovine serum albumin and lentil lectin labeled with fluorescein isothiocyanate) at transition from 5 to 25°C, showed high affinity of PNML coating to proteins at all investigated temperatures and pH. Thus, PNML coating have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). The high proliferation growth of the human embryonic kidney cell (HEK 293) onto PNML coating was demonstrated, indicating its excellent cytocompatibility. Copyright © 2014 Elsevier B.V. All rights reserved.

Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus

PubMed Central

Obermeier, Christian; Hosseini, Bashir; Friedt, Wolfgang; Snowdon, Rod

2009-01-01

Background Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana. Results Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development. Conclusion This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species. PMID:19575793

An Arabidopsis Prenylated Rab Acceptor 1 Isoform, AtPRA1.B6, Displays Differential Inhibitory Effects on Anterograde Trafficking of Proteins at the Endoplasmic Reticulum1[W][OA

PubMed Central

Lee, Myoung Hui; Jung, Chanjin; Lee, Junho; Kim, Soo Youn; Lee, Yongjik; Hwang, Inhwan

2011-01-01

Prenylated Rab acceptors (PRAs), members of the Ypt-interacting protein family of small membrane proteins, are thought to aid the targeting of prenylated Rabs to their respective endomembrane compartments. In plants, the Arabidopsis (Arabidopsis thaliana) PRA1 family contains 19 members that display varying degrees of sequence homology to animal PRA1 and localize to the endoplasmic reticulum (ER) and/or endosomes. However, the exact role of these proteins remains to be fully characterized. In this study, the effect of AtPRA1.B6, a member of the AtPRA1 family, on the anterograde trafficking of proteins targeted to various endomembrane compartments was investigated. High levels of AtPRA1.B6 resulted in differential inhibition of coat protein complex II vesicle-mediated anterograde trafficking. The trafficking of the vacuolar proteins sporamin:GFP (for green fluorescent protein) and AALP:GFP, the secretory protein invertase:GFP, and the plasma membrane proteins PMP:GFP and H+-ATPase:GFP was inhibited in a dose-dependent manner, while the trafficking of the Golgi-localized proteins ST:GFP and KAM1(ΔC):mRFP was not affected. Conversely, in RNA interference plants displaying lower levels of AtPRA1.B6 transcripts, the trafficking efficiency of sporamin:GFP and AALP:GFP to the vacuole was increased. Localization and N-glycan pattern analyses of cargo proteins revealed that AtPRA1.B6-mediated inhibition of anterograde trafficking occurs at the ER. In addition, AtPRA1.B6 levels were controlled by cellular processes, including 26S proteasome-mediated proteolysis. Based on these results, we propose that AtPRA1.B6 is a negative regulator of coat protein complex II vesicle-mediated anterograde trafficking for a subset of proteins at the ER. PMID:21828250

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

PubMed Central

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.

2014-01-01

SUMMARY Some capsid proteins built on the ubiquitous HK97-fold have accessory domains that impart specific functions. Bacteriophage P22 coat protein has a unique inserted I-domain. Two prior I-domain models from sub-nanometer cryoEM reconstructions differed substantially. Therefore, the NMR structure of the I-domain was determined, which also was used to improve cryoEM models of coat protein. The I-domain has an anti-parallel 6-stranded β-barrel fold, previously not observed in HK97-fold accessory domains. The D-loop, which is dynamic both in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. A newly described S-loop is important for capsid size determination, likely through intra-subunit interactions. Ten of eighteen 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. PMID:24836025

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

PubMed

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.

Protein-Coated Microcrystals, Combi-Protein-Coated Microcrystals, and Cross-Linked Protein-Coated Microcrystals of Enzymes for Use in Low-Water Media.

PubMed

Mukherjee, Joyeeta; Gupta, Munishwar N

2017-01-01

Protein-coated microcrystals (PCMC) are a high-activity preparation of enzymes for use in low-water media. The protocols for the preparation of PCMCs of Subtilisin Carlsberg and Candida antarctica lipase B (CAL B) are described. The combi-PCMC concept is useful both for cascade and non-cascade reactions. It can also be beneficial to combine two different specificities of a lipase when the substrate requires it. Combi-PCMC of CALB and Palatase used for the conversion of coffee oil present in spent coffee grounds to biodiesel is described. Cross-linked protein-coated microcrystals (CL-PCMC) in some cases can give better results than PCMC. Protocols for the CLPCMC of Subtilisin Carlsberg and Candida antarctica lipase B (CAL B) are described. A discussion of their applications is also provided.

High efficiency protein separation with organosilane assembled silica coated magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Chang, Jeong Ho; Kang, Ki Ho; Choi, Jinsub; Jeong, Young Keun

2008-10-01

This work describes the development of high efficiency protein separation with functionalized organosilanes on the surface of silica coated magnetic nanoparticles. The magnetic nanoparticles were synthesized with average particle size of 9 nm and silica coated magnetic nanoparticles were obtained by controlling the coating thicknesses on magnetic nanoparticles. The silica coating thickness could be uniformly sized with a diameter of 10-40 nm by a sol-gel approach. The surface modification was performed with four kinds of functionalized organosilanes such as carboxyl, aldehyde, amine, and thiol groups. The protein separation work with organosilane assembled silica coated magnetic nanoparticles was achieved for model proteins such as bovine serum albumin (BSA) and lysozyme (LSZ) at different pH conditions. Among the various functionalities, the thiol group showed good separation efficiency due to the change of electrostatic interactions and protein conformational structure. The adsorption efficiency of BSA and LSZ was up to 74% and 90% corresponding pH 4.65 and pH 11.

Heterologous Minor Coat Proteins of Citrus Tristeza Virus Strains Affect Encapsidation, but the Coexpression of HSP70h and p61 Restores Encapsidation to Wild-Type Levels

USDA-ARS?s Scientific Manuscript database

The long flexuous bipolar virions of Citrus tristeza virus (CTV), a Closterovirus, are encapsidated with two capsid proteins at opposite ends: the minor coat protein (CPm) encapsidates the 5’ 630 nts of the genomic RNA and the major coat protein encapsidates the remainder of the genome. In this stud...

Genetic engineering and chemical conjugation of potato virus X.

PubMed

Lee, Karin L; Uhde-Holzem, Kerstin; Fischer, Rainer; Commandeur, Ulrich; Steinmetz, Nicole F

2014-01-01

Here we report the genetic engineering and chemical modification of potato virus X (PVX) for the presentation of various peptides, proteins, and fluorescent dyes, or other chemical modifiers. Three different ways of genetic engineering are described and by these means, peptides are successfully expressed not only when the foot and mouth disease virus (FMDV) 2A sequence or a flexible glycine-serine linker is included, but also when the peptide is fused directly to the PVX coat protein. When larger proteins or unfavorable peptide sequences are presented, a partial fusion via the FMDV 2A sequence is preferable. When these PVX chimeras retain the ability to assemble into viral particles and are thus able to infect plants systemically, they can be utilized to inoculate susceptible plants for isolation of sufficient amounts of virus particles for subsequent chemical modification. Chemical modification is required for the display of nonbiological ligands such as fluorophores, polymers, and small drug compounds. We present three methods of chemical bioconjugation. For direct conjugation of small chemical modifiers to solvent exposed lysines, N-hydroxysuccinimide chemistry can be applied. Bio-orthogonal reactions such as copper-catalyzed azide-alkyne cycloaddition or hydrazone ligation are alternatives to achieve more efficient conjugation (e.g., when working with high molecular weight or insoluble ligands). Furthermore, hydrazone ligation offers an attractive route for the introduction of pH-cleavable cargos (e.g., therapeutic molecules).

DNA Photo Lithography with Cinnamate-based Photo-Bio-Nano-Glue

NASA Astrophysics Data System (ADS)

Feng, Lang; Li, Minfeng; Romulus, Joy; Sha, Ruojie; Royer, John; Wu, Kun-Ta; Xu, Qin; Seeman, Nadrian; Weck, Marcus; Chaikin, Paul

2013-03-01

We present a technique to make patterned functional surfaces, using a cinnamate photo cross-linker and photolithography. We have designed and modified a complementary set of single DNA strands to incorporate a pair of opposing cinnamate molecules. On exposure to 360nm UV, the cinnamate makes a highly specific covalent bond permanently linking only the complementary strands containing the cinnamates. We have studied this specific and efficient crosslinking with cinnamate-containing DNA in solution and on particles. UV addressability allows us to pattern surfaces functionally. The entire surface is coated with a DNA sequence A incorporating cinnamate. DNA strands A'B with one end containing a complementary cinnamated sequence A' attached to another sequence B, are then hybridized to the surface. UV photolithography is used to bind the A'B strand in a specific pattern. The system is heated and the unbound DNA is washed away. The pattern is then observed by thermo-reversibly hybridizing either fluorescently dyed B' strands complementary to B, or colloids coated with B' strands. Our techniques can be used to reversibly and/or permanently bind, via DNA linkers, an assortment of molecules, proteins and nanostructures. Potential applications range from advanced self-assembly, such as templated self-replication schemes recently reported, to designed physical and chemical patterns, to high-resolution multi-functional DNA surfaces for genetic detection or DNA computing.

C terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

South, T.L.; Blake, P.R.; Hare, D.R.

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retriviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 (Zn(HIV1-F2)). Unlike results obtained for the first retroviral-type zinc finger peptide, Zn (HIV1-F1) broad signals indicative of confomational lability were observed in the {sup 1}H NMR spectrum of An(HIV1-F2) at 25 C. The NMR signals narrowed upon cooling to {minus}2 C, enabling complete {sup 1}H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhausermore » effect (NOESY) data were sued to generate 30 distance geometry (DG) structures with penalties in the range 0.02-0.03 {angstrom}{sup 2}. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. These results indicate that the r.t. zinc finger sequences observed in retroviral NCPs, simple plant virus coat proteins, and in a human single-stranded nucleic acid binding protein share a common structural motif.« less

Identification of single nucleotide polymorphisms in the agouti signaling protein (ASIP) gene in some goat breeds in tropical and temperate climates.

PubMed

Adefenwa, Mufliat A; Peters, Sunday O; Agaviezor, Brilliant O; Wheto, Matthew; Adekoya, Khalid O; Okpeku, Moses; Oboh, Bola; Williams, Gabriel O; Adebambo, Olufunmilayo A; Singh, Mahipal; Thomas, Bolaji; De Donato, Marcos; Imumorin, Ikhide G

2013-07-01

The agouti-signaling protein (ASIP) plays a major role in mammalian pigmentation as an antagonist to melanocortin-1 receptor gene to stimulate pheomelanin synthesis, a major pigment conferring mammalian coat color. We sequenced a 352 bp fragment of ASIP gene spanning part of exon 2 and part of intron 2 in 215 animals representing six goat breeds from Nigeria and the United States: West African Dwarf, predominantly black; Red Sokoto, mostly red; and Sahel, mostly white from Nigeria; black and white Alpine, brown and white Spanish and white Saanen from the US. Twenty haplotypes from nine mutations representing three intronic, one silent and five missense (p.S19R, p.N35K, p.L36V, p.M42L and p.L45W) mutations were identified in Nigerian goats. Approximately 89 % of Nigerian goats carry haplotype 1 (TGCCATCCG) which seems to be the wild type configuration of mutations in this region of the gene. Although we found no association between these polymorphisms in the ASIP gene and coat color in Nigerian goats, in-silico functional analysis predicts putative deleterious functional impact of the p.L45W mutation on the basic amino-terminal domain of ASIP. In the American goats, two intronic mutations, g.293G>A and g.327C>A, were identified in the Alpine breed, although the g.293G>A mutation is common to American and Nigerian goat populations. All Sannen and Sahel goats in this study belong to haplotypes 1 of both populations which seem to be the wild-type composite ASIP haplotype. Overall, there was no clear association of this portion of the ASIP gene interrogated in this study with coat color variation. Therefore, additional genomic analyses of promoter sequence, the entire coding and non-coding regions of the ASIP gene will be required to obtain a definite conclusion.

Transfer of Fas (CD95) protein from the cell surface to the surface of polystyrene beads coated with anti-Fas antibody clone CH-11

PubMed Central

Sawai, H.; Domae, N.

2010-01-01

Mouse monoclonal anti-Fas (CD95) antibody clone CH-11 has been widely used in research on apoptosis. CH-11 has the ability to bind to Fas protein on cell surface and induce apoptosis. Here, we used polystyrene beads coated with CH-11 to investigate the role of lipid rafts in Fas-mediated apoptosis in SKW6.4 cells. Unexpectedly, by treatment of the cells with CH-11-coated beads Fas protein was detached from cell surface and transferred to the surface of CH-11-coated beads. Western blot analysis showed that Fas protein containing both extracellular and intracellular domains was attached to the beads. Fas protein was not transferred from the cells to the surface of the beads coated with other anti-Fas antibodies or Fas ligand. Similar phenomenon was observed in Jurkat T cells. Furthermore, CH-11-induced apoptosis was suppressed by pretreatment with CH-11-coated beads in Jurkat cells. These results suggest that CH-11 might possess distinct properties on Fas protein compared with other anti-Fas antibodies or Fas ligand, and also suggest that caution should be needed to use polystyrene beads coated with antibodies such as CH-11. PMID:20353915

Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

PubMed

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.

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

Code of Federal Regulations, 2014 CFR

2014-07-01

... Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. 174.514... Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. Residues of Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic...

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

Code of Federal Regulations, 2013 CFR

2013-07-01

... Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. 174.514... Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. Residues of Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic...

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

Code of Federal Regulations, 2011 CFR

2011-07-01

... Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. 174.514... Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. Residues of Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic...

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

Code of Federal Regulations, 2012 CFR

2012-07-01

... Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. 174.514... Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic Virus; exemption from the requirement for a tolerance. Residues of Coat Protein of Watermelon Mosaic Virus-2 and Zucchini Yellow Mosaic...

Evaluation of the Bacterial Diversity in the Human Tongue Coating Based on Genus-Specific Primers for 16S rRNA Sequencing.

PubMed

Sun, Beili; Zhou, Dongrui; Tu, Jing; Lu, Zuhong

2017-01-01

The characteristics of tongue coating are very important symbols for disease diagnosis in traditional Chinese medicine (TCM) theory. As a habitat of oral microbiota, bacteria on the tongue dorsum have been proved to be the cause of many oral diseases. The high-throughput next-generation sequencing (NGS) platforms have been widely applied in the analysis of bacterial 16S rRNA gene. We developed a methodology based on genus-specific multiprimer amplification and ligation-based sequencing for microbiota analysis. In order to validate the efficiency of the approach, we thoroughly analyzed six tongue coating samples from lung cancer patients with different TCM types, and more than 600 genera of bacteria were detected by this platform. The results showed that ligation-based parallel sequencing combined with enzyme digestion and multiamplification could expand the effective length of sequencing reads and could be applied in the microbiota analysis.

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

PubMed

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.

Molecular Characterization of a Novel Species of Capillovirus from Japanese Apricot (Prunus mume)

PubMed Central

Faure, Chantal; Theil, Sébastien; Candresse, Thierry

2018-01-01

With the increased use of high-throughput sequencing methods, new viruses infecting Prunus spp. are being discovered and characterized, especially in the family Betaflexiviridae. Double-stranded RNAs from symptomatic leaves of a Japanese apricot (Prunus mume) tree from Japan were purified and analyzed by Illumina sequencing. Blast comparisons of reconstructed contigs showed that the P. mume sample was infected by a putative novel virus with homologies to Cherry virus A (CVA) and to the newly described Currant virus A (CuVA), both members of genus Capillovirus. Completion of the genome showed the new agent to have a genomic organization typical of capilloviruses, with two overlapping open reading frames encoding a large replication-associated protein fused to the coat protein (CP), and a putative movement protein (MP). This virus shares only, respectively, 63.2% and 62.7% CP amino acid identity with the most closely related viruses, CVA and CuVA. Considering the species demarcation criteria in the family and phylogenetic analyses, this virus should be considered as representing a new viral species in the genus Capillovirus, for which the name of Mume virus A is proposed. PMID:29570605

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation.

PubMed

Dal Bosco, Daniela; Sinski, Iraci; Ritschel, Patrícia S; Camargo, Umberto A; Fajardo, Thor V M; Harakava, Ricardo; Quecini, Vera

2018-06-06

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

Physical Regulation of the Self-Assembly of Tobacco Mosaic Virus Coat Protein

PubMed Central

Kegel, Willem K.; van der Schoot, Paul

2006-01-01

We present a statistical mechanical model based on the principle of mass action that explains the main features of the in vitro aggregation behavior of the coat protein of tobacco mosaic virus (TMV). By comparing our model to experimentally obtained stability diagrams, titration experiments, and calorimetric data, we pin down three competing factors that regulate the transitions between the different kinds of aggregated state of the coat protein. These are hydrophobic interactions, electrostatic interactions, and the formation of so-called “Caspar” carboxylate pairs. We suggest that these factors could be universal and relevant to a large class of virus coat proteins. PMID:16731551

A novel surface modification approach for protein and cell microarrays

NASA Astrophysics Data System (ADS)

Kurkuri, Mahaveer D.; Driever, Chantelle; Thissen, Helmut W.; Voelcker, Nicholas H.

2007-01-01

Tissue engineering and stem cell technologies have led to a rapidly increasing interest in the control of the behavior of mammalian cells growing on tissue culture substrates. Multifunctional polymer coatings can assist research in this area in many ways, for example, by providing low non-specific protein adsorption properties and reactive functional groups at the surface. The latter can be used for immobilization of specific biological factors that influence cell behavior. In this study, glass slides were coated with copolymers of glycidyl methacrylate (GMA) and poly(ethylene glycol) methacrylate (PEGMA). The coatings were prepared by three different methods based on dip and spin coating as well as polymer grafting procedures. Coatings were characterized by X-ray photoelectron spectroscopy, surface sensitive infrared spectroscopy, ellipsometry and contact angle measurements. A fluorescently labelled protein was deposited onto reactive coatings using a contact microarrayer. Printing of a model protein (fluorescein labeled bovine serum albumin) was performed at different protein concentrations, pH, temperature, humidity and using different micropins. The arraying of proteins was studied with a microarray scanner. Arrays printed at a protein concentration above 50 μg/mL prepared in pH 5 phosphate buffer at 10°C and 65% relative humidity gave the most favourable results in terms of the homogeneity of the printed spots and the fluorescence intensity.

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

PubMed

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.

Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles

PubMed Central

Antrobus, Robin; Hirst, Jennifer; Bhumbra, Gary S.; Kozik, Patrycja; Jackson, Lauren P.; Sahlender, Daniela A.

2012-01-01

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions. PMID:22472443

Effect of manufacturing process sequence on the corrosion resistance characteristics of coated metallic bipolar plates

NASA Astrophysics Data System (ADS)

Dur, Ender; Cora, Ömer Necati; Koç, Muammer

2014-01-01

Metallic bipolar plate (BPP) with high corrosion and low contact resistance, durability, strength, low cost, volume, and weight requirements is one of the critical parts of the PEMFC. This study is dedicated to understand the effect of the process sequence (manufacturing then coating vs. coating then manufacturing) on the corrosion resistance of coated metallic bipolar plates. To this goal, three different PVD coatings (titanium nitride (TiN), chromium nitride (CrN), zirconium nitride (ZrN)), with three thicknesses, (0.1, 0.5, 1 μm) were applied on BPPs made of 316L stainless steel alloy before and after two types of manufacturing (i.e., stamping or hydroforming). Corrosion test results indicated that ZrN coating exhibited the best corrosion protection while the performance of TiN coating was the lowest among the tested coatings and thicknesses. For most of the cases tested, in which coating was applied before manufacturing, occurrence of corrosion was found to be more profound than the case where coating was applied after manufacturing. Increasing the coating thickness was found to improve the corrosion resistance. It was also revealed that hydroformed BPPs performed slightly better than stamped BPPs in terms of the corrosion behavior.

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

PubMed

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.

Oligovalent Fab display on M13 phage improved by directed evolution.

PubMed

Huovinen, Tuomas; Sanmark, Hanna; Ylä-Pelto, Jani; Vehniäinen, Markus; Lamminmäki, Urpo

2010-03-01

Efficient display of antibody on filamentous phage M13 coat is crucial for successful biopanning selections. We applied a directed evolution strategy to improve the oligovalent display of a poorly behaving Fab fragment fused to phage gene-3 for minor coat protein (g3p). The Fab displaying clones were enriched from a randomly mutated Fab gene library with polyclonal anti-mouse IgG antibodies. Contribution of each mutation to the improved phenotype of one selected mutant was studied. It was found out that two point mutations had significant contribution to the display efficiency of Fab clones superinfected with hyperphage. The most dramatic effect was connected to a start codon mutation, from AUG to GUG, of the PelB signal sequence preceding the heavy chain. The clone carrying this mutation, FabM(GUG), displayed Fab 19-fold better and yielded twofold higher phage titers than the original Fab.

Heterotopic bone formation around sintered porous-surfaced Ti-6Al-4V implants coated with native bone morphogenetic proteins.

PubMed

Simon, Ziv; Deporter, Douglas A; Pilliar, Robert M; Clokie, Cameron M

2006-09-01

Coating endosseous dental implants with growth factors such as bone morphogenetic proteins (BMPs) may be one way to accelerate and/or enhance the quality of osseointegration. The purpose of this study was to investigate in the murine muscle pouch model whether sintered porous-surfaced titanium alloy implants coated with BMPs would lead to heterotopic bone formation around and within the implant surface geometry. Porous-surfaced dental implants were coated with partially purified native human BMPs, with or without a carrier of Poloxamer 407 (BASF Corp., Parsippany, NJ), placed in gelatin capsules and implanted into the hindquarter muscles of mice. Mice were euthanized after 28 days. Sections of retrieved specimens were subsequently prepared for morphometric analysis of bone formation using backscatter electron microscopic images. Human BMPs, either with or without the carrier of Poloxamer 407, led to bone formation within and outside of the sintered porous implant surface. When the sintered implant surface region was subdivided into inner and outer halves, similar levels of bone ingrowth and contact were seen in the 2 halves. Evidence of bone formation to the depth of the solid implant core (i.e., the deepest level possible) also was seen. Sintered porous-surfaced dental implants can be used as substrate for partially purified BMPs in the murine muscle pouch model. With the addition of these osteoinductive factors, the porous implant surface supported bone formation within the surface porosity provided, in some instances, all the way to the solid implant core. The addition of growth factors to a sintered porous surface may be an efficient method for altering locally the healing sequence and quality of bone associated with osseointegration of bone-interfacing implants.

Role of Monomer Sequence, Hydrogen Bonding and Mesoscale Architecture in Marine Antifouling Coatings

NASA Astrophysics Data System (ADS)

Segalman, Rachel

Polypeptoids are non-natural, sequence specific polymers that offer the opportunity to probe the effect of monomer sequence, chirality, and chain shape on self-assembly and surface properties. Additionally, polypeptoid synthesis is more scaleable than traditional polypeptides suggesting their utility in large area applications. We have designed efficient marine anti-fouling coatings by using triblock copolymer scaffolds to which polypeptoids are tethered in order to tune both the modulus and surface energies with great precision. Surprisingly, when short sequences are tethered to a polymer backbone, polypeptoids consistently outperform analogous polypeptides in antifouling properties. We hypothesize that the hydrogen bonding inherent to the polypeptide backbone drives the observed differences in performance. We also find that the polymer scaffold housing the polypeptoids also plays a crucial role in directing surface presentation and therefore the overall coating properties.

Polyvalent immunoglobulin binding is an obstacle to accurate measurement of specific antibodies with ELISA despite inclusion of blocking agents.

PubMed

Loeffler, David A; Klaver, Andrea C

2017-11-01

Specific antibody concentrations are frequently measured in serum (and plasma and intravenous immunoglobulin) samples by enzyme-linked immunosorbent assay (ELISA). The standard negative control involves incubation of buffer alone on antigen-coated wells. The immunoreactivity that develops in antigen-coated wells in which diluted serum has been incubated is assumed to represent specific antibody binding. This approach can result in marked overestimation of specific antibody levels, because serum contains specific polyvalent antibodies which bind, primarily with low affinity, to multiple antigens (including those on ELISA plates) despite the use of blocking agents. Non-denaturing purification of serum IgG, followed by assessment of the antigen binding or antigen-binding affinity of this purified IgG, can reduce but not eliminate the problem of polyvalent antibody binding in indirect ELISAs. Alternatively, polyvalent antibody binding can be estimated by incubating a diluted serum sample on wells coated with an irrelevant protein (such as bovine serum albumin or a scrambled peptide sequence) or buffer alone, then subtracting this reactivity from the sample's binding to wells coated with the antigen of interest. Polyvalent binding of immunoglobulins must be accounted for in order to obtain accurate ELISA measurements of serum, plasma, or intravenous immunoglobulin antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

Alterations in nanoparticle protein corona by biological surfactants: impact of bile salts on β-lactoglobulin-coated gold nanoparticles.

PubMed

Winuprasith, Thunnalin; Chantarak, Sirinya; Suphantharika, Manop; He, Lili; McClements, David Julian

2014-07-15

The impact of biological surfactants (bile salts) on the protein (β-lactoglobulin) corona surrounding gold nanoparticles (200 nm) was studied using a variety of analytical techniques at pH 7: dynamic light scattering (DLS); particle electrophoresis (ζ-potential); UV-visible (UV) spectroscopy; transmission electron microscopy (TEM); and surface-enhanced Raman scattering (SERS). The bile salts adsorbed to the protein-coated nanoparticle surfaces and altered their interfacial composition, charge, and structure. SERS spectra of protein-coated nanoparticles after bile salt addition contained bands from both protein and bile salts, indicating that the protein was not fully displaced by the bile salts. UV, DLS and TEM techniques also indicated that the protein coating was not fully displaced from the nanoparticle surfaces. The impact of bile salts could be described by an orogenic mechanism: mixed interfaces were formed that consisted of islands of aggregated proteins surrounded by a sea of bile salts. This knowledge is useful for understanding the interactions of bile salts with protein-coated colloidal particles, which may be important for controlling the fate of colloidal delivery systems in the human gastrointestinal tract, or the gastrointestinal fate of ingested inorganic nanoparticles. Copyright © 2014 Elsevier Inc. All rights reserved.

The PsB glycoprotein complex is secreted as a preassembled precursor of the spore coat in Dictyostelium discoideum.

PubMed

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 assembly of specific extracellular matrices.

Field safety assessment of recombination in transgenic grapevines expressing the coat protein gene of Grapevine fanleaf virus.

PubMed

Vigne, Emmanuelle; Komar, Véronique; Fuchs, Marc

2004-04-01

One of the major environmental safety issues over transgenic crops containing virus-derived genes relates to the outcome of recombination events between viral transgene transcripts and RNAs from indigenous virus populations. We addressed this issue by assessing the emergence of viable Grapevine fanleaf virus (GFLV) recombinants in transgenic grapevines expressing the GFLV coat protein (CP) gene. Test plants consisted of nontransgenic scions grafted onto transgenic and nontransgenic rootstocks that were exposed over 3 years to nematode-mediated GFLV infection in two distinct vineyard sites. The CP gene of challenging GFLV isolates was amplified from scions by IC-RT-PCR, and characterized by RFLP and nucleotide sequencing using strain F13 as reference since it provided the CP transgene. Analysis of EcoRI and StyI RFLP banding patterns from 347 challenging GFLV isolates and sequence data from 85 variants revealed no characteristics similar to strain F13 and no difference in the molecular variability among isolates from 190 transgenic and 157 nontransgenic plants, or from plants within (253 individuals) or outside (94 individuals) of the two sites. Interestingly, five GFLV recombinants were identified in three nontransgenic plants located outside of the two field settings. This survey indicates that transgenic grapevines did not assist the emergence of viable GFLV recombinants to detectable levels nor did they affect the molecular diversity of indigenous GFLV populations during the trial period. This is the first report on safety assessment of recombination with a transgenic crop expressing a CP gene under field conditions of heavy disease pressure but low, if any, selection pressure against recombinant viruses.

Effect of chitosan coatings enriched with cinnamon oil on proximate composition of rainbow trout fillets

NASA Astrophysics Data System (ADS)

Yıldız, Pınar Oǧuzhan

2017-04-01

The effects of chitosan coating enriched with cinnamon oil on proximate composition of rainbow trout (Oncorhynchus mykiss) during storage at 4°C was investigated. The treatments included the following: C1 (control samples), C2 (chitosan coating) and C3 (chitosan + 1 % [v/w] cinnamon EO added). The control and the coated fish samples were analysed for chemical (moisture, protein, lipid and ash) composition. The mean of moisture, protein, lipid and ash in the control samples (C1) were 70.3%, 20.1%, 2.6% and 1.2%, in coated samples (C2) 69.70%, 24.21%, 2.4% and 2.2% and coated+cinnamon oil samples (C3) 69.70%, 25.05%, 2.5% and 2.2%, respectively. Moisture and lipid contents in control groups were higher than other groups, but protein and ash contents were lower. Significant increases (p<0.05) in protein content were observed between samples, which subsequently decreased the moisture content of these samples.

Nanohardness, corrosion and protein adsorption properties of CuAlO2 films deposited on 316L stainless steel for biomedical applications

NASA Astrophysics Data System (ADS)

Chang, Shih-Hang; Chen, Jian-Zhang; Hsiao, Sou-Hui; Lin, Guan-Wei

2014-01-01

This study preliminarily assesses the biomedical applications of CuAlO2 coatings according to nanoindentation, electrochemical, and protein adsorption tests. Nanoindentation results revealed that the surface hardness of 316L stainless steel increased markedly after coating with CuAlO2 films. Electrochemical tests of corrosion potential, breakdown potential, and corrosion current density showed that the corrosion resistance properties of 316L stainless steel are considerably improved by CuAlO2 coatings. Bicinchoninic acid (BCA) protein assay results revealed that the protein adsorption behavior of 316L stainless steel did not exhibit notable differences with or without CuAlO2 coatings. A CuAlO2 coating of 100 nm thickness improved the surface nanohardness and corrosion resistance ability of 316L stainless steel. CuAlO2 is a potential candidate for biomaterial coating applications, particularly for surface modification of fine, delicate implants.

Functional diversification upon leader protease domain duplication in the Citrus tristeza virus genome: Role of RNA sequences and the encoded proteins.

PubMed

Kang, Sung-Hwan; Atallah, Osama O; Sun, Yong-Duo; Folimonova, Svetlana Y

2018-01-15

Viruses from the family Closteroviridae show an example of intra-genome duplications of more than one gene. In addition to the hallmark coat protein gene duplication, several members possess a tandem duplication of papain-like leader proteases. In this study, we demonstrate that domains encoding the L1 and L2 proteases in the Citrus tristeza virus genome underwent a significant functional divergence at the RNA and protein levels. We show that the L1 protease is crucial for viral accumulation and establishment of initial infection, whereas its coding region is vital for virus transport. On the other hand, the second protease is indispensable for virus infection of its natural citrus host, suggesting that L2 has evolved an important adaptive function that mediates virus interaction with the woody host. Copyright © 2017 Elsevier Inc. All rights reserved.

A protein coated piezoelectric crystal detector

NASA Astrophysics Data System (ADS)

Suleiman, Ahmad; Pender, Marie; Ngeh-Ngwainbi, Jerome; Lubrano, Glenn; Guilbault, George

1990-05-01

The purpose of this project was to develop a protein coated, portable piezoelectric crystal detector for organophosphorus compounds. The performance of acetylcholinesterase, GD-1 anti-soman, anti-DMMP antibody, and bovine serum albumin (BSA) coatings was evaluated. Different immobilization methods were also tested. The responses obtained with the protein coatings immobilized via cross-linking with glutaraldehyde were acceptable, provided that the reference crystal was coated with dextran. The proposed coatings showed good stability and reasonable lifetimes that ranged from approximately three weeks in the case of the antibody coatings to several months in the case of BSA. Although moisture, gasoline, and sulfur are potential interferents, their effects on the sensor were eliminated by using a sodium sulfate scrubber which did not affect the performance of the detector towards organophosphates. A small, battery operated portable instrument capable of real time measurements with alarm function was produced. The instrument can be used in a wide range of applications, depending on the coatings applied to the crystals.

Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles.

PubMed

Norregaard, Kamilla; Andersson, Magnus; Nielsen, Peter Eigil; Brown, Stanley; Oddershede, Lene B

2014-09-01

This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes ~4 weeks.

Galectin-1 induces cell adhesion to the extracellular matrix and apoptosis of non-adherent human colon cancer Colo201 cells.

PubMed

Horiguchi, Natsuko; Arimoto, Kei-ichiro; Mizutani, Atsushi; Endo-Ichikawa, Yoko; Nakada, Hiroshi; Taketani, Shigeru

2003-12-01

To isolate cDNAs for molecules involved in cell adhesion to the extracellular matrix, expression cloning with non-adherent colon cancer Colo201 cells was carried out. Four positive clones were isolated and, when sequenced, one was found to be galectin-1, a beta-galactoside-binding protein. When cultured on fibronectin-, laminin-, and collagen-coated and non-coated dishes, the adherent galectin-1 cDNA-transfected Colo201 cells increased and spread somewhat. Immunofluorescence staining revealed that galectin-1 was expressed inside and outside of Colo201 cells. The adhesion was dependent on the carbohydrate-recognition domain of galectin-1 since lactose inhibited the adhesion and exogenously-added galectin-1 caused the adhesion. PD58059, an inhibitor of mitogen-activated protein kinase, or LY294002, a phosphoinositide 3-OH kinase inhibitor, decreased the adhesion. Furthermore, the expression of galectin-1 in Colo201 cells induced apoptotic cell death, while exogenously-added galectin-1 did not cause apoptosis. These results indicate that galectin-1 plays a role in both cell-matrix interactions and the inhibition of Colo201 cell proliferation, and suggest that galectin-1 expressed in cells could be associated with apoptosis.

Molecular adaptation within the coat protein-encoding gene of Tunisian almond isolates of Prunus necrotic ringspot virus.

PubMed

Boulila, Moncef; Ben Tiba, Sawssen; Jilani, Saoussen

2013-04-01

The sequence alignments of five Tunisian isolates of Prunus necrotic ringspot virus (PNRSV) were searched for evidence of recombination and diversifying selection. Since failing to account for recombination can elevate the false positive error rate in positive selection inference, a genetic algorithm (GARD) was used first and led to the detection of potential recombination events in the coat protein-encoding gene of that virus. The Recco algorithm confirmed these results by identifying, additionally, the potential recombinants. For neutrality testing and evaluation of nucleotide polymorphism in PNRSV CP gene, Tajima's D, and Fu and Li's D and F statistical tests were used. About selection inference, eight algorithms (SLAC, FEL, IFEL, REL, FUBAR, MEME, PARRIS, and GA branch) incorporated in HyPhy package were utilized to assess the selection pressure exerted on the expression of PNRSV capsid. Inferred phylogenies pointed out, in addition to the three classical groups (PE-5, PV-32, and PV-96), the delineation of a fourth cluster having the new proposed designation SW6, and a fifth clade comprising four Tunisian PNRSV isolates which underwent recombination and selective pressure and to which the name Tunisian outgroup was allocated.

Extractable Bacterial Surface Proteins in Probiotic–Host Interaction

PubMed Central

do Carmo, Fillipe L. R.; Rabah, Houem; De Oliveira Carvalho, Rodrigo D.; Gaucher, Floriane; Cordeiro, Barbara F.; da Silva, Sara H.; Le Loir, Yves; Azevedo, Vasco; Jan, Gwénaël

2018-01-01

Some Gram-positive bacteria, including probiotic ones, are covered with an external proteinaceous layer called a surface-layer. Described as a paracrystalline layer and formed by the self-assembly of a surface-layer-protein (Slp), this optional structure is peculiar. The surface layer per se is conserved and encountered in many prokaryotes. However, the sequence of the corresponding Slp protein is highly variable among bacterial species, or even among strains of the same species. Other proteins, including surface layer associated proteins (SLAPs), and other non-covalently surface-bound proteins may also be extracted with this surface structure. They can be involved a various functions. In probiotic Gram-positives, they were shown by different authors and experimental approaches to play a role in key interactions with the host. Depending on the species, and sometime on the strain, they can be involved in stress tolerance, in survival within the host digestive tract, in adhesion to host cells or mucus, or in the modulation of intestinal inflammation. Future trends include the valorization of their properties in the formation of nanoparticles, coating and encapsulation, and in the development of new vaccines. PMID:29670603

Heterogeneous electron transfer of a two-centered heme protein: redox and electrocatalytic properties of surface-immobilized cytochrome C(4).

PubMed

Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Di Rocco, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco

2009-10-15

The recombinant diheme cytochrome c(4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature a hydroxide ion axially bound to the heme iron at the N- and C-terminal domains, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups and changes in the net charge of the protein lobes yield two-centered protein systems with unprecedented properties in the electrode-immobilized state. The heterogeneous and intraheme electron transfer processes were characterized for these species in which the high- and low-potential heme groups are swapped over in the bilobal protein framework and experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode. The reduction thermodynamics for the native and mutated hemes were measured for the first time for a diheme cytochrome c. In the diffusing regime, they reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. T-dependent kinetic measurements show that the proteins are positioned approximately 7 A from the HOOC-terminated SAM-coated electrode. Protein-electrode orientation and efficient intraheme ET enable the His,OH(-)-ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes a novel two-centered heme-based biocatalytic interface to be exploited for "third-generation" amperometric biosensing.

Distinctive glial and neuronal interfacing on nanocrystalline diamond.

PubMed

Bendali, Amel; Agnès, Charles; Meffert, Simone; Forster, Valérie; Bongrain, Alexandre; Arnault, Jean-Charles; Sahel, José-Alain; Offenhäusser, Andreas; Bergonzo, Philippe; Picaud, Serge

2014-01-01

Direct electrode/neuron interfacing is a key challenge to achieve high resolution of neuronal stimulation required for visual prostheses. Neuronal interfacing on biomaterials commonly requires the presence of glial cells and/or protein coating. Nanocrystalline diamond is a highly mechanically stable biomaterial with a remarkably large potential window for the electrical stimulation of tissues. Using adult retinal cell cultures from rats, we found that glial cells and retinal neurons grew equally well on glass and nanocrystalline diamond. The use of a protein coating increased cell survival, particularly for glial cells. However, bipolar neurons appeared to grow even in direct contact with bare diamond. We investigated whether the presence of glial cells contributed to this direct neuron/diamond interface, by using purified adult retinal ganglion cells to seed diamond and glass surfaces with and without protein coatings. Surprisingly, these fully differentiated spiking neurons survived better on nanocrystalline diamond without any protein coating. This greater survival was indicated by larger cell numbers and the presence of longer neurites. When a protein pattern was drawn on diamond, neurons did not grow preferentially on the coated area, by contrast to their behavior on a patterned glass. This study highlights the interesting biocompatibility properties of nanocrystalline diamond, allowing direct neuronal interfacing, whereas a protein coating was required for glial cell growth.

Distinctive Glial and Neuronal Interfacing on Nanocrystalline Diamond

PubMed Central

Bendali, Amel; Agnès, Charles; Meffert, Simone; Forster, Valérie; Bongrain, Alexandre; Arnault, Jean-Charles; Sahel, José-Alain; Offenhäusser, Andreas; Bergonzo, Philippe; Picaud, Serge

2014-01-01

Direct electrode/neuron interfacing is a key challenge to achieve high resolution of neuronal stimulation required for visual prostheses. Neuronal interfacing on biomaterials commonly requires the presence of glial cells and/or protein coating. Nanocrystalline diamond is a highly mechanically stable biomaterial with a remarkably large potential window for the electrical stimulation of tissues. Using adult retinal cell cultures from rats, we found that glial cells and retinal neurons grew equally well on glass and nanocrystalline diamond. The use of a protein coating increased cell survival, particularly for glial cells. However, bipolar neurons appeared to grow even in direct contact with bare diamond. We investigated whether the presence of glial cells contributed to this direct neuron/diamond interface, by using purified adult retinal ganglion cells to seed diamond and glass surfaces with and without protein coatings. Surprisingly, these fully differentiated spiking neurons survived better on nanocrystalline diamond without any protein coating. This greater survival was indicated by larger cell numbers and the presence of longer neurites. When a protein pattern was drawn on diamond, neurons did not grow preferentially on the coated area, by contrast to their behavior on a patterned glass. This study highlights the interesting biocompatibility properties of nanocrystalline diamond, allowing direct neuronal interfacing, whereas a protein coating was required for glial cell growth. PMID:24664111

Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage

PubMed Central

Løset, Geir Åge; Roos, Norbert; Bogen, Bjarne; Sandlie, Inger

2011-01-01

Background Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display. Methodology/Principal Findings Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient. Conclusions/Significance Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before. PMID:21390283

Guiding plant virus particles to integrin-displaying cells

NASA Astrophysics Data System (ADS)

Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

2012-05-01

Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors. Electronic supplementary information (ESI) available: Synthetic procedures and compound characterization data; assay procedures; additional confocal micrographs at different time points. See DOI: 10.1039/c2nr30571b

Varieties of charge distributions in coat proteins of ssRNA+  viruses

NASA Astrophysics Data System (ADS)

Lošdorfer Božič, Anže; Podgornik, Rudolf

2018-01-01

A major part of the interactions involved in the assembly and stability of icosahedral, positive-sense single-stranded RNA (ssRNA+) viruses is electrostatic in nature, as can be inferred from the strong pH- and salt-dependence of their assembly phase diagrams. Electrostatic interactions do not act only between the capsid coat proteins (CPs), but just as often provide a significant contribution to the interactions of the CPs with the genomic RNA, mediated to a large extent by positively charged, flexible N-terminal tails of the CPs. In this work, we provide two clear and complementary definitions of an N-terminal tail of a protein, and use them to extract the tail sequences of a large number of CPs of ssRNA+  viruses. We examine the pH-dependent interplay of charge on both tails and CPs alike, and show that—in contrast to the charge on the CPs—the net positive charge on the N-tails persists even to very basic pH values. In addition, we note a limit to the length of the wild-type genomes of those viruses which utilize positively charged tails, when compared to viruses without charged tails and similar capsid size. At the same time, we observe no clear connection between the charge on the N-tails and the genome lengths of the viruses included in our study.

Identification and molecular characterization of a novel circular single-stranded DNA virus associated with yerba mate in Argentina.

PubMed

Bejerman, Nicolás; de Breuil, Soledad; Nome, Claudia

2018-06-06

A single-stranded DNA (ssDNA) virus was detected in Yerba mate samples showing chlorotic linear patterns, chlorotic rings and vein yellowing. The full-genome sequences of six different isolates of this ssDNA circular virus were obtained, which share > 99% sequence identity with each other. The newly identified virus has been tentatively named as yerba mate-associated circular DNA virus (YMaCV). The 2707 nt-long viral genome has two and three open reading frame on its complementary and virion-sense strands, respectively. The coat protein is more similar to that of mastreviruses (44% identity), whereas the replication-associated protein of YMaCV is more similar (49% identity) to that encoded by a recently described, unclassified ssDNA virus isolated on trees in Brazil. This is the first report of a circular DNA virus associated with yerba mate. Its unique genome organization and phylogenetic relationships indicates that YMaCV represents a distinct evolutionary lineage within the ssDNA viruses and therefore this virus should be classified as a member of a new species within an unassigned genus or family.

Flagellin based biomimetic coatings: From cell-repellent surfaces to highly adhesive coatings.

PubMed

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.

Bean common mosaic virus isolates causing different symptoms in asparagus bean in China differ greatly in the 5'-parts of their genomes.

PubMed

Zheng, Hongying; Chen, Jiong; Chen, Jianping; Adams, Michael J; Hou, Mingsheng

2002-06-01

Potyvirus isolates from asparagus bean ( Vigna sesquipedalis) plants in Zhejiang province, China, caused either rugose and vein banding mosaic symptoms (isolate R) or severe yellowing (isolate Y) in this host, but were otherwise similar in host range. Both isolates were completely sequenced and shown to be isolates of Bean common mosaic virus (BCMV). The complete sequences were 9992 (R) or 10062 (Y) nucleotides long and shared 91.7% identical nucleotides (93.2% identical amino acids) in their genomes and were more distantly related to the BCMV-Peanut stripe virus sequence (PStV). The isolates were much less similar to one another in the 5'-UTR and the N-terminal region of the P1 protein. In the P1, isolate Y was closer to PStV (76.1% identical amino acids) than to isolate R (64.8%). Phylogenetic analyses of the coat protein region showed that the new isolates grouped with other isolates from Vigna spp., forming the blackeye cowpea mosaic strain subgroup of BCMV with 94-98% nucleotides (96-99% amino acids) identical to one another and about 90% identity to other BCMV isolates. Other significant subgroupings amongst published BCMV isolates were detected.

Surface-independent antibacterial coating using silver nanoparticle-generating engineered mussel glue.

PubMed

Jo, Yun Kee; Seo, Jeong Hyun; Choi, Bong-Hyuk; Kim, Bum Jin; Shin, Hwa Hui; Hwang, Byeong Hee; Cha, Hyung Joon

2014-11-26

During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating strategy based on the fusion of MAP to a silver-binding peptide, which can synthesize silver nanoparticles having broad antibacterial activity. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface under mild condition. The biosynthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria and also revealed good cytocompatibility with mammalian cells. In this coating strategy, MAP-silver binding peptide fusion proteins provide hybrid environment incorporating inorganic silver nanoparticle and simultaneously mediate the interaction of silver nanoparticle with surroundings. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.

Direct demonstration of the lectin activity of gp90MEL, a lymphocyte homing receptor

PubMed Central

1990-01-01

Considerable evidence implicates gp90MEL as a lymphocyte homing receptor mediating lymphocyte attachment to high endothelial venules of lymph nodes in mouse. The protein appears to function as a calcium- dependent, lectin-like receptor as inferred primarily by the ability of specific carbohydrates to block its function and by the presence of a calcium-type lectin domain in its primary sequence. An ELISA assay is described which provides the first demonstration that the isolated protein has lectin activity and allows a further definition of its carbohydrate specificity. In addition to the monosaccharides mannose-6- phosphate and fructose-1-phosphate, ligand activity is shown for the sulfated glycolipid, sulfatide, and for two sulfated fucose-containing polysaccharides (fucoidin and egg jelly coat) from nonmammalian sources. PMID:2202735

Adapter-directed display: a modular design for shuttling display on phage surfaces.

PubMed

Wang, Kevin Caili; Wang, Xinwei; Zhong, Pingyu; Luo, Peter Peizhi

2010-02-05

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems. Copyright 2009 Elsevier Ltd. All rights reserved.

A novel subviral agent associated with a geminivirus: The first report of a DNA satellite

PubMed Central

Dry, Ian B.; Krake, Leslie R.; Rigden, Justin E.; Rezaian, M. Ali

1997-01-01

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem–loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication. PMID:9192696

Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

PubMed

Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

2016-10-01

A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

Identification and Characterization of Arabidopsis Seed Coat Mucilage Proteins.

PubMed

Tsai, Allen Yi-Lun; Kunieda, Tadashi; Rogalski, Jason; Foster, Leonard J; Ellis, Brian E; Haughn, George W

2017-02-01

Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features. © 2017 American Society of Plant Biologists. All Rights Reserved.

Identification and Characterization of Arabidopsis Seed Coat Mucilage Proteins1[OPEN

PubMed Central

Tsai, Allen Yi-Lun; Kunieda, Tadashi; Rogalski, Jason; Foster, Leonard J.; Ellis, Brian E.

2017-01-01

Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features. PMID:28003327

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

NASA Astrophysics Data System (ADS)

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 together, the data presented in this work indicates that the stability of the PEG coating and the many factors affecting it represent a fundamental limitation to the use of these particles.

Experimental rugged fitness landscape in protein sequence space.

PubMed

Hayashi, Yuuki; Aita, Takuyo; Toyota, Hitoshi; Husimi, Yuzuru; Urabe, Itaru; Yomo, Tetsuya

2006-12-20

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein D2 domain, which is essential for phage infection. After 20 cycles of random substitution at sites 12-130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.7x10(4)-fold increase in infectivity, defined as the number of infected cells per ml of phage suspension. Fitness was defined as the logarithm of infectivity, and we analyzed (1) the dependence of stationary fitness on library size, which increased gradually, and (2) the time course of changes in fitness in transitional phases, based on an original theory regarding the evolutionary dynamics in Kauffman's n-k fitness landscape model. In the landscape model, single mutations at single sites among n sites affect the contribution of k other sites to fitness. Based on the results of these analyses, k was estimated to be 18-24. According to the estimated parameters, the landscape was plotted as a smooth surface up to a relative fitness of 0.4 of the global peak, whereas the landscape had a highly rugged surface with many local peaks above this relative fitness value. Based on the landscapes of these two different surfaces, it appears possible for adaptive walks with only random substitutions to climb with relative ease up to the middle region of the fitness landscape from any primordial or random sequence, whereas an enormous range of sequence diversity is required to climb further up the rugged surface above the middle region.

Experimental Rugged Fitness Landscape in Protein Sequence Space

PubMed Central

Hayashi, Yuuki; Aita, Takuyo; Toyota, Hitoshi; Husimi, Yuzuru; Urabe, Itaru; Yomo, Tetsuya

2006-01-01

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein D2 domain, which is essential for phage infection. After 20 cycles of random substitution at sites 12–130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.7×104-fold increase in infectivity, defined as the number of infected cells per ml of phage suspension. Fitness was defined as the logarithm of infectivity, and we analyzed (1) the dependence of stationary fitness on library size, which increased gradually, and (2) the time course of changes in fitness in transitional phases, based on an original theory regarding the evolutionary dynamics in Kauffman's n-k fitness landscape model. In the landscape model, single mutations at single sites among n sites affect the contribution of k other sites to fitness. Based on the results of these analyses, k was estimated to be 18–24. According to the estimated parameters, the landscape was plotted as a smooth surface up to a relative fitness of 0.4 of the global peak, whereas the landscape had a highly rugged surface with many local peaks above this relative fitness value. Based on the landscapes of these two different surfaces, it appears possible for adaptive walks with only random substitutions to climb with relative ease up to the middle region of the fitness landscape from any primordial or random sequence, whereas an enormous range of sequence diversity is required to climb further up the rugged surface above the middle region. PMID:17183728

Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.

PubMed

Kibbe, M R; Murdock, A; Wickham, T; Lizonova, A; Kovesdi, I; Nie, S; Shears, L; Billiar, T R; Tzeng, E

2000-02-01

Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ineffective viral-target cell interactions. Viral attachment, which largely determines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while internalization follows binding of the adenoviral RGD motif to alpha(v)-integrin receptors. Modifications of the fiber coat protein sequence have been successful for targeting the adenovirus to more prevalent receptors in the vasculature, including heparan sulfate-containing receptors and alpha(v)-integrin receptors. Modified adenoviral vectors targeted to receptors more prevalent in the vasculature result in an increased transfer efficiency of the virus in vitro and in vivo even in the presence of clinically relevant doses of heparin. We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple positively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-binding sequence in the fiber coat protein that allows binding to alpha(v)-integrin receptors. The gene transfer efficiency of these modified viruses was compared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin during most vascular surgical procedures and the concern that heparin might interfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the effect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosidase expression compared with the unmodified adenovirus (mean +/- SEM, 1766.3 +/- 89.1 and 44.8 +/- 3.4 vs 10.1 +/- 0.7 mU per milligram of protein; P<.001). Following heparin administration, the gene transfer efficiency achieved with AdZ.F(pK7) diminished slightly in a concentration-dependent manner. However, the transfer efficiency was still greater than with the unmodified virus (mean +/- SEM, 1342.3 +/- 101.8 vs 4.8 +/- 0.4 mU per milligram of protein; P<.001). In vivo, following injury to the pig iliac artery with a 4F Fogarty balloon catheter, we found that AdZ.F(pK7) transduced the artery approximately 35-fold more efficiently than AdZ.F and 3-fold more efficiently than AdZ.F(RGD) following the administration of intravenous heparin, 100 U/kg body weight, and heparinized saline irrigation. Modifications of the adenovirus that lead to receptor targeting resulted in significantly improved gene transfer efficiencies. These improvements in transfer efficiencies observed with the modified vectors decreased slightly in the presence of heparin. However, AdZ.F(pK7) was still superior to AdZ.F(RGD) and AdZ.F despite heparin administration. These data demonstrate that modifications of adenoviral vectors that enhance binding to heparan sulfate receptors significantly improve gene transfer efficiency even in the presence of heparin and suggest an approach to optimize gene transfer into blood vessels.

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

NASA Astrophysics Data System (ADS)

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

2012-08-01

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.

M13 bacteriophage coat proteins engineered for improved phage display.

PubMed

Sidhu, Sachdev S; Feld, Birte K; Weiss, Gregory A

2007-01-01

This chapter describes a method for increasing levels of protein fusions displayed on the surfaces of M13 bacteriophage particles. By introducing mutations into the anchoring M13 coat protein, protein display levels can be increased by up to two orders of magnitude. Experimental methods are presented for the design, construction, and screening of phage-displayed libraries for improved protein display.

An Evolutionary Analysis of the Secoviridae Family of Viruses

PubMed Central

Thompson, Jeremy R.; Kamath, Nitin; Perry, Keith L.

2014-01-01

The plant-infecting Secoviridae family of viruses forms part of the Picornavirales order, an important group of non-enveloped viruses that infect vertebrates, arthropods, plants and algae. The impact of the secovirids on cultivated crops is significant, infecting a wide range of plants from grapevine to rice. The overwhelming majority are transmitted by ecdysozoan vectors such as nematodes, beetles and aphids. In this study, we have applied a variety of computational methods to examine the evolutionary traits of these viruses. Strong purifying selection pressures were calculated for the coat protein (CP) sequences of nine species, although for two species evidence of both codon specific and episodic diversifying selection were found. By using Bayesian phylogenetic reconstruction methods CP nucleotide substitution rates for four species were estimated to range from between 9.29×10−3 to 2.74×10−3 (subs/site/year), values which are comparable with the short-term estimates of other related plant- and animal-infecting virus species. From these data, we were able to construct a time-measured phylogeny of the subfamily Comovirinae that estimated divergence of ninety-four extant sequences occurred less than 1,000 years ago with present virus species diversifying between 50 and 250 years ago; a period coinciding with the intensification of agricultural practices in industrial societies. Although recombination (modularity) was limited to closely related taxa, significant and often unique similarities in the protein domains between secovirid and animal infecting picorna-like viruses, especially for the protease and coat protein, suggested a shared ancestry. We discuss our results in a wider context and find tentative evidence to indicate that some members of the Secoviridae might have their origins in insects, possibly colonizing plants in a number of founding events that have led to speciation. Such a scenario; virus infection between species of different taxonomic kingdoms, has significant implications for virus emergence. PMID:25180860

Lipid-coated mannitol core microparticles for sustained release of protein.

PubMed

Wang, Bifeng; Friess, Wolfgang

2018-07-01

Parenteral sustained release systems for proteins which provide therapeutic levels over a longer period avoiding frequent administration, which preserve protein stability during manufacturing, storage and application and which are biodegradable and highly biocompatible in the body are intensively sought after. The aim of this study was to generate and study mannitol core microparticles loaded with a monoclonal antibody IgG1 and coated with lipid either hard fat or glyceryl stearate at different coating levels. The protein was stabilized with 22.5 mg/mL sucrose, 0.1% PS 80, 10 mM methionine in 10 mM His buffer pH 7.2 during the spray loading process. 30 g protein-loaded mannitol carrier microparticles were coated with 5 g, 10 g, 20 g and 30 g of lipid, respectively. Placing more lipid onto the protein-loaded microparticles reduced both burst and release rate, and the particles maintained their geometric form during the release test. The IgG1 release from microparticles covered with a hard fat layer extended up to 6 weeks. The IgG1 was released in its monomeric form and maintained its secondary structure as shown by FTIR. Incomplete release of IgG1 from glyceryl stearate-coated microparticles was observed, which may be due to the small pore sizes of the glyceryl stearate layer or a detrimental surfactant character of glyceryl stearate to protein. Hence, these hard fat-coated mannitol core microparticles have high potential for protein delivery. Copyright © 2018 Elsevier B.V. All rights reserved.

Sequence Characterization of the MC1R Gene in Yak (Poephagus grunniens) Breeds with Different Coat Colors

PubMed Central

Chen, Shi-Yi; Huang, Yi; Zhu, Qing; Fontanesi, Luca; Yao, Yong-Gang; Liu, Yi-Ping

2009-01-01

Melanocortin 1 receptor (MC1R) gene plays a key role in determining coat color in several species, including the cattle. However, up to now there is no report regarding the MC1R gene and the potential association of its mutations with coat colors in yak (Poephagus grunniens). In this study, we sequenced the encoding region of the MC1R gene in three yak breeds with completely white (Tianzhu breed) or black coat color (Jiulong and Maiwa breeds). The predicted coding region of the yak MC1R gene resulted of 954 bp, the same to that of the wild-type cattle sequence, with >99% identity. None of the mutation events reported in cattle was found. Comparing the yak obtained sequences, five nucleotide substitutions were detected, which defined three haplotypes (EY1, EY2, and EY3). Of the five mutations, two, characterizing the EY1 haplotype, were nonsynonymous substitutions (c.340C>A and c.871G>A) causing amino acid changes located in the first extracellular loop (p.Q114K) and in the seventh transmembrane region (p.A291T). In silico prediction might indicate a functional effect of the latter substitution. However, all three haplotypes were present in the three yak breeds with relatively consistent frequency distribution, despite of their distinguished coat colors, which suggested that there was no across-breed association between haplotypes or genotypes and black/white phenotypes, at least in the investigated breeds. Other genes may be involved in affecting coat color in the analyzed yaks. PMID:19584942

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

PubMed

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.

Mutations in the gene encoding the Sigma 2 subunit of the adaptor protein 1 complex, AP1S2, cause X-linked mental retardation.

PubMed

Tarpey, Patrick S; Stevens, Claire; Teague, Jon; Edkins, Sarah; O'Meara, Sarah; Avis, Tim; Barthorpe, Syd; Buck, Gemma; Butler, Adam; Cole, Jennifer; Dicks, Ed; Gray, Kristian; Halliday, Kelly; Harrison, Rachel; Hills, Katy; Hinton, Jonathon; Jones, David; Menzies, Andrew; Mironenko, Tatiana; Perry, Janet; Raine, Keiran; Richardson, David; Shepherd, Rebecca; Small, Alexandra; Tofts, Calli; Varian, Jennifer; West, Sofie; Widaa, Sara; Yates, Andy; Catford, Rachael; Butler, Julia; Mallya, Uma; Moon, Jenny; Luo, Ying; Dorkins, Huw; Thompson, Deborah; Easton, Douglas F; Wooster, Richard; Bobrow, Martin; Carpenter, Nancy; Simensen, Richard J; Schwartz, Charles E; Stevenson, Roger E; Turner, Gillian; Partington, Michael; Gecz, Jozef; Stratton, Michael R; Futreal, P Andrew; Raymond, F Lucy

2006-12-01

In a systematic sequencing screen of the coding exons of the X chromosome in 250 families with X-linked mental retardation (XLMR), we identified two nonsense mutations and one consensus splice-site mutation in the AP1S2 gene on Xp22 in three families. Affected individuals in these families showed mild-to-profound mental retardation. Other features included hypotonia early in life and delay in walking. AP1S2 encodes an adaptin protein that constitutes part of the adaptor protein complex found at the cytoplasmic face of coated vesicles located at the Golgi complex. The complex mediates the recruitment of clathrin to the vesicle membrane. Aberrant endocytic processing through disruption of adaptor protein complexes is likely to result from the AP1S2 mutations identified in the three XLMR-affected families, and such defects may plausibly cause abnormal synaptic development and function. AP1S2 is the first reported XLMR gene that encodes a protein directly involved in the assembly of endocytic vesicles.

An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

PubMed

Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

2016-04-19

Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

PubMed Central

Swanson, Maud M.; Dawson, Lorna; Freitag, Thomas E.; Singh, Brajesh K.; Torrance, Lesley; Mushegian, Arcady R.

2015-01-01

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors. PMID:25841004

Metabolic markers and microecological characteristics of tongue coating in patients with chronic gastritis

PubMed Central

2013-01-01

Background In Traditional Chinese Medicine (TCM), tongue diagnosis has been an important diagnostic method for the last 3000 years. Tongue diagnosis is a non-invasive, simple and valuable diagnostic tool. TCM treats the tongue coating on a very sensitive scale that reflects physiological and pathological changes in the organs, especially the spleen and stomach. Tongue coating can diagnose disease severity and determine the TCM syndrome (“Zheng” in Chinese). The biological bases of different tongue coating appearances are still poorly understood and lack systematic investigation at the molecular level. Methods Tongue coating samples were collected from 70 chronic gastritis patients and 20 normal controls. 16S rRNA denatured gradient gel electrophoresis (16S rRNA–DGGE) and liquid chromatography and mass spectrometry (LC–MS) were designed to profile tongue coatings. The statistical techniques used were principal component analysis and partial least squares–discriminate analysis. Results Ten potential metabolites or markers were found in chronic gastritis patients, including UDP-D-galactose, 3-ketolactose, and vitamin D2, based on LC–MS. Eight significantly different strips were observed in samples from chronic gastritis patients based on 16S rRNA–DGGE. Two strips, Strips 8 and 10, were selected for gene sequencing. Strip 10 sequencing showed a 100% similarity to Rothia mucilaginosa. Strip 8 sequencing showed a 96.2% similarity to Moraxella catarrhalis. Conclusions Changes in glucose metabolism could possibly form the basis of tongue coating conformation in chronic gastritis patients. The study revealed important connections between metabolic components, microecological components and tongue coating in chronic gastritis patients. Compared with other diagnostic regimens, such as blood tests or tissue biopsies, tongue coating is more amenable to, and more convenient for, both patients and doctors. PMID:24041039

Bacterial adhesion to protein-coated surfaces: An AFM and QCM-D study

NASA Astrophysics Data System (ADS)

Strauss, Joshua; Liu, Yatao; Camesano, Terri A.

2009-09-01

Bacterial adhesion to biomaterials, mineral surfaces, or other industrial surfaces is strongly controlled by the way bacteria interact with protein layers or organic matter and other biomolecules that coat the materials. Despite this knowledge, many studies of bacterial adhesion are performed under clean conditions, instead of in the presence of proteins or organic molecules. We chose fetal bovine serum (FBS) as a model protein, and prepared FBS films on quartz crystals. The thickness of the FBS layer was characterized using atomic force microscopy (AFM) imaging under liquid and quartz crystal microbalance with dissipation (QCM-D). Next, we characterized how the model biomaterial surface would interact with the nocosomial pathogen Staphylococcus epidermidis. An AFM probe was coated with S. epidermidis cells and used to probe a gold slide that had been coated with FBS or another protein, fibronectin (FN). These experiments show that AFM and QCM-D can be used in complementary ways to study the complex interactions between bacteria, proteins, and surfaces.

Improvement and efficient display of Bacillus thuringiensis toxins on M13 phages and ribosomes.

PubMed

Pacheco, Sabino; Cantón, Emiliano; Zuñiga-Navarrete, Fernando; Pecorari, Frédéric; Bravo, Alejandra; Soberón, Mario

2015-12-01

Bacillus thuringiensis (Bt) produces insecticidal proteins that have been used worldwide in the control of insect-pests in crops and vectors of human diseases. However, different insect species are poorly controlled by the available Bt toxins or have evolved resistance to these toxins. Evolution of Bt toxicity could provide novel toxins to control insect pests. To this aim, efficient display systems to select toxins with increased binding to insect membranes or midgut proteins involved in toxicity are likely to be helpful. Here we describe two display systems, phage display and ribosome display, that allow the efficient display of two non-structurally related Bt toxins, Cry1Ac and Cyt1Aa. Improved display of Cry1Ac and Cyt1Aa on M13 phages was achieved by changing the commonly used peptide leader sequence of the coat pIII-fusion protein, that relies on the Sec translocation pathway, for a peptide leader sequence that relies on the signal recognition particle pathway (SRP) and by using a modified M13 helper phage (Phaberge) that has an amber mutation in its pIII genomic sequence and preferentially assembles using the pIII-fusion protein. Also, both Cry1Ac and Cyt1Aa were efficiently displayed on ribosomes, which could allow the construction of large libraries of variants. Furthermore, Cry1Ac or Cyt1Aa displayed on M13 phages or ribosomes were specifically selected from a mixture of both toxins depending on which antigen was immobilized for binding selection. These improved systems may allow the selection of Cry toxin variants with improved insecticidal activities that could counter insect resistances.

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901.

PubMed

Campos-Olivas, R; Hörr, I; Bormann, C; Jung, G; Gronenborn, A M

2001-05-11

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction. Copyright 2001 Academic Press.

Immunogenicity of HILDA/LIF either in a soluble or in a membrane anchored form expressed in vivo by recombinant vaccinia viruses.

PubMed

Taupin, J L; Acres, B; Dott, K; Schmitt, D; Kieny, M P; Gualde, N; Moreau, J F

1993-09-01

Insertion of various cDNAs in the genome of the vaccinia virus (VV) enables the in vivo and in vitro study of the functional role and/or the immunogenicity of the virally encoded recombinant proteins. We have prepared a recombinant VV expressing the cDNA of the human cytokine HILDA/LIF (human interleukin for DA cells/leukaemia inhibitory factor), and used this virus to immunize mice against this protein, which is very homologous to its murine counterpart (approximately 80% homology). We also constructed and expressed by the same system a chimeric gene encoding the HILDA/LIF protein fused to the 37 COOH-terminal amino-acids of the human decay accelerating factor (DAF). This sequence proved to be sufficient for the targeting of the fusion protein to the cell membrane, where it is linked to the phosphatidylinositols. Both recombinant VVs induced cytokine-specific antibodies in mice as analysed with an ELISA where the recombinant HILDA/LIF was plastic-coated and a cytofluorometric assay where the LIF-DAF molecule was present at the cell surface of stably transfected P815. In the latter case HILDA/LIF remained biologically active suggesting that it was expressed in its native form. The LIF-DAF fusion protein was found to exhibit a better capacity to elicit an antibody response against the native form of the cytokine as detected in cytofluorometric assays. Whatever the recombinant virus used to immunize the mice, the MoAbs obtained were positive either in the ELISA or in the cytofluorometric assays but one, which suggested that the plastic coating induced a conformational change of HILDA/LIF.

Previously unknown and highly divergent ssDNA viruses populate the oceans.

PubMed

Labonté, Jessica M; Suttle, Curtis A

2013-11-01

Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton.

Kinetic regulation of coated vesicle secretion

PubMed Central

Foret, Lionel; Sens, Pierre

2008-01-01

The secretion of vesicles for intracellular transport often relies on the aggregation of specialized membrane-bound proteins into a coat able to curve cell membranes. The nucleation and growth of a protein coat is a kinetic process that competes with the energy-consuming turnover of coat components between the membrane and the cytosol. We propose a generic kinetic description of coat assembly and the formation of coated vesicles and discuss its implication to the dynamics of COP vesicles that traffic within the Golgi and with the endoplasmic reticulum. We show that stationary coats of fixed area emerge from the competition between coat growth and the recycling of coat components, in a fashion resembling the treadmilling of cytoskeletal filaments. We further show that the turnover of coat components allows for a highly sensitive switching mechanism between a quiescent and a vesicle producing membrane, upon a slowing down of the exchange kinetics. We claim that the existence of this switching behavior, also triggered by factors, such as the presence of cargo and variation of the membrane mechanical tension, allows for efficient regulation of vesicle secretion. We propose a model, supported by different experimental observations, in which vesiculation of secretory membranes is impaired by the energy-consuming desorption of coat proteins, until the presence of cargo or other factors triggers a dynamical switch into a vesicle producing state. PMID:18824695

The dead seed coat functions as a long-term storage for active hydrolytic enzymes

PubMed Central

Raviv, Buzi; Aghajanyan, Lusine; Granot, Gila; Makover, Vardit; Frenkel, Omer; Gutterman, Yitzchak

2017-01-01

Seed development culminates in programmed cell death (PCD) and hardening of organs enclosing the embryo (e.g., pericarp, seed coat) providing essentially a physical shield for protection during storage in the soil. We examined the proposal that dead organs enclosing embryos are unique entities that store and release upon hydration active proteins that might increase seed persistence in soil, germination and seedling establishment. Proteome analyses of dead seed coats of Brassicaceae species revealed hundreds of proteins being stored in the seed coat and released upon hydration, many are stress-associated proteins such as nucleases, proteases and chitinases. Functional analysis revealed that dead seed coats function as long-term storage for multiple active hydrolytic enzymes (e.g., nucleases) that can persist in active forms for decades. Substances released from the dead seed coat of the annual desert plant Anastatica hierochuntica displayed strong antimicrobial activity. Our data highlighted a previously unrecognized feature of dead organs enclosing embryos (e.g., seed coat) functioning not only as a physical shield for embryo protection but also as a long-term storage for active proteins and other substances that are released upon hydration to the “seedsphere” and could contribute to seed persistence in the soil, germination and seedling establishment. PMID:28700755

The dead seed coat functions as a long-term storage for active hydrolytic enzymes.

PubMed

Raviv, Buzi; Aghajanyan, Lusine; Granot, Gila; Makover, Vardit; Frenkel, Omer; Gutterman, Yitzchak; Grafi, Gideon

2017-01-01

Seed development culminates in programmed cell death (PCD) and hardening of organs enclosing the embryo (e.g., pericarp, seed coat) providing essentially a physical shield for protection during storage in the soil. We examined the proposal that dead organs enclosing embryos are unique entities that store and release upon hydration active proteins that might increase seed persistence in soil, germination and seedling establishment. Proteome analyses of dead seed coats of Brassicaceae species revealed hundreds of proteins being stored in the seed coat and released upon hydration, many are stress-associated proteins such as nucleases, proteases and chitinases. Functional analysis revealed that dead seed coats function as long-term storage for multiple active hydrolytic enzymes (e.g., nucleases) that can persist in active forms for decades. Substances released from the dead seed coat of the annual desert plant Anastatica hierochuntica displayed strong antimicrobial activity. Our data highlighted a previously unrecognized feature of dead organs enclosing embryos (e.g., seed coat) functioning not only as a physical shield for embryo protection but also as a long-term storage for active proteins and other substances that are released upon hydration to the "seedsphere" and could contribute to seed persistence in the soil, germination and seedling establishment.

Promotion of pro-osteogenic responses by a bioactive ceramic coating.

PubMed

Aniket; Young, Amy; Marriott, Ian; El-Ghannam, Ahmed

2012-12-01

The objective of this study was to analyze the responses of bone-forming osteoblasts to Ti-6Al-4V implant material coated with silica-calcium phosphate nanocomposite (SCPC50). Osteoblast differentiation at the interface with SCPC50-coated Ti-6Al-4V was correlated to the adsorption of high amount of serum proteins, high surface affinity to fibronectin, Ca uptake from and P and Si release into the medium. SCPC50-coated Ti-6Al-4V adsorbed significantly more serum protein (p < 0.05) than control uncoated substrates. Moreover, Western blot analysis showed that the SCPC50 coating had a high affinity for serum fibronectin. Protein conformation analyses by FTIR showed that the ratio of the area under the peak for amide I/amide II bands was significantly higher (p < 0.05) on the surface of SCPC50-coated substrates than that on the surface of the control uncoated substrates. Moreover, ICP - OES analyses indicated that SCPC50-coated substrates withdrew Ca ions from, and released P and Si ions into, the tissue culture medium, respectively. In conjunction with the favorable protein adsorption and modifications in medium composition, MC3T3-E1 osteoblast-like cells attached to SCPC50-coated substrates expressed 10-fold higher level of mRNA encoding osteocalcin and had significantly higher production of osteopontin and osteocalcin proteins than cells attached to the uncoated Ti-6A1-4V substrates. In addition, osteoblast-like cells attached to the SCPC50-coated substrates produced significantly lower levels of the inflammatory and osteoclastogenic cytokines, IL-6, IL-12p40, and RANKL than those attached to uncoated Ti-6Al-4V substrates. These results suggest that SCPC50 coating could enhance bone integration with orthopedic and maxillofacial implants while minimizing the induction of inflammatory bone cell responses. Copyright © 2012 Wiley Periodicals, Inc.

Maintenance of coat protein N-terminal net charge and not primary sequence is essential for zucchini yellow mosaic virus systemic infectivity.

PubMed

Kimalov, Boaz; Gal-On, Amit; Stav, Ran; Belausov, Eduard; Arazi, Tzahi

2004-11-01

Zucchini yellow mosaic virus (ZYMV) surface exposed coat protein (CP) N-terminal domain (Nt) is 43 aa long and contains an equal number of positively and negatively charged amino acid residues (CP-Nt net charge = 0). A ZYMV-AGII truncation mutant lacking the first 20 aa of its CP-Nt (AGII-CP Delta 20; CP-Nt net charge = +2) was found to be systemically non-infectious even though AGII mutants harbouring larger CP-Nt deletions were previously demonstrated to be fully infectious. Nevertheless, AGII-CP Delta 20 infectivity was restored by fusion to its CP-Nt two Asp residues or a negatively charged Myc peptide, both predicted to neutralize CP-Nt net positive charge. To evaluate further the significance of CP-Nt net charge for AGII infectivity, a series of CP-Nt net charge mutants was generated and analysed for systemic infectivity of squash plants. AGII-CP(KKK) harbouring a CP-Nt amino fusion of three Lys residues (CP-Nt net charge = +3) was not systemically infectious. Addition of up to four Asp residues to CP-Nt did not abolish virus infectivity, although certain mutants were genetically unstable and had delayed infectivity. Addition of five negatively charged residues abolished infectivity (AGII-CP(DDDDD); CP-Nt net charge = -5) even though a recombinant CP(DDDDD) could assemble into potyviral-like particle in bacteria. Neutralization of CP-Nt net charge by fusing Asp or Lys residues recovered infectivity of AGII-CP(KKK) and AGII-CP(DDDDD). GFP-tagging of these mutants has demonstrated that both viruses have defective cell-to-cell movement. Together, these findings suggest that maintenance of CP-Nt net charge and not primary sequence is essential for ZYMV infectivity.

Design principles for robust vesiculation in clathrin-mediated endocytosis

PubMed Central

Hassinger, Julian E.; Oster, George; Drubin, David G.; Rangamani, Padmini

2017-01-01

A critical step in cellular-trafficking pathways is the budding of membranes by protein coats, which recent experiments have demonstrated can be inhibited by elevated membrane tension. The robustness of processes like clathrin-mediated endocytosis (CME) across a diverse range of organisms and mechanical environments suggests that the protein machinery in this process has evolved to take advantage of some set of physical design principles to ensure robust vesiculation against opposing forces like membrane tension. Using a theoretical model for membrane mechanics and membrane protein interaction, we have systematically investigated the influence of membrane rigidity, curvature induced by the protein coat, area covered by the protein coat, membrane tension, and force from actin polymerization on bud formation. Under low tension, the membrane smoothly evolves from a flat to budded morphology as the coat area or spontaneous curvature increases, whereas the membrane remains essentially flat at high tensions. At intermediate, physiologically relevant, tensions, the membrane undergoes a “snap-through instability” in which small changes in the coat area, spontaneous curvature or membrane tension cause the membrane to “snap” from an open, U-shape to a closed bud. This instability can be smoothed out by increasing the bending rigidity of the coat, allowing for successful budding at higher membrane tensions. Additionally, applied force from actin polymerization can bypass the instability by inducing a smooth transition from an open to a closed bud. Finally, a combination of increased coat rigidity and force from actin polymerization enables robust vesiculation even at high membrane tensions. PMID:28126722

Carbohydrate-protein interactions investigated on plastic chips statically coated with hydrophobically modified hydroxyethylcellulose.

PubMed

Dang, Fuquan; Maeda, Eiki; Osafune, Tomo; Nakajima, Kazuki; Kakehi, Kazuaki; Ishikawa, Mitsuru; Baba, Yoshinobu

2009-12-15

We developed a novel method for rapid screening of carbohydrate-protein interactions using poly(methyl methacrylate) (PMMA) channels statically coated with hydrophobically modified hydroxyethylcellulose (HM-HEC). We found that a self-assembled monolayer (SAM) of HM-HEC on a PMMA surface intact by water allows rapid and reproducible separations of glycan samples using a 20 mM phosphate without HM-HEC. The underlying mechanism for dynamic and static coatings on the PMMA surface is discussed. Simultaneous analysis of the molecular interaction between a complex mixture of carbohydrates from alpha1-acid glycoprotein and proteins has been successfully achieved in PMMA channels statically coated with a SAM of HM-HEC.

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

PubMed Central

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

2013-01-01

Background 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. Methods 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. Results 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. Conclusion Fluid-bed coating technology has potential for use in the solidification of drug nanosuspensions. PMID:23983465

Whey protein solution coating for fat-uptake reduction in deep-fried chicken breast strips.

PubMed

Dragich, Ann M; Krochta, John M

2010-01-01

This study investigated the use of whey protein, as an additional coating, in combination with basic, well-described predust, batter, and breading ingredients, for fat-uptake reduction in fried chicken. Chicken breasts were cut into strips (1 x 5 x 10 cm) and coated with wheat flour (WF) as a predust, dipped in batter, coated with WF as a breading, then dipped in 10% denatured whey protein isolate (DWPI) aqueous solution (wet basis). A WF-batter-WF treatment with no DWPI solution dip was included as a control. Coated chicken strips were deep-fried at 160 degrees C for 5 min. A Soxhlet-type extraction was performed to determine the fat content of the meat fraction of fried samples, the coating fraction of fried samples, raw chicken, and raw coating ingredients. The WF-batter-WF-10% DWPI solution had significantly lower fat uptake than the WF-batter-WF control, by 30.67% (dry basis). This article describes applied research involving fat reduction in coated deep-fried chicken. The methods used in this article were intended to achieve maximized fat reduction while maintaining a simple procedure applicable to actual food processing lines.

A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

PubMed Central

Umasankar, Perunthottathu K; Ma, Li; Thieman, James R; Jha, Anupma; Doray, Balraj; Watkins, Simon C; Traub, Linton M

2014-01-01

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization. DOI: http://dx.doi.org/10.7554/eLife.04137.001 PMID:25303365

An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems.

PubMed

Cheng, Hao-Wen; Chen, Kuan-Chun; Raja, Joseph A J; Li, Jian-Xian; Yeh, Shyi-Dong

2013-04-15

NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, "(109)KFTMHNQIF(117)", denoted as "nss", retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. Copyright © 2013 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

Rusen, L.; Mustaciosu, C.; Mitu, B.; Filipescu, M.; Dinescu, M.; Dinca, V.

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(ɛ-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-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.

Identification of a novel microRNA important for melanogenesis in alpaca (Vicugna pacos).

PubMed

Yang, S; Fan, R; Shi, Z; Ji, K; Zhang, J; Wang, H; Herrid, M; Zhang, Q; Yao, J; Smith, G W; Dong, C

2015-04-01

The molecular mechanisms underlying the formation of coat colors in animals are poorly understood. Recent studies have demonstrated that microRNA play important roles in the control of melanogenesis and coat color in mammals. In a previous study, we characterized the miRNA expression profiles in alpaca skin with brown and white coat color and identified a novel miRNA (named lpa-miR-nov-66) that is expressed significantly higher in white skin compared to brown skin. The present study was conducted to determine the functional roles of this novel miRNA in the regulation of melanogenesis in alpaca melanocytes. lpa-miR-nov-66 is predicted to target the soluble guanylate cyclase (sGC) gene based on presence of a binding site in the sGC coding sequence (CDS). Overexpression of lpa-miR-nov-66 in alpaca melanocyes upregulated the expression of sGC both at the mRNA and protein level. Overexpression of lpa-miR-nov-66 in melanocyes also resulted in decreased expression of key melanogenic genes including tyrosinase (TYR), tyrosinase related protein 1 (TYRP1), and microphthalmia transcription factor (MITF). Our ELISA assays showed increased cyclic guanosine monophosphate (cGMP) but decreased cyclic adenosine monophosphate (cAMP) production in melanocytes overexpressing lpa-miR-nov-66. In addition, overexpression of lpa-miR-nov-66 also reduced melanin production in cultured melanocytes. Results support a role of lpa-miR-nov-66 in melanocytes by directly or indirectly targeting , which regulates melanogenesis via the cAMP pathway.

Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly

PubMed Central

Burtey, Anne; Praefcke, Gerrit J. K; Peak-Chew, Sew-Yeu; Mills, Ian G; Benmerah, Alexandre; McMahon, Harvey T

2006-01-01

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors. PMID:16903783

Protein corona composition of gold nanoparticles/nanorods affects amyloid beta fibrillation process

NASA Astrophysics Data System (ADS)

Mirsadeghi, Somayeh; Dinarvand, Rassoul; Ghahremani, Mohammad Hossein; Hormozi-Nezhad, Mohammad Reza; Mahmoudi, Zohreh; Hajipour, Mohammad Javad; Atyabi, Fatemeh; Ghavami, Mahdi; Mahmoudi, Morteza

2015-03-01

Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades, nanoparticles (NPs) were recognized as one of the most promising tools for inhibiting the progress of the disease by controlling the fibrillation kinetic process; for instance, gold NPs have a strong capability to inhibit Aβ fibrillations. It is now well understood that a layer of biomolecules would cover the surface of NPs (so called ``protein corona'') upon the interaction of NPs with protein sources. Due to the fact that the biological species (e.g., cells and amyloidal proteins) ``see'' the protein corona coated NPs rather than the pristine coated particles, one should monitor the fibrillation process of amyloidal proteins in the presence of corona coated NPs (and not pristine coated ones). Therefore, the previously obtained data on NPs effects on the fibrillation process should be modified to achieve a more reliable and predictable in vivo results. Herein, we probed the effects of various gold NPs (with different sizes and shapes) on the fibrillation process of Aβ in the presence and absence of protein sources (i.e., serum and plasma). We found that the protein corona formed a shell at the surface of gold NPs, regardless of their size and shape, reducing the access of Aβ to the gold inhibitory surface and, therefore, affecting the rate of Aβ fibril formation. More specifically, the anti-fibrillation potencies of various corona coated gold NPs were strongly dependent on the protein source and their concentrations (10% serum/plasma (simulation of an in vitro milieu) and 100% serum/plasma (simulation of an in vivo milieu)).Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades, nanoparticles (NPs) were recognized as one of the most promising tools for inhibiting the progress of the disease by controlling the fibrillation kinetic process; for instance, gold NPs have a strong capability to inhibit Aβ fibrillations. It is now well understood that a layer of biomolecules would cover the surface of NPs (so called ``protein corona'') upon the interaction of NPs with protein sources. Due to the fact that the biological species (e.g., cells and amyloidal proteins) ``see'' the protein corona coated NPs rather than the pristine coated particles, one should monitor the fibrillation process of amyloidal proteins in the presence of corona coated NPs (and not pristine coated ones). Therefore, the previously obtained data on NPs effects on the fibrillation process should be modified to achieve a more reliable and predictable in vivo results. Herein, we probed the effects of various gold NPs (with different sizes and shapes) on the fibrillation process of Aβ in the presence and absence of protein sources (i.e., serum and plasma). We found that the protein corona formed a shell at the surface of gold NPs, regardless of their size and shape, reducing the access of Aβ to the gold inhibitory surface and, therefore, affecting the rate of Aβ fibril formation. More specifically, the anti-fibrillation potencies of various corona coated gold NPs were strongly dependent on the protein source and their concentrations (10% serum/plasma (simulation of an in vitro milieu) and 100% serum/plasma (simulation of an in vivo milieu)). Electronic supplementary information (ESI) available: Full characterization results of the nanoparticles, protein corona, and fibrillation process. See DOI: 10.1039/c4nr06009a

The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum.

PubMed

Srinivasan, S; Griffiths, K R; McGuire, V; Champion, A; Williams, K L; Alexander, S

2000-08-01

The terminal event of spore differentiation in the cellular slime mould Dictyostelium discoideum is the assembly of the spore coat, which surrounds the dormant amoeba and allows the organism to survive during extended periods of environmental stress. The spore coat is a polarized extracellular matrix composed of glycoproteins and cellulose. The process of spore coat formation begins by the regulated secretion of spore coat proteins from the prespore vesicles (PSVs). Four of the major spore coat proteins (SP96, PsB/SP85, SP70 and SP60) exist as a preassembled multiprotein complex within the PSVs. This complete complex has an endogenous cellulose-binding activity. Mutant strains lacking either the SP96 or SP70 proteins produce partial complexes that do not have cellulose-binding activity, while mutants lacking SP60 produce a partial complex that retains this activity. Using a combination of immunofluorescence microscopy and biochemical methods we now show that the lack of cellulose-binding activity in the SP96 and SP70 mutants results in abnormally assembled spore coats and spores with greatly reduced viability. In contrast, the SP60 mutant, in which the PsB complex retains its cellulose-binding activity, produces spores with apparently unaltered structure and viability. Thus, it is the loss of the cellulose-binding activity of the PsB complex, rather than the mere loss of individual spore coat proteins, that results in compromised spore coat structure. These results support the idea that the cellulose-binding activity associated with the complete PsB complex plays an active role in the assembly of the spore coat.

Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae.

PubMed

Juozapaitis, Mindaugas; Zvirbliene, Aurelija; Kucinskaite, Indre; Sezaite, Indre; Slibinskas, Rimantas; Coiras, Mayte; de Ory Manchon, Fernando; López-Huertas, María Rosa; Pérez-Breña, Pilar; Staniulis, Juozas; Narkeviciute, Irena; Sasnauskas, Kestutis

2008-05-01

Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.

Reverse transcription of phage RNA and its fragment directed by synthetic heteropolymeric primers

PubMed Central

Frolova, L. Yu.; Metelyev, V. G.; Ratmanova, K. I.; Smirnov, V. D.; Shabarova, Z. A.; Prokofyev, M. A.; Berzin, V. M.; Jansone, I. V.; Gren, E. J.; Kisselev, L. L.

1977-01-01

DNA synthesis catalysed by RNA-directed DNA-polymerase (reverse transcriptase) was found to proceed on the RNA template of an MS2 phage in the presence of heteropolymeric synthetic octa- and nonadeoxyribonucleotide primers complementary to the intercistronic region (coat protein binding site) and the region of the coat protein cistron, respectively. The product of synthesis consists of discrete DNA fractions of different length, including transcripts longer than 1,000 nucleotides. The coat protein inhibits DNA synthesis if it is initiated at its binding site, but has no effect on DNA synthesis initiated at the coat protein cistron. It has been suggested that, in this system, the initiation of DNA synthesis by synthetic primers is topographically specific. The MS2 coat protein binding site (an RNA fragment of 59 nucleotides) serves as a template for polydeoxyribonucleotide synthesis in the presence of octanucleotide primer and reverse transcriptase. The product of synthesis is homogenous and its length corresponds to the length of the template. The effective and complete copying of the fragment having a distinct secondary structure proves that the secondary structure does not interfere, in principle, with RNA being a template in the system of reverse transcription. PMID:71713

Proteins encoded by the gerP operon are localised to the inner coat in Bacilluscereus spores and are dependent on GerPA and SafA for assembly.

PubMed

Ghosh, Abhinaba; Manton, James D; Mustafa, Amin R; Gupta, Mudit; Ayuso-Garcia, Alejandro; Rees, Eric J; Christie, Graham

2018-05-04

Germination of Bacillus spores is triggered by certain amino acids and sugar molecules, which permeate through the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here we use the ellipsoid localisation microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex lytic enzymes within the inner coat. We reveal also that the GerPA protein alone can localise in the absence of all other GerP proteins, and that it has an essential role for the localisation of all other GerP proteins within the spore. The latter is also demonstrated to be SafA - but not CotE - dependent for localisation, which is consistent with an inner coat location. GerP null spores are shown also to have reduced permeability to fluorescently labelled dextran molecules compared to wild type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability. Importance The bacterial spore coat comprises a multi-layered proteinaceous structure that influences the distribution, survival and germination properties of spores in the environment. Results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ELM image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, healthcare and environmental sectors. Copyright © 2018 American Society for Microbiology.

An assessment of lactobiopolymer-montmorillonite composites for dip coating applications on fresh strawberries.

PubMed

Junqueira-Gonçalves, Maria Paula; Salinas, Gonzalo E; Bruna, Julio E; Niranjan, Keshavan

2017-04-01

The use of biopolymer coatings appears as a good alternative to preserve highly perishable fruits, as well as the environment. Proteins generally produce films with good mechanical properties, although their highly hydrophilic nature limits the use in many applications. Nanoparticles, such as nanoclays, can play a critical role in improving barrier properties. The present study evaluated the effect of the addition of montmorillonite (MMT)-nanoparticles to a lacto-biopolymer coating, focusing on: (i) the morphological, thermal and barrier properties of the material and (ii) the shelf life of coated fresh strawberries. The addition of MMT improved the water vapor barrier property. Morphological and thermal analysis indicated a good interaction between the milk protein and the nanoclay, which was intercalated within the milk protein base (MPB) matrix, offering a more tortuous path to diffusing migrants. The MMT-MPB coating helped to significantly (P ≤ 0.05) reduce the weight loss, as well as oxygen uptake and the release of carbon dioxide, and improved the fruit firmness and reduced mould and yeast load compared to the uncoated fruits. The addition of MMT gave statistical difference (P ≤ 0.05) in terms of weight loss, subjective global appearance and purchase intention of coated fresh strawberries. The addition of nanofillers, such as MMT, into protein-based coating could improve its water vapour barrier and could affect, positively, some parameters of the shelf life of coated strawberries. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Genetic diversity in the 3'-terminal region of papaya ringspot virus (PRSV-W) isolates from watermelon in Oklahoma.

PubMed

Abdalla, Osama A; Ali, Akhtar

2012-03-01

The 3'-terminal region (1191 nt) containing part of the NIb gene, complete coat protein (CP) and poly-A tail of 64 papaya ringspot virus (PRSV-W) isolates collected during 2008-2009 from watermelon in commercial fields of four different counties of Oklahoma were cloned and sequenced. Nucleotide and amino acid sequence identities ranged from 95.2-100% and 97.1-100%, respectively, among the Oklahoman PRSV-W isolates. Phylogenetic analysis showed that PRSW-W isolates clustered according to the locations where they were collected within Oklahoma, and each cluster contained two subgroups. All subgroups of Oklahoman PRSV-W isolates were on separate branches when compared to 35 known isolates originating from other parts of the world, including the one reported previously from the USA. This study helps in our understanding about the genetic diversity of PRSV-W isolates infecting cucurbits in Oklahoma.

Detection and partial molecular characterization of atypical plum pox virus isolates from naturally infected sour cherry.

PubMed

Chirkov, Sergei; Ivanov, Peter; Sheveleva, Anna

2013-06-01

Atypical isolates of plum pox virus (PPV) were discovered in naturally infected sour cherry in urban ornamental plantings in Moscow, Russia. The isolates were detected by polyclonal double antibody sandwich ELISA and RT-PCR using universal primers specific for the 3'-non-coding and coat protein (CP) regions of the genome but failed to be recognized by triple antibody sandwich ELISA with the universal monoclonal antibody 5B and by RT-PCR using primers specific to for PPV strains D, M, C and W. Sequence analysis of the CP genes of nine isolates revealed 99.2-100 % within-group identity and 62-85 % identity to conventional PPV strains. Phylogenetic analysis showed that the atypical isolates represent a group that is distinct from the known PPV strains. Alignment of the N-terminal amino acid sequences of CP demonstrated their close similarity to those of a new tentative PPV strain, CR.

Outbreak of betanodavirus infection in tilapia, Oreochromis niloticus (L.), in fresh water.

PubMed

Bigarré, L; Cabon, J; Baud, M; Heimann, M; Body, A; Lieffrig, F; Castric, J

2009-08-01

A betanodavirus associated with a massive mortality was isolated from larvae of tilapia, Oreochromis niloticus, maintained in fresh water at 30 degrees C. Histopathology revealed vacuolation of the nervous system, suggesting an infection by a betanodavirus. The virus was identified by indirect fluorescent antibody test in the SSN1 cell line and further characterized by sequencing of a PCR product. Sequencing of the T4 region of the coat protein gene indicated a phylogenetic clustering of this isolate within the red-spotted grouper nervous necrosis virus type. However, the tilapia isolate formed a unique branch distinct from other betanodavirus isolates. The disease was experimentally reproduced by bath infection of young tilapia at 30 degrees C. The reservoir of virus at the origin of the outbreak remains unidentified. To our knowledge, this is the first report of natural nodavirus infection in tilapia reared in fresh water.

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance

PubMed Central

Simberg, Dmitri; Park, Ji-Ho; Karmali, Priya P.; Zhang, Wan-Ming; Merkulov, Sergei; McCrae, Keith; Bhatia, Sangeeta; Sailor, Michael; Ruoslahti, Erkki

2009-01-01

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles. PMID:19394687

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance.

PubMed

Simberg, Dmitri; Park, Ji-Ho; Karmali, Priya P; Zhang, Wan-Ming; Merkulov, Sergei; McCrae, Keith; Bhatia, Sangeeta N; Sailor, Michael; Ruoslahti, Erkki

2009-08-01

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles.

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

NASA Astrophysics Data System (ADS)

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.

Architecture and assembly of the Bacillus subtilis spore coat.

PubMed

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 pattern in a biological organism.

Architecture and Assembly of the Bacillus subtilis Spore Coat

PubMed Central

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 pattern in a biological organism. PMID:25259857

Molecular cloning, mRNA expression and tissue distribution analysis of Slc7a11 gene in alpaca (Lama paco) skins associated with different coat colors.

PubMed

Tian, Xue; Meng, Xiaolin; Wang, Liangyan; Song, Yunfei; Zhang, Danli; Ji, Yuankai; Li, Xuejun; Dong, Changsheng

2015-01-25

Slc7a11 encoding solute carrier family 7 member 11 (amionic amino acid transporter light chain, xCT), has been identified to be a critical genetic regulator of pheomelanin synthesis in hair and melanocytes. To better understand the molecular characterization of Slc7a11 and the expression patterns in skin of white versus brown alpaca (lama paco), we cloned the full length coding sequence (CDS) of alpaca Slc7a11 gene and analyzed the expression patterns using Real Time PCR, Western blotting and immunohistochemistry. The full length CDS of 1512bp encodes a 503 amino acid polypeptide. Sequence analysis showed that alpaca xCT contains 12 transmembrane regions consistent with the highly conserved amino acid permease (AA_permease_2) domain similar to other vertebrates. Sequence alignment and phylogenetic analysis revealed that alpaca xCT had the highest identity and shared the same branch with Camelus ferus. Real Time PCR and Western blotting suggested that xCT was expressed at significantly high levels in brown alpaca skin, and transcripts and protein possessed the same expression pattern in white and brown alpaca skins. Additionally, immunohistochemical analysis further demonstrated that xCT staining was robustly increased in the matrix and root sheath of brown alpaca skin compared with that of white. These results suggest that Slc7a11 functions in alpaca coat color regulation and offer essential information for further exploration on the role of Slc7a11 in melanogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Seed Nutrition and Quality, Seed Coat Boron and Lignin Are Influenced by Delayed Harvest in Exotically-Derived Soybean Breeding Lines under High Heat.

PubMed

Bellaloui, Nacer; Smith, James R; Mengistu, Alemu

2017-01-01

The timing of harvest is a major factor affecting seed quality in soybean, particularly in Midsouthern USA, when rain during harvest period is not uncommon. The objective of this research was to evaluate the effects of time of harvest on soybean seed quality (seed composition, germination, seed coat boron, and lignin) in high germinability (HG) breeding lines (50% exotic) developed under high heat. The hypothesis was that seeds of HG lines possess physiological and genetic traits for a better seed quality at harvest maturity and delayed harvest. A 2-year field experiment was conducted under irrigated conditions. Results showed that, at harvest maturity, the exotic HG lines had higher seed protein, oleic acid, sugars, seed coat boron, and seed coat lignin, but lower seed oil compared with the non-exotic checks (Control), confirming our hypothesis. At 28 days after harvest maturity (delayed harvest), the content of seed protein, oleic acid, sugars, seed coat boron, and seed coat lignin were higher in some of the HG lines compared with the checks, indicating a possible involvement of these seed constituents, especially seed coat boron and seed coat lignin, in maintaining seed coat integrity and protecting seed coat against physical damage. Highly significant positive correlations were found between germination and seed protein, oleic acid, sugars, and seed coat boron and seed coat lignin. Highly significant negative correlation was found between germination and oil, linoleic acid, seed coat wrinkling, shattering, and hard seed. Yields of some HG lines were competitive with checks. This research demonstrated that time of harvesting is an important factor influencing seed protein and oil production. Also, since high oleic acid is desirable for oxidative stability, shelf-life and biodiesel properties, using HG lines could positively influence these important traits. This result should suggest to breeders of some of the advantages of selecting for high seed coat boron and lignin, and inform growers of the importance of timely harvest for maintaining high seed quality.

Protozoal Digestion of Coat-Defective Bacillus subtilis Spores Produces “Rinds” Composed of Insoluble Coat Protein▿

PubMed Central

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

2008-01-01

The Bacillus subtilis spore coat is a multilayer, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan Tetrahymena thermophila. When coat-defective spores are fed to Tetrahymena, the spores are readily digested. However, a residue termed a “rind” that looks like coat material remains. As observed with a phase-contrast microscope, the rinds are spherical or hemispherical structures that appear to be devoid of internal contents. Atomic force microscopy and chemical analyses showed that (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers, (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion, and (iii) the rinds and intact spores do not contain significant levels of silicon. PMID:18689521

Role of monomer sequence and backbone chemistry in polypeptoid copolymers for marine antifouling coatings

NASA Astrophysics Data System (ADS)

Patterson, Anastasia; Wenning, Brandon; Rizis, Georgios; Calabrese, David; Finlay, John; Franco, Sofia; Clare, Anthony; Kramer, Edward; Ober, Christopher; Segalman, Rachel

The design rules elucidated in this work suggest that antifouling coatings bearing pendant peptoid side chains perform better overall in marine fouling tests than those with peptide side chains, with extremely low attachment of N. incerta and high removal of U. linza. This difference in performance is likely due to the lack of a hydrogen bond donor in the peptoid backbone. Furthermore, we show that the bulk polymer material of these hierarchical coatings (based on PEO or PDMS) plays a key role in determining both surface presentation and fouling release performance. We demonstrate these trends utilizing a modular coating based on a triblock copolymer consisting of polystyrene and a vinyl-containing midblock, to which sequence-defined pendant oligomers (peptides or peptoids with sequences of oligo-PEO and fluoroalkyl groups) are attached via thiol-ene ``click'' chemistry. Surface presentation was analyzed with X-ray photoelectron spectroscopy and captive bubble water contact angle, and antifouling performance was evaluated with attachment and removal bioassays of the marine macroalga U. linza and diatom N. incerta. NSF GRFP and ONR PECASE.

Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution

PubMed Central

Jones, Justin A.; Harris, Thomas I.; Oliveira, Paula F.; Bell, Brianne E.; Alhabib, Abdulrahman; Lewis, Randolph V.

2016-01-01

The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution. PMID:27886066

Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.

PubMed

Jones, Justin A; Harris, Thomas I; Oliveira, Paula F; Bell, Brianne E; Alhabib, Abdulrahman; Lewis, Randolph V

2016-11-23

The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution.

Three-dimensional organization of three-domain copper oxidases: A review

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhukhlistova, N. E., E-mail: amm@ns.crys.ras.ru; Zhukova, Yu. N.; Lyashenko, A. V.

2008-01-15

'Blue' copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrenamore » maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.« less

Three-dimensional organization of three-domain copper oxidases: A review

NASA Astrophysics Data System (ADS)

Zhukhlistova, N. E.; Zhukova, Yu. N.; Lyashenko, A. V.; Zaĭtsev, V. N.; Mikhaĭlov, A. M.

2008-01-01

“Blue” copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrena maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.

The oculocerebrorenal syndrome gene product is a 105-kD protein localized to the Golgi complex.

PubMed Central

Olivos-Glander, I M; Jänne, P A; Nussbaum, R L

1995-01-01

The oculocerebrorenal syndrome of Lowe (OCRL) is a multisystem disorder affecting the lens, kidney, and CNS. The predicted amino acid sequence of the OCRL gene, OCRL-1, was used to develop antibodies against the OCRL-1 protein. Western blot analysis using affinity-purified serum against the amino terminus of the OCRL-1 gene product (ocrl-1) demonstrates a single protein of 105 kD in fibroblasts of a normal individual that is absent in fibroblasts of an OCRL patient who lacks OCRL-1 transcript. A single protein with the same electrophoretic mobility is found by western analysis in various human cultured cell lines, and approximately the same size protein is also found in all mouse tissues tested. Northern analysis of various human and mouse tissues demonstrate that OCRL-1 transcript is expressed in nearly all tissues examined. By immunofluorescence, the ocrl-1 antibody stains a juxtanuclear region in normal fibroblast cells, while no specific staining is evident in the OCRL patient who produces no transcript. Colocalization of the ocrl-1 protein to the Golgi complex was demonstrated using a known monoclonal antibody against a Golgi-specific coat protein, beta-COP (beta coatomer protein). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7573041

Sensing of heavy metal ions by intrinsic TMV coat protein fluorescence

NASA Astrophysics Data System (ADS)

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.

Clathrin-independent pathways do not contribute significantly to endocytic flux.

PubMed

Bitsikas, Vassilis; Corrêa, Ivan R; Nichols, Benjamin J

2014-09-17

Several different endocytic pathways have been proposed to function in mammalian cells. Clathrin-coated pits are well defined, but the identity, mechanism and function of alternative pathways have been controversial. Here we apply universal chemical labelling of plasma membrane proteins to define all primary endocytic vesicles, and labelling of specific proteins with a reducible SNAP-tag substrate. These approaches provide high temporal resolution and stringent discrimination between surface-connected and intracellular membranes. We find that at least 95% of the earliest detectable endocytic vesicles arise from clathrin-coated pits. GPI-anchored proteins, candidate cargoes for alternate pathways, are also found to enter the cell predominantly via coated pits. Experiments employing a mutated clathrin adaptor reveal distinct mechanisms for sorting into coated pits, and thereby explain differential effects on the uptake of transferrin and GPI-anchored proteins. These data call for a revision of models for the activity and diversity of endocytic pathways in mammalian cells.

Preventing Protein Adsorption and Macrophage Uptake of Gold Nanoparticles via a Hydrophobic Shield

PubMed Central

Larson, Timothy A.; Joshi, Pratixa P.; Sokolov, Konstantin

2012-01-01

Polyethylene glycol (PEG) surface coatings are widely used to render stealth properties to nanoparticles in biological applications. There is abundant literature on benefits of PEG coatings and their ability to reduce protein adsorption, to diminish non-specific interactions with cells, and to improve pharmacokinetics, but very little discussion of the limitations of PEG coatings. Here, we show that physiological concentrations of cysteine and cystine can displace methoxy-PEG-thiol molecules from the gold nanoparticle (GNP) surface that leads to protein adsorption and cell uptake in macrophages within 24 hours. Furthermore, we address this problem by incorporating an alkyl linker between the PEG and the thiol moieties that provides a hydrophobic shield layer between the gold surface and the hydrophilic outer PEG layer. The mPEG-alkyl-thiol coating greatly reduces protein adsorption on GNPs and their macrophage uptake. This has important implications for the design of GNP for biological systems. PMID:23009596

Exosome purification based on PEG-coated Fe3O4 nanoparticles.

PubMed

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.

Identification of trapped and boundary lipid binding sites in M13 coat protein/lipid complexes by deuterium NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Gorkom, L.C.; Horvath, L.I.; Hemminga, M.A.

The major coat protein of M13 bacteriophage has been incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the trimethyl segments of the choline headgroup (DMPC-d9). Two-component deuterium and phosphorus-31 NMR spectra have been observed from bilayer complexes containing the coat protein, indicating slow exchange (on the deuterium quadrupole anisotropy and phosphorus-31 chemical shift averaging time scales) of lipid molecules of less than 10(3) Hz between two motionally distinct environments in the complexes. The fraction of the isotropic spectral component increases with increasing M13 protein concentration, and this component is attributed to lipid headgroups, which are disordered relative to their order inmore » protein-free bilayers. The activation energy of the fast local motions of the trimethyl groups of the choline residue in the headgroup decreases from 23 kJ mol-1 in the pure lipid bilayers to 20 kJ mol-1 for the protein-associated lipid headgroups. The chemical exchange rate of lipid molecules between the two motionally distinct environments has been estimated to be 20-50 Hz by steady-state line-shape simulations of the deuterium spectra of DMPC-d9/M13 coat protein complexes using exchange-coupled modified Bloch equations. The off-rate was, as expected from one-to-one exchange, independent of the L/P ratio; tau off -1 = 0.23 kHz. It is suggested that the protein-associated lipid may be trapped between closely packed parallel aggregates of M13 coat protein and that the high local concentration of protein in a one-dimensional arrangement in lipid bilayers may be required for the fast reassembly of phage particles before release from an infected cell.« less

Physical, Chemical and Biochemical Modifications of Protein-Based Films and Coatings: An Extensive Review

PubMed Central

Zink, Joël; Wyrobnik, Tom; Prinz, Tobias; Schmid, Markus

2016-01-01

Protein-based films and coatings are an interesting alternative to traditional petroleum-based materials. However, their mechanical and barrier properties need to be enhanced in order to match those of the latter. Physical, chemical, and biochemical methods can be used for this purpose. The aim of this article is to provide an overview of the effects of various treatments on whey, soy, and wheat gluten protein-based films and coatings. These three protein sources have been chosen since they are among the most abundantly used and are well described in the literature. Similar behavior might be expected for other protein sources. Most of the modifications are still not fully understood at a fundamental level, but all the methods discussed change the properties of the proteins and resulting products. Mastering these modifications is an important step towards the industrial implementation of protein-based films. PMID:27563881

Synthetic Spores Give Insight into the Real Thing and Reveal Functional Applications | Center for Cancer Research

Cancer.gov

Spores from bacteria, such as Bacillus subtilis, are produced to allow the bacterium’s genetic material to survive harsh environments. When the bacterium senses nutrient depletion, it divides asymmetrically into a forespore and a mother cell. The mother cell engulfs the forespore, and coat proteins synthesized by the mother cell localize to the surface of the forespore. The mother cell eventually ruptures, releasing the mature spore, which is surrounded by a thick shell of approximately 70 different proteins. This protein coat is one of the most durable static biological structures, but, because of its complexity, detailed studies of how the coat forms have been lacking. Kumaran Ramamurthi, Ph.D., of CCR’s Laboratory of Molecular Biology, and his colleagues including postdoctoral fellow and lead author of the study I-Lin Wu, Ph.D., decided to investigate the assembly of the basement layer of the spore coat by decorating spherical membranes supported by silica beads with SpoIVA and SpoVM, proteins which are known to be required for coat assembly.

Binding of mouse immunoglobulin G to polylysine-coated glass substrate for immunodiagnosis

NASA Astrophysics Data System (ADS)

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.

Vanilla mosaic virus isolates from French Polynesia and the Cook Islands are Dasheen mosaic virus strains that exclusively infect vanilla.

PubMed

Farreyrol, K; Pearson, M N; Grisoni, M; Cohen, D; Beck, D

2006-05-01

Sequence was determined for the coat protein (CP) gene and 3' non-translated region (3'NTR) of two vanilla mosaic virus (VanMV) isolates from Vanilla tahitensis, respectively from the Cook Islands (VanMV-CI) and French Polynesia (VanMV-FP). Both viruses displayed distinctive features in the N-terminal region of their CPs; for VanMV-CI, a 16-amino-acid deletion including the aphid transmission-related DAG motif, and for VanMV-FP, a stretch of GTN repeats that putatively belongs to the class of natively unfolded proteins. VanMV-FP CP also has a novel DVG motif in place of the DAG motif, and an uncommon Q//V protease cleavage site. The sequences were compared to a range of Dasheen mosaic virus (DsMV) strains and to potyviruses infecting orchids. Identity was low to DsMV strains across the entire CP coding region and across the 3'NTR, but high across the CP core and the CI-6K2-NIa region. In accordance with current ICTV criteria for species demarcation within the family Potyviridae, VanMV-CI and VanMV-FP are strains of DsMV that exclusively infect vanilla.

Deep sequencing reveals microbiota dysbiosis of tongue coat in patients with liver carcinoma.

PubMed

Lu, Haifeng; Ren, Zhigang; Li, Ang; Zhang, Hua; Jiang, Jianwen; Xu, Shaoyan; Luo, Qixia; Zhou, Kai; Sun, Xiaoli; Zheng, Shusen; Li, Lanjuan

2016-09-08

Liver carcinoma (LC) is a common malignancy worldwide, associated with high morbidity and mortality. Characterizing microbiome profiles of tongue coat may provide useful insights and potential diagnostic marker for LC patients. Herein, we are the first time to investigate tongue coat microbiome of LC patients with cirrhosis based on 16S ribosomal RNA (rRNA) gene sequencing. After strict inclusion and exclusion criteria, 35 early LC patients with cirrhosis and 25 matched healthy subjects were enrolled. Microbiome diversity of tongue coat in LC patients was significantly increased shown by Shannon, Simpson and Chao 1 indexes. Microbiome on tongue coat was significantly distinguished LC patients from healthy subjects by principal component analysis. Tongue coat microbial profiles represented 38 operational taxonomic units assigned to 23 different genera, distinguishing LC patients. Linear discriminant analysis (LDA) effect size (LEfSe) reveals significant microbial dysbiosis of tongue coats in LC patients. Strikingly, Oribacterium and Fusobacterium could distinguish LC patients from healthy subjects. LEfSe outputs show microbial gene functions related to categories of nickel/iron_transport, amino_acid_transport, energy produced system and metabolism between LC patients and healthy subjects. These findings firstly identify microbiota dysbiosis of tongue coat in LC patients, may providing novel and non-invasive potential diagnostic biomarker of LC.

Deep sequencing reveals microbiota dysbiosis of tongue coat in patients with liver carcinoma

NASA Astrophysics Data System (ADS)

Lu, Haifeng; Ren, Zhigang; Li, Ang; Zhang, Hua; Jiang, Jianwen; Xu, Shaoyan; Luo, Qixia; Zhou, Kai; Sun, Xiaoli; Zheng, Shusen; Li, Lanjuan

2016-09-01

Liver carcinoma (LC) is a common malignancy worldwide, associated with high morbidity and mortality. Characterizing microbiome profiles of tongue coat may provide useful insights and potential diagnostic marker for LC patients. Herein, we are the first time to investigate tongue coat microbiome of LC patients with cirrhosis based on 16S ribosomal RNA (rRNA) gene sequencing. After strict inclusion and exclusion criteria, 35 early LC patients with cirrhosis and 25 matched healthy subjects were enrolled. Microbiome diversity of tongue coat in LC patients was significantly increased shown by Shannon, Simpson and Chao 1 indexes. Microbiome on tongue coat was significantly distinguished LC patients from healthy subjects by principal component analysis. Tongue coat microbial profiles represented 38 operational taxonomic units assigned to 23 different genera, distinguishing LC patients. Linear discriminant analysis (LDA) effect size (LEfSe) reveals significant microbial dysbiosis of tongue coats in LC patients. Strikingly, Oribacterium and Fusobacterium could distinguish LC patients from healthy subjects. LEfSe outputs show microbial gene functions related to categories of nickel/iron_transport, amino_acid_transport, energy produced system and metabolism between LC patients and healthy subjects. These findings firstly identify microbiota dysbiosis of tongue coat in LC patients, may providing novel and non-invasive potential diagnostic biomarker of LC.

Nucleotide sequence of a resistance breaking mutant of southern bean mosaic virus.

PubMed

Lee, L; Anderson, E J

1998-01-01

SBMV-S is a resistance-breaking mutant of an Arkansas isolate of the bean strain of southern bean mosaic virus (SBMV-BARK) that is able to move systemically in Phaseolus vulgaris cvs. Pinto and Great Northern, whereas the wild-type SBMV-BARK causes local necrotic lesions and is restricted to the inoculated leaves of these hosts. Sequence analysis of the 4136 nucleotide genomes of SBMV-BARK and SBMV-S revealed seven nucleotide differences, but only four deduced amino acid changes. A single amino acid change occurred in the C-terminal region of the putative RNA-dependent RNA polymerase and three differences were identified in the N-terminal portion of the virus coat protein. SBMV-BARK and SBMV-S were compared with other sobemoviruses and were found to contain a high level of nucleotide sequence identity (91.3%) to SBMV-B. Unlike SBMV-B however, SBMV-BARK and SBMV-S contained four putative overlapping open reading frames, making them more similar in genome organization to the cowpea strain, SBMV-C. The possibility exists that mutations or even errors, that resulted in mis-identification of open reading frames, occurred in previously published information on nucleotide sequence and genomic organization for SBMV-B.

Co-expression of hepatitis C virus polytope-HBsAg and p19-silencing suppressor protein in tobacco leaves.

PubMed

Mohammadzadeh, Sara; Roohvand, Farzin; Memarnejadian, Arash; Jafari, Anis; Ajdary, Soheila; Salmanian, Ali-Hatef; Ehsani, Parastoo

2016-01-01

Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition. We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine. A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector. Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants. The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

Detection of Intermediates And Kinetic Control During Assembly of Bacteriophage P22 Procapsid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tuma, R.; Tsuruta, H.; French, K.H.

2009-05-26

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter {approx} 50 nm). In this work, we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibriummore » (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering, which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild-type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly.« less

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

PubMed Central

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

2012-01-01

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. PMID:23015764

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

PubMed

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.

Improving biocompatibility by surface modification techniques on implantable bioelectronics.

PubMed

Lin, Peter; Lin, Chii-Wann; Mansour, Raafat; Gu, Frank

2013-09-15

For implantable bioelectronic devices, the interface between the device and the biological environment requires significant attention as it dictates the device performance in vivo. Non-specific protein adsorption onto the device surface is the initial stage of many degradation mechanisms that will ultimately compromise the functionality of the device. In order to preserve the functionality of any implanted bioelectronics overtime, protein adsorption must be controlled. This review paper outlines two major approaches to minimize protein adsorption onto the surface of implantable electronics. The first approach is surface coating, which minimizes close proximity interactions between proteins and device surfaces by immobilizing electrically neutral hydrophilic polymers as surface coating. These coatings reduce protein fouling by steric repulsion and formation of a hydration layer which acts as both a physical and energetic barrier that minimize protein adsorption onto the device. Relevant performances of various conventional hydrophilic coatings are discussed. The second approach is surface patterning using arrays of hydrophobic nanostructures through photolithography techniques. By establishing a large slip length via super hydrophobic surfaces, the amount of proteins adsorbed to the surface of the device can be reduced. The last section discusses emerging surface coating techniques utilizing zwitterionic polymers where ultralow-biofouling surfaces have been demonstrated. These surface modification techniques may significantly improve the long-term functionality of implantable bioelectronics, thus allowing researchers to overcome challenges to diagnose and treat chronic neurological and cardiovascular diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Expanding the genetic code for site-specific labelling of tobacco mosaic virus coat protein and building biotin-functionalized virus-like particles.

PubMed

Wu, F C; Zhang, H; Zhou, Q; Wu, M; Ballard, Z; Tian, Y; Wang, J Y; Niu, Z W; Huang, Y

2014-04-18

A method for site-specific and high yield modification of tobacco mosaic virus coat protein (TMVCP) utilizing a genetic code expanding technology and copper free cycloaddition reaction has been established, and biotin-functionalized virus-like particles were built by the self-assembly of the protein monomers.

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

PubMed

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 transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis , indicating that this protein would not be a good target for inhibiting spore formation.

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

PubMed Central

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

2017-01-01

ABSTRACT 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 transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation. PMID:28959733

Genetic structure and evolution of natural populations of viruses causing the tomato yellow leaf curl disease in Spain.

PubMed

Font, María Isabel; Rubio, Luis; Martínez-Culebras, Pedro Vicente; Jordá, Concepción

2007-09-01

The population structure and genetic variation of two begomoviruses: tomato yellow leaf curl Sardinia virus (TYLCSV) and tomato yellow leaf curl virus (TYLCV) in tomato crops of Spain were studied from 1997 until 2001. Restriction digestion of a genomic region comprised of the CP coat protein gene (CPR) of 358 TYLC virus isolates enabled us to classify them into 14 haplotypes. Nucleotide sequences of two genomic regions: CPR, and the surrounding intergenic region (SIR) were determined for at least two isolates per haplotype. SIR was more variable than CPR and showed multiple recombination events whereas no recombination was detected within CPR. In all geographic regions except Murcia, the population was, or evolved to be composed of one predominant haplotype with a low genetic diversity (<0.0180). In Murcia, two successive changes of the predominant haplotype were observed in the best studied population. Phylogenetic analysis showed that the TYLCSV sequences determined clustered with sequences obtained from the GenBank of other TYLCSV Spanish isolates which were clearly separated from TYLCSV Italian isolates. Most of our TYLCV sequences were similar to those of isolates from Japan and Portugal, and the sequences obtained from TYLCV isolates from the Canary island of Lanzarote were similar to those of Caribbean TYLCV isolates.

Surfactant-free Colloidal Particles with Specific Binding Affinity

PubMed Central

2017-01-01

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles. PMID:28847149

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

PubMed Central

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 <≈100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end group conversions prior to macrogelation, consistent with the formation of highly 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

Advanced stent coating for drug delivery and in vivo biocompatibility

NASA Astrophysics Data System (ADS)

Liu, Yi; Wang, Wuchen; Acharya, Gayathri; Shim, Yoon-Bo; Choe, Eun Sang; Lee, Chi H.

2013-10-01

As an effort to alleviate stent-induced cardiovascular injury including restenosis and thrombosis, advanced drug-eluting stent (ADES) with a bilayer construct composed of a top-coat made of collagen and a base-coat incorporated with N-nitrosomelatonin (NOMela)-loaded PLGA nanoparticles has been developed. NOMela is a hydrophobic prodrug of nitric oxide (NO) that is an endogenous anti-platelet compound. ADES was coated with PLGA nanoparticles via either electrophoretic deposition (EPD) technique or dip-coating technique, and their coating characteristics and efficacies were compared. The drug-loading efficacy and in vitro drug-release profiles from ADES were expressed with various variables including the additives to the collagen layer, the number of layers of the collagen top-coat, the hydrophobicity/hydrophilicity of the loaded drug, the coating technique of nanoparticles, and the concentration of coating emulsions in the EPD method. The morphological status of cross-section and surface of ADES was evaluated by laser scanning confocal microscope and scanning electronic microscope. The real-time release profiles of NO were assessed using the NO-microbiosensor. The anti-platelet activity of ADES was evaluated on the rabbit whole blood using an aggregometer. The intima formation and protein expression in aorta were examined using an in vivo rat model. Both collagen and PLGA used in ADES are biodegradable polymers that fully degrade and consequently produce less inflammation responses. NO released from ADES significantly reduced platelet aggregation in the rabbit blood as compared with those exposed to the control stents. ADES coated with a double layer consisted of collagen and PLGA and containing NOMela was less antigenic at the implanted sites and alleviating intima formation and thrombosis. An external exposure of aorta to NO elicits distinct and specific effects on mitogen-activated protein kinase (MAPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) activities which evoke the endoplasmic reticulum (ER) stress response. These findings elucidated that coordinate and reciprocal alterations in the protein kinases followed by the ER stress protein expression are an integral feature of the in-stent-mediated cardiovascular injury.

Polyhedron-like inclusion body formation by a mutant nucleopolyhedrovirus expressing the granulin gene from a granulovirus.

PubMed

Zhou, C E; Ko, R; Maeda, S

1998-01-20

The polyhedrin gene in Bombyx mori nucleopolyhedrovirus (BmNPV) was replaced with the granulin gene of Trichoplusia ni granulovirus (TnGV). The substitution was verified by Southern hybridization, and expression of granulin by the mutant virus, BmGran, was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by amino acid sequencing of the predominant protein of BmGran inclusion bodies (IBs). Light and electron microscopy examination of BmGran-infected B. mori and BmN cells revealed large, cuboidal, polyhedron-like IBs in the nucleus and cytoplasm, but granules were not seen. IBs contained small, parallel, electron-dense streaks, which defined the geometric pattern of crystallization. Geometric patterns of nuclear IBs were frequently disrupted by occlusion of polyhedron envelope fragments, resulting in IB instability and fracturing. Virions were not embedded in most of the polyhedron-like IBs, but accumulated with polyhedron envelope fragments. Some virions were coated with matrix protein and were partially wrapped by polyhedron envelope. These results suggested that (1) the amino acid sequence of granulin insufficient for determining IB morphology in TnGV-infected cells, and TnGV may have genes, not present in BmNPV, that control granule formation, and (2) interactions among the virion, the IB envelope, and the matrix protein may be important in virion occlusion and IB morphology and stability.

COPA mutations impair ER-Golgi transport causing hereditary autoimmune-mediated lung disease and arthritis

PubMed Central

Watkin, Levi B.; Jessen, Birthe; Wiszniewski, Wojciech; Vece, Timothy; Jan, Max; Sha, Youbao; Thamsen, Maike; Santos-Cortez, Regie L. P.; Lee, Kwanghyuk; Gambin, Tomasz; Forbes, Lisa; Law, Christopher S.; Stray-Petersen, Asbjørg; Cheng, Mickie H.; Mace, Emily M.; Anderson, Mark S.; Liu, Dongfang; Tang, Ling Fung; Nicholas, Sarah K.; Nahmod, Karen; Makedonas, George; Canter, Debra; Kwok, Pui-Yan; Hicks, John; Jones, Kirk D.; Penney, Samantha; Jhangiani, Shalini N.; Rosenblum, Michael D.; Dell, Sharon D.; Waterfield, Michael R.; Papa, Feroz R.; Muzny, Donna M.; Zaitlen, Noah; Leal, Suzanne M.; Gonzaga-Jauregui, Claudia; Boerwinkle, Eric; Eissa, N. Tony; Gibbs, Richard A.; Lupski, James R.; Orange, Jordan S.; Shum, Anthony K.

2015-01-01

Advances in genomics have allowed unbiased genetic studies of human disease with unexpected insights into the molecular mechanisms of cellular immunity and autoimmunity1. We performed whole exome sequencing (WES) and targeted sequencing in patients with an apparent Mendelian syndrome of autoimmune disease characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease (ILD). In five families, we identified four unique deleterious variants in the Coatomer subunit alpha (COPA) gene all located within the same functional domain. We hypothesized that mutant COPA leads to a defect in intracellular transport mediated by coat protein complex I (COPI)2–4. We show that COPA variants impair binding of proteins targeted for retrograde Golgi to ER transport and demonstrate that expression of mutant COPA leads to ER stress and the upregulation of Th17 priming cytokines. Consistent with this pattern of cytokine expression, patients demonstrated a significant skewing of CD4+ T cells toward a T helper 17 (Th17) phenotype, an effector T cell population implicated in autoimmunity5,6. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease. These findings provide a unique opportunity to understand how alterations in cellular homeostasis caused by a defect in the intracellular trafficking pathway leads to the generation of human autoimmune disease. PMID:25894502

Bufo arenarum egg jelly coat: purification and characterization of two highly glycosylated proteins.

PubMed Central

Arranz, S E; Albertali, I E; Cabada, M O

1997-01-01

Egg jelly coats from Bufo arenarum are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they transit along the different oviductal portions. In this study, we have isolated two highly glycosylated proteins of the jelly coat, which are secreted almost all the way along the oviduct. Both glycoproteins [designated as highly glycosylated protein (HGP) and low-molecular-mass highly glycosylated protein (L-HGP)] were purified to homogeneity, from the secretion of the caudal oviduct portion, by CsCl density gradient ultracentrifugation. HGP is a high-molecular-mass protein with mucin-like characteristics: high viscosity, a high content of serine and threonine, about 70% carbohydrate by weight, and a protease-resistant domain. Cleavage of disulphide bridges with reducing agents resulted in the release of a single subunit (300000 Da). L-HGP is also a disulphide-cross-linked protein with lower apparent monomeric molecular mass, in the range 100-120 kDa and containing 50% carbohydrate by weight. HGP contains galactose, fucose, N-acetylgalactosamine and sialic acid, but no mannose, suggesting the presence of O-linked oligosaccharides exclusively. The secretion ratio of HGP increases from cephalic (16% of total protein in pars preconvoluta) to caudal (40% of total protein in pars convoluta) oviductal portions. It appears to be the major structural component of the jelly coat. Our purification data suggest that HGP is non-covalently linked to the other egg jelly proteins. Polyclonal antiserum to each purified glycoprotein from secretion was raised in rabbits and used to localize both glycoproteins in the different oviductal portions, total egg jelly and the aqueous medium where oocyte strings were incubated. HGP forms a stable fibre matrix around the oocyte. L-HGP is present in the jelly coat and is released into the incubation medium. PMID:9173897

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments

PubMed Central

Dodonova, Svetlana O; Aderhold, Patrick; Kopp, Juergen; Ganeva, Iva; Röhling, Simone; Hagen, Wim J H; Sinning, Irmgard; Wieland, Felix; Briggs, John A G

2017-01-01

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. DOI: http://dx.doi.org/10.7554/eLife.26691.001 PMID:28621666

Antimicrobial edible films and coatings for fresh and minimally processed fruits and vegetables: a review.

PubMed

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.

The characterization of edible coating from tilapia surimi as a biodegradable packaging

NASA Astrophysics Data System (ADS)

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.

Plant and yeast cornichon possess a conserved acidic motif required for correct targeting of plasma membrane cargos.

PubMed

Rosas-Santiago, Paul; Lagunas-Gomez, Daniel; Yáñez-Domínguez, Carolina; Vera-Estrella, Rosario; Zimmermannová, Olga; Sychrová, Hana; Pantoja, Omar

2017-10-01

The export of membrane proteins along the secretory pathway is initiated at the endoplasmic reticulum after proteins are folded and packaged inside this organelle by their recruiting into the coat complex COPII vesicles. It is proposed that cargo receptors are required for the correct transport of proteins to its target membrane, however, little is known about ER export signals for cargo receptors. Erv14/Cornichon belong to a well conserved protein family in Eukaryotes, and have been proposed to function as cargo receptors for many transmembrane proteins. Amino acid sequence alignment showed the presence of a conserved acidic motif in the C-terminal in homologues from plants and yeast. Here, we demonstrate that mutation of the C-terminal acidic motif from ScErv14 or OsCNIH1, did not alter the localization of these cargo receptors, however it modified the proper targeting of the plasma membrane transporters Nha1p, Pdr12p and Qdr2p. Our results suggest that mistargeting of these plasma membrane proteins is a consequence of a weaker interaction between the cargo receptor and cargo proteins caused by the mutation of the C-terminal acidic motif. Copyright © 2017 Elsevier B.V. All rights reserved.

A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants.

PubMed

Kelloniemi, Jani; Mäkinen, Kristiina; Valkonen, Jari P T

2006-05-01

Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.

Protein conformation and disease : pathological consequences of analogous mutations in homologous proteins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stevens, F. J.; Pokkuluri, P. R.; Schiffer, M.

2000-12-19

The antibody light chain variable domain (V{sub L}){sup 1} and myelin protein zero (MPZ) are representatives of the functionally diverse immunoglobulin superfamily. The V{sub L} is a subunit of the antigen-binding component of antibodies, while MPZ is the major membrane-linked constituent of the myelin sheaths that coat peripheral nerves. Despite limited amino acid sequence homology, the conformations of the core structures of the two proteins are largely superimposable. Amino acid variations in V{sub L} account for various conformational disease outcomes, including amyloidosis. However, the specific amino acid changes in V{sub L} that are responsible for disease have been obscured bymore » multiple concurrent primary structure alterations. Recently, certain demyelination disorders have been linked to point mutations and single amino acid polymorphisms in MPZ. We demonstrate here that some pathogenic variations in MPZ correspond to changes suspected of determining amyloidosis in V{sub L}. This unanticipated observation suggests that studies of the biophysical origin of conformational disease in one member of a superfamily of homologous proteins may have implications throughout the superfamily. In some cases, findings may account for overt disease; in other cases, due to the natural repertoire of inherited polymorphisms, variations in a representative protein may predict subclinical impairment of homologous proteins.« less

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage.

PubMed

Ploss, Martin; Kuhn, Andreas

2011-09-26

Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

PubMed Central

2011-01-01

Background Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies. PMID:21943062

Polyurethane/poly(vinyl alcohol) hydrogel coating improves the cytocompatibility of neural electrodes

PubMed Central

Li, Mei; Zhou, Hai-han; Li, Tao; Li, Cheng-yan; Xia, Zhong-yuan; Duan, Yanwen Y.

2015-01-01

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. PMID:26889197

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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.

In Vivo Coating of Bacterial Magnetic Nanoparticles by Magnetosome Expression of Spider Silk-Inspired Peptides.

PubMed

Mickoleit, Frank; Borkner, Christian B; Toro-Nahuelpan, Mauricio; Herold, Heike M; Maier, Denis S; Plitzko, Jürgen M; Scheibel, Thomas; Schüler, Dirk

2018-03-12

Magnetosomes are natural magnetic nanoparticles with exceptional properties that are synthesized in magnetotactic bacteria by a highly regulated biomineralization process. Their usability in many applications could be further improved by encapsulation in biocompatible polymers. In this study, we explored the production of spider silk-inspired peptides on magnetosomes of the alphaproteobacterium Magnetospirillum gryphiswaldense. Genetic fusion of different silk sequence-like variants to abundant magnetosome membrane proteins enhanced magnetite biomineralization and caused the formation of a proteinaceous capsule, which increased the colloidal stability of isolated particles. Furthermore, we show that spider silk peptides fused to a magnetosome membrane protein can be used as seeds for silk fibril growth on the magnetosome surface. In summary, we demonstrate that the combination of two different biogenic materials generates a genetically encoded hybrid composite with engineerable new properties and enhanced potential for various applications.

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

NASA Astrophysics Data System (ADS)

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.

Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

PubMed Central

Sterk, Maaike; Romilly, Cédric; Wagner, E Gerhart H

2018-01-01

Abstract Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5′-tails—as standby sites—with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats—and to a lesser extent, AU-repeats—dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16–18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem–loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails. PMID:29420821

Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

PubMed

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 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.

Polyglycerol based coatings to reduce non-specific protein adsorption in sample vials and on SPR sensors.

PubMed

Becherer, Tobias; Grunewald, Christian; Engelschalt, Vivienne; Multhaup, Gerhard; Risse, Thomas; Haag, Rainer

2015-03-31

Coatings based on dendritic polyglycerol (dPG) were investigated for their use to control nonspecific protein adsorption in an assay targeted to analyze concentrations of a specific protein. We demonstrate that coating of the sample vial with dPG can significantly increase the recovery of an antibody after incubation. First, we determine the concentration dependent loss of an antibody due to nonspecific adsorption to glass via quartz crystal microbalance (QCM). Complementary to the QCM measurements, we applied the same antibody as analyte in an surface plasmon resonance (SPR) assay to determine the loss of analyte due to nonspecific adsorption to the sample vial. For this purpose, we used two different coatings based on dPG. For the first coating, which served as a matrix for the SPR sensor, carboxyl groups were incorporated into dPG as well as a dithiolane moiety enabling covalent immobilization to the gold sensor surface. This SPR-matrix exhibited excellent protein resistant properties and allowed the immobilization of amyloid peptides via amide bond formation. The second coating which was intended to prevent nonspecific adsorption to glass vials comprised a silyl moiety that allowed covalent grafting to glass. For demonstrating the impact of the vial coating on the accuracy of an SPR assay, we immobilized amyloid beta (Aβ) 1-40 and used an anti-Aβ 1-40 antibody as analyte. Alternate injection of analyte into the flow cell of the SPR device from uncoated and coated vials, respectively gave us the relative signal loss (1-RUuncoated/RUcoated) caused by the nonspecific adsorption. We found that the relative signal loss increases with decreasing analyte concentration. The SPR data correlate well with concentration dependent non-specific adsorption experiments of the analyte to glass surfaces performed with QCM. Our measurements show that rendering both the sample vial and the sensor surface is crucial for accurate results in protein assays. Copyright © 2015 Elsevier B.V. All rights reserved.

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

PubMed

Xu, Yi; Ju, Ho-Jong; DeBlasio, Stacy; Carino, Elizabeth J; Johnson, Richard; MacCoss, Michael J; Heck, Michelle; Miller, W Allen; Gray, Stewart M

2018-06-01

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein. IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed. Copyright © 2018 American Society for Microbiology.

Backbone dynamics of a model membrane protein: assignment of the carbonyl carbon /sup 13/C NMR resonances in detergent-solubilized M13 coat protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

The major coat protein of the filamentous bacteriophage M13 is a 50-residue amphiphilic polypeptide which is inserted, as an integral membrane-spanning protein, in the inner membrane of the Escherichia coli host during infection. /sup 13/C was incorporated biosynthetically into a total of 23 of the peptide carbonyls using labeled amino acids (alanine, glycine, lysine, phenylalanine, and proline). The structure and dynamics of carbonyl-labeled M13 coat protein were monitored by /sup 13/C nuclear magnetic resonance (NMR) spectroscopy. Assignment of many resonances was achieved by using protease digestion, pH titration, or labeling of the peptide bond with both /sup 13/C and /supmore » 15/N. The carbonyl region of the natural-abundance /sup 13/C NMR spectrum of M13 coat protein in sodium dodecyl sulfate solution shows approximately eight backbone carbonyl resonances with line widths much narrower than the rest. Three of these more mobile residues correspond to assigned peaks (glycine-3, lysine-48, and alanine-49) in the individual amino acid spectra, and another almost certainly arises from glutamic acid-2. A ninth residue, alanine-1, also gives rise to a very narrow carbonyl resonance if the pH is well above or below the pK/sub a/ of the terminal amino group. These data suggest that only about four residues at either end of the protein experience large-amplitude spatial fluctuations; the rest of the molecule is essentially rigid on the time scale of the overall rotational tumbling of the protein-detergent complex. The relative exposure of different regions of detergent-bound protein was monitored by limited digestion with proteinase K. Comparable spectra and digestion patterns were obtained when the protein was solubilized in sodium deoxycholate, suggesting that the coat protein binds both amphiphiles in a similar fashion.« less

Mono vs multilayer fibronectin coatings on polar/hydrophobic/ionic polyurethanes: Altering surface interactions with human monocytes.

PubMed

Gossart, Audrey; Battiston, Kyle G; Gand, Adeline; Pauthe, Emmanuel; Santerre, J Paul

2018-01-15

Monocyte interactions with materials that are biofunctionalized with fibronectin (Fn) are of interest because of the documented literature which associates this protein with white blood cell function at implant sites. A degradable-polar hydrophobic ionic polyurethane (D-PHI), has been reported to promote an anti-inflammatory response from human monocytes. The aim of the current work was to study the influence of intrinsic D-PHI material chemistry on Fn adsorption (mono and multi-layer structures), and to investigate the influence of such chemistry on the structural state of the Fn, as well as the latter's influence on the activity of human monocytes on the protein coated substrates. Significant differences in Fn adsorption, surface hydrophobicity and the availability of defined peptide sequences (N terminal, C terminal or Cell Binding Domain) for the Fn in mono vs multilayer structures were observed as a function of the changes in intrinsic material chemistry. A D-PHI-formulated polyurethane substrate with subtle changes in anionic and hydrophobic domain content relative to the polar non-ionic urethane/carbonate groups within the polymer matrix promoted the lowest activation of monocytes, in the presence of multi-layer Fn constructs. These results highlight the importance of chemical heterogeneity as a design parameter for biomaterial surfaces, and establishes a desired strategy for controlling human monocyte activity at the surface of devices, when these are coated with multi-layer Fn structures. The latter is an important step towards functionalizing the materials with multi-layer protein drug carriers as interventional therapeutic agents. The control of the behavior of monocytes, especially migration and activation, is of crucial interest to modulate the inflammatory response at the site of implanted biomaterial. Several studies report the influence of adsorbed serum proteins on the behavior of monocytes on biomaterials. However, few studies show the influence of surface chemical group distribution on the controlled adsorption and the subsequent induced conformation- of mono versus multi-layer assembled structures generated from specific proteins implicated in wound repair. The current research considered the role of Fn adsorption and conformation in thin films while interacting with the intrinsic chemistry of segmented block polyurethanes; and the influence of the former on modulation and activation of human monocytes. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Silica-gelatin hybrid sol-gel coatings: a proteomic study with biocompatibility implications.

PubMed

Araújo-Gomes, N; Romero-Gavilán, F; Lara-Sáez, I; Elortza, F; Azkargorta, M; Iloro, I; Martínez-Ibañez, M; Martín de Llano, J J; Gurruchaga, M; Goñi, I; Suay, J; Sánchez-Pérez, A M

2018-05-21

Osseointegration, including the foreign body reaction to biomaterials, is an immune-modulated, multifactorial, and complex healing process in which various cells and mediators are involved. The buildup of the osseointegration process is immunological and inflammation-driven, often triggered by the adsorption of proteins on the surfaces of the biomaterials and complement activation. New strategies for improving osseointegration use coatings as vehicles for osteogenic biomolecules delivery from implants. Natural polymers, such as gelatin, can mimic collagen I and enhance the biocompatibility of a material. In this experimental study, two different base sol-gel formulations and their combination with gelatin, were applied as coatings on sandblasted, acid-etched titanium (SAE-Ti) substrates and their biological potential as osteogenic biomaterials was tested. We examined the proteins adsorbed onto each surface and their in vitro and in vivo effects. In vitro results showed an improvement in cell proliferation and mineralization in gelatin-containing samples. In vivo testing showed the presence of a looser connective tissue layer in those coatings with substantially more complement activation proteins adsorbed, especially those containing gelatin. Vitronectin and FETUA, proteins associated with mineralization process, were significantly more adsorbed in gelatin coatings. This article is protected by copyright. All rights reserved.

Trypsin coatings on electrospun and alcohol-dispersed polymer nanofibers for trypsin digestion column

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jun, Seung-Hyun; Chang, Mun Seock; Kim, Byoung Chan

2010-09-15

The construction of a trypsin reactor in a chromatography column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coating, which was prepared by a two-step process of covalent attachment and enzyme crosslinking. In a comparative study with the trypsin coatings on asspun and non-dispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than twenty minutes. By applying themore » alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the enzymes were improved yielding more complete and reproducible digestions. Regardless of alcohol-dispersion or not, trypsin coating showed better digestion performance and improved performance stability under recycled uses than covalently-attached trypsin. The combination of highly stable trypsin coating and alcoholdispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for on-line and automated protein digestion.« less

Comprehensive profiling of lysine acetylation suggests the widespread function is regulated by protein acetylation in the silkworm, Bombyx mori.

PubMed

Nie, Zuoming; Zhu, Honglin; Zhou, Yong; Wu, Chengcheng; Liu, Yue; Sheng, Qing; Lv, Zhengbing; Zhang, Wenping; Yu, Wei; Jiang, Caiying; Xie, Longfei; Zhang, Yaozhou; Yao, Juming

2015-09-01

Lysine acetylation in proteins is a dynamic and reversible PTM and plays an important role in diverse cellular processes. In this study, using lysine-acetylation (Kac) peptide enrichment coupled with nano HPLC/MS/MS, we initially identified the acetylome in the silkworms. Overall, a total of 342 acetylated proteins with 667 Kac sites were identified in silkworm. Sequence motifs analysis around Kac sites revealed an enrichment of Y, F, and H in the +1 position, and F was also enriched in the +2 and -2 positions, indicating the presences of preferred amino acids around Kac sites in the silkworm. Functional analysis showed the acetylated proteins were primarily involved in some specific biological processes. Furthermore, lots of nutrient-storage proteins, such as apolipophorin, vitellogenin, storage proteins, and 30 K proteins, were highly acetylated, indicating lysine acetylation may represent a common regulatory mechanism of nutrient utilization in the silkworm. Interestingly, Ser2 proteins, the coating proteins of larval silk, were found to contain many Kac sites, suggesting lysine acetylation may be involved in the regulation of larval silk synthesis. This study is the first to identify the acetylome in a lepidoptera insect, and expands greatly the catalog of lysine acetylation substrates and sites in insects. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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.

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

PubMed

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

2018-04-15

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, Cd 2+ , Pb 2+ , Zn 2+ and Ni 2+ 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. Copyright © 2018 Elsevier B.V. All rights reserved.

Clathrin-independent pathways do not contribute significantly to endocytic flux

PubMed Central

Bitsikas, Vassilis; Corrêa, Ivan R; Nichols, Benjamin J

2014-01-01

Several different endocytic pathways have been proposed to function in mammalian cells. Clathrin-coated pits are well defined, but the identity, mechanism and function of alternative pathways have been controversial. Here we apply universal chemical labelling of plasma membrane proteins to define all primary endocytic vesicles, and labelling of specific proteins with a reducible SNAP-tag substrate. These approaches provide high temporal resolution and stringent discrimination between surface-connected and intracellular membranes. We find that at least 95% of the earliest detectable endocytic vesicles arise from clathrin-coated pits. GPI-anchored proteins, candidate cargoes for alternate pathways, are also found to enter the cell predominantly via coated pits. Experiments employing a mutated clathrin adaptor reveal distinct mechanisms for sorting into coated pits, and thereby explain differential effects on the uptake of transferrin and GPI-anchored proteins. These data call for a revision of models for the activity and diversity of endocytic pathways in mammalian cells. DOI: http://dx.doi.org/10.7554/eLife.03970.001 PMID:25232658

The use of phage display in neurobiology.

PubMed

Bradbury, Andrew R M

2010-04-01

Phage display has been extensively used to study protein-protein interactions, receptor- and antibody-binding sites, and immune responses, to modify protein properties, and to select antibodies against a wide range of different antigens. In the format most often used, a polypeptide is displayed on the surface of a filamentous phage by genetic fusion to one of the coat proteins, creating a chimeric coat protein, and coupling phenotype (the protein) to genotype (the gene within). As the gene encoding the chimeric coat protein is packaged within the phage, selection of the phage on the basis of the binding properties of the polypeptide displayed on the surface simultaneously results in the isolation of the gene encoding the polypeptide. This unit describes the background to the technique, and illustrates how it has been applied to a number of different problems, each of which has its neurobiological counterparts. Although this overview concentrates on the use of filamentous phage, which is the most popular platform, other systems are also described. (c) 2010 by John Wiley & Sons, Inc.

Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection.

PubMed

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.

Poly-2-methoxyethylacrylate-coated cardiopulmonary bypass circuit can reduce transfusion of platelet products compared to heparin-coated circuit during aortic arch surgery.

PubMed

Hosoyama, Katsuhiro; Ito, Koki; Kawamoto, Shunsuke; Kumagai, Kiichiro; Akiyama, Masatoshi; Adachi, Osamu; Kawatsu, Satoshi; Sasaki, Konosuke; Suzuki, Marina; Sugawara, Yumi; Shimizu, Yuya; Saiki, Yoshikatsu

2016-09-01

Several coating techniques for extracorporeal circulation have been developed to reduce the systemic inflammatory response during cardiopulmonary bypass (CPB). We compared the clinical effectiveness and biocompatibility of poly-2-methoxyethylacrylate (PMEA)- and heparin-coated CPB circuits in total aortic arch replacement (TAR) with the prolonged use of the bypass technique. Twenty patients who underwent elective TAR were divided randomly into two equal groups: group P (n = 10) to use PMEA-coated circuits and group H (n = 10) to use heparin-coated circuits. Clinical outcomes, hematological variables, and acute phase inflammatory response were analyzed perioperatively. Demographic, CPB, and clinical outcome data were similar for both groups. Hemoglobin and platelet count showed similar time-course curves. However, the amount of platelet products transfused intraoperatively was significantly larger in group H (group P 26.0 ± 7.0 units; group H 33.0 ± 6.7 units, p = 0.04). Total protein, and albumin levels were significantly higher in group P during and after the operation (total protein, p = 0.04; albumin, p = 0.02). The use of PMEA-coated circuit is associated with retainment of perioperative plasma proteins levels and may help to reduce transfusion of platelet products in TAR in comparison with the heparin-coated circuit.

Unfolding and inactivation of proteins by counterions in protein-nanoparticles interaction.

PubMed

Ghosh, Goutam; Gaikwad, Pallavi S; Panicker, Lata; Nath, Bimalendu B; Mukhopadhyaya, Rita

2016-09-01

In this work, the structure and activity of proteins; such as, hen egg lysozyme (HEWL) and calf intestine alkaline phosphatase (CIAP); have been investigated after incubation with surface coated iron oxide nanoparticles (IONPs) in water. IONPs were coated with counterions bound charge-ligands and were named as the charge-ligand counterions iron oxide nanoparticles (CLC-IONPs). The coating was done with tri-lithium citrate (TLC) and tri-potassium citrate (TKC) to have negative surface charge of CLC-IONPs and Li(+) and K(+), respectively, as counterions. To have positive surface charge, IONPs were coated with cetylpyridinium chloride (CPC) and cetylpyridinium iodide (CPI) having Cl(-) and I(-), respectively, as counterions. The secondary structure of proteins was measured using far ultraviolet circular dichroism (CD) spectroscopy which showed that both proteins were irreversibly unfolded after incubation with CLC-IONPs. The unfolded proteins were seen to be functionally inactive, as confirmed through their activity assays, i.e., HEWL with Escherichia coli (E. coli) and CIAP with para-nitrophenyl phosphate (pNPP). Additionally, we have observed that monomeric hemoglobin (Hb) from radio-resistant insect Chironomus ramosus (ChHb) was also partially unfolded upon interaction with CLC-IONPs. This work clearly shows the role of counterions in protein inactivation via protein-nanoparticles interaction and, therefore, CLC-IONPs could be used for therapeutic purpose. Copyright © 2016 Elsevier B.V. All rights reserved.

Interaction of S-layer proteins of Lactobacillus kefir with model membranes and cells.

PubMed

Hollmann, Axel; Delfederico, Lucrecia; Santos, Nuno C; Disalvo, E Anibal; Semorile, Liliana

2018-06-01

In previous works, it was shown that S-layer proteins from Lactobacillus kefir were able to recrystallize and stabilize liposomes, this feature reveling a great potential for developing liposomal-based carriers. Despite previous studies on this subject are important milestones, a number of questions remain unanswered. In this context, the feasibility of S-layer proteins as a biomaterial for drug delivery was evaluated in this work. First, S-layer proteins were fully characterized by electron microscopy, 2D-electrophoresis, and anionic exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Afterward, interactions of S-layer proteins with model lipid membranes were evaluated, showing that proteins adsorb to the lipid surface following a non-fickean or anomalous diffusion, when positively charged lipid were employed, suggesting that electrostatic interaction is a key factor in the recrystallization process on these proteins. Finally, the interaction of S-layer coated liposomes with Caco-2 cell line was assessed: First, cytotoxicity of formulations was tested showing no cytotoxic effects in S-layer coated vesicles. Second, by flow cytometry, it was observed an increased ability to transfer cargo molecules into Caco-2 cells from S-layer coated liposomes in comparison to control ones. All data put together, supports the idea that a combination of adhesive properties of S-layer proteins concomitant with higher stability of S-layer coated liposomes represents an exciting starting point in the development of new drug carriers.

Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

PubMed

Rahman, K Shamsur; Chowdhury, Erfan U; Poudel, Anil; Ruettger, Anke; Sachse, Konrad; Kaltenboeck, Bernhard

2015-05-01

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Defining Species-Specific Immunodominant B Cell Epitopes for Molecular Serology of Chlamydia Species

PubMed Central

Rahman, K. Shamsur; Chowdhury, Erfan U.; Poudel, Anil; Ruettger, Anke; Sachse, Konrad

2015-01-01

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. PMID:25761461

Polymer brush coatings for DNA: fundamental polymer physics and nanofabrication applications

NASA Astrophysics Data System (ADS)

de Vries, Renko

Recombinant DNA technology allows for the production of precisely defined self-assembling protein-based polymers. So far, the major applications for such protein-based polymers have been self-assembling hydrogels and micellar structures with biomedical application. Inspired by minimal models for the self-ssembly of rod-shaped viruses such as the tobacco mosaic virus, I have developed protein-polymers that co-assemble with DNA into rod-shaped virus-like particles, and protein-polymers that provide brush coatings around single DNA molecules. In this presentation I will focus on the latter, showing that on the one hand brush coated DNA is a rich model system for exploring the physics of bottle-brush polymers, while on the other hand brush coatings of DNA can also play an important practical role in nanofabrication. A key problem in the physics of bottle-brush polymers that I will address is the scale-dependence of bottle-brush elasticity. For long-wavelength thermal deformations probed by AFM imaging I will demonstrate that there is significant stiffening due to the brush coating, while for short wavelength thermal deformations probed by force spectroscopy, we find that stiffening due to the brush coating disappears completely. DNA brush coatings can also play an important practical role in nanofabrication by acting as a compatibilizer between chemically different building blocks. I will explore the example of DNA origami in combination with gold nanoparticles: while Mg2+ ions and high concentrations of monovalent salts are crucial for the stability of DNA origami, such solution conditions are typically incompatible with the colloidal stability of gold nanoparticles.I will show how DNA brush coatings can dramatically enhance the yield of formation of isolated DNA-gold nanoparticle composite nanostructures.

Genome characterization of sugarcane yellow leaf virus from China reveals a novel recombinant genotype.

PubMed

Lin, Yi-Hua; Gao, San-Ji; Damaj, Mona B; Fu, Hua-Ying; Chen, Ru-Kai; Mirkov, T Erik

2014-06-01

Sugarcane yellow leaf virus (SCYLV; genus Polerovirus, family Luteoviridae) is a recombinant virus associated with yellow leaf disease, a serious threat to sugarcane in China and worldwide. Among the nine known SCYLV genotypes existing worldwide, COL, HAW, REU, IND, CHN1, CHN2, BRA, CUB and PER, the last five have been reported in China. In this study, the complete genome sequences (5,880 nt) of GZ-GZ18 and HN-CP502 isolates from the Chinese provinces of Guizhou and Hainan, respectively, were cloned, sequenced and characterized. Phylogenetic analysis showed that, among 29 SCYLV isolates described worldwide, the two Chinese isolates clustered together into an independent clade based on the near-complete genome nucleotide (ORF0-ORF5) or amino acid sequences of individual genes, except for the MP protein (ORF4). We propose that the two isolates represent a novel genotype, CHN3, diverging from other genotypes by 1.7-13.6 % nucleotide differences in ORF0-ORF5, and 2.7-28.1 %, 1.8-20.4 %, 0.5-5.1 % and 2.7-15.9 % amino acid differences in P0 (ORF0), RdRp (RNA-dependent RNA polymerase) (ORF1+2), CP (coat protein) (ORF3) and RT (readthrough protein) (ORF3+5), respectively. CHN3 was closely related to the BRA, HAW and PER genotypes, differing by 1.7-3.8 % in the near-complete genome nucleotide sequence. Recombination analysis further identified CHN3 as a new recombinant strain, arising from the major parent CHN-HN1 and the minor parent CHN-GD-WY19. Recombination breakpoints were distributed mostly within the RdRp region in CHN3 and the four significant recombinant genotypes, IND, REU, CUB and BRA. Recombination is considered to contribute significantly to the evolution and emergence of such new SCYLV variants.

Mutant CCL2 Protein Coating Mitigates Wear Particle-Induced Bone Loss in a Murine Continuous Polyethylene Infusion Model

PubMed Central

Nabeshima, Akira; Pajarinen, Jukka; Lin, Tzu-hua; Jiang, Xinyi; Gibon, Emmanuel; Córdova, Luis A.; Loi, Florence; Lu, Laura; Jämsen, Eemeli; Egashira, Kensuke; Yang, Fan; Yao, Zhenyu; Goodman, Stuart B

2016-01-01

Wear particle-induced osteolysis limits the long-term survivorship of total joint replacement (TJR). Monocyte/macrophages are the key cells of this adverse reaction. Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) is the most important chemokine regulating trafficking of monocyte/macrophages in particle-induced inflammation. 7ND recombinant protein is a mutant of CCL2 that inhibits CCL2 signaling. We have recently developed a layer-by-layer (LBL) coating platform on implant surfaces that can release biologically active 7ND. In this study, we investigated the effect of 7ND on wear particle-induced bone loss using the murine continuous polyethylene (PE) particle infusion model with 7ND coating of a titanium rod as a local drug delivery device. PE particles were infused into hollow titanium rods with or without 7ND coating implanted in the distal femur for 4 weeks. Specific groups were also injected with RAW 264.7 as the reporter macrophages. Wear particle-induced bone loss and the effects of 7ND were evaluated by microCT, immunohistochemical staining, and bioluminescence imaging. Local delivery of 7ND using the LBL coating decreased systemic macrophage recruitment, the number of osteoclasts and wear particle-induced bone loss. The development of a novel orthopaedic implant coating with anti-CCL2 protein may be a promising strategy to mitigate peri-prosthetic osteolysis. PMID:27918885

A Novel Antimicrobial Coating Represses Biofilm and Virulence-Related Genes in Methicillin-Resistant Staphylococcus aureus

PubMed Central

Vaishampayan, Ankita; de Jong, Anne; Wight, Darren J.; Kok, Jan; Grohmann, Elisabeth

2018-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important cause of hospital-acquired infections worldwide. It is one of the most threatening pathogens due to its multi-drug resistance and strong biofilm-forming capacity. Thus, there is an urgent need for novel alternative strategies to combat bacterial infections. Recently, we demonstrated that a novel antimicrobial surface coating, AGXX®, consisting of micro-galvanic elements of the two noble metals, silver and ruthenium, surface-conditioned with ascorbic acid, efficiently inhibits MRSA growth. In this study, we demonstrated that the antimicrobial coating caused a significant reduction in biofilm formation (46%) of the clinical MRSA isolate, S. aureus 04-02981. To understand the molecular mechanism of the antimicrobial coating, we exposed S. aureus 04-02981 for different time-periods to the coating and investigated its molecular response via next-generation RNA-sequencing. A conventional antimicrobial silver coating served as a control. RNA-sequencing demonstrated down-regulation of many biofilm-associated genes and of genes related to virulence of S. aureus. The antimicrobial substance also down-regulated the two-component quorum-sensing system agr suggesting that it might interfere with quorum-sensing while diminishing biofilm formation in S. aureus 04-02981. PMID:29497410

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

NASA Astrophysics Data System (ADS)

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.

Variability and transmission by Aphis glycines of North American and Asian Soybean mosaic virus isolates.

PubMed

Domier, L L; Latorre, I J; Steinlage, T A; McCoppin, N; Hartman, G L

2003-10-01

The variability of North American and Asian strains and isolates of Soybean mosaic virus was investigated. First, polymerase chain reaction (PCR) products representing the coat protein (CP)-coding regions of 38 SMVs were analyzed for restriction fragment length polymorphisms (RFLP). Second, the nucleotide and predicted amino acid sequence variability of the P1-coding region of 18 SMVs and the helper component/protease (HC/Pro) and CP-coding regions of 25 SMVs were assessed. The CP nucleotide and predicted amino acid sequences were the most similar and predicted phylogenetic relationships similar to those obtained from RFLP analysis. Neither RFLP nor sequence analyses of the CP-coding regions grouped the SMVs by geographical origin. The P1 and HC/Pro sequences were more variable and separated the North American and Asian SMV isolates into two groups similar to previously reported differences in pathogenic diversity of the two sets of SMV isolates. The P1 region was the most informative of the three regions analyzed. To assess the biological relevance of the sequence differences in the HC/Pro and CP coding regions, the transmissibility of 14 SMV isolates by Aphis glycines was tested. All field isolates of SMV were transmitted efficiently by A. glycines, but the laboratory isolates analyzed were transmitted poorly. The amino acid sequences from most, but not all, of the poorly transmitted isolates contained mutations in the aphid transmission-associated DAG and/or KLSC amino acid sequence motifs of CP and HC/Pro, respectively.

A protocol for the production of gliadin-cyanoacrylate nanoparticles for hydrophilic coating

USDA-ARS?s Scientific Manuscript database

This article presents a protocol for the production of protein-based nanoparticles that change the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules...

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

PubMed

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.

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry.

PubMed

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.

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry

PubMed Central

2017-01-01

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. PMID:29064669

Evaluation of antimicrobial edible coatings from a whey protein isolate base to improve the shelf life of cheese.

PubMed

Ramos, Ó L; Pereira, J O; Silva, S I; Fernandes, J C; Franco, M I; Lopes-da-Silva, J A; Pintado, M E; Malcata, F X

2012-11-01

The objective of this work was to evaluate the effectiveness of antimicrobial edible coatings to wrap cheeses, throughout 60 d of storage, as an alternative to commercial nonedible coatings. Coatings were prepared using whey protein isolate, glycerol, guar gum, sunflower oil, and Tween 20 as a base matrix, together with several combinations of antimicrobial compounds-natamycin and lactic acid, natamycin and chitooligosaccharides (COS), and natamycin, lactic acid, and COS. Application of coating on cheese decreased water loss (~10%, wt/wt), hardness, and color change; however, salt and fat contents were not significantly affected. Moreover, the antimicrobial edible coatings did not permit growth of pathogenic or contaminant microorganisms, while allowing regular growth of lactic acid bacteria throughout storage. Commercial nonedible coatings inhibited only yeasts and molds. The antimicrobial edible coating containing natamycin and lactic acid was the best in sensory terms. Because these antimicrobial coatings are manufactured from food-grade materials, they can be consumed as an integral part of cheese, which represents a competitive advantage over nonedible coatings. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Antimicrobial paper based on a soy protein isolate or modified starch coating including carvacrol and cinnamaldehyde.

PubMed

Arfa, Afef Ben; Preziosi-Belloy, Laurence; Chalier, Pascale; Gontard, Nathalie

2007-03-21

Soy protein isolates (SPI) and octenyl-succinate (OSA) modified starch were used as paper coating and inclusion matrices of two antimicrobial compounds: cinnamaldehyde and carvacrol. Antimicrobial compound losses from the coated papers were evaluated after the coating and drying process, and the two matrices demonstrated retention ability that depended on the compound nature and concentration. Whereas carvacrol losses ranged between 12 and 45%, cinnamaldehyde losses varied from 43 to 76%. The losses were always higher from OSA-starch-coated papers than from SPI-coated papers. During storage in accelerated conditions, at 30 degrees C and 60% relative humidity, carvacrol retention from coated papers was found to be similar whatever the coating matrices and the carvacrol rate. In contrast, the retention from SPI-coated papers was particularly high for the cinnamaldehyde concentration of 30% (w/w) compared to the lowest (10% w/w) or highest concentration (60% w/w). Compared to carvacrol, faster release was observed, particurlarly when OSA-starch was used. The antimicrobial properties of the coated papers were shown against Escherichia coli and Botrytis cinerea and explained by favorable conditions of total release of the antimicrobial agents.

Folding and trimerization of clathrin subunits at the triskelion hub.

PubMed

Näthke, I S; Heuser, J; Lupas, A; Stock, J; Turck, C W; Brodsky, F M

1992-03-06

The triskelion shape of the clathrin molecule enables it to form the polyhedral protein network that covers clathrin-coated pits and vesicles. Domains within the clathrin heavy chain that are responsible for maintaining triskelion shape and function were identified and localized. Sequences that mediate trimerization are distal to the carboxyl terminus and are adjacent to a domain that mediates both light chain binding and clathrin assembly. Structural modeling predicts that within this domain, the region of heavy chain-light chain interaction is a bundle of three or four alpha helices. These studies establish a low resolution model of clathrin subunit folding in the central portion (hub) of the triskelion, thus providing a basis for future mutagenesis experiments.

Identification of small secreted peptides (SSPs) in maize and expression analysis of partial SSP genes in reproductive tissues.

PubMed

Li, Ye Long; Dai, Xin Ren; Yue, Xun; Gao, Xin-Qi; Zhang, Xian Sheng

2014-10-01

Maize 1,491 small secreted peptides were identified, which were classified according to the character of peptide sequences. Partial SSP gene expressions in reproductive tissues were determined by qRT-PCR. Small secreted peptides (SSPs) are important cell-cell communication messengers in plants. Most information on plant SSPs come from Arabidopsis thaliana and Oryza sativa, while little is known about the SSPs of other grass species such as maize (Zea mays). In this study, we identified 1,491 SSP genes from maize genomic sequences. These putative SSP genes were distributed throughout the ten maize chromosomes. Among them, 611 SSPs were classified into 198 superfamilies according to their conserved domains, and 725 SSPs with four or more cysteines at their C-termini shared similar cysteine arrangements with their counterparts in other plant species. Moreover, the SSPs requiring post-translational modification, as well as defensin-like (DEFL) proteins, were identified. Further, the expression levels of 110 SSP genes were analyzed in reproductive tissues, including male flower, pollen, silk, and ovary. Most of the genes encoding basal-layer antifungal peptide-like, small coat proteins-like, thioredoxin-like proteins, γ-thionins-like, and DEFL proteins showed high expression levels in the ovary and male flower compared with their levels in silk and mature pollen. The rapid alkalinization factor-like genes were highly expressed only in the mature ovary and mature pollen, and pollen Ole e 1-like genes showed low expression in silk. The results of this study provide basic information for further analysis of SSP functions in the reproductive process of maize.

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

PubMed

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.

Analysis of the epitope structure of Plum pox virus coat protein.

PubMed

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.

The prehistory of potyviruses: their initial radiation was during the dawn of agriculture.

PubMed

Gibbs, Adrian J; Ohshima, Kazusato; Phillips, Matthew J; Gibbs, Mark J

2008-06-25

Potyviruses are found world wide, are spread by probing aphids and cause considerable crop damage. Potyvirus is one of the two largest plant virus genera and contains about 15% of all named plant virus species. When and why did the potyviruses become so numerous? Here we answer the first question and discuss the other. We have inferred the phylogenies of the partial coat protein gene sequences of about 50 potyviruses, and studied in detail the phylogenies of some using various methods and evolutionary models. Their phylogenies have been calibrated using historical isolation and outbreak events: the plum pox virus epidemic which swept through Europe in the 20th century, incursions of potyviruses into Australia after agriculture was established by European colonists, the likely transport of cowpea aphid-borne mosaic virus in cowpea seed from Africa to the Americas with the 16th century slave trade and the similar transport of papaya ringspot virus from India to the Americas. Our studies indicate that the partial coat protein genes of potyviruses have an evolutionary rate of about 1.15x10(-4) nucleotide substitutions/site/year, and the initial radiation of the potyviruses occurred only about 6,600 years ago, and hence coincided with the dawn of agriculture. We discuss the ways in which agriculture may have triggered the prehistoric emergence of potyviruses and fostered their speciation.

The Prehistory of Potyviruses: Their Initial Radiation Was during the Dawn of Agriculture

PubMed Central

Gibbs, Adrian J.; Ohshima, Kazusato; Phillips, Matthew J.; Gibbs, Mark J.

2008-01-01

Background Potyviruses are found world wide, are spread by probing aphids and cause considerable crop damage. Potyvirus is one of the two largest plant virus genera and contains about 15% of all named plant virus species. When and why did the potyviruses become so numerous? Here we answer the first question and discuss the other. Methods and Findings We have inferred the phylogenies of the partial coat protein gene sequences of about 50 potyviruses, and studied in detail the phylogenies of some using various methods and evolutionary models. Their phylogenies have been calibrated using historical isolation and outbreak events: the plum pox virus epidemic which swept through Europe in the 20th century, incursions of potyviruses into Australia after agriculture was established by European colonists, the likely transport of cowpea aphid-borne mosaic virus in cowpea seed from Africa to the Americas with the 16th century slave trade and the similar transport of papaya ringspot virus from India to the Americas. Conclusions/Significance Our studies indicate that the partial coat protein genes of potyviruses have an evolutionary rate of about 1.15×10−4 nucleotide substitutions/site/year, and the initial radiation of the potyviruses occurred only about 6,600 years ago, and hence coincided with the dawn of agriculture. We discuss the ways in which agriculture may have triggered the prehistoric emergence of potyviruses and fostered their speciation. PMID:18575612

Interplay between grain structure and protein adsorption on functional response of osteoblasts: ultrafine-grained versus coarse-grained substrates.

PubMed

Misra, R D K; Nune, C; Pesacreta, T C; Somani, M C; Karjalainen, L P

2013-01-01

The rapid adsorption of proteins is the starting and primary biological response that occurs when a biomedical device is implanted in the physiological system. The biological response, however, depends on the surface characteristics of the device. Considering the significant interest in nano-/ultrafine surfaces and nanostructured coatings, we describe here, the interplay between grain structure and protein adsorption (bovine serum albumin: BSA) on osteoblasts functions by comparing nanograined/ultrafine-grained (NG/UFG) and coarse-grained (CG: grain size in the micrometer range) substrates by investigating cell-substrate interactions. The protein adsorption on NG/UFG surface was beneficial in favorably modulating biological functions including cell attachment, proliferation, and viability, whereas the effect was less pronounced on protein adsorbed CG surface. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on protein adsorbed NG/UFG surface. The functional response followed the sequence: NG/UFG(BSA) > NG/UFG > CG(BSA) > CG. The differences in the cellular response on bare and protein adsorbed NG/UFG and CG surfaces are attributed to cumulative contribution of grain structure and degree of hydrophilicity. The study underscores the potential advantages of protein adsorption on artificial biomedical devices to enhance the bioactivity and regulate biological functions. Copyright © 2012 Wiley Periodicals, Inc.

Specific trans-acting proteins interact with auxiliary RNA polyadenylation elements in the COX-2 3′-UTR

PubMed Central

Hall-Pogar, Tyra; Liang, Songchun; Hague, Lisa K.; Lutz, Carol S.

2007-01-01

Two cyclooxygenase (COX) enzymes, COX-1 and COX-2, are present in human cells. While COX-1 is constitutively expressed, COX-2 is inducible and up-regulated in response to many signals. Since increased transcriptional activity accounts for only part of COX-2 up-regulation, we chose to explore other RNA processing mechanisms in the regulation of this gene. Previously, we showed that COX-2 is regulated by alternative polyadenylation, and that the COX-2 proximal polyadenylation signal contains auxiliary upstream sequence elements (USEs) that are very important in efficient polyadenylation. To explore trans-acting protein factors interacting with these cis-acting RNA elements, we performed pull-down assays with HeLa nuclear extract and biotinylated RNA oligonucleotides representing COX-2 USEs. We identified PSF, p54nrb, PTB, and U1A as proteins specifically bound to the COX-2 USEs. We further explored their participation in polyadenylation using MS2 phage coat protein-MS2 RNA binding site tethering assays, and found that tethering any of these four proteins to the COX-2 USE mutant RNA can compensate for these cis-acting elements. Finally, we suggest that these proteins (p54nrb, PTB, PSF, and U1A) may interact as a complex since immunoprecipitations of the transfected MS2 fusion proteins coprecipitate the other proteins. PMID:17507659

Nucleotide sequence of RNA2 of Lettuce big-vein virus and evidence for a possible transcription termination/initiation strategy similar to that of rhabdoviruses.

PubMed

Sasaya, Takahide; Kusaba, Shinnosuke; Ishikawa, Koichi; Koganezawa, Hiroki

2004-09-01

Lettuce big-vein virus (LBVV) is the type species of the genus Varicosavirus and is a two-segmented negative-sense single-stranded RNA virus. The larger LBVV genome segment (RNA1) consists of 6797 nt and encodes an L polymerase that resembles that of rhabdoviruses. Here, the nucleotide sequence of the second LBVV genome segment (RNA2) is reported. LBVV RNA2 consisted of 6081 nt and contained antisense information for five major ORFs: ORF1 (nt 210-1403 on the viral RNA), ORF2 (nt 1493-2494), ORF3 (nt 2617-3489), ORF4 (nt 3843-4337) and ORF5 (nt 4530-5636), which had coding capacities of 44, 36, 32, 19 and 41 kDa, respectively. The gene at the 3' end of the viral RNA encoded a coat protein, while the other four genes encoded proteins of unknown functions. The 3'-terminal 11 nt of LBVV RNA2 were identical to those of LBVV RNA1, and the 5'-terminal regions of LBVV RNA1 and RNA2 contained a long common nucleotide stretch of about 100 nt. Northern blot analysis using probes specific to the individual ORFs revealed that LBVV transcribes monocistronic RNAs. Analysis of the terminal sequences, and primer extension and RNase H digestion analysis of LBVV mRNAs, suggested that LBVV utilizes a transcription termination/initiation strategy comparable with that of rhabdoviruses.

beta'-COP, a novel subunit of coatomer.

PubMed Central

Stenbeck, G; Harter, C; Brecht, A; Herrmann, D; Lottspeich, F; Orci, L; Wieland, F T

1993-01-01

Several lines of evidence favour the hypothesis that intracellular biosynthetic protein transport in eukaryotes is mediated by non-clathrin-coated vesicles (for a review see Rothman and Orci, 1992). The vesicles have been isolated and a set of their surface proteins has been characterized as coat proteins (COPs). These COPs exist in the cytosol as a preformed complex, the coatomer, which was prior to this study known to contain six subunits: four (alpha-, beta-, gamma- and delta-COP) with molecular weights between 160 and 58 kDa, and two additional proteins of approximately 36 and 20 kDa, epsilon- and xi-COP. Here we describe a novel subunit of the coatomer complex, beta'-COP. This subunit occurs in amounts stoichiometric to the established COPs both in the coatomer and in nonclathrin-coated vesicles and shows homology to the beta-subunits of trimeric G proteins. Images PMID:8334999

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

PubMed Central

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

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain*

PubMed Central

Hozumi, Kentaro; Ishikawa, Masaya; Hayashi, Takemitsu; Yamada, Yuji; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi

2012-01-01

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117–128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions. PMID:22654118

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

PubMed Central

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 prevents secondary invasions by identical or closely related viruses in the same host cells. Although known to occur in diverse viruses, SIE remains an enigma in terms of key molecular determinants and action mechanisms. In this study, we found that Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) encode two independently functioning cistrons that serve as effectors of SIE at the protein but not the RNA level. The coat protein and NIa-Pro encoded by these two viruses, when expressed from a heterologous virus, exerted SIE to the cognate viruses. The identification of virus-encoded effectors of SIE and their transgenic expression could potentially facilitate the development of virus-resistant crop plants. Additionally, functional conservation of SIE in diverse virus groups suggests that a better understanding of the underlying mechanisms of SIE could facilitate the development of novel antiviral therapies against viral diseases. PMID:27681136

Fish gelatin combined with chitosan coating inhibits myofibril degradation of golden pomfret (Trachinotus blochii) fillet during cold storage.

PubMed

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.

Polymer-Based Protein Engineering: Synthesis and Characterization of Armored, High Graft Density Polymer-Protein Conjugates.

PubMed

Carmali, Sheiliza; Murata, Hironobu; Cummings, Chad; Matyjaszewski, Krzysztof; Russell, Alan J

2017-01-01

Atom transfer radical polymerization (ATRP) from the surface of a protein can generate remarkably dense polymer shells that serve as armor and rationally tune protein function. Using straightforward chemistry, it is possible to covalently couple or display multiple small molecule initiators onto a protein surface. The chemistry is fine-tuned to be sequence specific (if one desires a single targeted site) at controlled density. Once the initiator is anchored on the protein surface, ATRP is used to grow polymers on protein surface, in situ. The technique is so powerful that a single-protein polymer conjugate molecule can contain more than 90% polymer coating by weight. If desired, stimuli-responsive polymers can be "grown" from the initiated sites to prepare enzyme conjugates that respond to external triggers such as temperature or pH, while still maintaining enzyme activity and stability. Herein, we focus mainly on the synthesis of chymotrypsin-polymer conjugates. Control of the number of covalently coupled initiator sites by changing the stoichiometric ratio between enzyme and the initiator during the synthesis of protein-initiator complexes allowed fine-tuning of the grafting density. For example, very high grafting density chymotrypsin conjugates were prepared from protein-initiator complexes to grow the temperature-responsive polymers, poly(N-isopropylacrylamide), and poly[N,N'-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate]. Controlled growth of polymers from protein surfaces enables one to predictably manipulate enzyme kinetics and stability without the need for molecular biology-dependent mutagenesis. © 2017 Elsevier Inc. All rights reserved.

On the influence of Ti-Al intermetallic coating architecture on mechanical properties and wear resistance of end mills

NASA Astrophysics Data System (ADS)

Vardanyan, E. L.; Budilov, V. V.; Ramazanov, K. N.; Ataullin, Z. R.

2017-07-01

Thin-film wear-resistant coatings are widely used to increase life and efficiency of metal cutting tools. This paper shows the results of a study on the influence of architecture (number, sequence and thickness of layers) of wear-resistant coatings on physical, mechanical and operational properties of end mills. Coatings consisting of alternating Ti-Al/Ti-Al-N layers of equal thickness demonstrated the best physical and mechanical properties. Durability of coated tools when processing materials from chromium-vanadium steel increased twice as compared to uncoated tools.

Intelligent Computation for Optimal Fabrication Condition of a Protein Chip with Ni-Co Alloy-Coated Surface.

PubMed

Chang, Yaw-Jen; Chang, Cheng-Hao

2016-06-01

Based on the principle of immobilized metal affinity chromatography (IMAC), it has been found that a Ni-Co alloy-coated protein chip is able to immobilize functional proteins with a His-tag attached. In this study, an intelligent computational approach was developed to promote the performance and repeatability of a Ni-Co alloy-coated protein chip. This approach was launched out of L18 experiments. Based on the experimental data, the fabrication process model of a Ni-Co protein chip was established by using an artificial neural network, and then an optimal fabrication condition was obtained using the Taguchi genetic algorithm. The result was validated experimentally and compared with a nitrocellulose chip. Consequentially, experimental outcomes revealed that the Ni-Co alloy-coated chip, fabricated using the proposed approach, had the best performance and repeatability compared with the Ni-Co chips of an L18 orthogonal array design and the nitrocellulose chip. Moreover, the low fluorescent background of the chip surface gives a more precise fluorescent detection. Based on a small quantity of experiments, this proposed intelligent computation approach can significantly reduce the experimental cost and improve the product's quality. © 2015 Society for Laboratory Automation and Screening.

MALDI Top-Down sequencing: calling N- and C-terminal protein sequences with high confidence and speed.

PubMed

Suckau, Detlev; Resemann, Anja

2009-12-01

The ability to match Top-Down protein sequencing (TDS) results by MALDI-TOF to protein sequences by classical protein database searching was evaluated in this work. Resulting from these analyses were the protein identity, the simultaneous assignment of the N- and C-termini and protein sequences of up to 70 residues from either terminus. In combination with de novo sequencing using the MALDI-TDS data, even fusion proteins were assigned and the detailed sequence around the fusion site was elucidated. MALDI-TDS allowed to efficiently match protein sequences quickly and to validate recombinant protein structures-in particular, protein termini-on the level of undigested proteins.

Coating nanoparticles with tunable surfactants facilitates control over the protein corona.

PubMed

Müller, J; Bauer, K N; Prozeller, D; Simon, J; Mailänder, V; Wurm, F R; Winzen, S; Landfester, K

2017-01-01

Nanoparticles with long blood circulation time are a prerequisite for targeted drug delivery. To make the nanoparticles invisible for phagocytizing cells, functional moieties on the particle surface are believed to be necessary to attract specific so-called 'stealth' proteins forming a protein 'corona'. Currently, covalent attachment of those moieties represents the only way to achieve that attraction. However, that approach requires a high synthetic effort and is difficult to control. Therefore, we present the coating of model nanoparticles with biodegradable polymeric surfactants as an alternative method. The thermodynamic parameters of the coating process can be tuned by adjusting the surfactants' block lengths and hydrophilicity. Consequently, the unspecific protein adsorption and aggregation tendency of the particles can be controlled, and stealth proteins inhibiting cell uptake are enriched on their surface. This non-covalent approach could be applied to any particle type and thus facilitates tuning the protein corona and its biological impact. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Design and characterization of calcium alginate microparticles coated with polycations as protein delivery system.

PubMed

Zarate, J; Virdis, L; Orive, G; Igartua, M; Hernández, R M; Pedraz, J L

2011-01-01

Bovine serum albumin (BSA) loaded calcium alginate microparticles (MPs) produced in this study by a w/o emulsification and external gelation method exhibited spherical and fairly smooth and porous morphology with 1.052 ± 0.057 µm modal particle size. The high permeability of the calcium alginate hydrogel lead to a potent burst effect and too fast protein release. To overcome these problems, MPs were coated with polycations, such as chitosan, poly-L-lysine and DEAE-dextran. Our results demonstrated that coated MPs showed slower release and were able to significantly reduce the release of BSA in the first hour. Therefore, this method can be applied to prepare coated alginate MPs which could be an optimal system for the controlled release of biotherapeutic molecules. Nevertheless, further studies are needed to optimize delivery properties which could provide a sustained release of proteins.

The improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein PhaP fused with cell adhesion motif RGD.

PubMed

Dong, Ying; Li, Ping; Chen, Chong-bo; Wang, Zhi-hui; Ma, Ping; Chen, Guo-Qiang

2010-12-01

Polyhydroxyalkanoates (PHA), a family of biopolyesters, have been studied as tissue engineering biomaterials due to their adjustable mechanical properties, biodegradability and tissue compatibility. Amphiphilic PHA granule binding protein PhaP has been shown to be able to bind to hydrophobic surfaces of polymers, especially PHA, via strong hydrophobic interaction. Genes of PhaP and RGD peptides, which are a cell adhesion motif recognized by many cell surface receptors, were successfully expressed and obtained as a pure fusion protein PhaP-RGD in Escherichia coli DH5α. When films of poly(3-hydroxybutyrate-co-3-hydroxy- hexanoate) (PHBHHx), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and polylactic acid (PLA) were coated with PhaP-RGD, their surface hydrophilicities were all increased compared with their corresponding naked (non-coated) films, respectively. Among the three biopolyesters, PHBHHx demonstrated the strongest affinity to PhaP. In vitro study showed that mouse fibroblasts L929 and mouse embryonic fibroblasts NIH/3T3 attached better and grew faster on all three PhaP-RGD coated films compared with their related behaviors on PhaP coated and non-coated films, respectively. Both fibroblasts attached and grew very well on PhaP-RGD coated PHBHHx, PHBV and PLA, even in their serum-free medium, while the non-coated and PhaP coated biopolyesters poorly supported the cell growth if the two fibroblasts were incubated in their serum free medium. These results indicated that PhaP-RGD could be used as a coating material to improve cell growth on hydrophobic biopolyesters for implant tissue engineering purposes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Real-Time Protein and Cell Binding Measurements on Hydroxyapatite Coatings

PubMed Central

Vilardell, A. M.; Cinca, N.; Jokinen, A.; Garcia-Giralt, N.; Dosta, S.; Cano, I. G.; Guilemany, J. M.

2016-01-01

Although a lot of in vitro and in vivo assays have been performed during the last few decades years for hydroxyapatite bioactive coatings, there is a lack of exploitation of real-time in vitro interaction measurements. In the present work, real-time interactions for a plasma sprayed hydroxyapatite coating were measured by a Multi-Parametric Surface Plasmon Resonance (MP-SPR), and the results were compared with standard traditional cell viability in vitro assays. MP-SPR is proven to be suitable not only for measurement of molecule–molecule interactions but also molecule–material interaction measurements and cell interaction. Although SPR is extensively utilized in interaction studies, recent research of protein or cell adsorption on hydroxyapatite coatings for prostheses applications was not found. The as-sprayed hydroxyapatite coating resulted in 62.4% of crystalline phase and an average thickness of 24 ± 6 μm. The MP-SPR was used to measure lysozyme protein and human mesenchymal stem cells interaction to the hydroxyapatite coating. A comparison between the standard gold sensor and Hydroxyapatite (HA)-plasma coated sensor denoted a clearly favourable cell attachment on HA coated sensor as a significantly higher signal of cell binding was detected. Moreover, traditional cell viability and proliferation tests showed increased activity with culture time indicating that cells were proliferating on HA coating. Cells show homogeneous distribution and proliferation along the HA surface between one and seven days with no significant mortality. Cells were flattened and spread on rough surfaces from the first day, with increasing cytoplasmatic extensions during the culture time. PMID:27618911

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 {sup 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 leastmore » 10{sup 5}-fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein the authors use {sup 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 {sup 15}N nucleus from a coupled proton; when {sup 15}N-labeled protonated protein is dissolved in {sup 2}H{sub 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{sup +} and OH{sup {minus}} ions. The time-dependent exchange-out experiment is suitable for slow exchange rates (k{sub 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.« less

Molecular Characterization of Geographically Different Banana bunchy top virus Isolates in India.

PubMed

Selvarajan, R; Mary Sheeba, M; Balasubramanian, V; Rajmohan, R; Dhevi, N Lakshmi; Sasireka, T

2010-10-01

Banana bunchy top disease (BBTD) caused by Banana bunchy top virus (BBTV) is one of the most devastating diseases of banana and poses a serious threat for cultivars like Hill Banana (Syn: Virupakshi) and Grand Naine in India. In this study, we have cloned and sequenced the complete genome comprised of six DNA components of BBTV infecting Hill Banana grown in lower Pulney hills, Tamil Nadu State, India. The complete genome sequence of this hill banana isolate showed high degree of similarity with the corresponding sequences of BBTV isolates originating from Lucknow, Uttar Pradesh State, India, and from Fiji, Egypt, Pakistan, and Australia. In addition, sixteen coat protein (CP) and thirteen replicase genes (Rep) sequences of BBTV isolates collected from different banana growing states of India were cloned and sequenced. The replicase sequences of 13 isolates showed high degree of similarity with that of South Pacific group of BBTV isolates. However, the CP gene of BBTV isolates from Shervroy and Kodaikanal hills of Tamil Nadu showed higher amino acid sequence variability compared to other isolates. Another hill banana isolate from Meghalaya state had 23 nucleotide substitutions in the CP gene but the amino acid sequence was conserved. This is the first report of the characterization of a complete genome of BBTV occurring in the high altitudes of India. Our study revealed that the Indian BBTV isolates with distinct geographical origins belongs to the South Pacific group, except Shervroy and Kodaikanal hill isolates which neither belong to the South Pacific nor the Asian group.

Recent progress in making protein microarray through BioLP

NASA Astrophysics Data System (ADS)

Yang, Rusong; Wei, Lian; Feng, Ying; Li, Xiujian; Zhou, Quan

2017-02-01

Biological laser printing (BioLP) is a promising biomaterial printing technique. It has the advantage of high resolution, high bioactivity, high printing frequency and small transported liquid amount. In this paper, a set of BioLP device is design and made, and protein microarrays are printed by this device. It's found that both laser intensity and fluid layer thickness have an influence on the microarrays acquired. Besides, two kinds of the fluid layer coating methods are compared, and the results show that blade coating method is better than well-coating method in BioLP. A microarray of 0.76pL protein microarray and a "NUDT" patterned microarray are printed to testify the printing ability of BioLP.

Detection of cell surface calreticulin as a potential cancer biomarker using near-infrared emitting gold nanoclusters

NASA Astrophysics Data System (ADS)

Subramaniyam Ramesh, Bala; Giorgakis, Emmanouil; Lopez-Davila, Victor; Kamali Dashtarzheneha, Ashkan; Loizidou, Marilena

2016-07-01

Calreticulin (CRT) is a cytoplasmic calcium-binding protein. The aim of this study was to investigate CRT presence in cancer with the use of fluorescent gold nanoclusters (AuNCs) and to explore AuNC synthesis using mercaptosuccinic acid (MSA) as a coating agent. MSA-coated AuNCs conferred well-dispersed, bio-stable, water-soluble nanoparticles with bioconjugation capacity and 800-850 nm fluorescence after broad-band excitation. Cell-viability assay revealed good AuNC tolerability. A native CRT amino-terminus corresponding peptide sequence was synthesised and used to generate rabbit site-specific antibodies. Target specificity was demonstrated with antibody blocking in colorectal and breast cancer cell models; human umbilical vein endothelial cells served as controls. We demonstrated a novel route of AuNC/MSA manufacture and CRT presence on colonic and breast cancerous cell surface. AuNCs served as fluorescent bio-probes specifically recognising surface-bound CRT. These results are promising in terms of AuNC application in cancer theranostics and CRT use as surface biomarker in human cancer.

Cerebroretinal microangiopathy with calcifications and cysts associated with CTC1 and NDP mutations.

PubMed

Romaniello, Romina; Arrigoni, Filippo; Citterio, Andrea; Tonelli, Alessandra; Sforzini, Cinzia; Rizzari, Carmelo; Pessina, Marco; Triulzi, Fabio; Bassi, Maria Teresa; Borgatti, Renato

2013-12-01

Mutations in the conserved telomere maintenance component 1 (CTC1) gene were recently described in Coats plus syndrome and in cerebroretinal microangiopathy with calcifications and cysts. Norrie disease protein (NDP) gene was found mutated in Norrie disease, in Familial Exudative Vitreoretinopathy, and in Coats syndrome. Here we describe a boy affected by Norrie disease who developed typical features of cerebroretinal microangiopathy with calcifications and cysts. Direct sequencing of the CTC1 and NDP genes in this patient shows the presence of compound heterozygosity for 2 mutations in CTC1 (c.775G>A, pV259M and a novel microdeletion c.1213delG) and a missense mutation in the NDP gene (c.182T>C, p.L61P). Based on these genetic findings and on the expression of both genes in endothelial cells, we postulate that microangiopathy might be a primary underlying pathologic abnormality in cerebroretinal microangiopathy with calcifications and cysts. This hypothesis is further supported by magnetic resonance imaging (MRI) data showing multiple minute calcifications in the deep gray nuclei and in terminal arteriolar zones.

Identification, Characterization and Full-Length Sequence Analysis of a Novel Polerovirus Associated with Wheat Leaf Yellowing Disease

PubMed Central

Zhang, Peipei; Liu, Yan; Liu, Wenwen; Cao, Mengji; Massart, Sebastien; Wang, Xifeng

2017-01-01

To identify the pathogens responsible for leaf yellowing symptoms on wheat samples collected from Jinan, China, we tested for the presence of three known barley/wheat yellow dwarf viruses (BYDV-GAV, -PAV, WYDV-GPV) (most likely pathogens) using RT-PCR. A sample that tested negative for the three viruses was selected for small RNA sequencing. Twenty-five million sequences were generated, among which 5% were of viral origin. A novel polerovirus was discovered and temporarily named wheat leaf yellowing-associated virus (WLYaV). The full genome of WLYaV corresponds to 5,772 nucleotides (nt), with six AUG-initiated open reading frames, one non-AUG-initiated open reading frame, and three untranslated regions, showing typical features of the family Luteoviridae. Sequence comparison and phylogenetic analyses suggested that WLYaV had the closest relationship with sugarcane yellow leaf virus (ScYLV), but the identities of full genomic nucleotides and deduced amino acid sequence of coat protein (CP) were 64.9 and 86.2%, respectively, below the species demarcation thresholds (90%) in the family Luteoviridae. Furthermore, agroinoculation of Nicotiana benthamiana leaves with a cDNA clone of WLYaV caused yellowing symptoms on the plant. Our study adds a new polerovirus that is associated with wheat leaf yellowing disease, which would help to identify and control pathogens of wheat. PMID:28932215

Identification, Characterization and Full-Length Sequence Analysis of a Novel Polerovirus Associated with Wheat Leaf Yellowing Disease.

PubMed

Zhang, Peipei; Liu, Yan; Liu, Wenwen; Cao, Mengji; Massart, Sebastien; Wang, Xifeng

2017-01-01

To identify the pathogens responsible for leaf yellowing symptoms on wheat samples collected from Jinan, China, we tested for the presence of three known barley/wheat yellow dwarf viruses (BYDV-GAV, -PAV, WYDV-GPV) (most likely pathogens) using RT-PCR. A sample that tested negative for the three viruses was selected for small RNA sequencing. Twenty-five million sequences were generated, among which 5% were of viral origin. A novel polerovirus was discovered and temporarily named wheat leaf yellowing-associated virus (WLYaV). The full genome of WLYaV corresponds to 5,772 nucleotides (nt), with six AUG-initiated open reading frames, one non-AUG-initiated open reading frame, and three untranslated regions, showing typical features of the family Luteoviridae . Sequence comparison and phylogenetic analyses suggested that WLYaV had the closest relationship with sugarcane yellow leaf virus (ScYLV), but the identities of full genomic nucleotides and deduced amino acid sequence of coat protein (CP) were 64.9 and 86.2%, respectively, below the species demarcation thresholds (90%) in the family Luteoviridae . Furthermore, agroinoculation of Nicotiana benthamiana leaves with a cDNA clone of WLYaV caused yellowing symptoms on the plant. Our study adds a new polerovirus that is associated with wheat leaf yellowing disease, which would help to identify and control pathogens of wheat.

The genomic and biological characterization of Citrullus lanatus cryptic virus infecting watermelon in China.

PubMed

Xin, Min; Cao, Mengji; Liu, Wenwen; Ren, Yingdang; Lu, Chuantao; Wang, Xifeng

2017-03-15

A dsRNA virus was detected in the watermelon (Citrullus lanatus) samples collected from Kaifeng, Henan province, China through the use of next generation sequencing of small RNAs. The complete genome of this virus is comprised of dsRNA-1 (1603nt) and dsRNA-2 (1466nt), both of which are single open reading frames and potentially encode a 54.2kDa RNA-dependent RNA polymerase (RdRp) and a 45.9kDa coat protein (CP), respectively. The RdRp and CP share the highest amino acid identities 85.3% and 75.4% with a previously reported Israeli strain Citrullus lanatus cryptic virus (CiLCV), respectively. Genome comparisons indicate that this virus is the same species with CiLCV, whereas the reported sequences of the Israeli strain of CiLCV are partial, and our newly identified sequences can represent the complete genome of CiLCV. Futhermore, phylogenetic tree analyses based on the RdRp sequences suggest that CiLCV is one member in the genus Deltapartitivirus, family Partitiviridae. In addition, field investigation and seed-borne bioassays show that CiLCV commonly occurs in many varieties and is transmitted though seeds at a very high rate. Copyright © 2017 Elsevier B.V. All rights reserved.

Chayote mosaic virus, a New Tymovirus Infecting Cucurbitaceae.

PubMed

Bernal, J J; Jiménez, I; Moreno, M; Hord, M; Rivera, C; Koenig, R; Rodríguez-Cerezo, E

2000-10-01

ABSTRACT Chayote mosaic virus (ChMV) is a putative tymovirus isolated from chayote crops in Costa Rica. ChMV was characterized at the host range, serological, and molecular levels. ChMV was transmitted mechanically and induced disease symptoms mainly in Cucurbitaceae hosts. Asymptomatic infections were detected in other host families. Serologically, ChMV is related to the Andean potato latent virus (APLV) and the Eggplant mosaic virus (EMV), both members of the genus Tymovirus infecting solanaceous hosts in the Caribbean Basin and South America. The sequence of the genomic RNA of ChMV was determined and its genetic organization was typical of tymoviruses. Comparisons with other tymoviral sequences showed that ChMV was a new member of the genus Tymovirus. The phylogenetic analyses of the coat protein gene were consistent with serological comparisons and positioned ChMV within a cluster of tymoviruses infecting mainly cucurbit or solanaceous hosts, including APLV and EMV. Phylogenetic analyses of the replicase protein gene confirmed the close relationship of ChMV and EMV. Our results suggest that ChMV is related to two tymoviruses (APLV and EMV) of proximal geographical provenance but with different natural host ranges. ChMV is the first cucurbit-infecting tymovirus to be fully characterized at the genomic level.

Characterization of New Isolates of Apricot vein clearing-associated virus and of a New Prunus-Infecting Virus: Evidence for Recombination as a Driving Force in Betaflexiviridae Evolution.

PubMed

Marais, Armelle; Faure, Chantal; Mustafayev, Eldar; Candresse, Thierry

2015-01-01

Double stranded RNAs from Prunus samples gathered from various surveys were analyzed by a deep-sequencing approach. Contig annotations revealed the presence of a potential new viral species in an Azerbaijani almond tree (Prunus amygdalus) and its genome sequence was completed. Its genomic organization is similar to that of the recently described Apricot vein clearing associated virus (AVCaV) for which two new isolates were also characterized, in a similar fashion, from two Japanese plums (Prunus salicina) from a French germplasm collection. The amino acid identity values between the four proteins encoded by the genome of the new virus have identity levels with those of AVCaV which fall clearly outside the species demarcation criteria. The new virus should therefore be considered as a new species for which the name of Caucasus prunus virus (CPrV) has been proposed. Phylogenetic relationships and nucleotide comparisons suggested that together with AVCaV, CPrV could define a new genus (proposed name: Prunevirus) in the family Betaflexiviridae. A molecular test targeting both members of the new genus was developed, allowing the detection of additional AVCaV isolates, and therefore extending the known geographical distribution and the host range of AVCaV. Moreover, the phylogenetic trees reconstructed with the amino acid sequences of replicase, movement and coat proteins of representative Betaflexiviridae members suggest that Citrus leaf blotch virus (CLBV, type member of the genus Citrivirus) may have evolved from a recombination event involving a Prunevirus, further highlighting the importance of recombination as a driving force in Betaflexiviridae evolution. The sequences reported in the present manuscript have been deposited in the GenBank database under accession numbers KM507061-KM504070.

Characterization of New Isolates of Apricot vein clearing-associated virus and of a New Prunus-Infecting Virus: Evidence for Recombination as a Driving Force in Betaflexiviridae Evolution

PubMed Central

Marais, Armelle; Faure, Chantal; Mustafayev, Eldar; Candresse, Thierry

2015-01-01

Double stranded RNAs from Prunus samples gathered from various surveys were analyzed by a deep-sequencing approach. Contig annotations revealed the presence of a potential new viral species in an Azerbaijani almond tree (Prunus amygdalus) and its genome sequence was completed. Its genomic organization is similar to that of the recently described Apricot vein clearing associated virus (AVCaV) for which two new isolates were also characterized, in a similar fashion, from two Japanese plums (Prunus salicina) from a French germplasm collection. The amino acid identity values between the four proteins encoded by the genome of the new virus have identity levels with those of AVCaV which fall clearly outside the species demarcation criteria. The new virus should therefore be considered as a new species for which the name of Caucasus prunus virus (CPrV) has been proposed. Phylogenetic relationships and nucleotide comparisons suggested that together with AVCaV, CPrV could define a new genus (proposed name: Prunevirus) in the family Betaflexiviridae. A molecular test targeting both members of the new genus was developed, allowing the detection of additional AVCaV isolates, and therefore extending the known geographical distribution and the host range of AVCaV. Moreover, the phylogenetic trees reconstructed with the amino acid sequences of replicase, movement and coat proteins of representative Betaflexiviridae members suggest that Citrus leaf blotch virus (CLBV, type member of the genus Citrivirus) may have evolved from a recombination event involving a Prunevirus, further highlighting the importance of recombination as a driving force in Betaflexiviridae evolution. The sequences reported in the present manuscript have been deposited in the GenBank database under accession numbers KM507061-KM504070. PMID:26086395

Surface modification of zinc oxide nanoparticles with amorphous silica alters their fate in the circulation.

PubMed

Konduru, Nagarjun V; Murdaugh, Kimberly M; Swami, Archana; Jimenez, Renato J; Donaghey, Thomas C; Demokritou, Philip; Brain, Joseph D; Molina, Ramon M

2016-08-01

Nanoparticle (NP) pharmacokinetics and biological effects are influenced by many factors, especially surface physicochemical properties. We assessed the effects of an amorphous silica coating on the fate of zinc after intravenous (IV) injection of neutron activated uncoated (65)ZnO or silica-coated (65)ZnO NPs in male Wistar Han rats. Groups of IV-injected rats were sequentially euthanized, and 18 tissues were collected and analyzed for (65)Zn radioactivity. The protein coronas on each ZnO NP after incubation in rat plasma were analyzed by SDS-PAGE gel electrophoresis and mass spectrometry of selected gel bands. Plasma clearance for both NPs was biphasic with rapid initial and slower terminal clearance rates. Half-lives of plasma clearance of silica-coated (65)ZnO were shorter (initial - <1 min; terminal - 2.5 min) than uncoated (65)ZnO (initial - 1.9 min; terminal - 38 min). Interestingly, the silica-coated (65)ZnO group had higher (65)Zn associated with red blood cells and higher initial uptake in the liver. The (65)Zn concentrations in all the other tissues were significantly lower in the silica-coated than uncoated groups. We also found that the protein corona formed on silica-coated ZnO NPs had higher amounts of plasma proteins, particularly albumin, transferrin, A1 inhibitor 3, α-2-hs-glycoprotein, apoprotein E and α-1 antitrypsin. Surface modification with amorphous silica alters the protein corona, agglomerate size, and zeta potential of ZnO NPs, which in turn influences ZnO biokinetic behavior in the circulation. This emphasizes the critical role of the protein corona in the biokinetics, toxicology and nanomedical applications of NPs.

Fine Structure Genetic and Physical Map of the Gene 3 to 10 Region of the Bacteriophage P22 Chromosome

PubMed Central

Casjens, S.; Eppler, K.; Sampson, L.; Parr, R.; Wyckoff, E.

1991-01-01

The mechanism by which dsDNA is packaged by viruses is not yet understood in any system. Bacteriophage P22 has been a productive system in which to study the molecular genetics of virus particle assembly and DNA packaging. Only five phage encoded proteins, the products of genes 3, 2, 1, 8 and 5, are required for packaging the virus chromosome inside the coat protein shell. We report here the construction of a detailed genetic and physical map of these genes, the neighboring gene 4 and a portion of gene 10, in which 289 conditional lethal amber, opal, temperature sensitive and cold sensitive mutations are mapped into 44 small (several hundred base pair) intervals of known sequence. Knowledge of missense mutant phenotypes and information on the location of these mutations allows us to begin the assignment of partial protein functions to portions of these genes. The map and mapping strains will be of use in the further genetic dissection of the P22 DNA packaging and prohead assembly processes. PMID:2029965

A Coincidence Detection Mechanism Controls PX-BAR Domain-Mediated Endocytic Membrane Remodeling via an Allosteric Structural Switch.

PubMed

Lo, Wen-Ting; Vujičić Žagar, Andreja; Gerth, Fabian; Lehmann, Martin; Puchkov, Dymtro; Krylova, Oxana; Freund, Christian; Scapozza, Leonardo; Vadas, Oscar; Haucke, Volker

2017-11-20

Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P 2 ) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P 2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei.

PubMed

Salmon, D; Geuskens, M; Hanocq, F; Hanocq-Quertier, J; Nolan, D; Ruben, L; Pays, E

1994-07-15

In T. brucei, a transferrin-binding protein has been found to share sequence homology with pESAG-7 and -6, the products of two related genes present in the VSG gene polycistronic transcription unit. When expressed in Xenopus oocytes, they appear as N-glycosylated proteins secreted in the medium (pESAG-7) and GPI anchored to the membrane (pESAG-6). These proteins are able to homo- or heterodimerize, probably through association in the same orientation. Only heterodimers can bind Tf, possibly two molecules per dimer. A comparison of Tf binding to pESAG-7/6-expressing oocytes and trypanosomes suggests that pESAG-7/6 is the Tf receptor of the parasite. In trypanosomes, the majority of pESAG-7/6 is released from the membrane and associates, together with Tf, with a glycosylated matrix present in the lumen of the flagellar pocket. Both pESAG-7/6 and Tf are internalized via coated pits and vesicles. These observations suggest a novel mode of Tf binding and uptake in trypanosomes.

Nanotube-assisted protein deactivation

NASA Astrophysics Data System (ADS)

Joshi, Amit; Punyani, Supriya; Bale, Shyam Sundhar; Yang, Hoichang; Borca-Tasciuc, Theodorian; Kane, Ravi S.

2008-01-01

Conjugating proteins onto carbon nanotubes has numerous applications in biosensing, imaging and cellular delivery. However, remotely controlling the activity of proteins in these conjugates has never been demonstrated. Here we show that upon near-infrared irradiation, carbon nanotubes mediate the selective deactivation of proteins in situ by photochemical effects. We designed nanotube-peptide conjugates to selectively destroy the anthrax toxin, and also optically transparent coatings that can self-clean following either visible or near-infrared irradiation. Nanotube-assisted protein deactivation may be broadly applicable to the selective destruction of pathogens and cells, and will have applications ranging from antifouling coatings to functional proteomics.

Occurrence and Partial Characterization of Lettuce big vein associated virus and Mirafiori lettuce big vein virus in Lettuce in Iran.

PubMed

Alemzadeh, E; Izadpanah, K

2012-12-01

Mirafiori lettuce big vein virus (MiLBVV) and lettuce big vein associated virus (LBVaV) were found in association with big vein disease of lettuce in Iran. Analysis of part of the coat protein (CP) gene of Iranian isolates of LBVaV showed 97.1-100 % nucleotide sequence identity with other LBVaV isolates. Iranian isolates of MiLBVV belonged to subgroup A and showed 88.6-98.8 % nucleotide sequence identity with other isolates of this virus when amplified by PCR primer pair MiLV VP. The occurrence of both viruses in lettuce crop was associated with the presence of resting spores and zoosporangia of the fungus Olpidium brassicae in lettuce roots under field and greenhouse conditions. Two months after sowing lettuce seed in soil collected from a lettuce field with big vein affected plants, all seedlings were positive for LBVaV and MiLBVV, indicating soil transmission of both viruses.

Characterization of a potyvirus associated with yellow mosaic disease of jasmine (Jasminum sambac L.) in Andhra Pradesh, India.

PubMed

Sudheera, Y; Vishnu Vardhan, G P; Hema, M; Krishna Reddy, M; Sreenivasulu, P

2014-01-01

A virus isolate associated with yellow mosaic disease was purified from commercially cultivated jasmine (Jasminum sambac) from Andhra Pradesh, India and it contained flexuous filamentous particles of ~720 × 13 nm. The denatured purified virus had single major polypeptide of molecular weight 32 kDa. Complementary DNA representing 1678 nucleotides (nt) of the 3' terminus of viral RNA was cloned and sequenced. Comparisons of complete coat protein (CP) gene nucleotide and amino acid sequences of the present virus isolate with certain reported potyviruses revealed 86.1 and 92.7 % identity, respectively with jasmine potyvirus T (JaVT) reported from Taiwan and less than 70 % with other potyviruses. Based on the phylogenetic analysis of 3' UTR and CP gene, the present virus isolate was identified as an isolate of JaVT that belongs to the genus Potyvirus and the name Jasmine yellow mosaic virus-Andhra Pradesh (JaYMV-AP) is proposed.

Occurrence of Squash yellow mild mottle virus and Pepper golden mosaic virus in Potential New Hosts in Costa Rica

PubMed Central

Castro, Ruth M.; Moreira, Lisela; Rojas, María R.; Gilbertson, Robert L.; Hernández, Eduardo; Mora, Floribeth; Ramírez, Pilar

2013-01-01

Leaf samples of Solanum lycopersicum, Capsicum annuum, Cucurbita moschata, Cucurbita pepo, Sechium edule and Erythrina spp. were collected. All samples were positive for begomoviruses using polymerase chain reaction and degenerate primers. A sequence of ∼1,100 bp was obtained from the genomic component DNA-A of 14 samples. In addition, one sequence of ∼580 bp corresponding to the coat protein (AV1) was obtained from a chayote (S. edule) leaf sample. The presence of Squash yellow mild mottle virus (SYMMoV) and Pepper golden mosaic virus (PepGMV) were confirmed. The host range reported for SYMMoV includes species of the Cucurbitaceae, Caricaceae and Fabaceae families. This report extends the host range of SYMMoV to include the Solanaceae family, and extends the host range of PepGMV to include C. moschata, C. pepo and the Fabaceae Erythrina spp. This is the first report of a begomovirus (PepGMV) infecting chayote in the Western Hemisphere. PMID:25288955

Occurrence of Squash yellow mild mottle virus and Pepper golden mosaic virus in Potential New Hosts in Costa Rica.

PubMed

Castro, Ruth M; Moreira, Lisela; Rojas, María R; Gilbertson, Robert L; Hernández, Eduardo; Mora, Floribeth; Ramírez, Pilar

2013-09-01

Leaf samples of Solanum lycopersicum, Capsicum annuum, Cucurbita moschata, Cucurbita pepo, Sechium edule and Erythrina spp. were collected. All samples were positive for begomoviruses using polymerase chain reaction and degenerate primers. A sequence of ∼1,100 bp was obtained from the genomic component DNA-A of 14 samples. In addition, one sequence of ∼580 bp corresponding to the coat protein (AV1) was obtained from a chayote (S. edule) leaf sample. The presence of Squash yellow mild mottle virus (SYMMoV) and Pepper golden mosaic virus (PepGMV) were confirmed. The host range reported for SYMMoV includes species of the Cucurbitaceae, Caricaceae and Fabaceae families. This report extends the host range of SYMMoV to include the Solanaceae family, and extends the host range of PepGMV to include C. moschata, C. pepo and the Fabaceae Erythrina spp. This is the first report of a begomovirus (PepGMV) infecting chayote in the Western Hemisphere.

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

USDA-ARS?s Scientific Manuscript database

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...

Evidence for lysine acetylation in the coat protein of a Polerovirus

USDA-ARS?s Scientific Manuscript database

Virions of the RPV strain of Cereal yellow dwarf virus (CYDV-RPV) were purified from infected oat tissue and analyzed by mass spectrometry. Two conserved residues, K147 and K181, residing in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional ...

Generation of PVY coat protein siRNAs in transgenic potatoes resistant to PVY.

USDA-ARS?s Scientific Manuscript database

Transgenic potatoes expressing the potato virus Y coat protein (PVY-CP) inverted hairpin RNA (ihRNA) construct driven by the Solanum bulbocastanum ubiquitin 409s promoter exhibited resistance to PVY in glass house studies using PVYNTN and PVYO as inocula and in field studies using naturally occurrin...

Chromosome localization of human genes for clathrin adaptor polypeptides AP2{beta} and AP50 and the clathrin-binding protein, VCP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Druck, T.; Gu, Y.; Prabhala, G.

1995-11-01

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed {open_quotes}assembly proteins,{close_quotes} or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2{alpha} and AP2{beta}, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2{beta} (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with A1`s. Chromosomal in situ hybridization of a human genomic clonemore » demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2{beta} fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34. 13 refs., 2 figs.« less

The C. elegans rab family: identification, classification and toolkit construction.

PubMed

Gallegos, Maria E; Balakrishnan, Sanjeev; Chandramouli, Priya; Arora, Shaily; Azameera, Aruna; Babushekar, Anitha; Bargoma, Emilee; Bokhari, Abdulmalik; Chava, Siva Kumari; Das, Pranti; Desai, Meetali; Decena, Darlene; Saramma, Sonia Dev Devadas; Dey, Bodhidipra; Doss, Anna-Louise; Gor, Nilang; Gudiputi, Lakshmi; Guo, Chunyuan; Hande, Sonali; Jensen, Megan; Jones, Samantha; Jones, Norman; Jorgens, Danielle; Karamchedu, Padma; Kamrani, Kambiz; Kolora, Lakshmi Divya; Kristensen, Line; Kwan, Kelly; Lau, Henry; Maharaj, Pranesh; Mander, Navneet; Mangipudi, Kalyani; Menakuru, Himabindu; Mody, Vaishali; Mohanty, Sandeepa; Mukkamala, Sridevi; Mundra, Sheena A; Nagaraju, Sudharani; Narayanaswamy, Rajhalutshimi; Ndungu-Case, Catherine; Noorbakhsh, Mersedeh; Patel, Jigna; Patel, Puja; Pendem, Swetha Vandana; Ponakala, Anusha; Rath, Madhusikta; Robles, Michael C; Rokkam, Deepti; Roth, Caroline; Sasidharan, Preeti; Shah, Sapana; Tandon, Shweta; Suprai, Jagdip; Truong, Tina Quynh Nhu; Uthayaruban, Rubatharshini; Varma, Ajitha; Ved, Urvi; Wang, Zeran; Yu, Zhe

2012-01-01

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).

The C. elegans Rab Family: Identification, Classification and Toolkit Construction

PubMed Central

Gallegos, Maria E.; Balakrishnan, Sanjeev; Chandramouli, Priya

2012-01-01

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB). PMID:23185324

Microstructure of Desmanthus illinoensis

NASA Astrophysics Data System (ADS)

Wood, Delilah F.; Orts, William J.; Glenn, Gregory M.

2010-06-01

Structure and histochemistry of mature seeds of Desmanthus illinoensis (Illinois bundle flower) show that the seed has typical legume structure. The seed can be separated into two major fractions including the seed coat/endosperm and the embryo. The seed coat consists of a cuticle, palisade sclereids, hour glass cells and mesophyll. Endosperm is attached to the inner portion of the seed coat and is thicker beneath the pleurogram in the center of the seed. The embryo consists mostly of two large cotyledons, the major storage structures of the seed. The cotyledons are high in protein which occurs in protein bodies. Protein bodies in the cotyledons include those without inclusions, those with phytin inclusions and those with calcium-rich crystals. The phytin inclusions are spherical and have high phosphorus and magnesium contents. The calcium-rich crystals are also included inside protein bodies and are druse-type crystals.

Bioactive potential of silica coatings and its effect on the adhesion of proteins to titanium implants.

PubMed

Romero-Gavilan, F; Araújo-Gomes, N; Sánchez-Pérez, A M; García-Arnáez, I; Elortza, F; Azkargorta, M; de Llano, J J Martín; Carda, C; Gurruchaga, M; Suay, J; Goñi, I

2018-02-01

There is an ever-increasing need to develop dental implants with ideal characteristics to achieve specific and desired biological response in the scope of improve the healing process post-implantation. Following that premise, enhancing and optimizing titanium implants through superficial treatments, like silica sol-gel hybrid coatings, are regarded as a route of future research in this area. These coatings change the physicochemical properties of the implant, ultimately affecting its biological characteristics. Sandblasted acid-etched titanium (SAE-Ti) and a silica hybrid sol-gel coating (35M35G30T) applied onto the Ti substrate were examined. The results of in vitro and in vivo tests and the analysis of the protein layer adsorbed to each surface were compared and discussed. In vitro analysis with MC3T3-E1 osteoblastic cells, showed that the sol-gel coating raised the osteogenic activity potential of the implants (the expression of osteogenic markers, the alkaline phosphatase (ALP) and IL-6 mRNAs, increased). In the in vivo experiments using as model rabbit tibiae, both types of surfaces promoted osseointegration. However, the coated implants demonstrated a clear increase in the inflammatory activity in comparison with SAE-Ti. Mass spectrometry (LC-MS/MS) analysis showed differences in the composition of protein layers formed on the two tested surfaces. Large quantities of apolipoproteins were found attached predominantly to SAE-Ti. The 35M35G30T coating adsorbed a significant quantity of complement proteins, which might be related to the material intrinsic bioactivity, following an associated, natural and controlled immune response. The correlation between the proteomic data and the in vitro and in vivo outcomes is discussed on this experimental work. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of multilayer rhBMP-2 DNA coating on the proliferation and differentiation of MC3T3-E1 cells seeded on roughed titanium surface.

PubMed

Jiang, Qiao-Hong; Liu, Li; Shen, Jian-Wei; Peel, Sean; Yang, Guo-Li; Zhao, Shi-Fang; He, Fu-Ming

2012-10-01

For bone morphogenetic protein (BMP) gene therapy to be a viable approach for enhancing implant osseointegration clinically, requires the development of efficient nonviral delivery vectors that can coat the implant. This study evaluated a multilayer cationic liposome-DNA complex (LDc) coating as a delivery vehicle for recombinant human BMP-2 (rhBMP-2). Multilayered coatings, comprising hyaluronic acid (HA) and LDc, were fabricated onto titanium using a layer-by-layer (LBL) assembly technique. Preosteoblastic MC3T3-E1 cells were cultured on the roughened titanium surfaces coated with multilayers of HA/LDc, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2 secreted by the MC3T3-E1 cells and the effect of the various surfaces on cell viability, proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and calcium deposition were evaluated. Messenger RNA levels of OC, ALP, Runx2, and Osx were also investigated. The results demonstrated that rhBMP-2 protein secreted into culture medium at 3 days was significantly higher than control groups. MC3T3-E1 cells cultured on the HA/LDc coating displayed significantly higher ALP activity and OC secretion at 7 days and 14 days culture, respectively. MC3T3-E1 cells cultured on HA/LDc upregulated expression of the osteoblast differentiation markers, especially on days 12 for OC and on days 6 and 12 for ALP and Osx. In conclusion, MC3T3-E1 cell cultured on the multilayer HA/LDc coating surface can secret rhBMP-2 protein and the protein levels were effective in inducing early osteogenic differentiation. Copyright © 2012 Wiley Periodicals, Inc.

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

NASA Astrophysics Data System (ADS)

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 multilayer flexible packaging films.

Impact of dietary fiber coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions.

PubMed

Tokle, Tanushree; Lesmes, Uri; Decker, Eric Andrew; McClements, David Julian

2012-01-01

Multilayer emulsions containing lipid droplets coated by lactoferrin (LF) - anionic polysaccharide layers have improved resistance to environmental stresses (such as pH, salt, and temperature), but their behavior within the gastrointestinal tract (GIT) is currently unknown. The objective of this research was therefore to monitor changes in the physicochemical properties and digestibility of these systems under simulated GIT conditions. Primary emulsions (5% corn oil, 0.5% LF) were prepared using a high-pressure homogenizer. Secondary emulsions (5% corn oil, 0.5% LF, 0.5% polysaccharide) were prepared by incorporating alginate, low methoxyl pectin (LMP) or high methoxyl pectin (HMP) into primary emulsions. Emulsions were then subjected to simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions in sequence. LF, LF-LMP and LF-HMP emulsions were stable to droplet aggregation in the stomach but aggregated in the small intestine, whereas LF-alginate emulsions aggregated in both the stomach and small intestine. The presence of a dietary fiber coating around the initial lipid droplets had little influence on the total extent of lipid digestion in SIF, but LF-alginate emulsions had a slower initial digestion rate than the other emulsions. These results suggest that the dietary fiber coatings may become detached in the small intestine, or that they were permeable to digestive enzymes. Pepsin was found to have little influence on the physical stability or digestibility of the emulsions. The knowledge obtained from this study is important for the design of delivery systems for encapsulation and release of lipophilic bioactive ingredients.

Chitosan-coated polystyrene microplate for covalent immobilization of enzyme.

PubMed

Zhang, Yaodong; Li, Li; Yu, Caihong; Hei, Tingting

2011-10-01

Microplates made of polystyrene have been widely used for immunoassays. Protein molecules that have been immobilized on a hydrophobic polystyrene microplate by passive adsorption lose their activity and suffer considerable denaturation. A new chitosan-coated microplate suitable for the covalent immobilization of enzymes has been developed. The primary amino groups of chitosan were exploited for this covalent coupling of proteins. The optical transmittance of the chitosan-coated microplate, at wavelengths of 400-800 nm, was estimated to be suitable for its application in chromogenic reaction-based bioassays. The immobilization efficiency of the chitosan-coated microplate was demonstrated to be far superior to that of a conventional microplate when tested using acetylcholinesterase (AChE) and β-glucosidase as model biomolecules, and the chitosan-coated microplate may thus have potential applications in biosensing and bioreactor systems. © Springer-Verlag 2011

A Combined Comparative Transcriptomic, Metabolomic, and Anatomical Analyses of Two Key Domestication Traits: Pod Dehiscence and Seed Dormancy in Pea (Pisum sp.)

PubMed Central

Hradilová, Iveta; Trněný, Oldřich; Válková, Markéta; Cechová, Monika; Janská, Anna; Prokešová, Lenka; Aamir, Khan; Krezdorn, Nicolas; Rotter, Björn; Winter, Peter; Varshney, Rajeev K.; Soukup, Aleš; Bednář, Petr; Hanáček, Pavel; Smýkal, Petr

2017-01-01

The origin of the agriculture was one of the turning points in human history, and a central part of this was the evolution of new plant forms, domesticated crops. Seed dispersal and germination are two key traits which have been selected to facilitate cultivation and harvesting of crops. The objective of this study was to analyze anatomical structure of seed coat and pod, identify metabolic compounds associated with water-impermeable seed coat and differentially expressed genes involved in pea seed dormancy and pod dehiscence. Comparative anatomical, metabolomics, and transcriptomic analyses were carried out on wild dormant, dehiscent Pisum elatius (JI64, VIR320) and cultivated, indehiscent Pisum sativum non-dormant (JI92, Cameor) and recombinant inbred lines (RILs). Considerable differences were found in texture of testa surface, length of macrosclereids, and seed coat thickness. Histochemical and biochemical analyses indicated genotype related variation in composition and heterogeneity of seed coat cell walls within macrosclereids. Liquid chromatography–electrospray ionization/mass spectrometry and Laser desorption/ionization–mass spectrometry of separated seed coats revealed significantly higher contents of proanthocyanidins (dimer and trimer of gallocatechin), quercetin, and myricetin rhamnosides and hydroxylated fatty acids in dormant compared to non-dormant genotypes. Bulk Segregant Analysis coupled to high throughput RNA sequencing resulted in identification of 770 and 148 differentially expressed genes between dormant and non-dormant seeds or dehiscent and indehiscent pods, respectively. The expression of 14 selected dormancy-related genes was studied by qRT-PCR. Of these, expression pattern of four genes: porin (MACE-S082), peroxisomal membrane PEX14-like protein (MACE-S108), 4-coumarate CoA ligase (MACE-S131), and UDP-glucosyl transferase (MACE-S139) was in agreement in all four genotypes with Massive analysis of cDNA Ends (MACE) data. In case of pod dehiscence, the analysis of two candidate genes (SHATTERING and SHATTERPROOF) and three out of 20 MACE identified genes (MACE-P004, MACE-P013, MACE-P015) showed down-expression in dorsal and ventral pod suture of indehiscent genotypes. Moreover, MACE-P015, the homolog of peptidoglycan-binding domain or proline-rich extensin-like protein mapped correctly to predicted Dpo1 locus on PsLGIII. This integrated analysis of the seed coat in wild and cultivated pea provides new insight as well as raises new questions associated with domestication and seed dormancy and pod dehiscence. PMID:28487704

Surface-Directed Assembly of Sequence-Defined Synthetic Polymers into Networks of Hexagonally Patterned Nanoribbons with Controlled Functionalities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Chun-Long; Zuckermann, Ronald N.; DeYoreo, James J.

The exquisite self-assembly of proteins and peptides in nature into highly ordered functional materials has inspired innovative approaches to biomimetic materials design and synthesis. Here we report the assembly of peptoids—a class of highly stable sequence-defined synthetic polymers—into biomimetic materials on mica surfaces. The assembling 12-mer peptoid contains alternating acidic and aromatic residues, and the presence of Ca2+ cations creates peptoid-peptoid and peptoid-mica interactions that drive assembly. In situ atomic force microscopy (AFM) shows that peptoids first assemble into discrete nanoparticles, these particles then transform into hexagonally-patterned nanoribbons on mica surfaces. AFM-based dynamic force spectroscopy (DFS) studies show that peptoid-micamore » interactions are much stronger than peptoidpeptoid interactions in the presence of Ca2+, illuminating the physical parameters that drive peptoid assembly. We further demonstrate the display of functional groups at the N-terminus of assembling peptoid sequence to produce biomimetic materials with similar hierarchical structures. This research demonstrates that surface-directed peptoid assembly can be used as a robust platform to develop biomimetic coating materials for applications.« less

Pepo aphid-borne yellows virus: a new species in the genus Polerovirus.

PubMed

Ibaba, Jacques D; Laing, Mark D; Gubba, Augustine

2017-02-01

Pepo aphid-borne yellows virus (PABYV) has been proposed as a putative representative of a new species in the genus Polerovirus in the family Luteoviridae. The genomes of two South African (SA) isolates of cucurbit-infecting PABYV were described in this record. Total RNA, extracted from a pattypan (Cucurbita pepo L.) and a baby marrow (C. pepo L.) leaf samples, was subjected to next-generation sequencing (NGS) on the HiSeq Illumina platform. Sanger sequencing was subsequently used to authenticate the integrity of PABYV's genome generated from de novo assembly of the NGS data. PABYV genome of SA isolates consists of 5813 nucleotides and displays an organisation typical of poleroviruses. Genome sequence comparisons of the SA PABYV isolates to other poleroviruses support the classification of PABYV as a new species in the genus Polerovirus. Recombination analyses showed that PABYV and Cucurbit aphid-borne yellows virus (CABYV) shared the same ancestor for the genome part situated between breaking points. Phylogenetic analyses of the RNA-dependent RNA polymerase and the coat protein genes showed that SA PABYV isolates shared distant relationship with CABYV and Suakwa aphid-borne yellows virus. Based on our results, we propose that PABYV is a distinct species in the genus Polerovirus.

Grapevine virus I, a putative new vitivirus detected in co-infection with grapevine virus G in New Zealand.

PubMed

Blouin, Arnaud G; Chooi, Kar Mun; Warren, Ben; Napier, Kathryn R; Barrero, Roberto A; MacDiarmid, Robin M

2018-05-01

A novel virus, with characteristics of viruses classified within the genus Vitivirus, was identified from a sample of Vitis vinifera cv. Chardonnay in New Zealand. The virus was detected with high throughput sequencing (small RNA and total RNA) and its sequence was confirmed by Sanger sequencing. Its genome is 7507 nt long (excluding the polyA tail) with an organisation similar to that described for other classifiable members of the genus Vitivirus. The closest relative of the virus is grapevine virus E (GVE) with 65% aa identity in ORF1 (65% nt identity) and 63% aa identity in the coat protein (66% nt identity). The relationship with GVE was confirmed with phylogenetic analysis, showing the new virus branching with GVE, Agave tequilina leaf virus and grapevine virus G (GVG). A limited survey revealed the presence of this virus in multiple plants from the same location where the newly described GVG was discovered, and in most cases both viruses were detected as co-infections. The genetic characteristics of this virus suggest it represents an isolate of a new species within the genus Vitivirus and following the current nomenclature, we propose the name "Grapevine virus I".

Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy.

PubMed

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 interactions. (c) 2009 Wiley Periodicals, Inc.

Silica coating influences the corona and biokinetics of cerium oxide nanoparticles.

PubMed

Konduru, Nagarjun V; Jimenez, Renato J; Swami, Archana; Friend, Sherri; Castranova, Vincent; Demokritou, Philip; Brain, Joseph D; Molina, Ramon M

2015-10-12

The physicochemical properties of nanoparticles (NPs) influence their biological outcomes. We assessed the effects of an amorphous silica coating on the pharmacokinetics and pulmonary effects of CeO2 NPs following intratracheal (IT) instillation, gavage and intravenous injection in rats. Uncoated and silica-coated CeO2 NPs were generated by flame spray pyrolysis and later neutron-activated. These radioactive NPs were IT-instilled, gavaged, or intravenously (IV) injected in rats. Animals were analyzed over 28 days post-IT, 7 days post-gavage and 2 days post-injection. Our data indicate that silica coating caused more but transient lung inflammation compared to uncoated CeO2. The transient inflammation of silica-coated CeO2 was accompanied by its enhanced clearance. Then, from 7 to 28 days, clearance was similar although significantly more (141)Ce from silica-coated (35%) was cleared than from uncoated (19%) (141)CeO2 in 28 days. The protein coronas of the two NPs were significantly different when they were incubated with alveolar lining fluid. Despite more rapid clearance from the lungs, the extrapulmonary (141)Ce from silica-coated (141)CeO2 was still minimal (<1%) although lower than from uncoated (141)CeO2 NPs. Post-gavage, nearly 100% of both NPs were excreted in the feces consistent with very low gut absorption. Both IV-injected (141)CeO2 NP types were primarily retained in the liver and spleen. The silica coating significantly altered the plasma protein corona composition and enhanced retention of (141)Ce in other organs except the liver. We conclude that silica coating of nanoceria alters the biodistribution of cerium likely due to modifications in protein corona formation after IT and IV administration.

Size-Dependent Protein-Nanoparticle Interactions in Citrate-Stabilized Gold Nanoparticles: The Emergence of the Protein Corona.

PubMed

Piella, Jordi; Bastús, Neus G; Puntes, Víctor

2017-01-18

Surface modifications of highly monodisperse citrate-stabilized gold nanoparticles (AuNPs) with sizes ranging from 3.5 to 150 nm after their exposure to cell culture media supplemented with fetal bovine serum were studied and characterized by the combined use of UV-vis spectroscopy, dynamic light scattering, and zeta potential measurements. In all the tested AuNPs, a dynamic process of protein adsorption was observed, evolving toward the formation of an irreversible hard protein coating known as Protein Corona. Interestingly, the thickness and density of this protein coating were strongly dependent on the particle size, making it possible to identify different transition regimes as the size of the particles increased: (i) NP-protein complexes (or incomplete corona), (ii) the formation of a near-single dense protein corona layer, and (iii) the formation of a multilayer corona. In addition, the different temporal patterns in the evolution of the protein coating came about more quickly for small particles than for the larger ones, further revealing the significant role that size plays in the kinetics of this process. Since the biological identity of the NPs is ultimately determined by the protein corona and different NP-biological interactions take place at different time scales, these results are relevant to biological and toxicological studies.

Historical overview of research on the tobacco mosaic virus genome: genome organization, infectivity and gene manipulation.

PubMed Central

Okada, Y

1999-01-01

Early in the development of molecular biology, TMV RNA was widely used as a mRNA [corrected] that could be purified easily, and it contributed much to research on protein synthesis. Also, in the early stages of elucidation of the genetic code, artificially produced TMV mutants were widely used and provided the first proof that the genetic code was non-overlapping. In 1982, Goelet et al. determined the complete TMV RNA base sequence of 6395 nucleotides. The four genes (130K, 180K, 30K and coat protein) could then be mapped at precise locations in the TMV genome. Furthermore it had become clear, a little earlier, that genes located internally in the genome were expressed via subgenomic mRNAs. The initiation site for assembly of TMV particles was also determined. However, although TMV contributed so much at the beginning of the development of molecular biology, its influence was replaced by that of Escherichia coli and its phages in the next phase. As recombinant DNA technology developed in the 1980s, RNA virus research became more detached from the frontier of molecular biology. To recover from this setback, a gene-manipulation system was needed for RNA viruses. In 1986, two such systems were developed for TMV, using full-length cDNA clones, by Dawson's group and by Okada's group. Thus, reverse genetics could be used to elucidate the basic functions of all proteins encoded by the TMV genome. Identification of the function of the 30K protein was especially important because it was the first evidence that a plant virus possesses a cell-to-cell movement function. Many other plant viruses have since been found to encode comparable 'movement proteins'. TMV thus became the first plant virus for which structures and functions were known for all its genes. At the birth of molecular plant pathology, TMV became a leader again. TMV has also played pioneering roles in many other fields. TMV was the first virus for which the amino acid sequence of the coat protein was determined and first virus for which cotranslational disassembly was demonstrated both in vivo and in vitro. It was the first virus for which activation of a resistance gene in a host plant was related to the molecular specificity of a product of a viral gene. Also, in the field of plant biotechnology, TMV vectors are among the most promising. Thus, for the 100 years since Beijerinck's work, TMV research has consistently played a leading role in opening up new areas of study, not only in plant pathology, but also in virology, biochemistry, molecular biology, RNA genetics and biotechnology. PMID:10212936

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

PubMed

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.

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

USDA-ARS?s Scientific Manuscript database

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...

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

USDA-ARS?s Scientific Manuscript database

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 ...

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

PubMed

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.

Algorithm, applications and evaluation for protein comparison by Ramanujan Fourier transform.

PubMed

Zhao, Jian; Wang, Jiasong; Hua, Wei; Ouyang, Pingkai

2015-12-01

The amino acid sequence of a protein determines its chemical properties, chain conformation and biological functions. Protein sequence comparison is of great importance to identify similarities of protein structures and infer their functions. Many properties of a protein correspond to the low-frequency signals within the sequence. Low frequency modes in protein sequences are linked to the secondary structures, membrane protein types, and sub-cellular localizations of the proteins. In this paper, we present Ramanujan Fourier transform (RFT) with a fast algorithm to analyze the low-frequency signals of protein sequences. The RFT method is applied to similarity analysis of protein sequences with the Resonant Recognition Model (RRM). The results show that the proposed fast RFT method on protein comparison is more efficient than commonly used discrete Fourier transform (DFT). RFT can detect common frequencies as significant feature for specific protein families, and the RFT spectrum heat-map of protein sequences demonstrates the information conservation in the sequence comparison. The proposed method offers a new tool for pattern recognition, feature extraction and structural analysis on protein sequences. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fabrication of robust hydrogel coatings on polydimethylsiloxane substrates using micropillar anchor structures with chemical surface modification.

PubMed

Zhang, Hongbin; Bian, Chao; Jackson, John K; Khademolhosseini, Farzad; Burt, Helen M; Chiao, Mu

2014-06-25

A durable hydrophilic and protein-resistant surface of polydimethylsiloxane (PDMS) based devices is desirable in many biomedical applications such as implantable and microfluidic devices. This paper describes a stable antifouling hydrogel coating on PDMS surfaces. The coating method combines chemical modification and surface microstructure fabrication of PDMS substrates. Three-(trimethoxysilyl)propyl methacrylates containing C═C groups were used to modify PDMS surfaces with micropillar array structures fabricated by a replica molding method. The micropillar structures increase the surface area of PDMS surfaces, which facilitates secure bonding with a hydrogel coating compared to flat PMDS surfaces. The adhesion properties of the hydrogel coating on PDMS substrates were characterized using bending, stretching and water immersion tests. Long-term hydrophilic stability (maintaining a contact angle of 55° for a month) and a low protein adsorption property (35 ng/cm(2) of adsorbed BSA-FITC) of the hydrogel coated PDMS were demonstrated. This coating method is suitable for PDMS modification with most crosslinkable polymers containing C═C groups, which can be useful for improving the anti-biofouling performance of PDMS-based biomedical microdevices.

Reactive polymer coatings: A robust platform towards sophisticated surface engineering for biotechnology

NASA Astrophysics Data System (ADS)

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 storage time prior to bonding, and (iii) well-defined surface functionalities for subsequent surface modifications. Finally, we have also prepared surface microstructures and surface patterns using reactive coatings via photopatterning, projection lithography, supramolecular nanostamping (SuNS), and vapor-assisted micropatterning in replica structures (VAMPIR). These patterning techniques can be complimentarily used and provide access to precisely confined microenvironments on flat and curved geometries. Reactive coatings provide a technology platform that creates active, long-term control and may lead to improved mimicry of biological systems for effective bio-functional modifications.

Kinetic Effects on Self-Assembly and Function of Protein-Polymer Bioconjugates in Thin Films Prepared by Flow Coating.

PubMed

Chang, Dongsook; Huang, Aaron; Olsen, Bradley D

2017-01-01

The self-assembly of nanostructured globular protein arrays in thin films is demonstrated using protein-polymer block copolymers based on a model protein mCherry and the polymer poly(oligoethylene glycol acrylate) (POEGA). Conjugates are flow coated into thin films on a poly(ethylene oxide) grafted Si surface, forming self-assembled cylindrical nanostructures with POEGA domains selectively segregating to the air-film interface. Long-range order and preferential arrangement of parallel cylinders templated by selective surfaces are demonstrated by controlling relative humidity. Long-range order increases with coating speed when the film thicknesses are kept constant, due to reduced nucleation per unit area of drying film. Fluorescence emission spectra of mCherry in films prepared at <25% relative humidity shows a small shift suggesting that proteins are more perturbed at low humidity than high humidity or the solution state. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Membrane Fission: Model for Intermediate Structures

PubMed Central

Kozlovsky, Yonathan; Kozlov, Michael M.

2003-01-01

Membrane budding-fission is a fundamental process generating intracellular carriers of proteins. Earlier works were focused only on formation of coated buds connected to the initial membrane by narrow membrane necks. We present the theoretical analysis of the whole pathway of budding-fission, including the crucial stage where the membrane neck undergoes fission and the carrier separates from the donor membrane. We consider two successive intermediates of the reaction: 1), a constricted membrane neck coming out of aperture of the assembling protein coat, and 2), hemifission intermediate resulting from self-fusion of the inner monolayer of the neck, while its outer monolayer remains continuous. Transformation of the constricted neck into the hemifission intermediate is driven by the membrane stress produced in the neck by the protein coat. Although apparently similar to hemifusion, the fission is predicted to have an opposite dependence on the monolayer spontaneous curvature. Analysis of the further stages of the process demonstrates that in all practically important cases the hemifission intermediate decays spontaneously into two separate membranes, thereby completing the fission process. We formulate the “job description” for fission proteins by calculating the energy they have to deliver and the radii of the protein coat aperture which have to be reached to drive the fission process. PMID:12829467

Coats' disease of the retina (unilateral retinal telangiectasis) caused by somatic mutation in the NDP gene: a role for norrin in retinal angiogenesis.

PubMed

Black, G C; Perveen, R; Bonshek, R; Cahill, M; Clayton-Smith, J; Lloyd, I C; McLeod, D

1999-10-01

Coats' disease is characterized by abnormal retinal vascular development (so-called 'retinal telangiectasis') which results in massive intraretinal and subretinal lipid accumulation (exudative retinal detachment). The classical form of Coats' disease is almost invariably isolated, unilateral and seen in males. A female with a unilateral variant of Coats' disease gave birth to a son affected by Norrie disease. Both carried a missense mutation within the NDP gene on chromosome Xp11.2. Subsequently analysis of the retinas of nine enucleated eyes from males with Coats' disease demonstrated in one a somatic mutation in the NDP gene which was not present within non-retinal tissue. We suggest that Coats' telangiectasis is secondary to somatic mutation in the NDP gene which results in a deficiency of norrin (the protein product of the NDP gene) within the developing retina. This supports recent observations that the protein is critical for normal retinal vasculogenesis.

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

PubMed

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).

Folding and Stabilization of Native-Sequence-Reversed Proteins

PubMed Central

Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

2016-01-01

Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

Folding and Stabilization of Native-Sequence-Reversed Proteins

NASA Astrophysics Data System (ADS)

Zhang, Yuanzhao; Weber, Jeffrey K.; Zhou, Ruhong

2016-04-01

Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols.

Phosphatidic acid phospholipase A1 mediates ER–Golgi transit of a family of G protein–coupled receptors

PubMed Central

Kunduri, Govind; Yuan, Changqing; Parthibane, Velayoudame; Nyswaner, Katherine M.; Kanwar, Ritu; Nagashima, Kunio; Britt, Steven G.; Mehta, Nickita; Kotu, Varshika; Porterfield, Mindy; Tiemeyer, Michael; Dolph, Patrick J.; Acharya, Usha

2014-01-01

The coat protein II (COPII)–coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein–coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking. PMID:25002678