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Sample records for hepadnavirus core proteins

  1. Cyclin-dependent kinase 2 phosphorylates s/t-p sites in the hepadnavirus core protein C-terminal domain and is incorporated into viral capsids.

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    Ludgate, Laurie; Ning, Xiaojun; Nguyen, David H; Adams, Christina; Mentzer, Laura; Hu, Jianming

    2012-11-01

    Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.

  2. Nuclear factor Y regulates ancient budgerigar hepadnavirus core promoter activity.

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    Shen, Zhongliang; Liu, Yanfeng; Luo, Mengjun; Wang, Wei; Liu, Jing; Liu, Wei; Pan, Shaokun; Xie, Youhua

    2016-09-16

    Endogenous viral elements (EVE) in animal genomes are the fossil records of ancient viruses and provide invaluable information on the origin and evolution of extant viruses. Extant hepadnaviruses include avihepadnaviruses of birds and orthohepadnaviruses of mammals. The core promoter (Cp) of hepadnaviruses is vital for viral gene expression and replication. We previously identified in the budgerigar genome two EVEs that contain the full-length genome of an ancient budgerigar hepadnavirus (eBHBV1 and eBHBV2). Here, we found eBHBV1 Cp and eBHBV2 Cp were active in several human and chicken cell lines. A region from nt -85 to -11 in eBHBV1 Cp was critical for the promoter activity. Bioinformatic analysis revealed a putative binding site of nuclear factor Y (NF-Y), a ubiquitous transcription factor, at nt -64 to -50 in eBHBV1 Cp. The NF-Y core binding site (ATTGG, nt -58 to -54) was essential for eBHBV1 Cp activity. The same results were obtained with eBHBV2 Cp and duck hepatitis B virus Cp. The subunit A of NF-Y (NF-YA) was recruited via the NF-Y core binding site to eBHBV1 Cp and upregulated the promoter activity. Finally, the NF-Y core binding site is conserved in the Cps of all the extant avihepadnaviruses but not of orthohepadnaviruses. Interestingly, a putative and functionally important NF-Y core binding site is located at nt -21 to -17 in the Cp of human hepatitis B virus. In conclusion, our findings have pinpointed an evolutionary conserved and functionally critical NF-Y binding element in the Cps of avihepadnaviruses.

  3. Identification and analysis of a new hepadnavirus in white storks.

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    Pult, I; Netter, H J; Bruns, M; Prassolov, A; Sirma, H; Hohenberg, H; Chang, S F; Frölich, K; Krone, O; Kaleta, E F; Will, H

    2001-10-10

    We identified, cloned, and functionally characterized a new avian hepadnavirus infecting storks (STHBV). STHBV has the largest DNA genome of all avian hepadnaviruses and, based on sequence and phylogenetic analysis, is most closely related to, but distinct from, heron hepatitis B virus (HHBV). Unique for STHBV among the other avian hepadnaviruses is a potential HNF1 binding site in the preS promoter. In common only with HHBV, STHBV has a myristylation signal on the S and not the preS protein, two C terminally located glycosylation sites on the precore/core proteins and lacks the phosphorylation site essential for the transcriptional transactivation activity of duck-HBV preS protein. The cloned STHBV genomes were competent in gene expression, replication, and viral particle secretion. STHBV infected primary duck hepatocytes very inefficiently suggesting a restricted host range, similar to other hepadnaviruses. This discovery of stork infections unravels novel evolutionary aspects of hepadnaviruses and provides new opportunities for hepadnavirus research.

  4. Characterization of a novel hepadnavirus in the white sucker (Catostomus commersonii) from the Great Lakes Region of the USA

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    Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Conway, Carla M.; Winton, James R.; Blazer, Vicki S.

    2015-01-01

    The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the USA. Dideoxysequencing confirmed the white sucker hepatitis B virus (WSHBV) has a circular genome (3542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase and surface proteins was low and ranged from 16-27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively non-coding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV other hepadnaviruses, and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, of which only 2.4% were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker.

  5. A new hepadnavirus endemic in arctic ground squirrels in Alaska.

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    Testut, P; Renard, C A; Terradillos, O; Vitvitski-Trepo, L; Tekaia, F; Degott, C; Blake, J; Boyer, B; Buendia, M A

    1996-07-01

    We present evidence for a novel member of the hepadnavirus family that is endemic in wild arctic ground squirrels (Spermophylus parryi kennicotti) in Alaska. This virus, designated arctic squirrel hepatitis virus (ASHV), was initially detected in the livers of animals bearing large hepatic nodules by nucleic acid hybridization with hepadnavirus probes and in plasma by cross-reactivity with antibodies to hepadnavirus surface and core antigens. The complete nucleotide sequence of the 3,302-bp-long ASHV genome was determined and compared with those of ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV); all sequences were organized into four open reading frames, designated pre-C/C, pre-S/S, pol, and X. Despite roughly equivalent variability among the three rodent hepadnaviruses (around 16% base and 19% amino acid exchanges), ASHV appeared to be more closely related to GSHV than to WHV in phylogenetic analysis. Accordingly, preliminary studies of the pathology of ASHV infection suggested that ASHV may be a less efficient oncogenic agent than WHV. About one-third of aged animals maintained in captivity, including virus-infected as well as uninfected squirrels, developed large liver nodules, consisting of hepatocellular adenomas or carcinomas or nonmalignant lesions characterized by drastic microvesicular steatosis. ASHV-infected arctic ground squirrels may serve as a new model with which to analyze the contribution of hepadnavirus- and host-specific determinants to liver pathology and tumorigenesis.

  6. Packing in protein cores

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    Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

    2017-07-01

    Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

  7. Endogenous hepadnaviruses, bornaviruses and circoviruses in snakes

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    Gilbert, C.; Meik, J. M.; Dashevsky, D.; Card, D. C.; Castoe, T. A.; Schaack, S.

    2014-01-01

    We report the discovery of endogenous viral elements (EVEs) from Hepadnaviridae, Bornaviridae and Circoviridae in the speckled rattlesnake, Crotalus mitchellii, the first viperid snake for which a draft whole genome sequence assembly is available. Analysis of the draft assembly reveals genome fragments from the three virus families were inserted into the genome of this snake over the past 50 Myr. Cross-species PCR screening of orthologous loci and computational scanning of the python and king cobra genomes reveals that circoviruses integrated most recently (within the last approx. 10 Myr), whereas bornaviruses and hepadnaviruses integrated at least approximately 13 and approximately 50 Ma, respectively. This is, to our knowledge, the first report of circo-, borna- and hepadnaviruses in snakes and the first characterization of non-retroviral EVEs in non-avian reptiles. Our study provides a window into the historical dynamics of viruses in these host lineages and shows that their evolution involved multiple host-switches between mammals and reptiles. PMID:25080342

  8. Genomic fossils calibrate the long-term evolution of hepadnaviruses.

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    Clément Gilbert

    Full Text Available Because most extant viruses mutate rapidly and lack a true fossil record, their deep evolution and long-term substitution rates remain poorly understood. In addition to retroviruses, which rely on chromosomal integration for their replication, many other viruses replicate in the nucleus of their host's cells and are therefore prone to endogenization, a process that involves integration of viral DNA into the host's germline genome followed by long-term vertical inheritance. Such endogenous viruses are highly valuable as they provide a molecular fossil record of past viral invasions, which may be used to decipher the origins and long-term evolutionary characteristics of modern pathogenic viruses. Hepadnaviruses (Hepadnaviridae are a family of small, partially double-stranded DNA viruses that include hepatitis B viruses. Here we report the discovery of endogenous hepadnaviruses in the genome of the zebra finch. We used a combination of cross-species analysis of orthologous insertions, molecular dating, and phylogenetic analyses to demonstrate that hepadnaviruses infiltrated repeatedly the germline genome of passerine birds. We provide evidence that some of the avian hepadnavirus integration events are at least 19 My old, which reveals a much deeper ancestry of Hepadnaviridae than could be inferred based on the coalescence times of modern hepadnaviruses. Furthermore, the remarkable sequence similarity between endogenous and extant avian hepadnaviruses (up to 75% identity suggests that long-term substitution rates for these viruses are on the order of 10(-8 substitutions per site per year, which is a 1,000-fold slower than short-term rates estimated based on the sequences of circulating hepadnaviruses. Together, these results imply a drastic shift in our understanding of the time scale of hepadnavirus evolution, and suggest that the rapid evolutionary dynamics characterizing modern avian hepadnaviruses do not reflect their mode of evolution on a deep

  9. Mechanism for CCC DNA synthesis in hepadnaviruses.

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    Ji A Sohn

    Full Text Available Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC DNA from the relaxed circular (RC viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT, or cellular DNA repair enzymes, or both. Physical mapping of the 5' and 3' ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5' ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1 invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2 predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5' end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.

  10. Hepadnaviruses and retroviruses share genome homology and features of replication.

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    Robinson, W S; Miller, R H; Marion, P L

    1987-01-01

    The hepadnavirus family includes hepatitis B virus (HBV), woodchuck hepatitis virus (WHV), ground squirrel hepatitis virus (GSHV) and duck hepatitis B virus (DHBV). These viruses share unique ultrastructural, molecular and biological features. HBV has great medical importance in many parts of the world. More important numerically than acute hepatitis B in high prevalence geographic regions is the liver disease associated with chronic infection. There appear to be more than 200 million chronically infected humans in the world, and these HBV infections appear to be the single most common cause of chronic liver disease and liver cancer in man. All hepadnaviruses share the propensity for silent infection in early life leading to persistence of the virus, and hepatocellular carcinoma (HCC) is clearly associated with long-standing persistent infection in man, woodchucks and ground squirrels. Although the viral DNA has been found to be integrated in cellular DNA of many HCC in man, woodchucks and ground squirrels, the precise role of the virus in tumor formation has not been defined. Hepadna viruses have an interesting molecular structure and mechanisms of replication, and they appear to share certain important features with retroviruses as reviewed here. It is of interest to define similarities and differences between hepadnaviruses and retroviruses in order to understand their evolutionary relationship and to determine whether they share a common oncogenic mechanism, since infection with members of both virus families is associated with neoplastic disease.

  11. Alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccDNA minichromosomes.

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    Fei Liu

    2013-09-01

    Full Text Available Covalently closed circular DNA (cccDNA of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9 and 27 (H3K27 in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection.

  12. Differential Stoichiometry among Core Ribosomal Proteins

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    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  13. Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

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    Rinker, Torri E; Philbrick, Brandon D; Temenoff, Johnna S

    2016-12-21

    Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation.

  14. The core and unique proteins of haloarchaea

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    Capes Melinda D

    2012-01-01

    Full Text Available Abstract Background Since the first genome of a halophilic archaeon was sequenced in 2000, biologists have been advancing the understanding of genomic characteristics that allow for survival in the harsh natural environments of these organisms. An increase in protein acidity and GC-bias in the genome have been implicated as factors in tolerance to extreme salinity, desiccation, and high solar radiation. However, few previous attempts have been made to identify novel genes that would permit survival in such extreme conditions. Results With the recent release of several new complete haloarchaeal genome sequences, we have conducted a comprehensive comparative genomic analysis focusing on the identification of unique haloarchaeal conserved proteins that likely play key roles in environmental adaptation. Using bioinformatic methods, we have clustered 31,312 predicted proteins from nine haloarchaeal genomes into 4,455 haloarchaeal orthologous groups (HOGs. We assigned likely functions by association with established COG and KOG databases in NCBI. After identifying homologs in four additional haloarchaeal genomes, we determined that there were 784 core haloarchaeal protein clusters (cHOGs, of which 83 clusters were found primarily in haloarchaea. Further analysis found that 55 clusters were truly unique (tucHOGs to haloarchaea and qualify as signature proteins while 28 were nearly unique (nucHOGs, the vast majority of which were coded for on the haloarchaeal chromosomes. Of the signature proteins, only one example with any predicted function, Ral, involved in desiccation/radiation tolerance in Halobacterium sp. NRC-1, was identified. Among the core clusters, 33% was predicted to function in metabolism, 25% in information transfer and storage, 10% in cell processes and signaling, and 22% belong to poorly characterized or general function groups. Conclusion Our studies have established conserved groups of nearly 800 protein clusters present in all

  15. Serological and molecular evidence of hepadnavirus infection in swine

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    Yasmine R Vieira

    2015-02-01

    Full Text Available [b]Introduction and objective[/b]. Recently, investigations in a swine herd identified evidence of the existence of a novel member of the Hepadnavirus family endemic in swine. The aim of this study was to investigate the serological and molecular markers of Hepadnavirus circulation in Brazilian domestic swine and wild boar herds, and to evaluate the identity with HBV and other Hepadnaviruses reported previously. [b]Materials and methods[/b]. For the study, 376 swine were screened for hepatitis B virus serological markers. Analyses were performed in serum samples using commercial enzyme-linked immunosorbent assay (ELISA kits (DiaSorin® for anti-HBc, HBsAg and anti-HBs. Reactive and undetermined swine serum samples were selected to perform DNA viral extraction (QIAamp DNA Mini Kit, Qiagen®, partial genome amplification and genome sequencing. [b]Results[/b]. From 376 swine samples analysed, 28 (7.45% were reactive to anti-HBc, 3 (0.80% to HBsAg and 6 (1.6% to anti-HBs. Besides, more 17 (4.52% swine samples analyzed were classified in the grey zone of the EIA test to anti-HBc and 2 (0.53% to HBsAg. From 49 samples molecularly analyzed after serological trial, 4 samples showed a positive result for the qualitative PCR for Hepadnavirus. Phylogenetic reconstruction using partial genome sequencing (360 bp of 3 samples showed similarity with HBV with 90.8–96.3% of identity. [b]Conclusions.[/b] Serological and molecular data showed evidence of the circulation of a virus similar to hepatitis B virus in swine.

  16. Chimeric hepatitis B virus core particles with parts or copies of the hepatitis C virus core protein.

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    Yoshikawa, A.; Tanaka, T; Hoshi, Y.; Kato, N; K. Tachibana; Iizuka, H; Machida, A; Okamoto, H; Yamasaki, M.; Miyakawa, Y

    1993-01-01

    Either parts or multiple copies of the core gene of hepatitis C virus (HCV) were fused to the 3' terminus of the hepatitis B virus (HBV) core gene with 34 codons removed. As many as four copies of HCV core protein (720 amino acids) were fused to the carboxy terminus of truncated HBV core protein (149 amino acids) without preventing the assembly of HBV core particles. Chimeric core particles were sandwiched between monoclonal antibody to HBV core and that to HCV core, thereby indicating that a...

  17. Chimeric hepatitis B virus core particles with parts or copies of the hepatitis C virus core protein.

    OpenAIRE

    Yoshikawa, A.; Tanaka, T; Hoshi, Y.; Kato, N.; Tachibana, K; Iizuka, H.; Machida, A; Okamoto, H; Yamasaki, M.; Miyakawa, Y

    1993-01-01

    Either parts or multiple copies of the core gene of hepatitis C virus (HCV) were fused to the 3' terminus of the hepatitis B virus (HBV) core gene with 34 codons removed. As many as four copies of HCV core protein (720 amino acids) were fused to the carboxy terminus of truncated HBV core protein (149 amino acids) without preventing the assembly of HBV core particles. Chimeric core particles were sandwiched between monoclonal antibody to HBV core and that to HCV core, thereby indicating that a...

  18. Purification and characterization of adenovirus core protein VII: a histone-like protein that is critical for adenovirus core formation.

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    Sharma, Gaurav; Moria, Nithesh; Williams, Martin; Krishnarjuna, Bankala; Pouton, Colin W

    2017-07-01

    Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

  19. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

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    Ngo, HT; Pham, Long; Kim, JW;

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray......, approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...

  20. Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

    Institute of Scientific and Technical Information of China (English)

    杨莉; 王春林; 汪垣; 李光地

    1999-01-01

    Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d

  1. Variability and conservation in hepatitis B virus core protein

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    Myers Richard

    2005-05-01

    Full Text Available Abstract Background Hepatitis B core protein (HBVc has been extensively studied from both a structural and immunological point of view, but the evolutionary forces driving sequence variation within core are incompletely understood. Results In this study, the observed variation in HBVc protein sequence has been examined in a collection of a large number of HBVc protein sequences from public sequence repositories. An alignment of several hundred sequences was carried out, and used to analyse the distribution of polymorphisms along the HBVc. Polymorphisms were found at 44 out of 185 amino acid positions analysed and were clustered predominantly in those parts of HBVc forming the outer surface and spike on intact capsid. The relationship between HBVc diversity and HBV genotype was examined. The position of variable amino acids along the sequence was examined in terms of the structural constraints of capsid and envelope assembly, and also in terms of immunological recognition by T and B cells. Conclusion Over three quarters of amino acids within the HBVc sequence are non-polymorphic, and variation is focused to a few amino acids. Phylogenetic analysis suggests that core protein specific forces constrain its diversity within the context of overall HBV genome evolution. As a consequence, core protein is not a reliable predictor of virus genotype. The structural requirements of capsid assembly are likely to play a major role in limiting diversity. The phylogenetic analysis further suggests that immunological selection does not play a major role in driving HBVc diversity.

  2. Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

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    Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

    1990-04-01

    The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

  3. Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus.

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    Deroubaix, Aurélie; Osseman, Quentin; Cassany, Aurélia; Bégu, Dominique; Ragues, Jessica; Kassab, Somar; Lainé, Sébastien; Kann, Michael

    2015-01-01

    Biopsies from patients show that hepadnaviral core proteins and capsids - collectively called core - are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that ϵ, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.

  4. C-terminal domain of hepatitis C virus core protein is essential for secretion

    Institute of Scientific and Technical Information of China (English)

    Soo-Ho Choi; Kyu-Jin Park; So-Yeon Kim; Dong-Hwa Choi; Jung-Min Park; Soon B. Hwang

    2005-01-01

    AIM: We have previously demonstrated that hepatitis C virus (HCV) core protein is efficiently released into the culture medium in insect cells. The objective of this study is to characterize the HCV core secretion in insect cells.METHODS: We constructed recombinant baculoviruses expressing various-length of mutant core proteins, expressed these proteins in insect cells, and examined core protein secretion in insect cells.RESULTS: Only wild type core was efficiently released into the culture medium, although the protein expression level of wild type core was lower than those of other mutant core proteins. We found that the shorter form of the core construct expressed the higher level of protein. However, if more than 18 amino acids of the core were truncated at the C-terminus,core proteins were no longer seareted into the culture medium.Membrane flotation data show that the secreted core proteins are associated with the cellular membrane protein, indicating that HCV core is secreted as a membrane complex.CONCLUSION: The C-terminal 18 amino acids of HCV core were crucial for core secretion into the culture media.Since HCV replication occurs on lipid raft membrane structure,these results suggest that HCV may utilize a unique core release mechanism to escape immune surveillance, thereby potentially representing the feature of HCV morphogenesis.

  5. Homogeneous protein analysis by magnetic core-shell nanorod probes

    KAUST Repository

    Schrittwieser, Stefan

    2016-03-29

    Studying protein interactions is of vital importance both to fundamental biology research and to medical applications. Here, we report on the experimental proof of a universally applicable label-free homogeneous platform for rapid protein analysis. It is based on optically detecting changes in the rotational dynamics of magnetically agitated core-shell nanorods upon their specific interaction with proteins. By adjusting the excitation frequency, we are able to optimize the measurement signal for each analyte protein size. In addition, due to the locking of the optical signal to the magnetic excitation frequency, background signals are suppressed, thus allowing exclusive studies of processes at the nanoprobe surface only. We study target proteins (soluble domain of the human epidermal growth factor receptor 2 - sHER2) specifically binding to antibodies (trastuzumab) immobilized on the surface of our nanoprobes and demonstrate direct deduction of their respective sizes. Additionally, we examine the dependence of our measurement signal on the concentration of the analyte protein, and deduce a minimally detectable sHER2 concentration of 440 pM. For our homogeneous measurement platform, good dispersion stability of the applied nanoprobes under physiological conditions is of vital importance. To that end, we support our measurement data by theoretical modeling of the total particle-particle interaction energies. The successful implementation of our platform offers scope for applications in biomarker-based diagnostics as well as for answering basic biology questions.

  6. The expanded FindCore method for identification of a core atom set for assessment of protein structure prediction.

    Science.gov (United States)

    Snyder, David A; Grullon, Jennifer; Huang, Yuanpeng J; Tejero, Roberto; Montelione, Gaetano T

    2014-02-01

    Maximizing the scientific impact of NMR-based structure determination requires robust and statistically sound methods for assessing the precision of NMR-derived structures. In particular, a method to define a core atom set for calculating superimpositions and validating structure predictions is critical to the use of NMR-derived structures as targets in the CASP competition. FindCore (Snyder and Montelione, Proteins 2005;59:673-686) is a superimposition independent method for identifying a core atom set and partitioning that set into domains. However, as FindCore optimizes superimposition by sensitively excluding not-well-defined atoms, the FindCore core may not comprise all atoms suitable for use in certain applications of NMR structures, including the CASP assessment process. Adapting the FindCore approach to assess predicted models against experimental NMR structures in CASP10 required modification of the FindCore method. This paper describes conventions and a standard protocol to calculate an "Expanded FindCore" atom set suitable for validation and application in biological and biophysical contexts. A key application of the Expanded FindCore method is to identify a core set of atoms in the experimental NMR structure for which it makes sense to validate predicted protein structure models. We demonstrate the application of this Expanded FindCore method in characterizing well-defined regions of 18 NMR-derived CASP10 target structures. The Expanded FindCore protocol defines "expanded core atom sets" that match an expert's intuition of which parts of the structure are sufficiently well defined to use in assessing CASP model predictions. We also illustrate the impact of this analysis on the CASP GDT assessment scores.

  7. Trafficking of hepatitis C virus core protein during virus particle assembly.

    Directory of Open Access Journals (Sweden)

    Natalie A Counihan

    2011-10-01

    Full Text Available Hepatitis C virus (HCV core protein is directed to the surface of lipid droplets (LD, a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly.

  8. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wen-Ta [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Li, Hui-Chun [Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China); Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Lo, Shih-Yen, E-mail: losylo@mail.tcu.edu.tw [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (China)

    2013-05-24

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

  9. Protein coalitions in a core mammalian biochemical network linked by rapidly evolving proteins

    Directory of Open Access Journals (Sweden)

    Tsoka Sophia

    2011-05-01

    Full Text Available Abstract Background Cellular ATP levels are generated by glucose-stimulated mitochondrial metabolism and determine metabolic responses, such as glucose-stimulated insulin secretion (GSIS from the β-cells of pancreatic islets. We describe an analysis of the evolutionary processes affecting the core enzymes involved in glucose-stimulated insulin secretion in mammals. The proteins involved in this system belong to ancient enzymatic pathways: glycolysis, the TCA cycle and oxidative phosphorylation. Results We identify two sets of proteins, or protein coalitions, in this group of 77 enzymes with distinct evolutionary patterns. Members of the glycolysis, TCA cycle, metabolite transport, pyruvate and NADH shuttles have low rates of protein sequence evolution, as inferred from a human-mouse comparison, and relatively high rates of evolutionary gene duplication. Respiratory chain and glutathione pathway proteins evolve faster, exhibiting lower rates of gene duplication. A small number of proteins in the system evolve significantly faster than co-pathway members and may serve as rapidly evolving adapters, linking groups of co-evolving genes. Conclusions Our results provide insights into the evolution of the involved proteins. We find evidence for two coalitions of proteins and the role of co-adaptation in protein evolution is identified and could be used in future research within a functional context.

  10. HSC90 is required for nascent hepatitis C virus core protein stability in yeast cells.

    Science.gov (United States)

    Kubota, Naoko; Inayoshi, Yasutaka; Satoh, Naoko; Fukuda, Takashi; Iwai, Kenta; Tomoda, Hiroshi; Kohara, Michinori; Kataoka, Kazuhiro; Shimamoto, Akira; Furuichi, Yasuhiro; Nomoto, Akio; Naganuma, Akira; Kuge, Shusuke

    2012-07-30

    Hepatitis C virus core protein (Core) contributes to HCV pathogenicity. Here, we demonstrate that Core impairs growth in budding yeast. We identify HSP90 inhibitors as compounds that reduce intracellular Core protein level and restore yeast growth. Our results suggest that HSC90 (Hsc82) may function in the protection of the nascent Core polypeptide against degradation in yeast and the C-terminal region of Core corresponding to the organelle-interaction domain was responsible for Hsc82-dependent stability. The yeast system may be utilized to select compounds that can direct the C-terminal region to reduce the stability of Core protein. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Directory of Open Access Journals (Sweden)

    Andrea Cerutti

    Full Text Available Hepatitis C virus (HCV infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS, but no nuclear export signal (NES has yet been identified.We show here that the aa(109-133 region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126 in the identified NES or in the sequence encoding the mature core aa(1-173 significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  12. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Science.gov (United States)

    Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

    2011-01-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  13. The retrovirus MA and PreTM proteins follow immature MVL cores

    DEFF Research Database (Denmark)

    Andersen, Klaus Bahl

    2013-01-01

    Detergent can dissolve retrovirus, exept the immature core. Here we show that the Matrix protein (MA) and the Transmembrane protein in its immature form (PreTM) bind to the retrovirus core. These attachments explain the attachment in the virus particle and the dynamics of the ability to fuse...

  14. The role of whey acidic protein four-disulfide-core proteins in respiratory health and disease.

    Science.gov (United States)

    Small, Donna M; Doherty, Declan F; Dougan, Caoifa M; Weldon, Sinéad; Taggart, Clifford C

    2017-04-01

    Members of the whey acidic protein (WAP) or WAP four-disulfide-core (WFDC) family of proteins are a relatively under-explored family of low molecular weight proteins. The two most prominent WFDC proteins, secretory leukocyte protease inhibitor (SLPI) and elafin (or the precursor, trappin-2), have been shown to possess multiple functions including anti-protease, anti-bacterial, anti-viral and anti-inflammatory properties. It is therefore of no surprise that both SLPI and elafin/trappin-2 have been developed as potential therapeutics. Given the abundance of SLPI and elafin/trappin-2 in the human lung, most work in the area of WFDC research has focused on the role of WFDC proteins in protecting the lung from proteolytic attack. In this review, we will outline the current evidence regarding the expanding role of WFDC protein function with a focus on WFDC activity in lung disease as well as emerging data regarding the function of some of the more recently described WFDC proteins.

  15. Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV core DII protein.

    Science.gov (United States)

    Lyn, Rodney K; Hope, Graham; Sherratt, Allison R; McLauchlan, John; Pezacki, John Paul

    2013-01-01

    Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein's lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV.

  16. HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Yongsheng, E-mail: yongshengtanwhu@126.com; Li, Yan, E-mail: liyansd2@163.com

    2015-10-23

    This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 {sup low} and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96{sup ®}Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 {sup low}, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases

  17. Similar Energetic Contributions of Packing in the Core of Membrane and Water-Soluble Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Joh, Nathan H.; Oberai, Amit; Yang, Duan; Whitelegge, Julian P.; Bowie, James U.; (UCLA)

    2009-09-15

    A major driving force for water-soluble protein folding is the hydrophobic effect, but membrane proteins cannot make use of this stabilizing contribution in the apolar core of the bilayer. It has been proposed that membrane proteins compensate by packing more efficiently. We therefore investigated packing contributions experimentally by observing the energetic and structural consequences of cavity creating mutations in the core of a membrane protein. We observed little difference in the packing energetics of water and membrane soluble proteins. Our results imply that other mechanisms are employed to stabilize the structure of membrane proteins.

  18. Abnormal distribution of calcium-handling proteins: a novel distinctive marker in core myopathies.

    Science.gov (United States)

    Herasse, Muriel; Parain, Karine; Marty, Isabelle; Monnier, Nicole; Kaindl, Angela M; Leroy, Jean-Paul; Richard, Pascale; Lunardi, Jöel; Romero, Norma B; Ferreiro, Ana

    2007-01-01

    Central core disease (CCD) and multi-minicore disease (MmD) are muscle disorders characterized by foci of mitochondria depletion and sarcomere disorganization ("cores") in muscle fibers. Although core myopathies are the most frequent congenital myopathies, their pathogenesis remains elusive and specific diagnostic markers are lacking. Core myopathies are mostly caused by mutations in 2 sarcoplasmic reticulum proteins: the massive Ca-release channel RyR1 or the selenoprotein N (SelN) of unknown function. To search for distinctive markers and to obtain further pathophysiological insight, we identified the molecular defects in 12 core myopathy patients and analyzed the immunolocalization of 6 proteins of the Ca-release complex in their muscle biopsies. In 7 cases with RYR1 mutations (6 CCD, one MmD), RyR1 was depleted from the cores; in contrast, the other proteins of the sarcoplasmic reticulum (calsequestrin, SERCA1/2, and triadin) and the T-tubule (dihydropyridine receptor-alpha1subunit) accumulated within or around the lesions, suggesting an original modification of the Ca-release complex protein arrangement. Conversely, all Ca-related proteins were distributed normally in 5 MmD cases with SelN mutations. Our results provide an appropriate tool to orientate the differential and molecular diagnosis of core myopathies and suggest that different pathophysiological mechanisms lead to core formation in SelN- and in RyR1-related core myopathies.

  19. Bidirectional Lipid Droplet Velocities Are Controlled by Differential Binding Strengths of HCV Core DII Protein

    Science.gov (United States)

    Lyn, Rodney K.; Hope, Graham; Sherratt, Allison R.; McLauchlan, John; Pezacki, John Paul

    2013-01-01

    Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV. PMID:24223760

  20. Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation

    Directory of Open Access Journals (Sweden)

    Hsu Chen-Ming

    2009-08-01

    Full Text Available Abstract Background The pathogenesis of inflammation and fibrosis in chronic hepatitis C virus (HCV infection remains unclear. Transgenic mice with constitutive HCV core over-expression display steatosis only. While the reasons for this are unclear, it may be important that core protein production in these models begins during gestation, in contrast to human hepatitis C virus infection, which occurs post-natally and typically in adults. AIMS: To more realistically model the effect of core protein production in the adult liver, we developed a mouse with conditional expression of HCV core and examined the effect of core protein production in the adult liver. Methods Liver biopsy samples from transgenic mice with tetracycline(tet-regulated conditional core protein expression were evaluated immunohistologically. Microarray analysis of HCV core transgenic mice with steatohepatitis pointed to a role of the complement pathway. This was further explored by blocking complement activation by in vivo administration of CD55 (decay accelerating factor for complement, which inhibits activation of C3. Results Transgenic mice exhibited low, intermediate, or high HCV core protein expression when fed a permissive diet of standard chow. Aside from hepatic steatosis, hepatic inflammation and fibrosis were seen in mice with intermediate levels of core protein. Microarray analyses of inflamed liver demonstrated activation of both the complement (C3 up-regulation and coagulation pathways (fibrinogen B up-regulation. Administration of CD55 reduced hepatic inflammation. Conclusion Transgenic mice that conditionally express intermediate HCV core protein develop inflammation, steatosis, and fibrosis. These effects mediated by HCV core are reduced by administration of CD55, a regulator of the complement pathway. The model may be valuable in investigating the pathogenesis of liver inflammation in chronic hepatitis C.

  1. The role of mutations in core protein of hepatitis B virus in liver fibrosis

    Directory of Open Access Journals (Sweden)

    Abbasi Shahsanam

    2009-11-01

    Full Text Available Abstract The core protein of hepatitis B virus encompasses B- and T-cell immunodominant epitopes and subdivided into two domains: the N-terminal and the functional C-terminal consisted phosphorylation sites. Mutations of the core gene may change the conformation of the core protein or cause alteration of important epitopes in the host immune response. In this study twenty nine men (mean age 40 ± 9 years old with chronic hepatitis B were recruited for direct sequencing of the core gene. Serum ALT and HBV DNA level were measured at the time of liver biopsy. The effects of core protein mutations on patients' characteristics and subsequently mutations in B cell, T helper and cytotoxic T lymphocyte (CTL epitopes and also C-terminal domain of core protein on the activity of liver disease was evaluated. Liver fibrosis was significantly increased in patients with core protein mutation (1.0 ± 0.8 vs 1.9 ± 1.4 for mean stage of fibrosis P = 0.05. Mutations in CTL epitopes and in phosphorylation sites of C-terminal domain of core protein also were associated with higher liver fibrosis (P = 0.003 and P = 0.04; Fisher's exact test for both. Patients with mutation in C-terminal domain had higher serum ALT (62 ± 17 vs 36 ± 12 IU/l, p = 0.02. Patients with mutations in B cell and T helper epitopes did not show significant difference in the clinical features. Our data suggests that core protein mutations in CTL epitopes and C-terminal domain accompanied with higher stage of liver fibrosis may be due to alterations in the function of core protein.

  2. The effect of HCV Core protein on the expression of miR-150

    Directory of Open Access Journals (Sweden)

    Sayad Khanizadeh

    2016-09-01

    Full Text Available Background : Hepatitis C virus (HCV is considered as one of the major pathogenic agents of chronic liver diseases. Previous studies have shown that HCV proteins can interaction with gene regulatory networks such as microRNAs. The aim of this study was to investigate the effect of HCV core protein on the expression of miR-150 in a cell culture model. Materials and Methods: Plasmids expressing full HCV core protein was transfected into Huh7 cell lines while a GFP expressing plasmid employed as negative control. Subsequently, total RNA extracted and Real-Time PCR performed to measure the expression level of miR-150 expression. Moreover, trypan blue exclusion assay was performed to investigate the effect of core protein on cell viability. Results: The gene expression analysis of miR-150 in Huh7 cells showed that endogenous HCV core protein could significantly down regulation of miR-150 when compared to GFP control plasmid and normal cells (P<0.01. Beside, core protein induced no significant proliferative or cytotoxic effects on hepatic cells as determined by trypan blue exclusion assay (P<0.05. Conclusion: Our study suggests that HCV core protein can led to down regulation of miR-150 expression. This data revealed that HCV protein interactions with cell regulatory machinery may contribute to pathogenesis of chronic liver diseases.

  3. Dynamics of lipid droplets induced by the hepatitis C virus core protein

    Energy Technology Data Exchange (ETDEWEB)

    Lyn, Rodney K. [Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Canada K1A 0R6 (Canada); Department of Chemistry, University of Ottawa, Ottawa (Canada); Kennedy, David C.; Stolow, Albert; Ridsdale, Andrew [Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Canada K1A 0R6 (Canada); Pezacki, John Paul, E-mail: john.pezacki@nrc.ca [Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Canada K1A 0R6 (Canada); Department of Chemistry, University of Ottawa, Ottawa (Canada)

    2010-09-03

    Research highlights: {yields} Hepatitis C virus uses lipid droplets (LD) onto which HCV core proteins bind. {yields} HCV core proteins on LDs facilitate viral particle assembly. {yields} We used a novel combination of CARS, two-photon fluorescence, and DIC microscopies. {yields} Particle tracking experiments show that core slowly affects LD localization. {yields} Particle tracking measured the change in speed and directionality of LD movement. -- Abstract: The hepatitis C virus (HCV) is a global health problem, with limited treatment options and no vaccine available. HCV uses components of the host cell to proliferate, including lipid droplets (LD) onto which HCV core proteins bind and facilitate viral particle assembly. We have measured the dynamics of HCV core protein-mediated changes in LDs and rates of LD movement on microtubules using a combination of coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), and differential interference contrast (DIC) microscopies. Results show that the HCV core protein induces rapid increases in LD size. Particle tracking experiments show that HCV core protein slowly affects LD localization by controlling the directionality of LD movement on microtubules. These dynamic processes ultimately aid HCV in propagating and the molecules and interactions involved represent novel targets for potential therapeutic intervention.

  4. Hepatitis C virus core protein induces neuroimmune activation and potentiates Human Immunodeficiency Virus-1 neurotoxicity.

    Directory of Open Access Journals (Sweden)

    Pornpun Vivithanaporn

    Full Text Available BACKGROUND: Hepatitis C virus (HCV genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection. METHODOLOGY: Human microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed. PRINCIPAL FINDINGS: HCV-encoded RNA as well as HCV core and non-structural 3 (NS3 proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1β, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05 but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8 expression was observed in both microglia and astrocytes (p<0.05. HCV core protein modulated neuronal membrane currents and reduced both β-III-tubulin and lipidated LC3-II expression (p<0.05. Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced β-III-tubulin expression (p<0.05. HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05. HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05. CONCLUSIONS: HCV core protein exposure caused neuronal injury

  5. 3.5A cryoEM structure of hepatitis B virus core assembled from full-length core protein.

    Directory of Open Access Journals (Sweden)

    Xuekui Yu

    Full Text Available The capsid shell of infectious hepatitis B virus (HBV is composed of 240 copies of a single protein called HBV core antigen (HBc. An atomic model of a core assembled from truncated HBc was determined previously by X-ray crystallography. In an attempt to obtain atomic structural information of HBV core in a near native, non-crystalline environment, we reconstructed a 3.5Å-resolution structure of a recombinant core assembled from full-length HBc by cryo electron microscopy (cryoEM and derived an atomic model. The structure shows that the 240 molecules of full-length HBc form a core with two layers. The outer layer, composed of the N-terminal assembly domain, is similar to the crystal structure of the truncated HBc, but has three differences. First, unlike the crystal structure, our cryoEM structure shows no disulfide bond between the Cys61 residues of the two subunits within the dimer building block, indicating such bond is not required for core formation. Second, our cryoEM structure reveals up to four more residues in the linker region (amino acids 140-149. Third, the loops in the cryoEM structures containing this linker region in subunits B and C are oriented differently (~30° and ~90° from their counterparts in the crystal structure. The inner layer, composed of the C-terminal arginine-rich domain (ARD and the ARD-bound RNAs, is partially-ordered and connected with the outer layer through linkers positioned around the two-fold axes. Weak densities emanate from the rims of positively charged channels through the icosahedral three-fold and local three-fold axes. We attribute these densities to the exposed portions of some ARDs, thus explaining ARD's accessibility by proteases and antibodies. Our data supports a role of ARD in mediating communication between inside and outside of the core during HBV maturation and envelopment.

  6. CORE

    DEFF Research Database (Denmark)

    Krigslund, Jeppe; Hansen, Jonas; Hundebøll, Martin

    2013-01-01

    different flows. Instead of maintaining these approaches separate, we propose a protocol (CORE) that brings together these coding mechanisms. Our protocol uses random linear network coding (RLNC) for intra- session coding but allows nodes in the network to setup inter- session coding regions where flows...... intersect. Routes for unicast sessions are agnostic to other sessions and setup beforehand, CORE will then discover and exploit intersecting routes. Our approach allows the inter-session regions to leverage RLNC to compensate for losses or failures in the overhearing or transmitting process. Thus, we...... increase the benefits of XORing by exploiting the underlying RLNC structure of individual flows. This goes beyond providing additional reliability to each individual session and beyond exploiting coding opportunistically. Our numerical results show that CORE outperforms both forwarding and COPE...

  7. CORE

    DEFF Research Database (Denmark)

    Krigslund, Jeppe; Hansen, Jonas; Hundebøll, Martin

    2013-01-01

    different flows. Instead of maintaining these approaches separate, we propose a protocol (CORE) that brings together these coding mechanisms. Our protocol uses random linear network coding (RLNC) for intra- session coding but allows nodes in the network to setup inter- session coding regions where flows...... intersect. Routes for unicast sessions are agnostic to other sessions and setup beforehand, CORE will then discover and exploit intersecting routes. Our approach allows the inter-session regions to leverage RLNC to compensate for losses or failures in the overhearing or transmitting process. Thus, we...... increase the benefits of XORing by exploiting the underlying RLNC structure of individual flows. This goes beyond providing additional reliability to each individual session and beyond exploiting coding opportunistically. Our numerical results show that CORE outperforms both forwarding and COPE...

  8. The Identification of Three Sizes of Core Proteins during the Establishment of Persistent Hepatitis C Virus Infection in vitro

    Institute of Scientific and Technical Information of China (English)

    Qingjiao Liao; Jiansheng Tian; Yang Wu; Xulin Chen

    2013-01-01

    Similar to Hepatitis C virus (HCV) infection in humans,HCVcc infection can also result in persistent and chronic infection.The core protein is a variable protein and exists in several sizes.Some sizes of core proteins have been reported to be related to chronic HCV infection.To study the possible role of the core protein in persistent HCV infection,a persistent HCVcc infection was established,and the expression of the core protein was analysed over the course of the infection.The results show that there are three sizes of core proteins (p24,p21 and p19) expressed during the establishment of persistent HCVcc infection.Of these,the p21 core protein is the mature form of the HCV core protein.The p24 core protein is the phosphorylated form of p21.The p19 core protein appears to be a functional by-product generated during the course of infection.These three core proteins are all localized in the cytoplasm and can be encapsidated into the HCV virion.The appearance of the p19 and p24 core proteins might be related to acute HCVcc infection and chronic infection respectively and may play an important role in the pathology of a HCV infection.

  9. Genes transactivated by hepatitis C virus core protein, a microarray assay

    Institute of Scientific and Technical Information of China (English)

    Min Liu; Shu-Lin Zhang; Jun Cheng; Yan Liu; Lin Wang; Qing Shao; Jian Zhang; Shu-Mei Lin

    2005-01-01

    AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection.METHODS: Reverse transcribed cDNA was subjected tomicroarray assay. The coding gene transactivated by HCV core protein was cloned and analyzed with bioinformatics methods.RESULTS: The expressive vector of pcDNA3.1(-)-core was constructed and confirmed by restriction enzyme digestion and DNA sequencing and approved correct. mRNA was purified from HepG2 and HepG2 cells transfected with pcDNA3.1(-)-core, respectively. The cDNA derived was subjected to microarray assay. A new gene namedHCTP4 was cloned with molecular biological method in combination with bioinformatics method.CONCLUSION: HCV core is a potential transactivator.Microarray is an efficient and convenient method for analysis of differentially expressed genes.

  10. Transactivating effect of hepatitis C virus core protein:A suppression subtractive hybridization study

    Institute of Scientific and Technical Information of China (English)

    Min Liu; Yan Liu; Jun Cheng; Shu-Lin Zhang; Lin Wang; Qing Shao; Jian Zhang; Qian Yang

    2004-01-01

    AIM: To investigate the transactivating effect of hepatitis C virus (HCV) core protein and to screen genes transactivated by HCV core protein.METHODS: pcDNA3.1(-)-core containing full-length HCV core gene was constructed by insertion of HCV core gene into EcoRI/BanHI site. HepG2 cells were cotransfected with pcDNA3.1(-)-core and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-gal by an enzyme-linked immunosorbent assay (ELISA) kit. HepG2 cells were transiently transfected with pcDNA3.1(-)-core using Lipofectamine reagent. Cells were collected and total mRNA was isolated. A subtracted cDNA library was generated and constructed into a pGEM-Teasy vector. The library was amplified with E. coli strain JM109. The cDNAs were sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR).RESULTS: The core mRNA and protein could be detected in HepG2 cell lysate which was transfected by the pcDNA3.1(-)-core. The activity of β-galactosidase in HepG2 cells transfected by the pcDNA3.1(-)-core was 5.4 times higher than that of HepG2 cells transfected by control plasmid. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233positive clones. Colony PCR showed that 2:13 clones contained 100-1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes. The full length sequences were obtained with bioinformatics method, accepted by GenBank. It was suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein.CONCLUSION: The core protein of HCV has transactivating effects on SV40 early promoter/enhancer. A total of 63 clones from cDNA library were randomly chosen and sequenced.Using the BLAST program at the National Center for Biotechnology Information, six of the sequences were unknown genes. The other 57 sequences were highly similar to known genes.

  11. HCV core protein represses the apoptosis and improves the autophagy of human hepatocytes

    Science.gov (United States)

    Liu, Changhong; Qu, Aihua; Han, Xiaochun; Wang, Yiguo

    2015-01-01

    Objectives: This study aims to investigate the influence on human hepatocytes apoptosis and autophagy by the hepatitis C virus (HCV) core protein. Methods: QSG-7701, a human-derived non-neoplastic liver cell line, was transfected with PIRES-core vector that was a eukaryotic vector to express HCV core protein. Fluorescence microscope was used to observe the changes of nuclei in apoptosis cells by Annex in V-FITC/PI double staining. Flow cytometry was applied to detect the rate of cell apoptosis. Western blotting was used to detect the expression of HCV core protein, transcription factor nuclear factor-kappa B (NF-κB), autophagic biomarker microtubule associated protein 1 light chain 3 (LC3), and Beclin-1. Results: The apoptosis rate was significantly lower (P < 0.05) in QSG7701/core group (transfected with PIRES-core vector, (1.34±0.07)%) than in QSG7701 group (no transfection, (2.35±0.11)%) and in QSG7701 QSG7701/pcDNA3.1 group (transfected with pcDNA3.1 vector, (2.58±0.1)%). NF-κB expression was up-expressed in QSG7701/core group than in QSG7701/pcDNA3.1 group and QSG7701 group (P < 0.05). LC3-II expression and Beclin-1 expression was significant higher in QSG7701/core group than in the QSG7701/pcDNA3.1 group and QSG7701 group (P < 0.05). Conclusion: HCV core protein can repress the apoptosis and improve the autophagy of QSG7701 through up-regulating NF-κB and Beclin-1 expression. PMID:26629077

  12. Hepatitis C virus core protein induces hepatic steatosis via Sirt1-dependent pathway.

    Science.gov (United States)

    Zhang, Chuanhai; Wang, Jingjing; Zhang, Hanlin; Liu, Shunai; Lee, Hyuek Jong; Jin, Wanzhu; Cheng, Jun

    2017-09-12

    Hepatic steatosis is a common feature of patients with chronic hepatitis C. Previous reports have shown that the overexpression of hepatitis C virus core-encoding sequences (hepatitis C virus genotypes 3a and 1b) significantly induces intracellular triglyceride accumulation. However, the underlying mechanism has not yet been revealed. To investigate whether Sirt1 is involved in hepatitis C virus-mediated hepatic steatosis, the overexpression of hepatitis C virus core 1b protein and Sirt1 and the knockdown of Sirt1 in HepG2 cells were performed. To confirm the results of the cellular experiment liver-specific Sirt1 KO mice with lentivirus-mediated hepatitis C virus core 1b overexpression were studied. Our results show that hepatitis C virus core 1b protein overexpression led to the accumulation of triglycerides in HepG2 cells. Notably the expression of PPARγ2 was dramatically increased at both the mRNA and protein levels by hepatitis C virus core 1b overexpression. The protein expression of Sirt1 is an upstream regulator of PPARγ2 and was also significantly increased after core 1b overexpression. In addition, the overexpression or knockdown of Sirt1 expression alone was sufficient to modulate p300-mediated PPARγ2 deacetylation. In vivo studies showed that hepatitis C virus core protein 1b-induced hepatic steatosis was attenuated in liver-specific Sirt1 KO mice by downregulation of PPARγ2 expression. Sirt1 mediates hepatitis C virus core protein 1b-induced hepatic steatosis by regulation of PPARγ2 expression. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Virus-producing cells determine the host protein profiles of HIV-1 virion cores

    Directory of Open Access Journals (Sweden)

    Santos Steven

    2012-08-01

    Full Text Available Abstract Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs, which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes, PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ, and non-activated THP1 cells (model of monocytes, mMN and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90 with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29, DNA binding (17, cytoskeleton (15, cytoskeleton regulation (21, chaperone (18, vesicular trafficking-associated (12 and ubiquitin-proteasome pathway-associated proteins (9 were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins

  14. Structures of the compact helical core domains of feline calicivirus and murine norovirus VPg proteins.

    Science.gov (United States)

    Leen, Eoin N; Kwok, K Y Rex; Birtley, James R; Simpson, Peter J; Subba-Reddy, Chennareddy V; Chaudhry, Yasmin; Sosnovtsev, Stanislav V; Green, Kim Y; Prater, Sean N; Tong, Michael; Young, Joanna C; Chung, Liliane M W; Marchant, Jan; Roberts, Lisa O; Kao, C Cheng; Matthews, Stephen; Goodfellow, Ian G; Curry, Stephen

    2013-05-01

    We report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases, the core of the protein adopts a compact helical structure flanked by flexible N and C termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases, the VPg cores are stabilized by networks of hydrophobic and salt bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilized the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined, but knowledge of its structure will facilitate future investigations.

  15. Discovery of protein complexes with core-attachment structures from Tandem Affinity Purification (TAP) data.

    Science.gov (United States)

    Wu, Min; Li, Xiao-Li; Kwoh, Chee-Keong; Ng, See-Kiong; Wong, Limsoon

    2012-09-01

    Many cellular functions involve protein complexes that are formed by multiple interacting proteins. Tandem Affinity Purification (TAP) is a popular experimental method for detecting such multi-protein interactions. However, current computational methods that predict protein complexes from TAP data require converting the co-complex relationships in TAP data into binary interactions. The resulting pairwise protein-protein interaction (PPI) network is then mined for densely connected regions that are identified as putative protein complexes. Converting the TAP data into PPI data not only introduces errors but also loses useful information about the underlying multi-protein relationships that can be exploited to detect the internal organization (i.e., core-attachment structures) of protein complexes. In this article, we propose a method called CACHET that detects protein complexes with Core-AttaCHment structures directly from bipartitETAP data. CACHET models the TAP data as a bipartite graph in which the two vertex sets are the baits and the preys, respectively. The edges between the two vertex sets represent bait-prey relationships. CACHET first focuses on detecting high-quality protein-complex cores from the bipartite graph. To minimize the effects of false positive interactions, the bait-prey relationships are indexed with reliability scores. Only non-redundant, reliable bicliques computed from the TAP bipartite graph are regarded as protein-complex cores. CACHET constructs protein complexes by including attachment proteins into the cores. We applied CACHET on large-scale TAP datasets and found that CACHET outperformed existing methods in terms of prediction accuracy (i.e., F-measure and functional homogeneity of predicted complexes). In addition, the protein complexes predicted by CACHET are equipped with core-attachment structures that provide useful biological insights into the inherent functional organization of protein complexes. Our supplementary material can

  16. HCV Core Protein Uses Multiple Mechanisms to Induce Oxidative Stress in Human Hepatoma Huh7 Cells

    Science.gov (United States)

    Ivanov, Alexander V.; Smirnova, Olga A.; Petrushanko, Irina Y.; Ivanova, Olga N.; Karpenko, Inna L.; Alekseeva, Ekaterina; Sominskaya, Irina; Makarov, Alexander A.; Bartosch, Birke; Kochetkov, Sergey N.; Isaguliants, Maria G.

    2015-01-01

    Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGFβ1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1α. The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein. PMID:26035647

  17. Technology and mechanism of a new protein-based core sand for aluminum casting

    Institute of Scientific and Technical Information of China (English)

    石晶玉; 黄天佑; 石红玉; 何镇明

    2001-01-01

    The protein-based binding material is from natural products, which is nontoxic and recyclable. This kind of green binder is earnestly needed by aluminum casting products. The new protein-based core possesses higher strength and easier shakeout. Its tensile strength is close to that of common resin sands. The micro-mechanism of protein binder was investigated by using infrared spectrum, chemical element analysis, SEM and thermal lost-mass analysis.

  18. Potent inhibition of late stages of hepadnavirus replication by a modified cell penetrating peptide

    DEFF Research Database (Denmark)

    Abdul, Fabien; Ndeboko, Bénédicte; Buronfosse, Thierry;

    2012-01-01

    by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)8-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral...... particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside...

  19. Hepatitis C virus core protein induces apoptosis-like caspase independent cell death

    Directory of Open Access Journals (Sweden)

    Gregor Michael

    2009-12-01

    Full Text Available Abstract Background Hepatitis C virus (HCV associated liver diseases may be related to apoptotic processes. Thus, we investigated the role of different HCV proteins in apoptosis induction as well as their potency to interact with different apoptosis inducing agents. Methods and Results The use of a tightly adjustable tetracycline (Tet-dependent HCV protein expression cell system with the founder osteosarcoma cell line U-2 OS allowed switch-off and on of the endogenous production of HCV proteins. Analyzed were cell lines expressing the HCV polyprotein, the core protein, protein complexes of the core, envelope proteins E1, E2 and p7, and non-structural proteins NS3 and NS4A, NS4B or NS5A and NS5B. Apoptosis was measured mainly by the detection of hypodiploid apoptotic nuclei in the absence or presence of mitomycin C, etoposide, TRAIL and an agonistic anti-CD95 antibody. To further characterize cell death induction, a variety of different methods like fluorescence microscopy, TUNEL (terminal deoxynucleotidyl transferase (TdT-catalyzed deoxyuridinephosphate (dUTP-nick end labeling assay, Annexin V staining, Western blot and caspase activation assays were included into our analysis. Two cell lines expressing the core protein but not the total polyprotein exerted a strong apoptotic effect, while the other cell lines did not induce any or only a slight effect by measuring the hypodiploid nuclei. Cell death induction was caspase-independent since it could not be blocked by zVAD-fmk. Moreover, caspase activity was absent in Western blot analysis and fluorometric assays while typical apoptosis-associated morphological features like the membrane blebbing and nuclei condensation and fragmentation could be clearly observed by microscopy. None of the HCV proteins influenced the apoptotic effect mediated via the mitochondrial apoptosis pathway while only the core protein enhanced death-receptor-mediated apoptosis. Conclusion Our data showed a caspase

  20. Structural proteins of ribonucleic acid tumor viruses. Purification of envelope, core, and internal components.

    Science.gov (United States)

    Strand, M; August, J T

    1976-01-25

    Murine type C virus structural proteins, the envelope glycopeptides, 30,000 dalton major core protein, and 15,000 dalton internal protein have each been purified to near homogeneity and in high yield from the smae batch of virus by use of phosphocellulose column chromatography and gel filtration procedures. Evidence that these proteins are specified by the viral genome was obtained by competition radioimmunoassay analysis, comparing these polypeptides from Rauscher virus cultivated in a variety of mammalian cell lines; all of the reactive antigenic determinants of these proteins appeared to be virus-specific.

  1. Evolution of Drosophila ribosomal protein gene core promoters.

    Science.gov (United States)

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2009-03-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module.

  2. Mitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein

    Energy Technology Data Exchange (ETDEWEB)

    Sekine, Shuichi; Ito, Konomi; Watanabe, Haruna; Nakano, Takafumi [Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 (Japan); Moriya, Kyoji; Shintani, Yoshizumi; Fujie, Hajime; Tsutsumi, Takeya; Miyoshi, Hideyuki; Fujinaga, Hidetake; Shinzawa, Seiko; Koike, Kazuhiko [Department of Internal Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Horie, Toshiharu, E-mail: t.horie@thu.ac.jp [Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 (Japan)

    2015-02-01

    Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ({sup 59}Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca{sup 2+} uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein. - Highlights: • Iron accumulation in the livers of patients with hepatitis C virus (HCV) infection is thought to exacerbate oxidative stress. • The impact of iron overload on mitochondrial damage and ROS production in HCV core protein-expressing cells were examined. • Mitochondrial iron uptake was increased in the livers of HCV core protein-expressing transgenic mice. • Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates

  3. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    Directory of Open Access Journals (Sweden)

    Rydzak Thomas

    2012-09-01

    Full Text Available Abstract Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative

  4. CORE: Common Region Extension Based Multiple Protein Structure Alignment for Producing Multiple Solution

    Institute of Scientific and Technical Information of China (English)

    Woo-Cheol Kim; Sanghyun Park; Jung-Im Won

    2013-01-01

    Over the past several decades,biologists have conducted numerous studies examining both general and specific functions of proteins.Generally,if similarities in either the structure or sequence of amino acids exist for two proteins,then a common biological function is expected.Protein function is determined primarily based on the structure rather than the sequence of amino acids.The algorithm for protein structure alignment is an essential tool for the research.The quality of the algorithm depends on the quality of the similarity measure that is used,and the similarity measure is an objective function used to determine the best alignment.However,none of existing similarity measures became golden standard because of their individual strength and weakness.They require excessive filtering to find a single alignment.In this paper,we introduce a new strategy that finds not a single alignment,but multiple alignments with different lengths.This method has obvious benefits of high quality alignment.However,this novel method leads to a new problem that the running time for this method is considerably longer than that for methods that find only a single alignment.To address this problem,we propose algorithms that can locate a common region (CORE) of multiple alignment candidates,and can then extend the CORE into multiple alignments.Because the CORE can be defined from a final alignment,we introduce CORE* that is similar to CORE and propose an algorithm to identify the CORE*.By adopting CORE* and dynamic programming,our proposed method produces multiple alignments of various lengths with higher accuracy than previous methods.In the experiments,the alignments identified by our algorithm are longer than those obtained by TM-align by 17% and 15.48%,on average,when the comparison is conducted at the level of super-family and fold,respectively.

  5. Molecular characterization of suppression of hepatitis B virus transcription by hepatitis C virus core protein

    Institute of Scientific and Technical Information of China (English)

    王海林; 颜子颖; 侯云德; 金冬雁

    1997-01-01

    To further elucidate the molecular mechanisms underlying the suppression of hepatitis B virus (HBV) expression by the hepatitis C virus (HCV) core protein, five molecular clones of HCV cDNA sequence con-taining the 5’ noncoding (5’NC) and the core regions have been isolated from Chinese HBV- and HCV-coinfected pa-tients. Sequence comparison and phylogenetic analysis showed that the HCV sequence cloned from coinfected individu-als is indistinguishable from that identified in other patients. Cotransfection assay confirmed that the core protein ex-pressed from one of the cloned sequence is capable of suppressing the expression of hepatitis B surface and e antigens (HBsAg and HBeAg, respectively). Deletion mapping revealed that the C-terminal hydrophobic region of the HCV core is necessary for the suppression. Results from reporter assays demonstrated that HCV core protein interacts with the HBV C promoter and enhancer II elements and down-regulates the transcription of HBV as well as other cellular and het

  6. Grafted block complex coacervate core micelles and their effect on protein adsorption on silica and polystyrene

    NARCIS (Netherlands)

    Brzozowska, Agata M.; de Keizer, Arie; Norde, Willem; Detrembleur, Christophe; Stuart, Martien Cohen

    2010-01-01

    We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins beta-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer

  7. Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Jian [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Zhang, Huaidong [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Gong, Rui, E-mail: gongr@wh.iov.cn [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China)

    2015-05-08

    Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.

  8. A core viral protein binds host nucleosomes to sequester immune danger signals.

    Science.gov (United States)

    Avgousti, Daphne C; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C; Blumenthal, Daniel; Paris, Andrew J; Reyes, Emigdio D; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H; Worthen, G Scott; Black, Ben E; Garcia, Benjamin A; Weitzman, Matthew D

    2016-07-01

    Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

  9. Toll-like receptor 2 senses hepatitis C virus core protein but not infectious viral particles

    Science.gov (United States)

    Hoffmann, Marco; Zeisel, Mirjam B.; Jilg, Nikolaus; Paranhos-Baccalà, Glaucia; Stoll-Keller, Françoise; Wakita, Takaji; Hafkemeyer, Peter; Blum, Hubert E.; Barth, Heidi; Henneke, Philipp; Baumert, Thomas F.

    2009-01-01

    Toll-like receptors (TLRs) are pathogen recognition molecules activating the innate immune system. Cell surface expressed TLRs, such as TLR2 and TLR4 have been shown to play an important role in human host defenses against viruses through sensing of viral structural proteins. In this study, we aimed to elucidate whether TLR2 and TLR4 participate in inducing antiviral immunity against hepatitis C virus by sensing viral structural proteins. We studied TLR2 and TLR4 activation by cell-culture derived infectious virions (HCVcc) and serum-derived virions in comparison to purified recombinant HCV structural proteins and enveloped virus-like particles. Incubation of TLR2 or TLR4 transfected cell lines with recombinant core protein resulted in activation of TLR2-dependent signaling. In contrast, neither infectious virions nor enveloped HCV-like particles triggered TLR2 and TLR4 signaling. These findings suggest that monomeric HCV core protein but not intact infectious particles are sensed by TLR2. Impairment of core-TLR interaction in infectious viral particles may contribute to escape from innate antiviral immune responses. PMID:20375602

  10. Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

    Science.gov (United States)

    Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

  11. Dengue virus capsid protein binds core histones and inhibits nucleosome formation in human liver cells.

    Directory of Open Access Journals (Sweden)

    Tonya M Colpitts

    Full Text Available Dengue virus (DENV is a member of the Flaviviridae and a globally (reemerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection.

  12. Control of vertebrate core planar cell polarity protein localization and dynamics by Prickle 2.

    Science.gov (United States)

    Butler, Mitchell T; Wallingford, John B

    2015-10-01

    Planar cell polarity (PCP) is a ubiquitous property of animal tissues and is essential for morphogenesis and homeostasis. In most cases, this fundamental property is governed by a deeply conserved set of 'core PCP' proteins, which includes the transmembrane proteins Van Gogh-like (Vangl) and Frizzled (Fzd), as well as the cytoplasmic effectors Prickle (Pk) and Dishevelled (Dvl). Asymmetric localization of these proteins is thought to be central to their function, and understanding the dynamics of these proteins is an important challenge in developmental biology. Among the processes that are organized by the core PCP proteins is the directional beating of cilia, such as those in the vertebrate node, airway and brain. Here, we exploit the live imaging capabilities of Xenopus to chart the progressive asymmetric localization of fluorescent reporters of Dvl1, Pk2 and Vangl1 in a planar polarized ciliated epithelium. Using this system, we also characterize the influence of Pk2 on the asymmetric dynamics of Vangl1 at the cell cortex, and we define regions of Pk2 that control its own localization and those impacting Vangl1. Finally, our data reveal a striking uncoupling of Vangl1 and Dvl1 asymmetry. This study advances our understanding of conserved PCP protein functions and also establishes a rapid, tractable platform to facilitate future in vivo studies of vertebrate PCP protein dynamics.

  13. High prevalence of antibodies to core+1/ARF protein in HCV-infected patients with advanced cirrhosis.

    Science.gov (United States)

    Kassela, Katerina; Karakasiliotis, Ioannis; Charpantidis, Stefanos; Koskinas, John; Mylopoulou, Theodora; Mimidis, Konstantinos; Sarrazin, Christoph; Grammatikos, Georgios; Mavromara, Penelope

    2017-07-01

    Hepatitis C virus (HCV) possesses a second open reading frame (ORF) within the core gene encoding an additional protein, known as the alternative reading frame protein (ARFP), F or core+1. The biological significance of the core+1/ARF protein remains elusive. However, several independent studies have shown the presence of core+1/ARFP antibodies in chronically HCV-infected patients. Furthermore, a higher prevalence of core+1/ARFP antibodies was detected in patients with HCV-associated hepatocellular carcinoma (HCC). Here, we investigated the incidence of core+1/ARFPantibodies in chronically HCV-infected patients at different stages of cirrhosis in comparison to chronically HCV-infected patients at earlier stages of disease. Using ELISA, we assessed the prevalence of anti-core+1 antibodies in 30 patients with advanced cirrhosis [model for end-stage liver disease (MELD) ≥15] in comparison with 50 patients with mild cirrhosis (MELD core+1 antibodies, in contrast with 16.5 % of non-cirrhotic HCV patients. Moreover, there was significantly higher positivity for anti-core+1 antibodies in HCV patients with advanced cirrhosis (36.7 %) compared to those with early cirrhosis (24 %) (Pcore+1 antibodies in HCV patients with HCC, suggest that core+1 protein may have a role in virus-associated pathogenesis, and provide evidence to suggest that the levels of anti-core+1 antibodies may serve as a marker for disease progression.

  14. Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein.

    Science.gov (United States)

    Buvoli, M; Cobianchi, F; Bestagno, M G; Mangiarotti, A; Bassi, M T; Biamonti, G; Riva, S

    1990-01-01

    The human hnRNP core protein A1 (34 kd) is encoded by a 4.6 kb gene split into 10 exons. Here we show that the A1 gene can be differentially spliced by the addition of an extra exon. The new transcript encodes a minor protein of the hnRNP complex, here defined A1B protein, with a calculated mol. wt of 38 kd, that coincides with a protein previously designated as B2 by some authors. In vitro translation of the mRNAs selected by hybridization with A1 cDNA produced two proteins of 34 and 38 kd; Northern blot analysis of poly(A)+ RNA from HeLa cells revealed that the abundance of the A1B mRNA was approximately 5% that of A1. The A1B protein was detected by Western blotting with an anti-A1 monoclonal antibody both in enriched preparations of basic hnRNP proteins and in 40S hnRNP particles. The A1B protein exhibits a significantly higher affinity than A1 for ssDNA. The recombinant A1B protein, expressed in Escherichia coli, shows the same electrophoretic mobility and charge as the cellular one. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1691095

  15. First principles design of a core bioenergetic transmembrane electron-transfer protein

    Energy Technology Data Exchange (ETDEWEB)

    Goparaju, Geetha; Fry, Bryan A.; Chobot, Sarah E.; Wiedman, Gregory; Moser, Christopher C.; Leslie Dutton, P.; Discher, Bohdana M.

    2016-05-01

    Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

  16. The hepatitis B virus core protein intradimer interface modulates capsid assembly and stability.

    Science.gov (United States)

    Selzer, Lisa; Katen, Sarah P; Zlotnick, Adam

    2014-09-02

    During the hepatitis B virus (HBV) life cycle, capsid assembly and disassembly must ensure correct packaging and release of the viral genome. Here we show that changes in the dynamics of the core protein play an important role in regulating these processes. The HBV capsid assembles from 120 copies of the core protein homodimer. Each monomer contains a conserved cysteine at position 61 that can form an intradimer disulfide that we use as a marker for dimer conformational states. We show that dimers in the context of capsids form intradimer disulfides relatively rapidly. Surprisingly, compared to reduced dimers, fully oxidized dimers assembled slower and into capsids that were morphologically similar but less stable. We hypothesize that oxidized protein adopts a geometry (or constellation of geometries) that is unfavorable for capsid assembly, resulting in weaker dimer-dimer interactions as well as slower assembly kinetics. Our results suggest that structural flexibility at the core protein intradimer interface is essential for regulating capsid assembly and stability. We further suggest that capsid destabilization by the C61-C61 disulfide has a regulatory function to support capsid disassembly and release of the viral genome.

  17. Structural insights into yeast histone chaperone Hif1: a scaffold protein recruiting protein complexes to core histones.

    Science.gov (United States)

    Liu, Hejun; Zhang, Mengying; He, Wei; Zhu, Zhongliang; Teng, Maikun; Gao, Yongxiang; Niu, Liwen

    2014-09-15

    Yeast Hif1 [Hat1 (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hif1 consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.

  18. Vaccine delivery system for tuberculosis based on nano-sized hepatitis B virus core protein particles

    Directory of Open Access Journals (Sweden)

    Dhanasooraj D

    2013-02-01

    Full Text Available Dhananjayan Dhanasooraj, R Ajay Kumar, Sathish MundayoorMycobacterium Research Group, Rajiv Gandhi Centre for Biotechnology, Kerala, IndiaAbstract: Nano-sized hepatitis B virus core virus-like particles (HBc-VLP are suitable for uptake by antigen-presenting cells. Mycobacterium tuberculosis antigen culture filtrate protein 10 (CFP-10 is an important vaccine candidate against tuberculosis. The purified antigen shows low immune response without adjuvant and tends to have low protective efficacy. The present study is based on the assumption that expression of these proteins on HBc nanoparticles would provide higher protection when compared to the native antigen alone. The cfp-10 gene was expressed as a fusion on the major immunodominant region of HBc-VLP, and the immune response in Balb/c mice was studied and compared to pure proteins, a mixture of antigens, and fusion protein-VLP, all without using any adjuvant. The humoral, cytokine, and splenocyte cell proliferation responses suggested that the HBc-VLP bearing CFP-10 generated an antigen-specific immune response in a Th1-dependent manner. By virtue of its self-adjuvant nature and ability to form nano-sized particles, HBc-VLPs are an excellent vaccine delivery system for use with subunit protein antigens identified in the course of recent vaccine research.Keywords: Mycobacterium tuberculosis, VLP, hepatitis B virus core particle, CFP-10, self-adjuvant, vaccine delivery

  19. High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein.

    Science.gov (United States)

    Klumpp, Klaus; Lam, Angela M; Lukacs, Christine; Vogel, Robert; Ren, Suping; Espiritu, Christine; Baydo, Ruth; Atkins, Kateri; Abendroth, Jan; Liao, Guochun; Efimov, Andrey; Hartman, George; Flores, Osvaldo A

    2015-12-01

    The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010-001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010-001-E2 binds at the dimer-dimer interface of the core proteins, forms a new interaction surface promoting protein-protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010-001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein-protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties.

  20. Effect of Hepatitis C Virus Core Protein on Interferon-Induced Antiviral Genes Expression and Its Mechanisms

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Emerging data indicated that HCV subverts the antiviral activity of interferon (IF); however,whether HCV core protein contributes to the process remains controversial. In the present study, we examined the effect of HCV core protein on interferon-induced antiviral gene expression and whether the effect is involved in the activation and negative regulation of the Jak/STAT signaling pathway. Our results showed that, following treatment with IFN-α, the transcription of PKR, MxA and 2'-5'OAS were down-regulated in HepG2 cells expressing the core protein. In the presence of HCV core protein,ISRE-dependent luciferase activity also decreased. Further study indicated that the core protein could inhibit the tyrosine phosphorylation of STAT1, whereas the level of STAT1 expression was unchanged.Accordingly, SOCS3, the negative regulator of the Jak/STAT pathway, was induced by HCV core protein. These results suggests that HCV core protein may interfere with the expression of some interferon-induced antiviral genes by inhibiting STAT1 phosphorylation and induction of SOCS3.

  1. Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways†

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A.; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-01-01

    Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome. PMID:16973564

  2. Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-10-01

    Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

  3. Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein.

    Science.gov (United States)

    Tych, Katarzyna M; Batchelor, Matthew; Hoffmann, Toni; Wilson, Michael C; Hughes, Megan L; Paci, Emanuele; Brockwell, David J; Dougan, Lorna

    2016-07-26

    Proteins from organisms that have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single-molecule force spectroscopy (SMFS), and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal β-strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (ΔGD-N) of the mutated proteins and show that there is a more significant reduction for TmCSP L40A (ΔΔG = 63%) than TmCSP V62A (ΔΔG = 47%), as might be expected on the basis of the relative reduction in the size of the side chain. By contrast, SMFS measures the mechanical stability (ΔG*) and shows a greater reduction for TmCSP V62A (ΔΔG* = 8.4%) than TmCSP L40A (ΔΔG* = 2.5%). While the impact on the mechanical stability is subtle, the results demonstrate the power of tuning noncovalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provide the opportunity to design proteins with optimized thermodynamic and mechanical properties.

  4. Hepatitis C virus core proteins derived from different quasispecies of genotype 1b inhibit the growth of Chang liver cells

    Institute of Scientific and Technical Information of China (English)

    Xue-Bing Yan; Lei Mei; Xia Feng; Mei-Rong Wan; Zhi Chen; Nicole Pavia; Christian Brechot

    2008-01-01

    AIM: To investigate the influence of different quasispecies of hepatitis C virus (HCV) genotype 1b core protein on growth of Chang liver cells.METHODS: Three eukaryotic expression plasmids (pEGFP-N1/core) that contained different quasispecies truncated core proteins of HCV genotype 1b were constructed. These were derived from tumor (T) and nontumor (NT) tissues of a patient infected with HCV and C191 (HCV-J6). The core protein expression plasmids were transiently transfected into Chang liver cells. At different times, the cell cycle and apoptosis was assayed by flow cytometry, and cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) assay.RESULTS: The proportion of S-phase Chang liver cells transfected with pEGFP-N1/core was significantly lower than that of cells transfected with blank plasmid at three different times after transfection (all P NT > C191), and apoptosis was increased in cells transfected with pEGFP-N1/core as the transfection time increased (72 h > 48 h > 24 h).CONCLUSION: These results suggest that HCV genotype 1b core protein induces apoptosis, and inhibits cellcycle progression and proliferation of Chang liver cells.Different quasispecies core proteins of HCV genotype 1b might have some differences in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.

  5. An α-helical core encodes the dual functions of the chlamydial protein IncA.

    Science.gov (United States)

    Ronzone, Erik; Wesolowski, Jordan; Bauler, Laura D; Bhardwaj, Anshul; Hackstadt, Ted; Paumet, Fabienne

    2014-11-28

    Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. An α-Helical Core Encodes the Dual Functions of the Chlamydial Protein IncA*

    Science.gov (United States)

    Ronzone, Erik; Wesolowski, Jordan; Bauler, Laura D.; Bhardwaj, Anshul; Hackstadt, Ted; Paumet, Fabienne

    2014-01-01

    Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2. PMID:25324548

  7. Hepatitis C virus quasispecies in cancerous and noncancerous hepatic lesions: the core protein-encoding region.

    Directory of Open Access Journals (Sweden)

    Alam,Shahjalal S.

    2002-06-01

    Full Text Available We have shown that highly proofreading DNA polymerase is required for the polymerase chain reaction in the genetic analysis of hepatitis C virus (HCV. To clarify the status of HCV quasispecies in hepatic tissue using proofreading DNA polymerase, we performed a genetic analysis of the HCV core protein-encoding region in cancerous and noncancerous lesions derived from 4 patients with hepatocellular carcinoma. In contrast to the previously published data, we observed neither deletions nor stop codons in the analyzed region and no significant difference in the complexity of HCV quasispecies between cancerous and noncancerous lesions. This result suggests that the HCV core gene is never structurally defective in hepatic tissues, including cancerous lesions. However, in 3 of the patients, the consensus HCV species differed between cancerous and noncancerous lesions, suggesting that the predominant replicating HCV species differs between these 2 types of lesions. Moreover, during the course of the study, we obtained several interesting variants possessing a substitution at codon 9 of the core gene, whose substitution has been shown to induce the production of the F protein synthesized by a - 2/+1 ribosomal frameshift.

  8. High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein

    Science.gov (United States)

    Klumpp, Klaus; Lam, Angela M.; Lukacs, Christine; Vogel, Robert; Ren, Suping; Espiritu, Christine; Baydo, Ruth; Atkins, Kateri; Abendroth, Jan; Liao, Guochun; Efimov, Andrey; Hartman, George; Flores, Osvaldo A.

    2015-01-01

    The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010–001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010–001-E2 binds at the dimer–dimer interface of the core proteins, forms a new interaction surface promoting protein–protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010–001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein–protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties. PMID:26598693

  9. Interaction of mitoxantrone, as an anticancer drug, with chromatin proteins, core histones and H1, in solution.

    Science.gov (United States)

    Hajihassan, Zahra; Rabbani-Chadegani, Azra

    2011-01-01

    In the present study, for the first time we have investigated the interaction of anticancer drug mitoxantrone with histone H1 and core histone proteins in solution using fluorescence, UV/Vis, CD spectroscopy and thermal denaturation techniques. The results showed that mitoxantrone reduced the absorbencies of H1 and core histone proteins at 210 nm (hypochromicity) and fluorescence emission intensity was decreased in a dose dependent. Binding of mitoxantrone changed secondary structures of the proteins as circular dichroism analysis confirmed it. Also, mitoxantrone increased the melting temperature of core histones at the final step of denaturation. The results suggest higher affinity of mitoxantrone to histone H1 compared to core histones providing histone proteins as a new target for mitoxantrone action at the chromatin level. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Controlled-release and preserved bioactivity of proteins from (self-assembled core-shell double-walled microspheres

    Directory of Open Access Journals (Sweden)

    Yuan W

    2012-01-01

    Full Text Available Weien Yuan1,2, Zhenguo Liu11Department of Neurology, Xinhua Hospital, affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 2School of Pharmacy, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaAbstract: In order to address preserved protein bioactivities and protein sustained-release problems, a method for preparing double-walled microspheres with a core (protein-loaded nanoparticles with a polymer-suspended granule system-formed core and a second shell (a polymer-formed shell for controlled drug release and preserved protein bioactivities has been developed using (solid-in-oil phase-in-hydrophilic oil-in-water (S/O/Oh/W phases. The method, based on our previous microsphere preparation method (solid-in-oil phase-in-hydrophilic oil-in-water (S/O/Oh/W, employs different concentric poly(D,L-lactide-co-glycolide, poly(D,L-lactide, and protein-loaded nanoparticles to produce a suspended liquid which then self-assembles to form shell-core microspheres in the hydrophilic oil phase, which are then solidified in the water phase. Variations in the preparation parameters allowed complete encapsulation by the shell phase, including the efficient formation of a poly(D,L-lactide shell encapsulating a protein-loaded nanoparticle-based poly(D,L-lactide-co-glycolide core. This method produces core-shell double-walled microspheres that show controlled protein release and preserved protein bioactivities for 60 days. Based upon these results, we concluded that the core-shell double-walled microspheres might be applied for tissue engineering and therapy for chronic diseases, etc.Keywords: protein delivery, protein stability, core-shell microspheres, dextran nanoparticles

  11. Hepatitis C virus core protein induces fibrogenic actions of hepatic stellate cells via toll-like receptor 2.

    Science.gov (United States)

    Coenen, Martin; Nischalke, Hans Dieter; Krämer, Benjamin; Langhans, Bettina; Glässner, Andreas; Schulte, Daniela; Körner, Christian; Sauerbruch, Tilman; Nattermann, Jacob; Spengler, Ulrich

    2011-09-01

    Hepatic stellate cells (HSCs) represent the main fibrogenic cell type accumulating extracellular matrix in the liver. Recent data suggest that hepatitis C virus (HCV) core protein may directly activate HSCs. Therefore, we examined the influence of recombinant HCV core protein on human HSCs. Primary human HSCs and the human HSC line LX-2 were stimulated with recombinant HCV proteins core and envelope 2 protein. Expression of procollagen type I α-1, α-smooth muscle actin, cysteine- and glycine-rich protein 2, glial fibrillary acidic protein, tissue growth factor β1, matrix metalloproteinases 2 (MMP2) and 13, tissue inhibitor of metalloproteinases 1 and 2 was investigated by real-time PCR. Intracellular signaling pathways of ERK1/2, p38 and, jun-amino-terminal kinase (JNK) were analyzed by western blot analysis. Recombinant HCV core protein induced upregulation of procollagen type I α-1, α-smooth muscle actin, MMP 2 and 13, tissue inhibitor of metalloproteinases 1 and 2, tissue growth factor β1, cysteine- and glycine-rich protein 2, and glial fibrillary acidic protein mRNA expression, whereas HCV envelope 2 protein did not exert any significant effect. Blocking of toll-like receptor 2 (TLR2) with a neutralizing antibody prevented mRNA upregulation by HCV core protein confirming that the TLR2 pathway was involved. Furthermore, western blot analysis revealed HCV-induced phosphorylation of the TLR2-dependent signaling molecules ERK1/2, p38 and JNK mitogen-activated kinases. Our in vitro results demonstrate a direct effect of HCV core protein on activation of HSCs toward a profibrogenic state, which is mediated via the TLR2 pathway. Manipulating the TLR2 pathway may thus provide a new approach for antifibrotic therapies in HCV infection.

  12. Cloning and characterization of a novel hepatitis B virus core binding protein C12

    Institute of Scientific and Technical Information of China (English)

    Yin-Ying Lu; Jun Cheng; Yong-Ping Yang; Yan Liu; Lin Wang; Ke Li; Ling-Xia Zhang

    2005-01-01

    AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade)containing X-α-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating.pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein.RESULTS: C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray,of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function

  13. The Amount of Hepatocyte Turnover That Occurred during Resolution of Transient Hepadnavirus Infections Was Lower When Virus Replication Was Inhibited with Entecavir▿ †

    Science.gov (United States)

    Mason, William S.; Xu, Chunxiao; Low, Huey Chi; Saputelli, Jeffry; Aldrich, Carol E.; Scougall, Catherine; Grosse, Arend; Colonno, Richard; Litwin, Sam; Jilbert, Allison R.

    2009-01-01

    Transient hepadnavirus infections can involve spread of virus to the entire hepatocyte population. In this situation hepatocytes present following recovery are derived from infected hepatocytes. During virus clearance antiviral cytokines are thought to block virus replication and formation of new covalently closed circular DNA (cccDNA), the viral transcriptional template. It remains unclear if existing cccDNA is eliminated noncytolytically or if hepatocyte death and proliferation, to compensate for killing of some of the infected hepatocytes, are needed to remove cccDNA from surviving infected hepatocytes. Interpreting the relationship between hepatocyte death and cccDNA elimination requires knowing both the amount of hepatocyte turnover and whether cccDNA synthesis is effectively blocked during the period of immune destruction of infected hepatocytes. We have addressed these questions by asking if treatment of woodchucks with the nucleoside analog inhibitor of viral DNA synthesis entecavir (ETV) reduced hepatocyte turnover during clearance of transient woodchuck hepatitis virus (WHV) infections. To estimate hepatocyte turnover, complexity analysis was carried out on virus-cell DNA junctions created by integration of WHV and present following recovery in the livers of WHV-infected control or ETV-treated woodchucks. We estimated that, on average, 2.2 to 4.8 times less hepatocyte turnover occurred during immune clearance in the ETV-treated woodchucks. Computer modeling of the complexity data suggests that mechanisms in addition to hepatocyte death were responsible for elimination of cccDNA during recovery from transient infections. PMID:19073743

  14. Rab18 is required for viral assembly of hepatitis C virus through trafficking of the core protein to lipid droplets.

    Science.gov (United States)

    Dansako, Hiromichi; Hiramoto, Hiroki; Ikeda, Masanori; Wakita, Takaji; Kato, Nobuyuki

    2014-08-01

    During persistent infection of HCV, the HCV core protein (HCV-JFH-1 strain of genotype 2a) is recruited to lipid droplets (LDs) for viral assembly, but the mechanism of recruitment of the HCV core protein is uncertain. Here, we demonstrated that one of the Ras-related small GTPases, Rab18, was required for trafficking of the core protein around LDs. The knockdown of Rab18 reduced intracellular and extracellular viral infectivity, but not intracellular viral replication in HCV-JFH-1-infected RSc cells (an HuH-7-derived cell line). Exogenous expression of Rab18 increased extracellular viral infectivity almost two-fold. Furthermore, Rab18 was co-localized with the core protein in HCV-JFH-1-infected RSc cells, and the knockdown of Rab18 blocked recruitment of the HCV-JFH-1 core protein to LDs. These results suggest that Rab18 has an important role in viral assembly through the trafficking of the core protein to LDs.

  15. Hepatitis C virus core protein impairs metabolic disorder of liver cell via HOTAIR-Sirt1 signalling

    Science.gov (United States)

    Li, Zhi-qin; Gu, Xin-yu; Hu, Jin-xing; Ping, Yu; Li, Hua; Yan, Jing-ya; Li, Juan; Sun, Ran; Yu, Zu-jing; Zhang, Yi

    2016-01-01

    It has been suggested that Hepatitis C virus (HCV) core protein is associated with metabolic disorders of liver cell. However, the precise mechanism is still unclear. The aim of the present study was to explore the impact of HCV core protein on hepatocyte metabolism by HepG2 and the possible involvement of long non-coding (lnc) RNAs in this process. The effect of HCV core protein on lncRNAs expression was examined with quantitative RT-PCR (qRT-PCR). Manipulation of HVC core protein and lncRNA HOTAIR was to evaluate the role of interaction between them on cell metabolism-related gene expression and cellular metabolism. The potential downstream Sirt1 signal was examined by western blotting and qRT-PCR. Our data suggested that suppression of HOTAIR abrogates HCV core protein-induced reduction in Sirt1 and differential expression of glucose- and lipid-metabolism-related genes. Also it benefits for metabolic homoeostasis of hepatocyte indicated by restoration of cellular reactive oxygen species (ROS) level and NAD/NADH ratio. By manipulation of HOTAIR, we concluded that HOTAIR negatively regulates Sirt1 expression through affecting its promotor methylation. Moreover, overexpression of Sirt1 reverses pcDNA-HOTAIR-induced glucose- and lipid-metabolism-related gene expression. Our study suggests that HCV core protein causes dysfunction of glucose and lipid metabolism in liver cells through HOTAIR-Sirt1 signalling pathway. PMID:27129296

  16. Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame.

    Science.gov (United States)

    Chang, S F; Netter, H J; Hildt, E; Schuster, R; Schaefer, S; Hsu, Y C; Rang, A; Will, H

    2001-01-01

    Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.

  17. Aquareovirus NS80 Initiates Efficient Viral Replication by Retaining Core Proteins within Replication-Associated Viral Inclusion Bodies

    OpenAIRE

    Liming Yan; Jie Zhang; Hong Guo; Shicui Yan; Qingxiu Chen; Fuxian Zhang; Qin Fang

    2015-01-01

    Viral inclusion bodies (VIBs) are specific intracellular compartments for reoviruses replication and assembly. Aquareovirus nonstructural protein NS80 has been identified to be the major constituent for forming globular VIBs in our previous study. In this study, we investigated the role of NS80 in viral structural proteins expression and viral replication. Immunofluorescence assays showed that NS80 could retain five core proteins or inner-capsid proteins (VP1-VP4 and VP6), but not outer-capsi...

  18. Interaction between hepatitis C virus core protein and translin protein- a possible molecular mechanism for hepatocellular carcinoma and lymphoma caused by hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Ke Li; Gang Wang; Li Li; Ju-Mei Chen; Lin Wang; Jun Cheng; Yin-Ying Lu; Ling-Xin Zhang; Jin-Song Mu; Yuan Hong; Yan Liu; Hui-Juan Duan

    2003-01-01

    AIM: To investigate the interaction between hepatitis C viruscore protein and translin protein and its role in thepathogenensis of hepatocellular carcinoma and lymphoma.METHODS: With the components of the yeast two hybridsystem 3, "bait" plasmids of HCV core the gene wasconstructed. After proving that hepatitis C virus core proteincould be firmly expressed in AH109 yeast strains, yeast two-hybrid screening was performed by mating AH109 with Y187that transformed with liver cDNA library plasmids - pACT2and then plated on quadrople dropout (QDO) medium andthen assayed for α-gal activity. Sequencing analysis of thegenes of library plasmids in yeast colonies that could growon QDO with α-gal activity was performed. The interactionbetween HCV core protein and the protein we obtained frompositive colony was further confirmed by repeating yeasttwo - hybrid analysis and coimmunoprecipitation in vitro.RESULTS: A gene from a positive colony was the gene oftranslin, a recombination hotspot binding protein. Theinteraction between HCV core protein and translin proteincould be proved not only in yeast, but also in vitro.CONCLUSION: The core protein of HCV can interact withtranslin protein. This can partly explain the molecularmechanism for hepatocellular carcinoma and lymphomacaused by HCV.

  19. Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins

    Energy Technology Data Exchange (ETDEWEB)

    Xing,Y.; Xu, Y.; Chen, Y.; Jeffrey, P.; Chao, Y.; Lin, Z.; Li, Z.; Strack, S.; Stock, J.; Shi, Y.

    2006-01-01

    The serine/threonine phosphatase protein phosphatase 2A (PP2A) plays an essential role in many aspects of cellular functions and has been shown to be an important tumor suppressor. The core enzyme of PP2A comprises a 65 kDa scaffolding subunit and a 36 kDa catalytic subunit. Here we report the crystal structures of the PP2A core enzyme bound to two of its inhibitors, the tumor-inducing agents okadaic acid and microcystin-LR, at 2.6 and 2.8 {angstrom} resolution, respectively. The catalytic subunit recognizes one end of the elongated scaffolding subunit by interacting with the conserved ridges of HEAT repeats 11-15. Formation of the core enzyme forces the scaffolding subunit to undergo pronounced structural rearrangement. The scaffolding subunit exhibits considerable conformational flexibility, which is proposed to play an essential role in PP2A function. These structures, together with biochemical analyses, reveal significant insights into PP2A function and serve as a framework for deciphering the diverse roles of PP2A in cellular physiology.

  20. Hepatitis C virus core protein induces energy metabolism disorders of hepatocytes by down-regulation of silent mating type information regulation 2 homolog-1 and adenosine monophosphate-acti vated protein kinase signaling pathway

    Institute of Scientific and Technical Information of China (English)

    于建武

    2013-01-01

    Objective To study the role of silent mating type information regulation2homotog-1(SIRT1)-adenosine monophosphate(AMP)-activated protein kinase(AMPK) signaling pathway in hepatitis C virus core protein(HCV-core)induced energy metabolism disorders

  1. Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min-Sung; Saunders, Adam M.; Hamaoka, Brent Y.; Beachy, Philip A.; Leahy, Daniel J. (Stanford-MED); (JHU)

    2011-09-20

    Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-{angstrom} crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, {alpha}-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.

  2. Immunochemical method for detection of antibody against HTLV-III core protein based upon recombinant HTLV-III gag gene encoded protein

    Energy Technology Data Exchange (ETDEWEB)

    Chang, N.T.; Ghrayeb, J.

    1989-02-28

    A method is described of detecting antibody against HTLV-III core protein in a biological fluid, comprising the steps of: a. providing an antigen immunoadsorbent comprising a solid phase to which is attached a HTLV-III core antigen which is a chimeric antigen comprising an amino acid sequence beginning at amino acid number 1 through 99, and extending to amino acid number 228, the chimeric antigen being immunoreactive with antibody against HTLV-III core protein; b. incubating the immunoadsorbent with a sample of the biological fluid to be tested under conditions which allow antibody in the sample to complex with the antigen immunoadsorbent; c. separating the immmunoadsorbent from the sample; and d. determining antibody bound to the iuumoadsorbent as an indication of antibody against HTLV-III core protein in the sample.

  3. Hepatitis C virus core protein abrogates the DDX3 function that enhances IPS-1-mediated IFN-beta induction.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Oshiumi

    Full Text Available The DEAD box helicase DDX3 assembles IPS-1 (also called Cardif, MAVS, or VISA in non-infected human cells where minimal amounts of the RIG-I-like receptor (RLR protein are expressed. DDX3 C-terminal regions directly bind the IPS-1 CARD-like domain as well as the N-terminal hepatitis C virus (HCV core protein. DDX3 physically binds viral RNA to form IPS-1-containing spots, that are visible by confocal microscopy. HCV polyU/UC induced IPS-1-mediated interferon (IFN-beta promoter activation, which was augmented by co-transfected DDX3. DDX3 spots localized near the lipid droplets (LDs where HCV particles were generated. Here, we report that HCV core protein interferes with DDX3-enhanced IPS-1 signaling in HEK293 cells and in hepatocyte Oc cells. Unlike the DEAD box helicases RIG-I and MDA5, DDX3 was constitutively expressed and colocalized with IPS-1 around mitochondria. In hepatocytes (O cells with the HCV replicon, however, DDX3/IPS-1-enhanced IFN-beta-induction was largely abrogated even when DDX3 was co-expressed. DDX3 spots barely merged with IPS-1, and partly assembled in the HCV core protein located near the LD in O cells, though in some O cells IPS-1 was diminished or disseminated apart from mitochondria. Expression of DDX3 in replicon-negative or core-less replicon-positive cells failed to cause complex formation or LD association. HCV core protein and DDX3 partially colocalized only in replicon-expressing cells. Since the HCV core protein has been reported to promote HCV replication through binding to DDX3, the core protein appears to switch DDX3 from an IFN-inducing mode to an HCV-replication mode. The results enable us to conclude that HCV infection is promoted by modulating the dual function of DDX3.

  4. Hepatitis C virus core protein abrogates the DDX3 function that enhances IPS-1-mediated IFN-beta induction.

    Science.gov (United States)

    Oshiumi, Hiroyuki; Ikeda, Masanori; Matsumoto, Misako; Watanabe, Ayako; Takeuchi, Osamu; Akira, Shizuo; Kato, Nobuyuki; Shimotohno, Kunitada; Seya, Tsukasa

    2010-12-08

    The DEAD box helicase DDX3 assembles IPS-1 (also called Cardif, MAVS, or VISA) in non-infected human cells where minimal amounts of the RIG-I-like receptor (RLR) protein are expressed. DDX3 C-terminal regions directly bind the IPS-1 CARD-like domain as well as the N-terminal hepatitis C virus (HCV) core protein. DDX3 physically binds viral RNA to form IPS-1-containing spots, that are visible by confocal microscopy. HCV polyU/UC induced IPS-1-mediated interferon (IFN)-beta promoter activation, which was augmented by co-transfected DDX3. DDX3 spots localized near the lipid droplets (LDs) where HCV particles were generated. Here, we report that HCV core protein interferes with DDX3-enhanced IPS-1 signaling in HEK293 cells and in hepatocyte Oc cells. Unlike the DEAD box helicases RIG-I and MDA5, DDX3 was constitutively expressed and colocalized with IPS-1 around mitochondria. In hepatocytes (O cells) with the HCV replicon, however, DDX3/IPS-1-enhanced IFN-beta-induction was largely abrogated even when DDX3 was co-expressed. DDX3 spots barely merged with IPS-1, and partly assembled in the HCV core protein located near the LD in O cells, though in some O cells IPS-1 was diminished or disseminated apart from mitochondria. Expression of DDX3 in replicon-negative or core-less replicon-positive cells failed to cause complex formation or LD association. HCV core protein and DDX3 partially colocalized only in replicon-expressing cells. Since the HCV core protein has been reported to promote HCV replication through binding to DDX3, the core protein appears to switch DDX3 from an IFN-inducing mode to an HCV-replication mode. The results enable us to conclude that HCV infection is promoted by modulating the dual function of DDX3.

  5. Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease.

    Science.gov (United States)

    Nassal, M; Galle, P R; Schaller, H

    1989-01-01

    The hepatitis B virus (HBV) C gene directs the synthesis of two major gene products: HBV core antigen (HBcAg[p21c]), which forms the nucleocapsid, and HBV e antigen (HBeAg [p17e]), a secreted antigen that is produced by several processing events during its maturation. These proteins contain an amino acid sequence similar to the active-site residues of aspartic acid and retroviral proteases. On the basis of this sequence similarity, which is highly conserved among mammalian hepadnaviruses, a model has been put forward according to which processing to HBeAg is due to self-cleavage of p21c involving the proteaselike sequence. Using site-directed mutagenesis in conjunction with transient expression of HBV proteins in the human hepatoma cell line HepG2, we tested this hypothesis. Our results with HBV mutants in which one or two of the conserved amino acids have been replaced by others suggest strongly that processing to HBeAg does not depend on the presence of an intact proteaselike sequence in the core protein. Attempts to detect an influence of this sequence on the processing of HBV P gene products into enzymatically active viral polymerase also gave no conclusive evidence for the existence of an HBV protease. Mutations replacing the putatively essential aspartic acid showed little effect on polymerase activity. Additional substitution of the likewise conserved threonine residue by alanine, in contrast, almost abolished the activity of the polymerase. We conclude that an HBV protease, if it exists, is functionally different from aspartic acid and retroviral proteases. Images PMID:2657101

  6. Phylogeny of whey acidic protein (WAP) four-disulfide core proteins and their role in lower vertebrates and invertebrates.

    Science.gov (United States)

    Smith, Valerie J

    2011-10-01

    Proteins containing WAP (whey acidic protein) domains with a characteristic WFDC (WAP four-disulfide core) occur not only in mammals (including marsupials and monotremes) but also in birds, reptiles, amphibians and fish. In addition, they are present in numerous invertebrates, from cnidarians to urochordates. Many of those from non-mammalian groups are poorly understood with respect to function or phylogeny. Those well characterized so far are waprins from snakes, perlwapins from bivalves and crustins from decapod crustaceans. Waprins are venom proteins with a single WAP domain at the C-terminus. They display antimicrobial, rather than proteinase inhibitory, activities. Perlwapins, in contrast, possess three WAP domains at the C-terminus and are expressed in the shell nacre of abalones. They participate in shell formation by inhibiting the growth of calcium crystals in the shell. The crustin group is the largest of all WFDC-containing proteins in invertebrates with the vast majority being highly expressed in the haemocytes. Most have a single WAP domain at the C-terminus. The presence and type of the domains between the signal sequence and the C-terminus WAP domain separate the different crustin types. Most of the Type I and II crustins are antimicrobial towards Gram-positive bacteria, whereas the Type III crustins tend to display protease inhibition. Expression studies show that at least some crustins have other important biological effects, as levels change with physiological stress, wound repair, tissue regeneration or ecdysis. Thus WAP domains are widely distributed and highly conserved, serving in diverse physiological processes (proteinase inhibition, bacterial killing or inhibition of calcium transport).

  7. A disulfide-bonded dimer of the core protein of hepatitis C virus is important for virus-like particle production.

    Science.gov (United States)

    Kushima, Yukihiro; Wakita, Takaji; Hijikata, Makoto

    2010-09-01

    Hepatitis C virus (HCV) core protein forms the nucleocapsid of the HCV particle. Although many functions of core protein have been reported, how the HCV particle is assembled is not well understood. Here we show that the nucleocapsid-like particle of HCV is composed of a disulfide-bonded core protein complex (dbc-complex). We also found that the disulfide-bonded dimer of the core protein (dbd-core) is formed at the endoplasmic reticulum (ER), where the core protein is initially produced and processed. Mutational analysis revealed that the cysteine residue at amino acid position 128 (Cys128) of the core protein, a highly conserved residue among almost all reported isolates, is responsible for dbd-core formation and virus-like particle production but has no effect on the replication of the HCV RNA genome or the several known functions of the core protein, including RNA binding ability and localization to the lipid droplet. The Cys128 mutant core protein showed a dominant negative effect in terms of HCV-like particle production. These results suggest that this disulfide bond is critical for the HCV virion. We also obtained the results that the dbc-complex in the nucleocapsid-like structure was sensitive to proteinase K but not trypsin digestion, suggesting that the capsid is built up of a tightly packed structure of the core protein, with its amino (N)-terminal arginine-rich region being concealed inside.

  8. Protein film voltammetry and co-factor electron transfer dynamics in spinach photosystem II core complex.

    Science.gov (United States)

    Zhang, Yun; Magdaong, Nikki; Frank, Harry A; Rusling, James F

    2014-05-01

    Direct protein film voltammetry (PFV) was used to investigate the redox properties of the photosystem II (PSII) core complex from spinach. The complex was isolated using an improved protocol not used previously for PFV. The PSII core complex had high oxygen-evolving capacity and was incorporated into thin lipid and polyion films. Three well-defined reversible pairs of reduction and oxidation voltammetry peaks were observed at 4 °C in the dark. Results were similar in both types of films, indicating that the environment of the PSII-bound cofactors was not influenced by film type. Based on comparison with various control samples including Mn-depleted PSII, peaks were assigned to chlorophyll a (Chl a) (Em = -0.47 V, all vs. NHE, at pH 6), quinones (-0.12 V), and the manganese (Mn) cluster (Em = 0.18 V). PFV of purified iron heme protein cytochrome b-559 (Cyt b-559), a component of PSII, gave a partly reversible peak pair at 0.004 V that did not have a potential similar to any peaks observed from the intact PSII core complex. The closest peak in PSII to 0.004 V is the 0.18 V peak that was found to be associated with a two-electron process, and thus is inconsistent with iron heme protein voltammetry. The -0.47 V peak had a peak potential and peak potential-pH dependence similar to that found for purified Chl a incorporated into DMPC films. The midpoint potentials reported here may differ to various extents from previously reported redox titration data due to the influence of electrode double-layer effects. Heterogeneous electron transfer (hET) rate constants were estimated by theoretical fitting and digital simulations for the -0.47 and 0.18 V peaks. Data for the Chl a peaks were best fit to a one-electron model, while the peak assigned to the Mn cluster was best fit by a two-electron/one-proton model.

  9. TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends.

    Science.gov (United States)

    Konishi, Akimitsu; Izumi, Takashi; Shimizu, Shigeomi

    2016-09-23

    Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Strategies for crystallizing a chromatin protein in complex with the nucleosome core particle.

    Science.gov (United States)

    Makde, Ravindra D; Tan, Song

    2013-11-15

    The molecular details of how chromatin factors and enzymes interact with the nucleosome are critical to understanding fundamental genetic processes including cell division and gene regulation. A structural understanding of such processes has been hindered by the difficulty in producing diffraction-quality crystals of chromatin proteins in complex with the nucleosome. We describe here the steps used to grow crystals of the 300-kDa RCC1 chromatin factor/nucleosome core particle complex that diffract to 2.9-Å resolution. These steps include both pre- and postcrystallization strategies potentially useful to other complexes. We screened multiple variant RCC1/nucleosome core particle complexes assembled using different RCC1 homologs and deletion variants, and nucleosomes containing nucleosomal DNA with different sequences and lengths, as well as histone deletion variants. We found that using RCC1 from different species produced different crystal forms of the RCC1/nucleosome complex consistent with key crystal packing interactions mediated by RCC1. Optimization of postcrystallization soaks to dehydrate the crystals dramatically improved the diffraction quality of the RCC1/nucleosome crystal from 5.0- to 2.9-Å resolution. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. The H1 linker histones: multifunctional proteins beyond the nucleosomal core particle.

    Science.gov (United States)

    Hergeth, Sonja P; Schneider, Robert

    2015-11-01

    The linker histone H1 family members are a key component of chromatin and bind to the nucleosomal core particle around the DNA entry and exit sites. H1 can stabilize both nucleosome structure and higher-order chromatin architecture. In general, H1 molecules consist of a central globular domain with more flexible tail regions at both their N- and C-terminal ends. The existence of multiple H1 subtypes and a large variety of posttranslational modifications brings about a considerable degree of complexity and makes studying this protein family challenging. Here, we review recent progress in understanding the function of linker histones and their subtypes beyond their role as merely structural chromatin components. We summarize current findings on the role of H1 in heterochromatin formation, transcriptional regulation and embryogenesis with a focus on H1 subtypes and their specific modifications. © 2015 The Authors.

  12. Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein, production of the viral particle and expression of host proteins involved in pathogenicity.

    Science.gov (United States)

    Otoguro, Teruhime; Tanaka, Tomohisa; Kasai, Hirotake; Yamashita, Atsuya; Moriishi, Kohji

    2016-11-01

    Hepatitis C virus (HCV) core protein is responsible for the formation of infectious viral particles and induction of pathogenicity. The C-terminal transmembrane region of the immature core protein is cleaved by signal peptide peptidase (SPP) for maturation of the core protein. SPP belongs to the family of presenilin-like aspartic proteases. Some presenilin inhibitors are expected to suppress HCV infection and production; however, this anti-HCV effect has not been investigated in detail. In this study, presenilin inhibitors were screened to identify anti-HCV compounds. Of the 13 presenilin inhibitors tested, LY411575 was the most potent inhibitor of SPP-dependent cleavage of HCV core protein. Production of intracellular core protein and supernatant infectious viral particles from HCV-infected cells was significantly impaired by LY411575 in a dose-dependent manner (half maximum inhibitory concentration = 0.27 μM, cytotoxic concentration of the extracts to cause death to 50% of viable cells > 10 μM). No effect of LY411575 on intracellular HCV RNA in the subgenomic replicon cells was detected. LY411575 synergistically promoted daclatasvir-dependent inhibition of viral production, but not that of viral replication. Furthermore, LY411575 inhibited HCV-related production of reactive oxygen species and expression of NADPH oxidases and vascular endothelial growth factor. Taken together, our data suggest that LY411575 suppresses HCV propagation through SPP inhibition and impairs host gene expressions related to HCV pathogenicity.

  13. Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein.

    Science.gov (United States)

    Angus, Allan G N; Dalrymple, David; Boulant, Steeve; McGivern, David R; Clayton, Reginald F; Scott, Martin J; Adair, Richard; Graham, Susan; Owsianka, Ania M; Targett-Adams, Paul; Li, Kui; Wakita, Takaji; McLauchlan, John; Lemon, Stanley M; Patel, Arvind H

    2010-01-01

    The cellular DEAD-box protein DDX3 was recently shown to be essential for hepatitis C virus (HCV) replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating the JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus life cycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core-DDX3 interaction is dispensable in the HCV life cycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.

  14. Hepatitis C virus NS5A and core proteins induce oxidative stress-mediated calcium signalling alterations in hepatocytes.

    Science.gov (United States)

    Dionisio, Natalia; Garcia-Mediavilla, Maria V; Sanchez-Campos, Sonia; Majano, Pedro L; Benedicto, Ignacio; Rosado, Juan A; Salido, Gines M; Gonzalez-Gallego, Javier

    2009-05-01

    The hepatitis C virus (HCV) structural core and non-structural NS5A proteins induce in liver cells a series of intracellular events, including elevation of reactive oxygen and nitrogen species (ROS/RNS). Since oxidative stress is associated to altered intracellular Ca(2+) homeostasis, we aimed to investigate the effect of these proteins on Ca(2+) mobilization in human hepatocyte-derived transfected cells, and the protective effect of quercetin treatment. Ca(2+) mobilization and actin reorganization were determined by spectrofluorimetry. Production of ROS/RNS was determined by flow cytometry. Cells transfected with NS5A and core proteins showed enhanced ROS/RNS production and resting cytosolic Ca(2+) concentration, and reduced Ca(2+) concentration into the stores. Phenylephrine-evoked Ca(2+) release, Ca(2+) entry and extrusion by the plasma membrane Ca(2+)-ATPase were significantly reduced in transfected cells. Similar effects were observed in cytokine-activated cells. Phenylephrine-evoked actin reorganization was reduced in the presence of core and NS5A proteins. These effects were significantly prevented by quercetin. Altered Ca(2+) mobilization and increased calpain activation were observed in replicon-containing cells. NS5A and core proteins induce oxidative stress-mediated Ca(2+) homeostasis alterations in human hepatocyte-derived cells, which might underlie the effects of both proteins in the pathogenesis of liver disorders associated to HCV infection.

  15. Grafted block complex coacervate core micelles and their effect on protein adsorption on silica and polystyrene.

    Science.gov (United States)

    Brzozowska, Agata M; de Keizer, Arie; Norde, Willem; Detrembleur, Christophe; Cohen Stuart, Martien A

    2010-07-01

    We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins beta-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer PAA(21)-b-PAPEO(14) (poly(acrylic acid)-b-poly(acrylate methoxy poly(ethylene oxide)), with a negatively charged PAA block and a neutral PAPEO block and a positively charged homopolymer P2MVPI (poly(N-methyl 2-vinyl pyridinium iodide). In solution, these C3Ms partly disintegrate at salt concentrations between 50 and 100 mM NaCl. Adsorption of C3Ms and proteins has been studied with fixed-angle optical reflectometry, at salt concentrations ranging from 1 to 100 mM NaCl. In comparison with the adsorption of PAA(21)-b-PAPEO(14) alone adsorption of C3Ms significantly increases the amount of PAA(21)-b-PAPEO(14) on the surface. This results in a higher surface density of PEO chains. The stability of the C3M coatings and their influence on protein adsorption are determined by the composition and the stability of the C3Ms in solution. A C3M-PAPEO(14)/P2MVPI(43) coating strongly suppresses the adsorption of all proteins on silica and polystyrene. The reduction of protein adsorption is the highest at 100 mM NaCl (>90%). The adsorbed C3M-PAPEO(14)/P2MVPI(43) layer is partly removed from the surface upon exposure to an excess of beta-lactoglobulin solution, due to formation of soluble aggregates consisting of beta-lactoglobulin and P2MVPI(43). In contrast, C3M-PAPEO(14)/P2MVPI(228) which has a fivefold longer cationic block enhances adsorption of the negatively charged proteins on both surfaces at salt concentrations above 1 mM NaCl. A single PAA(21)-b-PAPEO(14) layer causes only a moderate reduction of protein adsorption.

  16. Proteins from the organic matrix of core-top and fossil planktonic foraminifera

    Science.gov (United States)

    Robbins, L. L.; Brew, K.

    1990-08-01

    Organic constituents isolated from the tests (shells) of six species of core-top planktonic foraminifera, ranging in age between 2 and 4 Ka BP, consist of a heterogeneous mixture of proteins and polypeptides. At least seven discrete polypeptides are present as indicated by reverse phase HPLC and by gel electrophoresis. High percentages of aspartic acid and glutamic acid characterize one class of protein, while glycine, serine, and alanine-rich proteins dominate in a second class. Similar HPLC Chromatographie elution profiles are observed for all species analyzed, varying only in intensity of the peaks and in amino acid composition from species to species. The approximate molecular weights of two major fossil proteins ranged between 50,000 and 70,000 daltons. A comparison of 2-4 and 300 Ka Bp samples shows that while most of the polypeptides are present in both samples, some acidic polypeptides are not present in the older sample. These data suggest that some of the acidic polypeptides may be more soluble than other fractions and are lost more quickly from the test. The remaining hydrophobic, possibly more insoluble, polypeptides may be preserved in much older specimens and may be useful in tracing phylogeny of the planktonic foraminifera. Amino acid analyses of total test extracts before and after dialysis demonstrate that some acidic amino acids, particularly aspartic acid, and possibly peptides less than 6000-8000 daltons are lost during dialysis. Although a large percentage of these components are undoubtedly from the original organic matrix, at this point adsorbed components cannot be ruled out. These data caution against the use of total amino acid compositions in biogeochemical studies.

  17. Protein encapsulated core-shell structured particles prepared by coaxial electrospraying: investigation on material and processing variables.

    Science.gov (United States)

    Zamani, Maedeh; Prabhakaran, Molamma P; Thian, Eng San; Ramakrishna, Seeram

    2014-10-01

    Biodegradable polymeric particles have been extensively investigated for controlled drug delivery of various therapeutic agents. 'Coaxial' electrospraying was successfully employed in this study, to fabricate core-shell PLGA particles containing bovine serum albumin (BSA) as the model protein, and the results were also compared to particles prepared by 'emulsion' electrospraying. Two different molecular weights of PLGA were employed to encapsulate the protein. Solution properties and processing parameters were found to influence the morphology of the core-shell particles. Depending on the type of solvent used to dissolve the polymer as well as the polymer concentration and molecular weight, the mean diameter of the particles varied between 3.0 to 5.5 μm. Fluorescence microscopic analysis of the electrosprayed particles using FITC-conjugated BSA demonstrated the core-shell structure of the developed particles. The encapsulation efficiency and release behavior of BSA was influenced by shell:core feeding ratio, protein concentration, and the electrospraying method. The encapsulation efficiency of BSA within the core-shell particles of high and low molecular weight PLGA was found 15.7% and 25.1% higher than the emulsion electrosprayed particles, respectively. Moreover, the total amount of BSA released from low molecular weight PLGA particles was significantly higher than high molecular weight PLGA particles within 43 days of release studies, with negligible effect on encapsulation efficiency. The technique of coaxial electrospraying has high potential for encapsulation of susceptible protein-based therapeutic agents such as growth factors for multiple drug delivery applications.

  18. Bioengineered Vaults: Self-Assembling Protein Shell–Lipophilic Core Nanoparticles for Drug Delivery

    Science.gov (United States)

    2015-01-01

    We report a novel approach to a new class of bioengineered, monodispersed, self-assembling vault nanoparticles consisting of a protein shell exterior with a lipophilic core interior designed for drug and probe delivery. Recombinant vaults were engineered to contain a small amphipathic α-helix derived from the nonstructural protein 5A of hepatitis C virus, thereby creating within the vault lumen a lipophilic microenvironment into which lipophilic compounds could be reversibly encapsulated. Multiple types of electron microscopy showed that attachment of this peptide resulted in larger than expected additional mass internalized within the vault lumen attributable to incorporation of host lipid membrane constituents spanning the vault waist (>35 nm). These bioengineered lipophilic vaults reversibly associate with a sample set of therapeutic compounds, including all-trans retinoic acid, amphotericin B, and bryostatin 1, incorporating hundreds to thousands of drug molecules per vault nanoparticle. Bryostatin 1 is of particular therapeutic interest because of its ability to potently induce expression of latent HIV, thus representing a preclinical lead in efforts to eradicate HIV/AIDS. Vaults loaded with bryostatin 1 released free drug, resulting in activation of HIV from provirus latency in vitro and induction of CD69 biomarker expression following intravenous injection into mice. The ability to preferentially and reversibly encapsulate lipophilic compounds into these novel bioengineered vault nanoparticles greatly advances their potential use as drug delivery systems. PMID:25061969

  19. Methionine Oxidation Perturbs the Structural Core of the Prion Protein and Suggests a Generic Misfolding Pathway*

    Science.gov (United States)

    Younan, Nadine D.; Nadal, Rebecca C.; Davies, Paul; Brown, David R.; Viles, John H.

    2012-01-01

    Oxidative stress and misfolding of the prion protein (PrPC) are fundamental to prion diseases. We have therefore probed the effect of oxidation on the structure and stability of PrPC. Urea unfolding studies indicate that H2O2 oxidation reduces the thermodynamic stability of PrPC by as much as 9 kJ/mol. 1H-15N NMR studies indicate methionine oxidation perturbs key hydrophobic residues on one face of helix-C as follows: Met-205, Val-209, and Met-212 together with residues Val-160 and Tyr-156. These hydrophobic residues pack together and form the structured core of the protein, stabilizing its ternary structure. Copper-catalyzed oxidation of PrPC causes a more significant alteration of the structure, generating a monomeric molten globule species that retains its native helical content. Further copper-catalyzed oxidation promotes extended β-strand structures that lack a cooperative fold. This transition from the helical molten globule to β-conformation has striking similarities to a misfolding intermediate generated at low pH. PrP may therefore share a generic misfolding pathway to amyloid fibers, irrespective of the conditions promoting misfolding. Our observations support the hypothesis that oxidation of PrP destabilizes the native fold of PrPC, facilitating the transition to PrPSc. This study gives a structural and thermodynamic explanation for the high levels of oxidized methionine in scrapie isolates. PMID:22654104

  20. A polymer-protein core-shell nanomedicine for inhibiting cancer migration followed by photo-triggered killing.

    Science.gov (United States)

    Ramachandran, Ranjith; Malarvizhi, Giridharan Loghanathan; Chandran, Parwathy; Gupta, Neha; Menon, Deepthy; Panikar, Dilip; Nair, Shantikumar; Koyakutty, Manzoor

    2014-08-01

    Migratory capacity of cancer plays a critical role in the process of metastasis. Aberrant focal adhesions activated by the phosphorylation of Src kinase enables cancer cells to anchor on its micro-environment and migrate towards biochemically favorable niche, causing metastasis. Effective blocking of the migratory capacity of cancer cells by inhibiting protein kinases and subsequent application of cytotoxic stress may provide better therapeutic outcome. Here, we report a novel core-shell nanomedicine that inhibits cancer migration by nano-shell and impart reactive oxygen stress by laser assisted photosensitization of nano-core. For this, we have optimized a polymer-protein nanoconstruct where a photosensitizer (5,10,15, 20-tetrakis(meso-hydroxyphenyl)porphyrin (mTHPP) is loaded into poly(lactic-co-glycolic acid) (PLGA) nano-core and Src kinase inhibitor (dasatinib) is loaded into albumin nano-shell. The polymer-core was prepared by electrospray technique and albumin-shell was formed by alcohol coacervation. Transmission electron microscopy studies revealed the formation of - 80 nm sized nano-core decorated with - 10 nm size nano-shell. Successful incorporation of monomeric mTHPP in nano-core resulted improved photo-physical properties and singlet oxygen release under physiological conditions compared to free-mTHPP. Core-shell nanomedicine also showed dose and time dependent cellular uptake in U87MG glioma cells. Dasatinib released from nano-shell caused down regulation of phospho-Src leading to significant impairment of cancer migration and subsequent laser assisted photosensitization of nano-core resulted in the release of reactive oxygen stress leading to apoptosis of spatially confined cancer cells. In vivo studies on Wistar rats indicated the absence of any significant toxicity caused by the intravenous administration of nanomedicine. These results clearly show the advantage of core-shell nanomedicine mediated combinatorial approach for inhibiting important

  1. Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain.

    Science.gov (United States)

    Kimura, Tatsuji; Ohno, Nobuhiko; Terada, Nobuo; Rokuhara, Akinori; Matsumoto, Akihiro; Yagi, Shintaro; Tanaka, Eiji; Kiyosawa, Kendo; Ohno, Shinichi; Maki, Noboru

    2005-06-10

    DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.

  2. Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region*

    Science.gov (United States)

    Cobb, Nathan J.; Apostol, Marcin I.; Chen, Shugui; Smirnovas, Vytautas; Surewicz, Witold K.

    2014-01-01

    Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrPSc. One key operational parameter used to define differences between strains has been conformational stability of PrPSc as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrPSc, especially because large strain-specific differences in PrPSc stability are often observed despite a similar size of the PrPSc core region. PMID:24338015

  3. Conformational stability of mammalian prion protein amyloid fibrils is dictated by a packing polymorphism within the core region.

    Science.gov (United States)

    Cobb, Nathan J; Apostol, Marcin I; Chen, Shugui; Smirnovas, Vytautas; Surewicz, Witold K

    2014-01-31

    Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP(Sc). One key operational parameter used to define differences between strains has been conformational stability of PrP(Sc) as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP(Sc), especially because large strain-specific differences in PrP(Sc) stability are often observed despite a similar size of the PrP(Sc) core region.

  4. "Hot cores" in proteins: Comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms

    Directory of Open Access Journals (Sweden)

    Bossa Francesco

    2008-02-01

    Full Text Available Abstract Background A wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. These include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. It has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. In this work, protein crystal structures from hyper/thermophilic organisms and their mesophilic homologs have been compared, in order to quantify the difference of apolar contact area and to assess the role played by the hydrophobic contacts in the stabilization of the protein core, at high temperatures. Results The construction of two datasets was carried out so as to satisfy several restrictive criteria, such as minimum redundancy, resolution and R-value thresholds and lack of any structural defect in the collected structures. This approach allowed to quantify with relatively high precision the apolar contact area between interacting residues, reducing the uncertainty due to the position of atoms in the crystal structures, the redundancy of data and the size of the dataset. To identify the common core regions of these proteins, the study was focused on segments that conserve a similar main chain conformation in the structures analyzed, excluding the intervening regions whose structure differs markedly. The results indicated that hyperthermophilic proteins underwent a significant increase of the hydrophobic contact area contributed by those residues composing the alpha-helices of the structurally conserved regions. Conclusion This study indicates the decreased flexibility of alpha-helices in proteins core as a major factor contributing to the enhanced termostability of a number of hyperthermophilic proteins. This effect, in turn, may be due to an increased number of buried methyl groups in

  5. Designed armadillo repeat proteins as general peptide-binding scaffolds: consensus design and computational optimization of the hydrophobic core

    DEFF Research Database (Denmark)

    Parmeggiani, Fabio; Pellarin, Riccardo; Larsen, Anders Peter

    2007-01-01

    interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large...... number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences...

  6. Structural Rearrangement upon Fragmentation of the Stability Core of the ALS-Linked Protein TDP-43.

    Science.gov (United States)

    Morgan, Brittany R; Zitzewitz, Jill A; Massi, Francesca

    2017-08-08

    Amyotrophic lateral sclerosis (ALS) is the most common adult degenerative motor neuron disease. Experimental evidence indicates a direct role of transactive-response DNA-binding protein 43 (TDP-43) in the pathology of ALS and other neurodegenerative diseases. TDP-43 has been identified as a major component of cytoplasmic inclusions in patients with sporadic ALS; however, the molecular basis of the disease mechanism is not yet fully understood. Fragmentation within the second RNA recognition motif (RRM2) of TDP-43 has been observed in patient tissues and may play a role in the formation of aggregates in disease. To determine the structural and dynamical changes resulting from the truncation that could lead to aggregation and toxicity, we performed molecular dynamics simulations of the full-length RRM2 domain (the stability core of TDP-43) and of a truncated variant (where residues 189-207 are deleted to mimic a site of cleavage within RRM2 found in ALS patients). Our simulations show heterogeneous structural reorganization and decreased stability of the truncated RRM2 domain compared to the full-length domain, consistent with previous experimental results. The decreased stability and structural reorganization in the truncated RRM2 result in a higher probability of protein-protein interactions through altered electrostatic surface charges and increased accessibility of hydrophobic residues (including the nuclear export sequence), providing a rationale for the increased cytoplasmic aggregation of RRM2 fragments seen in sporadic ALS patients. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Protein-loaded microspheres prepared by sequential adsorption of dextran sulphate and protamine on melamine formaldehyde core.

    Science.gov (United States)

    Balabushevich, Nadezda G; Larionova, Natalia I

    2009-11-01

    Polyelectrolyte multilayer microspheres were fabricated by layer-by-layer self-assembly of a dextran sulphate and a protamine on melamine formaldehyde cores, followed by the partial decomposition of the core. Effects of pH on the encapsulation of proteins and enzymes with different physico-chemical properties (insulin, aprotinin, peroxidase, glucose oxidase (GOD), catalase (Cat)) in the prepared microspheres were then studied. This method of protein encapsulation demonstrated a high loading capacity and efficiency. The protein incorporation and release was regulated by the pH of the solution. Encapsulated enzymes retained a high specific activity depending on the amount of protein incorporated. Bienzyme system GOD/Cat immobilized in the microspheres was suitable for the glucose content assay.

  8. Screening and identification of interacting proteins with hepatitis B virus core protein in leukocytes and cloning of new gene C1

    Institute of Scientific and Technical Information of China (English)

    Shu-Mei Lin; Jun Cheng; Yin-Ying Lu; Shu-Lin Zhang; Qian Yang; Tian-Yan Chen; Min Liu; Lin Wang

    2006-01-01

    AIM: To investigate the biological function of HBcAg in pathogenesis of HBV replication in peripheral blood mononuclear cells (PBMCs).METHODS: HBcAg region was amplified by polymerase chain reaction (PCR) and HBV HBcAg bait plasmid pGBKT7-HBcAg was constructed by routine molecular biological methods. Then the recombinant plasmid DNA was transformed into yeast AH109. After the HBV core protein was expressed in AH10g yeast strains (Western blot analysis), yeast-two hybrid screening was performed by mating AH109 with Y187 containing leukocyte cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-HisAde) (QDO) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) (TDO). The second screening was performed with the LacZ report gene ( yeast cells were grown in QDO medium containing X-a-gal). The interaction between HBV core protein and the protein obtained from positive colonies was further confirmed by repeating yeast-two hybrid. After plasmid DNA was extracted from blue colonies and sequenced, the results were analyzed by bioinformatic methods.RESULTS: Eighteen colonies were obtained and sequenced, including hypermethylated in cancer 2 (3colones), eukaryotic translation elongation factor 2 (2colones), acetyl-coenzyme A synthetase 3 (1 colone),DNA polymerase gamma (1 colone), putative translation initiation factor (1 colone), chemokine (C-C motif)receptor 5 (1 colone), mitochondrial ribosomal protein L41 (1 colone), kyot binding protein genes (1 colone),RanBPM (1 colone), HBeAg-binding protein 3 (1 colone),programmed cell death 2 (1 colone). Four new genes with unknown function were identified.CONCLUSION: Successful cloning of genes of HBV core protein interacting proteins in leukocytes may provide some new clues for studying the biological functions of HBV core protein.

  9. HCV core protein promotes liver fibrogenesis via up-regulation of CTGF with TGF-beta1.

    Science.gov (United States)

    Shin, Ju Yeop; Hur, Wonhee; Wang, Jin Sang; Jang, Jeong Won; Kim, Chang Wook; Bae, Si Hyun; Jang, Sung Key; Yang, Se-Hwan; Sung, Young Chul; Kwon, Oh-Joo; Yoon, Seung Kew

    2005-04-30

    Liver cirrhosis is one of the major complications of hepatitis C virus (HCV) infection, but the mechanisms underlying HCV-related fibrogenesis are still not clear. Although the roles of HCV core protein remain poorly understood, it is supposed to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to examine the role of HCV core protein on the hepatic fibrogenesis. We established an in vitro co-culture system with primary hepatic stellate cell (HSC) isolated from rats, and a stable HepG2-HCV core cell line which had been transfected with HCV core gene. The expressions of fibrosis-related molecules transforming growth factor beta1 (TGF-beta1), transforming growth factor beta receptor II (TGFbetaRII), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were analyzed via histological or molecular methods. In addition, the expression levels of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) from the co-cultured media were measured by zymogram and ELISA, respectively. The expressions of alpha-SMA, TGF-beta1, Col I, TGFbetaRII and MMP-2 were significantly increased in the co-culture of stable HepG2-HCV core with HSC. Moreover, the significant increases of CTGF and TGF-beta1 in the HCV core-expressing cells were observed by either Northern or Western blot analysis. These results suggest that HCV core protein may contribute to the hepatic fibrogenesis via up-regulation of CTGF and TGF-beta1.

  10. Magnetic core/shell Fe3O4/Au nanoparticles for studies of quinolones binding to protein by fluorescence spectroscopy.

    Science.gov (United States)

    Jin, Rui; Song, Daqian; Xiong, Huixia; Ai, Lisha; Ma, Pinyi; Sun, Ying

    2016-03-01

    Magnetic core/shell Fe3O4/Au nanoparticles were used in the determination of drug binding to bovine serum albumin (BSA) using a fluorescence spectroscopic method. The binding constants and number of binding sites for protein with drugs were calculated using the Scatchard equation. Because of their superparamagnetic and biocompatible characteristics, magnetic core/shell Fe3O4/Au nanoparticles served as carrier proteins for fixing proteins. After binding of the protein to a drug, the magnetic core/shell Fe3O4/Au nanoparticles-protein-drug complex was separated from the free drug using an applied magnetic field. The free drug concentration was obtained directly by fluorescence spectrometry and the proteins did not influence the drug determination. So, the achieved number of binding sites should be reliable. The binding constant and site number for ciprofloxacin (CPFX) binding to BSA were 2.055 × 10(5) L/mol and 31.7, and the corresponding values for norfloxacin (NOR) binding to BSA were 1.383 × 10(5) L/mol and 38.8. Based on the achieved results, a suitable method was proposed for the determination of binding constants and the site number for molecular interactions. The method was especially suitable for studies on the interactions of serum albumin with the active ingredients of Chinese medicine.

  11. Molecular characterization and polyclonal antibody generation against core component CagX protein of Helicobacter pylori type IV secretion system

    Science.gov (United States)

    Gopal, Gopal Jee; Kumar, Awanish; Pal, Jagannath; Mukhopadhyay, Gauranga

    2014-01-01

    Gram-negative bacteria Helicobacter pylori cause gastric ulcer, duodenal cancer, and found in almost half of the world’s residents. The protein responsible for this disease is secreted through type IV secretion system (TFSS) of H. pylori. TFSS is encoded by 40-kb region of chromosomal DNA known as cag-pathogenicity island (PAI). TFSS comprises of three major components: cytoplasmic/inner membrane ATPase, transmembrane core-complex and outer membranous pilli, and associated subunits. Core complex consists of CagX, CagT, CagM, and Cag3(δ) proteins as per existing knowledge. In this study, we have characterized one of the important component of core-complex forming sub-unit protein, i.e., CagX. Complete ORF of CagX except signal peptide coding region was cloned and expressed in pET28a vector. Purification of CagX protein was performed, and polyclonal anti-sera against full-length recombinant CagX were raised in rabbit model. We obtained a very specific and high titer, CagX anti-sera that were utilized to characterize endogenous CagX. Surface localization of CagX was also seen by immunofluorescence microscopy. In short for the first time a full-length CagX was characterized, and we showed that CagX is the part of high molecular weight core complex, which is important for assembly and function of H. pylori TFSS. PMID:24637488

  12. Reduction of protein adsorption on silica and polystyrene surfaces due to coating with Complex Coacervate Core Micelles

    NARCIS (Netherlands)

    Brzozowska, A. M.; Hofs, B.; de Keizer, A.; Fokkink, R.; Stuart, Martien A. Cohen; Norde, W.

    2009-01-01

    The reduction of protein adsorption by a polymer brush formed upon adsorption of Complex Coacervate Core Micelles (C3Ms), consisting of a charged copolymer containing a neutral block and an oppositely charged homopolymer, on silica and polystyrene surfaces has been studied in situ using fixed angle

  13. DISTRIBUTION OF GBM HEPARAN-SULFATE PROTEOGLYCAN CORE PROTEIN AND SIDE-CHAINS IN HUMAN GLOMERULAR-DISEASES

    NARCIS (Netherlands)

    VANDENBORN, J; VANDENHEUVEL, LPWJ; BAKKER, MAH; VEERKAMP, JH; ASSMANN, KJM; WEENING, JJ; BERDEN, JHM

    1993-01-01

    Using monoclonal antibodies (mAbs) recognizing either the core protein or the heparan sulfate (HS) side chain of human GBM heparan sulfate proteoglycan (HSPG), we investigated their glomerular distribution on cryostat sections of human kidney tissues. The study involved 95 biopsies comprising twelve

  14. Aberrant expression of mucin core proteins and o-linked glycans associated with progression of pancreatic cancer

    DEFF Research Database (Denmark)

    Remmers, Neeley; Anderson, Judy M; Linde, Erin M;

    2013-01-01

    Mucin expression is a common feature of most adenocarcinomas and features prominently in current attempts to improve diagnosis and therapy for pancreatic cancer and other adenocarcinomas. We investigated the expression of a number of mucin core proteins and associated O-linked glycans expressed...

  15. pH/sugar dual responsive core-cross-linked PIC micelles for enhanced intracellular protein delivery.

    Science.gov (United States)

    Ren, Jie; Zhang, Yanxin; Zhang, Ju; Gao, Hongjun; Liu, Gan; Ma, Rujiang; An, Yingli; Kong, Deling; Shi, Linqi

    2013-10-14

    Herein, a series of biocompatible, robust, pH/sugar-sensitive, core-cross-linked, polyion complex (PIC) micelles based on phenylboronic acid-catechol interaction were developed for protein intracellular delivery. The rationally designed poly(ethylene glycol)-b-poly(glutamic acid-co-glutamicamidophenylboronic acid) (PEG-b-P(Glu-co-GluPBA)) and poly(ethylene glycol)-b-poly(l-lysine-co-ε-3,4-dihydroxyphenylcarboxyl-L-lysine) (PEG-b-P(Lys-co-LysCA)) copolymers were successfully synthesized and self-assembled under neutral aqueous condition to form uniform micelles. These micelles possessed a distinct core-cross-linked core-shell structure comprised of the PEG outer shell and the PGlu/PLys polyion complex core bearing boronate ester cross-linking bonds. The cross-linked micelles displayed superior physiological stabilities compared with their non-cross-linked counterparts while swelling and disassembling in the presence of excess fructose or at endosomal pH. Notably, either negatively or positively charged proteins can be encapsulated into the micelles efficiently under mild conditions. The in vitro release studies showed that the release of protein cargoes under physiological conditions was minimized, while a burst release occurred in response to excess fructose or endosomal pH. The cytotoxicity of micelles was determined by cck-8 assay in HepG2 cells. The cytochrome C loaded micelles could efficiently delivery proteins into HepG2 cells and exhibited enhanced apoptosis ability. Hence, this type of core-cross-linked PIC micelles has opened a new avenue to intracellular protein delivery.

  16. Stability of Retroviral Vectors Against Ultracentrifugation Is Determined by the Viral Internal Core and Envelope Proteins Used for Pseudotyping.

    Science.gov (United States)

    Kim, Soo-Hyun; Lim, Kwang-Il

    2017-05-31

    Retroviral and lentiviral vectors are mostly pseudotyped and often purified and concentrated via ultracentrifugation. In this study, we quantified and compared the stabilities of retroviral [murine leukemia virus (MLV)-based] and lentiviral [human immunodeficiency virus (HIV)-1-based] vectors pseudotyped with relatively mechanically stable envelope proteins, vesicular stomatitis virus glycoproteins (VSVGs), and the influenza virus WSN strain envelope proteins against ultracentrifugation. Lentiviral genomic and functional particles were more stable than the corresponding retroviral particles against ultracentrifugation when pseudotyped with VSVGs. However, both retroviral and lentiviral particles were unstable when pseudotyped with the influenza virus WSN strain envelope proteins. Therefore, the stabilities of pseudotyped retroviral and lentiviral vectors against ultracentrifugation process are a function of not only the type of envelope proteins, but also the type of viral internal core (MLV or HIV-1 core). In addition, the fraction of functional viral particles among genomic viral particles greatly varied at times during packaging, depending on the type of envelope proteins used for pseudotyping and the viral internal core.

  17. Papain digestion of crude Trichoderma reesei cellulase: Purification and properties of cellobiohydrolase I and II core proteins

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J.; Brown, J.P.; Evans, B.R.; Affholter, K.A.

    1992-01-01

    Papain digestion of a crude Trichoderma reesei cellulose preparation followed by gel filtration on a Superdex column resulted in the separation of cellobiohydrolase (CBH) I and II core proteins (cp). They were further purified to apparent homogeneity by chromatofocusing. N-terminal protein sequencing of the CBH II cp preparation confirmed its identity. A comparison of the catalytic activity and cellulose-binding ability of these core proteins was made. The major differences between them were the findings that CBH II cp possessed a sixfold higher specific activity toward p-nitrophenylcellobioside than the native CBH II preparation and still bound to microcrystalline cellulose, unlike CBH I cp. Neither CBH I cp nor CBH II cp had activity toward carboxymethylcellulose, but both were able to hydrolyze barley b-glucan. These data suggest that removal of the cellulose-binding domain and hinge region from CBH I and II have different effects on their properties.

  18. Papain digestion of crude Trichoderma reesei cellulase: Purification and properties of cellobiohydrolase I and II core proteins

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J.; Brown, J.P.; Evans, B.R.; Affholter, K.A.

    1992-12-01

    Papain digestion of a crude Trichoderma reesei cellulose preparation followed by gel filtration on a Superdex column resulted in the separation of cellobiohydrolase (CBH) I and II core proteins (cp). They were further purified to apparent homogeneity by chromatofocusing. N-terminal protein sequencing of the CBH II cp preparation confirmed its identity. A comparison of the catalytic activity and cellulose-binding ability of these core proteins was made. The major differences between them were the findings that CBH II cp possessed a sixfold higher specific activity toward p-nitrophenylcellobioside than the native CBH II preparation and still bound to microcrystalline cellulose, unlike CBH I cp. Neither CBH I cp nor CBH II cp had activity toward carboxymethylcellulose, but both were able to hydrolyze barley b-glucan. These data suggest that removal of the cellulose-binding domain and hinge region from CBH I and II have different effects on their properties.

  19. Mesitylene-Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Ribeiro, Orquidea; Du, Yang

    2016-01-01

    are limited in their ability to maintain the structural integrity of membrane proteins and thus there are major efforts underway to develop novel agents with improved properties. We prepared mesitylene-cored glucoside amphiphiles (MGAs) with three alkyl chains and compared these agents with previously...... developed xylene-linked maltoside agents (XMAs) with two alkyl chains and a conventional detergent (DDM). When these agents were evaluated for four membrane proteins including a G protein-coupled receptor (GPCR), some agents such as MGA-C13 and MGA-C14 resulted in markedly enhanced stability of membrane...

  20. The N-terminus of murine leukaemia virus p12 protein is required for mature core stability.

    Directory of Open Access Journals (Sweden)

    Darren J Wight

    2014-10-01

    Full Text Available The murine leukaemia virus (MLV gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

  1. The N-Terminus of Murine Leukaemia Virus p12 Protein Is Required for Mature Core Stability

    Science.gov (United States)

    Wight, Darren J.; Boucherit, Virginie C.; Wanaguru, Madushi; Elis, Efrat; Hirst, Elizabeth M. A.; Li, Wilson; Ehrlich, Marcelo; Bacharach, Eran; Bishop, Kate N.

    2014-01-01

    The murine leukaemia virus (MLV) gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices. PMID:25356837

  2. Progressive dry-core-wet-rim hydration trend in a nested-ring topology of protein binding interfaces

    Directory of Open Access Journals (Sweden)

    Li Zhenhua

    2012-03-01

    Full Text Available Abstract Background Water is an integral part of protein complexes. It shapes protein binding sites by filling cavities and it bridges local contacts by hydrogen bonds. However, water molecules are usually not included in protein interface models in the past, and few distribution profiles of water molecules in protein binding interfaces are known. Results In this work, we use a tripartite protein-water-protein interface model and a nested-ring atom re-organization method to detect hydration trends and patterns from an interface data set which involves immobilized interfacial water molecules. This data set consists of 206 obligate interfaces, 160 non-obligate interfaces, and 522 crystal packing contacts. The two types of biological interfaces are found to be drier than the crystal packing interfaces in our data, agreeable to a hydration pattern reported earlier although the previous definition of immobilized water is pure distance-based. The biological interfaces in our data set are also found to be subject to stronger water exclusion in their formation. To study the overall hydration trend in protein binding interfaces, atoms at the same burial level in each tripartite protein-water-protein interface are organized into a ring. The rings of an interface are then ordered with the core atoms placed at the middle of the structure to form a nested-ring topology. We find that water molecules on the rings of an interface are generally configured in a dry-core-wet-rim pattern with a progressive level-wise solvation towards to the rim of the interface. This solvation trend becomes even sharper when counterexamples are separated. Conclusions Immobilized water molecules are regularly organized in protein binding interfaces and they should be carefully considered in the studies of protein hydration mechanisms.

  3. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    Science.gov (United States)

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Hepatitis C virus RNA and core protein in kidney glomerular and tubular structures isolated with laser capture microdissection

    Science.gov (United States)

    Sansonno, D; Lauletta, G; Montrone, M; Grandaliano, G; Schena, F P; Dammacco, F

    2005-01-01

    The role of hepatits C virus (HCV) in the production of renal injury has been extensively investigated, though with conflicting results. Laser capture microdissection (LCM) was performed to isolate and collect glomeruli and tubules from 20 consecutive chronically HCV-infected patients, namely 6 with membranoproliferative glomerulonephritis, 4 with membranous glomerulonephritis, 7 with focal segmental glomerulosclerosis and 3 with IgA-nephropathy. RNA for amplification of specific viral sequences was provided by terminal continuation methodology and compared with the expression profile of HCV core protein. For each case two glomeruli and two tubular structures were microdissected and processed. HCV RNA sequences were demonstrated in 26 (65%) of 40 glomeruli, but in only 4 (10%) of the tubules (P viral sequences in the glomeruli and present in 31 of the 40 tubules. HCV RNA and/or HCV core protein was found in all four disease types. The immunohistochemical picture of HCV core protein was compared with the LCM-based immunoassays of the adjacent tissue sections. Immune deposits were detected in 7 (44%) of 16 biopsy samples shown to be positive by extraction methods. The present study indicates that LCM is a reliable method for measuring both HCV RNA genomic sequences and HCV core protein in kidney functional structures from chronically HCV-infected patients with different glomerulopathies and provides a useful baseline estimate to define the role of HCV in the production of renal injury. The different distribution of HCV RNA and HCV-related proteins may reflect a peculiar ‘affinity’ of kidney microenvironments for HCV and point to distinct pathways of HCV-related damage in glomeruli and tubules. PMID:15932511

  5. Decorin core protein (decoron shape complements collagen fibril surface structure and mediates its binding.

    Directory of Open Access Journals (Sweden)

    Joseph P R O Orgel

    Full Text Available Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM. With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein and binding sites in the d and e(1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1 bands. This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

  6. Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Hong-Chao Zhou; De-Zhong Xu; Xue-Ping Wang; Jing-Xia Zhang; Ying-Huang; Yong-Ping Yan; Yong Zhu; Bo-Quan Jin

    2001-01-01

    AIM To identify hepatitis C virus (HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS Utilizing the method of computer prediction followed by a 4 h 51 Cr-release assay confirmation. RESULTS The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVFAL (core TS0-158)".The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%,respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis.But blocking of CTL response with anti-CD8 mAb could abolish the Iysis. CONCLUSION The peptide (core 150 - 158 ) is the candidate epitope recognized by HLA-A2 restricted CTL.

  7. Crystal structure of the dimeric protein core of decorin, the archetypal small leucine-rich repeat proteoglycan.

    Science.gov (United States)

    Scott, Paul G; McEwan, Paul A; Dodd, Carole M; Bergmann, Ernst M; Bishop, Paul N; Bella, Jordi

    2004-11-02

    Decorin is a ubiquitous extracellular matrix proteoglycan with a variety of important biological functions that are mediated by its interactions with extracellular matrix proteins, cytokines, and cell surface receptors. Decorin is the prototype of the family of small leucine-rich repeat proteoglycans and proteins (SLRPs), characterized by a protein core composed of leucine-rich repeats (LRRs), flanked by two cysteine-rich regions. We report here the crystal structure of the dimeric protein core of decorin, the best characterized member of the SLRP family. Each monomer adopts the curved solenoid fold characteristic of LRR domains, with a parallel beta-sheet on the inside interwoven with loops containing short segments of beta-strands, 3(10) helices, and polyproline II helices on the outside. Two main features are unique to this structure. First, decorin dimerizes through the concave surfaces of the LRR domains, which have been implicated previously in protein-ligand interactions. The amount of surface buried in this dimer rivals the buried surfaces of some of the highest-affinity macromolecular complexes reported to date. Second, the C-terminal region adopts an unusual capping motif that involves a laterally extended LRR and a disulfide bond. This motif seems to be unique to SLRPs and has not been observed in any other LRR protein structure to date. Possible implications of these features for decorin ligand binding and SLRP function are discussed.

  8. Intrinsic hepatocyte dedifferentiation is accompanied by upregulation of mesenchymal markers, protein sialylation and core alpha 1,6 linked fucosylation

    Science.gov (United States)

    Mehta, Anand; Comunale, Mary Ann; Rawat, Siddhartha; Casciano, Jessica C.; Lamontagne, Jason; Herrera, Harmin; Ramanathan, Aarti; Betesh, Lucy; Wang, Mengjun; Norton, Pamela; Steel, Laura F.; Bouchard, Michael J.

    2016-01-01

    Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the β-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers. PMID:27328854

  9. Structural Transformation of the Amyloidogenic Core Region of TDP-43 Protein Initiates Its Aggregation and Cytoplasmic Inclusion*

    Science.gov (United States)

    Jiang, Lei-Lei; Che, Mei-Xia; Zhao, Jian; Zhou, Chen-Jie; Xie, Mu-Yun; Li, Hai-Yin; He, Jian-Hua; Hu, Hong-Yu

    2013-01-01

    TDP-43 (TAR DNA-binding protein of 43 kDa) is a major deposited protein in amyotrophic lateral sclerosis and frontotemporal dementia with ubiquitin. A great number of genetic mutations identified in the flexible C-terminal region are associated with disease pathologies. We investigated the molecular determinants of TDP-43 aggregation and its underlying mechanisms. We identified a hydrophobic patch (residues 318–343) as the amyloidogenic core essential for TDP-43 aggregation. Biophysical studies demonstrated that the homologous peptide formed a helix-turn-helix structure in solution, whereas it underwent structural transformation from an α-helix to a β-sheet during aggregation. Mutation or deletion of this core region significantly reduced the aggregation and cytoplasmic inclusions of full-length TDP-43 (or TDP-35 fragment) in cells. Thus, structural transformation of the amyloidogenic core initiates the aggregation and cytoplasmic inclusion formation of TDP-43. This particular core region provides a potential therapeutic target to design small-molecule compounds for mitigating TDP-43 proteinopathies. PMID:23689371

  10. Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X-ray crystallography.

    Science.gov (United States)

    Nkansah, Edwin; Shah, Rahi; Collie, Gavin W; Parkinson, Gary N; Palmer, Jonathan; Rahman, Khondaker M; Bui, Tam T; Drake, Alex F; Husby, Jarmila; Neidle, Stephen; Zinzalla, Giovanna; Thurston, David E; Wilderspin, Andrew F

    2013-04-02

    The STAT3 transcription factor plays a central role in a wide range of cancer types where it is over-expressed. Previously, phosphorylation of this protein was thought to be a prerequisite for direct binding to DNA. However, we have now shown complete binding of a purified unphosphorylated STAT3 (uSTAT3) core directly to M67 DNA, the high affinity STAT3 target DNA sequence, by a protein electrophoretic mobility shift assay (PEMSA). Binding to M67 DNA was inhibited by addition of increasing concentrations of a phosphotyrosyl peptide. X-ray crystallography demonstrates one mode of binding that is similar to that known for the STAT3 core phosphorylated at Y705.

  11. Affinity of anticancer drug, daunomycin, to core histones in solution:comparison of free and cross-linked proteins

    Institute of Scientific and Technical Information of China (English)

    Azra RABBANI; Sayeh ABDOSAMADI; Naghmeh SARI-SARAF

    2007-01-01

    Aim: The interaction of anthracyclinc anticancer drugs with chromatin, nuclco-somes and historic H1 has been extensively studied. In the present study, for the first time, we have investigated the binding of anthracycline antibiotic, daunomycin,to free and cross-linked thymus core histones (CL-core) in solution and in the absence of DNA. Methods: Fluorescence, UV/Vis spectroscopy and equilibrium dialysis techniques were used. Results: The UV spectroscopy results show that daunomycin induces hypochromicity in the absorption spectra of the core histones.Fluorescence emission intensity is decreased upon daunomycin binding and the process is concentration dependent. The equilibrium dialysis shows that the bind-ing is positive cooperative with the binding sites as Scatchard plot and Hill Coef-ficient confirm it. Conclusion: The results suggest that daunomycin shows much higher affinity to core histories free in solution than to CL-core, implying that the binding is most likely due to the accessibility of these proteins to the environment.It is suggested that daunomycin binds strongly to open state of histones, such as in tumor cells, rather than to their compact structure seen in normal chromatin.

  12. Memory T-Cell-Mediated Immune Responses Specific to an Alternative Core Protein in Hepatitis C Virus Infection

    Science.gov (United States)

    Bain, Christine; Parroche, Peggy; Lavergne, Jean Pierre; Duverger, Blandine; Vieux, Claude; Dubois, Valérie; Komurian-Pradel, Florence; Trépo, Christian; Gebuhrer, Lucette; Paranhos-Baccala, Glaucia; Penin, François; Inchauspé, Geneviève

    2004-01-01

    In vitro studies have described the synthesis of an alternative reading frame form of the hepatitis C virus (HCV) core protein that was named F protein or ARFP (alternative reading frame protein) and includes a domain coded by the +1 open reading frame of the RNA core coding region. The expression of this protein in HCV-infected patients remains controversial. We have analyzed peripheral blood from 47 chronically or previously HCV-infected patients for the presence of T lymphocytes and antibodies specific to the ARFP. Anti-ARFP antibodies were detected in 41.6% of the patients infected with various HCV genotypes. Using a specific ARFP 99-amino-acid polypeptide as well as four ARFP predicted class I-restricted 9-mer peptides, we show that 20% of the patients display specific lymphocytes capable of producing gamma interferon, interleukin-10, or both cytokines. Patients harboring three different viral genotypes (1a, 1b, and 3) carried T lymphocytes reactive to genotype 1b-derived peptides. In longitudinal analysis of patients receiving therapy, both core and ARFP-specific T-cell- and B-cell-mediated responses were documented. The magnitude and kinetics of the HCV antigen-specific responses differed and were not linked with viremia or therapy outcome. These observations provide strong and new arguments in favor of the synthesis, during natural HCV infection, of an ARFP derived from the core sequence. Moreover, the present data provide the first demonstration of the presence of T-cell-mediated immune responses directed to this novel HCV antigen. PMID:15367612

  13. Hepatitis C Virus Core Protein Abrogates the DDX3 Function That Enhances IPS-1-Mediated IFN–Beta Induction

    OpenAIRE

    2010-01-01

    The DEAD box helicase DDX3 assembles IPS-1 (also called Cardif, MAVS, or VISA) in non-infected human cells where minimal amounts of the RIG-I-like receptor (RLR) protein are expressed. DDX3 C-terminal regions directly bind the IPS-1 CARD-like domain as well as the N-terminal hepatitis C virus (HCV) core protein. DDX3 physically binds viral RNA to form IPS-1-containing spots, that are visible by confocal microscopy. HCV polyU/UC induced IPS-1-mediated interferon (IFN)-beta promoter activation,...

  14. Capsid proteins from human immunodeficiency virus type 1 and simian immunodeficiency virus SIVmac can coassemble into mature cores of infectious viruses.

    Science.gov (United States)

    Chen, Jianbo; Pathak, Vinay K; Peng, Weiqun; Hu, Wei-Shau

    2008-09-01

    We have recently shown that the Gag polyproteins from human immunodeficiency virus type 1 (HIV-1) and HIV-2 can coassemble and functionally complement each other. During virion maturation, the Gag polyproteins undergo proteolytic cleavage to release mature proteins including capsid (CA), which refolds and forms the outer shell of a cone-shaped mature core. Less than one-half of the CA proteins present within the HIV-1 virion are required to form the mature core. Therefore, it is unclear whether the mature core in virions containing both HIV-1 and HIV-2 Gag consists of CA proteins from a single virus or from both viruses. To determine whether CA proteins from two different viruses can coassemble into mature cores of infectious viruses, we exploited the specificity of the tripartite motif 5alpha protein from the rhesus monkey (rhTRIM5alpha) for cores containing HIV-1 CA (hCA) but not the simian immunodeficiency virus SIV(mac) CA protein (sCA). If hCA and sCA cannot coassemble into the same core when equal amounts of sCA and hCA are coexpressed, the infectivities of such virus preparations in cells should be inhibited less than twofold by rhTRIM5alpha. However, if hCA and sCA can coassemble into the same core structure to form a mixed core, rhTRIM5alpha would be able to recognize such cores and significantly restrict virus infectivity. We examined the restriction phenotypes of viruses containing both hCA and sCA. Our results indicate that hCA and sCA can coassemble into the same mature core to produce infectious virus. To our knowledge, this is the first demonstration of functional coassembly of heterologous CA protein into the retroviral core.

  15. Crystallization of the avian reovirus double-stranded RNA-binding and core protein σA

    Energy Technology Data Exchange (ETDEWEB)

    Hermo-Parrado, X. Lois; Guardado-Calvo, Pablo [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Llamas-Saiz, Antonio L. [Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Fox, Gavin C. [Spanish CRG Beamline BM16, European Synchrotron Radiation Facility (ESRF), 6 Rue Jules Horowitz, BP 220, F-38043 Grenoble (France); Vazquez-Iglesias, Lorena; Martínez-Costas, José; Benavente, Javier [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Raaij, Mark J. van, E-mail: vanraaij@usc.es [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain)

    2007-05-01

    The avian reovirus double-stranded RNA-binding and core protein σA has been crystallized in space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2°. A complete data set has been collected to 2.3 Å resolution and analyzed. The avian reovirus protein σA plays a dual role: it is a structural protein forming part of the transcriptionally active core, but it has also been implicated in the resistance of the virus to interferon by strongly binding double-stranded RNA and thus inhibiting the double-stranded RNA-dependent protein kinase. The σA protein has been crystallized from solutions containing ammonium sulfate at pH values around 6. Crystals belonging to space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2° were grown and a complete data set has been collected to 2.3 Å resolution. The self-rotation function suggests that σA may form symmetric arrangements in the crystals.

  16. Single molecule force spectroscopy reveals critical roles of hydrophobic core packing in determining the mechanical stability of protein GB1.

    Science.gov (United States)

    Bu, Tianjia; Wang, Hui-Chuan Eileen; Li, Hongbin

    2012-08-21

    Understanding molecular determinants of protein mechanical stability is important not only for elucidating how elastomeric proteins are designed and functioning in biological systems but also for designing protein building blocks with defined nanomechanical properties for constructing novel biomaterials. GB1 is a small α/β protein and exhibits significant mechanical stability. It is thought that the shear topology of GB1 plays an important role in determining its mechanical stability. Here, we combine single molecule atomic force microscopy and protein engineering techniques to investigate the effect of side chain reduction and hydrophobic core packing on the mechanical stability of GB1. We engineered seven point mutants and carried out mechanical φ-value analysis of the mechanical unfolding of GB1. We found that three mutations, which are across the surfaces of two subdomains that are to be sheared by the applied stretching force, in the hydrophobic core (F30L, Y45L, and F52L) result in significant decrease in mechanical unfolding force of GB1. The mechanical unfolding force of these mutants drop by 50-90 pN compared with wild-type GB1, which unfolds at around 180 pN at a pulling speed of 400 nm/s. These results indicate that hydrophobic core packing plays an important role in determining the mechanical stability of GB1 and suggest that optimizing hydrophobic interactions across the surfaces that are to be sheared will likely be an efficient method to enhance the mechanical stability of GB1 and GB1 homologues.

  17. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    Science.gov (United States)

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

  18. Hepatitis C virus core protein inhibits E6AP expression via DNA methylation to escape from ubiquitin-dependent proteasomal degradation.

    Science.gov (United States)

    Kwak, Juri; Shim, Joo Hee; Tiwari, Indira; Jang, Kyung Lib

    2016-09-28

    The E6-associated protein (E6AP) is a ubiquitin ligase that mediates ubiquitination and proteasomal degradation of hepatitis C virus (HCV) core protein. Given the role of HCV core protein as a major component of the viral nucleocapsid, as well as a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis, HCV has likely evolved a strategy to counteract the host anti-viral defense mechanism of E6AP and maximize its potential to produce infectious virus particles. In the present study, we found that HCV core protein derived from either ectopic expression or HCV infection inhibits E6AP expression via promoter hypermethylation in human hepatocytes. As a result, the potential of E6AP to ubiquitinate and degrade HCV core protein through the ubiquitin-proteasome system was severely impaired, which in turn led to stimulation of virus propagation. The effects of HCV core protein were almost completely abolished when the E6AP level was restored by ectopic expression of E6AP, treatment with a universal DNA methyltransferase (DNMT) inhibitor, 5-Aza-2'dC, or knock-down of DNMT1. In conclusion, HCV core protein inhibits E6AP expression via DNA methylation to protect itself from ubiquitin-dependent proteasomal degradation and stimulate virus propagation, providing a potential target for the development of anti-viral drugs against HCV.

  19. Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core.

    Science.gov (United States)

    Gates, Zachary P; Baxa, Michael C; Yu, Wookyung; Riback, Joshua A; Li, Hui; Roux, Benoît; Kent, Stephen B H; Sosnick, Tobin R

    2017-02-28

    The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

  20. Construct design, biophysical, and biochemical characterization of the fusion core from mouse hepatitis virus (a coronavirus) spike protein.

    Science.gov (United States)

    Xu, Yanhui; Cole, David K; Lou, Zhiyong; Liu, Yiwei; Qin, Lan; Li, Xu; Bai, Zhihong; Yuan, Fang; Rao, Zihe; Gao, George F

    2004-11-01

    Membrane fusion between virus and host cells is the key step for enveloped virus entry and is mediated by the viral envelope fusion protein. In murine coronavirus, mouse hepatitis virus (MHV), the spike (S) protein mediates this process. Recently, the formation of anti-parallel 6-helix bundle of the MHV S protein heptad repeat (HR) regions (HR1 and HR2) has been confirmed, implying coronavirus has a class I fusion protein. This bundle is also called fusion core. To facilitate the solution of the crystal structure of this fusion core, we deployed an Escherichia coli in vitro expression system to express the HR1 and HR2 regions linked together by a flexible linker as a single chain (named 2-helix). This 2-helix polypeptide subsequently assembled into a typical 6-helix bundle. This bundle has been analyzed by a series of biophysical and biochemical techniques and confirmed that the design technique can be used for coronavirus as we successfully used for members of paramyxoviruses.

  1. Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarA.

    Science.gov (United States)

    Cheung, Wai Ling; Chen, Maria Y; Maksimov, Mikhail O; Link, A James

    2016-10-26

    Lasso peptides are a member of the superclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Like all RiPPs, lasso peptides are derived from a gene-encoded precursor protein. The biosynthesis of lasso peptides requires two enzymatic activities: proteolytic cleavage between the leader peptide and the core peptide in the precursor protein, accomplished by the B enzymes, and ATP-dependent isopeptide bond formation, accomplished by the C enzymes. In a subset of lasso peptide biosynthetic gene clusters from Gram-positive organisms, the B enzyme is split between two proteins. One such gene cluster is found in the organism Rhodococcus jostii, which produces the antimicrobial lasso peptide lariatin. The B enzyme in R. jostii is split between two open reading frames, larB1 and larB2, both of which are required for lariatin biosynthesis. While the cysteine catalytic triad is found within the LarB2 protein, LarB1 is a PqqD homologue expected to bind to the lariatin precursor LarA based on its structural homology to other RiPP leader peptide binding domains. We show that LarB1 binds to the leader peptide of the lariatin precursor protein LarA with a sub-micromolar affinity. We used photocrosslinking with the noncanonical amino acid p-azidophenylalanine and mass spectrometry to map the interaction of LarA and LarB1. This analysis shows that the LarA leader peptide interacts with a conserved motif within LarB1 and, unexpectedly, the core peptide of LarA also binds to LarB1 in several positions. A Rosetta model built from distance restraints from the photocrosslinking experiments shows that the scissile bond between the leader peptide and core peptide in LarA is in a solvent-exposed loop.

  2. The F-box protein Ppa is a common regulator of core EMT factors Twist, Snail, Slug, and Sip1.

    Science.gov (United States)

    Lander, Rachel; Nordin, Kara; LaBonne, Carole

    2011-07-11

    A small group of core transcription factors, including Twist, Snail, Slug, and Sip1, control epithelial-mesenchymal transitions (EMTs) during both embryonic development and tumor metastasis. However, little is known about how these factors are coordinately regulated to mediate the requisite behavioral and fate changes. It was recently shown that a key mechanism for regulating Snail proteins is by modulating their stability. In this paper, we report that the stability of Twist is also regulated by the ubiquitin-proteasome system. We found that the same E3 ubiquitin ligase known to regulate Snail family proteins, Partner of paired (Ppa), also controlled Twist stability and did so in a manner dependent on the Twist WR-rich domain. Surprisingly, Ppa could also target the third core EMT regulatory factor Sip1 for proteasomal degradation. Together, these results indicate that despite the structural diversity of the core transcriptional regulatory factors implicated in EMT, a common mechanism has evolved for controlling their stability and therefore their function.

  3. Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150.

    Science.gov (United States)

    Elsworth, Brendan; Sanders, Paul R; Nebl, Thomas; Batinovic, Steven; Kalanon, Ming; Nie, Catherine Q; Charnaud, Sarah C; Bullen, Hayley E; de Koning Ward, Tania F; Tilley, Leann; Crabb, Brendan S; Gilson, Paul R

    2016-11-01

    The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.

  4. Additives and Protein-DNA Combinations Modulate the Humoral Immune Response Elicited by a Hepatitis C Virus Core-encoding Plasmid in Mice

    Directory of Open Access Journals (Sweden)

    Alvarez-Lajonchere Liz

    2002-01-01

    Full Text Available Humoral and cellular immune responses are currently induced against hepatitis C virus (HCV core following vaccination with core-encoding plasmids. However, the anti-core antibody response is frequently weak or transient. In this paper, we evaluated the effect of different additives and DNA-protein combinations on the anti-core antibody response. BALB/c mice were intramuscularly injected with an expression plasmid (pIDKCo, encoding a C-terminal truncated variant of the HCV core protein, alone or combined with CaCl2, PEG 6000, Freund's adjuvant, sonicated calf thymus DNA and a recombinant core protein (Co.120. Mixture of pIDKCo with PEG 6000 and Freund's adjuvant accelerated the development of the anti-core Ab response. Combination with PEG 6000 also induced a bias to IgG2a subclass predominance among anti-core antibodies. The kinetics, IgG2a/IgG1 ratio and epitope specificity of the anti-core antibody response elicited by Co.120 alone or combined with pIDKCo was different regarding that induced by the pIDKCo alone. Our data indicate that the antibody response induced following DNA immunization can be modified by formulation strategies.

  5. Alcohol Induced Hepatic Degeneration in a Hepatitis C Virus Core Protein Transgenic Mouse Model

    Directory of Open Access Journals (Sweden)

    Dong-Hyung Noh

    2014-03-01

    Full Text Available Hepatitis C virus (HCV has become a major public health issue. It is prevalent in most countries. HCV infection frequently begins without clinical symptoms, before progressing to persistent viremia, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC in the majority of patients (70% to 80%. Alcohol is an independent cofactor that accelerates the development of HCC in chronic hepatitis C patients. The purpose of the current study was to evaluate ethanol-induced hepatic changes in HCV core-Tg mice and mutant core Tg mice. Wild type (NTG, core wild-Tg mice (TG-K, mutant core 116-Tg mice (TG-116 and mutant core 99-Tg mice (TG-99 were used in this investigation. All groups were given drinking water with 10% ethanol and 5% sucrose for 13 weeks. To observe liver morphological changes, we performed histopathological and immunohistochemical examinations. Histopathologically, NTG, TG-K and TG-116 mice showed moderate centrilobular necrosis, while severe centrilobular necrosis and hepatocyte dissociation were observed in TG-99 mice with increasing lymphocyte infiltration and piecemeal necrosis. In all groups, a small amount of collagen fiber was found, principally in portal areas. None of the mice were found to have myofibroblasts based on immunohistochemical staining specific for α-SMA. CYP2E1-positive cells were clearly detected in the centrilobular area in all groups. In the TG-99 mice, we also observed cells positive for CK8/18, TGF-β1 and phosphorylated (p-Smad2/3 and p21 around the necrotic hepatocytes in the centrilobular area (p < 0.01. Based on our data, alcohol intake induced piecemeal necrosis and hepatocyte dissociation in the TG-99 mice. These phenomena involved activation of the TGF-β1/p-Smad2/3/p21 signaling pathway in hepatocytes. Data from this study will be useful for elucidating the association between alcohol intake and HCV infection.

  6. Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber.

    Science.gov (United States)

    U S, Dinish; Fu, Chit Yaw; Soh, Kiat Seng; Ramaswamy, Bhuvaneswari; Kumar, Anil; Olivo, Malini

    2012-03-15

    Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-μg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ∼100 pg in a sample volume of ∼10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases.

  7. Getting to the core of protein pharmaceuticals – comprehensive structure analysis by mass spectrometry

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Mistarz, Ulrik Hvid; Rand, Kasper Dyrberg

    2015-01-01

    Protein pharmaceuticals are the fastest growing class of novel therapeutic agents, and have been a major research and development focus in the (bio)pharmaceutical industry. Due to their large size and structural diversity, biopharmaceuticals represent a formidable challenge regarding analysis....... Mass spectrometry has evolved as a powerful tool for the characterization of both primary and higher order structures of protein pharmaceuticals. Furthermore, the chemical and physical stability of protein drugs, as well as their pharmacokinetics are nowadays routinely determined by mass spectrometry...

  8. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3.

    Science.gov (United States)

    Floor, Stephen N; Condon, Kendall J; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A

    2016-01-29

    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.

  9. Preliminary Analysis of Gene Expression Profiles in HepG2 Cell Line Induced by Different Genotype Core Proteins of HCV

    Institute of Scientific and Technical Information of China (English)

    Jun Dou; Pengbo Liu; Jing Wang; Xinjian Zhang

    2006-01-01

    In present investigation, we constructed recombinants expressing the HCV genotypes 1b, 2a, and 4d core proteins,and established human hepatocellular carcinoma (HepG2) cell line that expressed various genotype core proteins.The gene expression profiles in the cells expressing different HCV genotype core proteins were compared with those in the control by microarray analysis. In data analysis, a threshold was set to eliminate all genes that were not increased or decreased by 2.5-fold change in a comparison between the transfected cells and control cells. The preliminary microarray analysis suggests that the gene expression profiles regulated by three kinds of genotype core proteins are mainly involved in transport, signal transduction, regulation of transcription, protease activity, etc.,and that some pathogenesis/oncogenesis gene expressions are up/down- regulated simultaneously in the HepG2 cell line. The data suggest that each core protein has its gene expressions profile and that the profiles are implicated in HCV replication and pathogenesis, which may open up a novel way to understand the function of the HCV variant core proteins biological and their pathogenic mechanism.

  10. Localization of Core Planar Cell Polarity Proteins, PRICKLEs, in Ameloblasts of Rat Incisors: Possible Regulation of Enamel Rod Decussation.

    Science.gov (United States)

    Nishikawa, Sumio; Kawamoto, Tadafumi

    2015-04-28

    To confirm the possible involvement of planar cell polarity proteins in odontogenesis, one group of core proteins, PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, was examined in enamel epithelial cells and ameloblasts by immunofluorescence microscopy. PRICKLE1 and PRICKLE2 showed similar localization in the proliferation and secretory zones of the incisor. Immunoreactive dots and short rods in ameloblasts and stratum intermedium cells were evident in the proliferation to differentiation zone, but in the secretion zone, cytoplasmic dots decreased and the distal terminal web was positive for PRICKLE1 and PRICKLE2. PRICKLE3 and PRICKLE4 showed cytoplasmic labeling in ameloblasts and other enamel epithelial cells. Double labeling of PRICKLE2 with VANGL1, which is another planar cell polarity protein, showed partial co-localization. To examine the transport route of PRICKLE proteins, PRICKLE1 localization was examined after injection of a microtubule-disrupting reagent, colchicine, and was compared with CX43, which is a membrane protein transported as vesicles via microtubules. The results confirmed the retention of immunoreactive dots for PRICKLE1 in the cytoplasm of secretory ameloblasts of colchicine-injected animals, but fewer dots were observed in control animals. These results suggest that PRICKLE1 and PRICKLE2 are transported as vesicles to the junctional area, and are involved in pattern formation of distal junctional complexes and terminal webs of ameloblasts, further implying a role in the formed enamel rod arrangement.

  11. Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles

    Science.gov (United States)

    Weidner, A.; Gräfe, C.; von der Lühe, M.; Remmer, H.; Clement, J. H.; Eberbeck, D.; Ludwig, F.; Müller, R.; Schacher, F. H.; Dutz, S.

    2015-07-01

    Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40 °C. For incubation temperatures above 50 °C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on corona

  12. Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles.

    Science.gov (United States)

    Weidner, A; Gräfe, C; von der Lühe, M; Remmer, H; Clement, J H; Eberbeck, D; Ludwig, F; Müller, R; Schacher, F H; Dutz, S

    2015-12-01

    Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40 °C. For incubation temperatures above 50 °C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on

  13. Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails.

    Directory of Open Access Journals (Sweden)

    Aneta Tarczewska

    Full Text Available Two major lipophilic hormones, 20-hydroxyecdysone (20E and juvenile hormone (JH, govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64 and Tribolium castaneum (TcChd64. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS, we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV circular dichroism (CD spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG-like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs may be crucial for multiple interactions with many partners.

  14. Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

    Science.gov (United States)

    Abdallah, Cosette; Lejamtel, Charlène; Benzoubir, Nassima; Battaglia, Serena; Sidahmed-Adrar, Nazha; Desterke, Christophe; Lemasson, Matthieu; Rosenberg, Arielle R; Samuel, Didier; Bréchot, Christian; Pflieger, Delphine; Le Naour, François; Bourgeade, Marie-Françoise

    2017-08-22

    Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

  15. Hepatitis C virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor-7 and signal transducer and activator of transcription (STAT 1 protein expression.

    Directory of Open Access Journals (Sweden)

    Amy E L Stone

    Full Text Available Plasmacytoid Dendritic Cells (pDCs represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs through pattern recognition receptors (PRR. PRR/PAMP interactions trigger signaling events that induce interferon (IFN production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL IFNs in response to HCV RNA. Extracellular HCV core protein (Core is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.

  16. Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity

    NARCIS (Netherlands)

    Endoh, M.; Endo, T.A.; Endoh, T.; Fujimura, Y.; Ohara, O.; Toyoda, T.; Otte, A.P.; Okano, M.; Brockdorff, N.; Vidal, M.; Koseki, H.

    2008-01-01

    The Polycomb group (PcG) proteins mediate heritable silencing of developmental regulators in metazoans, participating in one of two distinct multimeric protein complexes, the Polycomb repressive complexes 1 (PRC1) and 2 (PRC2). Although PRC2 has been shown to share target genes with the core transcr

  17. Substitution in Amino Acid 70 of Hepatitis C Virus Core Protein Changes the Adipokine Profile via Toll-Like Receptor 2/4 Signaling.

    Directory of Open Access Journals (Sweden)

    Satoko Uraki

    Full Text Available It has been suggested that amino acid (aa substitution at position 70 from arginine (70R to glutamine (70Q in the genotype 1b hepatitis C virus (HCV core protein is associated with insulin resistance and worse prognosis. However, the precise mechanism is still unclear. The aim of this study was to investigate the impact of the substitution at position 70 in HCV core protein on adipokine production by murine and human adipocytes.The influence of treatment with HCV core protein (70R or 70Q on adipokine production by both 3T3-L1 and human adipocytes were examined with real-time PCR and enzyme-linked immunosorbent assay (ELISA, and triglyceride content was also analyzed. The effects of toll-like receptor (TLR2/4 inhibition on IL-6 production by 3T3-L1 induced by HCV core protein were examined.IL-6 production was significantly increased and adiponectin production was reduced without a change in triglyceride content by treatment with 70Q compared to 70R core protein in both murine and human adipocytes. IL-6 induction of 3T3-L1 cells treated by 70Q HCV core protein was significantly inhibited with anti-TLR2 antibody by 42%, and by TLR4 inhibitor by 40%.Our study suggests that extracellular HCV core protein with substitution at position 70 enhanced IL-6 production and reduced adiponectin production from visceral adipose tissue, which can cause insulin resistance, hepatic steatosis, and ultimately development of HCC.

  18. A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.

    Science.gov (United States)

    Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, T M; Meiler, Jens; Hamm, Heidi E

    2016-09-09

    G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding.

  19. Elastin-based protein polymer nanoparticles carrying drug at both corona and core suppress tumor growth in vivo.

    Science.gov (United States)

    Shi, Pu; Aluri, Suhaas; Lin, Yi-An; Shah, Mihir; Edman, Maria; Dhandhukia, Jugal; Cui, Honggang; MacKay, J Andrew

    2013-11-10

    Numerous nanocarriers of small molecules depend on either non-specific physical encapsulation or direct covalent linkage. In contrast, this manuscript explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for nanoparticulate drug delivery. To explore this approach, genetically engineered diblock copolymers were constructed from elastin-like polypeptides (ELPs) that assemble small (nanoparticles. ELPs are protein polymers of the sequence (Val-Pro-Gly-Xaa-Gly)n, where the identity of Xaa and n determine their assembly properties. Initially, a screening assay for model drug encapsulation in ELP nanoparticles was developed, which showed that Rose Bengal and Rapa have high non-specific encapsulation in the core of ELP nanoparticles with a sequence where Xaa=Ile or Phe. While excellent at entrapping these drugs, their release was relatively fast (2.2h half-life) compared to their intended mean residence time in the human body. Having determined that Rapa can be non-specifically entrapped in the core of ELP nanoparticles, FK506 binding protein 12 (FKBP), which is the cognate protein target of Rapa, was genetically fused to the surface of these nanoparticles (FSI) to enhance their avidity towards Rapa. The fusion of FKBP to these nanoparticles slowed the terminal half-life of drug release to 57.8h. To determine if this class of drug

  20. Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

    Science.gov (United States)

    Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

    2017-05-01

    Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

    Energy Technology Data Exchange (ETDEWEB)

    Simonetti, Angelita [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Marzi, Stefano [Architecture et Réactivité de l’ARN, UPR 9002 CNRS, IBMC (Institute of Molecular and Cellular Biology), 15 Rue R. Descartes, 67084 Strasbourg, France, Université de Strasbourg, 67000 Strasbourg (France); Fabbretti, Attilio [University of Camerino, 62032 Camerino (Monaco) (Italy); Hazemann, Isabelle; Jenner, Lasse [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale -INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Urzhumtsev, Alexandre [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Université de Lorraine, 54506 Vandoeuvre-lès-Nancy (France); Gualerzi, Claudio O. [University of Camerino, 62032 Camerino (Monaco) (Italy); Klaholz, Bruno P., E-mail: klaholz@igbmc.fr [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

    2013-06-01

    The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

  2. Regulated Transport into the Nucleus of Herpesviridae DNA Replication Core Proteins

    Directory of Open Access Journals (Sweden)

    Alessandro Ripalti

    2013-09-01

    Full Text Available The Herpesvirdae family comprises several major human pathogens belonging to three distinct subfamilies. Their double stranded DNA genome is replicated in the nuclei of infected cells by a number of host and viral products. Among the latter the viral replication complex, whose activity is strictly required for viral replication, is composed of six different polypeptides, including a two-subunit DNA polymerase holoenzyme, a trimeric primase/helicase complex and a single stranded DNA binding protein. The study of herpesviral DNA replication machinery is extremely important, both because it provides an excellent model to understand processes related to eukaryotic DNA replication and it has important implications for the development of highly needed antiviral agents. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. The nuclear transport process of these enzymes is controlled by three mechanisms, typifying the main processes through which protein nuclear import is generally regulated in eukaryotic cells. These include cargo post-translational modification-based recognition by the intracellular transporters, piggy-back events allowing coordinated nuclear import of multimeric holoenzymes, and chaperone-assisted nuclear import of specific subunits. In this review we summarize these mechanisms and discuss potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle by targeting nuclear import of the Herpesvirus DNA replicating enzymes.

  3. A novel protein, CHRONO, functions as a core component of the mammalian circadian clock.

    Directory of Open Access Journals (Sweden)

    Akihiro Goriki

    2014-04-01

    Full Text Available Circadian rhythms are controlled by a system of negative and positive genetic feedback loops composed of clock genes. Although many genes have been implicated in these feedback loops, it is unclear whether our current list of clock genes is exhaustive. We have recently identified Chrono as a robustly cycling transcript through genome-wide profiling of BMAL1 binding on the E-box. Here, we explore the role of Chrono in cellular timekeeping. Remarkably, endogenous CHRONO occupancy around E-boxes shows a circadian oscillation antiphasic to BMAL1. Overexpression of Chrono leads to suppression of BMAL1-CLOCK activity in a histone deacetylase (HDAC -dependent manner. In vivo loss-of-function studies of Chrono including Avp neuron-specific knockout (KO mice display a longer circadian period of locomotor activity. Chrono KO also alters the expression of core clock genes and impairs the response of the circadian clock to stress. CHRONO forms a complex with the glucocorticoid receptor and mediates glucocorticoid response. Our comprehensive study spotlights a previously unrecognized clock component of an unsuspected negative circadian feedback loop that is independent of another negative regulator, Cry2, and that integrates behavioral stress and epigenetic control for efficient metabolic integration of the clock.

  4. Functional Role of Histidine in the Conserved His-x-Asp Motif in the Catalytic Core of Protein Kinases.

    Science.gov (United States)

    Zhang, Lun; Wang, Jian-Chuan; Hou, Li; Cao, Peng-Rong; Wu, Li; Zhang, Qian-Sen; Yang, Huai-Yu; Zang, Yi; Ding, Jian-Ping; Li, Jia

    2015-05-11

    The His-x-Asp (HxD) motif is one of the most conserved structural components of the catalytic core of protein kinases; however, the functional role of the conserved histidine is unclear. Here we report that replacement of the HxD-histidine with Arginine or Phenylalanine in Aurora A abolishes both the catalytic activity and auto-phosphorylation, whereas the Histidine-to-tyrosine impairs the catalytic activity without affecting its auto-phosphorylation. Comparisons of the crystal structures of wild-type (WT) and mutant Aurora A demonstrate that the impairment of the kinase activity is accounted for by (1) disruption of the regulatory spine in the His-to-Arg mutant, and (2) change in the geometry of backbones of the Asp-Phe-Gly (DFG) motif and the DFG-1 residue in the His-to-Tyr mutant. In addition, bioinformatics analyses show that the HxD-histidine is a mutational hotspot in tumor tissues. Moreover, the H174R mutation of the HxD-histidine, in the tumor suppressor LKB1 abrogates the inhibition of anchorage-independent growth of A549 cells by WT LKB1. Based on these data, we propose that the HxD-histidine is involved in a conserved inflexible organization of the catalytic core that is required for the kinase activity. Mutation of the HxD-histidine may also be involved in the pathogenesis of some diseases including cancer.

  5. The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis.

    Science.gov (United States)

    McGlinchey, Ryan P; Shewmaker, Frank; McPhie, Peter; Monterroso, Begoña; Thurber, Kent; Wickner, Reed B

    2009-08-18

    Pmel17 is a melanocyte protein necessary for eumelanin deposition 1 in mammals and found in melanosomes in a filamentous form. The luminal part of human Pmel17 includes a region (RPT) with 10 copies of a partial repeat sequence, pt.e.gttp.qv., known to be essential in vivo for filament formation. We show that this RPT region readily forms amyloid in vitro, but only under the mildly acidic conditions typical of the lysosome-like melanosome lumen, and the filaments quickly become soluble at neutral pH. Under the same mildly acidic conditions, the Pmel filaments promote eumelanin formation. Electron diffraction, circular dichroism, and solid-state NMR studies of Pmel17 filaments show that the structure is rich in beta sheet. We suggest that RPT is the amyloid core domain of the Pmel17 filaments so critical for melanin formation.

  6. Conserved function of core clock proteins in the gymnosperm Norway spruce (Picea abies L. Karst).

    Science.gov (United States)

    Karlgren, Anna; Gyllenstrand, Niclas; Källman, Thomas; Lagercrantz, Ulf

    2013-01-01

    From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana), a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)) that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies) putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1) in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partly conserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of the three-loop network typical of the circadian clock in angiosperms were present before the split of gymnosperms and angiosperms.

  7. Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

    Energy Technology Data Exchange (ETDEWEB)

    Vazquez-Iglesias, Lorena; Lostale-Seijo, Irene; Martinez-Costas, Jose [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain); Benavente, Javier, E-mail: franciscojavier.benavente@usc.es [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain)

    2012-10-25

    A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

  8. Protein-ligand docking using hamiltonian replica exchange simulations with soft core potentials.

    Science.gov (United States)

    Luitz, Manuel P; Zacharias, Martin

    2014-06-23

    Molecular dynamics (MD) simulations in explicit solvent allow studying receptor-ligand binding processes including full flexibility of the binding partners and an explicit inclusion of solvation effects. However, in MD simulations, the search for an optimal ligand-receptor complex geometry is frequently trapped in locally stable non-native binding geometries. A Hamiltonian replica-exchange (H-REMD)-based protocol has been designed to enhance the sampling of putative ligand-receptor complexes. It is based on softening nonbonded ligand-receptor interactions along the replicas and one reference replica under the control of the original force field. The efficiency of the method has been evaluated on two receptor-ligand systems and one protein-peptide complex. Starting from misplaced initial docking geometries, the H-REMD method reached in each case the known binding geometry significantly faster than a standard MD simulation. The approach could also be useful to identify and evaluate alternative binding geometries in a given binding region with small relative differences in binding free energy.

  9. Structural Insight into the Core of CAD, the Multifunctional Protein Leading De Novo Pyrimidine Biosynthesis.

    Science.gov (United States)

    Moreno-Morcillo, María; Grande-García, Araceli; Ruiz-Ramos, Alba; Del Caño-Ochoa, Francisco; Boskovic, Jasminka; Ramón-Maiques, Santiago

    2017-06-06

    CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into ∼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. HCV core protein-induced down-regulation of microRNA-152 promoted aberrant proliferation by regulating Wnt1 in HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Shifeng Huang

    Full Text Available BACKGROUND: Hepatitis C virus (HCV has been reported to regulate cellular microRNAs (miRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC, but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152 by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells. METHODS: MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR. Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting. RESULTS: HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT-PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001, miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels. CONCLUSION: These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1

  11. Core Binding Factor β Protects HIV, Type 1 Accessory Protein Viral Infectivity Factor from MDM2-mediated Degradation.

    Science.gov (United States)

    Matsui, Yusuke; Shindo, Keisuke; Nagata, Kayoko; Yoshinaga, Noriyoshi; Shirakawa, Kotaro; Kobayashi, Masayuki; Takaori-Kondo, Akifumi

    2016-11-25

    HIV, type 1 overcomes host restriction factor apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor β (CBFβ). CBFβ is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing the steady-state level of Vif protein; however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Because we have reported previously that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFβ might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFβ preferentially interact with MDM2 and that overexpression of CBFβ disrupts the interaction between MDM2 and Vif. Knockdown of CBFβ reduced the steady-state level of Vif in MDM2-proficient cells but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFβ, degraded faster than wild-type Vif in MDM2-proficient cells but not in MDM2-null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than the parental virus. Combinatory substitution of Vif residues required for CBFβ binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFβ.

  12. Proteasomes regulate hepatitis B virus replication by degradation of viral core-related proteins in a two-step manner.

    Science.gov (United States)

    Zheng, Zi-Hua; Yang, Hui-Ying; Gu, Lin; Peng, Xiao-Mou

    2016-10-01

    The cellular proteasomes presumably inhibit the replication of hepatitis B virus (HBV) due to degradation of the viral core protein (HBcAg). Common proteasome inhibitors, however, either enhance or inhibit HBV replication. In this study, the exact degradation process of HBcAg and its influences on HBV replication were further studied using bioinformatic analysis, protease digestion assays of recombinant HBcAg, and proteasome inhibitor treatments of HBV-producing cell line HepG2.2.15. Besides HBcAg and hepatitis B e antigen precursor, common hepatitis B core-related antigens (HBcrAgs), the small and the large degradation intermediates of these HBcrAgs (HBcrDIs), were regularly found in cytosol of HepG2.2.15 cells. Further, the results of investigation reveal that the degradation process of cytosolic HBcrAgs in proteasomes consists of two steps: the limited proteolysis into HBcrDIs by the trypsin-like (TL) activity and the complete degradation of HBcrDIs by the chymotrypsin-like (chTL) activity. Concordantly, HBcrAgs and the large HBcrDI or HBcrDIs (including the small HBcrDI) were accumulated when the TL or chTL activity was inhibited, which generally correlated with enhancement and inhibition of HBV replication, respectively. The small HBcrDI inhibited HBV replication by assembling into the nucleocapsids and preventing the victim particles from being mature enough for envelopment. The two-step degradation manner may highlight some new anti-HBV strategies.

  13. Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Koike, Manabu, E-mail: m_koike@nirs.go.jp [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yutoku, Yasutomo [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Graduate School of Science, Chiba University, Chiba 263-8522 (Japan); Koike, Aki [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2011-08-19

    Highlights: {yields} p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. {yields} p21 colocalized with the DSB marker {gamma}-H2AX and the DSB sensor Ku80. {yields} Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. {yields} Accumulation activity of p21 was conserved among human and animal cells. {yields} p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker {gamma}-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful

  14. Protein-protein Förster resonance energy transfer analysis of nucleosome core particles containing H2A and H2A.Z.

    Science.gov (United States)

    Hoch, Duane A; Stratton, Jessica J; Gloss, Lisa M

    2007-08-24

    A protein-protein Förster resonance energy transfer (FRET) system, employing probes at multiple positions, was designed to specifically monitor the dissociation of the H2A-H2B dimer from the nucleosome core particle (NCP). Tryptophan donors and Cys-AEDANS acceptors were chosen because, compared to previous NCP FRET fluorophores, they: (1) are smaller and less hydrophobic, which should minimize perturbations of histone and NCP structure; and (2) have an R0 of 20 A, which is much less than the dimensions of the NCP (approximately 50 A width and approximately 100 A diameter). Equilibrium protein unfolding titrations indicate that the donor and acceptor moieties have minimal effects on the stability of the H2A-H2B dimer and (H3-H4)2 tetramer. NCPs containing the various FRET pairs were reconstituted with the 601 DNA positioning element. Equilibrium NaCl-induced dissociation of the modified NCPs showed that the 601 sequence stabilized the NCP to dimer dissociation relative to weaker positioning sequences. This finding implies a significant role for the H2A-H2B dimers in determining the DNA sequence dependence of NCP stability. The free energy of dissociation determined from reversible and well-defined sigmoidal transitions revealed two distinct phases reflecting the dissociation of individual H2A-H2B dimers, confirming cooperativity as suggested previously; these data allow quantitative description of the cooperativity. The FRET system was then used to study the effects of the histone variant H2A.Z on NCP stability; previous studies have reported both destabilizing and stabilizing effects. H2A.Z FRET NCP dissociation transitions suggest a slight increase in stability but a significant increase in cooperativity of the dimer dissociations. Thus, the utility of this protein-protein FRET system to monitor the effects of histone variants on NCP dynamics has been demonstrated, and the system appears equally well-suited for dissection of the kinetic processes of dimer

  15. Reduction of protein adsorption on silica and polysulfone surfaces coated with complex coacervate core micelles with poly(vinyl alcohol) as a neutral brush forming block

    NARCIS (Netherlands)

    Brzozowska, A. M.; Zhang, Q.; de Keizer, A.; Norde, W.; Stuart, M. A. Cohen

    2010-01-01

    We have studied the formation and stability of complex coacervate core micelles (C3Ms) in solution, and the influence of C3M coatings on the adsorption of the proteins beta-lactoglobulin (beta-lac), bovine serum albumin (BSA). and lysozyme (Lsz) on silica and polysulfone surfaces. The C3M5 consist o

  16. Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core*

    Science.gov (United States)

    Camus, Gregory; Herker, Eva; Modi, Ankit A.; Haas, Joel T.; Ramage, Holly R.; Farese, Robert V.; Ott, Melanie

    2013-01-01

    The triglyceride-synthesizing enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) plays a critical role in hepatitis C virus (HCV) infection by recruiting the HCV capsid protein core onto the surface of cellular lipid droplets (LDs). Here we find a new interaction between the non-structural protein NS5A and DGAT1 and show that the trafficking of NS5A to LDs depends on DGAT1 activity. DGAT1 forms a complex with NS5A and core and facilitates the interaction between both viral proteins. A catalytically inactive mutant of DGAT1 (H426A) blocks the localization of NS5A, but not core, to LDs in a dominant-negative manner and impairs the release of infectious viral particles, underscoring the importance of DGAT1-mediated translocation of NS5A to LDs in viral particle production. We propose a model whereby DGAT1 serves as a cellular hub for HCV core and NS5A proteins, guiding both onto the surface of the same subset of LDs, those generated by DGAT1. These results highlight the critical role of DGAT1 as a host factor for HCV infection and as a potential drug target for antiviral therapy. PMID:23420847

  17. Distribution, ultrastructural localization, and ontogeny of the core protein of a heparan sulfate proteoglycan in human skin and other basement membranes

    DEFF Research Database (Denmark)

    Horiguchi, Y; Couchman, J R; Ljubimov, A V;

    1989-01-01

    A variety of heparan sulfate proteoglycans (HSPG) have been identified on cell surfaces and in basement membrane (BM). To more fully characterize HSPG in human skin BM, we used two monoclonal antibodies (MAb) directed against epitopes of the core protein of a high molecular weight HSPG isolated...

  18. Liver cancer-derived hepatitis C virus core proteins shift TGF-beta responses from tumor suppression to epithelial-mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    Serena Battaglia

    Full Text Available BACKGROUND: Chronic hepatitis C virus (HCV infection and associated liver cirrhosis represent a major risk factor for hepatocellular carcinoma (HCC development. TGF-beta is an important driver of liver fibrogenesis and cancer; however, its actual impact in human cancer progression is still poorly known. The aim of this study was to investigate the role of HCC-derived HCV core natural variants on cancer progression through their impact on TGF-beta signaling. PRINCIPAL FINDINGS: We provide evidence that HCC-derived core protein expression in primary human or mouse hepatocyte alleviates TGF-beta responses in terms or growth inhibition or apoptosis. Instead, in these hepatocytes TGF-beta was still able to induce an epithelial to mesenchymal transition (EMT, a process that contributes to the promotion of cell invasion and metastasis. Moreover, we demonstrate that different thresholds of Smad3 activation dictate the TGF-beta responses in hepatic cells and that HCV core protein, by decreasing Smad3 activation, may switch TGF-beta growth inhibitory effects to tumor promoting responses. CONCLUSION/SIGNIFICANCE: Our data illustrate the capacity of hepatocytes to develop EMT and plasticity under TGF-beta, emphasize the role of HCV core protein in the dynamic of these effects and provide evidence for a paradigm whereby a viral protein implicated in oncogenesis is capable to shift TGF-beta responses from cytostatic effects to EMT development.

  19. Cross-linking of soybean protein isolate-chitosan coacervate with transglutaminase utilizing capsanthin as the model core.

    Science.gov (United States)

    Huang, G Q; Xiao, J X; Qiu, H W; Yang, J

    2014-01-01

    Transglutaminase (TG) is an alternative coacervate cross-linking agent to aldehydes due to its safety. In this work, the cross-linking conditions of soybean protein isolate (SPI)-chitosan coacervates with TG-utilizing capsanthin as the model core were optimized and its cross-linking effectiveness was compared with that of glutaraldehyde. Results indicated that the optimum capsanthin microcapsule cross-linking conditions were as follows: a suspension pH of 6.0, an incubation duration of 3 h, a TG concentration of 18.75 U/g SPI and a reaction temperature of 45 °C. Under these conditions, TG provided a cross-linking effectiveness comparable with that of glutaraldehyde in regards to microcapsule stability against swelling in 80 °C water and heating at 150 °C. Differential scanning calorimetry analysis revealed that TG cross-linking increased the integrity of the microcapsule walls. It was concluded that the SPI-chitosan coacervation pair has potential applications in the food industry in terms of cross-linker safety and effectiveness.

  20. Rapid Histone-Catalyzed DNA Lesion Excision and Accompanying Protein Modification in Nucleosomes and Nucleosome Core Particles.

    Science.gov (United States)

    Weng, Liwei; Greenberg, Marc M

    2015-09-01

    C5'-Hydrogen atoms are frequently abstracted during DNA oxidation. The oxidized abasic lesion 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) is an electrophilic product of the C5'-radical. DOB is a potent irreversible inhibitor of DNA polymerase β, and forms interstrand cross-links in free DNA. We examined the reactivity of DOB within nucleosomes and nucleosome core particles (NCPs), the monomeric component of chromatin. Depending upon the position at which DOB is generated within a NCP, it is excised from nucleosomal DNA at a rate 275-1500-fold faster than that in free DNA. The half-life of DOB (7.0-16.8 min) in NCPs is shorter than any other abasic lesion. DOB's lifetime in NCPs is also significantly shorter than the estimated lifetime of an abasic site within a cell, suggesting that the observed chemistry would occur intracellularly. Histones also catalyze DOB excision when the lesion is present in the DNA linker region of a nucleosome. Schiff-base formation between DOB and histone proteins is detected in nucleosomes and NCPs, resulting in pyrrolone formation at the lysine residues. The lysines modified by DOB are often post-translationally modified. Consequently, the histone modifications described herein could affect the regulation of gene expression and may provide a chemical basis for the cytotoxicity of the DNA damaging agents that produce this lesion.

  1. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R

    1997-01-01

    Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now...... obtained cDNA clones encoding the entire bamacan core protein of Mr = 138 kD, which reveal a five domain, head-rod-tail configuration. The head and tail are potentially globular, while the central large rod probably forms coiled-coil structures, with one large central and several very short interruptions....... This molecular architecture is novel for an extracellular matrix molecule, but it resembles that of a group of intracellular proteins, including some proposed to stabilize the mitotic chromosome scaffold. We have previously proposed a similar stabilizing role for bamacan in the basement membrane matrix...

  2. Decomposition of total solvation energy into core, side-chains and water contributions: Role of cross correlations and protein conformational fluctuations in dynamics of hydration layer

    Science.gov (United States)

    Mondal, Sayantan; Mukherjee, Saumyak; Bagchi, Biman

    2017-09-01

    Dynamical coupling between water and amino acid side-chain residues in solvation dynamics is investigated by selecting residues often used as natural probes, namely tryptophan, tyrosine and histidine, located at different positions on protein surface. Such differently placed residues are found to exhibit different timescales of relaxation. The total solvation response measured by the probe is decomposed in terms of its interactions with (i) protein core, (ii) side-chain and (iii) water. Significant anti cross-correlation among these contributions are observed. When the motion of the protein side-chains is quenched, solvation either becomes faster or slower depending on the location of the probe.

  3. Protein glycosylation in the phytopathogen Ustilago maydis: From core oligosaccharide synthesis to the ER glycoprotein quality control system, a genomic analysis.

    Science.gov (United States)

    Fernández-Alvarez, Alfonso; Elías-Villalobos, Alberto; Ibeas, José I

    2010-09-01

    The corn smut fungus Ustilago maydis has, over recent decades, become established as a robust pathogenic model for studying fungi-plant relationships. This use of U. maydis can be attributed to its biotrophic host interaction, easy culture and genetic manipulation in the laboratory, and the severe disease symptoms it induces in infected maize. Recent studies have shown that normal protein glycosylation is essential for pathogenic development, but dispensable for the saprophytic growth or mating. Given the relevance of protein glycosylation for U. maydis virulence, and consequently its role in the plant pathogenesis, here we review the main actors and events implicated in protein glycosylation. Furthermore, we describe the results of an in silico search, where we identify all the conserved members of the N- and O-glycosylation pathways in U. maydis at each stage: core oligosaccharide synthesis, addition of the core oligosaccharide to nascent target proteins, maturation and extension of the core oligosaccharide, and the quality control system used by the cell to avoid the presence of unfolded glycoproteins. Finally, we discuss how these genes could affect U. maydis virulence and their biotechnological implications.

  4. Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation.

    Science.gov (United States)

    Wittkop, Linda; Schwarz, Alexandra; Cassany, Aurelia; Grün-Bernhard, Stefanie; Delaleau, Mildred; Rabe, Birgit; Cazenave, Christian; Gerlich, Wolfram; Glebe, Dieter; Kann, Michael

    2010-07-01

    Capsids of hepatitis B virus and other hepadnaviruses contain a cellular protein kinase, which phosphorylates the capsid protein. Some phosphorylation sites are shown to be essential for distinct steps of viral replication as pregenome packaging or plus strand DNA synthesis. Although different protein kinases have been reported to phosphorylate the capsid protein, varying experimental approaches do not allow direct comparison. Furthermore, the activity of a specific protein kinase has not yet been correlated to steps in the hepadnaviral life cycle. In this study we show that capsids from various sources encapsidate active protein kinase Calpha, irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle.

  5. Hepatitis C virus Core protein stimulates cell growth by down-regulating p16 expression via DNA methylation.

    Science.gov (United States)

    Park, Sun-Hye; Lim, Joo Song; Lim, Su-Yeon; Tiwari, Indira; Jang, Kyung Lib

    2011-11-01

    Hepatitis C virus Core plays a vital role in the development of hepatocellular carcinoma; however, its action mechanism is still controversial. Here, we showed that Core down-regulated levels of p16, resulting in inactivation of Rb and subsequent activation of E2F1, which lead to growth stimulation of hepatocytes. For this effect, Core inhibited p16 expression by inducing promoter hypermethylation via up-regulation of DNA methyltransferase 1 (DNMT1) and DNMT3b. The growth stimulatory effect of Core was abolished when levels of p16 were restored by either exogenous complementation or treatment with 5-Aza-2'dC, indicating that the effect is critical for the stimulation of cell growth by Core. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  6. Protein-Assisted Assembly of Modular 3D Plasmonic Raspberry-like Core/Satellite Nanoclusters: Correlation of Structure and Optical Properties.

    Science.gov (United States)

    Höller, Roland P M; Dulle, Martin; Thomä, Sabrina; Mayer, Martin; Steiner, Anja Maria; Förster, Stephan; Fery, Andreas; Kuttner, Christian; Chanana, Munish

    2016-06-28

    We present a bottom-up assembly route for a large-scale organization of plasmonic nanoparticles (NPs) into three-dimensional (3D) modular assemblies with core/satellite structure. The protein-assisted assembly of small spherical gold or silver NPs with a hydrophilic protein shell (as satellites) onto larger metal NPs (as cores) offers high modularity in sizes and composition at high satellite coverage (close to the jamming limit). The resulting dispersions of metal/metal nanoclusters exhibit high colloidal stability and therefore allow for high concentrations and a precise characterization of the nanocluster architecture in dispersion by small-angle X-ray scattering (SAXS). Strong near-field coupling between the building blocks results in distinct regimes of dominant satellite-to-satellite and core-to-satellite coupling. High robustness against satellite disorder was proved by UV/vis diffuse reflectance (integrating sphere) measurements. Generalized multiparticle Mie theory (GMMT) simulations were employed to describe the electromagnetic coupling within the nanoclusters. The close correlation of structure and optical property allows for the rational design of core/satellite nanoclusters with tailored plasmonics and well-defined near-field enhancement, with perspectives for applications such as surface-enhanced spectroscopies.

  7. A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins

    Directory of Open Access Journals (Sweden)

    Reeja Maria Cherian

    2015-08-01

    Full Text Available Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b, CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1, core 3 (B3GNT6, core 4 (GCNT1 and B3GNT6, or extended core 1 (B3GNT3 chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc or type 2 (Galb4GlcNAc outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.

  8. Core-Shell Soy Protein-Soy Polysaccharide Complex (Nano)particles as Carriers for Improved Stability and Sustained Release of Curcumin.

    Science.gov (United States)

    Chen, Fei-Ping; Ou, Shi-Yi; Tang, Chuan-He

    2016-06-22

    Using soy protein isolate (SPI) and soy-soluble polysaccharides (SSPS) as polymer matrixes, this study reported a novel process to fabricate unique core-shell complex (nano)particles to perform as carriers for curcumin (a typical poorly soluble bioactive). In the process, curcumin-SPI nanocomplexes were first formed at pH 7.0 and then coated by SSPS. At this pH, the core-shell complex was formed in a way the SPI nanoparticles might be incorporated into the interior of SSPS molecules without distinctly affecting the size and morphology of particles. The core-shell structure was distinctly changed by adjusting pH from 7.0 to 4.0. At pH 4.0, SSPS was strongly bound to the surface of highly aggregated SPI nanoparticles, and as a consequence, much larger complexes were formed. The bioaccessibility of curcumin in the SPI-curcumin complexes was unaffected by the SSPS coating. However, the core-shell complex formation greatly improved the thermal stability and controlled release properties of encapsulated curcumin. The improvement was much better at pH 4.0 than that at pH 7.0. All of the freeze-dried core-shell complex preparations exhibited good redispersion behavior. The findings provide a simple approach to fabricate food-grade delivery systems for improved water dispersion, heat stability, and even controlled release of poorly soluble bioactives.

  9. Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein

    Directory of Open Access Journals (Sweden)

    Theo Luiz Ferraz de Souza

    2016-11-01

    Full Text Available Background Hepatitis C virus (HCV core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124 is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood. Methods Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs. Results The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12, indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs. Discussion Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

  10. Radioimmunoassay and enzyme-linked immunoassay of antibodies to the core protein (P24) of human T-lymphotropic virus (HTLV III). [Acquired immunodeficiency syndrome (AIDS)

    Energy Technology Data Exchange (ETDEWEB)

    Neurath, A.R.; Strick, N.; Sproul, P.

    1985-05-01

    Human T-cell lymphotropic viruses designated HTLV III or LAV are considered to represent the causative agents of the acquired immunodeficiency syndrome (AIDS). Therefore a simple direct RIA or ELISA method for antibodies to distinct epitopes of HTLV III/LAV structural components would be of great value. The authors describe RIA and ELISA assays which obviate the need for purified virus or virus proteins, do not utilize infected cells and thus do not diminish the source for continuous production of viral antigens and are specific for a major core protein of HTLV III/LAV.

  11. [The determination of the genotype of natural reassortant influenza A viruses according to the core protein genes by the methods of competitive dot hybridization and sequencing].

    Science.gov (United States)

    Grinbaum, E B; Zolotarev, F N; Petrov, N A; Litvinova, O M; Konovalenko, I B; Luzianina, T Ia; Golubev, D B

    1992-01-01

    Simultaneous circulation of different subtypes of influenza A viruses provides conditions for reassortant strains formation. A comparative investigation of genome of 47 influenza A virus strains (H1N1, H2N2, and H3N2) was carried out by competitive dot hybridization technique and sequence analysis of some of cDNA-copies of the virus genes. All the genes of 43 strains encoding nonglycolysed proteins corresponded to the serum subtype of surface glycoproteins. The reassortant pattern of genome for some genes of core proteins was revealed in 4 viruses. All the dot hybridization data were completely confirmed by sequence analysis of the genes.

  12. Decorin protein core affects the global gene expression profile of the tumor microenvironment in a triple-negative orthotopic breast carcinoma xenograft model.

    Directory of Open Access Journals (Sweden)

    Simone Buraschi

    Full Text Available Decorin, a member of the small leucine-rich proteoglycan gene family, exists and functions wholly within the tumor microenvironment to suppress tumorigenesis by directly targeting and antagonizing multiple receptor tyrosine kinases, such as the EGFR and Met. This leads to potent and sustained signal attenuation, growth arrest, and angiostasis. We thus sought to evaluate the tumoricidal benefits of systemic decorin on a triple-negative orthotopic breast carcinoma xenograft model. To this end, we employed a novel high-density mixed expression array capable of differentiating and simultaneously measuring gene signatures of both Mus musculus (stromal and Homo sapiens (epithelial tissue origins. We found that decorin protein core modulated the differential expression of 374 genes within the stromal compartment of the tumor xenograft. Further, our top gene ontology classes strongly suggests an unexpected and preferential role for decorin protein core to inhibit genes necessary for immunomodulatory responses while simultaneously inducing expression of those possessing cellular adhesion and tumor suppressive gene properties. Rigorous verification of the top scoring candidates led to the discovery of three genes heretofore unlinked to malignant breast cancer that were reproducibly found to be induced in several models of tumor stroma. Collectively, our data provide highly novel and unexpected stromal gene signatures as a direct function of systemic administration of decorin protein core and reveals a fundamental basis of action for decorin to modulate the tumor stroma as a biological mechanism for the ascribed anti-tumorigenic properties.

  13. Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD.

    Directory of Open Access Journals (Sweden)

    Heng-Li Chen

    Full Text Available The rise of multidrug-resistant (MDR pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc contains an arginine-rich domain (ARD at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV. In this study, we demonstrated that the peptide containing the full-length ARD I-IV (HBc147-183 has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II-IV (HBc153-176 and ARD I-III (HBc147-167 were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS in several in vitro binding assays. Peptide ARD I-IV (HBc147-183 had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p. inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that

  14. Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD).

    Science.gov (United States)

    Chen, Heng-Li; Su, Pei-Yi; Chang, Ya-Shu; Wu, Szu-Yao; Liao, You-Di; Yu, Hui-Ming; Lauderdale, Tsai-Ling; Chang, Kaichih; Shih, Chiaho

    2013-01-01

    The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I-IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II-IV (HBc153-176) and ARD I-III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I-IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that HBc ARD

  15. Downregulation of miRNA-30c and miR-203a is associated with hepatitis C virus core protein-induced epithelial–mesenchymal transition in normal hepatocytes and hepatocellular carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Dongjing [Hepatobiliary and Enteric Surgery Research Center, Xiangya Hospital, Central South University, Changsha 410008 (China); Wu, Jilin, E-mail: 6296082@qq.com [Hepatobiliary and Enteric Surgery Research Center, Xiangya Hospital, Central South University, Changsha 410008 (China); Liu, Meizhou [Department of Medical Service, Shenzhen Second People' s Hospital, Shenzhen, Guangdong 518035 (China); Yin, Hui [Staff' s Hospital, Central South University, Changsha, Hunan 410078 (China); He, Jiantai [Hepatobiliary and Enteric Surgery Research Center, Xiangya Hospital, Central South University, Changsha 410008 (China); Zhang, Bo, E-mail: zhangbo8095@126.com [Department of Ultrasonography, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China)

    2015-09-04

    Hepatitis C virus (HCV) Core protein has been demonstrated to induce epithelial–mesenchymal transition (EMT) and is associated with cancer progression of hepatocellular carcinoma (HCC). However, how the Core protein regulates EMT is still unclear. In this study, HCV Core protein was overexpressed by an adenovirus. The protein levels of EMT markers were measured by Western blot. The xenograft animal model was established by inoculation of HepG2 cells. Results showed that ectopic expression of HCV core protein induced EMT in L02 hepatocytes and HepG2 tumor cells by upregulating vimentin, Sanl1, and Snal2 expression and downregulating E-cadherin expression. Moreover, Core protein downregulated miR-30c and miR-203a levels in L02 and HepG2 cells, but artificial expression of miR-30c and miR-203a reversed Core protein-induced EMT. Further analysis showed that ectopic expression of HCV core protein stimulated cell proliferation, inhibited apoptosis, and increased cell migration, whereas artificial expression of miR-30c and miR-203a significantly reversed the role of Core protein in these cell functions in L02 and HepG2 cells. In the HepG2 xenograft tumor models, artificial expression of miR-30c and miR-203a inhibited EMT and tumor growth. Moreover, L02 cells overexpressing Core protein can form tumors in nude mice. In HCC patients, HCV infection significantly shortened patients' survival time, and loss of miR-30c and miR-203 expression correlated with poor survival. In conclusion, HCV core protein downregulates miR-30c and miR-203a expression, which results in activation of EMT in normal hepatocytes and HCC tumor cells. The Core protein-activated-EMT is involved in the carcinogenesis and progression of HCC. Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC. - Highlights: • HCV core protein downregulates miR-30c and miR-203a expression. • Downregulation of miR-30c and miR-203a activates EMT. • Activated-EMT is involved in the

  16. A C-terminal Hydrophobic, Solvent-protected Core and a Flexible N-terminus are Potentially Required for Human Papillomavirus 18 E7 Protein Functionality

    Energy Technology Data Exchange (ETDEWEB)

    Liu, S.; Tian, Y; Greenaway, F; Sun, M

    2010-01-01

    The oncogenic potential of the high-risk human papillomavirus (HPV) relies on the expression of genes specifying the E7 and E6 proteins. To investigate further the variation in oligomeric structure that has been reported for different E7 proteins, an HPV-18 E7 cloned from a Hispanic woman with cervical intraepithelial neoplasia was purified to homogeneity most probably as a stable monomeric protein in aqueous solution. We determined that one zinc ion is present per HPV-18 E7 monomer by amino acid and inductively coupled plasma-atomic emission spectroscopy analysis. Intrinsic fluorescence and circular dichroism spectroscopic results indicate that the zinc ion is important for the correct folding and thermal stability of HPV-18 E7. Hydroxyl radical mediated protein footprinting coupled to mass spectrometry and other biochemical and biophysical data indicate that near the C-terminus, the four cysteines of the two Cys-X{sub 2}-Cys motifs that are coordinated to the zinc ion form a solvent inaccessible core. The N-terminal LXCXE pRb binding motif region is hydroxyl radical accessible and conformationally flexible. Both factors, the relative flexibility of the pRb binding motif at the N-terminus and the C-terminal metal-binding hydrophobic solvent-protected core, combine together and facilitate the biological functions of HPV-18 E7.

  17. Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor.

    Science.gov (United States)

    Cook, Jonathan D; Soto-Montoya, Hazel; Korpela, Markus K; Lee, Jeffrey E

    2015-07-24

    Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion.

  18. Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-like globin gene cluster

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681--10970 bp) in the locus control region (LCR) of the human b-like globin gene cluster has been examined by using both the in vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using the in vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstituted in vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or the in vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human β-like globin gene's expression.

  19. Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human b-like globin gene cluster

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681--10970 bp) in the locus control region (LCR) of the human b-like globin gene cluster has been examined by using both the in vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using the in vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstituted in vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or the in vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human b-like globin gene's expression.

  20. HCV core protein binds to gC1qR to induce A20 expression and inhibit cytokine production through MAPKs and NF-κB signaling pathways.

    Science.gov (United States)

    Song, Xiaotian; Yao, Zhiyan; Yang, Jianling; Zhang, Zhengzheng; Deng, Yuqing; Li, Miao; Ma, Cuiqing; Yang, Lijuan; Gao, Xue; Li, Wenjian; Liu, Jianguo; Wei, Lin

    2016-06-07

    Hepatitis C virus (HCV) infection is characterized by a strong propensity toward chronicity. During chronic HCV infection, HCV core protein is implicated in deregulating cytokine expression that associates with chronic inflammation. A20 is known as a powerful suppressor in cytokine signaling, in this study, we explored the A20 expression in macrophages induced by HCV core protein and the involved signaling pathways. Results demonstrated that HCV core protein induced A20 expression in macrophages. Silencing A20 significantly enhanced the secretion of IL-6, IL-1β and TGF-β1, but not IL-8 and TNF. Additionally, HCV core protein interacted with gC1qR, but not TLR2, TLR3 and TLR4 in pull-down assay. Silencing gC1qR abrogated core-induced A20 expression. Furthermore, HCV core protein activated MAPK, NF-κB and PI3K/AKT pathways in macrophages. Inhibition of P38, JNK and NF-κB but not ERK and AKT activities greatly reduced the A20 expression. In conclusion, the study suggests that HCV core protein ligates gC1qR to induce A20 expression in macrophages via P38, JNK and NF-κB signaling pathways, which leads to a low-grade chronic inflammation during HCV infection. It represents a novel mechanism by which HCV usurps the host for persistence.

  1. Colloidal Gold--Collagen Protein Core--Shell Nanoconjugate: One-Step Biomimetic Synthesis, Layer-by-Layer Assembled Film, and Controlled Cell Growth.

    Science.gov (United States)

    Xing, Ruirui; Jiao, Tifeng; Yan, Linyin; Ma, Guanghui; Liu, Lei; Dai, Luru; Li, Junbai; Möhwald, Helmuth; Yan, Xuehai

    2015-11-11

    The biogenic synthesis of biomolecule-gold nanoconjugates is of key importance for a broad range of biomedical applications. In this work, a one-step, green, and condition-gentle strategy is presented to synthesize stable colloidal gold-collagen core-shell nanoconjugates in an aqueous solution at room temperature, without use of any reducing agents and stabilizing agents. It is discovered that electrostatic binding between gold ions and collagen proteins and concomitant in situ reduction by hydroxyproline residues are critically responsible for the formation of the core-shell nanoconjugates. The film formed by layer-by-layer assembly of such colloidal gold-collagen nanoconjugates can notably improve the mechanical properties and promote cell adhesion, growth, and differentiation. Thus, the colloidal gold-collagen nanoconjugates synthesized by such a straightforward and clean manner, analogous to a biomineralization pathway, provide new alternatives for developing biologically based hybrid biomaterials toward a range of therapeutic and diagnostic applications.

  2. The hepatitis C virus core protein inhibits adipose triglyceride lipase (ATGL)-mediated lipid mobilization and enhances the ATGL interaction with comparative gene identification 58 (CGI-58) and lipid droplets.

    Science.gov (United States)

    Camus, Gregory; Schweiger, Martina; Herker, Eva; Harris, Charles; Kondratowicz, Andrew S; Tsou, Chia-Lin; Farese, Robert V; Herath, Kithsiri; Previs, Stephen F; Roddy, Thomas P; Pinto, Shirly; Zechner, Rudolf; Ott, Melanie

    2014-12-26

    Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.

  3. Biomagnetic of Apatite-Coated Cobalt Ferrite: A Core-Shell Particle for Protein Adsorption and pH-Controlled Release.

    Science.gov (United States)

    Tang, I-Ming; Krishnamra, Nateetip; Charoenphandhu, Narattaphol; Hoonsawat, Rassmidara; Pon-On, Weeraphat

    2011-12-01

    Magnetic nanoparticle composite with a cobalt ferrite (CoFe2O4, (CF)) core and an apatite (Ap) coating was synthesized using a biomineralization process in which a modified simulated body fluid (1.5SBF) solution is the source of the calcium phosphate for the apatite formation. The core-shell structure formed after the citric acid-stabilized cobalt ferrite (CFCA) particles were incubated in the 1.5 SBF solution for 1 week. The mean particle size of CFCA-Ap is about 750 nm. A saturation magnetization of 15.56 emug(-1) and a coercivity of 1808.5 Oe were observed for the CFCA-Ap obtained. Bovine serum albumin (BSA) was used as the model protein to study the adsorption and release of the proteins by the CFCA-Ap particles. The protein adsorption by the CFCA-Ap particles followed a more typical Freundlich than Langmuir adsorption isotherm. The BSA release as a function of time became less rapid as the CFCA-Ap particles were immersed in higher pH solution, thus indicating that the BSA release is dependent on the local pH.

  4. Expression, localization and functional divergence of alphaB-crystallin and heat shock protein 27 in core myopathies and neurogenic atrophy.

    Science.gov (United States)

    Fischer, Dirk; Matten, Jens; Reimann, Jens; Bönnemann, Carsten; Schröder, Rolf

    2002-09-01

    AlphaB-crystallin (alphaBC) and heat shock protein 27 (hsp 27) are members of the family of small heat shock proteins (shsps), which exert a role as molecular chaperones by binding unfolded or denatured proteins, thereby suppressing irreversible protein aggregation and consecutive cell damage. The essential role of shsps in human neuromuscular disorders is highlighted by the observation that a mutation of the human alphaBC gene causes an autosomal dominant "myofibrillar myopathy" characterized by alphaBC and desmin accumulation. Furthermore, an aberrant immunostaining of alphaBC was recently reported in sporadic inclusion body myositis. In the present study we analyzed the expression and localization of alphaB-crystallin and hsp 27 in various congenital myopathies by means of indirect immunofluorescence, immunogold electron microscopy and Western blotting. We demonstrate an increased immunoreactivity of alphaBC and hsp 27 in central and minicore lesions as well as in target fibers, which renders both shsps as reliable, but nonspecific, markers for core and target structures. In contrast, Western blotting demonstrated a normal expression level of alphaBC and hsp 27, which indicates that the increased immunostaining is not the result of an enhanced protein expression. Furthermore, thiocyanate-induced degradation of actin filaments led to a dramatic decrease of hsp 27 immunostaining in core and target lesions, whereas the increased alphaBC and desmin immunostaining was found to be even more enhanced. The latter findings imply a functional diversity of both shsps with a preferential association of hsp 27 with the actin microfilament system and alphaBC with the intermyofibrillar desmin cytoskeleton in human skeletal muscle.

  5. Structure of the cytoplasmic domain of TcpE, the inner membrane core protein required for assembly of the Vibrio cholerae toxin-coregulated pilus.

    Science.gov (United States)

    Kolappan, Subramaniapillai; Craig, Lisa

    2013-04-01

    Type IV pili are long thin surface-displayed polymers of the pilin subunit that are present in a diverse group of bacteria. These multifunctional filaments are critical to virulence for pathogens such as Vibrio cholerae, which use them to form microcolonies and to secrete the colonization factor TcpF. The type IV pili are assembled from pilin subunits by a complex inner membrane machinery. The core component of the type IV pilus-assembly platform is an integral inner membrane protein belonging to the GspF superfamily of secretion proteins. These proteins somehow convert chemical energy from ATP hydrolysis by an assembly ATPase on the cytoplasmic side of the inner membrane to mechanical energy for extrusion of the growing pilus filament out of the inner membrane. Most GspF-family inner membrane core proteins are predicted to have N-terminal and central cytoplasmic domains, cyto1 and cyto2, and three transmembrane segments, TM1, TM2 and TM3. Cyto2 and TM3 represent an internal repeat of cyto1 and TM1. Here, the 1.88 Å resolution crystal structure of the cyto1 domain of V. cholerae TcpE, which is required for assembly of the toxin-coregulated pilus, is reported. This domain folds as a monomeric six-helix bundle with a positively charged membrane-interaction face at one end and a hydrophobic groove at the other end that may serve as a binding site for partner proteins in the pilus-assembly complex.

  6. Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17.

    Science.gov (United States)

    Czerny, Daniel D; Padmanaban, Senthilkumar; Anishkin, Andriy; Venema, Kees; Riaz, Zoya; Sze, Heven

    2016-09-01

    Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na(+)/H(+) antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 and aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. These results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates in ion recognition, coordination and/or translocation, similar to characterized cation/H(+) exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family.

  7. Reference: -300CORE [PLACE

    Lifescience Database Archive (English)

    Full Text Available -300CORE Forde BG, Heyworth A, Pywell J, Kreis M Nucleotide sequence of a B1 hordein gene and the identifica...tion of possible upstream regulatory elements in endosperm storage protein genes fr

  8. Lateral diffusion of peripheral membrane proteins on supported lipid bilayers is controlled by the additive frictional drags of (1) bound lipids and (2) protein domains penetrating into the bilayer hydrocarbon core.

    Science.gov (United States)

    Ziemba, Brian P; Falke, Joseph J

    2013-01-01

    Peripheral membrane proteins bound to lipids on bilayer surfaces play central roles in a wide array of cellular processes, including many signaling pathways. These proteins diffuse in the plane of the bilayer and often undergo complex reactions involving the binding of regulatory and substrate lipids and proteins they encounter during their 2D diffusion. Some peripheral proteins, for example pleckstrin homology (PH) domains, dock to the bilayer in a relatively shallow position with little penetration into the bilayer. Other peripheral proteins exhibit more complex bilayer contacts, for example classical protein kinase C isoforms (PKCs) bind as many as six lipids in stepwise fashion, resulting in the penetration of three PKC domains (C1A, C1B, C2) into the bilayer headgroup and hydrocarbon regions. A molecular understanding of the molecular features that control the diffusion speeds of proteins bound to supported bilayers would enable key molecular information to be extracted from experimental diffusion constants, revealing protein-lipid and protein-bilayer interactions difficult to study by other methods. The present study investigates a range of 11 different peripheral protein constructs comprised by 1-3 distinct domains (PH, C1A, C1B, C2, anti-lipid antibody). By combining these constructs with various combinations of target lipids, the study measures 2D diffusion constants on supported bilayers for 17 different protein-lipid complexes. The resulting experimental diffusion constants, together with the known membrane interaction parameters of each complex, are used to analyze the molecular features correlated with diffusional slowing and bilayer friction. The findings show that both (1) individual bound lipids and (2) individual protein domains that penetrate into the hydrocarbon core make additive contributions to the friction against the bilayer, thereby defining the 2D diffusion constant. An empirical formula is developed that accurately estimates the diffusion

  9. Selective enrichment of metal-binding proteins based on magnetic core/shell microspheres functionalized with metal cations.

    Science.gov (United States)

    Fang, Caiyun; Zhang, Lei; Zhang, Xiaoqin; Lu, Haojie

    2015-06-21

    Metal binding proteins play many important roles in a broad range of biological processes. Characterization of metal binding proteins is important for understanding their structure and biological functions, thus leading to a clear understanding of metal associated diseases. The present study is the first to investigate the effectiveness of magnetic microspheres functionalized with metal cations (Ca(2+), Cu(2+), Zn(2+) and Fe(3+)) as the absorbent matrix in IMAC technology to enrich metal containing/binding proteins. The putative metal binding proteins in rat liver were then globally characterized by using this strategy which is very easy to handle and can capture a number of metal binding proteins effectively. In total, 185 putative metal binding proteins were identified from rat liver including some known less abundant and membrane-bound metal binding proteins such as Plcg1, Acsl5, etc. The identified proteins are involved in many important processes including binding, catalytic activity, translation elongation factor activity, electron carrier activity, and so on.

  10. A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

    Science.gov (United States)

    Settem, R P; Honma, K; Nakajima, T; Phansopa, C; Roy, S; Stafford, G P; Sharma, A

    2013-03-01

    Tannerella forsythia is a pathogen implicated in periodontitis, an inflammatory disease of the tooth-supporting tissues often leading to tooth loss. This key periodontal pathogen is decorated with a unique glycan core O-glycosidically linked to the bacterium's proteinaceous surface (S)-layer lattice and other glycoproteins. Herein, we show that the terminal motif of this glycan core acts to modulate dendritic cell effector functions to suppress T-helper (Th)17 responses. In contrast to the wild-type bacterial strain, infection with a mutant strain lacking the complete S-layer glycan core induced robust Th17 and reduced periodontal bone loss in mice. Our findings demonstrate that surface glycosylation of this pathogen may act to ensure its persistence in the host likely through suppression of Th17 responses. In addition, our data suggest that the bacterium then induces the Toll-like receptor 2-Th2 inflammatory axis that has previously been shown to cause bone destruction. Our study provides a biological basis for pathogenesis and opens opportunities in exploiting bacterial glycans as therapeutic targets against periodontitis and a range of other infectious diseases.

  11. Genome-wide annotation, expression profiling, and protein interaction studies of the core cell-cycle genes in Phalaenopsis aphrodite.

    Science.gov (United States)

    Lin, Hsiang-Yin; Chen, Jhun-Chen; Wei, Miao-Ju; Lien, Yi-Chen; Li, Huang-Hsien; Ko, Swee-Suak; Liu, Zin-Huang; Fang, Su-Chiung

    2014-01-01

    Orchidaceae is one of the most abundant and diverse families in the plant kingdom and its unique developmental patterns have drawn the attention of many evolutionary biologists. Particular areas of interest have included the co-evolution of pollinators and distinct floral structures, and symbiotic relationships with mycorrhizal flora. However, comprehensive studies to decipher the molecular basis of growth and development in orchids remain scarce. Cell proliferation governed by cell-cycle regulation is fundamental to growth and development of the plant body. We took advantage of recently released transcriptome information to systematically isolate and annotate the core cell-cycle regulators in the moth orchid Phalaenopsis aphrodite. Our data verified that Phalaenopsis cyclin-dependent kinase A (CDKA) is an evolutionarily conserved CDK. Expression profiling studies suggested that core cell-cycle genes functioning during the G1/S, S, and G2/M stages were preferentially enriched in the meristematic tissues that have high proliferation activity. In addition, subcellular localization and pairwise interaction analyses of various combinations of CDKs and cyclins, and of E2 promoter-binding factors and dimerization partners confirmed interactions of the functional units. Furthermore, our data showed that expression of the core cell-cycle genes was coordinately regulated during pollination-induced reproductive development. The data obtained establish a fundamental framework for study of the cell-cycle machinery in Phalaenopsis orchids.

  12. Hepatitis B virus core protein with hot-spot mutations inhibit MxA gene transcription but has no effect on inhibition of virus replication by interferon α.

    Science.gov (United States)

    Zhijian, Yu; Zhen, Huang; Fan, Zhang; Jin, Yang; Qiwen, Deng; Zhongming, Zeng

    2010-10-20

    It has been reported that hepatitis B virus (HBV) core protein (HBc) can inhibit the transcription of human interferon-induced MxA gene. In this study, we investigated whether HBc protein mutations at hot spots (L60V, S87G and I97L) could still inhibit MxA transcription and the potential significance of this inhibition in virus replication in vitro. Our data indicated that the IFN-induced MxA mRNA expression level and MxA promoter activity was significantly down-regulated by mutant protein of HBc(I97L), compared to WT and the other two mutated HBc proteins(L60V or S87G). However, in Huh7 cells stably expressing WT or the mutated HBc proteins (L60V, S87G or I97L), IFN-α could inhibit the extra- and intracellular HBV DNA level and HBsAg secretion to a similar level compared to that in cells transfected with control plasmids. In conclusion, HBc protein with I97L mutation may play an special role in suppressing the transcription of MxA gene. Moreover, the inhibitory effect on MxA gene transcription by the WT or mutated HBc proteins (L60V, S87G and I97L) has no impact on inhibition of HBV replication by IFN-α in Huh7 cells. The clinical significance of the inhibitory effect of MxA gene transcription by HBc protein requires further study.

  13. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in a...

  14. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in...

  15. Enhancement of cytotoxic T lymphocyte activity by dendritic cells loaded with Tat-protein transduction domain-fused hepatitis B virus core antigen

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The protein transduction domain (PTD) of human immuno-deficiency virus-1-Tat protein has a unique potency to pen-etrate the cellular membranes. To synthesize the sequence of Tat-PTD and hepatitis B virus core antigen (HBcAg), we spliced these sequences and linked a fusion gene into the pMAL-c2x vector. The fusion proteins were purified by affin-ity chromatography and pulsed with bone marrow -derived den-dritic cells (DCs), and the transduction of recombinant pro-tein was detected by immunofluorescence antibody assay.Results showed that recombinant PTD-HBcAg could pen-etrate into DC cytoplasm while recombinant HBcAg was de-tected on the surface of cells. The percentage of DC surface molecules, such as CD80, CD86 and major histocompatibii-ity complex Ⅱ, and production of cytokine (IL-12pT0) induced by recombinant PTD-HBcAg were significantly higher than those induced by recombinant HBcAg or tumor necrosis fac-tor-α. DCs treated with PTD-HBcAg induced T cells to dif-ferentiate into specific cytotoxic T lymphocytes (CTLs) and enhanced the CTL killing response. In conclusion, the ex-pressed and purified PTD-HBcAg fusion protein could pen-etrate into cells through the plasma membrane, promote DC maturation, and enhance T cells response to generate HBcAg-specific CTLs efficiently.

  16. A truncated fragment of Ov-ASP-1 consisting of the core pathogenesis-related-1 (PR-1) domain maintains adjuvanticity as the full-length protein.

    Science.gov (United States)

    Guo, Jingjing; Yang, Yi; Xiao, Wenjun; Sun, Weilai; Yu, Hong; Du, Lanying; Lustigman, Sara; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

    2015-04-15

    The Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has good adjuvanticity for a variety of antigens and vaccines, probably due to its ability activate antigen-processing cells (APCs). However, the functional domain of Ov-ASP-1 as an adjuvant is not clearly defined. Based on the structural prediction of this protein family, we constructed a 16-kDa recombinant protein of Ov-ASP-1 that contains only the core pathogenesis-related-1 (PR-1) domain (residues 10-153), designated ASPPR. We found that ASPPR exhibits adjuvanticity similar to that of the full-length Ov-ASP-1 (residues 10-220) for various antigens, including ovalbumin (OVA), HBsAg protein antigen, and the HIV peptide 5 (Pep5) antigen, but it is more suitable for vaccine design in ASPPR-antigen fusion proteins, and more stable in PBS than Ov-ASP-1 stored at -70 °C. These results suggest that ASPPR might be the functional region of Ov-ASP-1 as an adjuvant, and therefore could be developed as an adjuvant for human use. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Evaluation of a new wide pore core-shell material (Aeris WIDEPORE) and comparison with other existing stationary phases for the analysis of intact proteins.

    Science.gov (United States)

    Fekete, Szabolcs; Berky, Róbert; Fekete, Jenő; Veuthey, Jean-Luc; Guillarme, Davy

    2012-05-04

    The separation of large biomolecules such as proteins or monoclonal antibodies (mAbs) by RPLC can be drastically enhanced thanks to the use of columns packed with wide-pore porous sub-2 μm particles or shell particles. In this context, a new wide-pore core-shell material has been recently released under the trademark Aeris WIDEPORE. It is made of a 3.2 μm solid inner core surrounded by a 0.2 μm porous layer (total particle size of 3.6 μm). The aim of this study was to evaluate the performance of this new material, compare it to other recently developed and older conventional wide-pore columns and demonstrate its applicability to real-life separations of proteins and mAbs. At first, the traditional h(min) values of the Aeris WIDEPORE column were determined for small model compounds. The h(min) values were equal to 1.7-1.8 and 1.4 for the 2.1 and 4.6 mm I.D. columns, respectively, which are in agreement with the values reported for other core-shell materials. In the case of a small protein Insulin (5.7 kDa), the achievable lowest h value was below 2 and this impressive result confirms that the Aeris WIDEPORE material should be dedicated to protein analysis. This column was then compared with five other commercially available wide-pore and medium-pore stationary phases, in the gradient elution mode, using various flow rates, gradient steepness and model proteins of MW=5.7-66.8 kDa. The Aeris WIDEPORE material often provided the best performance, in terms of peak capacity, peak capacity per time and pressure unit (PPT) and also based on the gradient kinetic plot representation. Finally, real separations of filgrastim (18.8 kDa) and its oxidized and reduced forms were performed on the different columns and the Aeris WIDEPORE material provided the most impressive performance (peak capacity>100 for t(grad)material was also evaluated on digested and reduced mAb and powerful, high-throughput separations were also attained.

  18. Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-Mike globin gene cluster

    Institute of Scientific and Technical Information of China (English)

    赵晖; 张树冰; 蒋俶; 钱若兰

    2000-01-01

    HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNasel hypersensitive site 2 (HS2core DNA sequence, -10681-10970 bp) in the locus control region (LCR) of the human β-like globin gene cluster has been examined by using both the in vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG 14/17 cannot. Using the in vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG 1/2 cannot bind to the nucleosomes reconstituted in vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and t

  19. Antibodies to the core proteins of Nairobi sheep disease virus/Ganjam virus reveal details of the distribution of the proteins in infected cells and tissues.

    Science.gov (United States)

    Lasecka, Lidia; Bin-Tarif, Abdelghani; Bridgen, Anne; Juleff, Nicholas; Waters, Ryan A; Baron, Michael D

    2015-01-01

    Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.

  20. Antibodies to the core proteins of Nairobi sheep disease virus/Ganjam virus reveal details of the distribution of the proteins in infected cells and tissues.

    Directory of Open Access Journals (Sweden)

    Lidia Lasecka

    Full Text Available Nairobi sheep disease virus (NSDV; also called Ganjam virus in India is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV. Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N and polymerase (L and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN, where NSDV appeared to target monocytes and/or macrophages.

  1. Electroacupuncture Suppresses Discrete Cue-Evoked Heroin-Seeking and Fos Protein Expression in the Nucleus Accumbens Core in Rats

    Directory of Open Access Journals (Sweden)

    Sheng Liu

    2012-01-01

    Full Text Available Relapse to drug seeking was studied using a rodent model of reinstatement induced by exposure to drug-related cues. Here, we used intravenous drug self-administration procedures in rats to further investigate the beneficial effects of electroacupuncture (EA on heroin-seeking behavior in a reinstatement model of relapse. We trained Sprague-Dawley rats to nose-poke for i.v. heroin either daily for 4 h or 25 infusions for 14 consecutive days. Then the rats were abstinent from heroin for two weeks. 2 Hz EA stimulation was conducted once daily for 14 days during heroin abstinence. We tested these animals for contextual and discrete cue-induced reinstatement of active responses. We also applied immunohistochemistry to detect Fos-positive nuclei in the nucleus accumbens (NACc core and shell after reinstatement test. We found that active responses elicited by both contextual cues and discrete cues were high in the rats trained with heroin than in saline controls. EA treatment significantly reduced active responses elicited by discrete cues. EA stimulation attenuated Fos expression in the core but not the shell of the NACc. Altogether, these results highlight the therapeutic benefit of EA in preventing relapse to drug addiction.

  2. Time-resolved quantitative proteomics implicates the core snRNP protein SmB together with SMN in neural trafficking.

    Science.gov (United States)

    Prescott, Alan R; Bales, Alexandra; James, John; Trinkle-Mulcahy, Laura; Sleeman, Judith E

    2014-02-15

    The biogenesis of splicing snRNPs (small nuclear ribonucleoproteins) is a complex process, beginning and ending in the nucleus of the cell but including key stages that take place in the cytoplasm. In particular, the SMN (survival motor neuron) protein complex is required for addition of the core Sm proteins to the snRNP. Insufficiency of SMN results in the inherited neurodegenerative condition, spinal muscular atrophy (SMA). Details of the physical organization of the cytoplasmic stages of snRNP biogenesis are unknown. Here, we use time-resolved quantitative proteomics to identify proteins that associate preferentially with either newly assembled or mature splicing snRNPs. We identified highly mobile SmB protein-trafficking vesicles in neural cells, which are dependent on the cellular levels of SMN and SmB for their morphology and mobility. We propose that these represent a family of related vesicles, some of which play a role in snRNP biogenesis and some that might play more diverse roles in cellular RNA metabolism.

  3. Hepatitis C virus core protein down-regulates p21(Waf1/Cip1 and inhibits curcumin-induced apoptosis through microRNA-345 targeting in human hepatoma cells.

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    Tzu-Yue Shiu

    Full Text Available BACKGROUND: Hepatitis C virus (HCV has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates p21(Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21(Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21(Waf1/Cip1 expression in human hepatoma cells. METHODS: Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay. RESULTS: HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed p21(Waf1/Cip1 gene expression through targeting its 3' untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21(Waf1/Cip1-targeting microRNA-345 in Huh7 cells. CONCLUSION AND SIGNIFICANCE: HCV core protein enhances the expression of microRNA-345 which then down-regulates p21(Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21(Waf1/Cip1 gene expression through miR-345 targeting.

  4. Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation

    Science.gov (United States)

    Ludgate, Laurie; Liu, Kuancheng; Luckenbaugh, Laurie; Streck, Nicholas; Eng, Stacey; Voitenleitner, Christian; Delaney, William E.

    2016-01-01

    ABSTRACT Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors. IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have

  5. Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7

    DEFF Research Database (Denmark)

    Galli, Andrea; Scheel, Troels K H; Prentoe, Jannick C

    2013-01-01

    JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A...

  6. Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

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    Aris Haryanto

    2015-10-01

    Full Text Available Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC and typically requires a specific nuclear localization signal (NLS. In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc, phosphorylated recombinant HBV core (rHBc and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

  7. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K

    2011-01-01

    To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A...... domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (¿40) or 25-aa (¿25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. ¿40 conferred efficient growth characteristics...... deletions in EGFP, while 2a(J6)¿40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A¿40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those...

  8. Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein

    Science.gov (United States)

    Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.

    2015-01-01

    ABSTRACT The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg2+-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg2+-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg2+ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm. PMID:25944861

  9. Crystal Structure of a Complex of Surfactant Protein D (SP-D) and Haemophilus influenzae Lipopolysaccharide Reveals Shielding of Core Structures in SP-D-Resistant Strains.

    Science.gov (United States)

    Clark, Howard W; Mackay, Rose-Marie; Deadman, Mary E; Hood, Derek W; Madsen, Jens; Moxon, E Richard; Townsend, J Paul; Reid, Kenneth B M; Ahmed, Abdul; Shaw, Amy J; Greenhough, Trevor J; Shrive, Annette K

    2016-05-01

    The carbohydrate recognition domains (CRDs) of lung collectin surfactant protein D (SP-D) recognize sugar patterns on the surface of lung pathogens and promote phagocytosis. Using Haemophilus influenzae Eagan strains expressing well-characterized lipopolysaccharide (LPS) surface structures of various levels of complexity, we show that bacterial recognition and binding by SP-D is inversely related to LPS chain extent and complexity. The crystal structure of a biologically active recombinant trimeric SP-D CRD complexed with a delipidated Eagan 4A LPS suggests that efficient LPS recognition by SP-D requires multiple binding interactions utilizing the three major ligand-binding determinants in the SP-D binding pocket, with Ca-dependent binding of inner-core heptose accompanied by interaction of anhydro-Kdo (4,7-anhydro-3-deoxy-d-manno-oct-2-ulosonic acid) with Arg343 and Asp325. Combined with enzyme-linked immunosorbent assays (ELISAs) and fluorescence-activated cell sorter (FACS) binding analyses, our results show that extended LPS structures previously thought to be targets for collectins are important in shielding the more vulnerable sites in the LPS core, revealing a mechanism by which pathogens with complex LPS extensions efficiently evade a first-line mucosal innate immune defense. The structure also reveals for the first time the dominant form of anhydro-Kdo.

  10. Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro▿

    Science.gov (United States)

    Boregowda, Rajeev K.; Lin, Li; Zhu, Qin; Tian, Fang; Hu, Jianming

    2011-01-01

    Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a trans-complementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming. PMID:21593164

  11. Ice cores

    DEFF Research Database (Denmark)

    Svensson, Anders

    2014-01-01

    Ice cores from Antarctica, from Greenland, and from a number of smaller glaciers around the world yield a wealth of information on past climates and environments. Ice cores offer unique records on past temperatures, atmospheric composition (including greenhouse gases), volcanism, solar activity......, dustiness, and biomass burning, among others. In Antarctica, ice cores extend back more than 800,000 years before present (Jouzel et al. 2007), whereas. Greenland ice cores cover the last 130,000 years...

  12. Ice cores

    DEFF Research Database (Denmark)

    Svensson, Anders

    2014-01-01

    Ice cores from Antarctica, from Greenland, and from a number of smaller glaciers around the world yield a wealth of information on past climates and environments. Ice cores offer unique records on past temperatures, atmospheric composition (including greenhouse gases), volcanism, solar activity......, dustiness, and biomass burning, among others. In Antarctica, ice cores extend back more than 800,000 years before present (Jouzel et al. 2007), whereas. Greenland ice cores cover the last 130,000 years...

  13. Modeling of the catalytic core of Arabidopsis thaliana Dicer-like 4 protein and its complex with double-stranded RNA.

    Science.gov (United States)

    Mickiewicz, Agnieszka; Sarzyńska, Joanna; Miłostan, Maciej; Kurzyńska-Kokorniak, Anna; Rybarczyk, Agnieszka; Łukasiak, Piotr; Kuliński, Tadeusz; Figlerowicz, Marek; Błażewicz, Jacek

    2017-02-01

    Plant Dicer-like proteins (DCLs) belong to the Ribonuclease III (RNase III) enzyme family. They are involved in the regulation of gene expression and antiviral defense through RNA interference pathways. A model plant, Arabidopsis thaliana encodes four DCL proteins (AtDCL1-4) that produce different classes of small regulatory RNAs. Our studies focus on AtDCL4 that processes double-stranded RNAs (dsRNAs) into 21 nucleotide trans-acting small interfering RNAs. So far, little is known about the structures of plant DCLs and the complexes they form with dsRNA. In this work, we present models of the catalytic core of AtDCL4 and AtDCL4-dsRNA complex constructed by computational methods. We built a homology model of the catalytic core of AtDCL4 comprising Platform, PAZ, Connector helix and two RNase III domains. To assemble the AtDCL4-dsRNA complex two modeling approaches were used. In the first method, to establish conformations that allow building a consistent model of the complex, we used Normal Mode Analysis for both dsRNA and AtDCL4. The second strategy involved template-based approach for positioning of the PAZ domain and manual arrangement of the Connector helix. Our results suggest that the spatial orientation of the Connector helix, Platform and PAZ relative to the RNase III domains is crucial for measuring dsRNA of defined length. The modeled complexes provide information about interactions that may contribute to the relative orientations of these domains and to dsRNA binding. All these information can be helpful for understanding the mechanism of AtDCL4-mediated dsRNA recognition and binding, to produce small RNA of specific size.

  14. Effect of a buried ion pair in the hydrophobic core of a protein: An insight from constant pH molecular dynamics study.

    Science.gov (United States)

    Pathak, Arup K

    2015-03-01

    Constant pH molecular dynamics (CpHMD) is a commonly used sampling method, which incorporates the coupling of conformational flexibility and protonation state of a protein during the simulation by using pH as an external parameter. The effects on the structure and stability of a hyperstable variant of staphylococcal nuclease (Δ+PHS) protein of an artificial charge pair buried in its hydrophobic core are investigated by applying both CpHMD and accelerated molecular dynamics coupled with constant pH (CpHaMD) methods. Generalized Born electrostatics is used to model the solvent water. Two sets of starting coordinates of V23E/L36K variant of Δ+PHS, namely, Maestro generated coordinates from Δ+PHS and crystal structure coordinates of the same are considered for detail investigations. On the basis of root mean square displacement (RMSD) and root mean square fluctuations (RMSF) calculations, it is observed that this variant is stable over a wide range of pH. The calculated pKa values for aspartate and glutamate residues based on both CpHMD and CpHaMD simulations are consistent with the reported experimental values (within ± 0.5 to ± 1.5 pH unit), which clearly indicates that the local chemical environment of the carboxylic acids in V23E/L36K variant are comparable to the parent form. The strong salt bridge interaction between the mutated pair, E23/K36 and additional hydrogen bonds formed in the V23E/L36K variant, may help to compensate for the unfavorable self-energy experienced by the burial of these residues in the hydrophobic core. However, from RMSD, RMSF, and pKa analysis, no significant change in the global conformation of V23E/L36K variant with respect to the parent form, Δ+PHS is noticed.

  15. Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli

    Directory of Open Access Journals (Sweden)

    Gómez-Lim Miguel A

    2009-02-01

    Full Text Available Abstract Background Gag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB when fused to p24. Results A fusion between ricin toxin B subunit and p24 HIV (RTB/p24 was expressed in E. coli. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin. Conclusion In this work, we report the expression in E. coli of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when

  16. Age-dependent preferential dense-core vesicle exocytosis in neuroendocrine cells revealed by newly developed monomeric fluorescent timer protein.

    Science.gov (United States)

    Tsuboi, Takashi; Kitaguchi, Tetsuya; Karasawa, Satoshi; Fukuda, Mitsunori; Miyawaki, Atsushi

    2010-01-01

    Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K(+)] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

  17. Variability within a pea core collection of LEAM and HSP22, two mitochondrial seed proteins involved in stress tolerance.

    Science.gov (United States)

    Avelange-Macherel, Marie-Hélène; Payet, Nicole; Lalanne, David; Neveu, Martine; Tolleter, Dimitri; Burstin, Judith; Macherel, David

    2015-07-01

    LEAM, a late embryogenesis abundant protein, and HSP22, a small heat shock protein, were shown to accumulate in the mitochondria during pea (Pisum sativum L.) seed development, where they are expected to contribute to desiccation tolerance. Here, their expression was examined in seeds of 89 pea genotypes by Western blot analysis. All genotypes expressed LEAM and HSP22 in similar amounts. In contrast with HSP22, LEAM displayed different isoforms according to apparent molecular mass. Each of the 89 genotypes harboured a single LEAM isoform. Genomic and RT-PCR analysis revealed four LEAM genes differing by a small variable indel in the coding region. These variations were consistent with the apparent molecular mass of each isoform. Indels, which occurred in repeated domains, did not alter the main properties of LEAM. Structural modelling indicated that the class A α-helix structure, which allows interactions with the mitochondrial inner membrane in the dry state, was preserved in all isoforms, suggesting functionality is maintained. The overall results point out the essential character of LEAM and HSP22 in pea seeds. LEAM variability is discussed in terms of pea breeding history as well as LEA gene evolution mechanisms.

  18. The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

    Science.gov (United States)

    Roth, Braden M; Ishimaru, Daniella; Hennig, Mirko

    2013-09-13

    MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

  19. Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

    Science.gov (United States)

    Anand, Sanjeev K; Gaba, Amit; Singh, Jaswant; Tikoo, Suresh K

    2014-02-01

    Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

  20. The snRNP core protein SmB and tissue-specific SmN protein are differentially distributed between snRNP particles.

    Science.gov (United States)

    Huntriss, J D; Latchman, D S; Williams, D G

    1993-01-01

    The SmN protein is a tissue specific component of the small nuclear ribonucleoprotein particle which is closely related to the ubiquitously expressed SmB protein but is expressed only in the brain and heart. To investigate the function of SmN, its localisation within different snRNP particles was investigated using a range of anti-snRNP monoclonal antibodies. SmN and SmB were found to exhibit different patterns of association with snRNP particles in two cell lines, ND7 and F9 which express SmN. In both cases, SmN was found to be present in the U-2 snRNP but was excluded from the U-1 snRNPs whereas SmB was present in both U-1 and U-2 snRNPs. Data from transfected 3T3 mouse fibroblasts cell lines artificially expressing a low level of SmN also confirm this observation. In contrast, SmN was found to be an integral component of both the U-1 and U-2 snRNPs in both 3T3 cells artificially expressing high levels of SmN and in adult rat brain which has a naturally high level of SmN expression. Taken together, the results suggest that the pre-U1 snRNP particle has a lower affinity for SmN than for SmB. Thus, SmN expressed at low levels incorporates into U2, but SmN expressed at high levels incorporates into both U1 and U2 snRNPs and replaces SmB. The significance of these effects is discussed in terms of the potential role played by SmN in constitutive and alternative splicing pathways in neuronal cells. Images PMID:8371979

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

    Directory of Open Access Journals (Sweden)

    Lijun Chao

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

  2. Expression and Characterization of an Ice Binding Protein from a Bacterium Isolated at a Depth of 3,519 Meters in the Vostok Ice Core, Antarctica

    Science.gov (United States)

    Christner, B. C.; Achberger, A.; Brox, T. I.; Skidmore, M. L.

    2011-12-01

    The cryopreservation of microorganisms in ancient glacial ice is possible if lethal levels of macromolecular damage are not incurred and cellular integrity is not compromised via intracellular ice formation or recrystallization. There are numerous examples of cold-adapted species that prevent or limit ice crystal growth by producing ice-binding proteins (IBP). Previously, a bacterium (isolate 3519-10; Flavobacteriaceae family) recovered from a depth of 3,519 meters below the surface in the Vostok ice core was shown to produce and secrete an IBP that inhibits the recrystallization of ice. To explore the phenotypic advantage that IBPs confer to ice-entrapped cells, experiments were designed to examine the expression of 3519-10's IBP gene and protein at different temperatures, assess the effect of the IBP on bacterial viability in ice, and determine how the IBP influences the physical structure of the ice. Total RNA isolated from aerobic cultures grown at temperatures between 4C to 25C and analyzed by reverse transcription-PCR indicated constitutive expression of the IBP gene. Additionally, SDS-PAGE analysis of 3519-10's extracellular proteins revealed a polypeptide corresponding to the predicted size of the 54 kDa IBP at all temperatures tested. The total extracellular protein fraction was subsequently used in assays with Escherichia coli to examine the effect of the IBP on bacterial survival in warm ice (-5C) and after freeze-thaw cycling. In the presence of 100 μg mL-1 of extracellular protein from 3519-10, the survival of E. coli was increased by greater than 100-fold; however, the survival of E. coli suspensions containing the same concentration of bovine serum albumin was not significantly different than controls (pcells and non-IBP producing bacteria, and 10 times as many crystals as in particle-free deionized water. Presumably, the effect that the IBP has on bacterial viability and ice crystal structure is due to its activity as an inhibitor of ice

  3. Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

    Science.gov (United States)

    Yang, Ching-Chun; Huang, Er-Yi; Li, Hung-Cheng; Su, Pei-Yi; Shih, Chiaho

    2014-01-01

    Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

  4. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A.

    Science.gov (United States)

    Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K; Meuleman, Philip; Serre, Stephanie B N; Lademann, Jacob B; Ghanem, Lubna; Scheel, Troels K H; Leroux-Roels, Geert; Bukh, Jens

    2011-09-01

    To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (Δ40) or 25-aa (Δ25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. Δ40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with Δ40 or Δ25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFPΔ40 acquired various deletions in EGFP, while 2a(J6)Δ40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5AΔ40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log(10) FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.

  5. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  6. Comparative Analysis of the 15.5kD Box C/D snoRNP Core Protein in the Primitive Eukaryote Giardia lamblia Reveals Unique Structural and Functional Features

    Energy Technology Data Exchange (ETDEWEB)

    Biswas, Shyamasri; Buhrman, Greg; Gagnon, Keith; Mattos, Carla; Brown, II, Bernard A.; Maxwell, E. Stuart (NCSU); (UTSMC)

    2012-07-11

    Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 {angstrom}. The Giardia 15.5kD protein exhibits the typical {alpha}-{beta}-{alpha} sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.

  7. Transformer core

    NARCIS (Netherlands)

    Mehendale, A.; Hagedoorn, Wouter; Lötters, Joost Conrad

    2010-01-01

    A transformer core includes a stack of a plurality of planar core plates of a magnetically permeable material, which plates each consist of a first and a second sub-part that together enclose at least one opening. The sub-parts can be fitted together via contact faces that are located on either side

  8. Transformer core

    NARCIS (Netherlands)

    Mehendale, A.; Hagedoorn, Wouter; Lötters, Joost Conrad

    2008-01-01

    A transformer core includes a stack of a plurality of planar core plates of a magnetically permeable material, which plates each consist of a first and a second sub-part that together enclose at least one opening. The sub-parts can be fitted together via contact faces that are located on either side

  9. A network of HSPG core proteins and HS modifying enzymes regulates netrin-dependent guidance of D-type motor neurons in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Stephan Gysi

    Full Text Available Heparan sulfate proteoglycans (HSPGs are proteins with long covalently attached sugar side chains of the heparan sulfate (HS type. Depending on the cellular context HS chains carry multiple structural modifications such as sulfate residues or epimerized sugars allowing them to bind to a wide range of molecules. HSPGs have been found to play extremely diverse roles in animal development and were shown to interact with certain axon guidance molecules. In this study we describe the role of the Caenorhabditis elegans HSPG core proteins Syndecan (SDN-1 and Glypican (LON-2 and the HS modifying enzymes in the dorsal guidance of D-type motor axons, a process controlled mainly by the conserved axon guidance molecule UNC-6/Netrin. Our genetic analysis established the specific HS code relevant for this axon guidance event. Using two sensitized genetic backgrounds, we isolated novel components influencing D-type motor axon guidance with a link to HSPGs, as well as new alleles of several previously characterized axon guidance genes. Interestingly, the dorsal axon guidance defects induced by mutations in zfp-1 or lin-35 depended on the transgene oxIs12 used to visualize the D-type motor neurons. oxIs12 is a large multi-copy transgene that enlarges the X chromosome by approximately 20%. In a search for genes with a comparable phenotype we found that a mutation in the known dosage compensation gene dpy-21 showed similar axon guidance defects as zfp-1 or lin-35 mutants. Thus, derepression of genes on X, where many genes relevant for HS dependent axon guidance are located, might also influence axon guidance of D-type motor neurons.

  10. A Network of HSPG Core Proteins and HS Modifying Enzymes Regulates Netrin-Dependent Guidance of D-Type Motor Neurons in Caenorhabditis elegans

    Science.gov (United States)

    Gysi, Stephan; Rhiner, Christa; Flibotte, Stephane; Moerman, Donald G.; Hengartner, Michael O.

    2013-01-01

    Heparan sulfate proteoglycans (HSPGs) are proteins with long covalently attached sugar side chains of the heparan sulfate (HS) type. Depending on the cellular context HS chains carry multiple structural modifications such as sulfate residues or epimerized sugars allowing them to bind to a wide range of molecules. HSPGs have been found to play extremely diverse roles in animal development and were shown to interact with certain axon guidance molecules. In this study we describe the role of the Caenorhabditis elegans HSPG core proteins Syndecan (SDN-1) and Glypican (LON-2) and the HS modifying enzymes in the dorsal guidance of D-type motor axons, a process controlled mainly by the conserved axon guidance molecule UNC-6/Netrin. Our genetic analysis established the specific HS code relevant for this axon guidance event. Using two sensitized genetic backgrounds, we isolated novel components influencing D-type motor axon guidance with a link to HSPGs, as well as new alleles of several previously characterized axon guidance genes. Interestingly, the dorsal axon guidance defects induced by mutations in zfp-1 or lin-35 depended on the transgene oxIs12 used to visualize the D-type motor neurons. oxIs12 is a large multi-copy transgene that enlarges the X chromosome by approximately 20%. In a search for genes with a comparable phenotype we found that a mutation in the known dosage compensation gene dpy-21 showed similar axon guidance defects as zfp-1 or lin-35 mutants. Thus, derepression of genes on X, where many genes relevant for HS dependent axon guidance are located, might also influence axon guidance of D-type motor neurons. PMID:24066155

  11. Mast cell differentiation and activation is closely linked to expression of genes coding for the serglycin proteoglycan core protein and a distinct set of chondroitin sulfate and heparin sulfotransferases.

    Science.gov (United States)

    Duelli, Annette; Rönnberg, Elin; Waern, Ida; Ringvall, Maria; Kolset, Svein O; Pejler, Gunnar

    2009-12-01

    Serglycin (SG) proteoglycan consists of a small core protein to which glycosaminoglycans of chondroitin sulfate or heparin type are attached. SG is crucial for maintaining mast cell (MC) granule homeostasis through promoting the storage of various basic granule constituents, where the degree of chondroitin sulfate/heparin sulfation is essential for optimal SG functionality. However, the regulation of the SG core protein expression and of the various chondroitin sulfate/heparin sulfotransferases during MC differentiation and activation are poorly understood. Here we addressed these issues and show that expression of the SG core protein, chondroitin 4-sulfotransferase (C4ST)-1, and GalNAc(4S)-6-O-sulfotransferase (GalNAc4S6ST) are closely linked to MC maturation. In contrast, the expression of chondroitin 6-sulfotransferase correlated negatively with MC maturation. The expression of N-deacetylase/N-sulfotransferase (NDST)-2, a key enzyme in heparin synthesis, also correlated strongly with MC maturation, whereas the expression of the NDST-1 isoform was approximately equal at all stages of maturation. MC activation by either calcium ionophore or IgE ligation caused an up-regulated expression of the SG core protein, C4ST-1, and GalNAc4S6ST, accompanied by increased secretion of chondroitin sulfate as shown by biosynthetic labeling experiments. In contrast, NDST-2 was down-regulated after MC activation, suggesting that MC activation modulates the nature of the glycosaminoglycan chains attached to the SG core protein. Taken together, these data show that MC maturation is associated with the expression of a distinct signature of genes involved in SG proteoglycan synthesis, and that MC activation modulates their expression.

  12. Increased outer arm and core fucose residues on the N-glycans of mutated alpha-1 antitrypsin protein from alpha-1 antitrypsin deficient individuals.

    Science.gov (United States)

    McCarthy, Cormac; Saldova, Radka; O'Brien, M Emmet; Bergin, David A; Carroll, Tomás P; Keenan, Joanne; Meleady, Paula; Henry, Michael; Clynes, Martin; Rudd, Pauline M; Reeves, Emer P; McElvaney, Noel G

    2014-02-01

    Alpha-1 antitrypsin (AAT) is the major physiological inhibitor of a range of serine proteases, and in the lung, it maintains a protease-antiprotease balance. AAT deficiency (AATD) is an autosomal co-dominant condition with the Z mutation being the most common cause. Individuals homozygous for Z (PiZZ) have low levels of circulating mutant Z-AAT protein leading to premature emphysematous lung disease. Extensive glycoanalysis has been performed on normal AAT (M-AAT) from healthy individuals and the importance of glycosylation in affecting the immune modulatory roles of AAT is documented. However, no glycoanalysis has been carried out on Z-AAT from deficient individuals to date. In this study, we investigate whether the glycans present on Z-AAT differ to those found on M-AAT from healthy controls. Plasma AAT was purified from 10 individuals: 5 AATD donors with the PiZZ phenotype and 5 PiMM healthy controls. Glycoanalysis was performed employing N-glycan release, exoglycosidase digestion and UPLC analysis. No difference in branched glycans was identified between AATD and healthy controls. However, a significant increase in both outer arm (α1-3) (p = 0.04) and core (α1-6) fucosylated glycans (p < 0.0001) was found on Z-AAT compared to M-AAT. This study has identified increased fucosylation on N-glycans of Z-AAT indicative of ongoing inflammation in AATD individuals with implications for early therapeutic intervention.

  13. Hamming distance geometry of a protein conformational space. Application to the clustering of a 4 ns molecular dynamics trajectory of the HIV-1 integrase catalytic core

    CERN Document Server

    Laboulais, C; Le Bret, M; Gabarro-Arpa, J; Laboulais, Cyril; Ouali, Mohammed; Bret, Marc Le; Gabarro-Arpa, Jacques

    2001-01-01

    Protein structures can be encoded into binary sequences, these are used to define a Hamming distance in conformational space: the distance between two different molecular conformations is the number of different bits in their sequences. Each bit in the sequence arises from a partition of conformational space in two halves. Thus, the information encoded in the binary sequences is also used to characterize the regions of conformational space visited by the system. We apply this distance and their associated geometric structures, to the clustering and analysis of conformations sampled during a 4 ns molecular dynamics simulation of the HIV-1 integrase catalytic core. The cluster analysis of the simulation shows a division of the trajectory into two segments of 2.6 and 1.4 ns length, which are qualitatively different: the data points to the fact that equilibration is only reached at the end of the first segment. Some length of the paper is devoted to compare the Hamming distance to the r.m.s. deviation measure. Th...

  14. Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

    Science.gov (United States)

    Mitchell, Kathryn J.; Pinton, Paolo; Varadi, Aniko; Tacchetti, Carlo; Ainscow, Edward K.; Pozzan, Tullio; Rizzuto, Rosario; Rutter, Guy A.

    2001-01-01

    The role of dense core secretory vesicles in the control of cytosolic-free Ca2+ concentrations ([Ca2+]c) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2–synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet β-cells: (a) increases in [Ca2+]c cause a prompt increase in intravesicular-free Ca2+ concentration ([Ca2+]SV), which is mediated by a P-type Ca2+-ATPase distinct from the sarco(endo) plasmic reticulum Ca2+-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca2+ pumps; (b) steady state Ca2+ concentrations are 3–5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca2+; (c) inositol (1,4,5) trisphosphate has no impact on [Ca2+]SV in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca2+]SV. Thus, secretory vesicles represent a dynamic Ca2+ store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca2+-induced Ca2+ release from vesicles docked at the plasma membrane could participate in triggering exocytosis. PMID:11571310

  15. Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.

    Science.gov (United States)

    Mitchell, K J; Pinton, P; Varadi, A; Tacchetti, C; Ainscow, E K; Pozzan, T; Rizzuto, R; Rutter, G A

    2001-10-01

    The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

  16. Expression and partial characterization of an ice binding protein from a bacterium isolated at a depth of 3,519 meters in the Vostok ice core, Antarctica

    Directory of Open Access Journals (Sweden)

    Amanda Marie Achberger

    2011-12-01

    Full Text Available Cryopreservation of microorganisms in ancient glacial ice is possible if lethal levels of macromolecular damage are not incurred and cellular integrity is not compromised via intracellular ice formation or recrystallization. Previously, a bacterium (isolate 3519-10 recovered from a depth of 3,519 meters below the surface in the Vostok ice core was shown to secrete an IBP that inhibits the recrystallization of ice. To explore the advantage that IBPs confer to ice-entrapped cells, experiments were designed to examine the expression of 3519-10’s IBP gene and protein at different temperatures, assess the effect of the IBP on bacterial viability in ice, and determine how the IBP influences the physical structure of the ice. Total RNA isolated from cultures grown between 4 to 25⁰C and analyzed by reverse transcription-PCR indicated constitutive expression of the IBP gene. SDS-PAGE analysis of 3519-10’s extracellular proteins also revealed a polypeptide of the predicted size of the 54 kDa IBP at all temperatures tested. In the presence of 100 µg mL-1 of extracellular protein from 3519-10, the survival of Escherichia coli was increased by greater than 34-fold after freeze-thaw cycling. Microscopic analysis of ice formed in the presence of the IBP indicated that per mm2 field of view, there were ~5 times as many crystals as in ice formed in the presence of washed 3519-10 cells and non-IBP producing bacteria, and ~10 times as many crystals as in filtered deionized water. Presumably, the effect that the IBP has on bacterial viability and ice crystal structure is due to its activity as an inhibitor of ice recrystallization. A myriad of molecular adaptations are likely to play a role in bacterial persistence under frozen conditions, but the ability of 3519-10’s IBP to control ice crystal structure, and thus the liquid vein network within the ice, may provide one explanation for its successful survival deep within the Antarctic ice sheet for

  17. Hepatitis C virus core protein increases Snail expression and induces epithelial-mesenchymal transition through the signal transducer and activator of transcription 3 pathway in hepatoma cells.

    Science.gov (United States)

    Zhou, Jia-Jia; Meng, Zhe; He, Xiao-Yu; Cheng, Di; Ye, Hui-Lin; Deng, Xiao-Geng; Chen, Ru-Fu

    2017-05-01

    Aberrant expression of Snail, a mediator of epithelial-mesenchymal transition (EMT), is crucial for cancer invasiveness and metastasis. Although hepatitis C virus (HCV) core protein has been implicated in hepatocarcinogenesis, the relationship between HCV core and Snail expression has not been clarified. HepG2 and Huh7 stable cell lines were established by transfection with pcDNA-HCVc. HepG2-HCVc and Huh7-HCVc cells were co-administered with AG490. Cell migration and invasiveness were tested. STAT3 and Snail expression was analyzed by Real-time PCR and Western blot. We found that HCV core is capable of increasing Snail expression and inducing EMT in hepatoma cells. HCV core-induced Snail expression was accompanied by activation of signal transducer and activator of transcription 3 (STAT3), inhibition of STAT3 abrogated HCV core-induced Snail expression and EMT. Furthermore, chromatin immunoprecipitation showed that phosphorylated STAT3 directly binds to the Snail promoter. Collectively, these results suggest that HCV core would play a role in hepatocellular carcinoma invasiveness and metastasis by activating the STAT3 pathway, increasing Snail expression and subsequently triggering EMT. These findings would advance the understanding of HCV-mediated invasiveness and metastasis, and might provide a new potential therapeutic target for HCV-related hepatocellular carcinoma. © 2016 The Japan Society of Hepatology.

  18. Ice Cores

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Records of past temperature, precipitation, atmospheric trace gases, and other aspects of climate and environment derived from ice cores drilled on glaciers and ice...

  19. Core BPEL

    DEFF Research Database (Denmark)

    Hallwyl, Tim; Højsgaard, Espen

    extensions. Combined with the fact that the language definition does not provide a formal semantics, it is an arduous task to work formally with the language (e.g. to give an implementation). In this paper we identify a core subset of the language, called Core BPEL, which has fewer and simpler constructs......, does not allow omissions, and does not contain ignorable elements. We do so by identifying syntactic sugar, including default values, and ignorable elements in WS-BPEL. The analysis results in a translation from the full language to the core subset. Thus, we reduce the effort needed for working...... formally with WS-BPEL, as one, without loss of generality, need only consider the much simpler Core BPEL. This report may also be viewed as an addendum to the WS-BPEL standard specification, which clarifies the WS-BPEL syntax and presents the essential elements of the language in a more concise way...

  20. Core BPEL

    DEFF Research Database (Denmark)

    Hallwyl, Tim; Højsgaard, Espen

    extensions. Combined with the fact that the language definition does not provide a formal semantics, it is an arduous task to work formally with the language (e.g. to give an implementation). In this paper we identify a core subset of the language, called Core BPEL, which has fewer and simpler constructs......, does not allow omissions, and does not contain ignorable elements. We do so by identifying syntactic sugar, including default values, and ignorable elements in WS-BPEL. The analysis results in a translation from the full language to the core subset. Thus, we reduce the effort needed for working...... formally with WS-BPEL, as one, without loss of generality, need only consider the much simpler Core BPEL. This report may also be viewed as an addendum to the WS-BPEL standard specification, which clarifies the WS-BPEL syntax and presents the essential elements of the language in a more concise way...

  1. Core benefits

    National Research Council Canada - National Science Library

    Keith, Brian W

    2010-01-01

    This SPEC Kit explores the core employment benefits of retirement, and life, health, and other insurance -benefits that are typically decided by the parent institution and often have significant governmental regulation...

  2. Interaction of a C-terminal Truncated Hepatitis C Virus Core Protein with Plasmid DNA Vaccine Leads to in vitro Assembly of Heterogeneous Virus-like Particles

    Directory of Open Access Journals (Sweden)

    Nelson Acosta-Rivero

    2005-01-01

    Full Text Available Recently, it has been shown that HCV core proteins (HCcAg with C-terminal deletions assemble in vitro into virus-like particles (VLPs in the presence of structured RNA molecules. Results presented in this work showed that a truncated HCcAg variant covering the first 120 aa (HCcAg.120 with a 32 aa N-terminal fusion peptide (6xHistag-XpressTMepitope interacts with plasmid DNA vaccine. Interestingly, the buoyant density of VLPs containing HCcAg.120 in CsCl gradients changed from 1.15-1,17 g mLˉ1 to 1.30-1.34 g mLˉ1 after addition of plasmid DNA to assembly reactions. In addition, a delay in electrophoretic mobility of HCcAg.120-plasmid samples on agarose gels was observed indicating a direct interaction between VLPs and nucleic acids. Remarkably, addition of either plasmid DNA or tRNA to assembly reactions leaded to heterogeneous and larger VLPs formation than those observed in HCcAg.120 assembly reactions. VLPs containing HCcAg.120 induced a specific IgG antibodies in mice that reacted with hepatocytes from HCV-infected patients. VLPs obtained in this work would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure. Besides, the capacity of particles containing HCcAg.120 to interact with nucleic acids could be used in the development of DNA vaccines and viral vectors based on these particles.

  3. DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic.

    Directory of Open Access Journals (Sweden)

    Ying Yin

    Full Text Available BACKGROUND: Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4 primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV clearance. PRINCIPAL FINDINGS: Plasmids expressing HBV core protein (HBcAg or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc, CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI of pAAV/HBV1.2. HBV surface antigen (HBsAg and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance. CONCLUSION: Viral clearance could be efficiently achieved by Th1/Th2-balanced

  4. Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database

    Energy Technology Data Exchange (ETDEWEB)

    Omenn, Gilbert; States, David J.; Adamski, Marcin; Blackwell, Thomas W.; Menon, Rajasree; Hermjakob, Henning; Apweiler, Rolf; Haab, Brian B.; Simpson, Richard; Eddes, James; Kapp, Eugene; Moritz, Rod; Chan, Daniel W.; Rai, Alex J.; Admon, Arie; Aebersold, Ruedi; Eng, Jimmy K.; Hancock, William S.; Hefta, Stanley A.; Meyer, Helmut; Paik, Young-Ki; Yoo, Jong-Shin; Ping, Peipei; Pounds, Joel G.; Adkins, Joshua N.; Qian, Xiaohong; Wang, Rong; Wasinger, Valerie; Wu, Chi Yue; Zhao, Xiaohang; Zeng, Rong; Archakov, Alexander; Tsugita, Akira; Beer, Ilan; Pandey, Akhilesh; Pisano, Michael; Andrews, Philip; Tammen, Harald; Speicher, David W.; Hanash, Samir M.

    2005-08-13

    HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anticoagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics. med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan

  5. Core Java

    CERN Document Server

    Horstmann, Cay S

    2013-01-01

    Fully updated to reflect Java SE 7 language changes, Core Java™, Volume I—Fundamentals, Ninth Edition, is the definitive guide to the Java platform. Designed for serious programmers, this reliable, unbiased, no-nonsense tutorial illuminates key Java language and library features with thoroughly tested code examples. As in previous editions, all code is easy to understand, reflects modern best practices, and is specifically designed to help jumpstart your projects. Volume I quickly brings you up-to-speed on Java SE 7 core language enhancements, including the diamond operator, improved resource handling, and catching of multiple exceptions. All of the code examples have been updated to reflect these enhancements, and complete descriptions of new SE 7 features are integrated with insightful explanations of fundamental Java concepts.

  6. Development of a water-in-oil-in-water multiple emulsion system integrating biomimetic aqueous-core lipid nanodroplets for protein entity stabilization. Part II: process and product characterization.

    Science.gov (United States)

    Glasser, Cássia A; Vila, Marta M D C; Pereira, Júlio C; Chaud, Marco V; Oliveira Júnior, José M; Tubino, Matthieu; Balcão, Victor M

    2016-12-01

    The aqueous-core enclosed in lipid nanoballoons integrating multiple emulsions of the type water-in-oil-in-water mimic, at least in theory, the environment within viable cells, thus being suitable for housing hydrophilic protein entities such as bioactive proteins, peptides and bacteriophage particles. This study reports a complete physicochemical characterization of optimized biomimetic aqueous-core lipid nanoballoons housing hydrophilic (BSA) protein entities, evolved from a statistical 2(3)×3(1) factorial design study (three variables at two levels and one variable at three levels) that was the subject of the first paper of a series of three, aiming at complete stabilization of the three-dimensional structure of protein entities attempted via housing the said molecular entities within biomimetic aqueous-core lipid nanoballoons integrating a multiple (W/O/W) emulsion. The statistical factorial design followed led to the production of an optimum W/O/W multiple emulsion possessing quite homogeneous particles with an average hydrodynamic size of (186.2 ± 2.6) nm and average Zeta potential of (-36.5 ± 0.9) mV, and exhibiting a polydispersity index of 0.206 ± 0.014. Additionally, the results obtained for the diffusion coefficient of the lipid nanoballoons integrating the optimized W/O/W multiple emulsion were comparable and of the same order of magnitude (10(-12) m(2) s(-1)) as those published by other authors since, typically, diffusion coefficients for molecules range from 10(-10) to 10(-7) m(2) s(-1), but diffusion coefficients for nanoparticles are typically of the order of magnitude of 10(-12) m(2) s(-1).

  7. An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

    DEFF Research Database (Denmark)

    Hägglund, Per; Matthiesen, R.; Elortza, F.;

    2007-01-01

    between the two N-acetylglucosamine (GlcNAc) residues in the conserved N-glycan core structure, leaving single GlcNAc residues with putative fucosyl side chains attached to the peptide. To enable digestion of complex and hybrid type N-glycans, a number of exoglycosidases (β-galactosidase, neuraminidase...

  8. Functional evolution of the photolyase/cryptochrome protein family: importance of the C terminus of mammalian CRY1 for circadian core oscillator performance.

    NARCIS (Netherlands)

    I. Chaves (Ines); K. Yagita (Kazuhiro); S. Barnhoorn (Sander); H. Okamura (Hitoshi); G.T.J. van der Horst (Gijsbertus); F. Tamanini (Filippo)

    2006-01-01

    textabstractCryptochromes (CRYs) are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of th

  9. Reduction of protein adsorption to a solid surface by a coating composed of polymeric micelles with a glass-like core

    NARCIS (Netherlands)

    Hofs, B.; Brzozowska, A.; de Keizer, A.; Norde, W.; Stuart, Martien A. Cohen

    2008-01-01

    Adsorption studies by optical reflectometry show that complex coacervate core micelles (C3Ms) composed of poly([4-(2-amino-ethylthio)-butylene]hydrochloride)(49)-block-poly(ethylene oxide)(212) and poly([4-(2carboxy-ethylthio)-butylene] sodium salt)(47)-block-poly(ethylene oxide)(212) adsorb in equa

  10. The core 2 beta-1,6-N-acetylglucosaminyltransferase-M encoded by bovine herpesvirus 4 is not essential for virus replication despite contributing to post-translational modifications of structural proteins.

    Science.gov (United States)

    Markine-Goriaynoff, Nicolas; Gillet, Laurent; Karlsen, Odd A; Haarr, Lars; Minner, Frédéric; Pastoret, Paul-Pierre; Fukuda, Minoru; Vanderplasschen, Alain

    2004-02-01

    The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only virus gene known to date that encodes a homologue of the cellular core 2 beta-1,6-N-acetylglucosaminyltransferase-mucine type (C2GnT-M). Recently, our phylogenetic study revealed that the Bo17 gene has been acquired from an ancestor of the African buffalo around 1.5 million years ago. Despite this recent origin, the Bo17 sequence has spread to fixation in the virus population possibly by natural selection. Supporting the latter hypothesis, it has been shown by our group for the V. test strain that Bo17 is expressed during BoHV-4 replication in vitro, and that Bo17 expression product (pBo17) has all three enzymic activities exhibited by cellular C2GnT-M, i.e. core 2, core 4 and I branching activities. In the present study, firstly it was investigated whether encoding a functional C2GnT-M is a general property of BoHV-4 strains. Analysis of nine representative strains of the BoHV-4 species revealed that all of them express the Bo17 gene and the associated core 2 branching activity during virus replication in vitro. Secondly, in order to investigate the roles of Bo17, its kinetic class of expression was analysed and a deleted recombinant strain was produced. These experiments revealed that Bo17 is expressed as an early gene which is not essential for virus replication in vitro. However, comparison of the structural proteins, produced by the wild-type, the revertant and the deleted viruses, by 2D gels demonstrated that pBo17 contributes to the post-translational modifications of structural proteins. Possible roles of Bo17 in vivo are discussed.

  11. Effect of recombinant human bone morphogenetic protein 2/poly-lactide-co-glycolic acid (rhBMP-2/PLGA) with core decompression on repair of rabbit femoral head necrosis

    Institute of Scientific and Technical Information of China (English)

    Zhao-Xun Pan; Hong-Xin Zhang; Ye-Xin Wang; Long-Di Zhai; Wei Du

    2014-01-01

    Objective:To observe the effect of recombinant human bone morphogenetic protein 2/poly-lactide-co-glycolic acid (rhBMP-2/PLGA) with core decompression on repair of rabbit femoral head necrosis. Methods: Bilateral femoral head necrosis models of rabbit were established by steroid injection. A total of 48 rabbits (96 femoral head necrosis) were randomly divided into 4 groups: Group A, control group with12 rabbits, 24 femoral head necrosis;Group B, treated with rhBMP-2/PLGA implantation after core depression, with 12 rabbits, 24 femoral head necrosis;Group C, treated with rhBMP-2 implantation after core depression, with 12 rabbits, 24 femoral head necrosis;Group D treated with core depression group without implantation, with 12 rabbits, 24 femoral head necrosis. All animals were sacrificed after 12 weeks. The ability of repairing bone defect was evaluated by X-ray radiograph. Bone mineral density analysis of the defect regions were used to evaluate the level of ossification. The morphologic change and bone formation was assessed by HE staining. The angiogenesis was evaluated by VEGF immunohistochemistry. Results: The osteogenetic ability and quality of femoral head necrosis in group B were better than those of other groups after 12 weeks by X-ray radiograph and morphologic investigation. And the angiogenesis in group B was better than other groups. Group C had similar osteogenetic quality of femoral head necrosis and angiogenesis with group D. Conclusions:The treatment of rhBMP-2/PLGA implantation after core depression can promote the repair of rabbit femoral head necrosis. It is a promising and efficient synthetic bone material to treat the femoral head necrosis.

  12. Crystal structure of YwpF from Staphylococcus aureus reveals its architecture comprised of a β-barrel core domain resembling type VI secretion system proteins and a two-helix pair.

    Science.gov (United States)

    Lee, Sang Jae; Lee, Kyu-Yeon; Lee, Ki-Young; Kim, Dong-Gyun; Kim, Soon-Jong; Lee, Bong-Jin

    2015-04-01

    The ywpF gene (SAV2097) of the Staphylococcus aureus strain Mu50 encodes the YwpF protein, which may play a role in antibiotic resistance. Here, we report the first crystal structure of the YwpF superfamily from S. aureus at 2.5-Å resolution. The YwpF structure consists of two regions: an N-terminal core β-barrel domain that shows structural similarity to type VI secretion system (T6SS) proteins (e.g., Hcp1, Hcp3, and EvpC) and a C-terminal two-helix pair. Although the monomer structure of S. aureus YwpF resembles those of T6SS proteins, the dimer/tetramer model of S. aureus YwpF is distinct from the functionally important hexameric ring of T6SS proteins. We therefore suggest that the S. aureus YwpF may have a different function compared to T6SS proteins. © 2015 Wiley Periodicals, Inc.

  13. Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells.

    Science.gov (United States)

    Mariati; Yeo, Jessna H M; Koh, Esther Y C; Ho, Steven C L; Yang, Yuansheng

    2014-01-01

    The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild-type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation. © 2014 American Institute of Chemical Engineers.

  14. Central core disease

    Directory of Open Access Journals (Sweden)

    Jungbluth Heinz

    2007-05-01

    Full Text Available Abstract Central core disease (CCD is an inherited neuromuscular disorder characterised by central cores on muscle biopsy and clinical features of a congenital myopathy. Prevalence is unknown but the condition is probably more common than other congenital myopathies. CCD typically presents in infancy with hypotonia and motor developmental delay and is characterized by predominantly proximal weakness pronounced in the hip girdle; orthopaedic complications are common and malignant hyperthermia susceptibility (MHS is a frequent complication. CCD and MHS are allelic conditions both due to (predominantly dominant mutations in the skeletal muscle ryanodine receptor (RYR1 gene, encoding the principal skeletal muscle sarcoplasmic reticulum calcium release channel (RyR1. Altered excitability and/or changes in calcium homeostasis within muscle cells due to mutation-induced conformational changes of the RyR protein are considered the main pathogenetic mechanism(s. The diagnosis of CCD is based on the presence of suggestive clinical features and central cores on muscle biopsy; muscle MRI may show a characteristic pattern of selective muscle involvement and aid the diagnosis in cases with equivocal histopathological findings. Mutational analysis of the RYR1 gene may provide genetic confirmation of the diagnosis. Management is mainly supportive and has to anticipate susceptibility to potentially life-threatening reactions to general anaesthesia. Further evaluation of the underlying molecular mechanisms may provide the basis for future rational pharmacological treatment. In the majority of patients, weakness is static or only slowly progressive, with a favourable long-term outcome.

  15. C-terminal region of human T cell lymphotrophic virus type I (HTLV) p19 core protein is immunogenic in humans and contains an HTLV/sub I/-specific epitope

    Energy Technology Data Exchange (ETDEWEB)

    Palker, T.J.; Scearce, R.M.; Copeland, T.D.; Oroszlan, S.; Haynes, B.F.

    1986-04-01

    To study the human host response to viral structural proteins during HTLV type I infection, five synthetic peptides matching the N-terminal and C-terminal regions of HTLV/sub I/ p19 core protein were used to identify antigenic sites on p19 that were immunogenic in man. In radioimmunoassay and immunoprecipitation experiments, antibodies in 16 of 18 HTLV/sub I//sup +/ patient sera reacted with a synthetic peptide matching the C-terminal 11-amino acid sequence of p19, whereas only two sera contained antibodies that reacted with other N- or C-terminal region p19 synthetic peptides. Polyclonal rabbit antisera to N- and C-terminal peptides reacted with a native viral protein of 19,000 daltons and with gagencoded precursors of p19. Six monoclonal antibodies against native viral p19 were screened for reactivity to the five synthetic peptides. One of six antibodies (13B12) reacted with the C-terminal synthetic peptide of p19. Antibody 13B12 did not react with HTLV/sub II/ or HTLV/sub III/ proteins or with HTLV/sub III/-infected cells, nor did it cross-react with a wide variety of HTLV-uninfected normal host tissues. Thus, the C-terminus of p19 contains an antigen that is highly immunogenic in most HTLV/sub 1/-infected patients and is HTLV/sub I/ specific.

  16. Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder?

    Science.gov (United States)

    Schubert, K O; Föcking, M; Prehn, J H M; Cotter, D R

    2012-07-01

    Clathrin-mediated endocytosis (CME) is the best-characterized mechanism governing cellular membrane and protein trafficking. In this hypothesis review, we integrate recent evidence implicating CME and related cellular trafficking mechanisms in the pathophysiology of psychotic disorders such as schizophrenia and bipolar disorder. The evidence includes proteomic and genomic findings implicating proteins and genes of the clathrin interactome. Additionally, several important candidate genes for schizophrenia, such as dysbindin, are involved in processes closely linked to CME and membrane trafficking. We discuss that key aspects of psychosis neuropathology such as synaptic dysfunction, white matter changes and aberrant neurodevelopment are all influenced by clathrin-dependent processes, and that other cellular trafficking mechanisms previously linked to psychoses interact with the clathrin interactome in important ways. Furthermore, many antipsychotic drugs have been shown to affect clathrin-interacting proteins. We propose that the targeted pharmacological manipulation of the clathrin interactome may offer fruitful opportunities for novel treatments of schizophrenia.

  17. Reference: GT1CORE [PLACE

    Lifescience Database Archive (English)

    Full Text Available GT1CORE Green PJ, Yong M-H, Cuozzo M, Kano-Murakami Y, Silverstein P, Chua N-H Binding site require...ments for pea nuclear protein factor GT-1 correlate with sequences required for light-depend

  18. Dual-core antiresonant hollow core fibers.

    Science.gov (United States)

    Liu, Xuesong; Fan, Zhongwei; Shi, Zhaohui; Ma, Yunfeng; Yu, Jin; Zhang, Jing

    2016-07-25

    In this work, dual-core antiresonant hollow core fibers (AR-HCFs) are numerically demonstrated, based on our knowledge, for the first time. Two fiber structures are proposed. One is a composite of two single-core nested nodeless AR-HCFs, exhibiting low confinement loss and a circular mode profile in each core. The other has a relatively simple structure, with a whole elliptical outer jacket, presenting a uniform and wide transmission band. The modal couplings of the dual-core AR-HCFs rely on a unique mechanism that transfers power through the air. The core separation and the gap between the two cores influence the modal coupling strength. With proper designs, both of the dual-core fibers can have low phase birefringence and short modal coupling lengths of several centimeters.

  19. Tandem assays of protein and glucose with functionalized core/shell particles based on magnetic separation and surface-enhanced Raman scattering.

    Science.gov (United States)

    Kong, Xianming; Yu, Qian; Lv, Zhongpeng; Du, Xuezhong

    2013-10-11

    Tandem assays of protein and glucose in combination with mannose-functionalized Fe3 O4 @SiO2 and Ag@SiO2 tag particles have promising potential in effective magnetic separation and highly sensitive and selective SERS assays of biomaterials. It is for the first time that tandem assay of glucose is developed using SERS based on the Con A-sandwiched microstructures between the functionalized magnetic and tag particles.

  20. Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder?

    LENUS (Irish Health Repository)

    2012-02-01

    Clathrin-mediated endocytosis (CME) is the best-characterized mechanism governing cellular membrane and protein trafficking. In this hypothesis review, we integrate recent evidence implicating CME and related cellular trafficking mechanisms in the pathophysiology of psychotic disorders such as schizophrenia and bipolar disorder. The evidence includes proteomic and genomic findings implicating proteins and genes of the clathrin interactome. Additionally, several important candidate genes for schizophrenia, such as dysbindin, are involved in processes closely linked to CME and membrane trafficking. We discuss that key aspects of psychosis neuropathology such as synaptic dysfunction, white matter changes and aberrant neurodevelopment are all influenced by clathrin-dependent processes, and that other cellular trafficking mechanisms previously linked to psychoses interact with the clathrin interactome in important ways. Furthermore, many antipsychotic drugs have been shown to affect clathrin-interacting proteins. We propose that the targeted pharmacological manipulation of the clathrin interactome may offer fruitful opportunities for novel treatments of schizophrenia.Molecular Psychiatry advance online publication, 11 October 2011; doi:10.1038\\/mp.2011.123.

  1. Tailor-made Au@Ag core-shell nanoparticle 2D arrays on protein-coated graphene oxide with assembly enhanced antibacterial activity

    Science.gov (United States)

    Wang, Huiqiao; Liu, Jinbin; Wu, Xuan; Tong, Zhonghua; Deng, Zhaoxiang

    2013-05-01

    Water-dispersible two-dimensional (2D) assemblies of Au@Ag core-shell nanoparticles are obtained through a highly selective electroless silver deposition on pre-assembled gold nanoparticles on bovine serum albumin (BSA)-coated graphene oxide (BSA-GO). While neither BSA-GO nor AuNP-decorated BSA-GO shows any antibacterial ability, the silver-coated GO@Au nanosheets (namely GO@Au@Ag) exhibit an enhanced antibacterial activity against Gram-negative Escherichia coli (E. coli) bacteria, superior to unassembled Au@Ag nanoparticles and even ionic Ag. Such an improvement may be attributed to the increased local concentration of silver nanoparticles around a bacterium and a polyvalent interaction with the bacterial surface. In addition, the colloidal stability of this novel nano-antimicrobial against the formation of random nanoparticle aggregates guarantees a minimized activity loss of the Au@Ag nanoparticles. The antibacterial efficacy of GO@Au@Ag is less sensitive to the existence of Cl-, in comparison with silver ions, providing another advantage for wound dressing applications. Our research unambiguously reveals a strong and very specific interaction between the GO@Au@Ag nanoassembly and E. coli, which could be an important clue toward a rational design, synthesis and assembly of innovative and highly active antibacterial nanomaterials.

  2. Animal MRI Core

    Data.gov (United States)

    Federal Laboratory Consortium — The Animal Magnetic Resonance Imaging (MRI) Core develops and optimizes MRI methods for cardiovascular imaging of mice and rats. The Core provides imaging expertise,...

  3. Modulation of the Unfolded Protein Response Is the Core of MicroRNA-122-Involved Sensitivity to Chemotherapy in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Fu Yang

    2011-07-01

    Full Text Available The loss of microRNA-122 (miR-122 expression correlates to many characteristic properties of hepatocellular carcinoma (HCC cells, including clonogenic survival, anchorage-independent growth, migration, invasion, epithelial-mesenchymal transition, and tumorigenesis. However, all of these findings do not sufficiently explain the oncogenic potential of miR-122. In the current study, we used two-dimensional differential in-gel electrophoresis to measure changes in the expression of thousands of proteins in response to the inhibition of miR-122 in human hepatoma cells. Several proteins that were upregulated on miR-122 inhibition were involved in the unfolded protein response (UPR pathway. The overexpression of miR-122 resulted in the repression of UPR pathway activation. Therefore, miR-122 may act as an inhibitor of the chaperone gene expression and negatively regulate the UPR pathway in HCC. We further showed that the miR-122 inhibitor enhanced the stability of the 26S proteasome non-ATPase regulatory subunit 10 (PSMD10 through the up-regulation of its target gene cyclin-dependent kinase 4 (CDK4. This process may activate the UPR pathway to prevent chemotherapy-mediated tumor cell apoptosis. The current study suggests that miR-122 negatively regulates the UPR through the CDK4-PSMD10 pathway. The down-regulation of miR-122 activated the CDK4-PSMD10-UPR pathway to decrease tumor cell anticancer drug-mediated apoptosis. We identified a new HCC therapeutic target and proclaimed the potential risk of the therapeutic use of miR-122 silencing.

  4. Bound or free: interaction of the C-terminal domain of Escherichia coli single-stranded DNA-binding protein (SSB) with the tetrameric core of SSB.

    Science.gov (United States)

    Su, Xun-Cheng; Wang, Yao; Yagi, Hiromasa; Shishmarev, Dmitry; Mason, Claire E; Smith, Paul J; Vandevenne, Marylène; Dixon, Nicholas E; Otting, Gottfried

    2014-04-01

    Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

  5. Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

    Directory of Open Access Journals (Sweden)

    Lopes EPA

    2000-01-01

    Full Text Available This study was undertaken to evaluate an enzyme immunoassay (EIA for hepatitis C virus antibody detection (anti-HCV, using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm. Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott®, specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95% sera from patients in the study group and negative in 38 of the 39 (97% sera from those in the control group, showing an accuracy of 96%. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

  6. Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway-dependent gene signatures in colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Karsten Jürchott

    2010-12-01

    Full Text Available Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1 by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

  7. Protein X of hepatitis B virus: origin and structure similarity with the central domain of DNA glycosylase.

    Directory of Open Access Journals (Sweden)

    Formijn J van Hemert

    Full Text Available Orthohepadnavirus (mammalian hosts and avihepadnavirus (avian hosts constitute the family of Hepadnaviridae and differ by their capability and inability for expression of protein X, respectively. Origin and functions of X are unclear. The evolutionary analysis at issue of X indicates that present strains of orthohepadnavirus started to diverge about 25,000 years ago, simultaneously with the onset of avihepadnavirus diversification. These evolutionary events were preceded by a much longer period during which orthohepadnavirus developed a functional protein X while avihepadnavirus evolved without X. An in silico generated 3D-model of orthohepadnaviral X protein displayed considerable similarity to the tertiary structure of DNA glycosylases (key enzymes of base excision DNA repair pathways. Similarity is confined to the central domain of MUG proteins with the typical DNA-binding facilities but without the capability of DNA glycosylase enzymatic activity. The hypothetical translation product of a vestigial X reading frame in the genome of duck hepadnavirus could also been folded into a DNA glycosylase-like 3D-structure. In conclusion, the most recent common ancestor of ortho- and avihepadnavirus carried an X sequence with orthology to the central domain of DNA glycosylase.

  8. Delipidation of a hepadnavirus: Viral inactivation and vaccine development.

    Science.gov (United States)

    Cham, B E; Vickery, K; Tohidi-Esfahani, R; Cossart, Y

    2006-10-01

    Many viruses including HIV, hepatitis C and hepatitis B, have an outer lipid envelope which maintains inserted viral peptides in the "correct" functional conformation and orientation. Disruption of the lipid envelope by most solvents destroys infectivity and often results in a loss of antigenicity. This communication outlines a novel approach to viral inactivation by specific solvent delipidation which modifies the whole virion rendering it non-infective, but antigenic. Duck hepatitis B virus (DHBV) was delipidated using a diisopropylether (DIPE) and butanol mixture and residual infectivity tested by inoculation into day-old ducks. Delipidation completely inactivated the DHBV (p vaccinate ducks. Three doses of delipidated DHBV induced anti-DHBs antibody production and prevented high dose challenge infection in five out of six ducks. In comparison, five of six ducks vaccinated with undelipidated DHBV and four of four ducks vaccinated with glutaraldehyde inactivated DHBV were unprotected (p inactivated DHBV, viral antigens were retained in an appropriate form to induce immunity. Delipidation of enveloped viruses with specific organic solvents has potential as the basis for development of vaccines.

  9. Complex coacervate core micelles.

    Science.gov (United States)

    Voets, Ilja K; de Keizer, Arie; Cohen Stuart, Martien A

    2009-01-01

    In this review we present an overview of the literature on the co-assembly of neutral-ionic block, graft, and random copolymers with oppositely charged species in aqueous solution. Oppositely charged species include synthetic (co)polymers of various architectures, biopolymers - such as proteins, enzymes and DNA - multivalent ions, metallic nanoparticles, low molecular weight surfactants, polyelectrolyte block copolymer micelles, metallo-supramolecular polymers, equilibrium polymers, etcetera. The resultant structures are termed complex coacervate core/polyion complex/block ionomer complex/interpolyelectrolyte complex micelles (or vesicles); i.e., in short C3Ms (or C3Vs) and PIC, BIC or IPEC micelles (and vesicles). Formation, structure, dynamics, properties, and function will be discussed. We focus on experimental work; theory and modelling will not be discussed. Recent developments in applications and micelles with heterogeneous coronas are emphasized.

  10. Time-Resolved Fluorescence Anisotropy Study of the Interaction Between DNA and a Peptide Truncated from the p53 Protein Core Domain.

    Science.gov (United States)

    Liu, Chengxuan; Liang, Gaiting; Liu, Zhen; Zu, Lily

    2014-03-01

    Time-resolved fluorescence anisotropy spectroscopy was applied to study the interaction between a peptide truncated from the binding site of tumor suppressor p53 protein and the DNAs covalently labeled with 6-carboxyfluorescein (FAM) dye. Fluorescence intensity quenching and changes of anisotropy decay lifetime were monitored when FAM labeled DNA formed complex with the peptide. The results demonstrated that the sequence of DNA could not define the binding specificity between the peptide and DNA. But the anisotropy decay of FAM can be used to examine the binding affinity of the peptide to DNA. The fluorescent dynamics of FAM can also be used to represent the rigidity of the complex formed between the peptide and DNA.

  11. Inferring Gene Networks for Strains of Dehalococcoides Highlights Conserved Relationships between Genes Encoding Core Catabolic and Cell-Wall Structural Proteins.

    Science.gov (United States)

    Mansfeldt, Cresten B; Heavner, Gretchen W; Rowe, Annette R; Hayete, Boris; Church, Bruce W; Richardson, Ruth E

    2016-01-01

    The interpretation of high-throughput gene expression data for non-model microorganisms remains obscured because of the high fraction of hypothetical genes and the limited number of methods for the robust inference of gene networks. Therefore, to elucidate gene-gene and gene-condition linkages in the bioremediation-important genus Dehalococcoides, we applied a Bayesian inference strategy called Reverse Engineering/Forward Simulation (REFS™) on transcriptomic data collected from two organohalide-respiring communities containing different Dehalococcoides mccartyi strains: the Cornell University mixed community D2 and the commercially available KB-1® bioaugmentation culture. In total, 49 and 24 microarray datasets were included in the REFS™ analysis to generate an ensemble of 1,000 networks for the Dehalococcoides population in the Cornell D2 and KB-1® culture, respectively. Considering only linkages that appeared in the consensus network for each culture (exceeding the determined frequency cutoff of ≥ 60%), the resulting Cornell D2 and KB-1® consensus networks maintained 1,105 nodes (genes or conditions) with 974 edges and 1,714 nodes with 1,455 edges, respectively. These consensus networks captured multiple strong and biologically informative relationships. One of the main highlighted relationships shared between these two cultures was a direct edge between the transcript encoding for the major reductive dehalogenase (tceA (D2) or vcrA (KB-1®)) and the transcript for the putative S-layer cell wall protein (DET1407 (D2) or KB1_1396 (KB-1®)). Additionally, transcripts for two key oxidoreductases (a [Ni Fe] hydrogenase, Hup, and a protein with similarity to a formate dehydrogenase, "Fdh") were strongly linked, generalizing a strong relationship noted previously for Dehalococcoides mccartyi strain 195 to multiple strains of Dehalococcoides. Notably, the pangenome array utilized when monitoring the KB-1® culture was capable of resolving signals from multiple

  12. Inferring Gene Networks for Strains of Dehalococcoides Highlights Conserved Relationships between Genes Encoding Core Catabolic and Cell-Wall Structural Proteins

    Science.gov (United States)

    Mansfeldt, Cresten B.; Heavner, Gretchen W.; Rowe, Annette R.; Hayete, Boris; Church, Bruce W.; Richardson, Ruth E.

    2016-01-01

    The interpretation of high-throughput gene expression data for non-model microorganisms remains obscured because of the high fraction of hypothetical genes and the limited number of methods for the robust inference of gene networks. Therefore, to elucidate gene-gene and gene-condition linkages in the bioremediation-important genus Dehalococcoides, we applied a Bayesian inference strategy called Reverse Engineering/Forward Simulation (REFS™) on transcriptomic data collected from two organohalide-respiring communities containing different Dehalococcoides mccartyi strains: the Cornell University mixed community D2 and the commercially available KB-1® bioaugmentation culture. In total, 49 and 24 microarray datasets were included in the REFS™ analysis to generate an ensemble of 1,000 networks for the Dehalococcoides population in the Cornell D2 and KB-1® culture, respectively. Considering only linkages that appeared in the consensus network for each culture (exceeding the determined frequency cutoff of ≥ 60%), the resulting Cornell D2 and KB-1® consensus networks maintained 1,105 nodes (genes or conditions) with 974 edges and 1,714 nodes with 1,455 edges, respectively. These consensus networks captured multiple strong and biologically informative relationships. One of the main highlighted relationships shared between these two cultures was a direct edge between the transcript encoding for the major reductive dehalogenase (tceA (D2) or vcrA (KB-1®)) and the transcript for the putative S-layer cell wall protein (DET1407 (D2) or KB1_1396 (KB-1®)). Additionally, transcripts for two key oxidoreductases (a [Ni Fe] hydrogenase, Hup, and a protein with similarity to a formate dehydrogenase, “Fdh”) were strongly linked, generalizing a strong relationship noted previously for Dehalococcoides mccartyi strain 195 to multiple strains of Dehalococcoides. Notably, the pangenome array utilized when monitoring the KB-1® culture was capable of resolving signals from

  13. k-core covers and the core

    NARCIS (Netherlands)

    Sanchez-Rodriguez, E.; Borm, Peter; Estevez-Fernandez, A.; Fiestras-Janeiro, G.; Mosquera, M.A.

    2015-01-01

    This paper extends the notion of individual minimal rights for a transferable utility game (TU-game) to coalitional minimal rights using minimal balanced families of a specific type, thus defining a corresponding minimal rights game. It is shown that the core of a TU-game coincides with the core of

  14. Academic Rigor: The Core of the Core

    Science.gov (United States)

    Brunner, Judy

    2013-01-01

    Some educators see the Common Core State Standards as reason for stress, most recognize the positive possibilities associated with them and are willing to make the professional commitment to implementing them so that academic rigor for all students will increase. But business leaders, parents, and the authors of the Common Core are not the only…

  15. k-core covers and the core

    NARCIS (Netherlands)

    Sanchez-Rodriguez, E.; Borm, Peter; Estevez-Fernandez, A.; Fiestras-Janeiro, G.; Mosquera, M.A.

    This paper extends the notion of individual minimal rights for a transferable utility game (TU-game) to coalitional minimal rights using minimal balanced families of a specific type, thus defining a corresponding minimal rights game. It is shown that the core of a TU-game coincides with the core of

  16. Proteomic analysis of small acid soluble proteins in the spore core of Bacillus subtilis ΔprpE and 168 strains with predictions of peptides liquid chromatography retention times as an additional tool in protein identification

    Directory of Open Access Journals (Sweden)

    Obuchowski Michał

    2010-11-01

    Full Text Available Abstract Background Sporulation, characteristic for some bacteria such as Bacillus subtilis, has not been entirely defined yet. Protein phosphatase E (PrpE and small, acid soluble spore proteins (SASPs influence this process. Nevertheless, direct result of PrpE interaction on SASPs content in spore coat of B. subtilis has not been evidenced so far. As proteomic approach enables global analysis of occurring proteins, therefore it was chosen in this experiment to compare SASPs occurrence in two strains of B. subtilis, standard 168 and ΔprpE, lacking PrpE phosphatase. Proteomic analysis is still a challenge, and despite of big approach in mass spectrometry (MS field, the identification reliability remains unsatisfactory. Therefore there is a rising interest in new methods, particularly bioinformatic tools that would harden protein identification. Most of currently applied algorithms are based on MS-data. Information from separation steps is not still in routine usage, even though they also provide valuable facts about analyzed structures. The aim of this research was to apply a model for peptides retention times prediction, based on quantitative structure-retention relationships (QSRR in SASPs analysis, obtained from two strains of B. subtilis proteome digests after separation and identification of the peptides by LC-ESI-MS/MS. The QSRR approach was applied as the additional constraint in proteomic research verifying results of MS/MS ion search and confirming the correctness of the peptides identifications along with the indication of the potential false positives and false negatives. Results In both strains of B. subtilis, peptides characteristic for SASPs were found, however their identification confidence varied. According to the MS identity parameter Xcorr and difference between predicted and experimental retention times (ΔtR four groups could be distinguished: correctly and incorrectly identified, potential false positives and false

  17. Structural Comparison of Toxic Core of Cry1 A?Type Insecticidal Crystal Proteins%Cry 1 A型杀虫晶体蛋白活性区的空间结构比较分析

    Institute of Scientific and Technical Information of China (English)

    刘肖萍; 林毅

    2016-01-01

    The 3?D structure of the toxic core for all available 9 typical Cry1 A proteins were constructed by the method of homology modeling.The structural differences among different Cry1 A proteins indicated that Domain Ⅰ and Domain Ⅲ were more conservative than Domain Ⅱ.Structures of Cry1 A Domain Ⅰ were al-most identical except Cry1 Aa and Cry1 Af which had one more helix.The differences among the Cry1 A Domain II were mainly located in loops.Structures of DomainⅢ of Cry1 Ab,Cry1 Ad,Cry1 Ae and Cry1 Af were con-sistent,thus these four Cry1 A proteins and the other five were divided into two different subgroups.The re-sults confirm that the key residues and motifs are important for the insecticidal activites of Cry1 A proteins.%对9种代表性Cry1 A型杀虫晶体蛋白成员进行活性区空间结构的同源模建与比较分析。分析结果表明:Domain Ⅰ和Domain Ⅲ相对保守,其中,Domain Ⅰ整体走向及结构基本重叠,只有 Cry1 Aa和 Cry1 Af多了一个螺旋;DomainⅡ的主要差异体现在loop上;将DomainⅢ结构一致的成员Cry1 Ab,Cry1 Ad,Cry1 Ae和Cry1 Af归为一个亚型,其他5种成员归为另一个亚型。研究确定了影响 Cry1 A型杀虫晶体蛋白结构差异的关键氨基酸及关键结构片段。

  18. Comodules over semiperfect corings

    CERN Document Server

    Caenepeel, S

    2011-01-01

    We discuss when the Rat functor associated to a coring satisfying the left $\\alpha$-condition is exact. We study the category of comodules over a semiperfect coring. We characterize semiperfect corings over artinian rings and over qF-rings.

  19. Coring Sample Acquisition Tool

    Science.gov (United States)

    Haddad, Nicolas E.; Murray, Saben D.; Walkemeyer, Phillip E.; Badescu, Mircea; Sherrit, Stewart; Bao, Xiaoqi; Kriechbaum, Kristopher L.; Richardson, Megan; Klein, Kerry J.

    2012-01-01

    A sample acquisition tool (SAT) has been developed that can be used autonomously to sample drill and capture rock cores. The tool is designed to accommodate core transfer using a sample tube to the IMSAH (integrated Mars sample acquisition and handling) SHEC (sample handling, encapsulation, and containerization) without ever touching the pristine core sample in the transfer process.

  20. Mx1 and Mx2 key antiviral proteins are surprisingly lost in toothed whales.

    Science.gov (United States)

    Braun, Benjamin A; Marcovitz, Amir; Camp, J Gray; Jia, Robin; Bejerano, Gill

    2015-06-30

    Viral outbreaks in dolphins and other Delphinoidea family members warrant investigation into the integrity of the cetacean immune system. The dynamin-like GTPase genes Myxovirus 1 (Mx1) and Mx2 defend mammals against a broad range of viral infections. Loss of Mx1 function in human and mice enhances infectivity by multiple RNA and DNA viruses, including orthomyxoviruses (influenza A), paramyxoviruses (measles), and hepadnaviruses (hepatitis B), whereas loss of Mx2 function leads to decreased resistance to HIV-1 and other viruses. Here we show that both Mx1 and Mx2 have been rendered nonfunctional in Odontoceti cetaceans (toothed whales, including dolphins and orcas). We discovered multiple exon deletions, frameshift mutations, premature stop codons, and transcriptional evidence of decay in the coding sequence of both Mx1 and Mx2 in four species of Odontocetes. We trace the likely loss event for both proteins to soon after the divergence of Odontocetes and Mystocetes (baleen whales) ∼33-37 Mya. Our data raise intriguing questions as to what drove the loss of both Mx1 and Mx2 genes in the Odontoceti lineage, a double loss seen in none of 56 other mammalian genomes, and suggests a hitherto unappreciated fundamental genetic difference in the way these magnificent mammals respond to viral infections.

  1. Banded transformer cores

    Science.gov (United States)

    Mclyman, C. W. T. (Inventor)

    1974-01-01

    A banded transformer core formed by positioning a pair of mated, similar core halves on a supporting pedestal. The core halves are encircled with a strap, selectively applying tension whereby a compressive force is applied to the core edge for reducing the innate air gap. A dc magnetic field is employed in supporting the core halves during initial phases of the banding operation, while an ac magnetic field subsequently is employed for detecting dimension changes occurring in the air gaps as tension is applied to the strap.

  2. The core paradox.

    Science.gov (United States)

    Kennedy, G. C.; Higgins, G. H.

    1973-01-01

    Rebuttal of suggestions from various critics attempting to provide an escape from the seeming paradox originated by Higgins and Kennedy's (1971) proposed possibility that the liquid in the outer core was thermally stably stratified and that this stratification might prove a powerful inhibitor to circulation of the outer core fluid of the kind postulated for the generation of the earth's magnetic field. These suggestions are examined and shown to provide no reasonable escape from the core paradox.

  3. K-core inflation

    OpenAIRE

    Alexander L. Wolman

    2011-01-01

    K-core inflation is a new class of underlying inflation measures. The two most popular measures of underlying inflation are core inflation and trimmed mean inflation. The former removes fixed categories of goods and services (food and energy) from the inflation calculation, and the latter removes fixed percentiles of the weighted distribution of price changes. In contrast, k-core inflation specifies a size of relative price change to be removed from the inflation calculation. Thus, the catego...

  4. Sediment Core Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — FUNCTION: Provides instrumentation and expertise for physical and geoacoustic characterization of marine sediments. DESCRIPTION: The multisensor core logger measures...

  5. Sediment Core Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — FUNCTION: Provides instrumentation and expertise for physical and geoacoustic characterization of marine sediments.DESCRIPTION: The multisensor core logger measures...

  6. Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions.

    Science.gov (United States)

    Walzer, Sascha A; Egerer-Sieber, Claudia; Sticht, Heinrich; Sevvana, Madhumati; Hohl, Katharina; Milbradt, Jens; Muller, Yves A; Marschall, Manfred

    2015-11-13

    Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1-175 and 50-292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and β secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59-87) embraces pUL50 and contributes 1510 Å(2) to the total interface area (1880 Å(2)). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy.

  7. Ice Core Investigations

    Science.gov (United States)

    Krim, Jessica; Brody, Michael

    2008-01-01

    What can glaciers tell us about volcanoes and atmospheric conditions? How does this information relate to our understanding of climate change? Ice Core Investigations is an original and innovative activity that explores these types of questions. It brings together popular science issues such as research, climate change, ice core drilling, and air…

  8. Making an Ice Core.

    Science.gov (United States)

    Kopaska-Merkel, David C.

    1995-01-01

    Explains an activity in which students construct a simulated ice core. Materials required include only a freezer, food coloring, a bottle, and water. This hands-on exercise demonstrates how a glacier is formed, how ice cores are studied, and the nature of precision and accuracy in measurement. Suitable for grades three through eight. (Author/PVD)

  9. Ice Core Investigations

    Science.gov (United States)

    Krim, Jessica; Brody, Michael

    2008-01-01

    What can glaciers tell us about volcanoes and atmospheric conditions? How does this information relate to our understanding of climate change? Ice Core Investigations is an original and innovative activity that explores these types of questions. It brings together popular science issues such as research, climate change, ice core drilling, and air…

  10. Iowa Core Annual Report

    Science.gov (United States)

    Iowa Department of Education, 2015

    2015-01-01

    One central component of a great school system is a clear set of expectations, or standards, that educators help all students reach. In Iowa, that effort is known as the Iowa Core. The Iowa Core represents the statewide academic standards, which describe what students should know and be able to do in math, science, English language arts, and…

  11. Mercury's core evolution

    Science.gov (United States)

    Deproost, Marie-Hélène; Rivoldini, Attilio; Van Hoolst, Tim

    2016-10-01

    Remote sensing data of Mercury's surface by MESSENGER indicate that Mercury formed under reducing conditions. As a consequence, silicon is likely the main light element in the core together with a possible small fraction of sulfur. Compared to sulfur, which does almost not partition into solid iron at Mercury's core conditions and strongly decreases the melting temperature, silicon partitions almost equally well between solid and liquid iron and is not very effective at reducing the melting temperature of iron. Silicon as the major light element constituent instead of sulfur therefore implies a significantly higher core liquidus temperature and a decrease in the vigor of compositional convection generated by the release of light elements upon inner core formation.Due to the immiscibility in liquid Fe-Si-S at low pressure (below 15 GPa), the core might also not be homogeneous and consist of an inner S-poor Fe-Si core below a thinner Si-poor Fe-S layer. Here, we study the consequences of a silicon-rich core and the effect of the blanketing Fe-S layer on the thermal evolution of Mercury's core and on the generation of a magnetic field.

  12. Comparison of HCV core and coreE1E2 virus-like particles generated by stably transfected Leishmania tarentolae for stimulation of Th1 immune responses in mice.

    Science.gov (United States)

    Bolhassani, Azam; Davoudi, Noushin; Agi, Elnaz; Motevalli, Fatemeh

    2017-01-25

    Virus-like particles (VLPs) could be improved into successful immunogens as well as a potent delivery vehicle, but however, the current expression systems for VLPs production have some limitations. Recently, we developed a novel strategy to produce two HCV VLPs containing core or coreE1E2 proteins using stably transfected Leishmania tarentolae promastigotes. Then, BALB/c mice were injected by both viral like particles in different immunization strategies such as homologous DNA-, homologous VLP-, and heterologous DNA/ VLP-based immunizations. TEM microscopy indicated HCV core and HCV coreE1E2 VLP assembly with average size of 30-40 and 40-60 nm after purification, respectively. Our results showed that homologous immunizations with both HCV core or coreE1E2 VLPs significantly induced anti-core or anti-coreE1E2 antibody responses, respectively as well as secretion of IFN-γ cytokine as compared to other strategies. Moreover, DNA-prime/VLP-boost regimens significantly elicited higher levels of IFN-γ and antibody responses in comparison with homologous DNA/DNA regimens. The groups immunized with homologous or heterologous coreE1E2 VLPs showed markedly higher immune responses as compared to groups immunized with core VLP regimens against coreE1E2 protein. The crude HCV VLPs generated by Leishmania expression system could elicit a Th1-type response as a promising vaccine candidate against HCV infections.

  13. Mars' core and magnetism.

    Science.gov (United States)

    Stevenson, D J

    2001-07-12

    The detection of strongly magnetized ancient crust on Mars is one of the most surprising outcomes of recent Mars exploration, and provides important insight about the history and nature of the martian core. The iron-rich core probably formed during the hot accretion of Mars approximately 4.5 billion years ago and subsequently cooled at a rate dictated by the overlying mantle. A core dynamo operated much like Earth's current dynamo, but was probably limited in duration to several hundred million years. The early demise of the dynamo could have arisen through a change in the cooling rate of the mantle, or even a switch in convective style that led to mantle heating. Presently, Mars probably has a liquid, conductive outer core and might have a solid inner core like Earth.

  14. The t-core of an s-core

    OpenAIRE

    Fayers, Matthew

    2010-01-01

    We consider the $t$-core of an $s$-core partition, when $s$ and $t$ are coprime positive integers. Olsson has shown that the $t$-core of an $s$-core is again an $s$-core, and we examine certain actions of the affine symmetric group on $s$-cores which preserve the $t$-core of an $s$-core. Along the way, we give a new proof of Olsson's result. We also give a new proof of a result of Vandehey, showing that there is a simultaneous $s$- and $t$-core which contains all others.

  15. The t-core of an s-core

    OpenAIRE

    Fayers, Matthew

    2010-01-01

    We consider the $t$-core of an $s$-core partition, when $s$ and $t$ are coprime positive integers. Olsson has shown that the $t$-core of an $s$-core is again an $s$-core, and we examine certain actions of the affine symmetric group on $s$-cores which preserve the $t$-core of an $s$-core. Along the way, we give a new proof of Olsson's result. We also give a new proof of a result of Vandehey, showing that there is a simultaneous $s$- and $t$-core which contains all others.

  16. Korrelasjon mellom core styrke, core stabilitet og utholdende styrke i core

    OpenAIRE

    Berg-Olsen, Andrea Marie; Fugelsøy, Eivor; Maurstad, Ann-Louise

    2010-01-01

    Formålet med studien var å se hvilke korrelasjon det er mellom core styrke, core stabilitet og utholdende styrke i core. Testingen bestod av tre hoveddeler hvor vi testet core styrke, core stabilitet og utholdende styrke i core. Innenfor core styrke og utholdende styrke i core ble tre ulike tester utført. Ved måling av core stabilitet ble det gjennomført kun en test. I core styrke ble isometrisk abdominal fleksjon, isometrisk rygg ekstensjon og isometrisk lateral fleksjon testet. Sit-ups p...

  17. Korrelasjon mellom core styrke, core stabilitet og utholdende styrke i core

    OpenAIRE

    Berg-Olsen, Andrea Marie; Fugelsøy, Eivor; Maurstad, Ann-Louise

    2010-01-01

    Formålet med studien var å se hvilke korrelasjon det er mellom core styrke, core stabilitet og utholdende styrke i core. Testingen bestod av tre hoveddeler hvor vi testet core styrke, core stabilitet og utholdende styrke i core. Innenfor core styrke og utholdende styrke i core ble tre ulike tester utført. Ved måling av core stabilitet ble det gjennomført kun en test. I core styrke ble isometrisk abdominal fleksjon, isometrisk rygg ekstensjon og isometrisk lateral fleksjon testet. Sit-ups p...

  18. Earth's inner core: Innermost inner core or hemispherical variations?

    NARCIS (Netherlands)

    Lythgoe, K. H.; Deuss, A.; Rudge, J. F.; Neufeld, J. A.

    2014-01-01

    The structure of Earth's deep inner core has important implications for core evolution, since it is thought to be related to the early stages of core formation. Previous studies have suggested that there exists an innermost inner core with distinct anisotropy relative to the rest of the inner core.

  19. Earth's inner core: Innermost inner core or hemispherical variations?

    NARCIS (Netherlands)

    Lythgoe, K. H.; Deuss, A.; Rudge, J. F.; Neufeld, J. A.

    2014-01-01

    The structure of Earth's deep inner core has important implications for core evolution, since it is thought to be related to the early stages of core formation. Previous studies have suggested that there exists an innermost inner core with distinct anisotropy relative to the rest of the inner core.

  20. Finding your next core business.

    Science.gov (United States)

    Zook, Chris

    2007-04-01

    How do you know when your core needs to change? And how do you determine what should replace it? From an in-depth study of 25 companies, the author, a strategy consultant, has discovered that it's possible to measure the vitality of a business's core. If it needs reinvention, he says, the best course is to mine hidden assets. Some of the 25 companies were in deep crisis when they began the process of redefining themselves. But, says Zook, management teams can learn to recognize early signs of erosion. He offers five diagnostic questions with which to evaluate the customers, key sources of differentiation, profit pools, capabilities, and organizational culture of your core business. The next step is strategic regeneration. In four-fifths of the companies Zook examined, a hidden asset was the centerpiece of the new strategy. He provides a map for identifying the hidden assets in your midst, which tend to fall into three categories: undervalued business platforms, untapped insights into customers, and underexploited capabilities. The Swedish company Dometic, for example, was manufacturing small absorption refrigerators for boats and RVs when it discovered a hidden asset: its understanding of, and access to, customers in the RV market. The company took advantage of a boom in that market to refocus on complete systems for live-in vehicles. The Danish company Novozymes, which produced relatively low-tech commodity enzymes such as those used in detergents, realized that its underutilized biochemical capability in genetic and protein engineering was a hidden asset and successfully refocused on creating bioengineered specialty enzymes. Your next core business is not likely to announce itself with fanfare. Use the author's tools to conduct an internal audit of possibilities and pinpoint your new focus.

  1. IGCSE core mathematics

    CERN Document Server

    Wall, Terry

    2013-01-01

    Give your core level students the support and framework they require to get their best grades with this book dedicated to the core level content of the revised syllabus and written specifically to ensure a more appropriate pace. This title has been written for Core content of the revised Cambridge IGCSE Mathematics (0580) syllabus for first teaching from 2013. ? Gives students the practice they require to deepen their understanding through plenty of practice questions. ? Consolidates learning with unique digital resources on the CD, included free with every book. We are working with Cambridge

  2. Core shroud corner joints

    Science.gov (United States)

    Gilmore, Charles B.; Forsyth, David R.

    2013-09-10

    A core shroud is provided, which includes a number of planar members, a number of unitary corners, and a number of subassemblies each comprising a combination of the planar members and the unitary corners. Each unitary corner comprises a unitary extrusion including a first planar portion and a second planar portion disposed perpendicularly with respect to the first planar portion. At least one of the subassemblies comprises a plurality of the unitary corners disposed side-by-side in an alternating opposing relationship. A plurality of the subassemblies can be combined to form a quarter perimeter segment of the core shroud. Four quarter perimeter segments join together to form the core shroud.

  3. 丙型肝炎病毒核心蛋白通过FOXO1/PGC-1α途径上调磷酸烯醇式丙酮酸羧基酶的转录%Hepatitis C virus core protein upregulates the transcription of PCK1 through FOXO1/PGC-1α pathway

    Institute of Scientific and Technical Information of China (English)

    陈继征; 王倩; 徐松

    2012-01-01

    [目的]分析丙型肝炎病毒(HCV)核心蛋白(CORE)稳定表达对磷酸烯醇式丙酮酸羧基酶(PCK1)转录水平的影响,并分析HCV CORE调控PCK1转录的分子机制,为进一步阐明HCV感染致2型糖尿病机理的探讨提供新的思路.[方法]利用反转录病毒表达系统构建稳定表达HCV CORE的HuhT-lunet-core细胞系.采用Real-time PCR和萤光素酶报告基因技术检测Huh7-1unet-core细胞系中PCK1、FOXO1以及PGC-1α转录水平变化,并结合Western blot分析FOXO1的活性变化.[结果]HCV CORE的稳定表达显著增强PCK1的转录水平,HCV CORE不影响FOXO1的转录和表达水平,但降低FOXO1的磷酸化水平,激活了FOXO1的转录活性,并增强PGC-1α的mRNA表达水平.[结论]HCV CORE在Huh7-1unet细胞中的稳定表达激活FOXO1的转录活性,并与PGC-1α协同作用,上调PCK1的转录,从而导致肝糖异生过度发生,对HCV CORE调控PCK1转录的分子机制的揭示可能为HCV感染相关的糖尿病的治疗提供新的靶点.%[Objective] We analyzed the effect of the stable expressed Hepatitis C virus core protein on PCK1 mRNA expression level and the molecular mechanisms involved in Huh7-lunet cells. [Methods] A retroviral vector mediated mammalian cell expression cell line of the HCV core protein was constructed. The mRNA and protein levels of PCK1 , FOXO1 and PGC-1α were analyzed by Real-time PCR and luciferase assay in Huh7-lunet-core cells. [Results] HCV CORE upregulated the mRNA levels of PCK1 significantly. Both the mRNA and protein levels of F0X01 were not affected in Huh7-lunet-core cells, whereas a decreased phosphorylation status of FOXO1 was exhibited. Moreover, activation of FOXO1 by HCV CORE was detected. Further, the mRNA level of PGC-la was found to be significantly elevated in Huh7-lunet-core cells. [Conclusion] Our results revealed for the first time that HCV core protein expression-mediated F0X01 activation and the increased PGC-lα leaded to the elevation of PCK1 at

  4. Biospecimen Core Resource - TCGA

    Science.gov (United States)

    The Cancer Genome Atlas (TCGA) Biospecimen Core Resource centralized laboratory reviews and processes blood and tissue samples and their associated data using optimized standard operating procedures for the entire TCGA Research Network.

  5. NICHD Zebrafish Core

    Data.gov (United States)

    Federal Laboratory Consortium — The core[HTML_REMOVED]s goal is to help researchers of any expertise perform zebrafish experiments aimed at illuminating basic biology and human disease mechanisms,...

  6. iPSC Core

    Data.gov (United States)

    Federal Laboratory Consortium — The induced Pluripotent Stem Cells (iPSC) Core was created in 2011 to accelerate stem cell research in the NHLBI by providing investigators consultation, technical...

  7. INTEGRAL core programme

    Science.gov (United States)

    Gehrels, N.; Schoenfelder, V.; Ubertini, P.; Winkler, C.

    1997-01-01

    The International Gamma Ray Astrophysics Laboratory (INTEGRAL) mission is described with emphasis on the INTEGRAL core program. The progress made in the planning activities for the core program is reported on. The INTEGRAL mission has a nominal lifetime of two years with a five year extension option. The observing time will be divided between the core program (between 30 and 35 percent during the first two years) and general observations. The core program consists of three main elements: the deep survey of the Galactic plane in the central radian of the Galaxy; frequent scans of the Galactic plane in the search for transient sources, and pointed observations of several selected sources. The allocation of the observation time is detailed and the sensitivities of the observations are outlined.

  8. Focusing on Core Business

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    China is regulating state-owned enterprises that are investing outside of their core business realms, concerned that poor investment decisions could lead to loss of state-owned assets, but some doubt the effect of the new regulation

  9. Organizing Core Tasks

    DEFF Research Database (Denmark)

    Boll, Karen

    Civil servants conduct the work which makes welfare states functions on an everyday bases: Police men police, school teachers teach, and tax inspectors inspect. Focus in this paper is on the core tasks of tax inspectors. The paper argues that their core task of securing the collection of revenue...... has remained much the same within the last 10 years. However, how the core task has been organized has changed considerable under the influence of various “organizing devices”. The paper focusses on how organizing devices such as risk assessment, output-focus, effect orientation, and treatment...... projects influence the organization of core tasks within the tax administration. The paper shows that the organizational transformations based on the use of these devices have had consequences both for the overall collection of revenue and for the employees’ feeling of “making a difference”. All in all...

  10. Structure and dynamics of core-periphery networks

    CERN Document Server

    Csermely, Peter; Wu, Ling-Yun; Uzzi, Brian

    2013-01-01

    Recent studies uncovered important core/periphery network structures characterizing complex sets of cooperative and competitive interactions between network nodes, be they proteins, cells, species or humans. Better characterization of the structure, dynamics and function of core/periphery networks is a key step of our understanding cellular functions, species adaptation, social and market changes. Here we summarize the current knowledge of the structure and dynamics of "traditional" core/periphery networks, rich-clubs, nested, bow-tie and onion networks. Comparing core/periphery structures with network modules, we discriminate between global and local cores. The core/periphery network organization lies in the middle of several extreme properties, such as random/condensed structures, clique/star configurations, network symmetry/asymmetry, network assortativity/disassortativity, as well as network hierarchy/anti-hierarchy. These properties of high complexity together with the large degeneracy of core pathways e...

  11. Encapsulation into complex coacervate core micelles promotes EGFP dimerization

    NARCIS (Netherlands)

    Nolles, A.; Dongen, Van N.J.E.; Westphal, A.H.; Visser, A.J.W.G.; Kleijn, J.M.; Berkel, Van W.J.H.; Borst, J.W.

    2017-01-01

    Complex coacervate core micelles (C3Ms) are colloidal structures useful for encapsulation of biomacromolecules. We previously demonstrated that enhanced green fluorescent protein (EGFP) can be encapsulated into C3Ms using the diblock copolymer

  12. Inner core structure behind the PKP core phase triplication

    NARCIS (Netherlands)

    Blom, Nienke A.; Deuss, Arwen; Paulssen, Hanneke; Waszek, Lauren

    2015-01-01

    The structure of the Earth's inner core is not well known between depths of ∼100–200 km beneath the inner core boundary. This is a result of the PKP core phase triplication and the existence of strong precursors to PKP phases, which hinder the measurement of inner core compressional PKIKP waves at e

  13. Inner core structure behind the PKP core phase triplication

    NARCIS (Netherlands)

    Blom, Nienke A.; Deuss, Arwen; Paulssen, Hanneke; Waszek, Lauren

    The structure of the Earth's inner core is not well known between depths of ∼100–200 km beneath the inner core boundary. This is a result of the PKP core phase triplication and the existence of strong precursors to PKP phases, which hinder the measurement of inner core compressional PKIKP waves at

  14. Pressure Core Characterization

    Science.gov (United States)

    Santamarina, J. C.

    2014-12-01

    Natural gas hydrates form under high fluid pressure and low temperature, and are found in permafrost, deep lakes or ocean sediments. Hydrate dissociation by depressurization and/or heating is accompanied by a multifold hydrate volume expansion and host sediments with low permeability experience massive destructuration. Proper characterization requires coring, recovery, manipulation and testing under P-T conditions within the stability field. Pressure core technology allows for the reliable characterization of hydrate bearing sediments within the stability field in order to address scientific and engineering needs, including the measurement of parameters used in hydro-thermo-mechanical analyses, and the monitoring of hydrate dissociation under controlled pressure, temperature, effective stress and chemical conditions. Inherent sampling effects remain and need to be addressed in test protocols and data interpretation. Pressure core technology has been deployed to study hydrate bearing sediments at several locations around the world. In addition to pressure core testing, a comprehensive characterization program should include sediment analysis, testing of reconstituted specimens (with and without synthetic hydrate), and in situ testing. Pressure core characterization technology can be used to study other gas-charged formations such as deep sea sediments, coal bed methane and gas shales.

  15. GREEN CORE HOUSE

    Directory of Open Access Journals (Sweden)

    NECULAI Oana

    2017-05-01

    Full Text Available The Green Core House is a construction concept with low environmental impact, having as main central element a greenhouse. The greenhouse has the innovative role to use the biomass energy provided by plants to save energy. Although it is the central piece, the greenhouse is not the most innovative part of the Green Core House, but the whole building ensemble because it integrates many other sustainable systems as "waste purification systems", "transparent photovoltaic panels" or "double skin façades".

  16. Birefringent hollow core fibers

    DEFF Research Database (Denmark)

    Roberts, John

    2007-01-01

    Hollow core photonic crystal fiber (HC-PCF), fabricated according to a nominally non-birefringent design, shows a degree of un-controlled birefringence or polarization mode dispersion far in excess of conventional non polarization maintaining fibers. This can degrade the output pulse in many...... and an increased overlap between the polarization modes at the glass interfaces. The interplay between these effects leads to a wavelength for optimum polarization maintenance, lambda(PM), which is detuned from the wavelength of highest birefringence. By a suitable fiber design involving antiresonance of the core...

  17. Birefringent hollow core fibers

    DEFF Research Database (Denmark)

    Roberts, John

    2007-01-01

    Hollow core photonic crystal fiber (HC-PCF), fabricated according to a nominally non-birefringent design, shows a degree of un-controlled birefringence or polarization mode dispersion far in excess of conventional non polarization maintaining fibers. This can degrade the output pulse in many...... and an increased overlap between the polarization modes at the glass interfaces. The interplay between these effects leads to a wavelength for optimum polarization maintenance, lambda(PM), which is detuned from the wavelength of highest birefringence. By a suitable fiber design involving antiresonance of the core...

  18. Investigation of EAS cores

    Science.gov (United States)

    Shaulov, S. B.; Beyl, P. F.; Beysembaev, R. U.; Beysembaeva, E. A.; Bezshapov, S. P.; Borisov, A. S.; Cherdyntceva, K. V.; Chernyavsky, M. M.; Chubenko, A. P.; Dalkarov, O. D.; Denisova, V. G.; Erlykin, A. D.; Kabanova, N. V.; Kanevskaya, E. A.; Kotelnikov, K. A.; Morozov, A. E.; Mukhamedshin, R. A.; Nam, R. A.; Nesterova, N. M.; Nikolskaya, N. M.; Pavluchenko, V. P.; Piskal, V. V.; Puchkov, V. S.; Pyatovsky, S. E.; Ryabov, V. A.; Sadykov, T. Kh.; Schepetov, A. L.; Smirnova, M. D.; Stepanov, A. V.; Uryson, A. V.; Vavilov, Yu. N.; Vildanov, N. G.; Vildanova, L. I.; Zayarnaya, I. S.; Zhanceitova, J. K.; Zhukov, V. V.

    2017-06-01

    The development of nuclear-electromagnetic cascade models in air in the late forties have shown informational content of the study of cores of extensive air showers (EAS). These investigations were the main goal in different experiments which were carried out over many years by a variety of methods. Outcomes of such investigations obtained in the HADRON experiment using an X-ray emulsion chamber (XREC) as a core detector are considered. The Ne spectrum of EAS associated with γ-ray families, spectra of γ-rays (hadrons) in EAS cores and the Ne dependence of the muon number, ⟨Nμ⟩, in EAS with γ-ray families are obtained for the first time at energies of 1015-1017 eV with this method. A number of new effects were observed, namely, an abnormal scaling violation in hadron spectra which are fundamentally different from model predictions, an excess of muon number in EAS associated with γ-ray families, and the penetrating component in EAS cores. It is supposed that the abnormal behavior of γ-ray spectra and Ne dependence of the muon number are explained by the emergence of a penetrating component in the 1st PCR spectrum `knee' range. Nuclear and astrophysical explanations of the origin of the penetrating component are discussed. The necessity of considering the contribution of a single close cosmic-ray source to explain the PCR spectrum in the knee range is noted.

  19. Some Core Contested Concepts

    Science.gov (United States)

    Chomsky, Noam

    2015-01-01

    Core concepts of language are highly contested. In some cases this is legitimate: real empirical and conceptual issues arise. In other cases, it seems that controversies are based on misunderstanding. A number of crucial cases are reviewed, and an approach to language is outlined that appears to have strong conceptual and empirical motivation, and…

  20. Looking for Core Values

    Science.gov (United States)

    Carter, Margie

    2010-01-01

    People who view themselves as leaders, not just managers or teachers, are innovators who focus on clarifying core values and aligning all aspects of the organization with these values to grow their vision. A vision for an organization can't be just one person's idea. Visions grow by involving people in activities that help them name and create…

  1. Nucleosome Core Particle

    Science.gov (United States)

    1997-01-01

    Nucleosome Core Particle grown on STS-81. The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication. Principal Investigator's are Dr. Dan Carter and Dr. Gerard Bunick of New Century Pharmaceuticals.

  2. Investigation of EAS cores

    Directory of Open Access Journals (Sweden)

    Shaulov S.B.

    2017-01-01

    Full Text Available The development of nuclear-electromagnetic cascade models in air in the late forties have shown informational content of the study of cores of extensive air showers (EAS. These investigations were the main goal in different experiments which were carried out over many years by a variety of methods. Outcomes of such investigations obtained in the HADRON experiment using an X-ray emulsion chamber (XREC as a core detector are considered. The Ne spectrum of EAS associated with γ-ray families, spectra of γ-rays (hadrons in EAS cores and the Ne dependence of the muon number, ⟨Nμ⟩, in EAS with γ-ray families are obtained for the first time at energies of 1015–1017 eV with this method. A number of new effects were observed, namely, an abnormal scaling violation in hadron spectra which are fundamentally different from model predictions, an excess of muon number in EAS associated with γ-ray families, and the penetrating component in EAS cores. It is supposed that the abnormal behavior of γ-ray spectra and Ne dependence of the muon number are explained by the emergence of a penetrating component in the 1st PCR spectrum ‘knee’ range. Nuclear and astrophysical explanations of the origin of the penetrating component are discussed. The necessity of considering the contribution of a single close cosmic-ray source to explain the PCR spectrum in the knee range is noted.

  3. Modeling Core Collapse Supernovae

    Science.gov (United States)

    Mezzacappa, Anthony

    2017-01-01

    Core collapse supernovae, or the death throes of massive stars, are general relativistic, neutrino-magneto-hydrodynamic events. The core collapse supernova mechanism is still not in hand, though key components have been illuminated, and the potential for multiple mechanisms for different progenitors exists. Core collapse supernovae are the single most important source of elements in the Universe, and serve other critical roles in galactic chemical and thermal evolution, the birth of neutron stars, pulsars, and stellar mass black holes, the production of a subclass of gamma-ray bursts, and as potential cosmic laboratories for fundamental nuclear and particle physics. Given this, the so called ``supernova problem'' is one of the most important unsolved problems in astrophysics. It has been fifty years since the first numerical simulations of core collapse supernovae were performed. Progress in the past decade, and especially within the past five years, has been exponential, yet much work remains. Spherically symmetric simulations over nearly four decades laid the foundation for this progress. Two-dimensional modeling that assumes axial symmetry is maturing. And three-dimensional modeling, while in its infancy, has begun in earnest. I will present some of the recent work from the ``Oak Ridge'' group, and will discuss this work in the context of the broader work by other researchers in the field. I will then point to future requirements and challenges. Connections with other experimental, observational, and theoretical efforts will be discussed, as well.

  4. The Earth's Core.

    Science.gov (United States)

    Jeanloz, Raymond

    1983-01-01

    The nature of the earth's core is described. Indirect evidence (such as that determined from seismological data) indicates that it is an iron alloy, solid toward its center but otherwise liquid. Evidence also suggests that it is the turbulent flow of the liquid that generates the earth's magnetic field. (JN)

  5. Some Core Contested Concepts

    Science.gov (United States)

    Chomsky, Noam

    2015-01-01

    Core concepts of language are highly contested. In some cases this is legitimate: real empirical and conceptual issues arise. In other cases, it seems that controversies are based on misunderstanding. A number of crucial cases are reviewed, and an approach to language is outlined that appears to have strong conceptual and empirical motivation, and…

  6. Languages for Dublin Core.

    Science.gov (United States)

    Baker, Thomas

    1998-01-01

    Focusing on languages for the Dublin Core, examines the experience of some related ways to seek semantic interoperability through simplicity: planned languages, interlingua constructs, and pidgins. Also defines the conceptual and organizational problem of maintaining a metadata standard in multiple languages. (AEF)

  7. Core calculations of JMTR

    Energy Technology Data Exchange (ETDEWEB)

    Nagao, Yoshiharu [Japan Atomic Energy Research Inst., Oarai, Ibaraki (Japan). Oarai Research Establishment

    1998-03-01

    In material testing reactors like the JMTR (Japan Material Testing Reactor) of 50 MW in Japan Atomic Energy Research Institute, the neutron flux and neutron energy spectra of irradiated samples show complex distributions. It is necessary to assess the neutron flux and neutron energy spectra of an irradiation field by carrying out the nuclear calculation of the core for every operation cycle. In order to advance core calculation, in the JMTR, the application of MCNP to the assessment of core reactivity and neutron flux and spectra has been investigated. In this study, in order to reduce the time for calculation and variance, the comparison of the results of the calculations by the use of K code and fixed source and the use of Weight Window were investigated. As to the calculation method, the modeling of the total JMTR core, the conditions for calculation and the adopted variance reduction technique are explained. The results of calculation are shown. Significant difference was not observed in the results of neutron flux calculations according to the difference of the modeling of fuel region in the calculations by K code and fixed source. The method of assessing the results of neutron flux calculation is described. (K.I.)

  8. The core and cosmopolitans

    DEFF Research Database (Denmark)

    Dahlander, Linus; Frederiksen, Lars

    2012-01-01

    Users often interact and help each other solve problems in communities, but few scholars have explored how these relationships provide opportunities to innovate. We analyze the extent to which people positioned within the core of a community as well as people that are cosmopolitans positioned...

  9. Schumpeter's core works revisited

    DEFF Research Database (Denmark)

    Andersen, Esben Sloth

    2012-01-01

    This paper organises Schumpeter’s core books in three groups: the programmatic duology,the evolutionaryeconomic duology,and the socioeconomic synthesis. By analysing these groups and their interconnections from the viewpoint of modern evolutionaryeconomics,the paper summarises resolved problems...

  10. Adult educators' core competences

    DEFF Research Database (Denmark)

    Wahlgren, Bjarne

    2016-01-01

    environment as well as the kind of adult education concerned (e.g. basic education, work-related education etc.). However, it seems that it is possible to identify certain competence requirements which transcend national, cultural and functional boundaries. This research note summarises these common or “core...

  11. Adult educators' core competences

    DEFF Research Database (Denmark)

    Wahlgren, Bjarne

    2016-01-01

    environment as well as the kind of adult education concerned (e.g. basic education, work-related education etc.). However, it seems that it is possible to identify certain competence requirements which transcend national, cultural and functional boundaries. This research note summarises these common or “core...

  12. Evidence for three distinct D proteins, which react differentially with anti-Sm autoantibodies, in the cores of the major snRNPs U1, U2, U4/U6 and U5.

    Science.gov (United States)

    Lehmeier, T; Foulaki, K; Lührmann, R

    1990-11-25

    Electrophoresis of the mixture of proteins from purified snRNPs U1, U2, U4/U6 and U5 on SDS-polyacrylamide gels that had been allowed to polymerise in the presence of high TEMED concentrations have revealed the presence of proteins in the snRNPs that previously had eluded detection. The most striking case is that of protein D, heretofore generally observed as a single broad band; in high-TEMED gels, this splits into three clearly-separated bands, identified as three distinct proteins. We have denoted these proteins D1 (16 kDa), D2 (16.5 kDa) and D3 (18 kDa). Chemical and immunological studies have shown that D1 is identical with the common snRNP protein D, whose structure was recently resolved by cDNA cloning (Rokeach et al. (1988), Proc. Natl. Acad. Sci. USA, 85, 4832-4836) and that D2 and D3 are clearly distinct from D1 and very probably from each other. In addition to D1, proteins D2 and D3 are present in purified U1, U2, U4/U6 and U5 snRNPs isolated from HeLa cells, so these also belong to the group of common snRNP proteins. They are also found in snRNPs isolated from mouse cells, indicating that the role of these proteins in the structure and/or function of UsnRNPs has been conserved in evolution. Interestingly, patients with systemic lupus erythematosus produce populations of anti-Sm autoantibodies that react differentially with the D proteins; some recognise all of them and others only a subset. The high-TEMED gels allow improved resolution not only of the D proteins, but also of some of the U5-specific proteins contained in 20S U5 snRNPs, in particular the 15-kDa protein. In addition, under these conditions, the common G protein, previously observed as a single band, appears as a doublet. Whether the additional band represents a distinct common snRNP protein or a post-translationally modified version of G is not yet known.

  13. Application of Core Dynamics Modeling to Core-Mantle Interactions

    Science.gov (United States)

    Kuang, Weijia

    2003-01-01

    Observations have demonstrated that length of day (LOD) variation on decadal time scales results from exchange of axial angular momentum between the solid mantle and the core. There are in general four core-mantle interaction mechanisms that couple the core and the mantle. Of which, three have been suggested likely the dominant coupling mechanism for the decadal core-mantle angular momentum exchange, namely, gravitational core-mantle coupling arising from density anomalies in the mantle and in the core (including the inner core), the electromagnetic coupling arising from Lorentz force in the electrically conducting lower mantle (e.g. D-layer), and the topographic coupling arising from non-hydrostatic pressure acting on the core-mantle boundary (CMB) topography. In the past decades, most effort has been on estimating the coupling torques from surface geomagnetic observations (kinematic approach), which has provided insights on the core dynamical processes. In the meantime, it also creates questions and concerns on approximations in the studies that may invalidate the corresponding conclusions. The most serious problem is perhaps the approximations that are inconsistent with dynamical processes in the core, such as inconsistencies between the core surface flow beneath the CMB and the CMB topography, and that between the D-layer electric conductivity and the approximations on toroidal field at the CMB. These inconsistencies can only be addressed with numerical core dynamics modeling. In the past few years, we applied our MoSST (Modular, Scalable, Self-consistent and Three-dimensional) core dynamics model to study core-mantle interactions together with geodynamo simulation, aiming at assessing the effect of the dynamical inconsistencies in the kinematic studies on core-mantle coupling torques. We focus on topographic and electromagnetic core-mantle couplings and find that, for the topographic coupling, the consistency between the core flow and the CMB topography is

  14. USGS Core Research Center (CRC) Collection of Core

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — The Core Research Center (CRC) was established in 1974 by the U.S. Geological Survey (USGS) to preserve valuable rock cores for use by scientists and educators from...

  15. Ice Cores of the National Ice Core Laboratory

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — The U.S. National Ice Core Laboratory (NICL) is a facility for storing, curating, and studying ice cores recovered from the polar regions of the world. It provides...

  16. Core Outlet Temperature Study

    Energy Technology Data Exchange (ETDEWEB)

    Moisseytsev, A. [Argonne National Laboratory (ANL), Argonne, IL (United States); Hoffman, E. [Argonne National Laboratory (ANL), Argonne, IL (United States); Majumdar, S. [Argonne National Laboratory (ANL), Argonne, IL (United States)

    2008-07-28

    It is a known fact that the power conversion plant efficiency increases with elevation of the heat addition temperature. The higher efficiency means better utilization of the available resources such that higher output in terms of electricity production can be achieved for the same size and power of the reactor core or, alternatively, a lower power core could be used to produce the same electrical output. Since any nuclear power plant, such as the Advanced Burner Reactor, is ultimately built to produce electricity, a higher electrical output is always desirable. However, the benefits of the higher efficiency and electricity production usually come at a price. Both the benefits and the disadvantages of higher reactor outlet temperatures are analyzed in this work.

  17. Geomagnetism of earth's core

    Science.gov (United States)

    Benton, E. R.

    1983-01-01

    Instrumentation, analytical methods, and research goals for understanding the behavior and source of geophysical magnetism are reviewed. Magsat, launched in 1979, collected global magnetometer data and identified the main terrestrial magnetic fields. The data has been treated by representing the curl-free field in terms of a scalar potential which is decomposed into a truncated series of spherical harmonics. Solutions to the Laplace equation then extend the field upward or downward from the measurement level through intervening spaces with no source. Further research is necessary on the interaction between harmonics of various spatial scales. Attempts are also being made to analytically model the main field and its secular variation at the core-mantle boundary. Work is also being done on characterizing the core structure, composition, thermodynamics, energetics, and formation, as well as designing a new Magsat or a tethered satellite to be flown on the Shuttle.

  18. Geomagnetism of earth's core

    Science.gov (United States)

    Benton, E. R.

    1983-01-01

    Instrumentation, analytical methods, and research goals for understanding the behavior and source of geophysical magnetism are reviewed. Magsat, launched in 1979, collected global magnetometer data and identified the main terrestrial magnetic fields. The data has been treated by representing the curl-free field in terms of a scalar potential which is decomposed into a truncated series of spherical harmonics. Solutions to the Laplace equation then extend the field upward or downward from the measurement level through intervening spaces with no source. Further research is necessary on the interaction between harmonics of various spatial scales. Attempts are also being made to analytically model the main field and its secular variation at the core-mantle boundary. Work is also being done on characterizing the core structure, composition, thermodynamics, energetics, and formation, as well as designing a new Magsat or a tethered satellite to be flown on the Shuttle.

  19. Dynamics of core accretion

    Science.gov (United States)

    Nelson, Andrew F.; Ruffert, Maximilian

    2013-02-01

    We perform three-dimensional hydrodynamic simulations of gas flowing around a planetary core of mass Mpl = 10M⊕ embedded in a near Keplerian background flow, using a modified shearing box approximation. We assume an ideal gas behaviour following an equation of state with a fixed ratio of the specific heats, γ = 1.42, consistent with the conditions of a moderate-temperature background disc with solar composition. No radiative heating or cooling is included in the models. We employ a nested grid hydrodynamic code implementing the `Piecewise Parabolic Method' with as many as six fixed nested grids, providing spatial resolution on the finest grid comparable to the present-day diameters of Neptune and Uranus. We find that a strongly dynamically active flow develops such that no static envelope can form. The activity is not sensitive to plausible variations in the rotation curve of the underlying disc. It is sensitive to the thermodynamic treatment of the gas, as modelled by prescribed equations of state (either `locally isothermal' or `locally isentropic') and the temperature of the background disc material. The activity is also sensitive to the shape and depth of the core's gravitational potential, through its mass and gravitational softening coefficient. Each of these factors influences the magnitude and character of hydrodynamic feedback of the small-scale flow on the background, and we conclude that accurate modelling of such feedback is critical to a complete understanding of the core accretion process. The varying flow pattern gives rise to large, irregular eruptions of matter from the region around the core which return matter to the background flow: mass in the envelope at one time may not be found in the envelope at any later time. No net mass accretion into the envelope is observed over the course of the simulation and none is expected, due to our neglect of cooling. Except in cases of very rapid cooling however, as defined by locally isothermal or

  20. Some core contested concepts.

    Science.gov (United States)

    Chomsky, Noam

    2015-02-01

    Core concepts of language are highly contested. In some cases this is legitimate: real empirical and conceptual issues arise. In other cases, it seems that controversies are based on misunderstanding. A number of crucial cases are reviewed, and an approach to language is outlined that appears to have strong conceptual and empirical motivation, and to lead to conclusions about a number of significant issues that differ from some conventional beliefs.

  1. Core-seis: a code for LMFBR core seismic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Chellapandi, P.; Ravi, R.; Chetal, S.C.; Bhoje, S.B. [Indira Gandhi Centre for Atomic Research, Kalpakkam (India). Reactor Group

    1995-12-31

    This paper deals with a computer code CORE-SEIS specially developed for seismic analysis of LMFBR core configurations. For demonstrating the prediction capability of the code, results are presented for one of the MONJU reactor core mock ups which deals with a cluster of 37 subassemblies kept in water. (author). 3 refs., 7 figs., 2 tabs.

  2. Core Exercises: Why You Should Strengthen Your Core Muscles

    Science.gov (United States)

    Healthy Lifestyle Fitness You know core exercises are good for you — but do you include core exercises in your fitness routine? Here's why ... 18, 2014 Original article: http://www.mayoclinic.org/healthy-lifestyle/fitness/in-depth/core-exercises/art-20044751 . Mayo Clinic Footer Legal Conditions and ...

  3. ICF Core Sets for stroke

    National Research Council Canada - National Science Library

    Szilvia Geyh; Alarcos Cieza; Jan Schouten; Hugh Dickson; Peter Frommelt; Zaliha Omar; Nenad Kostanjsek; Haim Ring; Gerold Stucki

    2004-01-01

    Objective: To report on the results of the consensus process integrating evidence from preliminary studies to develop the first version of the Comprehensive ICF Core Set and the Brief ICF Core Set for stroke. Methods...

  4. Hollow-Core Fiber Lamp

    Science.gov (United States)

    Yi, Lin (Inventor); Tjoelker, Robert L. (Inventor); Burt, Eric A. (Inventor); Huang, Shouhua (Inventor)

    2016-01-01

    Hollow-core capillary discharge lamps on the millimeter or sub-millimeter scale are provided. The hollow-core capillary discharge lamps achieve an increased light intensity ratio between 194 millimeters (useful) and 254 millimeters (useless) light than conventional lamps. The capillary discharge lamps may include a cone to increase light output. Hollow-core photonic crystal fiber (HCPCF) may also be used.

  5. On core stability and extendability

    OpenAIRE

    Shellshear, Evan

    2007-01-01

    This paper investigates conditions under which the core of a TU cooperative game is stable. In particular the author extends the idea of extendability to find new conditions under which the core is stable. It is also shown that these new conditions are not necessary for core stability.

  6. Dual-core Itanium Processor

    CERN Multimedia

    2006-01-01

    Intel’s first dual-core Itanium processor, code-named "Montecito" is a major release of Intel's Itanium 2 Processor Family, which implements the Intel Itanium architecture on a dual-core processor with two cores per die (integrated circuit). Itanium 2 is much more powerful than its predecessor. It has lower power consumption and thermal dissipation.

  7. On core stability and extendability

    OpenAIRE

    Shellshear, Evan

    2007-01-01

    This paper investigates conditions under which the core of a TU cooperative game is stable. In particular the author extends the idea of extendability to find new conditions under which the core is stable. It is also shown that these new conditions are not necessary for core stability.

  8. Winning Cores in Parity Games

    DEFF Research Database (Denmark)

    Vester, Steen

    2016-01-01

    We introduce the novel notion of winning cores in parity games and develop a deterministic polynomial-time under-approximation algorithm for solving parity games based on winning core approximation. Underlying this algorithm are a number properties about winning cores which are interesting...... in their own right. In particular, we show that the winning core and the winning region for a player in a parity game are equivalently empty. Moreover, the winning core contains all fatal attractors but is not necessarily a dominion itself. Experimental results are very positive both with respect to quality...

  9. Models of the earth's core

    Science.gov (United States)

    Stevenson, D. J.

    1981-01-01

    Combined inferences from seismology, high-pressure experiment and theory, geomagnetism, fluid dynamics, and current views of terrestrial planetary evolution lead to models of the earth's core with five basic properties. These are that core formation was contemporaneous with earth accretion; the core is not in chemical equilibrium with the mantle; the outer core is a fluid iron alloy containing significant quantities of lighter elements and is probably almost adiabatic and compositionally uniform; the more iron-rich inner solid core is a consequence of partial freezing of the outer core, and the energy release from this process sustains the earth's magnetic field; and the thermodynamic properties of the core are well constrained by the application of liquid-state theory to seismic and labroatory data.

  10. 乙型肝炎病毒包膜区和核心区CTL抗原表位变异对感染慢性化及疾病转归的影响%Impact of CTL epitope mutations of hepatitis B virus envelope protein and core protein on chronicity and disease prognosis in patients with hepatitis B

    Institute of Scientific and Technical Information of China (English)

    哈明昊; 佘会元; 吴建秋; 孙莉萍; 沈文娟; 黄钟鸣; 陈晓兰; 单文艳; 何慧芳

    2015-01-01

    Objective To investigate the impact of cytotoxic T lymphocyte (CTL) epitope mutation of HBV envelope and core protein on disease progress in patients with hepatitis B. Methods 38 individuals with asymptomatic HBsAg carrier,35 patients with chronic hepatitis B and 23 with liver cirrhosis were recruited in this study. Gene sequencing analysis of HBV core region and envelope region was carried out in all enrolled patients to unveil any possible CTL epitope mutation. Results The mutation rates of locus 41~49,88~96,97~108,172~180 and 185~194 of CTL epitope in HBV envelope region were 48(55.2%),13(14.9%),11(12.6%),9(10.3%) and 6 (6.9%),respectively;60% of mutation at locus 41~49 in patients with chronic hepatitis B and 65.2% in with liver cirrhosis were significantly higher than 31.6% in asymptomatic HBsAg carriers(P<0.05);the mutation rates at 18~27 and 91~95 locus of CTL epitope in HBV core region were 56.2% and 43.8%,respectively;the mutation rates at locus 18~27 were also higher in patients with CHB (20.0%) and LC (30.4%) than that in asymptomatic HBsAg carriers (10.5%,P<0.05);Multivariate analysis showed that locus 41~49 mutation was an independent risk factor for the progress of CHB to LC. Conclusion CTL epitope mutation of HBV is a significant cause for the chronicity of hepatitis B virus infection.%目的:探讨HBV包膜区和核心区细胞毒性T淋巴细胞(CTL)抗原表位变异的规律及对疾病发展转归的影响。方法本研究选择无症状HBsAg携带者(ASC)38例,慢性乙型肝炎(CHB)35例,乙型肝炎肝硬化(LC)23例。采用DNA测序法检测HBV包膜区和核心区病毒序列及表位变异情况,并分析其与临床转归的关系。结果在入组患者中,HBV包膜区CTL抗原表位41~49、88~96、97~108、172~180和185~194变异率分别为48个(55.2%)、13个(14.9%)、11个(12.6%)、9个(10.3%)和6个(6.9%);HBV CTL 41~49位变异率在CHB和肝硬化患者中的变异率分别为60.0%

  11. 丙型肝炎病毒核心蛋白通过自诱导方式可溶性表达及其生物学功能鉴定%The biological function of auto-induced expression of the hepatitis C virus soluble core protein

    Institute of Scientific and Technical Information of China (English)

    龚旭阳; 马其欢; 杜茜; 胡接力; 蔡雪飞; 黄爱龙

    2013-01-01

    Objective To investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro ceil-based system.Methods The PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector.The recombinant plasmid was transformed into BL21 (DE3)pLysS E.coli to achieve high-concentration expression of the recombinant C protein by auto-induction.The recombinant protein C was purified by NiNTA affinity chromatography,and tested in a protein binding assay for its ability to bind the HCV NS3 protein.Results The transformed E.coli produced a large amount of recombinant protein C,as detected in the sonicated supernatant of the bacteria culture.The antigenic reactivity of the recombinant protein C was confirmed by western blotting.However,the recombinant protein C could not be purified by Ni-NTA affinity chromatography,but co-precipitated with the HCV NS3 protein.Conclusion Soluble recombinant protein C was successfully expressed by auto-induction,and shown to interact with the HCV NS3 protein,which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes.Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.%目的 构建包含HCV核心蛋白全长基因的重组原核表达质粒,经转化表达型大肠杆菌后自诱导培养以获取可溶性重组核心蛋白,并检测其生物学功能. 方法 以H/FL质粒为模板,通过PCR扩增全长HCV核心蛋白DNA.PCR产物经双酶切后定向克隆到pET28a原核表达载体中.将重组的原核表达载体转化表达型大肠杆菌BL21 (DE3) pLysS,并通过自诱导培养的方式使其在高浓度状态下表达重组核心蛋白.重组核心蛋白经Western blot检测鉴定生物学功能,并与HCV NS3蛋白进行相

  12. Analysis of protein interaction networks related to core transcription factors target genes in embryonic stem cells%胚胎干细胞核心转录因子靶基因集蛋白互作网络特征分析

    Institute of Scientific and Technical Information of China (English)

    左长清; 汪宗桂; 吴铁; 崔燎

    2011-01-01

    BACKGROUND: It is sufficient to reprogram somatic cells, through transduction of some core transcription factors, into pluripotent stem cells (iPS) that exhibit the essential characteristics of embryonic stem (ES) cells. At present, the complex mechanism is not yet fully understood.OBJECTIVE: To study the protein interaction networks related to core transcription factors target genes in embryonic stem cells and to obtain regulatory mechanism controlled "stemness".METHODS: Non-redundant protein interaction data (NRPD) were obtained after removal of redundant data in BioGRID database.Protein interaction pairs, formed by the target genes, were extracted by perl program and the largest continuous protein interaction networks were obtained through searching NRPD using breadth-first search algorithm. At the same time, 1 000 random networks were analyzed and compared. At last, network visualization was analyzed through the Cytoscape software and network characteristics were explained using complex scale-free network model of Barabasi-Albert.RESULTS AND CONCLUSION : More protein interaction pairs and larger continuous protein network, statistically significant difference compared with random genes, were formed by core transcription factor target genes. The continuous protein network is scale-free characteristics of complex networks. This study has suggested that target genes may regulate synergistically "stemness" characteristics of embryonic stem cells through close interaction and forming a network module.%背景:外源性核心转录因子导入终末分化细胞能产生具有与胚胎干细胞特性相似的诱导多潜能干细胞,其复杂机制目前尚未完全阐明.目的:分析核心转录因子靶基因集蛋白互作网络特征,获得其影响"干性"特征的调控机制.方法:去除BioGRID数据库中的冗余数据,获得非冗余蛋白互作数据库.perl程序搜索靶基因集组成的蛋白互作对,广度优先算法搜索非冗余蛋白互作数

  13. Understanding core conductor fabrics

    Energy Technology Data Exchange (ETDEWEB)

    Swenson, D E, E-mail: deswenson@affinity-esd.com [Affinity Static Control Consulting, LLC 2609 Quanah Drive, Round Rock, Texas, 78681 (United States)

    2011-06-23

    ESD Association standard test method ANSI/ESD STM2.1 - Garments (STM2.1), provides electrical resistance test procedures that are applicable for materials and garments that have surface conductive or surface dissipative properties. As has been reported in other papers over the past several years{sup 1} fabrics are now used in many industries for electrostatic control purposes that do not have surface conductive properties and therefore cannot be evaluated using the procedures in STM2.1{sup 2}. A study was conducted to compare surface conductive fabrics with samples of core conductor fibre based fabrics in order to determine differences and similarities with regards to various electrostatic properties. This work will be used to establish a new work item proposal within WG-2, Garments, in the ESD Association Standards Committee in the USA.

  14. Growth outside the core.

    Science.gov (United States)

    Zook, Chris; Allen, James

    2003-12-01

    Growth in an adjacent market is tougher than it looks; three-quarters of the time, the effort fails. But companies can change those odds dramatically. Results from a five-year study of corporate growth conducted by Bain & Company reveal that adjacency expansion succeeds only when built around strong core businesses that have the potential to become market leaders. And the best place to look for adjacency opportunities is inside a company's strongest customers. The study also found that the most successful companies were able to consistently, profitably outgrow their rivals by developing a formula for pushing out the boundaries of their core businesses in predictable, repeatable ways. Companies use their repeatability formulas to expand into any number of adjacencies. Some companies make repeated geographic moves, as Vodafone has done in expanding from one geographic market to another over the past 13 years, building revenues from $1 billion in 1990 to $48 billion in 2003. Others apply a superior business model to new segments. Dell, for example, has repeatedly adapted its direct-to-customer model to new customer segments and new product categories. In other cases, companies develop hybrid approaches. Nike executed a series of different types of adjacency moves: it expanded into adjacent customer segments, introduced new products, developed new distribution channels, and then moved into adjacent geographic markets. The successful repeaters in the study had two common characteristics. First, they were extraordinarily disciplined, applying rigorous screens before they made an adjacency move. This discipline paid off in the form of learning curve benefits, increased speed, and lower complexity. And second, in almost all cases, they developed their repeatable formulas by studying their customers and their customers' economics very, very carefully.

  15. Complex coacervate core micelles

    NARCIS (Netherlands)

    Voets, I.K.; Keizer, de A.; Cohen Stuart, M.A.

    2009-01-01

    In this review we present an overview of the literature on the co-assembly of neutral-ionic block, graft, and random copolymers with oppositely charged species in aqueous solution. Oppositely charged species include synthetic (co)polymers of various architectures, biopolymers - such as proteins, enz

  16. Complex coacervate core micelles

    NARCIS (Netherlands)

    Voets, I.K.; Keizer, de A.; Cohen Stuart, M.A.

    2009-01-01

    In this review we present an overview of the literature on the co-assembly of neutral-ionic block, graft, and random copolymers with oppositely charged species in aqueous solution. Oppositely charged species include synthetic (co)polymers of various architectures, biopolymers - such as proteins,

  17. Advances in core drilling technology

    Science.gov (United States)

    Holdsworth, G.

    Some notable technical advances in drill design were reported at the meeting, held in Canada August 30-September 1, 1982, at the University of Calgary. Chief amongst these was a battery powered, computer assisted electromechanical core drill which has recently been used by the Danes in Greenland to continuously core to the base of the ice sheet at 2038 m. This is the deepest coring operation so far on the Greenland ice sheet. (The record for deep glacier drilling is held by the U.S. Army Cold Regions Research and Engineering Laboratory for the continuous coring through 2164 m of ice to bedrock at Byrd Station, Antarctica, in 1968). In early 1982, a current Soviet core drilling operation was reported to be at a depth of 2000 m at Vostok station, Antarctica, where the total ice thickness is about 4000 m; the goal of core drilling the entire ice thickness there could be achieved before the end of 1983.

  18. 芹菜素在静水压下对人体腰椎间盘髓核蛋白多糖核心蛋白基因表达的影响%Effects of apigenin on proteoglycan core protein' s gene expression in the human lumbar disc nucleus pulposus in response to hydrostatic pressure

    Institute of Scientific and Technical Information of China (English)

    柳根哲; 乔卫平; 徐林; 李春根; 孙旗; 曲弋; 陈江

    2011-01-01

    [ Objective ] To investigate the effects of apigenin on proteoglycan core protein gene expression in the human lumbar intervertebral disc nucleus pulposus in response to hydrostatic pressure. [ Methods] Thirty -two lumbar intervertebral disc nucleus pulposus specimens were obtained from patients who had undergone posterior discectomy. The syringes were placed in a water filled pressure vessel kept at 37℃. Hydrostatic pressures of 1 ( control), 3 and 30 atmospheres (atm) were applied. After incubation of 2 hours, each nucleus pulposus specimen was embedded in paraffin, sliced for situ hybridization to study the effect of the apigenin of the intervertebral disc nucleus pulposus proteoglycan core protein gene' s transcription. As a competitive inhibitor of NO synthases, NG - Monomethy - L - arginine ( L - NMMA, 500 μmol) and as an organic donor of NO, S - nitroso - N - acetylpenicillamine ( SNAP, 200 μmol) were used. [ Results] The results of apigenin 3 atm group and the L - NMMA 3 atm group in situ hybridization to proteoglycan core protein showed the largest number of positive nucleus pulposus cells, with a largest average optical density. [ Conclusion] For the degenerative intervertebral disc nucleus pulposus, apigenin plays an important role in the promotion of proteoglycan core protein mRNA gene' s transcription levels.%[目的]探讨中药提取物芹菜素在静水压下对体外培养的人体腰椎间盘髓核蛋白多糖核心蛋白基因表达的影响.[方法]32例后路腰椎间盘髓核摘除术的志愿患者中获取的32个新鲜椎间盘髓核样品,每1例髓核样品被切成1~2 mm3大小的碎块,与1 ml培养基DMEM一同装入2.5 ml的注射器中.培养基中分别加入芹菜素(Apigenin)、一氧化氮合成酶的竞争性抑制剂NG-Monomethyl-L-arginine(简称L-NMMA)及一氧化氮的供体Sodium Nitroprusside(简称SNAP),在静水压装置中培养2 h后,对每组中的髓核样品进行石蜡包埋、切片,通过原位杂交来研

  19. Core TuLiP

    OpenAIRE

    Czenko, M.R.; Etalle, Sandro

    2007-01-01

    We propose CoreTuLiP - the core of a trust management language based on Logic Programming. CoreTuLiP is based on a subset of moded logic programming, but enjoys the features of TM languages such as RT; in particular clauses are issued by different authorities and stored in a distributed manner. We present a lookup and inference algorithm which we prove to be correct and complete w.r.t. the declarative semantics. CoreTuLiP enjoys uniform syntax and the well-established semantics and is express...

  20. Biochemistry Instrumentation Core Technology Center

    Data.gov (United States)

    Federal Laboratory Consortium — The UCLA-DOE Biochemistry Instrumentation Core Facility provides the UCLA biochemistry community with easy access to sophisticated instrumentation for a wide variety...

  1. Waves in the core and mechanical core-mantle interactions

    DEFF Research Database (Denmark)

    Jault, D.; Finlay, Chris

    2015-01-01

    the motions in the direction parallel to the Earth'srotation axis. This property accounts for the signicance of the core-mantle topography.In addition, the stiening of the uid in the direction parallel to the rotation axis gives riseto a magnetic diusion layer attached to the core-mantle boundary, which would...

  2. Characterizing the Core via K-Core Covers

    NARCIS (Netherlands)

    Sanchez, S.M.; Borm, P.E.M.; Estevez, A.

    2013-01-01

    This paper extends the notion of individual minimal rights for a transferable utility game (TU-game) to coalitional minimal rights using minimal balanced families of a specific type, thus defining a corresponding minimal rights game. It is shown that the core of a TU-game coincides with the core of

  3. Characterizing the Core via K-Core Covers

    NARCIS (Netherlands)

    Sanchez, S.M.; Borm, P.E.M.; Estevez, A.

    2013-01-01

    This paper extends the notion of individual minimal rights for a transferable utility game (TU-game) to coalitional minimal rights using minimal balanced families of a specific type, thus defining a corresponding minimal rights game. It is shown that the core of a TU-game coincides with the core of

  4. Relativistic frozen core potential scheme with relaxation of core electrons

    Science.gov (United States)

    Nakajima, Yuya; Seino, Junji; Hayami, Masao; Nakai, Hiromi

    2016-10-01

    This letter proposes a relaxation scheme for core electrons based on the frozen core potential method at the infinite-order Douglas-Kroll-Hess level, called FCP-CR. The core electrons are self-consistently relaxed using frozen molecular valence potentials after the valence SCF calculation is performed. The efficiency of FCP-CR is confirmed by calculations of gold clusters. Furthermore, FCP-CR reproduces the results of the all-electron method for the energies of coinage metal dimers and the core ionization energies and core level shifts of vinyl acetate and three tungsten complexes at the Hartree-Fock and/or symmetry-adapted cluster configuration interaction levels.

  5. HCV Core Residues Critical for Infectivity Are Also Involved in Core-NS5A Complex Formation

    Science.gov (United States)

    Gawlik, Katarzyna; Baugh, James; Chatterji, Udayan; Lim, Precious J.; Bobardt, Michael D.; Gallay, Philippe A.

    2014-01-01

    Hepatitis C virus (HCV) infection is a major cause of liver disease. The molecular machinery of HCV assembly and particle release remains obscure. A better understanding of the assembly events might reveal new potential antiviral strategies. It was suggested that the nonstructural protein 5A (NS5A), an attractive recent drug target, participates in the production of infectious particles as a result of its interaction with the HCV core protein. However, prior to the present study, the NS5A-binding site in the viral core remained unknown. We found that the D1 domain of core contains the NS5A-binding site with the strongest interacting capacity in the basic P38-K74 cluster. We also demonstrated that the N-terminal basic residues of core at positions 50, 51, 59 and 62 were required for NS5A binding. Analysis of all substitution combinations of R50A, K51A, R59A, and R62A, in the context of the HCVcc system, showed that single, double, triple, and quadruple mutants were fully competent for viral RNA replication, but deficient in secretion of viral particles. Furthermore, we found that the extracellular and intracellular infectivity of all the mutants was abolished, suggesting a defect in the formation of infectious particles. Importantly, we showed that the interaction between the single and quadruple core mutants and NS5A was impaired in cells expressing full-length HCV genome. Interestingly, mutations of the four basic residues of core did not alter the association of core or NS5A with lipid droplets. This study showed for the first time that basic residues in the D1 domain of core that are critical for the formation of infectious extracellular and intracellular particles also play a role in core-NS5A interactions. PMID:24533158

  6. Adult educators' core competences

    Science.gov (United States)

    Wahlgren, Bjarne

    2016-06-01

    Which competences do professional adult educators need? This research note discusses the topic from a comparative perspective, finding that adult educators' required competences are wide-ranging, heterogeneous and complex. They are subject to context in terms of national and cultural environment as well as the kind of adult education concerned (e.g. basic education, work-related education etc.). However, it seems that it is possible to identify certain competence requirements which transcend national, cultural and functional boundaries. This research note summarises these common or "core" requirements, organising them into four thematic subcategories: (1) communicating subject knowledge; (2) taking students' prior learning into account; (3) supporting a learning environment; and (4) the adult educator's reflection on his or her own performance. At the end of his analysis of different competence profiles, the author notes that adult educators' ability to train adult learners in a way which then enables them to apply and use what they have learned in practice (thus performing knowledge transfer) still seems to be overlooked.

  7. Core of Cloud Computing

    Directory of Open Access Journals (Sweden)

    Prof. C.P.Chandgude

    2017-04-01

    Full Text Available Advancement in computing facilities marks back from 1960’s with introduction of mainframes. Each of the computing has one or the other issues, so keeping this in mind cloud computing was introduced. Cloud computing has its roots in older technologies such as hardware virtualization, distributed computing, internet technologies, and autonomic computing. Cloud computing can be described with two models, one is service model and second is deployment model. While providing several services, cloud management’s primary role is resource provisioning. While there are several such benefits of cloud computing, there are challenges in adopting public clouds because of dependency on infrastructure that is shared by many enterprises. In this paper, we present core knowledge of cloud computing, highlighting its key concepts, deployment models, service models, benefits as well as security issues related to cloud data. The aim of this paper is to provide a better understanding of the cloud computing and to identify important research directions in this field

  8. Maximum stellar iron core mass

    Indian Academy of Sciences (India)

    F W Giacobbe

    2003-03-01

    An analytical method of estimating the mass of a stellar iron core, just prior to core collapse, is described in this paper. The method employed depends, in part, upon an estimate of the true relativistic mass increase experienced by electrons within a highly compressed iron core, just prior to core collapse, and is significantly different from a more typical Chandrasekhar mass limit approach. This technique produced a maximum stellar iron core mass value of 2.69 × 1030 kg (1.35 solar masses). This mass value is very near to the typical mass values found for neutron stars in a recent survey of actual neutron star masses. Although slightly lower and higher neutron star masses may also be found, lower mass neutron stars are believed to be formed as a result of enhanced iron core compression due to the weight of non-ferrous matter overlying the iron cores within large stars. And, higher mass neutron stars are likely to be formed as a result of fallback or accretion of additional matter after an initial collapse event involving an iron core having a mass no greater than 2.69 × 1030 kg.

  9. The INTEGRAL Core Observing Programme

    OpenAIRE

    Winkler, C; Gehrels, N.; Lund, N.; Schoenfelder, V.; Ubertini, P.

    1999-01-01

    The Core Programme of the INTEGRAL mission is defined as the portion of the scientific programme covering the guaranteed time observations for the INTEGRAL Science Working Team. This paper describes the current status of the Core Programme preparations and summarizes the key elements of the observing programme.

  10. The INTEGRAL Core Observing Programme

    DEFF Research Database (Denmark)

    Winkler, C.; Gehrels, N.; Lund, Niels

    1999-01-01

    The Core Programme of the INTEGRAL mission is defined as the portion of the scientific programme covering the guaranteed time observations for the INTEGRAL Science Working Team. This paper describes the current status of the Core Programme preparations and summarizes the key elements...

  11. Complicated Politics to the Core

    Science.gov (United States)

    McGuinn, Patrick

    2015-01-01

    People dislike the Common Core for several different reasons, and so it is important to disaggregate the sources of opposition and to assess and then to dispel some of the myths that have built up around it. It also is important to understand the unusual political alliances that have emerged in opposition to Common Core implementation and how they…

  12. Common Core: Victory Is Yours!

    Science.gov (United States)

    Fink, Jennifer L. W.

    2012-01-01

    In this article, the author discusses how to implement the Common Core State Standards in the classroom. She presents examples and activities that will leave teachers feeling "rosy" about tackling the new standards. She breaks down important benchmarks and shows how other teachers are doing the Core--and loving it!

  13. The INTEGRAL Core Observing Programme

    DEFF Research Database (Denmark)

    Winkler, C.; Gehrels, N.; Lund, Niels

    1999-01-01

    The Core Programme of the INTEGRAL mission is defined as the portion of the scientific programme covering the guaranteed time observations for the INTEGRAL Science Working Team. This paper describes the current status of the Core Programme preparations and summarizes the key elements...

  14. Complicated Politics to the Core

    Science.gov (United States)

    McGuinn, Patrick

    2015-01-01

    People dislike the Common Core for several different reasons, and so it is important to disaggregate the sources of opposition and to assess and then to dispel some of the myths that have built up around it. It also is important to understand the unusual political alliances that have emerged in opposition to Common Core implementation and how they…

  15. [Alternative scaffold proteins].

    Science.gov (United States)

    Petrovskaia, L E; Shingarova, L N; Dolgikh, D A; Kirpichnikov, M P

    2011-01-01

    Review is devoted to the challenging direction in modem molecular biology and bioengineering - the properties of alternative scaffold proteins (ASP) and methods for obtaining ASP binding molecules. ASP molecules incorporate conservative protein core and hypervariable regions, providing for the binding function. Structural classification of ASP includes several types which differ also in their molecular targets and potential applications. Construction of artificial binding proteins on the ASP basis implies a combinatorial library design with subsequent selection of specific binders with the use of phage display or the modem cell-free systems. Alternative binding proteins on non-immunoglobulin scaffolds find broad applications in different fields ofbiotechnology and molecular medicine.

  16. Core body temperature in obesity.

    Science.gov (United States)

    Heikens, Marc J; Gorbach, Alexander M; Eden, Henry S; Savastano, David M; Chen, Kong Y; Skarulis, Monica C; Yanovski, Jack A

    2011-05-01

    A lower core body temperature set point has been suggested to be a factor that could potentially predispose humans to develop obesity. We tested the hypothesis that obese individuals have lower core temperatures than those in normal-weight individuals. In study 1, nonobese [body mass index (BMI; in kg/m(2)) temperature-sensing capsules, and we measured core temperatures continuously for 24 h. In study 2, normal-weight (BMI of 18-25) and obese subjects swallowed temperature-sensing capsules to measure core temperatures continuously for ≥48 h and kept activity logs. We constructed daily, 24-h core temperature profiles for analysis. Mean (±SE) daily core body temperature did not differ significantly between the 35 nonobese and 46 obese subjects (36.92 ± 0.03°C compared with 36.89 ± 0.03°C; P = 0.44). Core temperature 24-h profiles did not differ significantly between 11 normal-weight and 19 obese subjects (P = 0.274). Women had a mean core body temperature ≈0.23°C greater than that of men (36.99 ± 0.03°C compared with 36.76 ± 0.03°C; P body temperature. It may be necessary to study individuals with function-altering mutations in core temperature-regulating genes to determine whether differences in the core body temperature set point affect the regulation of human body weight. These trials were registered at clinicaltrials.gov as NCT00428987 and NCT00266500.

  17. The core proteome and pan proteome of Salmonella Paratyphi A epidemic strains.

    Directory of Open Access Journals (Sweden)

    Li Zhang

    Full Text Available Comparative proteomics of the multiple strains within the same species can reveal the genetic variation and relationships among strains without the need to assess the genomic data. Similar to comparative genomics, core proteome and pan proteome can also be obtained within multiple strains under the same culture conditions. In this study we present the core proteome and pan proteome of four epidemic Salmonella Paratyphi A strains cultured under laboratory culture conditions. The proteomic information was obtained using a Two-dimensional gel electrophoresis (2-DE technique. The expression profiles of these strains were conservative, similar to the monomorphic genome of S. Paratyphi A. Few strain-specific proteins were found in these strains. Interestingly, non-core proteins were found in similar categories as core proteins. However, significant fluctuations in the abundance of some core proteins were also observed, suggesting that there is elaborate regulation of core proteins in the different strains even when they are cultured in the same environment. Therefore, core proteome and pan proteome analysis of the multiple strains can demonstrate the core pathways of metabolism of the species under specific culture conditions, and further the specific responses and adaptations of the strains to the growth environment.

  18. Mobilities of the inner three core residues and the Man(alpha 1--6) branch of the glycan at Asn78 of the alpha-subunit of human chorionic gonadotropin are restricted by the protein.

    Science.gov (United States)

    van Zuylen, C W; de Beer, T; Leeflang, B R; Boelens, R; Kaptein, R; Kamerling, J P; Vliegenthart, J F

    1998-02-17

    Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone involved in the maintenance of the corpus luteum in early pregnancy. The free alpha-subunit of hCG has a biological activity of its own, namely, stimulation of prolactin secretion from term pregnancy decidual cells [Blithe, D. L., et al. (1991) Endocrinology 129, 2257-2259]. Glycosylation at Asn78 of the alpha-subunit is required for the stability of the protein, but the exact nature of the stabilizing effect is not known. In our previous study, it was indicated that GlcNAc-1 at Asn78 has a reduced mobility, whereas the glycan at Asn52 is highly mobile [De Beer, T., et al. (1996) Eur. J. Biochem. 241, 229-242]. In the present investigation, it is shown that the PNGase F susceptibility of the Asn52-linked glycan in the free alpha-subunit is absent in the heterodimer. Thus, the high mobility of the glycan at Asn52 may be characteristic for the free alpha-subunit. For accurate modeling of alpha hCG, knowledge of the behavior of each of the glycans is essential. In this context, the mobility of the glycans and their interactions with the protein are explored by NMR spectroscopy using desialylated, partially deglycosylated free alpha-subunit (as-pd alpha) carrying glycans at Asn78 only. NOEs between GlcNAc-2 and several amino acid residues indicate that GlcNAc-2 is involved in stabilizing alpha hCG. From the values of 13C relaxation parameters T2 and T1 rho of the constituting monosaccharide residues, it was concluded that the inner three residues have a severely restricted mobility. The Man-4 and Man-4' residues of the diantennary oligosaccharide exhibit a similar relaxation behavior, suggesting that the Man-4' branch occurs in a single conformation of the C5-C6 linkage of Man-3 instead of in rapidly interconverting conformations that are known to exist for this linkage for the free oligosaccharide.

  19. Multiple structural alignment and core detection by geometric hashing.

    Science.gov (United States)

    Leibowitz, N; Fligelman, Z Y; Nussinov, R; Wolfson, H J

    1999-01-01

    A Multiple Structural Alignment algorithm is presented. The algorithm accepts an ensemble of protein structures and finds the largest substructure (core) of C alpha atoms whose geometric configuration appear in all the molecules of the ensemble (core). Both the detection of this core and the resulting structural alignment are done simultaneously. Other large enough multistructural superimpositions are detected as well. Our method is based on the Geometric Hashing paradigm and a superimposition clustering technique which represents superimpositions by sets of matching atoms. The algorithm proved to be efficient on real data in a series of experiments. The same method can be applied to any ensemble of molecules (not necessarily proteins) since our basic technique is sequence order independent.

  20. HBc二聚体组装核衣壳相关功能域突变对HBV复制的影响%The effect of mutation of hepatitis B virus core protein dimer interface domain related to nucleocapsid formation on HBV replication

    Institute of Scientific and Technical Information of China (English)

    邓凯; 蒋栋; 韩进超; 潘孝本; 王豪; 魏来

    2010-01-01

    Objective To investigate the effect of hepatitis B virus core protein (HBc) dimer interfaces amino acids mutation on nucleocapsid assembly and HBV DNA replication. Methods Based on HBc three dimension structure, four HBc dimer interfaces domain mutation plasmids, pHBc14-18M,pHBc120-135M,pHBc23-39M and pHBc122-139M were constructed in pcDNA3.1 vector by PCR site-directed mutagenesis, there was a flag-tag at the C-terminal of all mutants for easy detection. Wild type core protein plasmid 1-183flag was also constructed as a positive control. The 4 mutants were cotransfected HepG2 cells with pHBV1.2 core negative plasmid (pHBV1.2-core-) ,which contained 1.2 copies of HBV whole genome but the core protein would not express due to a stop codon. The capsid formation, HBV pregenome(pgRNA) and HBV DNA replication mediate were analyzed by native agarose gel electrophoresis and Western blot, Northern blot and Southern blot , respectively. The 4 mutants were also cotransfected HepG2 cells with HBV wild type plasmid pHBV1.2 and examined by Southern blot. Virions in the medium were determined by native agarose gel electrophoresis and Western blot. Results Cotransfecting HepG2 cells with pHBV1.2-core- plasmid, pHBc14-18M,pHBc120-135M and pHBc122-139M mutant groups formed nucleocapsid-like structure but pHBc23-39M could not, Northern and Southern blot displayed no signal in all mutants except 1-183flag conrol group. In pHBV1.2 cotransfection experiment, HBV DNA replication was blocked in pHBc14-18M, pHBc120-135M and pHBc122-139M mutant groups, sharply decreased in pHBc120-135M and pHBc122-139M groups, correspondingly virons production in medium were also inhibited. pHBc23-39M mutant exerted no influence on HBV replication. Conclusion pHBc23-39M mutant can neither form nucleocapsid-like structure nor interact with wild type HBc dimmer to interfere HBV replication.On the contrast, pHBc14-18M, pHBc120-135M and pHBc122-139M mutants can form nucleocapsid-like structure by themselves

  1. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the best-studied

  2. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the

  3. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the best-studied

  4. Genome evolution in cyanobacteria: the stable core and the variable shell.

    Science.gov (United States)

    Shi, Tuo; Falkowski, Paul G

    2008-02-19

    Cyanobacteria are the only known prokaryotes capable of oxygenic photosynthesis, the evolution of which transformed the biology and geochemistry of Earth. The rapid increase in published genomic sequences of cyanobacteria provides the first opportunity to reconstruct events in the evolution of oxygenic photosynthesis on the scale of entire genomes. Here, we demonstrate the overall phylogenetic incongruence among 682 orthologous protein families from 13 genomes of cyanobacteria. However, using principal coordinates analysis, we discovered a core set of 323 genes with similar evolutionary trajectories. The core set is highly conserved in amino acid sequence and contains genes encoding the major components in the photosynthetic and ribosomal apparatus. Many of the key proteins are encoded by genome-wide conserved small gene clusters, which often are indicative of protein-protein, protein-prosthetic group, and protein-lipid interactions. We propose that the macromolecular interactions in complex protein structures and metabolic pathways retard the tempo of evolution of the core genes and hence exert a selection pressure that restricts piecemeal horizontal gene transfer of components of the core. Identification of the core establishes a foundation for reconstructing robust organismal phylogeny in genome space. Our phylogenetic trees constructed from 16S rRNA gene sequences, concatenated orthologous proteins, and the core gene set all suggest that the ancestral cyanobacterium did not fix nitrogen and probably was a thermophilic organism.

  5. COSIS: COre State Indication System

    Energy Technology Data Exchange (ETDEWEB)

    Cho, J. Y.; Lee, K. B.; Koo, B. S.; Lee, W. K.; Lee, C. C; Zee, S. Q

    2006-02-15

    COSIS (COre State Indication System) which implemented in the SMART research reactor plays a role to supply the core state parameters or graphs for the operator to recognize the core state effectively. The followings are the main functions of COSIS. (1) Validity Check for the Process Signals and Determination of the COSIS Inputs (SIGVAL) (2) Coolant Flow Rate Calculation (FLOW) (3) Core Thermal Power Calculation (COREPOW) (4) In-core 3-Dimensional Power Distribution Calculation and Peaking Parameters Generation (POWER3D) (5) Azimuthal Tilt Calculation (AZITILT). This report describes the methodology of COSIS which produces the core state parameters using the process and detector signals. In the SIGVAL module, COSIS checks most signals except for the CEA position and determines the input signals. In the FLOW module, the corelation coefficient between the RPM signal and coolant flow is updated from the energy balance at the steam generator, and the coolant flow rate is calculated using the RPM signal. In the COREPOW module, the secondary calorimetric power, the primary {delta}T power and the ex-core power are calculated, and the final core thermal power and biased core power are determined. In the POWER3D module, the 3-dimensional power distribution is calculated using the in-core detector signal, and the 3-D peaking factor, 2-D radial peaking factor, axial offset, maximum linear power density are produced. In the AZITILT module, the arithmetic averaged and vector averaged azimuthal tilts are calculated, and the final tilt is determined. The COSIS performance test of the COSIS is performed for the temperature compensation method, the COREPOW and the POWER3D modules. The test for the temperature compensation method is performed for the temperature variations of the linear, parabolic, exponential, sine function. The test shows that the implemented temperature compensation method works soundly. The COREPOW test is performed by varying the core power from the initial

  6. Epistemology and ontology in core ontologies: FOLaw and LRI-Core, two core ontologies for law

    NARCIS (Netherlands)

    Breukers, J.A.P.J.; Hoekstra, R.J.

    2004-01-01

    For more than a decade constructing ontologies for legal domains, we, at the Leibniz Center for Law, felt really the need to develop a core ontology for law that would enable us to re-use the common denominator of the various legal domains. In this paper we present two core ontologies for law. The

  7. Characterization of nonconventional hepatitis B viruses lacking the core promoter.

    Science.gov (United States)

    Chang, Shau-Feng; Chang, Shih-Hsuan; Li, Bi-Chen; Will, Hans; Netter, Hans Jürgen

    2004-12-20

    The core gene (C-gene) promoter and regulatory sequences play a central role in the hepatitis B virus (HBV) life cycle. They are essential for the synthesis of the pregenomic and precore mRNA. The pregenomic RNA is the template required for replication and also the template for the synthesis of the core protein and polymerase. Here, we report the in vivo existence and functional characterization of HBV variants that lack the C-gene promoter region and the regulatory sequences located therein. HBV promoter fragments were isolated by PCR from sera of chronic carriers and characterized. Truncated promoter elements were identified, and then tested in the context of wild-type genomes in the HuH-7 cell line. The expression of the recombinant HBV genome resulted in the synthesis of surface proteins, and low level of core protein as well as a transcript pattern similar to, but smaller in size to wild-type virus. The recombinant HBV genome with the truncated promoter region produced pregenomic RNA-like transcripts. These transcripts were encapsidated and reverse transcribed when complemented by sufficient core and polymerase protein. These date provide an explanation as to why such deletion mutants of HBV can be produced at all, they highlight the functional potentials of viral sequences activated by mutations and may be of relevance for viral evolution and persistence.

  8. Fluorophore-cored dendrimers for patterns in metalloprotein sensing.

    Science.gov (United States)

    Jiwpanich, Siriporn; Sandanaraj, Britto S; Thayumanavan, S

    2009-02-21

    In fluorophore-cored dendrimers with peripheral binding functionalities, the effect of generation upon protein binding-induced fluorescence quenching can be unpredictable; this is because the increase in fluorophore-binding functionality distance with generation is also accompanied by an increase in the number of binding moieties and the interplay between the two features is utilized to create patterns for metalloprotein sensing.

  9. Regulatory Enhancer-Core-Promoter Communication via Transcription Factors and Cofactors.

    Science.gov (United States)

    Zabidi, Muhammad A; Stark, Alexander

    2016-12-01

    Gene expression is regulated by genomic enhancers that recruit transcription factors and cofactors to activate transcription from target core promoters. Over the past years, thousands of enhancers and core promoters in animal genomes have been annotated, and we have learned much about the domain structure in which regulatory genomes are organized in animals. Enhancer-core-promoter targeting occurs at several levels, including regulatory domains, DNA accessibility, and sequence-encoded core-promoter specificities that are likely mediated by different regulatory proteins. We review here current knowledge about enhancer-core-promoter targeting, regulatory communication between enhancers and core promoters, and the protein factors involved. We conclude with an outlook on open questions that we find particularly interesting and that will likely lead to additional insights in the upcoming years.

  10. Multi-Core Cache Hierarchies

    CERN Document Server

    Balasubramonian, Rajeev

    2011-01-01

    A key determinant of overall system performance and power dissipation is the cache hierarchy since access to off-chip memory consumes many more cycles and energy than on-chip accesses. In addition, multi-core processors are expected to place ever higher bandwidth demands on the memory system. All these issues make it important to avoid off-chip memory access by improving the efficiency of the on-chip cache. Future multi-core processors will have many large cache banks connected by a network and shared by many cores. Hence, many important problems must be solved: cache resources must be allocat

  11. Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes.

    Science.gov (United States)

    Teichmann, Martin; Dumay-Odelot, Hélène; Fribourg, Sébastien

    2012-01-01

    The winged helix (WH) domain is found in core components of transcription systems in eukaryotes and prokaryotes. It represents a sub-class of the helix-turn-helix motif. The WH domain participates in establishing protein-DNA and protein-protein-interactions. Here, we discuss possible explanations for the enrichment of this motif in transcription systems.

  12. Characterizing Facesheet/Core Disbonding in Honeycomb Core Sandwich Structure

    Science.gov (United States)

    Rinker, Martin; Ratcliffe, James G.; Adams, Daniel O.; Krueger, Ronald

    2013-01-01

    Results are presented from an experimental investigation into facesheet core disbonding in carbon fiber reinforced plastic/Nomex honeycomb sandwich structures using a Single Cantilever Beam test. Specimens with three, six and twelve-ply facesheets were tested. Specimens with different honeycomb cores consisting of four different cell sizes were also tested, in addition to specimens with three different widths. Three different data reduction methods were employed for computing apparent fracture toughness values from the test data, namely an area method, a compliance calibration technique and a modified beam theory method. The compliance calibration and modified beam theory approaches yielded comparable apparent fracture toughness values, which were generally lower than those computed using the area method. Disbonding in the three-ply facesheet specimens took place at the facesheet/core interface and yielded the lowest apparent fracture toughness values. Disbonding in the six and twelve-ply facesheet specimens took place within the core, near to the facesheet/core interface. Specimen width was not found to have a significant effect on apparent fracture toughness. The amount of scatter in the apparent fracture toughness data was found to increase with honeycomb core cell size.

  13. Introduction to the Core Curriculum in GIS

    OpenAIRE

    2015-01-01

    This short narrative introduces the Core Curriculum in GIS and provides a historical overview of the Core Curriculum Project, including the later Core Curriculum in GIScience and Core Curriculum for Technical Programs. Appended to this description is an original pamphlet advertising the Core Curriculum in GIS.

  14. Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis

    Science.gov (United States)

    Pène, Véronique; Lemasson, Matthieu; Harper, Francis; Pierron, Gérard; Rosenberg, Arielle R.

    2017-01-01

    In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before

  15. Assessing Core Competencies

    Science.gov (United States)

    Narayanan, M.

    2004-12-01

    Catherine Palomba and Trudy Banta offer the following definition of assessment, adapted from one provided by Marches in 1987. Assessment in the systematic collection, review, and use of information about educational programs undertaken for the purpose of improving student learning and development. (Palomba and Banta 1999). It is widely recognized that sophisticated computing technologies are becoming a key element in today's classroom instructional techniques. Regardless, the Professor must be held responsible for creating an instructional environment in which the technology actually supplements learning outcomes of the students. Almost all academic disciplines have found a niche for computer-based instruction in their respective professional domain. In many cases, it is viewed as an essential and integral part of the educational process. Educational institutions are committing substantial resources to the establishment of dedicated technology-based laboratories, so that they will be able to accommodate and fulfill students' desire to master certain of these specific skills. This type of technology-based instruction may raise some fundamental questions about the core competencies of the student learner. Some of the most important questions are : 1. Is the utilization of these fast high-powered computers and user-friendly software programs creating a totally non-challenging instructional environment for the student learner ? 2. Can technology itself all too easily overshadow the learning outcomes intended ? 3. Are the educational institutions simply training students how to use technology rather than educating them in the appropriate field ? 4. Are we still teaching content-driven courses and analysis oriented subject matter ? 5. Are these sophisticated modern era technologies contributing to a decline in the Critical Thinking Capabilities of the 21st century technology-savvy students ? The author tries to focus on technology as a tool and not on the technology

  16. Viral Evolution Core | FNLCR Staging

    Science.gov (United States)

    Brandon F. Keele, Ph.D. PI/Senior Principal Investigator, Retroviral Evolution Section Head, Viral Evolution Core Leidos Biomedical Research, Inc. Frederick National Laboratory for Cancer Research Frederick, MD 21702-1201 Tel: 301-846-173

  17. Lunar Core Drive Tubes Summary

    Data.gov (United States)

    National Aeronautics and Space Administration — Contains a brief summary and high resolution imagery from various lunar rock and core drive tubes collected from the Apollo and Luna missions to the moon.

  18. Viscosity of the earth's core.

    Science.gov (United States)

    Gans, R. F.

    1972-01-01

    Calculation of the viscosity of the core at the boundary of the inner and outer core. It is assumed that this boundary is a melting transition and the viscosity limits of the Andrade (1934,1952) hypothesis (3.7 to 18.5 cp) are adopted. The corresponding kinematic viscosities are such that the precessional system explored by Malkus (1968) would be unstable. Whether it would be sufficiently unstable to overcome a severely subadiabatic temperature gradient cannot be determined.

  19. Nanoporous polymer liquid core waveguides

    DEFF Research Database (Denmark)

    Gopalakrishnan, Nimi; Christiansen, Mads Brøkner; Ndoni, Sokol

    2010-01-01

    We demonstrate liquid core waveguides defined by UV to enable selective water infiltration in nanoporous polymers, creating an effective refractive index shift Δn=0.13. The mode confinement and propagation loss in these waveguides are presented.......We demonstrate liquid core waveguides defined by UV to enable selective water infiltration in nanoporous polymers, creating an effective refractive index shift Δn=0.13. The mode confinement and propagation loss in these waveguides are presented....

  20. Core-Shell-structured Dendritic Mesoporous Silica Nanoparticles for Combined Photodynamic Therapy and Antibody Delivery.

    Science.gov (United States)

    Abbaraju, Prasanna Lakshmi; Yang, Yannan; Yu, Meihua; Fu, Jianye; Xu, Chun; Yu, Chengzhong

    2017-07-04

    Multifunctional core-shell-structured dendritic mesoporous silica nanoparticles with a fullerene-doped silica core, a dendritic silica shell and large pores have been prepared. The combination of photodynamic therapy and antibody therapeutics significantly inhibits the cancer cell growth by effectively reducing the level of anti-apoptotic proteins. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Crystallization in Earth's Core after High-Temperature Core Formation

    Science.gov (United States)

    Hirose, K.; Morard, G.; Hernlund, J. W.; Helffrich, G. R.; Ozawa, H.

    2015-12-01

    Recent core formation models based on the metal-silicate partitioning of siderophile elements suggest that the Earth's core was formed by metal segregation at high pressure and high temperature in a deep magma ocean. It is also thought that the simultaneous solubility of silicon and oxygen in liquid iron are strongly enhanced at high pressure and high temperature, such that at the end of accretion the core was rich in both silicon and oxygen. Here we performed crystallization experiments on the Fe-Si binary and Fe-Si-O ternary systems up to core pressure in a laser-heated diamond-anvil cell. The starting material for the latter was a homogeneous mixture of fine-grain Fe-Si and SiO2 (sustain without extreme degrees of secular cooling. However, even for modest degrees of joint Si-O incorporation into the early core, the buoyancy released by crystallization of SiO2 is sufficient to overcome thermal stratification and sustain the geodynamo.

  2. Core formation in silicate bodies

    Science.gov (United States)

    Nimmo, F.; O'Brien, D. P.; Kleine, T.

    2008-12-01

    Differentiation of a body into a metallic core and silicate mantle occurs most efficiently if temperatures are high enough to allow at least the metal to melt [1], and is enhanced if matrix deformation occurs [2]. Elevated temperatures may occur due to either decay of short-lived radio-isotopes, or gravitational energy release during accretion [3]. For bodies smaller than the Moon, core formation happens primarily due to radioactive decay. The Hf-W isotopic system may be used to date core formation; cores in some iron meteorites and the eucrite parent body (probably Vesta) formed within 1 My and 1-4~My of solar system formation, respectively [4]. These formation times are early enough to ensure widespread melting and differentiation by 26Al decay. Incorporation of Fe60 into the core, together with rapid early mantle solidification and cooling, may have driven early dynamo activity on some bodies [5]. Iron meteorites are typically depleted in sulphur relative to chondrites, for unknown reasons [6]. This depletion contrasts with the apparently higher sulphur contents of cores in larger planetary bodies, such as Mars [7], and also has a significant effect on the timing of core solidification. For bodies of Moon-size and larger, gravitational energy released during accretion is probably the primary cause of core formation [3]. The final stages of accretion involve large, stochastic collisions [8] between objects which are already differentiated. During each collision, the metallic cores of the colliding objects merge on timescales of a few hours [9]. Each collision will reset the Hf-W isotopic signature of both mantle and core, depending on the degree to which the impactor core re-equilibrates with the mantle of the target [10]. The re-equilibration efficiency depends mainly on the degree to which the impactor emulsifies [11], which is very uncertain. Results from N-body simulations [8,12] suggest that significant degrees of re- equilibration are required [4,10]. Re

  3. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... for the vegetarian proteins, whether they have carbohydrate. Protein Choices Plant-Based Proteins Plant-based protein foods ...

  4. Viscosity of Earth's Outer Core

    CERN Document Server

    Smylie, D E

    2007-01-01

    A viscosity profile across the entire fluid outer core is found by interpolating between measured boundary values, using a differential form of the Arrhenius law governing pressure and temperature dependence. The discovery that both the retrograde and prograde free core nutations are in free decay (Palmer and Smylie, 2005) allows direct measures of viscosity at the top of the outer core, while the reduction in the rotational splitting of the two equatorial translational modes of the inner core allows it to be measured at the bottom. We find 2,371 plus/minus 1,530 Pa.s at the top and 1.247 plus/minus 0.035 x 10^11 Pa.s at the bottom. Following Brazhkin (1998) and Brazhkin and Lyapin (2000) who get 10^2 Pa.s at the top, 10^11 Pa.s at the bottom, by an Arrhenius extrapolation of laboratory experiments, we use a differential form of the Arrhenius law to interpolate along the melting temperature curve to find a viscosity profile across the outer core. We find the variation to be closely log-linear between the meas...

  5. Is a malleable protein necessarily highly dynamic?

    DEFF Research Database (Denmark)

    Kjærgaard, Magnus; Poulsen, Flemming Martin; Teilum, Kaare

    2012-01-01

    core of NCBD in the ligand-free state and in a well-folded complex with the ligand activator for thyroid hormone and retinoid receptors using multiple NMR methods including methyl chemical shifts, coupling constants, and methyl order parameters. From all NMR measures, the aliphatic side chains...... in the hydrophobic core are slightly more dynamic in the free protein than in the complex, but have mobility comparable to the hydrophobic cores of average folded proteins. Urea titration monitored by NMR reveals that all parts of the protein, including the side-chain packing in the hydrophobic core, denatures...... in a single cooperative process. The molten globule characteristics of NCBD are thus restricted to a slowly fluctuating tertiary structure. Consequently, the conformational plasticity of the protein is most likely related to its low overall stability rather than an intrinsically flexible protein structure...

  6. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  7. Duck hepatitis B virus replication in primary bile duct epithelial cells.

    Science.gov (United States)

    Lee, J Y; Culvenor, J G; Angus, P; Smallwood, R; Nicoll, A; Locarnini, S

    2001-08-01

    Primary cultures of intrahepatic bile duct epithelial (IBDE) cells isolated from duckling livers were successfully grown for studies of duck hepatitis B virus (DHBV). The primary IBDE cells were characterized by immunohistochemistry using CAM 5.2, a cytokeratin marker which was shown to react specifically to IBDE cells in duck liver tissue sections and in primary cultures of total duck liver cells. Immunofluorescence assay using anti-duck albumin, a marker for hepatocytes, revealed that these IBDE cultures did not appear to contain hepatocytes. A striking feature of these cultures was the duct-like structures present within each cell colony of multilayered IBDE cells. Normal duck serum in the growth medium was found to be essential for the development of these cells into duct-like structures. When the primary cultures of duck IBDE cells were acutely infected with DHBV, dual-labeled confocal microscopy using a combination of anti-DHBV core proteins and CAM 5.2 or a combination of anti-pre-S1 proteins and CAM 5.2 revealed that the IBDE cell colonies contained DHBV proteins. Immunoblot analysis of these cells showed that the DHBV pre-S1 and core proteins were similar to their counterparts in infected primary duck hepatocyte cultures. Southern blot analysis of infected IBDE preparations using a digoxigenin-labeled positive-sense DHBV riboprobe revealed the presence of hepadnavirus covalently closed circular (CCC) DNA, minus-sense single-stranded (SS) DNA, double-stranded linear DNA, and relaxed circular DNA. The presence of minus-sense SS DNA in the acutely infected IBDE cultures is indicative of DHBV reverse transcriptase activity, while the establishment of a pool of viral CCC DNA reveals the ability of these cells to maintain persistent infection. Taken collectively, the results from this study demonstrated that primary duck IBDE cells supported hepadnavirus replication as shown by the de novo synthesis of DHBV proteins and DNA replicative intermediates.

  8. SMART core protection system design

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J. K.; Park, H. Y.; Koo, I. S. [KAERI, Taejon (Korea, Republic of); Park, H. S.; Kim, J. S.; Son, C. H. [Samchang Enterprise Co., Ltd., Taejon (Korea, Republic of)

    2003-10-01

    SMART COre Protection System(SCOPS) is designed with real-tims Digital Signal Processor(DSP) board and Network Interface Card(NIC) board. SCOPS has a Control Rod POSition (CRPOS) software module while Core Protection Calculator System(CPCS) consists of Core Protection Calculators(CPCs) and Control Element Assembly(CEA) Calculators(CEACs) in the commercial nuclear plant. It's not necessary to have a independent cabinets for SCOPS because SCOPS is physically very small. Then SCOPS is designed to share the cabinets with Plant Protection System(PPS) of SMART. Therefor it's very easy to maintain the system because CRPOS module is used instead of the computer with operating system.

  9. Atmospheric Methane in Ice Cores

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The reconstruction of air trapped in ice cores provides us the most direct information about atmospheric CH4 variations in the past history. Ice core records from the "Three Poles (Antarctica, Greenland and Tibetan Plateau)" reveal the detailed fluctuations of atmospheric CH4 concentration with time and are allowed to quantify the CH4 differences among latitudes. These data are indispensably in the farther study of the relationship between greenhouse gases and climatic change, and of the past changes in terrestrial CH4 emissions. Ice cores reconstruction indicates that atmospheric CH4 concentration has increased quickly since industrialization, and the present day's level of atmospheric CH4 (1800 ppbv) is unprecedented during the past Glacial-Interglacial climate cycles.

  10. Distributed k-Core Decomposition

    CERN Document Server

    Montresor, Alberto; Miorandi, Daniele

    2011-01-01

    Among the novel metrics used to study the relative importance of nodes in complex networks, k-core decomposition has found a number of applications in areas as diverse as sociology, proteinomics, graph visualization, and distributed system analysis and design. This paper proposes new distributed algorithms for the computation of the k-core decomposition of a network, with the purpose of (i) enabling the run-time computation of k-cores in "live" distributed systems and (ii) allowing the decomposition, over a set of connected machines, of very large graphs, that cannot be hosted in a single machine. Lower bounds on the algorithms complexity are given, and an exhaustive experimental analysis on real-world graphs is provided.

  11. Identifying ELIXIR Core Data Resources.

    Science.gov (United States)

    Durinx, Christine; McEntyre, Jo; Appel, Ron; Apweiler, Rolf; Barlow, Mary; Blomberg, Niklas; Cook, Chuck; Gasteiger, Elisabeth; Kim, Jee-Hyub; Lopez, Rodrigo; Redaschi, Nicole; Stockinger, Heinz; Teixeira, Daniel; Valencia, Alfonso

    2016-01-01

    The core mission of ELIXIR is to build a stable and sustainable infrastructure for biological information across Europe. At the heart of this are the data resources, tools and services that ELIXIR offers to the life-sciences community, providing stable and sustainable access to biological data. ELIXIR aims to ensure that these resources are available long-term and that the life-cycles of these resources are managed such that they support the scientific needs of the life-sciences, including biological research. ELIXIR Core Data Resources are defined as a set of European data resources that are of fundamental importance to the wider life-science community and the long-term preservation of biological data. They are complete collections of generic value to life-science, are considered an authority in their field with respect to one or more characteristics, and show high levels of scientific quality and service. Thus, ELIXIR Core Data Resources are of wide applicability and usage. This paper describes the structures, governance and processes that support the identification and evaluation of ELIXIR Core Data Resources. It identifies key indicators which reflect the essence of the definition of an ELIXIR Core Data Resource and support the promotion of excellence in resource development and operation. It describes the specific indicators in more detail and explains their application within ELIXIR's sustainability strategy and science policy actions, and in capacity building, life-cycle management and technical actions. The identification process is currently being implemented and tested for the first time. The findings and outcome will be evaluated by the ELIXIR Scientific Advisory Board in March 2017. Establishing the portfolio of ELIXIR Core Data Resources and ELIXIR Services is a key priority for ELIXIR and publicly marks the transition towards a cohesive infrastructure.

  12. Potent inhibition of late stages of hepadnavirus replication by a modified cell penetrating peptide

    DEFF Research Database (Denmark)

    Abdul, Fabien; Ndeboko, Bénédicte; Buronfosse, Thierry

    2012-01-01

    Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication...... been so far described for other enveloped viruses....

  13. Earth's core and the geodynamo

    Science.gov (United States)

    Buffett

    2000-06-16

    Earth's magnetic field is generated by fluid motion in the liquid iron core. Details of how this occurs are now emerging from numerical simulations that achieve a self-sustaining magnetic field. Early results predict a dominant dipole field outside the core, and some models even reproduce magnetic reversals. The simulations also show how different patterns of flow can produce similar external fields. Efforts to distinguish between the various possibilities appeal to observations of the time-dependent behavior of the field. Important constraints will come from geological records of the magnetic field in the past.

  14. Magnetic Probing of Core Geodynamics

    Science.gov (United States)

    Voorhies, Coerte V.

    2004-01-01

    To better understand geomagnetic theory and observation, we can use spatial magnetic spectra for the main field and secular variation to test core dynmcal hypotheses against seismology. The hypotheses lead to theoretical spectra which are fitted to observational spectra. Each fit yields an estimate of the radius of Earth's core and uncertainty. If this agrees with the seismologic value, then the hypothes pass the test. A new way to obtain theoretical spectra extends the hydromagnetic scale analysis of Benton to scale-variant field and flow. For narrow scale flow and a dynamically weak field by the top of Earth's core, this yields a generalized Stevenson-McLeod spectrum for the core-source field, and a secular variation spectrum modulated by a cubic polynomial in spherical harmonic degree n. The former passes the tests. The latter passes many tests, but does not describe rapid dipole decline and quadrupole rebound; some tests suggest it is a bit hard, or rich in narrow scale change. In a core geodynamo, motion of the fluid conductor does work against the Lorentz force. This converts kinetic into magnetic energy which, in turn, is lost to heat via Ohmic dissipation. In the analysis at lentgh-scale l/k, if one presumes kinetic energy is converted in either eddy- overturning or magnetic free-decay time-scales, then Kolmogorov or other spectra in conflict with observational spectra can result. Instead, the rate work is done roughly balances the dissipation rate, which is consistent with small scale flow. The conversion time-scale depends on dynamical constraints. These are summarized by the magneto-geostrophic vertical vorticity balance by the top of the core, which includes anisotropic effects of rotation, the magnetic field, and the core- mantle boundary. The resulting theoretical spectra for the core-source field and its SV are far more compatible with observation. The conversion time-scale of order l20 years is pseudo-scale-invarient. Magnetic spectra of other

  15. Core competencies in internal medicine.

    Science.gov (United States)

    Porcel, José Manuel; Casademont, Jordi; Conthe, Pedro; Pinilla, Blanca; Pujol, Ramón; García-Alegría, Javier

    2012-06-01

    The working group on Competencies of Internal Medicine from the Spanish Society of Internal Medicine (SEMI) proposes a series of core competencies that we consider should be common to all European internal medicine specialists. The competencies include aspects related to patient care, clinical knowledge, technical skills, communication skills, professionalism, cost-awareness in medical care and academic activities. The proposal could be used as a working document for the Internal Medicine core curriculum in the context of the educational framework of medical specialties in Europe.

  16. Core Task and Organizational Reality

    DEFF Research Database (Denmark)

    Vikkelsø, Signe

    2015-01-01

    of core objects such as ‘task’ and ‘coordination,’ contemporary organization studies emphasize, much like other social science disciplines, broader topics such as ‘network,’ ‘identity,’ and ‘change.’ The paper argues that this altered focus and vocabulary is accompanied by a diminished ability to specify...... and intervene into the practical reality of organizations. It further argues that a discipline's core objects are not anachronisms to be discarded with, but crucial for specifying reality in ways that have proven practically relevant and still are....

  17. Producing gapped-ferrite transformer cores

    Science.gov (United States)

    Mclyman, W. T.

    1980-01-01

    Improved manufacturing techniques make reproducible gaps and minimize cracking. Molded, unfired transformer cores are cut with thin saw and then fired. Hardened semicircular core sections are bonded together, placed in aluminum core box, and fluidized-coated. After winding is run over box, core is potted. Economical method significantly reduces number of rejects.

  18. Multi-core Architectures and Streaming Applications

    NARCIS (Netherlands)

    Smit, Gerard J.M.; Kokkeler, André B.J.; Wolkotte, Pascal T.; Burgwal, van de Marcel D.; Mandoiu, I.; Kennings, A.

    2008-01-01

    In this paper we focus on algorithms and reconfigurable multi-core architectures for streaming digital signal processing (DSP) applications. The multi-core concept has a number of advantages: (1) depending on the requirements more or fewer cores can be switched on/off, (2) the multi-core structure f

  19. Core Stability Training for Injury Prevention

    OpenAIRE

    2013-01-01

    Context: Enhancing core stability through exercise is common to musculoskeletal injury prevention programs. Definitive evidence demonstrating an association between core instability and injury is lacking; however, multifaceted prevention programs including core stabilization exercises appear to be effective at reducing lower extremity injury rates. Evidence Acquisition: PubMed was searched for epidemiologic, biomechanic, and clinical studies of core stability for injury prevention (keywords: ...

  20. Core proteome of the minimal cell: comparative proteomics of three mollicute species.

    Directory of Open Access Journals (Sweden)

    Gleb Y Fisunov

    Full Text Available Mollicutes (mycoplasmas have been recognized as highly evolved prokaryotes with an extremely small genome size and very limited coding capacity. Thus, they may serve as a model of a 'minimal cell': a cell with the lowest possible number of genes yet capable of autonomous self-replication. We present the results of a comparative analysis of proteomes of three mycoplasma species: A. laidlawii, M. gallisepticum, and M. mobile. The core proteome components found in the three mycoplasma species are involved in fundamental cellular processes which are necessary for the free living of cells. They include replication, transcription, translation, and minimal metabolism. The members of the proteome core seem to be tightly interconnected with a number of interactions forming core interactome whether or not additional species-specific proteins are located on the periphery. We also obtained a genome core of the respective organisms and compared it with the proteome core. It was found that the genome core encodes 73 more proteins than the proteome core. Apart of proteins which may not be identified due to technical limitations, there are 24 proteins that seem to not be expressed under the optimal conditions.

  1. Visual Feedback for Rover-based Coring

    Science.gov (United States)

    Backes, Paul; Helmick, Daniel; Bajracharya, Max

    2008-01-01

    Technology for coring from a low-mass rover has been developed to enable core sample acquisition where a planetary rover experiences moderate slip during the coring operation. A new stereo vision technique, Absolute Motion Visual Odometry, is used to measure rover slip during coring and the slip is accommodated through corresponding arm pose updating. Coring rate is controlled by feedback of themeasured force of the coring tool against the environment. Test results in the JPL Marsyard show for the first time that coring from a low-mass rover with slip is feasible.

  2. Core Competence Development : paradigm and practical implementations

    OpenAIRE

    Koay, Ze Wei; E.Markov, Denis

    2011-01-01

    The theory of core competence has drawn a large amount of attention in the academic field as well as of practitioners in the corporate world. Theory asserts that long-term value creation and competitiveness of the corporation relies on full-scale exploitation and timely development of company Core Competences; business strategies should be built around the core competencies of a firm. Identification and exploitation of Core Competences as well as essential elements comprising Core Competences...

  3. Fuzzy Cores and Fuzzy Balancedness

    NARCIS (Netherlands)

    van Gulick, G.; Norde, H.W.

    2011-01-01

    We study the relation between the fuzzy core and balancedness for fuzzy games. For regular games, this relation has been studied by Bondareva (1963) and Shapley (1967). First, we gain insight in this relation when we analyse situations where the fuzzy game is continuous. Our main result shows that a

  4. Competition for cores in remanufacturing

    NARCIS (Netherlands)

    Bulmus, Serra Caner; Zhu, Stuart X.; Teunter, Ruud

    2014-01-01

    We study competition between an original equipment manufacturer (OEM) and an independently operating remanufacturer (IO). Different from the existing literature, the OEM and IO compete not only for selling their products but also for collecting returned products (cores) through their acquisition pri

  5. One Health Core Competency Domains.

    Science.gov (United States)

    Frankson, Rebekah; Hueston, William; Christian, Kira; Olson, Debra; Lee, Mary; Valeri, Linda; Hyatt, Raymond; Annelli, Joseph; Rubin, Carol

    2016-01-01

    The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting "One Health" approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills, and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education, as they describe the knowledge, skills, and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches.

  6. Fuzzy Cores and Fuzzy Balancedness

    NARCIS (Netherlands)

    van Gulick, G.; Norde, H.W.

    2011-01-01

    We study the relation between the fuzzy core and balancedness for fuzzy games. For regular games, this relation has been studied by Bondareva (1963) and Shapley (1967). First, we gain insight in this relation when we analyse situations where the fuzzy game is continuous. Our main result shows that

  7. Common Core: Solve Math Problems

    Science.gov (United States)

    Strom, Erich

    2012-01-01

    The new common core standards for mathematics demand that students (and teachers!) exhibit deeper conceptual understanding. That's music to the ears of education professor John Tapper, who says teachers have overemphasized teaching procedures--and getting right answers. In his new book, "Solving for Why," he makes a powerful case for moving beyond…

  8. One Health Core Competency Domains

    Science.gov (United States)

    Frankson, Rebekah; Hueston, William; Christian, Kira; Olson, Debra; Lee, Mary; Valeri, Linda; Hyatt, Raymond; Annelli, Joseph; Rubin, Carol

    2016-01-01

    The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting “One Health” approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills, and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education, as they describe the knowledge, skills, and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches. PMID:27679794

  9. Stability of Molten Core Materials

    Energy Technology Data Exchange (ETDEWEB)

    Layne Pincock; Wendell Hintze

    2013-01-01

    The purpose of this report is to document a literature and data search for data and information pertaining to the stability of nuclear reactor molten core materials. This includes data and analysis from TMI-2 fuel and INL’s LOFT (Loss of Fluid Test) reactor project and other sources.

  10. One Health Core Competency Domains

    Directory of Open Access Journals (Sweden)

    Rebekah Frankson

    2016-09-01

    Full Text Available The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting ‘One Health’ approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education as they describe the knowledge, skills and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches.

  11. Earth rotation and core topography

    Science.gov (United States)

    Hager, Bradford H.; Clayton, Robert W.; Spieth, Mary Ann

    1988-01-01

    The NASA Geodynamics program has as one of its missions highly accurate monitoring of polar motion, including changes in length of day (LOD). These observations place fundamental constraints on processes occurring in the atmosphere, in the mantle, and in the core of the planet. Short-timescale (t less than or approx 1 yr) variations in LOD are mainly the result of interaction between the atmosphere and the solid earth, while variations in LOD on decade timescales result from the exchange of angular momentum between the mantle and the fluid core. One mechanism for this exchange of angular momentum is through topographic coupling between pressure variations associated with flow in the core interacting with topography at the core-mantel boundary (CMB). Work done under another NASA grant addressing the origin of long-wavelength geoid anomalies as well as evidence from seismology, resulted in several models of CMB topography. The purpose of work supported by NAG5-819 was to study further the problem of CMB topography, using geodesy, fluid mechanics, geomagnetics, and seismology. This is a final report.

  12. CopperCore Service Integration

    Science.gov (United States)

    Vogten, Hubert; Martens, Harrie; Nadolski, Rob; Tattersall, Colin; van Rosmalen, Peter; Koper, Rob

    2007-01-01

    In an e-learning environment there is a need to integrate various e-learning services like assessment services, collaboration services, learning design services and communication services. In this article we present the design and implementation of a generic integrative service framework, called CopperCore Service Integration (CCSI). We will…

  13. Core shift effect in blazars

    Science.gov (United States)

    Agarwal, A.; Mohan, P.; Gupta, Alok C.; Mangalam, A.; Volvach, A. E.; Aller, M. F.; Aller, H. D.; Gu, M. F.; Lähteenmäki, A.; Tornikoski, M.; Volvach, L. N.

    2017-07-01

    We studied the pc-scale core shift effect using radio light curves for three blazars, S5 0716+714, 3C 279 and BL Lacertae, which were monitored at five frequencies (ν) between 4.8 and 36.8 GHz using the University of Michigan Radio Astronomical Observatory (UMRAO), the Crimean Astrophysical Observatory (CrAO) and Metsähovi Radio Observatory for over 40 yr. Flares were Gaussian fitted to derive time delays between observed frequencies for each flare (Δt), peak amplitude (A) and their half width. Using A ∝ να, we infer α in the range of -16.67-2.41 and using Δ t ∝ ν ^{1/k_r}, we infer kr ∼ 1, employed in the context of equipartition between magnetic and kinetic energy density for parameter estimation. From the estimated core position offset (Ωrν) and the core radius (rcore), we infer that opacity model may not be valid in all cases. The mean magnetic field strengths at 1 pc (B1) and at the core (Bcore) are in agreement with previous estimates. We apply the magnetically arrested disc model to estimate black hole spins in the range of 0.15-0.9 for these blazars, indicating that the model is consistent with expected accretion mode in such sources. The power-law-shaped power spectral density has slopes -1.3 to -2.3 and is interpreted in terms of multiple shocks or magnetic instabilities.

  14. Structural characterization of core-bradavidin in complex with biotin

    Science.gov (United States)

    Agrawal, Nitin; Määttä, Juha A. E.; Kulomaa, Markku S.; Hytönen, Vesa P.; Johnson, Mark S.; Airenne, Tomi T.

    2017-01-01

    Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin. PMID:28426764

  15. Magnectic Probing of Core Geodynamics

    Science.gov (United States)

    Voorhies, Coerte

    2004-01-01

    To better understand geomagnetic theory and observation, we can use spatial magnetic spectra for the main field and secular variation to test core dynamical hypotheses against seismology. The hypotheses lead to theoretical spectra which are fitted to observational spectra. Each fit yields an estimate of the radius of Earth s core and uncertainty. If this agrees with the seismologic value, then the hypotheses pass the test. A new way to obtain theoretical spectra extends the hydromagnetic scale analysis of Benton to scale-variant field and flow. For narrow scale flow and a dynamically weak field by the top of Earth s core, this yields a JGR-PI, and a secular variation spectrum modulated by a cubic polynomial in spherical harmonic degree n. The former passes the tests. The latter passes many tests, but does not describe rapid dipole decline and quadrupole rebound; some tests suggest it is a bit hard, or rich in narrow scale change.In a core geodynamo, motion of the fluid conductor does work against the Lorentz force. This converts kinetic into magnetic energy which, in turn, is lost to heat via Ohmic dissipation. In the analysis at length- scale l/k, if one presumes kinetic energy is converted in either eddy- overturning or magnetic free-decay time-scales, then Kolmogorov or other spectra in conflict with observational spectra can result. Instead, the rate work is done roughly balances the dissipation rate, which is consistent with small scale flow. The conversion time-scale depends on dynamical constraints. These are summarized by the magneto- geostrophic vertical vorticity balance by the top of the core, which includes anisotropic effects of rotation, the magnetic field, and the core-mantle boundary. The resulting theoretical spectra for the core- source field and its SV are far more compatible with observation. The conversion time-scale of order 120 years is pseudo-scale-invariant. Magnetic spectra of other planets may differ; however, if a transition to non

  16. High throughput protein-protein interaction data: clues for the architecture of protein complexes

    Directory of Open Access Journals (Sweden)

    Pang Chi

    2008-11-01

    Full Text Available Abstract Background High-throughput techniques are becoming widely used to study protein-protein interactions and protein complexes on a proteome-wide scale. Here we have explored the potential of these techniques to accurately determine the constituent proteins of complexes and their architecture within the complex. Results Two-dimensional representations of the 19S and 20S proteasome, mediator, and SAGA complexes were generated and overlaid with high quality pairwise interaction data, core-module-attachment classifications from affinity purifications of complexes and predicted domain-domain interactions. Pairwise interaction data could accurately determine the members of each complex, but was unexpectedly poor at deciphering the topology of proteins in complexes. Core and module data from affinity purification studies were less useful for accurately defining the member proteins of these complexes. However, these data gave strong information on the spatial proximity of many proteins. Predicted domain-domain interactions provided some insight into the topology of proteins within complexes, but was affected by a lack of available structural data for the co-activator complexes and the presence of shared domains in paralogous proteins. Conclusion The constituent proteins of complexes are likely to be determined with accuracy by combining data from high-throughput techniques. The topology of some proteins in the complexes will be able to be clearly inferred. We finally suggest strategies that can be employed to use high throughput interaction data to define the membership and understand the architecture of proteins in novel complexes.

  17. Interleaved Core Assignment for Bidirectional Transmission in Multi-Core Fibers

    DEFF Research Database (Denmark)

    Ye, Feihong; Morioka, Toshio

    2013-01-01

    We study interleaved core assignment for bidirectional transmission in multi-core fibers. By combining it with heterogeneous core structure in an 18-core fiber, the transmission distance is extended by 10 times compared to homogeneous core structure with unidirectional transmission, achieving...

  18. Interleaved Core Assignment for Bidirectional Transmission in Multi-Core Fibers

    OpenAIRE

    2013-01-01

    We study interleaved core assignment for bidirectional transmission in multi-core fibers. By combining it with heterogeneous core structure in an 18-core fiber, the transmission distance is extended by 10 times compared to homogeneous core structure with unidirectional transmission, achieving a total capacity of 1 Pb/s per direction.

  19. Interleaved Core Assignment for Bidirectional Transmission in Multi-Core Fibers

    DEFF Research Database (Denmark)

    Ye, Feihong; Morioka, Toshio

    2013-01-01

    We study interleaved core assignment for bidirectional transmission in multi-core fibers. By combining it with heterogeneous core structure in an 18-core fiber, the transmission distance is extended by 10 times compared to homogeneous core structure with unidirectional transmission, achieving a t...

  20. Core promoter recognition complex changes accompany liver development

    Science.gov (United States)

    D’Alessio, Joseph A.; Ng, Raymond; Willenbring, Holger; Tjian, Robert

    2011-01-01

    Recent studies of several key developmental transitions have brought into question the long held view of the basal transcriptional apparatus as ubiquitous and invariant. In an effort to better understand the role of core promoter recognition and coactivator complex switching in cellular differentiation, we have examined changes in transcription factor IID (TFIID) and cofactor required for Sp1 activation/Mediator during mouse liver development. Here we show that the differentiation of fetal liver progenitors to adult hepatocytes involves a wholesale depletion of canonical cofactor required for Sp1 activation/Mediator and TFIID complexes at both the RNA and protein level, and that this alteration likely involves silencing of transcription factor promoters as well as protein degradation. It will be intriguing for future studies to determine if a novel and as yet unknown core promoter recognition complex takes the place of TFIID in adult hepatocytes and to uncover the mechanisms that down-regulate TFIID during this critical developmental transition. PMID:21368148

  1. Cores to the rescue: how old cores enable new science

    Science.gov (United States)

    Ito, E.; Noren, A. J.; Brady, K.

    2016-12-01

    The value of archiving scientific specimens and collections for the purpose of enabling further research using new analytical techniques, resolving conflicting results, or repurposing them for entirely new research, is often discussed in abstract terms. We all agree that samples with adequate metadata ought to be archived systematically for easy access, for a long time and stored under optimal conditions. And yet, as storage space fills, there is a temptation to cull the collection, or when a researcher retires, to discard the collection unless the researcher manages to make his or her own arrangement for the collection to be accessioned elsewhere. Nobody has done anything with these samples in over 20 years! Who would want them? It turns out that plenty of us do want them, if we know how to find them and if they have sufficient metadata to assess past work and suitability for new analyses. The LacCore collection holds over 33 km of core from >6700 sites in diverse geographic locations worldwide with samples collected as early as 1950s. From these materials, there are many examples to illustrate the scientific value of archiving geologic samples. One example that benefitted Ito personally were cores from Lakes Mirabad and Zeribar, Iran, acquired in 1963 by Herb Wright and his associates. Several doctoral and postdoctoral students generated and published paleoecological reconstructions based on cladocerans, diatoms, pollen or plant macrofossils, mostly between 1963 and 1967. The cores were resampled in 1990s by a student being jointly advised by Wright and Ito for oxygen isotope analysis of endogenic calcite. The results were profitably compared with pollen and the results published in 2001 and 2006. From 1979 until very recently, visiting Iran for fieldwork was not pallowed for US scientists. Other examples will be given to further illustrate the power of archived samples to advance science.

  2. Hydrologic characterization of four cores from the Geysers Coring Project

    Energy Technology Data Exchange (ETDEWEB)

    Persoff, Peter; Hulen, Jeffrey B.

    1996-01-24

    Results of hydrologic tests conducted on four representative core plugs from Geysers Coring Project drill hole SB-15-D have been related to detailed mineralogic and textural characterization of the plugs to yield new information about permeability, porosity, and capillary-pressure characteristics of the uppermost Geysers steam reservoir and its immediately overlying caprock. The core plugs are all fine- to medium-grained, Franciscan-assemblage (late Mesozoic) metagraywacke with sparse Franciscan metamorphic quartz-calcite veins and late Cenozoic, hydrothermal quartz-calcite-pyrite veins. The matrices of three plugs from the caprock are rich in metamorphic mixed-layer illite/smectite and disseminated hydrothermal pyrite; the reservoir plug instead contains abundant illite and only minor pyrite. The reservoir plug and one caprock plug are sparsely disrupted by latest-stage, unmineralized microfractures which both follow and crosscut veinlets but which could be artifacts. Porosities of the plugs, measured by Boyles-law gas expansion, range between 1.9 and 2.5%. Gas permeability and Klinkenberg slip factor were calculated from gas-pressure-pulse-decay measurements using a specially designed permeameter with small (2 mL) reservoirs. Matrix permeabilities in the range 10-21 m² ( = 1 nanodarcy) were measured for two plugs that included mineral-filled veins but no unfilled microfractures. Greater permeabilities were measured on plugs that contained microfractures; at 500 psi net confining pressure, an effective aperture of 1.6 µm was estimated for one plug. Capillary pressure curves were determined for three cores by measuring saturation as weight gain of plugs equilibrated with atmospheres in which the relative humidity was controlled by saturated brines.

  3. Core-shell Hydroxyapatite Combined with Bone Morphogenetic Protein 2 in Periodontal Regeneration Treatmentin Dogs%贝壳多孔羟基磷灰石基骨修复材料和骨形成蛋白-2联合应用引导犬牙周组织再生效果初步评估

    Institute of Scientific and Technical Information of China (English)

    陈斌; 吴文蕾; 孙卫斌; 韩方凯; 张其清

    2011-01-01

    目的 初步评估贝壳多孔羟基磷灰石基骨修复材料及该材料和骨形成蛋白-2联合应用引导比格犬牙周组织再生的效果.方法 选取18月龄比格犬6只,牙周基础治疗后1周,在下颌第二、三、四前磨牙,建立急性牙周骨缺损模型,依照分组情况进行不同治疗.实验组(T组)植入骨修复材料和骨形成蛋白-2;阴性对照组(NC组)植入骨修复材料;空白对照组(BC组)不植入任何材料.实验设计采取同颌同名牙对照,同一只比格犬的3对同颌同名牙分别为:空白对照组和阴性对照组,阴性对照组和实验组,空白对照组和实验组.术后12周,处死动物,Micro-CT检查并对数据进行统计学分析.结果 材料植入后,未见材料溢出,植入局部和全身都未见明显不良反应.3组缺损都有一定程度骨再生,以T组再生组织量最多,BC组最少.Micro-CT结果显示:T组、NC组和BC组的骨再生平均高度为(4.50±0.47) mm、(1.75±0.42) mm和(0.87±0.31) mm.NC组和BC组相比,差异有统计学意义(P<0.05).T组与NC组和BC组相比,差异均有统计学意义(P<0.05),且有临床意义.结论 贝壳多孔羟基磷灰石基骨修复材料和骨形成蛋白-2联合应用于比格犬,可以获得更好的引导组织再生效果.%Objective To evaluate the ability of core-shell hydroxyapatite bone graft material alone and combined with bone morphogenetic protein 2 ( BMP-2) in periodontal regeneration treatment in dogs. Methods Thirty-six defects were created in six 18-months male beagle dogs at the sites of the second, third and fourth mandibular premolars one week later after the dogs were treated with non-surgical periodontal therapy. Different treatments were carried out according to which group the teeth belonged. There are 3 groups. The test group (group T) was treated with core-shell hydroxyapatite bone graft material combined with BMP-2; the negative control group (group NC) was treated with core-shell hydroxyapatite

  4. Hydrologic characterization of four cores from the Geysers Coring Project

    Energy Technology Data Exchange (ETDEWEB)

    Persoff, P. [Lawrence Berkeley National Lab., CA (United States); Hulen, J.B. [Univ. of Utah, Salt Lake City, UT (United States). Earth Sciences and Resources Institute

    1996-01-01

    Results of hydrologic tests on 4 representative core plugs from Geysers Coring Project drill hole SB-15-D were related to mineralogy and texture. Permeability measurements were made on 3 plugs from caprock and one plug from the steam reservoir. Late-stage microfractures present in 2 of the plugs contributed to greater permeability, but the values for the 2 other plugs indicate a typical matrix permeability of 1 to 2 {times} 10{sup {minus}21}m{sup 2}. Klinkenberg slip factor b for these plugs is generally consistent with the inverse relation between slip factor and permeability observed by Jones (1972) for plugs of much more permeable material. The caprock and reservoir samples are nearly identical metagraywackes with slight mineralogical differences which appear to have little effect on hydrology. The late stage microfractures are suspected of being artifacts. The capillary pressure curves for 3 cores are fit by power-law relations which can be used to estimate relative permeability curves for the matrix rocks.

  5. Logging-while-coring method and apparatus

    Science.gov (United States)

    Goldberg, David S.; Myers, Gregory J.

    2007-11-13

    A method and apparatus for downhole coring while receiving logging-while-drilling tool data. The apparatus includes core collar and a retrievable core barrel. The retrievable core barrel receives core from a borehole which is sent to the surface for analysis via wireline and latching tool The core collar includes logging-while-drilling tools for the simultaneous measurement of formation properties during the core excavation process. Examples of logging-while-drilling tools include nuclear sensors, resistivity sensors, gamma ray sensors, and bit resistivity sensors. The disclosed method allows for precise core-log depth calibration and core orientation within a single borehole, and without at pipe trip, providing both time saving and unique scientific advantages.

  6. Conceptual study of advanced PWR core design

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young Jin; Chang, Moon Hee; Kim, Keung Ku; Joo, Hyung Kuk; Kim, Young Il; Noh, Jae Man; Hwang, Dae Hyun; Kim, Taek Kyum; Yoo, Yon Jong

    1997-09-01

    The purpose of this project is for developing and verifying the core design concepts with enhanced safety and economy, and associated methodologies for core analyses. From the study of the sate-of-art of foreign advanced reactor cores, we developed core concepts such as soluble boron free, high convertible and enhanced safety core loaded semi-tight lattice hexagonal fuel assemblies. To analyze this hexagonal core, we have developed and verified some neutronic and T/H analysis methodologies. HELIOS code was adopted as the assembly code and HEXFEM code was developed for hexagonal core analysis. Based on experimental data in hexagonal lattices and the COBRA-IV-I code, we developed a thermal-hydraulic analysis code for hexagonal lattices. Using the core analysis code systems developed in this project, we designed a 600 MWe core and studied the feasibility of the core concepts. Two additional scopes were performed in this project : study on the operational strategies of soluble boron free core and conceptual design of large scale passive core. By using the axial BP zoning concept and suitable design of control rods, this project showed that it was possible to design a soluble boron free core in 600 MWe PWR. The results of large scale core design showed that passive concepts and daily load follow operation could be practiced. (author). 15 refs., 52 tabs., 101 figs.

  7. The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

    Science.gov (United States)

    Topalidou, Irini; Cattin-Ortolá, Jérôme; Pappas, Andrea L; Cooper, Kirsten; Merrihew, Gennifer E; MacCoss, Michael J; Ailion, Michael

    2016-05-01

    The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.

  8. Whole Core Transport Calculation Methodology for a Hexagonal Core

    Energy Technology Data Exchange (ETDEWEB)

    Cho, J. Y.; Kim, K. S.; Lee, C. C.; Zee, S. Q.; Joo, H. G

    2007-07-15

    This report discusses the hexagonal module implemented to the DeCART code and the performance of them. The implemented hexagonal module includes the hexagonal ray tracing and the CMFD acceleration modules. The performance of the implemented hexagonal module is examined for 4 tests of: (1) CMFD acceleration test, (2) the accuracy test of the hexagonal module, (3) the performance test for 2-D NGNP problem and (4) the applicability test for 3-D NGNP problem. The features of the implemented hexagonal modules are: (1) The Modular ray tracing scheme based on a hexagonal assembly and a path linking scheme between the modular rays. (2) Segment generation based on the structure unit. (3) Cell ray approximation: This feature is developed to reduce the memory required to store the segment information. (4) Modified cycle ray scheme that begins the ray tracing at a given surface and finishes if the reflected ray meets the starting surface. This feature is developed to reduce the memory required for the angular flux at the core boundary. (5) Fixed assembly geometry. The pin geometry of the single pin per assembly problem is different from that of the multi-pin problem. The core geometry of a single assembly problem is also different from that of the multi-assembly problem. (6) CMFD module based on unstructured cell. This feature is to deal with the irregular gap cells that are positioned at the assembly boundaries. The examination results of the 4 tests can be summarized as: (1) The CMFD acceleration test shows that the CMFD module speedups about greater than 200 for the core problem. (2) The accuracy test shows that the hexagonal MOC module produces an accurate solution of less than 60 pcm of eigenvalue and less than 2 % of local pin power errors. (3) The performance test for 2-D NGNP problem shows that the implemented hexagonal module works soundly and produces a reasonable solution by cooperating with the existing DeCART library and the other modules. (4) The applicability

  9. Divergent Chemoenzymatic Synthesis of Asymmetrical-Core-Fucosylated and Core-Unmodified N-Glycans

    NARCIS (Netherlands)

    Li, Tiehai; Huang, Min; Liu, Lin; Wang, Shuo; Moremen, Kelley W; Boons, Geert-Jan|info:eu-repo/dai/nl/088245489

    2016-01-01

    A divergent chemoenzymaytic approach for the preparation of core-fucosylated and core-unmodified asymmetrical N-glycans from a common advances precursor is described. An undecasaccharide was synthesized by sequential chemical glycosylations of an orthogonally protected core fucosylated

  10. Evidence for core 2 to core 1 O-glycan remodeling during the recycling of MUC1.

    Science.gov (United States)

    Razawi, Hanieh; Kinlough, Carol L; Staubach, Simon; Poland, Paul A; Rbaibi, Youssef; Weisz, Ora A; Hughey, Rebecca P; Hanisch, Franz-Georg

    2013-08-01

    The apical transmembrane glycoprotein MUC1 is endocytosed to recycle through the trans-Golgi network (TGN) or Golgi complex to the plasma membrane. We followed the hypothesis that not only the known follow-up sialylation of MUC1 in the TGN is associated with this process, but also a remodeling of O-glycan core structures, which would explain the previously described differential core 2- vs core 1-based O-glycosylation of secreted, single Golgi passage and recycling membrane MUC1 isoforms (Engelmann K, Kinlough CL, Müller S, Razawi H, Baldus SE, Hughey RP, Hanisch F-G. 2005. Glycobiology. 15:1111-1124). Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles. To address this novel observation, we used recombinant epitope-tagged MUC1 (MUC1-M) and mutant forms with abrogated clathrin-mediated endocytosis (MUC1-M-Y20,60N) or blocked recycling (palmitoylation-defective MUC1-M-CQC/AQA). We show that the CQC/AQA mutant transits the TGN at significantly lower levels, concomitant with a strongly reduced shedding from the plasma membrane and its accumulation in endosomal compartments. Intriguingly, the O-glycosylation of the shed MUC1 ectodomain subunit changes from preponderant sialylated core 1 (MUC1-M) to core 2 glycans on the non-recycling CQC/AQA mutant. The O-glycoprofile of the non-recycling CQC/AQA mutant resembles the core 2 glycoprofile on a secretory MUC1 probe that transits the Golgi complex only once. In contrast, the MUC1-M-Y20,60N mutant recycles via flotillin-dependent pathways and shows the wild-type phenotype with dominant core 1 expression. Differential radiolabeling of protein with [(35)S]Met/Cys or glycans with [(3)H]GlcNH2 in pulse-chase experiments of surface biotinylated MUC1 revealed a significantly shorter half-life of [(3)H]MUC1 when compared with [(35)S]MUC1, whereas the same ratio for the CQC/AQA mutant was close to one. This finding further supports the novel possibility of a recycling-associated O

  11. Sediments at the top of Earth's core.

    Science.gov (United States)

    Buffett, B A; Garnero, E J; Jeanloz, R

    2000-11-17

    Unusual physical properties at the core-mantle boundary have been inferred from seismic and geodetic observations in recent years. We show how both types of observations can be explained by a layer of silicate sediments, which accumulate at the top of the core as Earth cools. Compaction of the sediments expels most of the liquid iron but leaves behind a small amount of core material, which is entrained in mantle convection and may account for the isotopic signatures of core material in some hot spot plumes. Extraction of light elements from the liquid core also enhances the vigor of convection in the core and may increase the power available to the geodynamo.

  12. Core and Lumbopelvic Stabilization in Runners.