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Sample records for dpv ul46m protein

  1. A proteomic perspective of inbuilt viral protein regulation: pUL46 tegument protein is targeted for degradation by ICP0 during herpes simplex virus type 1 infection.

    Science.gov (United States)

    Lin, Aaron E; Greco, Todd M; Döhner, Katinka; Sodeik, Beate; Cristea, Ileana M

    2013-11-01

    Much like the host cells they infect, viruses must also regulate their life cycles. Herpes simples virus type 1 (HSV-1), a prominent human pathogen, uses a promoter-rich genome in conjunction with multiple viral trans-activating factors. Following entry into host cells, the virion-associated outer tegument proteins pUL46 and pUL47 act to increase expression of viral immediate-early (α) genes, thereby helping initiate the infection life cycle. Because pUL46 has gone largely unstudied, we employed a hybrid mass spectrometry-based approach to determine how pUL46 exerts its functions during early stages of infection. For a spatio-temporal characterization of pUL46, time-lapse microscopy was performed in live cells to define its dynamic localization from 2 to 24 h postinfection. Next, pUL46-containing protein complexes were immunoaffinity purified during infection of human fibroblasts and analyzed by mass spectrometry to investigate virus-virus and virus-host interactions, as well as post-translational modifications. We demonstrated that pUL46 is heavily phosphorylated in at least 23 sites. One phosphorylation site matched the consensus 14-3-3 phospho-binding motif, consistent with our identification of 14-3-3 proteins and host and viral kinases as specific pUL46 interactions. Moreover, we determined that pUL46 specifically interacts with the viral E3 ubiquitin ligase ICP0. We demonstrated that pUL46 is partially degraded in a proteasome-mediated manner during infection, and that the catalytic activity of ICP0 is responsible for this degradation. This is the first evidence of a viral protein being targeted for degradation by another viral protein during HSV-1 infection. Together, these data indicate that pUL46 levels are tightly controlled and important for the temporal regulation of viral gene expression throughout the virus life cycle. The concept of a structural virion protein, pUL46, performing nonstructural roles is likely to reflect a theme common to many viruses

  2. Identification of a novel linear B-cell epitope in the UL26 and UL26.5 proteins of Duck Enteritis Virus

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    Kong Xiangang

    2010-09-01

    Full Text Available Abstract Background The Unique Long 26 (UL26 and UL26.5 proteins of herpes simplex virus are known to function during the assembly of the viruses. However, for duck enteritis virus (DEV, which is an unassigned member of the family Herpesviridae, little information is available about the function of the two proteins. In this study, the C-terminus of DEV UL26 protein (designated UL26c, which contains the whole of UL26.5, was expressed, and the recombinant UL26c protein was used to immunize BALB/c mice to generate monoclonal antibodies (mAb. The mAb 1C8 was generated against DEV UL26 and UL26.5 proteins and used subsequently to map the epitope in this region. Both the mAb and its defined epitope will provide potential tools for further study of DEV. Results A mAb (designated 1C8 was generated against the DEV UL26c protein, and a series of 17 partially overlapping fragments that spanned the DEV UL26c were expressed with GST tags. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA and western blotting analysis using mAb 1C8 to identify the epitope. A linear motif, 520IYYPGE525, which was located at the C-terminus of the DEV UL26 and UL26.5 proteins, was identified by mAb 1C8. The result of the ELISA showed that this epitope could be recognized by DEV-positive serum from mice. The 520IYYPGE525 motif was the minimal requirement for reactivity, as demonstrated by analysis of the reactivity of 1C8 with several truncated peptides derived from the motif. Alignment and comparison of the 1C8-defined epitope sequence with those of other alphaherpesviruses indicated that the motif 521YYPGE525 in the epitope sequence was conserved among the alphaherpesviruses. Conclusion A mAb, 1C8, was generated against DEV UL26c and the epitope-defined minimal sequence obtained using mAb 1C8 was 520IYYPGE525. The mAb and the identified epitope may be useful for further study of the design of diagnostic reagents for DEV.

  3. Identification and characterization of the pseudorabies virus UL43 protein

    International Nuclear Information System (INIS)

    Klupp, Barbara G.; Altenschmidt, Jan; Granzow, Harald; Fuchs, Walter; Mettenleiter, Thomas C.

    2005-01-01

    Among the least characterized herpesvirus membrane proteins are the homologs of UL43 of herpes simplex virus 1 (HSV-1). To identify and characterize the UL43 protein of pseudorabies virus (PrV), part of the open reading frame was expressed in Escherichia coli and used for immunization of a rabbit. The antiserum recognized in Western blots a 34-kDa protein in lysates of PrV infected cells and purified virions, demonstrating that the UL43 protein is a virion component. In indirect immunofluorescence analysis, the antiserum labeled vesicular structures in PrV infected cells which also contained glycoprotein B. To functionally analyze UL43, a deletion mutant was constructed lacking amino acids 23-332 of the 373aa protein. This mutant was only slightly impaired in replication as assayed by one-step growth kinetics, measurement of plaque sizes, and electron microscopy. Interestingly, the PrV UL43 protein was able to inhibit fusion induced by PrV glycoproteins in a transient expression-fusion assay to a similar extent as gM. Double mutant viruses lacking, in addition to UL43, the multiply membrane spanning glycoproteins K or M did not show a phenotype beyond that observed in the gK and gM single deletion mutants

  4. Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93.

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    Köppen-Rung, Pánja; Dittmer, Alexandra; Bogner, Elke

    2016-07-01

    DNA packaging into procapsids is a common multistep process during viral maturation in herpesviruses. In human cytomegalovirus (HCMV), the proteins involved in this process are terminase subunits pUL56 and pUL89, which are responsible for site-specific cleavage and insertion of the DNA into the procapsid via portal protein pUL104. However, additional viral proteins are required for the DNA packaging process. We have shown previously that the plasmid that encodes capsid-associated pUL77 encodes another potential player during capsid maturation. Pulse-chase experiments revealed that pUL77 is stably expressed during HCMV infection. Time course analysis demonstrated that pUL77 is expressed in the early late part of the infectious cycle. The sequence of pUL77 was analyzed to find nuclear localization sequences (NLSs), revealing monopartite NLSm at the N terminus and bipartite NLSb in the middle of pUL77. The potential NLSs were inserted into plasmid pHM829, which encodes a chimeric protein with β-galactosidase and green fluorescent protein. In contrast to pUL56, neither NLSm nor NLSb was sufficient for nuclear import. Furthermore, we investigated by coimmunoprecipitation whether packaging proteins, as well as pUL93, the homologue protein of herpes simplex virus 1 pUL17, are interaction partners of pUL77. The interactions between pUL77 and packaging proteins, as well as pUL93, were verified. We showed that the capsid-associated pUL77 is another potential player during capsid maturation of HCMV. Protein UL77 (pUL77) is a conserved core protein of HCMV. This study demonstrates for the first time that pUL77 has early-late expression kinetics during the infectious cycle and an intrinsic potential for nuclear translocation. According to its proposed functions in stabilization of the capsid and anchoring of the encapsidated DNA during packaging, interaction with further DNA packaging proteins is required. We identified physical interactions with terminase subunits pUL56 and pUL

  5. HSV-1 nucleocapsid egress mediated by UL31 in association with UL34 is impeded by cellular transmembrane protein 140

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    Guan, Ying [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China); Yunnan Academy of Tobacco Science, Kunming, Yunnan 650106 (China); Guo, Lei; Yang, Erxia; Liao, Yun; Liu, Longding; Che, Yanchun; Zhang, Ying; Wang, Lichun; Wang, Jingjing [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China); Li, Qihan, E-mail: imbcams.lq@gmail.com [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China)

    2014-09-15

    During HSV-1 infection, the viral UL31 protein forms a complex with the UL34 protein at the cellular nuclear membrane, where both proteins play important roles in the envelopment of viral nucleocapsids and their egress into the cytoplasm. To characterize the mechanism of HSV-1 nucleocapsid egress, we screened host proteins to identify proteins that interacted with UL31 via yeast two-hybrid analysis. Transmembrane protein 140 (TMEM140), was identified and confirmed to bind to and co-localize with UL31 during viral infection. Further studies indicated that TMEM140 inhibits HSV-1 proliferation through selectively blocking viral nucleocapsid egress during the viral assembly process. The blockage function of TMEM140 is mediated by impeding the formation of the UL31–UL34 complex due to competitive binding to UL31. Collectively, these data suggest the essentiality of the UL31–UL34 interaction in the viral nucleocapsid egress process and provide a new anti-HSV-1 strategy in viral assembly process of nucleocapsid egress. - Highlights: • Cellular TMEM140 protein interacts with HSV-1 UL31 protein during viral infection. • Increasing expression of TMEM140 leads to inhibition of HSV-1 proliferation. • Increasing expression of TMEM140 blocks HSV-1 nucleocapsid egress process. • Binding to UL31 of TMEM140 impedes formation of HSV-1 UL31–UL34 complex.

  6. The herpes simplex virus 1 UL51 protein interacts with the UL7 protein and plays a role in its recruitment into the virion.

    Science.gov (United States)

    Roller, Richard J; Fetters, Rachel

    2015-03-01

    The alphaherpesvirus UL51 protein is a tegument component that interacts with the viral glycoprotein E and functions at multiple steps in virus assembly and spread in epithelial cells. We show here that pUL51 forms a complex in infected cells with another conserved tegument protein, pUL7. This complex can form in the absence of other viral proteins and is largely responsible for recruitment of pUL7 to cytoplasmic membranes and into the virion tegument. Incomplete colocalization of pUL51 and pUL7 in infected cells, however, suggests that a significant fraction of the population of each protein is not complexed with the other and that they may accomplish independent functions. The ability of herpesviruses to spread from cell to cell in the face of an immune response is critical for disease and shedding following reactivation from latency. Cell-to-cell spread is a conserved ability of herpesviruses, and the identification of conserved viral genes that mediate this process will aid in the design of attenuated vaccines and of novel therapeutics. The conserved UL51 gene of herpes simplex virus 1 plays important roles in cell-to-cell spread and in virus assembly in the cytoplasm, both of which likely depend on specific interactions with other viral and cellular proteins. Here we identify one of those interactions with the product of another conserved herpesvirus gene, UL7, and show that formation of this complex mediates recruitment of UL7 to membranes and to the virion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Expression and characterization of the UL31 protein from duck enteritis virus

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    Zhu Dekang

    2009-02-01

    Full Text Available Abstract Background Previous studies indicate that the UL31 protein and its homology play similar roles in nuclear egress of all herpesviruses. However, there is no report on the UL31 gene product of DEV. In this study, we expressed and presented the basic properties of the DEV UL31 product. Results The entire ORF of the UL31 was cloned into pET 32a (+ prokaryotic expression vector. Escherichia coli BL21(DE3 competent cells were transformed with the construct followed by the induction of protein expression by the addition of IPTG. Band corresponding to the predicted sizes (55 kDa was produced on the SDS-PAGE. Over expressed 6×His-UL31 fusion protein was purified by nickel affinity chromatography. The DEV UL31 gene product has been identified by using a rabbit polyclonal antiserum raised against the purified protein. A protein of approximate 35 kDa that reacted with the antiserum was detected in immunoblots of DEV-infected cellular lysates, suggesting that the 35 kDa protein was the primary translation product of the UL31 gene. RT-PCR analyses revealed that the UL31 gene was transcribed most abundantly during the late phase of replication. Subsequently, Immunofluorescence analysis revealed that the protein was widespread speckled structures in the nuclei of infected cells. Western blotting of purified virion preparations showed that UL31 was a component of intracellular virions but was absent from mature extracellular virions. Finally, an Immunofluorescence assay was established to study the distribution of the UL31 antigen in tissues of artificially DEV infected ducks. The results showed that the UL31 antigen was primarily located in the cells of digestive organs and immunological organs. Conclusion In this work, we present the basic properties of the DEV UL31 product. The results indicate that DEV UL31 shares many similarities with its HSV or PRV homolog UL31 and suggest that functional cross-complementation is possible between members of the

  8. Nuclear body formation and PML body remodeling by the human cytomegalovirus protein UL35

    International Nuclear Information System (INIS)

    Salsman, Jayme; Wang Xueqi; Frappier, Lori

    2011-01-01

    The human cytomegalovirus (HCMV) UL35 gene encodes two proteins, UL35 and UL35a. Expression of UL35 in transfected cells results in the formation of UL35 nuclear bodies that associate with promyelocytic leukemia (PML) protein. PML forms the basis for PML nuclear bodies that are important for suppressing viral lytic gene expression. Given the important relationship between PML and viral infection, we have further investigated the association of UL35 with PML bodies. We demonstrate that UL35 bodies form independently of PML and subsequently recruit PML, Sp100 and Daxx. In contrast, UL35a did not form bodies; however, it could bind UL35 and inhibit the formation of UL35 bodies. The HCMV tegument protein pp71 promoted the formation of UL35 bodies and the cytoplasmic localization of UL35a. Similarly, UL35a shifted pp71 to the cytoplasm. These results indicate that the interplay between UL35, UL35a and pp71 affects their subcellular localization and likely their functions throughout infection.

  9. Identification of interaction domains within the UL37 tegument protein of herpes simplex virus type 1.

    Science.gov (United States)

    Bucks, Michelle A; Murphy, Michael A; O'Regan, Kevin J; Courtney, Richard J

    2011-07-20

    Herpes simplex virus type 1 (HSV-1) UL37 is a 1123 amino acid tegument protein that self-associates and binds to the tegument protein UL36 (VP1/2). Studies were undertaken to identify regions of UL37 involved in these protein-protein interactions. Coimmunoprecipitation assays showed that residues within the carboxy-terminal half of UL37, amino acids 568-1123, are important for interaction with UL36. Coimmunoprecipitation assays also revealed that amino acids 1-300 and 568-1123 of UL37 are capable of self-association. UL37 appears to self-associate only under conditions when UL36 is not present or is present in low amounts, suggesting UL36 and UL37 may compete for binding. Transfection-infection experiments were performed to identify domains of UL37 that complement the UL37 deletion virus, K∆UL37. The carboxy-terminal region of UL37 (residues 568-1123) partially rescues the K∆UL37 infection. These results suggest the C-terminus of UL37 may contribute to its essential functional role within the virus-infected cell. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene

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    M. Zeeshan Chaudhry

    2017-07-01

    Full Text Available Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV UL36 gene, pUL36 (also known as vICA, binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV homolog of the UL36 gene is called M36, and its protein product (pM36 is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo.

  11. Identification of structural protein-protein interactions of herpes simplex virus type 1.

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    Lee, Jin H; Vittone, Valerio; Diefenbach, Eve; Cunningham, Anthony L; Diefenbach, Russell J

    2008-09-01

    In this study we have defined protein-protein interactions between the structural proteins of herpes simplex virus type 1 (HSV-1) using a LexA yeast two-hybrid system. The majority of the capsid, tegument and envelope proteins of HSV-1 were screened in a matrix approach. A total of 40 binary interactions were detected including 9 out of 10 previously identified tegument-tegument interactions (Vittone, V., Diefenbach, E., Triffett, D., Douglas, M.W., Cunningham, A.L., and Diefenbach, R.J., 2005. Determination of interactions between tegument proteins of herpes simplex virus type 1. J. Virol. 79, 9566-9571). A total of 12 interactions involving the capsid protein pUL35 (VP26) and 11 interactions involving the tegument protein pUL46 (VP11/12) were identified. The most significant novel interactions detected in this study, which are likely to play a role in viral assembly, include pUL35-pUL37 (capsid-tegument), pUL46-pUL37 (tegument-tegument) and pUL49 (VP22)-pUS9 (tegument-envelope). This information will provide further insights into the pathways of HSV-1 assembly and the identified interactions are potential targets for new antiviral drugs.

  12. Identification of host cell proteins which interact with herpes simplex virus type 1 tegument protein pUL37.

    Science.gov (United States)

    Kelly, Barbara J; Diefenbach, Eve; Fraefel, Cornel; Diefenbach, Russell J

    2012-01-20

    The herpes simplex virus type 1 (HSV-1) structural tegument protein pUL37, which is conserved across the Herpesviridae family, is known to be essential for secondary envelopment during the egress of viral particles. To shed light on additional roles of pUL37 during viral replication a yeast two-hybrid screen of a human brain cDNA library was undertaken. This screen identified ten host cell proteins as potential pUL37 interactors. One of the interactors, serine threonine kinase TAOK3, was subsequently confirmed to interact with pUL37 using an in vitro pulldown assay. Such host cell/pUL37 interactions provide further insights into the multifunctional role of this herpesviral tegument protein. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. The Ribosomal Protein uL22 Modulates the Shape of the Protein Exit Tunnel

    DEFF Research Database (Denmark)

    Wekselman, Itai; Zimmerman, Ella; Davidovich, Chen

    2017-01-01

    Erythromycin is a clinically useful antibiotic that binds to an rRNA pocket in the ribosomal exit tunnel. Commonly, resistance to erythromycin is acquired by alterations of rRNA nucleotides that interact with the drug. Mutations in the β hairpin of ribosomal protein uL22, which is rather distal...... of the β hairpin of the mutated uL22 toward the interior of the exit tunnel, triggering a cascade of structural alterations of rRNA nucleotides that propagate to the erythromycin binding pocket. Our findings support recent studies showing that the interactions between uL22 and specific sequences within...

  14. Embedded 32-bit Differential Pulse Voltammetry (DPV) Technique for 3-electrode Cell Sensing

    Science.gov (United States)

    N, Aqmar N. Z.; Abdullah, W. F. H.; Zain, Z. M.; Rani, S.

    2018-03-01

    This paper addresses the development of differential pulse voltammetry (DPV) embedded algorithm using an ARM cortex processor with new developed potentiostat circuit design for in-situ 3-electrode cell sensing. This project is mainly to design a low cost potentiostat for the researchers in laboratories. It is required to develop an embedded algorithm for analytical technique to be used with the designed potentiostat. DPV is one of the most familiar pulse technique method used with 3-electrode cell sensing in chemical studies. Experiment was conducted on 10mM solution of Ferricyanide using the designed potentiostat and the developed DPV algorithm. As a result, the device can generate an excitation signal of DPV from 0.4V to 1.2V and produced a peaked voltammogram with relatively small error compared to the commercial potentiostat; which is only 6.25% difference in peak potential reading. The design of potentiostat device and its DPV algorithm is verified.

  15. Cyclin A degradation by primate cytomegalovirus protein pUL21a counters its innate restriction of virus replication.

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    Nicolas Caffarelli

    Full Text Available Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC, but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus' ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.

  16. Herpes simplex virus type 1 gene UL14: phenotype of a null mutant and identification of the encoded protein.

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    Cunningham, C; Davison, A J; MacLean, A R; Taus, N S; Baines, J D

    2000-01-01

    Herpes simplex virus type 1 (HSV-1) gene UL14 is located between divergently transcribed genes UL13 and UL15 and overlaps the promoters for both of these genes. UL14 also exhibits a substantial overlap of its coding region with that of UL13. It is one of the few HSV-1 genes for which a phenotype and protein product have not been described. Using mass spectrometric and immunological approaches, we demonstrated that the UL14 protein is a minor component of the virion tegument of 32 kDa which is expressed late in infection. In infected cells, the UL14 protein was detected in the nucleus at discrete sites within electron-dense nuclear bodies and in the cytoplasm initially in a diffuse distribution and then at discrete sites. Some of the UL14 protein was phosphorylated. A mutant with a 4-bp deletion in the central region of UL14 failed to produce the UL14 protein and generated small plaques. The mutant exhibited an extended growth cycle at low multiplicity of infection and appeared to be compromised in efficient transit of virus particles from the infected cell. In mice injected intracranially, the 50% lethal dose of the mutant was reduced more than 30,000-fold. Recovery of the mutant from the latently infected sacral ganglia of mice injected peripherally was significantly less than that of wild-type virus, suggesting a marked defect in the establishment of, or reactivation from, latent infection.

  17. Interaction and interdependent packaging of tegument protein UL11 and glycoprotein e of herpes simplex virus.

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    Han, Jun; Chadha, Pooja; Meckes, David G; Baird, Nicholas L; Wills, John W

    2011-09-01

    The UL11 tegument protein of herpes simplex virus plays a critical role in the secondary envelopment; however, the mechanistic details remain elusive. Here, we report a new function of UL11 in the budding process in which it directs efficient acquisition of glycoprotein E (gE) via a direct interaction. In vitro binding assays showed that the interaction required only the first 28, membrane-proximal residues of the cytoplasmic tail of gE, and the C-terminal 26 residues of UL11. A second, weaker binding site was also found in the N-terminal half of UL11. The significance of the gE-UL11 interaction was subsequently investigated with viral deletion mutants. In the absence of the gE tail, virion packaging of UL11, but not other tegument proteins such as VP22 and VP16, was reduced by at least 80%. Reciprocally, wild-type gE packaging was also drastically reduced by about 87% in the absence of UL11, and this defect could be rescued in trans by expressing U(L)11 at the U(L)35 locus. Surprisingly, a mutant that lacks the C-terminal gE-binding site of UL11 packaged nearly normal amounts of gE despite its strong interaction with the gE tail in vitro, indicating that the interaction with the UL11 N terminus may be important. Mutagenesis studies of the UL11 N terminus revealed that the association of UL11 with membrane was not required for this function. In contrast, the UL11 acidic cluster motif was found to be critical for gE packaging and was not replaceable with foreign acidic clusters. Together, these results highlight an important role of UL11 in the acquisition of glycoprotein-enriched lipid bilayers, and the findings may also have important implications for the role of UL11 in gE-mediated cell-to-cell spread.

  18. Down-regulation of human cytomegalovirus UL138, a novel latency ...

    Indian Academy of Sciences (India)

    2013-07-12

    Jul 12, 2013 ... observed a 74.6% decrease in luciferase activity and a 46.2% decrease in HCMV UL138 protein expression. Our .... Trizol agent (Takara, Dalian, China), according to the man- ... hybrid primer and polyA structure were found in the se- quence. ..... resolution profiling and analysis of viral and host small RNAs.

  19. Identification of binding domains in the herpes simplex virus type 1 small capsid protein pUL35 (VP26).

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    Apcarian, Arin; Cunningham, Anthony L; Diefenbach, Russell J

    2010-11-01

    In this study, fragments of the small capsid protein pUL35 (VP26) from herpes simplex virus type 1 (HSV-1) were generated to identify binding domains for a number of known ligands. Analysis of the binding of dynein light chain subunits, DYNLT1 and DYNLT3, as well the HSV-1 structural proteins pUL19 (VP5) and pUL37 was then undertaken using the LexA yeast two-hybrid assay. The N-terminal half of pUL35, in particular residues 30-43, was identified as a common region for the binding of DYNLT1 and DYNLT3. Additional distinct regions in the C terminus of pUL35 also contribute to the binding of DYNLT1 and DYNLT3. In contrast, only the C-terminal half of pUL35 was found to mediate the binding of pUL19 and pUL37 through distinct regions. The relevance of this information to the role of pUL35 in viral transport and assembly is discussed.

  20. Analysis of contributions of herpes simplex virus type 1 UL43 protein ...

    African Journals Online (AJOL)

    Purpose: To investigate whether UL43 protein, which is highly conserved in alpha- and gamma herpes viruses, and a non-glycosylated transmembrane protein, is involved in virus entry and virus-induced cell fusion. Methods: Mutagenesis was accomplished by a markerless two-step Red recombination mutagenesis system ...

  1. Novel Structure and Unexpected RNA-Binding Ability of the C-Terminal Domain of Herpes Simplex Virus 1 Tegument Protein UL21

    Energy Technology Data Exchange (ETDEWEB)

    Metrick, Claire M.; Heldwein, Ekaterina E. (Tufts-MED)

    2016-04-06

    Proteins forming the tegument layers of herpesviral virions mediate many essential processes in the viral replication cycle, yet few have been characterized in detail. UL21 is one such multifunctional tegument protein and is conserved among alphaherpesviruses. While UL21 has been implicated in many processes in viral replication, ranging from nuclear egress to virion morphogenesis to cell-cell spread, its precise roles remain unclear. Here we report the 2.7-Å crystal structure of the C-terminal domain of herpes simplex virus 1 (HSV-1) UL21 (UL21C), which has a unique α-helical fold resembling a dragonfly. Analysis of evolutionary conservation patterns and surface electrostatics pinpointed four regions of potential functional importance on the surface of UL21C to be pursued by mutagenesis. In combination with the previously determined structure of the N-terminal domain of UL21, the structure of UL21C provides a 3-dimensional framework for targeted exploration of the multiple roles of UL21 in the replication and pathogenesis of alphaherpesviruses. Additionally, we describe an unanticipated ability of UL21 to bind RNA, which may hint at a yet unexplored function.

    IMPORTANCEDue to the limited genomic coding capacity of viruses, viral proteins are often multifunctional, which makes them attractive antiviral targets. Such multifunctionality, however, complicates their study, which often involves constructing and characterizing null mutant viruses. Systematic exploration of these multifunctional proteins requires detailed road maps in the form of 3-dimensional structures. In this work, we determined the crystal structure of the C-terminal domain of UL21, a multifunctional tegument protein that is conserved among alphaherpesviruses. Structural analysis pinpointed surface areas of potential functional importance that provide a starting point for mutagenesis. In addition, the unexpected RNA-binding ability of UL21 may expand its functional repertoire

  2. Labeling and localization of the herpes simplex virus capsid protein UL25 and its interaction with the two triplexes closest to the penton

    Science.gov (United States)

    Conway, James F.; Cockrell, Shelley K.; Copeland, Anna Maria; Newcomb, William W.; Brown, Jay C.; Homa, Fred L.

    2010-01-01

    The herpes simplex virus type 1 (HSV-1) UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component or CCSC. Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid binding domain of UL25 located on the surface of the mature DNA-containing capsid. PMID:20109467

  3. Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1

    Directory of Open Access Journals (Sweden)

    Wang Yu

    2011-04-01

    Full Text Available Abstract Background In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV, the causative agent of duck viral enteritis (DVE, the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. Results DEV UL15 consists of two exons with a 3.5 kilobases (kb inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa, whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s in the cytoplasm at 6 h post infection (h p. i. and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s in the cytoplasm in the absence of any other viral protein. Conclusions DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15

  4. Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1.

    Science.gov (United States)

    Zhu, Hongwei; Li, Huixin; Han, Zongxi; Shao, Yuhao; Wang, Yu; Kong, Xiangang

    2011-04-06

    In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV), the causative agent of duck viral enteritis (DVE), the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. DEV UL15 consists of two exons with a 3.5 kilobases (kb) inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa), whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp) ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s) in the cytoplasm at 6 h post infection (h p. i.) and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s) in the cytoplasm in the absence of any other viral protein. DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15 are very similar to those of alphaherpesviruses and

  5. The human cytomegalovirus UL11 protein interacts with the receptor tyrosine phosphatase CD45, resulting in functional paralysis of T cells.

    Directory of Open Access Journals (Sweden)

    Ildar Gabaev

    2011-12-01

    Full Text Available Human cytomegalovirus (CMV exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV.

  6. The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seongman; Chul Ahn, Byung; O' Callaghan, Dennis J. [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932 (United States); Kim, Seong Kee, E-mail: skim1@lsuhsc.edu [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932 (United States)

    2012-10-25

    The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

  7. The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

    International Nuclear Information System (INIS)

    Kim, Seongman; Chul Ahn, Byung; O’Callaghan, Dennis J.; Kim, Seong Kee

    2012-01-01

    The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)–UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

  8. Involvement of the UL24 protein in herpes simplex virus 1-induced dispersal of B23 and in nuclear egress

    International Nuclear Information System (INIS)

    Lymberopoulos, Maria H.; Bourget, Amelie; Abdeljelil, Nawel Ben; Pearson, Angela

    2011-01-01

    UL24 of herpes simplex virus 1 (HSV-1) is widely conserved within the Herpesviridae family. Herein, we tested the hypothesis that UL24, which we have previously shown to induce the redistribution of nucleolin, also affects the localization of the nucleolar protein B23. We found that HSV-1-induced dispersal of B23 was dependent on UL24. The conserved N-terminal portion of UL24 was sufficient to induce the redistribution of B23 in transient transfection assays. Mutational analysis revealed that the endonuclease motif of UL24 was important for B23 dispersal in both transfected and infected cells. Nucleolar protein relocalization during HSV-1 infection was also observed in non-immortalized cells. Analysis of infected cells by electron microscopy revealed a decrease in the ratio of cytoplasmic versus nuclear viral particles in cells infected with a UL24-deficient strain compared to KOS-infected cells. Our results suggest that UL24 promotes nuclear egress of nucleocapsids during HSV-1 infection, possibly though effects on nucleoli.

  9. A Tyrosine-Based Trafficking Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary Envelopment.

    Science.gov (United States)

    Dietz, Andrea N; Villinger, Clarissa; Becker, Stefan; Frick, Manfred; von Einem, Jens

    2018-01-01

    The human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi compartment-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 proteins fused to enhanced green fluorescent protein (eGFP) identified amino acids 23 to 34 as important determinants for its Golgi complex localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (YXXΦ) in pUL71. This led us to hypothesize a requirement of the YXXΦ motif for the function of pUL71 in infection. Mutation of both the tyrosine residue and the entire YXXΦ motif resulted in an altered distribution of mutant pUL71 at the plasma membrane and in the cytoplasm during infection. Both YXXΦ mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant-virus-infected cells exhibited impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed, similar to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the YXXΦ motif for targeting pUL71 to the Golgi complex was further demonstrated when clathrin-mediated endocytosis was inhibited either by coexpression of the C-terminal part of cellular AP180 (AP180-C) or by treatment with methyl-β-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and that is furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC. IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can

  10. Expression and characterization of UL16 gene from duck enteritis virus

    Directory of Open Access Journals (Sweden)

    Wang Mingshu

    2011-08-01

    Full Text Available Abstract Background Previous studies have indicated that the UL16 protein and its homologs from herpesvirus were conserved and played similar roles in viral DNA packaging, virion assembly, budding, and egress. However, there was no report on the UL16 gene product of duck enteritis virus (DEV. In this study, we analyzed the amino acid sequence of UL16 using bioinformatics tools and expressed in Escherichia coli Rosetta (DE3 induced by isopropy1-β-D-thiogalactopyranoside (IPTG. The recombinant protein was produced, purified using a Ni-NTA column and used to generate the polyclonal antibody against UL16. The intracellular distribution of the DEV UL16 product was carried out using indirect immunofluorescence assay. Results In our study, UL16 gene of DEV was composed of 1089 nucleotides, which encoded 362 amino acids. Multiple sequence alignment suggested that the UL16 gene was highly conserved in herpesvirus family. The UL16 gene was cloned into a pET prokaryotic expression vector and transformed into Escherichia coli Rossetta (DE3 induced by IPTG. A 60kDa fusion protein band corresponding to the predicted size was produced on the SDS-PAGE, purified using a Ni-NTA column. Anti-UL16 polyclonal sera was prepared by immunizing rabbits, and reacted with a band in the IPTG induced cell lysates with an apparent molecular mass of 60 kDa. In vivo expression of the UL16 protein in DEV infected duck embryo fibroblast cells (DEFs was localized mostly around perinuclear cytoplasmic area and in cytosol using indirect immunofluorescence assay. Conclusions The UL16 gene of DEV was successfully cloned, expressed and detected in DEV infected DEFs for the first time. The UL16 protein localized mostly around perinuclear cytoplasmic area and in cytosol in DEV infected DEFs. DEV UL16 shared high similarity with UL16 family members, indicating that DEV UL16 many has similar function with its homologs. All these results may provide some insight for further research about

  11. Identification of conserved amino acids in the herpes simplex virus type 1 UL8 protein required for DNA synthesis and UL52 primase interaction in the virus replisome.

    Science.gov (United States)

    Muylaert, Isabella; Zhao, Zhiyuan; Andersson, Torbjörn; Elias, Per

    2012-09-28

    We have used oriS-dependent transient replication assays to search for species-specific interactions within the herpes simplex virus replisome. Hybrid replisomes derived from herpes simplex virus type 1 (HSV-1) and equine herpesvirus type 1 (EHV-1) failed to support DNA replication in cells. Moreover, the replisomes showed a preference for their cognate origin of replication. The results demonstrate that the herpesvirus replisome behaves as a molecular machine relying on functionally important interactions. We then searched for functional interactions in the replisome context by subjecting HSV-1 UL8 protein to extensive mutagenesis. 52 mutants were made by replacing single or clustered charged amino acids with alanines. Four mutants showed severe replication defects. Mutant A23 exhibited a lethal phenotype, and mutants A49, A52 and A53 had temperature-sensitive phenotypes. Mutants A49 and A53 did not interact with UL52 primase as determined by co-immunoprecipitation experiments. Using GFP-tagged UL8, we demonstrate that all mutants were unable to support formation of ICP8-containing nuclear replication foci. Extended mutagenesis suggested that a highly conserved motif corresponding to mutant A49 serves an important role for establishing a physical contact between UL8 and UL52. The replication-defective mutations affected conserved amino acids, and similar phenotypes were observed when the corresponding mutations were introduced into EHV-1 UL8.

  12. The ribosomal protein uL22 modulates the shape of the nascent protein exit tunnel

    DEFF Research Database (Denmark)

    Wekselman, I.; Zimmerman, E.; Davidovich, C.

    2017-01-01

    in the entrance of theribosomal exit tunnel and interferes with the progression of nas-cent chains. Commonly, resistance to erythromycin is acquiredby alterations of rRNA nucleotides that interact with the drug.Mutations in theb-hairpin of ribosomal protein uL22, which israther distal to the erythromycin binding...... to erythromycin binding pocket and increases its flexi-bility. Based on our results, we suggest a feasble mechanism thatexplains how nanscent proteins can be translated when ery-thromycin is bound to the ribosome. Furthermore, our findingssupport recent studies showing that the interactions betweenuL22...

  13. Advanced dependent pressure vessel (DPV) nickel-hydrogen spacecraft battery design

    Energy Technology Data Exchange (ETDEWEB)

    Coates, D.K.; Grindstaff, B.; Swaim, O.; Fox, C. [Eagle-Picher Industries, Inc., Joplin, MO (United States). Advanced Systems Operation

    1995-12-31

    The dependent pressure vessel (DPV) nickel-hydrogen (NiH{sub 2}) battery is being developed as a potential spacecraft battery design for both military and commercial satellites. The limitations of standard NiH{sub 2} individual pressure vessel (IPV) flight battery technology are primarily related to the internal cell design and the battery packaging issues associated with grouping multiple cylindrical cells. The DPV cell design offers higher energy density and reduced cost, while retaining the established IPV technology flight heritage and database. The advanced cell design offers a more efficient mechanical, electrical and thermal cell configuration and a reduced parts count. The geometry of the DPV cell promotes compact, minimum volume packaging and weight efficiency. The DPV battery design offers significant cost and weight savings advantages while providing minimal design risks.

  14. Advanced nickel/hydrogen dependent pressure vessel (DPV) cell and battery concepts

    Energy Technology Data Exchange (ETDEWEB)

    Caldwell, D.B. [Technologies Div., Eagle Picher Industries, Inc., Joplin, MO (United States); Fox, C.L. [Technologies Div., Eagle Picher Industries, Inc., Joplin, MO (United States); Miller, L.E. [Technologies Div., Eagle Picher Industries, Inc., Joplin, MO (United States)

    1997-03-01

    The dependent pressure vessel (DPV) nickel/hydrogen (NiH{sub 2}) design is being developed by Eagle-Picher industries, Inc. (EPI) as an advanced battery for military and commercial aerospace and terrestrial applications. The DPV cell design offers high specific energy and energy density as well as reduced cost, while retaining the established individual pressure vessel (IPV) technology, flight heritage and database. This advanced DPV design also offers a more efficient mechanical, electrical and thermal cell and battery configuration and a reduced parts count. The DPV battery design promotes compact, minimum volume packaging and weight efficiency, and delivers cost and weight savings with minimal design risks. (orig.)

  15. Transcription pattern of UL131A-128 mRNA in clinical strains of ...

    Indian Academy of Sciences (India)

    Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by ...

  16. The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

    Science.gov (United States)

    Biswas, N; Weller, S K

    2001-05-18

    Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been

  17. Human Cytomegalovirus pUL47 Modulates Tegumentation and Capsid Accumulation at the Viral Assembly Complex

    Science.gov (United States)

    Cappadona, Ilaria; Villinger, Clarissa; Schutzius, Gabi; Mertens, Thomas

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in the tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop-infected cells clearly showed cytoplasmic accumulations of nonenveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop-infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in the absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells, whereas it was found exclusively at the vAC in the context of virus infection. Colocalization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids. IMPORTANCE Generation of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for

  18. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Christina Funk

    2015-06-01

    Full Text Available Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary

  19. Residues of the UL25 Protein of Herpes Simplex Virus That Are Required for Its Stable Interaction with Capsids ▿

    Science.gov (United States)

    Cockrell, Shelley K.; Huffman, Jamie B.; Toropova, Katerina; Conway, James F.; Homa, Fred L.

    2011-01-01

    The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes. PMID:21411517

  20. A polymerase chain reaction assay for detection of virulent and attenuated strains of duck plague virus.

    Science.gov (United States)

    Xie, Liji; Xie, Zhixun; Huang, Li; Wang, Sheng; Huang, Jiaoling; Zhang, Yanfang; Zeng, Tingting; Luo, Sisi

    2017-11-01

    Sequence analysis of duck plague virus (DPV) revealed that there was a 528bp (B fragment) deletion within the UL2 gene of DPV attenuated vaccine strain in comparison with field virulent strains. The finding of gene deletion provides a potential differentiation test between DPV virulent strain and attenuated strain based on their UL2 gene sizes. Thus we developed a polymerase chain reaction (PCR) assay targeting to the DPV UL2 gene for simultaneous detection of DPV virulent strain and attenuated strain, 827bp for virulent strain and 299bp for attenuated strain. This newly developed PCR for DPV was highly sensitive and specific. It detected as low as 100fg of DNA on both DPV virulent and attenuated strains, no same size bands were amplified from other duck viruses including duck paramyxovirus, duck tembusu virus, duck circovirus, Muscovy duck parvovirus, duck hepatitis virus type I, avian influenza virus and gosling plague virus. Therefore, this PCR assay can be used for the rapid, sensitive and specific detection of DPV virulent and attenuated strains affecting ducks. Copyright © 2017. Published by Elsevier B.V.

  1. Dynamic and nucleolin-dependent localization of human cytomegalovirus UL84 to the periphery of viral replication compartments and nucleoli.

    Science.gov (United States)

    Bender, Brian J; Coen, Donald M; Strang, Blair L

    2014-10-01

    Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin. Importance: The protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and cellular

  2. pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

    Science.gov (United States)

    Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

    2018-05-01

    The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Cellular homeoproteins, SATB1 and CDP, bind to the unique region between the human cytomegalovirus UL127 and major immediate-early genes

    International Nuclear Information System (INIS)

    Lee Jialing; Klase, Zachary; Gao Xiaoqi; Caldwell, Jeremy S.; Stinski, Mark F.; Kashanchi, Fatah; Chao, S.-H.

    2007-01-01

    An AT-rich region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region (UR). It has been shown that the UR represses activation of transcription from the UL127 promoter and functions as a boundary between the divergent UL127 and MIE genes during human CMV infection [Angulo, A., Kerry, D., Huang, H., Borst, E.M., Razinsky, A., Wu, J., Hobom, U., Messerle, M., Ghazal, P., 2000. Identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription of the divergent UL127 promoter. J. Virol. 74 (6), 2826-2839; Lundquist, C.A., Meier, J.L., Stinski, M.F., 1999. A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer. J. Virol. 73 (11), 9039-9052]. A putative forkhead box-like (FOX-like) site, AAATCAATATT, was identified in the UR and found to play a key role in repression of the UL127 promoter in recombinant virus-infected cells [Lashmit, P.E., Lundquist, C.A., Meier, J.L., Stinski, M.F., 2004. Cellular repressor inhibits human cytomegalovirus transcription from the UL127 promoter. J. Virol. 78 (10), 5113-5123]. However, the cellular factors which associate with the UR and FOX-like region remain to be determined. We reported previously that pancreatic-duodenal homeobox factor-1 (PDX1) bound to a 45-bp element located within the UR [Chao, S.H., Harada, J.N., Hyndman, F., Gao, X., Nelson, C.G., Chanda, S.K., Caldwell, J.S., 2004. PDX1, a Cellular Homeoprotein, Binds to and Regulates the Activity of Human Cytomegalovirus Immediate Early Promoter. J. Biol. Chem. 279 (16), 16111-16120]. Here we demonstrate that two additional cellular homeoproteins, special AT-rich sequence binding protein 1 (SATB1) and CCAAT displacement protein (CDP), bind to the human CMV UR in vitro and in vivo. Furthermore, CDP is identified as a FOX-like binding protein

  4. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

    Science.gov (United States)

    Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

    2018-01-13

    The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  5. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97

    Directory of Open Access Journals (Sweden)

    Jens Milbradt

    2018-01-01

    Full Text Available The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  6. End-joining inhibition at telomeres requires the translocase and polySUMO-dependent ubiquitin ligase Uls1.

    Science.gov (United States)

    Lescasse, Rachel; Pobiega, Sabrina; Callebaut, Isabelle; Marcand, Stéphane

    2013-03-20

    In eukaryotes, permanent inhibition of the non-homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non-essential Swi2/Snf2-related translocase and a Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere-telomere fusions. Uls1 requirement is alleviated by the absence of poly-SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly-SUMO conjugates. We propose that one of Uls1 functions is to clear non-functional poly-SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly-SUMOylated proteins on DNA in eukaryotes.

  7. Involvement of UL24 in herpes-simplex-virus-1-induced dispersal of nucleolin

    International Nuclear Information System (INIS)

    Lymberopoulos, Maria H.; Pearson, Angela

    2007-01-01

    UL24 of herpes simplex virus 1 is important for efficient viral replication, but its function is unknown. We generated a recombinant virus, vHA-UL24, encoding UL24 with an N-terminal hemagglutinin tag. By indirect immunofluorescence at 9 h post-infection (hpi), we detected HA-UL24 in nuclear foci and in cytoplasmic speckles. HA-UL24 partially co-localized with nucleolin, but not with ICP8 or coilin, markers for nucleoli, viral replication compartments, and Cajal bodies respectively. HA-UL24 staining was often juxtaposed to that of another nucleolar protein, fibrillarin. Analysis of HSV-1-induced nucleolar modifications revealed that by 18 hpi, nucleolin staining had dispersed, and fibrillarin staining went from clusters of small spots to a few separate but prominent spots. Fibrillarin redistribution appeared to be independent of UL24. In contrast, cells infected with a UL24-deficient virus retained foci of nucleolin staining. Our results demonstrate involvement of UL24 in dispersal of nucleolin during infection

  8. 75 FR 54381 - Charles M. Russell National Wildlife Refuge and UL Bend National Wildlife Refuge, MT

    Science.gov (United States)

    2010-09-07

    ... DEPARTMENT OF THE INTERIOR Fish and Wildlife Service [FWS-R6-R-2010-N078; 60138-1261-6CCP-S3] Charles M. Russell National Wildlife Refuge and UL Bend National Wildlife Refuge, MT AGENCY: Fish and Wildlife Service, Interior. ACTION: Notice of availability: Draft comprehensive conservation plan and draft...

  9. 75 FR 67095 - Charles M. Russell National Wildlife Refuge and UL Bend National Wildlife Refuge, Montana

    Science.gov (United States)

    2010-11-01

    ... DEPARTMENT OF THE INTERIOR Fish and Wildlife Service [FWS-R6-R-2010-N215; 60138-1261-6CCP-S3] Charles M. Russell National Wildlife Refuge and UL Bend National Wildlife Refuge, Montana AGENCY: Fish and Wildlife Service, Interior (DOI). ACTION: Notice; extension of comment period. SUMMARY: We, the U.S. Fish...

  10. Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA

    Directory of Open Access Journals (Sweden)

    Dixon Melissa

    2005-07-01

    Full Text Available Abstract Background Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks. Results A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus. Conclusion These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.

  11. Screening and identification of host factors interacting with UL14 of herpes simplex virus 1.

    Science.gov (United States)

    Wu, Fuqing; Xing, Junji; Wang, Shuai; Li, Meili; Zheng, Chunfu

    2011-08-01

    The UL14 protein of herpes simplex virus type 1 (HSV-1) is highly conserved in herpesvirus family. However, its exact function during the HSV-1 replication cycle is little known. In the present study, a high throughput yeast two-hybrid system was employed to screen the cellular factors interacting with UL14, and five target candidates were yielded: (1) TSC22 domain family protein 3 (TSC22D3); (2) Mediator of RNA polymerase II transcription subunit 8 isoform 1(MED8); (3) Runt-related transcription factor 3 (RUNX3); (4) Arrestin beta-2 (ARRB2); (5) Cereblon (CRBN). Indirect immunofluorescent assay showed that both TSC22D3 and MED8 co-localized with UL14. Co-immunoprecipitation assay demonstrated that UL14 could be immunoprecipitated by TSC22D3, suggesting that UL14 interacted with TSC22D3 under physiological condition. In summary, this study opened up new avenues toward delineating the function and physiological significance of UL14 during the HSV-1 replication cycle.

  12. Localization of HCMV UL33 and US27 in endocytic compartments and viral membranes

    DEFF Research Database (Denmark)

    Fraile-Ramos, Alberto; Pelchen-Matthews, Annegret; Kledal, Thomas N

    2002-01-01

    and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor-like proteins, UL33 and US27, in transfected and human cytomegalovirus-infected cells. Immunofluorescence staining indicated that UL33 and US27......The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor-like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles......27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus-infected cells showed that most virus particles wrapped individually...

  13. Conserved retinoblastoma protein-binding motif in human cytomegalovirus UL97 kinase minimally impacts viral replication but affects susceptibility to maribavir

    Directory of Open Access Journals (Sweden)

    Chou Sunwen

    2009-01-01

    Full Text Available Abstract The UL97 kinase has been shown to phosphorylate and inactivate the retinoblastoma protein (Rb and has three consensus Rb-binding motifs that might contribute to this activity. Recombinant viruses containing mutations in the Rb-binding motifs generally replicated well in human foreskin fibroblasts with only a slight delay in replication kinetics. Their susceptibility to the specific UL97 kinase inhibitor, maribavir, was also examined. Mutation of the amino terminal motif, which is involved in the inactivation of Rb, also renders the virus hypersensitive to the drug and suggests that the motif may play a role in its mechanism of action.

  14. Crystal Structure of the N-Terminal Half of the Traffic Controller UL37 from Herpes Simplex Virus 1

    Energy Technology Data Exchange (ETDEWEB)

    Koenigsberg, Andrea L.; Heldwein, Ekaterina E.; Sandri-Goldin, Rozanne M.

    2017-08-02

    Inner tegument protein UL37 is conserved among all three subfamilies of herpesviruses. Studies of UL37 homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), have suggested that UL37 plays an essential albeit poorly defined role in intracellular capsid trafficking. At the same time, HSV and PRV homologs cannot be swapped, which suggests that in addition to a conserved function, UL37 homologs also have divergent virus-specific functions. Accurate dissection of UL37 functions requires detailed maps in the form of atomic-resolution structures. Previously, we reported the crystal structure of the N-terminal half of UL37 (UL37N) from PRV. Here, we report the crystal structure of HSV-1 UL37N. Comparison of the two structures reveals that UL37 homologs differ in their overall shapes, distributions of surface charges, and locations of projecting loops. In contrast, the previously identified R2 surface region is structurally conserved. We propose that within the N-terminal half of UL37, functional conservation is centered within the R2 surface region, whereas divergent structural elements pinpoint regions mediating virus-specific functions and may engage different binding partners. Together, the two structures can now serve as templates for a structure-guided exploration of both conserved and virus-specific functions of UL37.

    IMPORTANCEThe ability to move efficiently within host cell cytoplasm is essential for replication in all viruses. It is especially important in the neuroinvasive alphaherpesviruses, such as human herpes simplex virus 1 (HSV-1), HSV-2, and veterinarian pseudorabies virus (PRV), that infect the peripheral nervous system and have to travel long distances along axons. Capsid movement in these viruses is controlled by capsid-associated tegument proteins, yet their specific roles have not yet been defined. Systematic exploration of the roles of tegument proteins in capsid trafficking requires

  15. A Simple, Cost-Effective Sensor for Detecting Lead Ions in Water Using Under-Potential Deposited Bismuth Sub-Layer with Differential Pulse Voltammetry (DPV)

    Science.gov (United States)

    Dai, Yifan; Liu, Chung Chiun

    2017-01-01

    This research has developed a simple to use, cost effective sensor system for the detection of lead ions in tap water. An under-potential deposited bismuth sub-layer on a thin gold film based electrochemical sensor was designed, manufactured, and evaluated. Differential pulse voltammetry (DPV) measurement technique was employed in this detection. Tap water from the Cleveland, OH, USA regional water district was the test medium. Concentrations of lead ion in the range of 8 × 10−7 M to 5 × 10−4 M were evaluated, showing a good sensitivity over this concentration range. The calibration curve for the DPV measurements of lead ions in tap water showed excellent reproducibility with R2 value of 0.970. This DPV detection system required 3–6 min to complete the detection measurement. A longer measurement time of 6 min was used for the lower lead ion concentration. The selectivity of this lead ion sensor was very good, and Fe III, Cu II, Ni II, and Mg II at a concentration level of 5 × 10−4 M did not interfere with the lead ion measurement. PMID:28441356

  16. Further Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and its Interaction with ICP8, the Major DNA-Binding Protein of Herpes Simplex Virus

    Science.gov (United States)

    1994-01-01

    Baringer, J.R. 1974. Recovery of herpes simplex virus from human sacral ganglions. N. Eng!. J. Med. 291:828-830. Baringer, J.R. 1976. The biology of herpes ...UL37 Protein of Herpes Simplex Virus Type 1 and its Interaction with [CPS, the Major DNA~Binding Protein of Herpes Simplex Virus" beyond brief...Protein of Herpes Simplex Virus Type 1 and its Interaction with [CPS, the Major DNA-Binding Protein of Herpes Simplex Virus Allen G. Albright Doctor of

  17. Nano-hole induction by nanodiamond and nanoplatinum liquid, DPV576, reverses multidrug resistance in human myeloid leukemia (HL60/AR

    Directory of Open Access Journals (Sweden)

    Ghoneum A

    2013-07-01

    Full Text Available Alia Ghoneum,1,2 Shivani Sharma,1,3 James Gimzewsk1,3 1Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, 2Department of Otalaryngology, Drew University of Medicine and Science, Los Angeles, 3California Nanosystems Institute (CNSI at University of California, Los Angeles, Los Angeles, CA, USA Abstract: Recently nanoparticles have been extensively studied and have proven to be a promising candidate for cancer treatment and diagnosis. In the current study, we examined the chemo-sensitizing activity of a mixture of nanodiamond (ND and nanoplatinum (NP solution known as DPV576, against multidrug-resistant (MDR human myeloid leukemia (HL60/AR and MDR-sensitive cells (HL60. Cancer cells were cultured with different concentrations of daunorubicin (DNR (1 × 10-9–1 × 10-6 M in the presence of selected concentrations of DPV576 (2.5%–10% v/v. Cancer cell survival was determined by MTT assay, drug accumulation by flow cytometry and confocal laser scanning microscopy (CLSM, and holes and structural changes by atomic force microscopy (AFM. Co-treatment of HL60/AR cells with DNR plus DPV576 resulted in the reduction of the IC50 to 1/4th. This was associated with increased incidences of holes inside the cells as compared with control untreated cells. On the other hand, HL60 cells did not show changes in their drug accumulation post-treatment with DPV576 and DNR. We conclude that DPV576 is an effective chemo-sensitizer as indicated by the reversal of HL60/AR cells to DNR and may represent a potential novel adjuvant for the treatment of chemo-resistant human myeloid leukemia. Keywords: nanodiamond, nanoplatinum, daunorubicin, flow cytometry, AFM

  18. The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

    Directory of Open Access Journals (Sweden)

    Laura Graf

    2013-12-01

    Full Text Available The human cytomegalovirus (HCMV-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.

  19. Antibodies against human cytomegalovirus late protein UL94 in the pathogenesis of scleroderma-like skin lesions in chronic graft-versus-host disease.

    Science.gov (United States)

    Pastano, Rocco; Dell'Agnola, Chiara; Bason, Caterina; Gigli, Federica; Rabascio, Cristina; Puccetti, Antonio; Tinazzi, Elisa; Cetto, Gianluigi; Peccatori, Fedro; Martinelli, Giovanni; Lunardi, Claudio

    2012-09-01

    Human cytomegalovirus (hCMV) infection and its reactivation correlate both with the increased risk and with the worsening of graft-versus-host disease (GVHD). Because scleroderma-like skin lesions can occur in chronic GVHD (cGVHD) in allogeneic stem-cell transplant (HCT) patients and hCMV is relevant in the pathogenesis of systemic sclerosis (SSc), we evaluated the possible pathogenetic link between hCMV and skin cGVHD. Plasma from 18 HCT patients was tested for anti-UL94 and/or anti-NAG-2 antibodies, identified in SSc patients, by direct ELISA assays. Both donors and recipients were anti-hCMV IgG positive, without autoimmune diseases. Patients' purified anti-UL94 and anti-NAG-2 IgG binding to human umbilical endothelial cells (HUVECs) and fibroblasts was performed by FACS analysis and ELISA test. HUVECs apoptosis and fibroblasts proliferation induced by patients' anti-NAG-2 antibodies were measured by DNA fragmentation and cell viability, respectively. About 11/18 patients developed cGVHD and all of them showed skin involvement, ranging from diffuse SSc-like lesions to limited erythema. Eight of eleven cGVHD patients were positive for anti-UL94 and/or anti-NAG-2 antibodies. Remarkably, 4/5 patients who developed diffuse or limited SSc-like lesions had antibodies directed against both UL94 and NAG-2; their anti-NAG-2 IgG-bound HUVECs and fibroblasts induce both endothelial cell apoptosis and fibroblasts proliferation, similar to that induced by purified anti-UL94 and anti-NAG-2 antibodies obtained from SSc patients. In conclusion, our data suggest a pathogenetic link between hCMV infection and scleroderma-like skin cGVHD in HCT patients through a mechanism of molecular mimicry between UL94 viral protein and NAG-2 molecule, as observed in patients with SSc.

  20. Effect of Nanodiamond and Nanoplatinum Liquid, DPV576, on Human Primary Keratinocytes.

    Science.gov (United States)

    Ghoneum, Mamdooh H; Katano, Hideki; Agrawal, Sudhanshu; Ganguly, Sreerupa; Agrawal, Anshu

    2017-01-01

    Nanofabrics are now being used in a wide range of products that come into direct contact with skin, including carpet, clothing, and medical fabrics. In the current study, we examined the effect of a dispersed aqueous mixture of nanodiamond (ND) and nanoplatinum (NP) (DPV576) on human primary keratinocytes with respect to transient receptor potential vanilloid (TRPV) channel expression, secretion of cytokines and production of nerve growth factor (NGF). Keratinocytes were treated with DPV576 at concentrations of 1:10 and 1:100 dilutions for 24 hours in vitro, and their activation of was determined by production of cytokines TNF-α, IL-1β, and prostaglandin (PGE2), and by production of NGF. Inhibitor experiments were carried out by incubating keratinocytes with the TRPV4-selective antagonist HC-067047. TRPV receptor expression (TRPV1, TRPV3 and TRPV4) on keratinocytes as well as changes in Ca2+ potential were examined by flow cytometry. DPV576 treatment of keratinocytes resulted in the following effects: (1) stimulation of keratinocytes as indicated by the significant secretion of cytokines IL-1β, TNF-α, and PGE2, an effect noted only at higher concentration (1:10); (2) significant decrease in the expression of TRPV4 on keratinocytes with a spike in the calcium flux, but no change in the expression of TRPV1 and TRPV3; (3) induction of cytokine secretion independent of TRPV4, as the addition of TRPV4 inhibitor had no significant effect on the cytokine production from keratinocytes; (4) induction of NGF secretion by keratinocytes. These results demonstrate that DPV576 activates keratinocytes via multiple signaling pathways which may reduce stress associated with inflammation, pain, and circadian rhythms. ND/NP-coated fabrics that target the modulation of local inflammation, pain, and circadian rhythms could potentially be of benefit to humans.

  1. Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

    International Nuclear Information System (INIS)

    Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana G.

    2006-01-01

    Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (ΔUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the ΔUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or ΔUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites

  2. Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

    Science.gov (United States)

    Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

    2005-07-01

    Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

  3. Bovine herpesvirus type-1 glycoprotein K (gK) interacts with UL20 and is required for infectious virus production

    Energy Technology Data Exchange (ETDEWEB)

    Haque, Muzammel; Stanfield, Brent; Kousoulas, Konstantin G.

    2016-12-15

    We have previously shown that the HSV-1 gK and UL20 proteins interact and function in virion envelopment, membrane fusion, and neuronal entry. Alignment of the predicted secondary structures of gKs encoded by BoHV-1, HSV-1, HSV-2, EHV-1 and VZV indicated a high degree of domain conservation. Two BoHV-1 gK-null mutant viruses were created by either gK gene deletion or stop codon insertion. In addition, a V5 epitope-tag was inserted at the carboxyl terminus of gK gene to detect gK. The engineered gK-null mutant viruses failed to replicate and produce viral plaques. Co-immunoprecipitation of gK and UL20 expressed via different methods revealed that gK and UL20 physically interacted in the presence or absence of other viral proteins. Confocal microscopy showed that gK and UL20 colocalized in infected cells. These results indicate that BoHV-1 gK and UL20 may function in a similar manner to other alphaherpesvirus orthologues specified by HSV-1, PRV and EHV-1. -- Highlights: •Glycoprotein K(gK) is conserved among alphaherpesviruses and serves similar functions. •The bovine herpesvirus-1 gK and UL20 proteins physically interact in a similar manner to herpes simplex virus type 1 and equine herpesvirus-1. •The bovine herpesvirus-1 (BoHV-1) gK interacts with UL20 and is essential for virus replication and spread.

  4. Bovine herpesvirus type-1 glycoprotein K (gK) interacts with UL20 and is required for infectious virus production

    International Nuclear Information System (INIS)

    Haque, Muzammel; Stanfield, Brent; Kousoulas, Konstantin G.

    2016-01-01

    We have previously shown that the HSV-1 gK and UL20 proteins interact and function in virion envelopment, membrane fusion, and neuronal entry. Alignment of the predicted secondary structures of gKs encoded by BoHV-1, HSV-1, HSV-2, EHV-1 and VZV indicated a high degree of domain conservation. Two BoHV-1 gK-null mutant viruses were created by either gK gene deletion or stop codon insertion. In addition, a V5 epitope-tag was inserted at the carboxyl terminus of gK gene to detect gK. The engineered gK-null mutant viruses failed to replicate and produce viral plaques. Co-immunoprecipitation of gK and UL20 expressed via different methods revealed that gK and UL20 physically interacted in the presence or absence of other viral proteins. Confocal microscopy showed that gK and UL20 colocalized in infected cells. These results indicate that BoHV-1 gK and UL20 may function in a similar manner to other alphaherpesvirus orthologues specified by HSV-1, PRV and EHV-1. -- Highlights: •Glycoprotein K(gK) is conserved among alphaherpesviruses and serves similar functions. •The bovine herpesvirus-1 gK and UL20 proteins physically interact in a similar manner to herpes simplex virus type 1 and equine herpesvirus-1. •The bovine herpesvirus-1 (BoHV-1) gK interacts with UL20 and is essential for virus replication and spread.

  5. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    Energy Technology Data Exchange (ETDEWEB)

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela, E-mail: angela.pearson@iaf.inrs.ca

    2013-09-15

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.

  6. Biophysical and morphological effects of nanodiamond/nanoplatinum solution (DPV576) on metastatic murine breast cancer cells in vitro

    International Nuclear Information System (INIS)

    Ghoneum, Alia; Zhu, Huanqi; Woo, JungReem; Zabinyakov, Nikita; Sharma, Shivani; Gimzewski, James K

    2014-01-01

    Nanoparticles have recently gained increased attention as drug delivery systems for the treatment of cancer due to their minute size and unique chemical properties. However, very few studies have tested the biophysical changes associated with nanoparticles on metastatic cancer cells at the cellular and sub-cellular scales. Here, we investigated the mechanical and morphological properties of cancer cells by measuring the changes in cell Young’s Modulus using AFM, filopodial retraction (FR) by time lapse optical light microscopy imaging and filopodial disorganization by high resolution AFM imaging of cells upon treatment with nanoparticles. In the current study, nanomechanical changes in live murine metastatic breast cancer cells (4T1) post exposure to a nanodiamond/nanoplatinum mixture dispersed in aqueous solution (DPV576), were monitored. Results showed a decrease in Young’s modulus at two hours post treatment with DPV576 in a dose dependent manner. Partial FR at 20 min and complete FR at 40 min were observed. Moreover, analysis of the retraction distance (in microns) measured over time (minutes), showed that a DPV576 concentration of 15%v/v yielded the highest FR rate. In addition, DPV576 treated cells showed early signs of filopodial disorganization and disintegration. This study demonstrates the changes in cell stiffness and tracks early structural alterations of metastatic breast cancer cells post treatment with DPV576, which may have important implications in the role of nanodiamond/nanoplatinum based cancer cell therapy and sensitization to chemotherapy drugs. (paper)

  7. Analysis of the herpes simplex virus type 1 UL6 gene in patients with stromal keratitis

    International Nuclear Information System (INIS)

    Ellison, Aaron R.; Yang Li; Cevallos, A. Vicky; Margolis, Todd P.

    2003-01-01

    Recent work suggests that herpes simplex virus (HSV) stromal keratitis in the mouse is caused by autoreactive T lymphocytes triggered by a 16 amino acid region of the HSV UL6 protein (aa299-314) , Science 279, 1344-1347). In the present study we sought to determine whether genetic variation of this presumed autoreactive UL6 epitope is responsible for different pathogenic patterns of human HSV keratitis. To accomplish this, we sequenced the HSV UL6 gene from ocular isolates of 10 patients with necrotizing stromal keratitis, 7 patients with recurrent epithelial keratitis, and 8 patients with other forms of HSV keratitis. The sequences obtained predicted identical UL6(299-314) epitopes for all 25 viral isolates. Furthermore, the upstream sequence of all isolates was free of insertions, deletions, and stop codons. We conclude that different pathogenic patterns of human HSV keratitis occur independent of genetic variation of the HSV UL6 (299-314) epitope

  8. Multiple phosphorylation sites at the C-terminus regulate nuclear import of HCMV DNA polymerase processivity factor ppUL44

    International Nuclear Information System (INIS)

    Alvisi, Gualtiero; Marin, Oriano; Pari, Gregory; Mancini, Manuela; Avanzi, Simone; Loregian, Arianna; Jans, David A.; Ripalti, Alessandro

    2011-01-01

    The processivity factor of human cytomegalovirus DNA polymerase, phosphoprotein ppUL44, is essential for viral replication. During viral infection ppUL44 is phosphorylated by the viral kinase pUL97, but neither the target residues on ppUL44 nor the effect of phosphorylation on ppUL44's activity are known. We report here that ppUL44 is phosphorylated when transiently expressed in mammalian cells and coimmunoprecipitates with cellular kinases. Of three potential phosphorylation sites (S413, S415, S418) located upstream of ppUL44's nuclear localization signal (NLS) and one (T427) within the NLS itself, protein kinase CK2 (CK2) specifically phosphorylates S413, to trigger a cascade of phosphorylation of S418 and S415 by CK1 and CK2, respectively. Negative charge at the CK2/CK1 target serine residues facilitates optimal nuclear accumulation of ppUL44, whereas negative charge on T427, a potential cyclin-dependent 1 phosphorylation site, strongly decreases nuclear accumulation. Thus, nuclear transport of ppUL44 is finely tuned during viral infection through complex phosphorylation events.

  9. Identification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and Demonstration that it Interacts with ICP8, the Major DNA Binding Protein of Herpes Simplex Virus

    Science.gov (United States)

    1992-10-20

    R . 1974 . Recovery of herpes simplex virus from human sacral gangl ions. N. Engl. J. Med. 291 :828-830. Baringer, J.R . 1975. Herpes simplex virus...AII’I fORCE MEDICAL C(NTEIt Title of Dissertation : "Ideatification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and...Demonstration that It Interacts with reps. the Major DNA Binding Protein of Herpes Simplex Virus" Name of Candidate: Lisa Shelton Doctor of

  10. Diabetes in Patients with ß-thalassemia or other Hemoglobinopathies - Analysis from the DPV Database.

    Science.gov (United States)

    Warncke, K; Konrad, K; Kohne, E; Hammer, E; Ohlenschläger, U; Herrlinger, S; Jäger, A; Holl, R W

    2016-11-01

    Background: Diabetes mellitus is a common endocrinopathy in patients with thalassemia major, but the occurrence of hemoglobinopathies is rare in Germany and Western Europe. The longitudinal German-Austrian DPV (Diabetes Patienten Verlaufsdokumentation) registry allows a comprehensive characterization of this group of patients. Patients/methods: Patients from the DPV-registry agedthalassemia major or other hemoglobinopathies were compared to patients with type 1 diabetes (T1D) and type 2 diabetes (T2D) using the statistical software SAS 9.4. Results: 94 patients (0.13% of patients) with hemoglobinopathies are registered in DPV. 82.4% of 17 patients with thalassemia major, 100% of 12 patients with sickle cell disease (SCD) and >90% of 65 patients with other hemoglobinopathies receive insulin treatment. In the majority of patients with thalassemia major, hemosiderosis is documented. Patients with thalassemia major developed diabetes at a median age of 14.6 [IQR 8.4-18.0] years (9.0 years [5.3-12.5] in T1D; 18.7 years [14.2-25.6] in TD2; both pthalassemia major is probably caused by hemosiderosis due to polytransfusion, while patients with SCD/thalassemia minor are most likely affected by T1D. The high rate of hypoglycemia in patients with ß-thalassemia major may be caused by liver fibrosis and a lack of hepatic glycogen stores. © Georg Thieme Verlag KG Stuttgart · New York.

  11. CRISPR/Cas9 Mutagenesis of UL21 in Multiple Strains of Herpes Simplex Virus Reveals Differential Requirements for pUL21 in Viral Replication

    Directory of Open Access Journals (Sweden)

    Renée L. Finnen

    2018-05-01

    Full Text Available Studies from multiple laboratories using different strains or species of herpes simplex virus (HSV with deletions in UL21 have yielded conflicting results regarding the necessity of pUL21 in HSV infection. To resolve this discrepancy, we utilized CRISPR/Cas9 mutagenesis to isolate pUL21 deficient viruses in multiple HSV backgrounds, and performed a side-by-side comparison of the cell-to-cell spread and replication phenotypes of these viruses. These analyses confirmed previous studies implicating the involvement of pUL21 in cell-to-cell spread of HSV. Cell-to-cell spread of HSV-2 was more greatly affected by the lack of pUL21 than HSV-1, and strain-specific differences in the requirement for pUL21 in cell-to-cell spread were also noted. HSV-2 strain 186 lacking pUL21 was particularly crippled in both cell-to-cell spread and viral replication in non-complementing cells, in comparison to other HSV strains lacking pUL21, suggesting that the strict requirement for pUL21 by strain 186 may not be representative of the HSV-2 species as a whole. This work highlights CRISPR/Cas9 technology as a useful tool for rapidly constructing deletion mutants of alphaherpesviruses, regardless of background strain, and should find great utility whenever strain-specific differences need to be investigated.

  12. Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

    International Nuclear Information System (INIS)

    Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank

    2013-01-01

    Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

  13. Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank, E-mail: fkempken@bot.uni-kiel.de

    2013-08-30

    Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

  14. UL146 variability among clinical isolates of Human Cytomegalovirus from Japan

    Directory of Open Access Journals (Sweden)

    Francisco Aguayo

    2010-01-01

    Full Text Available Human Cytomegalovirus (HCMV is a herpesvirus associated with serious diseases in immunocompromised subjects. The region between ORF UL133 and UL151 from HCMV, named ULb' is frequently deleted in attenuated AD169 and in highly passaged laboratory strains. However, this region is conserved in low-passaged and more virulent HCMV, like the Toledo strain. The UL146 gene, which is located in the ULb' region, encodes a CXC-chemokine analogue. The diversity of UL146 gene was evaluated among fifty-six clinical isolates of HCMV from Japan. Results show that UL146 gene was successfully amplified by the polymerase chain reaction (PCR in only 17/56 strains (30%, while the success rate for UL145/UL147 gene was 18/56 strains (32%. After DNA sequencing, the 35 amplified strains were classified into 8 groups. When compared, variability of UL146 ranged from 25.1% to 52.9% at the DNA level and from 34.5% to 67% at the amino acid level. Seven groups had the interleukin-8 (IL-8 motif ERL (Glu-Leu-Arg CXC and one group had only the CXC motif, suggesting the absence of the IL-8 function of UL146. In conclusion, we found that UL146 gene of HCMV is hyper-variable in clinical strains from Japan suggesting the possibility of a different function in each sequence group.

  15. Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

    Science.gov (United States)

    Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

    2018-01-15

    Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways. IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4 + T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

  16. Search for inclusive b→ulν decays

    International Nuclear Information System (INIS)

    Roberts, S.E.; Battle, M.O.; Thorndike, E.H.

    1993-01-01

    The authors have performed an inclusive analysis of the decay B→Xlν, measuring the lepton and neutrino momentum. They open-quotes detectclose quotes neutrinos via the missing 4-momentum in events, taking advantages of the hermeticity of the CLEO-II detector. The Υ(4S) resonance produces B mesons nearly at rest, and this, coupled with measurements of the lepton and neutrino momenta, provides a reasonable measurement of M X . By studying the distribution of M X they attempt a separation of semileptonic B decays into b→clν and b→ulν components, and infer a value of |V ub /V cb |

  17. Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

    International Nuclear Information System (INIS)

    Wei Yi; Shen Enzhi; Zhao Na; Liu Qian; Fan Jinling; Marc, Jan; Wang Yongchao; Sun Le; Liang Qianjin

    2008-01-01

    A novel centrosome-related protein Crp F46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein Crp F46 has an apparent molecular mass of ∼ 60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-Crp F46 monoclonal antibody revealed that Crp F46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using Crp F46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense Crp F46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that Crp F46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism

  18. 46 CFR 108.463 - Foam rate: Protein.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Foam rate: Protein. 108.463 Section 108.463 Shipping... EQUIPMENT Fire Extinguishing Systems Foam Extinguishing Systems § 108.463 Foam rate: Protein. (a) If the outlets of a protein foam extinguishing system are in a space, the foam rate at each outlet must be at...

  19. Identification of a Genetic Variation in ERAP1 Aminopeptidase that Prevents Human Cytomegalovirus miR-UL112-5p-Mediated Immunoevasion

    Directory of Open Access Journals (Sweden)

    Paolo Romania

    2017-07-01

    Full Text Available Herein, we demonstrate that HCMV miR-UL112-5p targets ERAP1, thereby inhibiting the processing and presentation of the HCMV pp65495-503 peptide to specific CTLs. In addition, we show that the rs17481334 G variant, naturally occurring in the ERAP1 3′ UTR, preserves ERAP1 from miR-UL112-5p-mediated degradation. Specifically, HCMV miR-UL112-5p binds the 3′ UTR of ERAP1 A variant, but not the 3′ UTR of ERAP1 G variant, and, accordingly, ERAP1 expression is reduced both at RNA and protein levels only in human fibroblasts homozygous for the A variant. Consistently, HCMV-infected GG fibroblasts were more efficient in trimming viral antigens and being lysed by HCMV-peptide-specific CTLs. Notably, a significantly decreased HCMV seropositivity was detected among GG individuals suffering from multiple sclerosis, a disease model in which HCMV is negatively associated with adult-onset disorder. Overall, our results identify a resistance mechanism to HCMV miR-UL112-5p-based immune evasion strategy with potential implications for individual susceptibility to infection and other diseases.

  20. Pendidikan Hati Perspektif al-Ghazālī dalam Kitab Ihya’ Ulūm al-Dīn

    Directory of Open Access Journals (Sweden)

    Muhammad Hasyim

    2017-09-01

    Full Text Available Al-Ghazālī in Sufism has had big influenced of his thought in the world. He was Syafi’i follower in fiqih law and Asy’ariyah in theology, when he was in Baghdad he hung out with many mazhabs. Ihya’ Ulūm al-Dīn is outlined with sharp thought method. The discussion is divided to systematic parts. It combined outward and inner fiqh, in explaining the difficult terms, this book uses tamstil. Al Ghazali has written heart diseases and its medicine. It also inserted many advantage stories as refreshing phase. Heart education is the soul controlling process. This soul consequently is able to encourage the physical activity in daily life appropriate with syari’ah of Allah through riyadhoh, fasting, praying or other worships. It also makes person becomes obidient people from birth to death, through many education and teaching and learning gradually, in which the learning peocess is parents and society responsibility toward a pious (shalih person. The purpose of psychology according to Ghazali is forming pious or shalih people.

  1. Lean diabetes in middle-aged adults: A joint analysis of the German DIVE and DPV registries

    OpenAIRE

    Hartmann, Bettina; Lanzinger, Stefanie; Bramlage, Peter; Gro?, Felix; Danne, Thomas; Wagner, Siegfried; Krakow, Dietmar; Zimmermann, Artur; Malcharzik, Christian; Holl, Reinhard W.

    2017-01-01

    Aims To assess differences in demographics, treatment and outcome of lean (LD) compared to overweight and obese people with diabetes clinically classified as type 2 diabetes mellitus (T2DM). Materials and methods We combined data from the German DIVE (Diabetes Versorgungs-Evaluation) and DPV (Diabetes-Patienten-Verlaufsdokumentation) databases to produce a large cohort of people with T2DM. The characteristics of people with Body Mass Index (BMI)

  2. Development and evaluation of an immunochromatographic strip test based on the recombinant UL51 protein for detecting antibody against duck enteritis virus

    Directory of Open Access Journals (Sweden)

    Sun Tao

    2010-10-01

    Full Text Available Abstract Background Duck enteritis virus (DEV infection causes substantial economic losses to the worldwide duck-producing areas. The monitoring of DEV-specific antibodies is a key to evaluate the effect of DEV vaccine and develop rational immunization programs. Thus, in this study, an immunochromatographic strip (ICS test was developed for detecting DEV serum antibodies. Results The ICS test is based on membrane chromatography, and uses both the purified recombinant UL51 protein conjugated with colloidal gold and goat anti-rabbit IgG conjugated with colloidal gold as tracers, the purified recombinant UL51 protein as the capture reagent at the test line, and rabbit IgG as the capture reagent at the control line. The specificity of the ICS was evaluated by sera against DEV, Duck hepatitis virus (DHV, Riemerella anatipestifer (RA, Duck E. coli, Muscovy duck parvovirus (MPV, or Duck Influenza viruses (DIV. Only sera against DEV showed the strong positive results. In order to determine the sensitivity of the ICS, anti-DEV serum diluted serially was tested, and the minimum detection limit of 1:128 was obtained. The ICS components, which are provided in a sealed package, require no refrigeration and are stable for 12 months. To evaluate the effect of the ICS, 110 duck serum samples collected from several non-immune duck flocks were simultaneously tested by the ICS test, enzyme-linked immunosorbent assay (ELISA and neutralization test (NT. The results showed that the sensitivity of the ICS test was almost consistent with ELISA and much higher than NT, has low cost, and is rapid (15 min and easy to perform with no requirement of specialized equipment, reagent or technicians. Conclusions In this work, we successfully developed a simple and rapid ICS test for detecting DEV serum antibodies for the first time. The ICS test was high specific and sensitive for the rapid detection of anti-DEV antibodies, and has great potential to be used for the serological

  3. Production and Its Anti-hyperglycemic Effects of γ-Aminobutyric Acid from the Wild Yeast Strain Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1.

    Science.gov (United States)

    Han, Sang-Min; Lee, Jong-Soo

    2017-09-01

    This study was done to produce γ-aminobutyric acid (GABA) from wild yeast as well as investigate its anti-hyperglycemic effects. Among ten GABA-producing yeast strains, Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1 produced high GABA concentration of 134.4 µg/mL and 179.2 µg/mL, respectively. P. silvicola UL6-1 showed a maximum GABA yield of 136.5 µg/mL and 200.8 µg/mL from S. carnicolor 402-JB-1 when they were cultured for 30 hr at 30℃ in yeast extract-peptone-dextrose medium. The cell-free extract from P. silvicola UL6-1 and S. carnicolor 402-JB-1 showed very high anti-hyperglycemic α-glucosidase inhibitory activity of 72.3% and 69.9%, respectively. Additionally, their cell-free extract-containing GABA showed the anti-hyperglycemic effect in streptozotocin-induced diabetic Sprague-Dawley rats.

  4. Crystallization and preliminary crystallographic analysis of tRNA (m7G46) methyltransferase from Escherichia coli

    International Nuclear Information System (INIS)

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun; Niu, Liwen

    2008-01-01

    tRNA (m 7 G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m 7 G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N 7 -methylguanosine (m 7 G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His 6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2 1

  5. Resistance to maribavir is associated with the exclusion of pUL27 from nucleoli during human cytomegalovirus infection

    Science.gov (United States)

    Hakki, Morgan; Drummond, Coyne; Houser, Benjamin; Marousek, Gail; Chou, Sunwen

    2011-01-01

    Select mutations in the human cytomegalovirus (HCMV) gene UL27 confer low-grade resistance to the HCMV UL97 kinase inhibitor maribavir (MBV). It has been reported that the 608-amino acid UL27 gene product (pUL27) normally localizes to cell nuclei and nucleoli, whereas its truncation at codon 415, as found in a MBV-resistant mutant, results in cytoplasmic localization. We now show that in the context of full-length pUL27, diverse single amino acid substitutions associated with MBV resistance result in loss of its nucleolar localization when visualized after transient transfection, whereas substitutions representing normal interstrain polymorphism had no such effect. The same differences in localization were observed during a complete infection cycle with recombinant HCMV strains over-expressing full-length fluorescent pUL27 variants. Nested UL27 C-terminal truncation expression plasmids showed that amino acids 596–599 were required for the nucleolar localization of pUL27. These results indicate that the loss of a nucleolar function of pUL27 may contribute to MBV resistance, and that the nucleolar localization of pUL27 during HCMV infection depends not only on a carboxy-terminal domain but also on a property of pUL27 that is affected by MBV-resistant mutations, such as an interaction with component(s) of the nucleolus. PMID:21906628

  6. Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

    Science.gov (United States)

    Mostafa, Heba H; Thompson, Thornton W; Konen, Adam J; Haenchen, Steve D; Hilliard, Joshua G; Macdonald, Stuart J; Morrison, Lynda A; Davido, David J

    2018-04-01

    In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39 , which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo , we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39 mut ), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection. IMPORTANCE HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in

  7. Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

    Directory of Open Access Journals (Sweden)

    Sofiya Fedosyuk

    2016-12-01

    Full Text Available Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83 structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240, we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.

  8. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells

    International Nuclear Information System (INIS)

    Haering, H.U.; White, M.F.; Machicao, F.; Ermel, B.; Schleicher, E.; Obermaier, B.

    1987-01-01

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, the authors labeled intact rat fat cells with [ 32 P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, they found, in addition to the 95-kDa β-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor β-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32 P incorporation into a 116-kDa band, a 62 kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. They suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission

  9. The Cytomegalovirus UL146 Gene Product vCXCL1 Targets Both CXCR1 and CXCR2 as an Agonist

    DEFF Research Database (Denmark)

    Luttichau, H.R.

    2010-01-01

    Large DNA viruses, such as herpesvirus and poxvirus, encode proteins that target and exploit the chemokine system of their host. UL146 and UL147 in the cytomegalovirus (CMV) genome encode the two CXC chemokines vCXCL1 and vCXCL2. In this study, vCXCL1 was probed against a panel of the 18 classified...... human chemokine receptors. In calcium mobilization assays vCXCL1 acted as an agonist on both CXCR1 and CXCR2 but did not activate or block any of the other 16 chemokine receptors. vCXCL1 was characterized and compared with CXCL1/GRO alpha, CXCL2/GRO beta, CXCL3/GRO gamma, CXCL5/ENA-78, CXCL6/GCP2, CXCL7...

  10. The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Nemčovičová, Ivana [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States); Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava (Slovakia); Zajonc, Dirk M., E-mail: dzajonc@liai.org [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States)

    2014-03-01

    The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host

  11. The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

    International Nuclear Information System (INIS)

    Nemčovičová, Ivana; Zajonc, Dirk M.

    2014-01-01

    The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X 6 G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor interactions

  12. Down-regulation of MHC class I by the Marek's disease virus (MDV) UL49.5 gene product mildly affects virulence in a haplotype-specific fashion.

    Science.gov (United States)

    Jarosinski, Keith W; Hunt, Henry D; Osterrieder, Nikolaus

    2010-09-30

    Marek's disease is a devastating neoplastic disease of chickens caused by Marek's disease virus (MDV). MDV down-regulates surface expression of MHC class I molecules, although the mechanism has remained elusive. MDV harbors a UL49.5 homolog that has been shown to down-regulate MHC class I expression in other Varicelloviruses. Using in vitro assays, we showed that MDV pUL49.5 down-regulates MHC class I directly and identified its cytoplasmic tail as essential for this function. In vivo, viruses lacking the cytoplasmic tail of pUL49.5 showed no differences in MD pathogenesis compared to revertant viruses in highly susceptible chickens of the B(19)B(19) MHC class I haplotype, while there was a mild reduction in pathogenic potential of the deletion viruses in chickens more resistant to MD pathogenesis (MHC:B(21)B(21)). We concluded that the pathogenic effect of MHC class I down-regulation mediated by pUL49.5 is small because virus immune evasion possibly requires more than one viral protein. Copyright 2010 Elsevier Inc. All rights reserved.

  13. 78 FR 28812 - Energy Efficiency Program for Industrial Equipment: Petition of UL Verification Services Inc. for...

    Science.gov (United States)

    2013-05-16

    ... are engineers. UL today is comprised of five businesses, Product Safety, Verification Services, Life..., Director--Global Technical Research, UL Verification Services. Subscribed and sworn to before me this 20... (431.447(c)(4)) General Personnel Overview UL is a global independent safety science company with more...

  14. Crystallization and preliminary crystallographic analysis of tRNA (m{sup 7}G46) methyltransferase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    2008-08-01

    tRNA (m{sup 7}G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m{sup 7}G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N{sup 7}-methylguanosine (m{sup 7}G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His{sub 6} tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2{sub 1}.

  15. AWI Moored ULS Data, Weddell Sea (1990-1998)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set consists of moored Upward Looking Sonar (ULS) data from 14 stations in the Weddell Sea. Parameters in the processed data files are water pressure,...

  16. Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane.

    Science.gov (United States)

    Bailer, Susanne M.

    2017-11-25

    Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress.

  17. Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

    Science.gov (United States)

    Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

    2017-02-01

    The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

  18. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

    Science.gov (United States)

    Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-03-10

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

  19. Application of ascorbic acid 2-phosphate as a new voltammetric substrate for alkaline phosphatase determination in human serum

    Directory of Open Access Journals (Sweden)

    Wei Sun

    2005-12-01

    Full Text Available An electrochemical assay of the enzyme alkaline phosphatase (ALP using ascorbic acid 2-phosphate (AAP as a new voltammetric substrate has been described in this paper. In the alkaline buffer solution the ALP enzymatic hydrolysis product of AAP was ascorbic acid (AA, which was an electro-active substance and had a sensitive differential pulse voltammetric (DPV oxidative response on glassy carbon electrode (GCE at +380 mV (versus Ag/AgCl, so the activity of ALP could be monitored voltammetrically of the oxidative peak current of AA. The electrochemical behaviours of AA were carefully studied and the AA standard solution could be measured by DPV method in the linear range from 10.0 to 1000.0 μmol/L with the detection limit of 8.0 μmol/L. The optimal conditions for ALP enzymatic reaction and the voltammetric detection were optimized. Under the optimal conditions the calibration curve for ALP assay exhibited a linear range from 0.4 to 2000.0 U/L with a detection limit of 0.3 U/L. This proposed method was further applied to determine the ALP content in healthy human serum and the results were in good agreement with the traditional p-nitrophenyl phosphate spectrophotometric method. The kinetic constants of enzymatic reaction were also investigated with the apparent kinetic constant Km as 2.77 mmol/L and the maximum velocity Vmax as 0.33 mol/min.

  20. Nuclear trafficking of the human cytomegalovirus pp71 (ppUL82) tegument protein

    International Nuclear Information System (INIS)

    Shen Weiping; Westgard, Elizabeth; Huang Liqun; Ward, Michael D.; Osborn, Jodi L.; Chau, Nha H.; Collins, Lindsay; Marcum, Benjamin; Koach, Margaret A.; Bibbs, Jennifer; Semmes, O. John; Kerry, Julie A.

    2008-01-01

    The human cytomegalovirus tegument protein pp71 localizes to the nucleus immediately upon infection, and functions to initiate viral gene expression. Analysis of a series of random insertion mutations revealed that sequences within the mid region (MR) of pp71 are important for localization to the nucleus. Fusion of MR sequences with eGFP revealed that amino acids 94 to 300 were sufficient to target proteins to the nucleus. Random substitution mutagenesis within this domain resulted in two double substitution mutants, pp71P203T/T223M and pp71T228M/L275Q, with a predominantly cytoplasmic localization. Disruption of nuclear targeting resulted in relocalization of the fusion proteins to a distinct perinuclear region. Using tandem mass spectrometry, we determined that threonine 223 can be phosphorylated. Mutation of this residue to a phosphomimetic amino acid resulted in abrogation of nuclear targeting. These results strongly suggest that the intracellular trafficking of pp71 is regulated by phosphorylation

  1. Simultaneous DPV determination of morphine and codeine using dsDNA modified screen printed electrode strips coupled with electromembrane extraction

    Directory of Open Access Journals (Sweden)

    Rouhollah Feizbakhsh

    2016-01-01

    Full Text Available In this work a sensitive electrochemical sensor for simultaneous determination of morphine and codeine constructed by application of disposable screen printed carbon electrode strips (SPCE modified by double strand (ds calf thymus DNA. According to the results of the modified SPCE strips and experimented parameters, we observed a considerable shift between potentials of morphine and codeine current peaks. Related to these observed shifts, we studied on the effect of the concentration of modifier and pH value on the anodic oxidation pattern of morphine and codeine in the case of optimize the method to get better signals with maximum potential distance. Also to boosting the LODs of this electroanalytical method coupled with an electro-membrane preconcentration (EME step. The calibration curve which was plotted by the variation of differential pulse voltammetry (DPV currents as a function of different morphine and codeine concentration were linear within the range of 0.7– 40 µM and 2.3- 40 µM for morphine and codeine respectively. Also the limits of detection were 0.07 µM and 0.23 µM, respectively. Finally, the proposed method was able to determine morphine and codeine simultaneously and effectively in urinary real samples

  2. ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

    International Nuclear Information System (INIS)

    Zhang Yongfang; Xia Zongqin; Hu Ya'er

    2010-01-01

    Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

  3. WATER QUALITY EVALUATION OF CRIŞUL ALB AND CRIŞUL NEGRU RIVERS CATCHMENTS, FROM CODRU-MOMA MOUNTAINS (WEST OF ROMANIA, USING BENTHIC INVERTEBRATES COMMUNITIES

    Directory of Open Access Journals (Sweden)

    Andreea VARGA

    2010-01-01

    Full Text Available Water quality evaluation of the two watersheds involved the collection of thirteen samples from the tributaries of Crişul Alb and Crişul Negru rivers. The samples were collected in june 2010 with a benthic net, which had the mesh size of 250 µm, by disturbing the substrate, being thus qualitative samples. To get an overview, a series of physical-chemical parameters (water temperature, pH, oxygen, conductivity, cyanide, nitrates, nitrites, phosphates was studied in parallel with the study of benthic community. In most of the sampling points the major group of benthic macroinvertebrates were found and in some EPT group (Ephemeroptera, Plecoptera, Trichoptera prevailed even, which is known as a clean freshwater group, sensitive to pollution and human impact.

  4. Properties of virion transactivator proteins encoded by primate cytomegaloviruses

    Directory of Open Access Journals (Sweden)

    Barry Peter A

    2009-05-01

    Full Text Available Abstract Background Human cytomegalovirus (HCMV is a betaherpesvirus that causes severe disease in situations where the immune system is immature or compromised. HCMV immediate early (IE gene expression is stimulated by the virion phosphoprotein pp71, encoded by open reading frame (ORF UL82, and this transactivation activity is important for the efficient initiation of viral replication. It is currently recognized that pp71 acts to overcome cellular intrinsic defences that otherwise block viral IE gene expression, and that interactions of pp71 with the cell proteins Daxx and ATRX are important for this function. A further property of pp71 is the ability to enable prolonged gene expression from quiescent herpes simplex virus type 1 (HSV-1 genomes. Non-human primate cytomegaloviruses encode homologs of pp71, but there is currently no published information that addresses their effects on gene expression and modes of action. Results The UL82 homolog encoded by simian cytomegalovirus (SCMV, strain Colburn, was identified and cloned. This ORF, named S82, was cloned into an HSV-1 vector, as were those from baboon, rhesus monkey and chimpanzee cytomegaloviruses. The use of an HSV-1 vector enabled expression of the UL82 homologs in a range of cell types, and permitted investigation of their abilities to direct prolonged gene expression from quiescent genomes. The results show that all UL82 homologs activate gene expression, and that neither host cell type nor promoter target sequence has major effects on these activities. Surprisingly, the UL82 proteins specified by non-human primate cytomegaloviruses, unlike pp71, did not direct long term expression from quiescent HSV-1 genomes. In addition, significant differences were observed in the intranuclear localization of the UL82 homologs, and in their effects on Daxx. Strikingly, S82 mediated the release of Daxx from nuclear domain 10 substructures much more rapidly than pp71 or the other proteins tested. All

  5. Herpes Simplex Virus 1 UL24 Abrogates the DNA Sensing Signal Pathway by Inhibiting NF-κB Activation.

    Science.gov (United States)

    Xu, Haiyan; Su, Chenhe; Pearson, Angela; Mody, Christopher H; Zheng, Chunfu

    2017-04-01

    Cyclic GMP-AMP synthase (cGAS) is a newly identified DNA sensor that recognizes foreign DNA, including the genome of herpes simplex virus 1 (HSV-1). Upon binding of viral DNA, cGAS produces cyclic GMP-AMP, which interacts with and activates stimulator of interferon genes (STING) to trigger the transcription of antiviral genes such as type I interferons (IFNs), and the production of inflammatory cytokines. HSV-1 UL24 is widely conserved among members of the herpesviruses family and is essential for efficient viral replication. In this study, we found that ectopically expressed UL24 could inhibit cGAS-STING-mediated promoter activation of IFN-β and interleukin-6 (IL-6), and UL24 also inhibited interferon-stimulatory DNA-mediated IFN-β and IL-6 production during HSV-1 infection. Furthermore, UL24 selectively blocked nuclear factor κB (NF-κB) but not IFN-regulatory factor 3 promoter activation. Coimmunoprecipitation analysis demonstrated that UL24 bound to the endogenous NF-κB subunits p65 and p50 in HSV-1-infected cells, and UL24 was also found to bind the Rel homology domains (RHDs) of these subunits. Furthermore, UL24 reduced the tumor necrosis factor alpha (TNF-α)-mediated nuclear translocation of p65 and p50. Finally, mutational analysis revealed that the region spanning amino acids (aa) 74 to 134 of UL24 [UL24(74-134)] is responsible for inhibiting cGAS-STING-mediated NF-κB promoter activity. For the first time, UL24 was shown to play an important role in immune evasion during HSV-1 infection. IMPORTANCE NF-κB is a critical component of the innate immune response and is strongly induced downstream of most pattern recognition receptors (PRRs), leading to the production of IFN-β as well as a number of inflammatory chemokines and interleukins. To establish persistent infection, viruses have evolved various mechanisms to counteract the host NF-κB pathway. In the present study, for the first time, HSV-1 UL24 was demonstrated to inhibit the activation of NF

  6. The DEAD-box protein MEL-46 is required in the germ line of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Minasaki, Ryuji; Puoti, Alessandro; Streit, Adrian

    2009-06-17

    In the hermaphrodite of the nematode Caenorhabditis elegans, the first germ cells differentiate as sperm. Later the germ line switches to the production of oocytes. This process requires the activity of a genetic regulatory network that includes among others the fem, fog and mog genes. The function of some of these genes is germline specific while others also act in somatic tissues. DEAD box proteins have been shown to be involved in the control of gene expression at different steps such as transcription and pre-mRNA processing. We show that the Caenorhabditis elegans gene mel-46 (maternal effect lethal) encodes a DEAD box protein that is related to the mammalian DDX20/Gemin3/DP103 genes. mel-46 is expressed throughout development and mutations in mel-46 display defects at multiple developmental stages. Here we focus on the role of mel-46 in the hermaphrodite germ line. mel-46(yt5) mutant hermaphrodites are partially penetrant sterile and fully penetrant maternal effect lethal. The germ line of mutants shows variable defects in oogenesis. Further, mel-46(yt5) suppresses the complete feminization caused by mutations in fog-2 and fem-3, two genes that are at the top and the center, respectively, of the genetic germline sex determining cascade, but not fog-1 that is at the bottom of this cascade. The C. elegans gene mel-46 encodes a DEAD box protein that is required maternally for early embryogenesis and zygotically for postembryonic development. In the germ line, it is required for proper oogenesis. Although it interacts genetically with genes of the germline sex determination machinery its primary function appears to be in oocyte differentiation rather than sex determination.

  7. Synthesis and luminescent properties of novel red-emitting M7Sn(PO46:Eu3+ (M = Sr, Ba phosphors

    Directory of Open Access Journals (Sweden)

    Guo Feng

    2018-03-01

    Full Text Available Novel Eu3+-activated M7Sn(PO46 (where M = Sr, Ba red-emitting phosphors were synthesized via conventional solid-state reaction method at 1200 °C for 2 h. The luminescence properties of the prepared samples and quenching concentration of Sr7-xSn(PO46:xEu3+ and Ba7-xSn(PO46:xEu3+ were investigated. These phosphors can be efficiently excited by UV (395 nm and visible blue (465 nm light nicely matching the output wavelengths of the near-UV LEDs and InGaN blue LED chips and emit the red light. The critical concentrations of the Eu3+ activator were found to be 0.175 mol and 0.21 mol per formula unit for Sr7-xSn(PO46:xEu3+ and Ba7-xSn(PO46:xEu3+, respectively. The M7-xSn(PO46:xEu3+ (M = Sr, Ba phosphor may be a good candidate for light-emitting diodes application.

  8. [Prevalence of upper limb work-related musculoskeletal disorders (UL-WMSDs) in workers of the upholstered furniture industry].

    Science.gov (United States)

    Nicoletti, S; Carino, M; Di Leone, G; Trani, G; Carella, F; Rubino, G; Leone, E; Popolizio, R; Colafiglio, S; Ambrosi, L

    2008-01-01

    The upholstered furniture industry, the so-called "triangle of the sofa industry", is a geographic area of national and strategic economic importance in southern Italy. The single tasks are carried out mostly manually, with the characteristics of a handicraft approach. The aim of the survey was to assess the prevalence of upper limb work-related musculoskeletal disorders (UL-WMSDs) in 30 factories of the sofa industry located in a large geographic area of the Puglia and Basilicata Regions. In the period 1 January-31 December 2003 a network of occupational physicians investigated a population of 5.477 subjects (exposed n=3481, controls n=1996, M=3865, F=1612) in 30 different factories of the area. More than 60 percent of the total workforce studied was employed in large-sized companies (>500 employees). The following work tasks were considered: filling preparation workers, leather-cutting operators, sewing and upholstery-assembly workers. Case-definition was assessed through standardized procedures: symptoms by questionnaire plus physical and laboratory/imaging findings. Cumulative prevalence rates of UL-WMSDs as at 31 December 2003 reached values of up to 30% in high risk groups. Prevalence rates showed good correlation with the concise OCRA index used for assessment of exposure to repetitive strain and movements of the upper limb. The most frequently occurring disorders were tendon-related cysts and wrist tendonitis. Shoulder disorders were more frequent in male and female leather-cutting operators. This survey showed a significantly high prevalence of UL-WMSDs in sofa industry workers. It did not seem to be confirmed in this study that there was a greater female susceptibility to UL-WMSDs with the exception of carpal tunnel syndrome: gender difference seems to be less relevant at increasing levels of occupational exposure to repetitive movements and exertion of the upper limbs.

  9. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    Science.gov (United States)

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  10. Comparative immunoblot analysis with 10 different, partially overlapping recombinant fusion proteins derived from 5 different cytomegalovirus proteins

    NARCIS (Netherlands)

    van Zanten, J.; LAZZAROTTO, T; CAMPISI, B; VORNHAGEN, R; JAHN, G; LANDINI, MP; The, T. Hauw

    Ten fusion proteins derived from five various CMV encoded proteins were used for the detection of specific antibody response by immunoblot technique in sera from renal transplant recipients. The fusion proteins were derived from the following CMV specific proteins: the assembly protein ppUL80a with

  11. Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

    Science.gov (United States)

    Balachandran, Manasi; Giannone, Richard J; Bemis, David A; Kania, Stephen A

    2017-01-01

    Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

  12. Partial functional complementation between human and mouse cytomegalovirus chemokine receptor homologues

    DEFF Research Database (Denmark)

    Farrell, Helen E; Abraham, Alexander M; Cardin, Rhonda D

    2011-01-01

    The human cytomegalovirus (CMV) proteins US28 and UL33 are homologous to chemokine receptors (CKRs). Knockout of the mouse CMV M33 protein (UL33 homologue) results in substantial attenuation of salivary gland infection/replication and reduced efficiency of reactivation from tissue explants. M33-m...

  13. Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

    Directory of Open Access Journals (Sweden)

    Manasi Balachandran

    Full Text Available Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A gene and binds to immunoglobulin (Ig. The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

  14. The Murine Natural Cytotoxic Receptor NKp46/NCR1 Controls TRAIL Protein Expression in NK Cells and ILC1s

    Directory of Open Access Journals (Sweden)

    Sam Sheppard

    2018-03-01

    Full Text Available Summary: TRAIL is an apoptosis-inducing ligand constitutively expressed on liver-resident type 1 innate lymphoid cells (ILC1s and a subset of natural killer (NK cells, where it contributes to NK cell anti-tumor, anti-viral, and immunoregulatory functions. However, the intrinsic pathways involved in TRAIL expression in ILCs remain unclear. Here, we demonstrate that the murine natural cytotoxic receptor mNKp46/NCR1, expressed on ILC1s and NK cells, controls TRAIL protein expression. Using NKp46-deficient mice, we show that ILC1s lack constitutive expression of TRAIL protein and that NK cells activated in vitro and in vivo fail to upregulate cell surface TRAIL in the absence of NKp46. We show that NKp46 regulates TRAIL expression in a dose-dependent manner and that the reintroduction of NKp46 in mature NK cells deficient for NKp46 is sufficient to restore TRAIL surface expression. These studies uncover a link between NKp46 and TRAIL expression in ILCs with potential implications in pathologies involving NKp46-expressing cells. : Sheppard et al. find that mice deficient in the activating receptor NCR1/NKp46 (Ncr1−/− fail to express the apoptosis-inducing ligand TRAIL at the surface of group 1 innate lymphoid cells (ILC1s. Keywords: NK cell, natural killer cell, NKp46, ILC1, TRAIL, IL-15, IL-2

  15. Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting

    Science.gov (United States)

    Müller, Oliver; Ivanova, Lyudmila; Bialy, Dagmara; Pohlmann, Anja; Binz, Anne; Hegemann, Maike; Viejo-Borbolla, Abel; Rosenhahn, Bodo; Bauerfeind, Rudolf; Sodeik, Beate

    2017-01-01

    Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells. PMID:29284065

  16. Multiple 5' ends of human cytomegalovirus UL57 transcripts identify a complex, cycloheximide-resistant promoter region that activates oriLyt

    International Nuclear Information System (INIS)

    Kiehl, Anita; Huang, Lili; Franchi, David; Anders, David G.

    2003-01-01

    The human cytomegalovirus (HCMV) UL57 gene lies adjacent to HCMV oriLyt, from which it is separated by an organizationally conserved, mostly noncoding region that is thought to both regulate UL57 expression and activate oriLyt function. However, the UL57 promoter has not been studied. We determined the 5' ends of UL57 transcripts toward an understanding of the potential relationship between UL57 expression and oriLyt activation. The results presented here identified three distinct 5' ends spread over 800 bp, at nt 90302, 90530, and 91138; use of these sites exhibited differential sensitivity to phosphonoformic acid treatment. Interestingly, a 10-kb UL57 transcript accumulated in cycloheximide-treated infected cells, even though other early transcripts were not detectable. However, the 10-kb transcript did not accumulate in cells treated with the more stringent translation inhibitor anisomycin. Consistent with the notion that the identified 5' ends arise from distinct transcription start sites, the sequences upstream of sites I and II functioned as promoters responsive to HCMV infection in transient assays. However, the origin-proximal promoter region III required downstream sequences for transcriptional activity. Mutation of candidate core promoter elements suggested that promoter III is regulated by an initiator region (Inr) and a downstream promoter element. Finally, a 42-bp sequence containing the candidate Inr activated a minimal oriLyt core construct in transient replication assays. Thus, these studies showed that a large, complex promoter region with novel features controls UL57 expression, and identified a sequence that regulates both UL57 transcription and oriLyt activation

  17. Human cytomegalovirus UL145 gene is highly conserved among ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    capable of causing infections that persist lifelong, and normally ... 1 Virus Laboratory, Affiliated ShengJing Hospital, China Medical University, Shenyang 110004, P. R. China. 2Department of .... Elmer, USA), and negative controls were included in each round of .... variability of the UL145 gene in field isolates. To answer this.

  18. Contributions of herpes simplex virus type 1 envelope proteins to entry by endocytosis

    Science.gov (United States)

    Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly acidic pH. Thus, the ...

  19. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Arsheed Hussain Sheikh

    2016-02-01

    Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

  20. AWI Moored ULS Data, Greenland Sea and Fram Strait, 1991-2002

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set consists of Upward Looking Sonar (ULS) data from 11 moorings in the Greenland Sea. Parameters in the processed data files include ice draft, water...

  1. Lean diabetes in middle-aged adults: A joint analysis of the German DIVE and DPV registries.

    Directory of Open Access Journals (Sweden)

    Bettina Hartmann

    Full Text Available To assess differences in demographics, treatment and outcome of lean (LD compared to overweight and obese people with diabetes clinically classified as type 2 diabetes mellitus (T2DM.We combined data from the German DIVE (Diabetes Versorgungs-Evaluation and DPV (Diabetes-Patienten-Verlaufsdokumentation databases to produce a large cohort of people with T2DM. The characteristics of people with Body Mass Index (BMI <25 kg/m2, ≥25-30 kg/m2 and ≥30 kg/m2 aged 30 to 50 years were compared, including demographics, cardiovascular (CV risk factors, comorbidities and outcomes.A total of 37,870 people were included in the analysis, 3,191 of these (8.4% had a BMI < 25 kg/m2. LD reported more nicotine (41.6% of 2,070 vs. 38.1% of 6,070 and 33.4% of 16,823; P<0.001and alcohol consumption (12.0% of 1,282, 10.3% of 3,594 and 6.6% of 9,418; P<0.001compared to overweight and obese people. More LD were treated with insulin in comparison to the other subgroups (short acting insulin 33.1% of 3,191 vs. 28.4% of 9,234 and 28.0% of 25,445; P <0.001; long acting insulin 31.3% of 3,191 vs. 28.9% of 9,234 and 29.3% of 25,445; P = 0.043. Regression models adjusted for age, gender and diabetes duration showed a 2.50 times higher odds ratio (OR for hypoglycemia and a 2.52 higher OR for mortality in LD compared to the BMI subgroup ≥30 kg/m2.LD is associated with an increased risk of hypoglycaemia and death. Patients are characterized by male gender, lifestyle habits as smoking and alcohol consumption while cardiovascular comorbidities are less important. In comparison to patients of the other weight groups they are treated with insulin more often and considerably less with metformin.

  2. La expedición a Veracruz y la defensa de San Juan de Ulúa (1819-1825

    Directory of Open Access Journals (Sweden)

    Manuel Escalona Jiménez

    2000-01-01

    Full Text Available En 1819 una expedición militar al mando del teniente general Manuel Cagigal partió de Cádiz hacia La Habana. En 1821 el cuerpo expedicionario, compuesto de unos 200 hombres, ocupó Veracruz, pero pronto se vio obligado a replegarse a la isla de San Juan de Ulúa, donde consiguió resistir hasta 1825, a pesar de estar sometido al severo bloqueo de las fuerzas independentistas mexicanas y combatir en las condiciones más precarias, de tal modo que este episodio militar se ganó la admiración general.In 1819, a military expedition, under the command of Lieutenant General Manuel Cagigal, sailed from Cádiz bound to La Habana. In 1821, the army corps, around 200 soldiers, got hold of Veracruz, but was soon obliged to retire to the island of San Juan de Ulúa, where they managed to resist untill 1825, in spite of the blockade by the Mexican independentist Mexican force and the arduous circumstances of the fight to the extent of getting a general admiration.

  3. Vaccination with a HSV-2 UL24 mutant induces a protective immune response in murine and guinea pig vaginal infection models.

    Science.gov (United States)

    Visalli, Robert J; Natuk, Robert J; Kowalski, Jacek; Guo, Min; Blakeney, Susan; Gangolli, Seema; Cooper, David

    2014-03-10

    The rational design and development of genetically attenuated HSV-2 mutant viruses represent an attractive approach for developing both prophylactic and therapeutic vaccines for genital herpes. Previously, HSV-2 UL24 was shown to be a virulence determinant in both murine and guinea pig vaginal infection models. An UL24-βgluc insertion mutant produced syncytial plaques and replicated to nearly wild type levels in tissue culture, but induced little or no pathological effects in recipient mice or guinea pigs following vaginal infection. Here we report that immunization of mice or guinea pigs with high or low doses of UL24-βgluc elicited a highly protective immune response. UL24-βgluc immunization via the vaginal or intramuscular routes was demonstrated to protect mice from a lethal vaginal challenge with wild type HSV-2. Moreover, antigen re-stimulated splenic lymphocytes harvested from immunized mice exhibited both HSV-2 specific CTL activity and IFN-γ expression. Humoral anti-HSV-2 responses in serum were Th1-polarized (IgG2a>IgG1) and contained high-titer anti-HSV-2 neutralizing activity. Guinea pigs vaccinated subcutaneously with UL24-βgluc or the more virulent parental strain (186) were challenged with a heterologous HSV-2 strain (MS). Acute disease scores were nearly indistinguishable in guinea pigs immunized with either virus. Recurrent disease scores were reduced in UL24-βgluc immunized animals but not to the same extent as those immunized with strain 186. In addition, challenge virus was not detected in 75% of guinea pigs subcutaneously immunized with UL24-βgluc. In conclusion, disruption of the UL24 gene is a prime target for the development of a genetically attenuated live HSV-2 vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. NMR Determination of Protein Partitioning into Membrane Domains with Different Curvatures and Application to the Influenza M2 Peptide

    Science.gov (United States)

    Wang, Tuo; Cady, Sarah D.; Hong, Mei

    2012-01-01

    The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use 31P and 13C solid-state NMR to examine M2-induced membrane curvature. M2(22–46), which includes only the transmembrane (TM) helix, and M2(21–61), which contains an additional amphipathic helix, are studied. 31P chemical shift lineshapes indicate that M2(21–61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22–46) has minimal effect. The lamellar and isotropic domains have distinct 31P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. 31P- and 13C-detected 1H T2 relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21–61) preferentially binds to the high-curvature domain. 31P linewidths indicate that the isotropic vesicles induced by M2(21–61) are 10–35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides. PMID:22385849

  5. Tokat’ın 17. Yüzyıl Âlim Şairlerinden İshak Bin Hasan Tokatî ve Nazmu’l-Ulum Adlı Mesnevîsi Poetry in Rhymed Couplets of Tokatlı İshâk Efendi Nazmu'l-Ulûm

    Directory of Open Access Journals (Sweden)

    Bayram ÖZFIRAT

    2013-03-01

    Full Text Available As manuscript works, which constitute some indispensabletreasure with respect to history of our literature, come back into light,our knowledge on religious literary genres, as well as other, no doubtincreases. Such works added to literature thanks to efforts byresearchers give us the opportunity of close acquaintance with religiousworks that are considered as the most significant and the most prolificproducts of Divan Poetry, and of more accurate interpretationsregarding the genre of such works. Apart from works such as aqâid, fiqhand catechism (ilm-i hal in verse, siyer (life of Muhammad in verse,exegeses (tafsir and hadithes in verse that are generally written inpoetic structure for these disciplines, there are also some poetic piecesabout a subdivision or important question of abovementioned Islamicsciences. Faraiz, Hegira of Muhammad, miraj-name, rules of hajj,miracles of Prophet, all in verse, can be mentioned among suchexamples. In the wake of our researches, we found out that thesecatechisms in verse constitute a notable part of our religious literature.Tokatlı İshâk Efendi was an important poet and writer who lived in 17th century. Biographic sources dores not mention him alot. The poetryin rhymed couplets, Nazmu’l-Ulûm, which we are working on shows ushow important poet he is. At his Nazmu’l-Ulûm İshâk Efendi givesinformation about different sciences and wants them to learn. Becauseof these properties Nazmu’l-Ulûm is a written verse advice. Nazmu’l-Ulûm have got strong rhymes. That is possible to say poetry in rhymedcouplets have fascinating expressions rather than bare tellings. Somany telling arts and describtions were given place in that book. Tokatlı İshâk Efendi on yedinci yüzyılda yaşamış önemli bir şâir ve yazardır. Biyografik kaynaklar kendisinden fazla bahsetmez. Ama eserlerinden yola çıktığımızda Tokatlı İshâk Efendi’nin yetenekli bir şâir olduğunu rahatlıkla söyleyebiliriz.

  6. QUALITATIVE ASPECTS OF CRIŞUL REPEDE RIVER

    Directory of Open Access Journals (Sweden)

    VIGH MELINDA

    2012-03-01

    Full Text Available Qualitative aspects of Crisul Repede River. The evolution of water quality over the Crişul Repede River is atypical because of natural and anthropic factors. The most significant factors are the geological substrate, and the settlements with their agricultural and industrial activity. The study was performed at three gauging stations for the period 1996-2006, taking into account the annual average values. The considered elements were: the discharge values, temperature, suspensions, oxygen regime, nutrients and phosphorus, taking into consideration their temporal and spatial variation. By comparing them with the admitted limits we could enroll them in various quality classes.

  7. Experimental Study of an Offshore Wind Turbine TLP in ULS Conditions

    DEFF Research Database (Denmark)

    Wehmeyer, Christof; Ferri, Francesco; Skourup, Jesper

    2013-01-01

    An extensive model test program has been carried out in order to assess the behavior of a tension leg moored substructure as support of a floating offshore wind turbine (FOWT). The floater was inspired by an industrial design. The tests focused on the ultimate limit state (ULS) behavior, therefor...

  8. Pioneering the human development revolution: Analysing the trajectory of Mahbub ul Haq

    NARCIS (Netherlands)

    D.R. Gasper (Des)

    2011-01-01

    textabstractMahbub ul Haq's work to coordinate, establish and propagate the human development approach offers an example of effective leadership in promoting more ethical socio-economic development. This article reviews Pioneering the Human Development Revolution-An Intellectual Biography of Mahbub

  9. Effect of caffeine on the expression of a major X-ray induced protein in human tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Hughes, E.N.; Boothman, D.A. (Univ. of Pennsylvania School of Medicine, Philadelphia (USA))

    1991-03-01

    We have examined the effect of caffeine on the concomitant processes of the repair of potentially lethal damage (PLD) and the synthesis of X-ray-induced proteins in the human malignant melanoma cell line, Ul-Mel. Caffeine administered at a dose of 5mM after X radiation not only inhibited PLD repair but also markedly reduced the level of XIP269, a major X-ray-induced protein whose expression has been shown to correlate with the capacity to repair PLD. The expression of the vast majority of other cellular proteins, including seven other X-ray-induced proteins, remained unchanged following caffeine treatment. A possible role for XIP269 in cell cycle delay following DNA damage by X irradiation is discussed.

  10. AWI Moored ULS Data, Greenland Sea and Fram Strait, 1991-2002, Version 1

    Data.gov (United States)

    National Aeronautics and Space Administration — This data set consists of Upward Looking Sonar (ULS) data from 11 moorings in the Greenland Sea. Parameters in the processed data files include ice draft, water...

  11. Lean diabetes in middle-aged adults: A joint analysis of the German DIVE and DPV registries.

    Science.gov (United States)

    Hartmann, Bettina; Lanzinger, Stefanie; Bramlage, Peter; Groß, Felix; Danne, Thomas; Wagner, Siegfried; Krakow, Dietmar; Zimmermann, Artur; Malcharzik, Christian; Holl, Reinhard W

    2017-01-01

    To assess differences in demographics, treatment and outcome of lean (LD) compared to overweight and obese people with diabetes clinically classified as type 2 diabetes mellitus (T2DM). We combined data from the German DIVE (Diabetes Versorgungs-Evaluation) and DPV (Diabetes-Patienten-Verlaufsdokumentation) databases to produce a large cohort of people with T2DM. The characteristics of people with Body Mass Index (BMI) demographics, cardiovascular (CV) risk factors, comorbidities and outcomes. A total of 37,870 people were included in the analysis, 3,191 of these (8.4%) had a BMI < 25 kg/m2. LD reported more nicotine (41.6% of 2,070 vs. 38.1% of 6,070 and 33.4% of 16,823; P<0.001)and alcohol consumption (12.0% of 1,282, 10.3% of 3,594 and 6.6% of 9,418; P<0.001)compared to overweight and obese people. More LD were treated with insulin in comparison to the other subgroups (short acting insulin 33.1% of 3,191 vs. 28.4% of 9,234 and 28.0% of 25,445; P <0.001; long acting insulin 31.3% of 3,191 vs. 28.9% of 9,234 and 29.3% of 25,445; P = 0.043). Regression models adjusted for age, gender and diabetes duration showed a 2.50 times higher odds ratio (OR) for hypoglycemia and a 2.52 higher OR for mortality in LD compared to the BMI subgroup ≥30 kg/m2. LD is associated with an increased risk of hypoglycaemia and death. Patients are characterized by male gender, lifestyle habits as smoking and alcohol consumption while cardiovascular comorbidities are less important. In comparison to patients of the other weight groups they are treated with insulin more often and considerably less with metformin.

  12. A Monte Carlo study comparing PIV, ULS and DWLS in the estimation of dichotomous confirmatory factor analysis.

    Science.gov (United States)

    Nestler, Steffen

    2013-02-01

    We conducted a Monte Carlo study to investigate the performance of the polychoric instrumental variable estimator (PIV) in comparison to unweighted least squares (ULS) and diagonally weighted least squares (DWLS) in the estimation of a confirmatory factor analysis model with dichotomous indicators. The simulation involved 144 conditions (1,000 replications per condition) that were defined by a combination of (a) two types of latent factor models, (b) four sample sizes (100, 250, 500, 1,000), (c) three factor loadings (low, moderate, strong), (d) three levels of non-normality (normal, moderately, and extremely non-normal), and (e) whether the factor model was correctly specified or misspecified. The results showed that when the model was correctly specified, PIV produced estimates that were as accurate as ULS and DWLS. Furthermore, the simulation showed that PIV was more robust to structural misspecifications than ULS and DWLS. © 2012 The British Psychological Society.

  13. [A case of IgA2-lambda type M-protein that IgA concentration differs from the values of M-protein by serum protein electrophoresis].

    Science.gov (United States)

    Fukushima, M; Sugano, M; Ichikawa, T; Honda, T; Totsuka, M; Katsuyama, T; Fujita, K

    2001-07-01

    We report an IgA-lambda type M-protein in which the IgA concentration differed from the values of M-protein by serum protein electrophoresis found in a 53-year-old man with multiple myeloma. The M-protein value as determined by serum protein electrophoresis was 6,170 mg/dl. However, the serum IgA concentration was 3,052 mg/dl by turbidimetric immunoassay. Immuno-fixation electrophoresis using IgA subclass antisera revealed that this M-protein was the IgA2-lambda type. Western blotting analysis showed that the IgA2 molecules were composed of two approximately 68 kDa alpha 2 chains and two 28 kDa lambda chains. In addition the free lambda chain band was detected at the position of 28 kDa without 2-mercaptoethanol(2-ME) even though the patient IgA was purified. Since it is known that IgA2m(1) allotype easily release light chains from the IgA molecules in SDS-PAGE without 2-ME, we speculated that in this patient the IgA was the IgA2m(1) allotype. After peripheral blood stem cell transplantation(PBSCT), immunofixation electrophoresis of the patient serum revealed not only the bands of IgA2-lambda type M-protein, but also three bands of IgG1-kappa type M-protein in the gamma region.

  14. Identification by Mass Spectrometry and Immune Response Analysis of Guinea Pig Cytomegalovirus (GPCMV Pentameric Complex Proteins GP129, 131 and 133

    Directory of Open Access Journals (Sweden)

    Josephine S. Gnanandarajah

    2014-02-01

    Full Text Available Development of a vaccine against congenital infection with human cytomegalovirus (HCMV is a major public health priority. A potential vaccine target receiving considerable recent attention is the pentameric complex (PC of HCMV proteins consisting of gL, gH, UL128, UL130, and UL131, since some antibodies against these target proteins are capable of potently neutralizing virus at epithelial and endothelial cell surfaces. Recently, homologous proteins have been described for guinea pig cytomegalovirus (GPCMV, consisting of gH, gL, and the GPCMV proteins GP129, GP131, and GP133. To investigate these proteins as potential vaccine targets, expression of GP129-GP133 transcripts was confirmed by reverse-transcriptase PCR. Mass spectrometry combined with western blot assays demonstrated the presence of GP129, GP131, and GP133 proteins in virus particles. Recombinant proteins corresponding to these PC proteins were generated in baculovirus, and as GST fusion proteins. Recombinant proteins were noted to be immunoreactive with convalescent sera from infected animals, suggesting that these proteins are recognized in the humoral immune response to GPCMV infection. These analyses support the study of PC-based recombinant vaccines in the GPCMV congenital infection model.

  15. Haematological reference for red-browed parrot ( Amazona rhodocorytha , Salvadori, 1890 captive in the Atlantic Forest in Eastern Brazil

    Directory of Open Access Journals (Sweden)

    R.H. Mello

    Full Text Available ABSTRACT To conduct the survey were used 35 (thirty-five red-browned parrots (A. rhodocorytha, adults, captive, of both genders and clinically healthy, belonging to the live collection of the Museum of Biology Teacher Mello Leitao, located in Santa Teresa, Espírito Santo, Brazil. Harvests were performed in the morning, by puncture of the brachial vein getting 0.5mL of blood stored in EDTA for a period no longer than 6 hours. Blood smears of fresh material were made at collection, stained using the method of May-Grunwald-Giemsa. Analysis of blood elements was done by cell counting in a mirrored Neubauer chamber using Natt and Herrick solution at a ratio of 2:200 as diluent. For the analysis of the methodology, homoglobinometry cyanide hemoglobin using commercial kits by colorimetry on a semi-automatic biochemical analyzer was used. After completion of the statistical data the following parameters were obtained (mean±standard deviation: Erythrocytes (x106/μl: 2.68±0.56; Hemoglobin (g/dl: 14.27±0.69; Hematocrit (%: 53±3.38; Mean corpuscular volume (fl: 206.7±45.82; Mean corpuscular hemoglobin (pg: 56.4±14.46; Mean corpuscular hemoglobin concentration (%: 27.5±1.19; Thrombocytes (x3/μl: 25.8 ± 10.5; Total plasma protein (g/dl 5.4±0.5; Leukocytes (x103/dl: 3.1±2; Heterophile (/uL: 1937±1676; Lymphocytes (/uL: 1144±599; Monocytes (/uL: 24.4 ± 28.2; Basophils (/uL: 42.2±46.2; Eosinophils (/uL: 11.7±19.9. In the relation between males and females, no significant differences were found in any hematological parameter evaluated.

  16. Crystallization and preliminary X-ray analysis of coagulation factor IX-binding protein from habu snake venom at pH 6.5 and 4.6

    International Nuclear Information System (INIS)

    Suzuki, Nobuhiro; Shikamoto, Yasuo; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

    2004-01-01

    Crystals of habu coagulation factor IX-binding protein have been obtained at pH 6.5 and 4.6 and characterized by X-ray diffraction. Coagulation factor IX-binding protein isolated from Trimeresurus flavoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca 2+ -binding site with differing affinity (K d values of 14 and 130 µM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the affinity of the low-affinity site was reduced considerably. In order to identify which site has high affinity and also to investigate the Ca 2+ -releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29 Å resolution, respectively; the former crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 60.7, b = 63.5, c = 66.9 Å, β = 117.0°, while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8 Å, β = 110.4°

  17. Single-Use Disposable Electrochemical Label-Free Immunosensor for Detection of Glycated Hemoglobin (HbA1c Using Differential Pulse Voltammetry (DPV

    Directory of Open Access Journals (Sweden)

    Alireza Molazemhosseini

    2016-07-01

    Full Text Available A single-use disposable in vitro electrochemical immunosensor for the detection of HbA1c in undiluted human serum using differential pulse voltammetry (DPV was developed. A three-electrode configuration electrochemical biosensor consisted of 10-nm-thin gold film working and counter electrodes and a thick-film printed Ag/AgCl reference electrode was fabricated on a polyethylene terephthalate (PET substrate. Micro-fabrication techniques including sputtering vapor deposition and thick-film printing were used to fabricate the biosensor. This was a roll-to-roll cost-effective manufacturing process making the single-use disposable in vitro HbA1c biosensor a reality. Self-assembled monolayers of 3-Mercaptopropionic acid (MPA were employed to covalently immobilize anti-HbA1c on the surface of gold electrodes. Electrochemical impedance spectroscopy (EIS and X-ray photoelectron spectroscopy (XPS confirmed the excellent coverage of MPA-SAM and the upward orientation of carboxylic groups. The hindering effect of HbA1c on the ferricyanide/ferrocyanide electron transfer reaction was exploited as the HbA1c detection mechanism. The biosensor showed a linear range of 7.5–20 µg/mL of HbA1c in 0.1 M PBS. Using undiluted human serum as the test medium, the biosensor presented an excellent linear behavior (R2 = 0.999 in the range of 0.1–0.25 mg/mL of HbA1c. The potential application of this biosensor for in vitro measurement of HbA1c for diabetic management was demonstrated.

  18. Single-Use Disposable Electrochemical Label-Free Immunosensor for Detection of Glycated Hemoglobin (HbA1c) Using Differential Pulse Voltammetry (DPV).

    Science.gov (United States)

    Molazemhosseini, Alireza; Magagnin, Luca; Vena, Pasquale; Liu, Chung-Chiun

    2016-07-01

    A single-use disposable in vitro electrochemical immunosensor for the detection of HbA1c in undiluted human serum using differential pulse voltammetry (DPV) was developed. A three-electrode configuration electrochemical biosensor consisted of 10-nm-thin gold film working and counter electrodes and a thick-film printed Ag/AgCl reference electrode was fabricated on a polyethylene terephthalate (PET) substrate. Micro-fabrication techniques including sputtering vapor deposition and thick-film printing were used to fabricate the biosensor. This was a roll-to-roll cost-effective manufacturing process making the single-use disposable in vitro HbA1c biosensor a reality. Self-assembled monolayers of 3-Mercaptopropionic acid (MPA) were employed to covalently immobilize anti-HbA1c on the surface of gold electrodes. Electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS) confirmed the excellent coverage of MPA-SAM and the upward orientation of carboxylic groups. The hindering effect of HbA1c on the ferricyanide/ferrocyanide electron transfer reaction was exploited as the HbA1c detection mechanism. The biosensor showed a linear range of 7.5-20 µg/mL of HbA1c in 0.1 M PBS. Using undiluted human serum as the test medium, the biosensor presented an excellent linear behavior (R² = 0.999) in the range of 0.1-0.25 mg/mL of HbA1c. The potential application of this biosensor for in vitro measurement of HbA1c for diabetic management was demonstrated.

  19. Debittering of Protein Hydrolysates by Lactobacillus LBL-4 Aminopeptidase

    Directory of Open Access Journals (Sweden)

    Bozhidar Tchorbanov

    2011-01-01

    Full Text Available Yoghurt strain Lactobacillus LBL-4 cultivated for 8–10 h at pH ~6.0 was investigated as a considerable food-grade source of intracellular aminopeptidase. Cell-free extract manifesting >200 AP U/l was obtained from cells harvested from 1 L culture media. Subtilisin-induced hydrolysates of casein, soybean isolate, and Scenedesmus cell protein with degree of hydrolysis 20–22% incubated at 45∘C for 10 h by 10 AP U/g peptides caused an enlarging of DH up to 40–42%, 46–48%, and 38–40% respectively. The DH increased rapidly during the first 4 h, but gel chromatography studies on BioGel P-2 showed significant changes occurred during 4–10 h of enzyme action when the DH increased gradually. After the digestion, the remained AP activity can be recovered by ultrafiltration (yield 40–50%. Scenedesmus protein hydrolysate with DH 20% was inoculated by Lactobacillus LBL-4 cells, and after 72 h cultivation the DH reached 32%. The protein hydrolysates (DH above 40% obtained from casein and soybean isolate (high Q value demonstrated a negligible bitterness while Scenedesmus protein hydrolysates (low Q value after both treatments were free of bitterness.

  20. Linear uranium complexes X2UL5 with L=cyanide, iso-cyanate: DFT evidence for similarities between uranyl (X = O) and uranocene (X = Cp) derivatives

    International Nuclear Information System (INIS)

    Iche-Tarrat, N.; Marsden, Colin J.; Barros, N.; Maron, L.; Barros, N.

    2008-01-01

    A DFT study of the isostructural compounds [UO 2 L 5 ] n- with n=3-5 and linear [Cp 2 UL 5 ] m- with m=1-3 has been carried out for two different anionic ligands. Structurally stable structures are obtained for all systems. The coordination competition between cyanide (CN - ) and isocyanide (NC - ) as well as between cyanate (OCN - ) and iso-cyanate (NCO - ) has been studied in the uranyl case. A clear preference for cyanide and iso-cyanate complexes is reported. The coordination of five ligands in the equatorial plane is rationalized by the analysis of the MO diagram of both systems. Moreover, the qualitative comparison of the two MO diagrams shows a high similarity in agreement with the isolobality concept. The existence of linear [Cp 2 UL 5 ] - organometallic U(VI) complexes is thus proposed, as well as the possibility of obtaining complexes of both types for U(VI) and U(V) with OCN - ligands. In addition, the U(IV) linear metallocene is calculated to be stable for the latter ligand. (authors)

  1. A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens.

    Science.gov (United States)

    Awasthi, Sita; Mahairas, Gregory G; Shaw, Carolyn E; Huang, Meei-Li; Koelle, David M; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M

    2015-08-01

    We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4(+) and CD8(+) T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of transmission and

  2. Monolayer Assemblies of a De Novo Designed 4-alpha-Helix Bundle Carboprotein and Its Sulfur Anchor Fragment on Au(111) Surfaces Addressed by Voltammetry and In Situ Scanning Tunneling Microscopy

    DEFF Research Database (Denmark)

    Brask, Jesper; Wackerbarth, Hainer; Jensen, Knud J.

    2003-01-01

    carboprotein without thiol anchor have been prepared and investigated for comparison. Cyclic and differential pulse voltammetry (DPV) of the proteins show desorption peaks around -750 mV (SCE), whereas the thiol anchor desorption peak is at -685 mV. The peaks are by far the highest for thiol monomeric 4-R...

  3. Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes

    Directory of Open Access Journals (Sweden)

    Pascal Viens

    2015-10-01

    Full Text Available Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC or chitosan oligosaccharides (CHOS from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

  4. 46 CFR 120.410 - Lighting fixtures.

    Science.gov (United States)

    2010-10-01

    ...) Each table lamp, desk lamp, floor lamp, or similar equipment must be secured in place so that it cannot... Fixtures,” UL 1571, “Incandescent Lighting Fixtures,” UL 1572, “High Intensity Discharge Lighting Fixtures...

  5. Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens.

    Directory of Open Access Journals (Sweden)

    B David Persson

    2010-09-01

    Full Text Available The human membrane cofactor protein (MCP, CD46 is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6, Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4 that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.

  6. Human cytomegaloviruses expressing yellow fluorescent fusion proteins--characterization and use in antiviral screening.

    Directory of Open Access Journals (Sweden)

    Sarah Straschewski

    Full Text Available Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity. We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP fused with the viral proteins IE-2, ppUL32 (pp150, and ppUL83 (pp65. In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI. The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus-infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.

  7. Health Information in Modern Standard Arabic (al-ʻArabīyat ul-fuṣḥá)

    Science.gov (United States)

    ... Your Recovery After Cesarean Birth (Part 1) - English MP3 Your Recovery After Cesarean Birth (Part 1) - al-ʻArabīyat ul-fuṣḥá (Modern Standard Arabic) MP3 Your Recovery After Cesarean Birth (Part 1) - English ...

  8. Analysis of irradiated materials in Ul-chin unit 5

    International Nuclear Information System (INIS)

    Jung, Y. H.; Yoo, B. O.; Kim, H. M.; Joo, Y. S.

    2007-02-01

    The microstructure examination, the fracture surface observation, the composition analysis and the micro-hardness measurement were carried out for investigation of debris apart from structure in Ul-chin uint 5. As the results of investigation, those of debris were found out screw bolts and the washer. The screw bolts and the washer were coincident with materials from ASTM A-193 by quantitative analysis. The screw bolts and the washer were made by STS 304. Finally, all of screw bolts were parts of the LPSI pump case even though one of them was found in different place. The washer was part of the heat exchanger

  9. Gene Therapy for Fracture Repair

    Science.gov (United States)

    2007-05-01

    modulator-1; c-myc binding protein [ Homo sapiens ]. regulation of transcription, DNA dependent NM_012488 1.55 2.53 Rattus norvegicus α-2-macroglobulin...myc binding protein [ Homo sapiens ] Regulation of transcription, DNA dependent NM_012488 1.55 2.53 Rattus norvegicus α-2-macroglobulin (A2m) Protease...1-HIV LTR-MLV Promoter EG FP -C el l N um be r (M ea n) 10 ul Vector 50 ul Vector 7 Periosteal/Endosteal Cell Transduction 0 200 400 600 800

  10. Plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by HCMV US2 in cooperation with UL141.

    Directory of Open Access Journals (Sweden)

    Jye-Lin Hsu

    2015-04-01

    Full Text Available Human cytomegalovirus (HCMV US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I. Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2's impact on HCMV pathogenesis.

  11. Alternative Mode of E-Site tRNA Binding in the Presence of a Downstream mRNA Stem Loop at the Entrance Channel.

    Science.gov (United States)

    Zhang, Yan; Hong, Samuel; Ruangprasert, Ajchareeya; Skiniotis, Georgios; Dunham, Christine M

    2018-03-06

    Structured mRNAs positioned downstream of the ribosomal decoding center alter gene expression by slowing protein synthesis. Here, we solved the cryo-EM structure of the bacterial ribosome bound to an mRNA containing a 3' stem loop that regulates translation. Unexpectedly, the E-site tRNA adopts two distinct orientations. In the first structure, normal interactions with the 50S and 30S E site are observed. However, in the second structure, although the E-site tRNA makes normal interactions with the 50S E site, its anticodon stem loop moves ∼54 Å away from the 30S E site to interact with the 30S head domain and 50S uL5. This position of the E-site tRNA causes the uL1 stalk to adopt a more open conformation that likely represents an intermediate state during E-site tRNA dissociation. These results suggest that structured mRNAs at the entrance channel restrict 30S subunit movement required during translation to slow E-site tRNA dissociation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Recombinant HT{sub m4} gene, protein and assays

    Science.gov (United States)

    Lim, B.; Adra, C.N.; Lelias, J.M.

    1996-09-03

    The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

  13. ANP32B is a nuclear target of henipavirus M proteins.

    Directory of Open Access Journals (Sweden)

    Anja Bauer

    Full Text Available Membrane envelopment and budding of negative strand RNA viruses (NSVs is mainly driven by viral matrix proteins (M. In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed.

  14. Analysis of p130 protein and mRNA expression in ten patients with uterine papillary serous carcinoma

    Directory of Open Access Journals (Sweden)

    Shao-ting XU

    2011-11-01

    Full Text Available Objective To examine p130 protein and mRNA expression in uterine papillary serous carcinoma(UPSC and their clinical and pathologic significance.Methods A total of 10 UPSC patients(Stage I were included,with 10 cases of high-level endometrial carcinoma of the same stage taken as the control group and 10 cases of normal proliferative stage endometrium(EM taken as the disease control group.The level of p130 protein expression was determined by hematoxylin and eosin staining,microscopic observation,and immunohistochemistry,whereas the p130 mRNA levels were examined through real-time quantitative reverse transcriptase polymerase chain reaction.The clinicopathologic analysis was carried out in combination with clinical data.Results The p130 protein and p130 mRNA expression levels in the UPSC group(0.46±0.01 and 0.56±0.06,respectively were apparently less than that of the normal proliferative stage endometrium group(0.91±0.04 and 2.81±0.40,respectively;P < 0.01 and also less than those in high-level endometrial carcinoma(P < 0.05.Clinicopathologic analysis shows that all patients are post-menopausal women with symptoms of irregular vaginal bleeding and the average tumor size was 7.5cm(range: 1.2-14.8cm.The pathologic features are same as that of high-level ovarian papillary serous carcinoma.Conclusion Reduced p130 protein and p130 mRNA expression in UPSC might correlate with poor prognosis in UPSC patients.

  15. 46 CFR 125.180 - Incorporation by reference.

    Science.gov (United States)

    2010-10-01

    ... publish notice of change in the Federal Register and the material must be available to the public. All... Discharge Lighting Fixtures 129.410 UL 1573-1995, Stage and Studio Lighting Units 129.410 UL 1574-1995...

  16. Solid-phase immunoradiometric assay for serum amyloid A protein using magnetisable cellulose particles

    International Nuclear Information System (INIS)

    De Beer, F.C.; Dyck, R.F.; Pepys, M.B.

    1982-01-01

    An immunoradiometric assay for human serum amyloid A protein (SAA) was developed using magnetisable cellulose particles as the solid phase. Rabbit antiserum to SAA was raised by immunization with SAA isolated from acute-phase serum by gel filtration in formic acid. The antiserum was rendered monospecific for SAA by solid-phase immunoabsorption with normal human serum, which contains only traces of SAA, and some was coupled covalently to the cellulose particles. Immunopurified anti-SAA antibodies were isolated from the monospecific anti-SAA serum by binding to, and elution from insolubilized acute-phase serum and were radiolabelled with 125 I. The assay was calibrated with an acute phase serum which contained 6000 times more SAA than normal sera with the lowest detectable level of SAA, and an arbitrary value of 6000 U/l was assigned to this standard. Sera were tested in the native, undenatured state and there was no increase in SAA immunoreactivity following alkali treatment or heating. The assay range was from 1-2000 U/l so that all SAA levels above 6 U/l could be measured on a single (1:6) dilution of serum. The intra- and interassay coefficients of variation were 11.7 and 15.0% respectively. Among 100 healthy normal subjects (50 male, 50 female) the median SAA level was 9 U/l, range <1-100, with 93% below 20 U/l and only 2% below the lower limit of sensitivity of the assay (1 U/l). (Auth.)

  17. Use of activated protein C has no avail in the early phase of acute pancreatitis.

    Science.gov (United States)

    Akay, Sinan; Ozutemiz, Omer; Yenisey, Cigdem; Simsek, Nilufer Genc; Yuce, Gul; Batur, Yucel

    2008-01-01

    Sepsis and acute pancreatitis have similar pathogenetic mechanisms that have been implicated in the progression of multiple organ failure. Drotrecogin alfa, an analogue of endogenous protein C, reduces mortality in clinical sepsis. Our objective was to evaluate the early therapeutic effects of activated protein C (APC) in a rat model of acute necrotizing pancreatitis. Acute necrotizing pancreatitis was induced by intraductal injection of 5% Na taurocholate. Hourly bolus injections of saline or recombinant human APC (drotrecogin alfa) was commenced via femoral venous catheter four hours after the induction of acute pancreatitis. The experiment was terminated nine hours after pancreatitis induction. Animals in group one (n=20) had a sham operation while animals in group two (n=20) received saline and animals in group three (n=20) received drotrecogin alfa boluses after acute pancreatitis induction. Pancreatic tissue for histopathologic scores and myeloperoxidase, glutathione reductase, glutathione peroxidase, and catalase activities were collected, and blood for serum amylase, urea, creatinine, and interleukin-6 measurements was withdrawn. Serum amylase activity was significantly lower in the APC treated group than the untreated group (17,435+/-432 U/L vs. 27,426+/-118 U/L, respectively). While the serum interleukin-6 concentration in the APC untreated group was significantly lower than the treated group (970+/-323 pg/mL vs. 330+/-368 pg/mL, respectively). In the early phase of acute pancreatitis, drotrecogin alfa treatment did not result in a significant improvement in oxidative and inflammatory parameters or renal functions.

  18. Resource Allocation and Cluster Formation for Imperfect NOMA in DL/UL Decoupled HetNets

    KAUST Repository

    Celik, Abdulkadir; Radaydeh, Redha; Al-Qahtani, Fawaz; Abd El-Malek, Ahmed H.; Alouini, Mohamed-Slim

    2017-01-01

    Being capable of serving multiple users with the same radio resource, non-orthogonal multiple access (NOMA) can provide desirable performance enhancements in a fair and spectral efficient manner. In this paper, we investigate the resource allocation (RA) and cluster formation (CF) aspects of NOMA for downlink (DL) uplink (UL) decoupled (DUDe) heterogeneous networks (HetNets). A non-ideal NOMA scheme is considered with power disparity and sensitivity constraints (PDSCs), delay tolerance, and residual interference after cancellation. Taking the PDSCs into account, we analytically show that using the DL decoding order limits UL-NOMA performance by that of OMA, while employing an inverse order result in a performance gain that is mainly determined by the channel gain disparity of users. Thereafter, a generic CF method is proposed for any type of user graph, which iteratively forms clusters using Blossom algorithm. Finally, highly non-convex RA problem is converted into a convex form by employing geometric programming (GP) where power and bandwidth are optimized to maximize network sumrate and max-min fairness objectives.

  19. Resource Allocation and Cluster Formation for Imperfect NOMA in DL/UL Decoupled HetNets

    KAUST Repository

    Celik, Abdulkadir

    2017-04-15

    Being capable of serving multiple users with the same radio resource, non-orthogonal multiple access (NOMA) can provide desirable performance enhancements in a fair and spectral efficient manner. In this paper, we investigate the resource allocation (RA) and cluster formation (CF) aspects of NOMA for downlink (DL) uplink (UL) decoupled (DUDe) heterogeneous networks (HetNets). A non-ideal NOMA scheme is considered with power disparity and sensitivity constraints (PDSCs), delay tolerance, and residual interference after cancellation. Taking the PDSCs into account, we analytically show that using the DL decoding order limits UL-NOMA performance by that of OMA, while employing an inverse order result in a performance gain that is mainly determined by the channel gain disparity of users. Thereafter, a generic CF method is proposed for any type of user graph, which iteratively forms clusters using Blossom algorithm. Finally, highly non-convex RA problem is converted into a convex form by employing geometric programming (GP) where power and bandwidth are optimized to maximize network sumrate and max-min fairness objectives.

  20. Arabidopsis thaliana mTERF proteins: evolution and functional classification

    Directory of Open Access Journals (Sweden)

    Tatjana eKleine

    2012-10-01

    Full Text Available Organellar gene expression (OGE is crucial for plant development, photosynthesis and respiration, but our understanding of the mechanisms that control it is still relatively poor. Thus, OGE requires various nucleus-encoded proteins that promote transcription, splicing, trimming and editing of organellar RNAs, and regulate translation. In metazoans, proteins of the mitochondrial Transcription tERmination Factor (mTERF family interact with the mitochondrial chromosome and regulate transcriptional initiation and termination. Sequencing of the Arabidopsis thaliana genome led to the identification of a diversified MTERF gene family but, in contrast to mammalian mTERFs, knowledge about the function of these proteins in photosynthetic organisms is scarce. In this hypothesis article, I show that tandem duplications and one block duplication contributed to the large number of MTERF genes in A. thaliana, and propose that the expansion of the family is related to the evolution of land plants. The MTERF genes - especially the duplicated genes - display a number of distinct mRNA accumulation patterns, suggesting functional diversification of mTERF proteins to increase adaptability to environmental changes. Indeed, hypothetical functions for the different mTERF proteins can be predicted using co-expression analysis and gene ontology annotations. On this basis, mTERF proteins can be sorted into five groups. Members of the chloroplast and chloroplast-associated clusters are principally involved in chloroplast gene expression, embryogenesis and protein catabolism, while representatives of the mitochondrial cluster seem to participate in DNA and RNA metabolism in that organelle. Moreover, members of the mitochondrion-associated cluster and the low expression group may act in the nucleus and/or the cytosol. As proteins involved in OGE and presumably nuclear gene expression, mTERFs are ideal candidates for the coordination of the expression of organelle and nuclear

  1. The Epstein-Barr virus BFRF1 and BFLF2 proteins interact and coexpression alters their cellular localization

    International Nuclear Information System (INIS)

    Lake, Cathleen M.; Hutt-Fletcher, Lindsey M.

    2004-01-01

    The BFRF1 protein of Epstein-Barr virus (EBV) is a recently identified membrane protein that is the homolog of the alphaherpesvirus UL34 gene product. We report here that a yeast two-hybrid screen identified the BFLF2 gene product, a homolog of alphaherpesvirus UL31, as a protein that interacts with BFRF1. Expression of BFLF2 in mammalian cells revealed a protein of approximately 28 kDa that associated with BFRF1 in a noncovalently linked complex. When expressed alone, the BFRF1 protein was found in the cytoplasm and perinuclear region. BFLF2 was found diffusely in the nucleus in the absence of BFRF1, but coexpression of BFRF1 and BFLF2 resulted in colocalization of the two proteins at the nuclear rim. These data recapitulate the behavior of the alphaherpesvirus homologs of BFRF1 and BFLF2 and suggest that functional as well as structural and positional homology may be conserved

  2. Membrane-Dependent Effects of a Cytoplasmic Helix on the Structure and Drug Binding of the Influenza Virus M2 Protein

    Science.gov (United States)

    Cady, Sarah; Wang, Tuo; Hong, Mei

    2011-01-01

    The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22 to 46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45 to 62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21–61). 13C-2H distance measurements of 13C-labeled protein and 2H-labeled amantadine showed that in DMPC bilayers, the first equivalent of drug bound S31 inside the M2(21–61) pore, similar to the behavior of M2TM in DMPC bilayers. The non-specific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21–61) is S31 in the TM pore. Interestingly, when M2(21–61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, while M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state, but did not rescue drug binding to M2(21–61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helices to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein. PMID:21661724

  3. Altered binding of 125I-labeled calmodulin to a 46.5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

    International Nuclear Information System (INIS)

    Tallant, E.A.; Wallace, R.W.

    1987-01-01

    The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca 2+ /calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of 125 I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis

  4. Functional analysis of virion host shutoff protein of pseudorabies virus

    International Nuclear Information System (INIS)

    Lin, H.-W.; Chang, Y.-Y.; Wong, M.-L.; Lin, J.-W.; Chang, T.-J.

    2004-01-01

    During lytic infection, the virion host shutoff (vhs) protein of alphaherpesviruses causes the degradation of mRNAs nonspecifically. In this work, we cloned the vhs gene (UL41 open reading frame) of pseudorabies virus (PRV; TNL strain) by PCR, and its nucleotide sequences were determined. The PCR product of vhs gene was subcloned into the prokaryotic pET32b expression vector, and production of the recombinant vhs protein was examined by SDS-PAGE. Result of Western blotting demonstrated that our recombinant vhs protein reacted with antiserum against a synthetic peptide of 17 amino acids of the vhs protein. After purification with nickel-chelate affinity chromatography, the purified recombinant vhs protein exhibited in vitro ribonuclease activity as expected. We further cloned the vhs gene into eukaryotic expression vectors and investigated the intracellular function of vhs protein by DNA transfection. By transient trasfection and CAT assay, we found the CAT activity was reduced in the presence of vhs, indicating that degradation of mRNA of the CAT gene was caused by the vhs. Furthermore, our results showed that the plaque formation of pseudorabies virus was blocked by exogenous vhs. Taken together, we have cloned the vhs gene of pseudorabies virus (TNL strain) and conducted functional analysis of the recombinant vhs protein in vitro as well as in vivo

  5. Reduction of carbon tetrachloride-induced rat liver injury by IRFI 042, a novel dual vitamin E-like antioxidant.

    Science.gov (United States)

    Campo, G M; Squadrito, F; Ceccarelli, S; Calò, M; Avenoso, A; Campo, S; Squadrito, G; Altavilla, D

    2001-04-01

    Carbon tetrachloride (CCl4 )-induced hepatotoxicity is likely the result of a CCl4 -induced free radical production which causes membrane lipid peroxidation and activation of transcription factors regulating both the TNF-alpha gene and the early-immediate genes involved in tissue regeneration. IRFI 042 is a novel vitamin E-like compound having a masked sulphydryl group in the aliphatic side chain. We studied the effect of IRFI 042 on CCl4 -induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of CCl4 (1 ml/kg in vegetal oil). Serum alanine aminotransferase (ALT) activity, liver malondialdehyde (MAL), hydroxyl radical formation (OH*), calculated indirectly by a trapping agent, hepatic reduced glutathione (GSH) concentration, plasma TNF-alpha, liver histology and hepatic mRNA levels for TNF-alpha were evaluated 48 h after CCl4 administration. Hepatic vitamin E (VE) levels were evaluated, in a separate group of animals, 2 h after CCl4 injection. A control group with vitamin E (100 mg/kg) was also treated in order to evaluate the differences versus the analogue treated groups. Intraperitoneal injection of carbon tetrachloride produced a marked increase in serum ALT activity (CCl4 = 404.61 +/- 10.33 U/L; Controls= 28.54 +/- 4.25 U/L), liver MAL (CCl4 = 0.67 +/- 0.16 nmol/mg protein; Controls= 0.13 +/- 0.06 nmol/mg protein), OH(7) levels assayed as 2,3-DHBA (CCl4 = 8.73 +/- 1.46 microM; Controls= 0.45 +/- 0.15 microM) and 2,5-DHBA (CCl4 = 24.61 +/- 3.32 microM; Controls= 2.75 +/- 0.93 microM), induced a severe depletion of GSH (CCl4 = 3.26 +/- 1.85 micromol/g protein; Controls= 17.82 +/- 3.13 micromol/g protein) and a marked decrease in VE levels (CCl4 = 5.67 +/- 1.22 nmol/g tissue; Controls= 13.47 +/- 3.21 nmol/g tissue), caused liver necrosis, increased plasma TNF-alpha levels (CCl4 = 57.36 +/- 13.24 IU/ml; Controls= 7.26 +/- 2.31 IU/ml) and enhanced hepatic mRNA for TNF-alpha (CCl4 = 19.22 +/- 4.38 a.u.; Controls= 0.76 +/- 0.36 a

  6. Protein Structure and the Sequential Structure of mRNA

    DEFF Research Database (Denmark)

    Brunak, Søren; Engelbrecht, Jacob

    1996-01-01

    entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment, By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets, These signals do not originate from......A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting...... protein, The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain, A complete search for GenBank nucleotide sequences coding for structural...

  7. Fusobacterium nucleatum binding to complement regulatory protein CD46 modulates the expression and secretion of cytokines and matrix metalloproteinases by oral epithelial cells.

    Science.gov (United States)

    Mahtout, Hayette; Chandad, Fatiha; Rojo, Jose M; Grenier, Daniel

    2011-02-01

    Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum-bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.

  8. The nuclear import of ribosomal proteins is regulated by mTOR

    Science.gov (United States)

    Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

    2014-01-01

    Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

  9. Structural changes in the hot Algol OGLE-LMC-DPV-097 and its disc related to its long cycle

    Science.gov (United States)

    Garcés L, J.; Mennickent, R. E.; Djurašević, G.; Poleski, R.; Soszyński, I.

    2018-06-01

    Double Periodic Variables (DPVs) are hot Algols showing a long photometric cycle of uncertain origin. We report the discovery of changes in the orbital light curve of OGLE-LMC-DPV-097 which depend on the phase of its long photometric cycle. During the ascending branch of the long cycle the brightness at the first quadrature is larger than during the second quadrature, during the maximum of the long cycle the brightness is basically the same at both quadratures, during the descending branch the brightness at the second quadrature is larger than during the first quadrature, and during the minimum of the long cycle the secondary minimum disappears. We model the light curve at different phases of the long cycle and find that the data are consistent with changes in the properties of the accretion disc and two disc spots. The disc's size and temperature change with the long-cycle period. We find a smaller and hotter disc at minimum, and larger and cooler disc at maximum. The spot temperatures, locations, and angular sizes also show variability during the long cycle.

  10. SEASONAL AVERAGE FLOW IN RÂUL NEGRU HYDROGRAPHIC BASIN

    Directory of Open Access Journals (Sweden)

    VIGH MELINDA

    2015-03-01

    Full Text Available The Râul Negru hydrographic basin is a well individualised physical-geographical unit inside the Braşov Depression. The flow is controlled by six hydrometric stations placed on the main river and on two important tributaries. The data base for seasonal flow analysis contains the discharges from 1950-2012. The results of data analysis show that there significant space-time differences between multiannual seasonal averages. Some interesting conclusions can be obtained by comparing abundant and scarce periods. Flow analysis was made using seasonal charts Q = f(T. The similarities come from the basin’s relative homogeneity, and the differences from flow’s evolution and trend. Flow variation is analysed using variation coefficient. In some cases appear significant Cv values differences. Also, Cv values trends are analysed according to basins’ average altitude.

  11. Production Engineering Program to Develop Improved Mass-Production Process for M42/M46 Grenade Bodies

    Science.gov (United States)

    1978-03-01

    j~l~05- 4.00 ".490-:010 I MAT’L:- AISI 4140 , SPEC ASTM A507 I DOME, GRENADE M46 (For Inertia Welding Process) Figure 1 31...piece assembly consisting of a cylinder fabricated from embossed 4140 steel in a tube mill and attached by furnace brazing to an embossed dome...that the following three processes would receive in-depth study and inve.stigation: I. Emboss coiled 4140 steel flat stock in a Fenn mill to give the

  12. Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein.

    Science.gov (United States)

    Rebelein, Johannes G; Lee, Chi Chung; Newcomb, Megan; Hu, Yilin; Ribbe, Markus W

    2018-03-13

    The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase. IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid. The "normalization" of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe

  13. Plasma proteins of Barbus holubi and Clarias gariepinus

    African Journals Online (AJOL)

    The plasma proteins of the teleosts, Barbus holubl and ClariDs gariepllUl3 were investigated by means of cel1u1ose-acetate and polyacrylamide gel electrophoresis. Characteristic patterns were obtained with both methods for each species. It was found that the application of human nomenclature to the patterns obtained in ...

  14. Analysis of protein-protein docking decoys using interaction fingerprints: application to the reconstruction of CaM-ligand complexes

    Directory of Open Access Journals (Sweden)

    Uchikoga Nobuyuki

    2010-05-01

    Full Text Available Abstract Background Protein-protein docking for proteins with large conformational changes was analyzed by using interaction fingerprints, one of the scales for measuring similarities among complex structures, utilized especially for searching near-native protein-ligand or protein-protein complex structures. Here, we have proposed a combined method for analyzing protein-protein docking by taking large conformational changes into consideration. This combined method consists of ensemble soft docking with multiple protein structures, refinement of complexes, and cluster analysis using interaction fingerprints and energy profiles. Results To test for the applicability of this combined method, various CaM-ligand complexes were reconstructed from the NMR structures of unbound CaM. For the purpose of reconstruction, we used three known CaM-ligands, namely, the CaM-binding peptides of cyclic nucleotide gateway (CNG, CaM kinase kinase (CaMKK and the plasma membrane Ca2+ ATPase pump (PMCA, and thirty-one structurally diverse CaM conformations. For each ligand, 62000 CaM-ligand complexes were generated in the docking step and the relationship between their energy profiles and structural similarities to the native complex were analyzed using interaction fingerprint and RMSD. Near-native clusters were obtained in the case of CNG and CaMKK. Conclusions The interaction fingerprint method discriminated near-native structures better than the RMSD method in cluster analysis. We showed that a combined method that includes the interaction fingerprint is very useful for protein-protein docking analysis of certain cases.

  15. The Potential of Flexible UL/DL Slot Assignment in 5G Systems

    DEFF Research Database (Denmark)

    Catania, Davide; Gatnau, Marta; Cattoni, Andrea Fabio

    2014-01-01

    5th Generation (5G) small cells are expected to satisfy the increasing demand for wireless data traffic. In the presence of large scale dense and randomly deployed cells, autonomous and distributed configuration mechanisms are highly desirable. However, small cells typically serve a small number...... of users, such that sudden traffic imbalances between downlink (DL) and uplink (UL) are expected in the new 5G system. We exploit the flexibility of time-division duplex (TDD) to deal with such imbalances by adapting swiftly to instantaneously varying traffic needs. In this paper we propose a distributed...

  16. mKikGR, a monomeric photoswitchable fluorescent protein.

    Directory of Open Access Journals (Sweden)

    Satoshi Habuchi

    Full Text Available The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

  17. Selective dansylation of M protein within intact influenza virions

    Energy Technology Data Exchange (ETDEWEB)

    Robertson, B.H.; Bennett, J.C.; Compans, R.W.

    1982-12-01

    Exposure of purified influenza virions to (/sup 14/C)dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.

  18. Selective dansylation of M protein within intact influenza virions

    International Nuclear Information System (INIS)

    Robertson, B.H.; Bennett, J.C.; Compans, R.W.

    1982-01-01

    Exposure of purified influenza virions to [ 14 C]dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride

  19. GenBank blastx search result: AK288311 [KOME

    Lifescience Database Archive (English)

    Full Text Available (UL26), virion scaffolding protein (UL26.5), virion membrane glycoprotein B (gB), DNA cleavage/packaging pro... subunit (pol), nuclear phosphoprotein (UL31), DNA cleavage/packaging (UL32), DNA cleavage/packaging protein

  20. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

    Directory of Open Access Journals (Sweden)

    Mo Min

    2008-05-01

    Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

  1. Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

    Science.gov (United States)

    Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

    2007-11-01

    We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

  2. Cell lines that support replication of a novel herpes simplex virus 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31

    International Nuclear Information System (INIS)

    Liang Li; Tanaka, Michiko; Kawaguchi, Yasushi; Baines, Joel D.

    2004-01-01

    Previous results indicated that the herpes simplex virus 1 (HSV-1) U L 31 gene is necessary and sufficient for localization of the U L 34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U L 31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U L 31 gene. The replication of the U L 31 deletion virus was restored on U L 31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U L 34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U L 34 protein localized at the nuclear membrane in rabbit skin cells, and U L 31 complementing CV1 cells infected with the U L 31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U L 34 protein to the nuclear membrane. We speculate that this function partially complements that of U L 31 and may explain why U L 31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells

  3. mTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation via PRAS40 to Prevent Accumulation of Protein Stress.

    Science.gov (United States)

    Yun, Young Sung; Kim, Kwan Hyun; Tschida, Barbara; Sachs, Zohar; Noble-Orcutt, Klara E; Moriarity, Branden S; Ai, Teng; Ding, Rui; Williams, Jessica; Chen, Liqiang; Largaespada, David; Kim, Do-Hyung

    2016-02-18

    Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Energy Technology Data Exchange (ETDEWEB)

    Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  5. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    International Nuclear Information System (INIS)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress

  6. Direct quantification of human cytomegalovirus immediate-early and late mRNA levels in blood of lung transplant recipients by competitive nucleic acid sequence-based amplification

    NARCIS (Netherlands)

    Greijer, AE; Verschuuren, EAM; Harmsen, MC; Dekkers, CAJ; Adriaanse, HMA; The, TH; Middeldorp, JM

    The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels In the blood of patients after lung transplantation. RNA was isolated from 339

  7. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Köster, Tino

    2017-04-13

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  8. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

    2017-01-01

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  9. Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

    OpenAIRE

    Gadir, Noga; Haim-Vilmovsky, Liora; Kraut-Cohen, Judith; Gerst, Jeffrey E.

    2011-01-01

    Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with m...

  10. Krymskokaraimska wersja Melukhat Sha’ul. Wydanie krytyczne i analiza językoznawcza

    OpenAIRE

    Smętek, Dorota

    2012-01-01

    Wydział Neofilologii: Katedra Studiów Azjatyckich Rozprawa doktorska udostępnia w wydaniu krytycznym dramat p.t. Melukhat Sha’ul znajdujący się w krymskokaraimskim rękopisie zwanym medżumą i jest pierwszym opracowaniem naukowym tego dzieła. Oryginał rękopisu, o numerze VI-3/22, znajduje się na Krymie. Został on spisany przez Samuela Kohena w drugiej połowie dziewiętnastego wieku, to znaczy w roku 1876 z krótkimi fragmentami zapisanymi w 1875 i 1879. Rękopis zawiera turkijskie tłumaczenie d...

  11. Detection of 17 β-Estradiol in Environmental Samples and for Health Care Using a Single-Use, Cost-Effective Biosensor Based on Differential Pulse Voltammetry (DPV).

    Science.gov (United States)

    Dai, Yifan; Liu, Chung Chiun

    2017-03-29

    Environmental estrogen pollution and estrogen effects on the female reproductive system are well recognized scientifically. Among the estrogens, 17 β-estradiol is a priority in environmental estrogen pollution, and it is also a major contributor to estrogen which regulates the female reproductive system. 17 β-estradiol is carcinogenic and has a tumor promotion effect relating to breast cancer, lung cancer and others. It also affects psychological well-being such as depression, fatigue and others. Thus, a simple method of detecting 17 β-estradiol will be important for both environmental estrogen pollution and health care. This study demonstrates a single-use, cost-effective 17 β-estradiol biosensor system which can be used for both environmental and health care applications. The bio-recognition mechanism is based on the influence of the redox couple, K₃Fe(CN)₆/K₄Fe(CN)₆ by the interaction between 17 β-estradiol antigen and its α-receptor (ER-α; α-estrogen antibody). The transduction mechanism is an electrochemical analytical technique, differential pulse voltammetry (DPV). The levels of 17 β-estradiol antigen studied were between 2.25 pg/mL and 2250 pg/mL; Phosphate buffered saline (PBS), tap water from the Cleveland regional water district, and simulated urine were used as the test media covering the potential application areas for 17 β-estradiol detection. An interference study by testosterone, which has a similar chemical structure and molecular weight as those of 17 β-estradiol, was carried out, and this 17 β-estradiol biosensor showed excellent specificity without any interference by similar chemicals.

  12. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Science.gov (United States)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

  13. MONTHLY AVERAGE FLOW IN RÂUL NEGRU HYDROGRAPHIC BASIN

    Directory of Open Access Journals (Sweden)

    VIGH MELINDA

    2014-03-01

    Full Text Available Râul Negru hydrographic basin represents a well individualised and relatively homogenous physical-geographical unity from Braşov Depression. The flow is controlled by six hydrometric stations placed on the main collector and on two of the most powerful tributaries. Our analysis period is represented by the last 25 years (1988 - 2012 and it’s acceptable for make pertinent conclusions. The maximum discharge month is April, that it’s placed in the high flow period: March – June. Minimum discharges appear in November - because of the lack of pluvial precipitations; in January because of high solid precipitations and because of water volume retention in ice. Extreme discharge frequencies vary according to their position: in the mountain area – small basin surface; into a depression – high basin surface. Variation coefficients point out very similar variation principles, showing a relative homogeneity of flow processes.

  14. Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

    Directory of Open Access Journals (Sweden)

    Deutsch Eric W

    2008-05-01

    Full Text Available Abstract Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63. Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50 but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.

  15. Association of CAD, a multifunctional protein involved in pyrimidine synthesis, with mLST8, a component of the mTOR complexes

    Science.gov (United States)

    2013-01-01

    Background mTOR is a genetically conserved serine/threonine protein kinase, which controls cell growth, proliferation, and survival. A multifunctional protein CAD, catalyzing the initial three steps in de novo pyrimidine synthesis, is regulated by the phosphorylation reaction with different protein kinases, but the relationship with mTOR protein kinase has not been known. Results CAD was recovered as a binding protein with mLST8, a component of the mTOR complexes, from HEK293 cells transfected with the FLAG-mLST8 vector. Association of these two proteins was confirmed by the co-immuoprecipitaiton followed by immunoblot analysis of transfected myc-CAD and FLAG-mLST8 as well as that of the endogenous proteins in the cells. Analysis using mutant constructs suggested that CAD has more than one region for the binding with mLST8, and that mLST8 recognizes CAD and mTOR in distinct ways. The CAD enzymatic activity decreased in the cells depleted of amino acids and serum, in which the mTOR activity is suppressed. Conclusion The results obtained indicate that mLST8 bridges between CAD and mTOR, and plays a role in the signaling mechanism where CAD is regulated in the mTOR pathway through the association with mLST8. PMID:23594158

  16. Stringent homology-based prediction of H. sapiens-M. tuberculosis H37Rv protein-protein interactions.

    Science.gov (United States)

    Zhou, Hufeng; Gao, Shangzhi; Nguyen, Nam Ninh; Fan, Mengyuan; Jin, Jingjing; Liu, Bing; Zhao, Liang; Xiong, Geng; Tan, Min; Li, Shijun; Wong, Limsoon

    2014-04-08

    H. sapiens-M. tuberculosis H37Rv protein-protein interaction (PPI) data are essential for understanding the infection mechanism of the formidable pathogen M. tuberculosis H37Rv. Computational prediction is an important strategy to fill the gap in experimental H. sapiens-M. tuberculosis H37Rv PPI data. Homology-based prediction is frequently used in predicting both intra-species and inter-species PPIs. However, some limitations are not properly resolved in several published works that predict eukaryote-prokaryote inter-species PPIs using intra-species template PPIs. We develop a stringent homology-based prediction approach by taking into account (i) differences between eukaryotic and prokaryotic proteins and (ii) differences between inter-species and intra-species PPI interfaces. We compare our stringent homology-based approach to a conventional homology-based approach for predicting host-pathogen PPIs, based on cellular compartment distribution analysis, disease gene list enrichment analysis, pathway enrichment analysis and functional category enrichment analysis. These analyses support the validity of our prediction result, and clearly show that our approach has better performance in predicting H. sapiens-M. tuberculosis H37Rv PPIs. Using our stringent homology-based approach, we have predicted a set of highly plausible H. sapiens-M. tuberculosis H37Rv PPIs which might be useful for many of related studies. Based on our analysis of the H. sapiens-M. tuberculosis H37Rv PPI network predicted by our stringent homology-based approach, we have discovered several interesting properties which are reported here for the first time. We find that both host proteins and pathogen proteins involved in the host-pathogen PPIs tend to be hubs in their own intra-species PPI network. Also, both host and pathogen proteins involved in host-pathogen PPIs tend to have longer primary sequence, tend to have more domains, tend to be more hydrophilic, etc. And the protein domains from both

  17. Intergenotypic replacement of lyssavirus matrix proteins demonstrates the role of lyssavirus M proteins in intracellular virus accumulation.

    Science.gov (United States)

    Finke, Stefan; Granzow, Harald; Hurst, Jose; Pollin, Reiko; Mettenleiter, Thomas C

    2010-02-01

    Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

  18. Structure and Mechanism of Proton Transport Through the Transmembrane Tetrameric M2 Protein Bundle of the Influenza A Virus

    Energy Technology Data Exchange (ETDEWEB)

    R Acharya; V Carnevale; G Fiorin; B Levine; A Polishchuk; V Balannick; I Samish; R Lamb; L Pinto; et al.

    2011-12-31

    The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2{sup +} and 3{sup +} with a pK{sub a} near 6. A 1.65 {angstrom} resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

  19. Pharmacodynamic Activity of Dapivirine and Maraviroc Single Entity and Combination Topical Gels for HIV-1 Prevention.

    Science.gov (United States)

    Dezzutti, Charlene S; Yandura, Sarah; Wang, Lin; Moncla, Bernard; Teeple, Elizabeth A; Devlin, Brid; Nuttall, Jeremy; Brown, Elizabeth R; Rohan, Lisa C

    2015-11-01

    Dapivirine (DPV), a non-nucleoside reverse transcriptase inhibitor, and maraviroc (MVC), a CCR5 antagonist, were formulated into aqueous gels designed to prevent mucosal HIV transmission. 0.05% DPV, 0.1% MVC, 0.05% DPV/0.1% MVC and placebo gels were evaluated for pH, viscosity, osmolality, and in vitro release. In vitro assays and mucosal tissues were used to evaluate anti-HIV activity. Viability (Lactobacilli only) and epithelial integrity in cell lines and mucosal tissues defined safety. The gels were acidic and viscous. DPV gel had an osmolality of 893 mOsm/kg while the other gels had an osmolality of <100 mOsm/kg. MVC release was similar from the single and combination gels (~5 μg/cm(2)/min(1/2)), while DPV release was 10-fold less from the single as compared to the combination gel (0.4331 μg/cm(2)/min(1/2)). Titrations of the gels showed 10-fold more drug was needed to protect ectocervical than colonic tissue. The combination gel showed ~10- and 100-fold improved activity as compared to DPV and MVC gel, respectively. All gels were safe. The DPV/MVC gel showed a benefit blocking HIV infection of mucosal tissue compared to the single entity gels. Combination products with drugs affecting unique steps in the viral replication cycle would be advantageous for HIV prevention.

  20. Generation of 2.5 μm and 4.6 μm Dispersive Waves in Kagome Photonic Crystal Fiber with Plasma Production

    Institute of Scientific and Technical Information of China (English)

    Tian-Qi Zhao; Meng Li; Dong Wei; Xin Ding; Gui-Zhong Zhang; Jian-Quan Yao

    2017-01-01

    We report our numerical simulation on dispersive waves (DWs) generated in the Kr-filled Kagome hollow-core photonic crystal fiber,by deploying the unidirectional pulse propagation equation.Relatively strong dispersive waves are simultaneously generated at 2.5μm and 4.6μm.It is deciphered that the interplay between plasma currents due to Kr ionization and nonlinear effects plays a key role in DW generation.Remarkably,this kind of DW generation is corroborated by the plasma-corrected phase-matching condition.

  1. Whey Protein Ingestion Activates mTOR-dependent Signalling after Resistance Exercise in Young Men: A Double-Blinded Randomized Controlled Trial

    Directory of Open Access Journals (Sweden)

    David Cameron-Smith

    2009-12-01

    Full Text Available The effect of resistance exercise with the ingestion of supplementary protein on the activation of the mTOR cascade, in human skeletal muscle has not been fully elucidated. In this study, the impact of a single bout of resistance exercise, immediately followed by a single dose of whey protein isolate (WPI or placebo supplement, on the activation of mTOR signalling was analyzed. Young untrained men completed a maximal single-legged knee extension exercise bout and were randomized to ingest either WPI supplement (n = 7 or the placebo (n = 7. Muscle biopsies were taken from the vastus lateralis before, and 2, 4 and 24 hr post-exercise. WPI or placebo ingestion consumed immediately post-exercise had no impact on the phosphorylation of Akt (Ser473. However, WPI significantly enhanced phosphorylation of mTOR (Ser2448, 4E-BP1 (Thr37/46 and p70S6K (Thr389 at 2 hr post-exercise. This study demonstrates that a single dose of WPI, when consumed in modest quantities, taken immediately after resistance exercise elicits an acute and transient activation of translation initiation within the exercised skeletal muscle.

  2. Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

    International Nuclear Information System (INIS)

    Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

    1988-01-01

    The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated 32 P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. 32 P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice

  3. Galectin-1 Is an Interactive Protein of Selenoprotein M in the Brain

    Directory of Open Access Journals (Sweden)

    Qiong Liu

    2013-11-01

    Full Text Available Selenium, an essential trace element for human health, mainly exerts its biological function through selenoproteins. Selenoprotein M (SelM is one of the highly expressed selenoproteins in the brain, but its biological effect and molecular mechanism remain unclear. Thus, the interactive protein of SelM was investigated in this paper to guide further study. In order to avoid protein translational stop, the selenocysteine-encoding UGA inside the open reading frame of SelM was site-directly changed to the cysteine-encoding UGC to generate the SelM' mutant. Meanwhile, its N terminal transmembrane signal peptide was also cut off. This truncated SelM' was used to screen a human fetal brain cDNA library by the yeast two-hybrid system. A new interactive protein of SelM' was found to be galectin-1 (Gal-1. This protein-protein interaction was further verified by the results of fluorescence resonance energy transfer techniques, glutathione S-transferase pull-down and co-immunoprecipitation assays. As Gal-1 plays important roles in preventing neurodegeneration and promoting neuroprotection in the brain, the interaction between SelM' and Gal-1 displays a new direction for studying the biological function of SelM in the human brain.

  4. Calmodulin and calmodulin-binding proteins in cystic fibrosis and normal human fibroblasts

    International Nuclear Information System (INIS)

    Tallant, E.A.; Wallace, R.W.

    1986-01-01

    The authors have investigated the possibility that a lesion in a calmodulin (CaM)-dependent regulatory mechanism may be involved in cystic fibrosis (CF). The level of CaM, CaM-binding proteins (CaM-BP) and a CaM-dependent phosphatase (CaM-Ptase) have been compared in cultured fibroblasts from CF patients versus age- and sex-matched control subjects. The CaM concentration, measured by radioimmunoassay, ranged from 0.20 to 0.76 μg/mg protein (n=8); there was no significant difference in the average CaM concentration from CF patients vs controls. Using Western blotting techniques with 125 I-CaM, they detected at least ten distinct CaM-BPs in fibroblasts with molecular weights ranging from 230K to 37K; the only consistent difference between control and CF cell lines was in a 46.5K CaM-BP, which was depressed in all three CF samples. The 46.5 K CaM-BP was found only in the particulate fraction. A 59K CaM-BP was identified as a CaM-Ptase by its crossreactivity with an antibody against a brain CaM-Ptase. There was no significant difference in CaM-Ptase activity or in the amount of the phosphatase as determined by radioimmunoassay in CF vs. normal samples (n=8). Thus, the level of CaM as well as its various enzymes and proteins do not appear to be altered in CF fibroblasts except for a CaM-BP of 46.5K, the identity of which is currently being investigated

  5. Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus.

    Science.gov (United States)

    Marin, Milenen Hernández; Rodríguez-Tanty, Chryslaine; Higginson-Clarke, David; Bocalandro, Yadaris Márquez; Peña, Lilliam Pozo

    2005-10-28

    Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.

  6. [Effect of recombinant human growth hormone therapy on metabolic parameters in patients with craniopharyngioma].

    Science.gov (United States)

    Mao, J F; Wang, X; Xiong, S Y; Zheng, J J; Yu, B Q; Nie, M; Wu, X Y; Qi, S T

    2017-11-14

    Objective: To investigate the effects of recombinant human growth hormone (rhGH) on metabolic parameters in patients with craniopharyngioma surgeries. Methods: Totallys 30 patients with craniopharyngioma were included in this retrospective study. They were divided into growth hormone (GH) group and control group according to whether they received rhGH therapy or not. The following parameters, including body mass index (BMI), weight, waist circumstance, transaminase, fasting blood glucose, lipid profile and high-sensitivity C-reactive protein (hsCRP) were compared after rhGH therapy for 4-6 months. Results: In GH group, patients were 18-46 (30.0±8.8) years old. The duration after craniopharyngioma surgery was (12.9±5.4) years. Before rhGH therapy, they had got sufficient thyroid and glucocorticoid hormone replacement. After rhGH therapy, the body weight decreased from (92.3±20.1) to (87.6 ±14.6) kg ( P =0.190), with a reduction of BMI from (30.1±5.9) to (28.2±3.7) kg/m(2) ( P =0.120). The waist circumference decreased from (104.4±9.4) cm to (98.8±10.6) cm ( P =0.002). Alanine aminotransferase (ALT) decreased from (52±34) to (28±19) U/L ( P =0.029), with a reduction of aspartate transaminase (AST) from (46±21) to (33±18) U/L ( P =0.035) and γ-glutamyl transpeptadase (GGT) from (59±42) to (29±15) U/L ( P =0.02). hsCRP decreased from (5.3±4.9) to (2.3±2.8) mg/L ( P =0.006) and triglyceride (TG) decreased from (1.8±0.7) to (1.5±0.6) mmol/L ( P =0.028). Fasting blood glucose, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and free fat acid (FFA) were not significantly changed(all P >0.05). In the control group, the above mentioned parameters did not changed significantly during 4-6 months of observational period(all P >0.05). Conclusion: rhGH therapy improves metabolic parameters in patients after craniopharyngioma surgery by decreasing body weight, waist circumstance and fat deposit in liver, as well as

  7. Connecting protein and mRNA burst distributions for stochastic models of gene expression

    International Nuclear Information System (INIS)

    Elgart, Vlad; Jia, Tao; Fenley, Andrew T; Kulkarni, Rahul

    2011-01-01

    The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distribution. Additionally, if gene expression is repressed such that observed protein bursts arise only from single mRNAs, we show how observations of protein burst distributions (repressed and unrepressed) can be used to completely determine the mRNA burst distribution. Assuming independent contributions from individual bursts, we derive analytical expressions connecting means and variances for burst and steady-state protein distributions. Finally, we validate our general analytical results by considering a specific reaction scheme involving regulation of protein bursts by small RNAs. For a range of parameters, we derive analytical expressions for regulated protein distributions that are validated using stochastic simulations. The analytical results obtained in this work can thus serve as useful inputs for a broad range of studies focusing on stochasticity in gene expression

  8. Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue shifted intermediate

    NARCIS (Netherlands)

    Derix, N.M.; Wechselberger, R.W.|info:eu-repo/dai/nl/304829005; van der Horst, M.A.; Hellingwerf, K.J.; Boelens, R.|info:eu-repo/dai/nl/070151407; Kaptein, R.|info:eu-repo/dai/nl/074334603; van Nuland, N.A.J.

    2003-01-01

    The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB

  9. Rehabilitation of patients with primary liver cancer after transhepatic arterial chemoembolization%肝癌患者接受肝动脉灌注化疗栓塞术后护理康复效果分析

    Institute of Scientific and Technical Information of China (English)

    程光荣; 郭丽萍

    2016-01-01

    Objective We explored the effect of nursing care and its impact on rehabilitation in patients with primary liver cancer (PLC) after transhepatic arterial chemoembolization (TACE) in order to provide guidance for improving the life quality of patients. Methods 50 patients with moderate and advanced PLC underwent TACE with hepatic artery perfusion of raltitrexed,and we carried out holistic nursing,including psychological,febrile,dietary,abdominal and local nursing post-operatively for them. We also measured blood liver function indexes and alpha-fetoprotein (AFP) before and after the treatment. Results Four weeks after TACE, serum alanine transaminase,aspartic transaminae,total bilirubin and albumin levels decreased from (43.58±10.15)U/L,(41.25±11.34)U/L,(22.46±6.49)μmol/L and (35.32±4.12) g/L at admission to (38.23±7.86) U/L,(38.56±8.49) U/L,(16.84±10.16)μmol/L and(31.49±5.48) g/L,post-operatively,all no significant difference(P>0.05);serum AFP decreased from(1215.46±125.14) ng/mL to(811.27±165.28) ng/mL(P0.05);血清AFP水平由治疗前的(1215.46±125.14) ng/mL降至(811.27±165.28) ng/mL,显著降低(P<0.01);同时,不良反应情况得到巨大改善,发生白细胞降低和发热者均为2例(4%),心理紧张3例(6%),出现疼痛6例(14%),恶心或呕吐8例(16%)。结论肝动脉灌注雷替曲塞化疗术可有效治疗肝癌,术后护理可以预防并发症的发生,大大提高术后康复效果。

  10. Effect of quality protein maize diet on liver integrity and serum ...

    African Journals Online (AJOL)

    The study was designed to evaluate the effect of quality protein maize (QPM) diet on the histology of the liver and on the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in albino wistar rats. The AST level in rats fed QPM diet was 57.4 ± 8.92U/L which compared favourably with that ...

  11. Simultaneous isolation of mRNA and native protein from minute samples of cells

    DEFF Research Database (Denmark)

    Petersen, Tonny Studsgaard; Andersen, Claus Yding

    2014-01-01

    Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native...... in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization...... proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins...

  12. Stringent DDI-based prediction of H. sapiens-M. tuberculosis H37Rv protein-protein interactions.

    Science.gov (United States)

    Zhou, Hufeng; Rezaei, Javad; Hugo, Willy; Gao, Shangzhi; Jin, Jingjing; Fan, Mengyuan; Yong, Chern-Han; Wozniak, Michal; Wong, Limsoon

    2013-01-01

    H. sapiens-M. tuberculosis H37Rv protein-protein interaction (PPI) data are very important information to illuminate the infection mechanism of M. tuberculosis H37Rv. But current H. sapiens-M. tuberculosis H37Rv PPI data are very scarce. This seriously limits the study of the interaction between this important pathogen and its host H. sapiens. Computational prediction of H. sapiens-M. tuberculosis H37Rv PPIs is an important strategy to fill in the gap. Domain-domain interaction (DDI) based prediction is one of the frequently used computational approaches in predicting both intra-species and inter-species PPIs. However, the performance of DDI-based host-pathogen PPI prediction has been rather limited. We develop a stringent DDI-based prediction approach with emphasis on (i) differences between the specific domain sequences on annotated regions of proteins under the same domain ID and (ii) calculation of the interaction strength of predicted PPIs based on the interacting residues in their interaction interfaces. We compare our stringent DDI-based approach to a conventional DDI-based approach for predicting PPIs based on gold standard intra-species PPIs and coherent informative Gene Ontology terms assessment. The assessment results show that our stringent DDI-based approach achieves much better performance in predicting PPIs than the conventional approach. Using our stringent DDI-based approach, we have predicted a small set of reliable H. sapiens-M. tuberculosis H37Rv PPIs which could be very useful for a variety of related studies. We also analyze the H. sapiens-M. tuberculosis H37Rv PPIs predicted by our stringent DDI-based approach using cellular compartment distribution analysis, functional category enrichment analysis and pathway enrichment analysis. The analyses support the validity of our prediction result. Also, based on an analysis of the H. sapiens-M. tuberculosis H37Rv PPI network predicted by our stringent DDI-based approach, we have discovered some

  13. A multi-directional rapidly exploring random graph (mRRG) for protein folding

    KAUST Repository

    Nath, Shuvra Kanti; Thomas, Shawna; Ekenna, Chinwe; Amato, Nancy M.

    2012-01-01

    Modeling large-scale protein motions, such as those involved in folding and binding interactions, is crucial to better understanding not only how proteins move and interact with other molecules but also how proteins misfold, thus causing many devastating diseases. Robotic motion planning algorithms, such as Rapidly Exploring Random Trees (RRTs), have been successful in simulating protein folding pathways. Here, we propose a new multi-directional Rapidly Exploring Random Graph (mRRG) specifically tailored for proteins. Unlike traditional RRGs which only expand a parent conformation in a single direction, our strategy expands the parent conformation in multiple directions to generate new samples. Resulting samples are connected to the parent conformation and its nearest neighbors. By leveraging multiple directions, mRRG can model the protein motion landscape with reduced computational time compared to several other robotics-based methods for small to moderate-sized proteins. Our results on several proteins agree with experimental hydrogen out-exchange, pulse-labeling, and F-value analysis. We also show that mRRG covers the conformation space better as compared to the other computation methods. Copyright © 2012 ACM.

  14. The cytoplasmic tails of infectious bronchitis virus E and M proteins mediate their interaction

    International Nuclear Information System (INIS)

    Corse, Emily; Machamer, Carolyn E.

    2003-01-01

    Virus-like particle (VLP) formation by the coronavirus E and M proteins suggests that interactions between these proteins play a critical role in coronavirus assembly. We studied interactions between the infectious bronchitis virus (IBV) E and M proteins using in vivo crosslinking and VLP assembly assays. We show that IBV E and M can be crosslinked to each other in IBV-infected and transfected cells, indicating that they interact. The cytoplasmic tails of both proteins are important for this interaction. We also examined the ability of the mutant and chimeric E and M proteins to form VLPs. IBV M proteins that are missing portions of their cytoplasmic tails or transmembrane regions were not able to support VLP formation, regardless of their ability to be crosslinked to IBV E. Interactions between the E and M proteins and the membrane bilayer are likely to play an important role in VLP formation and virus budding

  15. Herpesvirus capsid assembly and DNA packaging

    Science.gov (United States)

    Heming, Jason D.; Conway, James F.; Homa, Fred L.

    2017-01-01

    Herpes simplex virus type I (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. During productive lytic infection, over 80 viral proteins are expressed in a highly regulated manner, resulting in the replication of viral genomes and assembly of progeny virions. The virion of all herpesviruses consists of an external membrane envelope, a proteinaceous layer called the tegument, and an icosahedral capsid containing the double-stranded linear DNA genome. The capsid shell of HSV-1 is built from four structural proteins: a major capsid protein, VP5, which forms the capsomers (hexons and pentons), the triplex consisting of VP19C and VP23 found between the capsomers, and VP26 which binds to VP5 on hexons but not pentons. In addition, the dodecameric pUL6 portal complex occupies one of the 12 capsid vertices, and the capsid vertex specific component (CVSC), a heterotrimer complex of pUL17, pUL25 and pUL36 binds specifically to the triplexes adjacent to each penton. The capsid is assembled in the nucleus where the viral genome is packaged into newly assembled closed capsid shells. Cleavage and packaging of replicated, concatemeric viral DNA requires the seven viral proteins encoded by the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes. Considerable advances have been made in understanding the structure of the herpesvirus capsid and the function of several of the DNA packaging proteins by applying biochemical, genetic, and structural techniques. This review is a summary of recent advances with respect to the structure of the HSV-1 virion capsid and what is known about the function of the seven packaging proteins and their interactions with each other and with the capsid shell. PMID:28528442

  16. A Structural analysis of M protein in coronavirus assembly and morphology

    DEFF Research Database (Denmark)

    W. Neuman, Benjamin; Kiss, Gabriella; H. Kunding, Andreas

    2011-01-01

    The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy...... protein functions to promote virus assembly....

  17. Protein functional features are reflected in the patterns of mRNA translation speed.

    Science.gov (United States)

    López, Daniel; Pazos, Florencio

    2015-07-09

    The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

  18. KC-46A Tanker Modernization (KC-46A)

    Science.gov (United States)

    2015-12-01

    Program Startup Workshop with the assistance from Defense Acquisition University at Wright-Patterson Air Force Base, Ohio. July 8 - 10, 2013: The KC-46A...Selected Acquisition Report (SAR) RCS: DD-A&T(Q&A)823-387 KC-46A Tanker Modernization (KC-46A) As of FY 2017 President’s Budget Defense Acquisition ...Deliveries and Expenditures 48 Operating and Support Cost 49 Common Acronyms and Abbreviations for MDAP Programs Acq O&M - Acquisition

  19. Translational regulation of ribosomal protein S15 drives characteristic patterns of protein-mRNA epistasis.

    Science.gov (United States)

    Mallik, Saurav; Basu, Sudipto; Hait, Suman; Kundu, Sudip

    2018-04-21

    Do coding and regulatory segments of a gene co-evolve with each-other? Seeking answers to this question, here we analyze the case of Escherichia coli ribosomal protein S15, that represses its own translation by specifically binding its messenger RNA (rpsO mRNA) and stabilizing a pseudoknot structure at the upstream untranslated region, thus trapping the ribosome into an incomplete translation initiation complex. In the absence of S15, ribosomal protein S1 recognizes rpsO and promotes translation by melting this very pseudoknot. We employ a robust statistical method to detect signatures of positive epistasis between residue site pairs and find that biophysical constraints of translational regulation (S15-rpsO and S1-rpsO recognition, S15-mediated rpsO structural rearrangement, and S1-mediated melting) are strong predictors of positive epistasis. Transforming the epistatic pairs into a network, we find that signatures of two different, but interconnected regulatory cascades are imprinted in the sequence-space and can be captured in terms of two dense network modules that are sparsely connected to each other. This network topology further reflects a general principle of how functionally coupled components of biological networks are interconnected. These results depict a model case, where translational regulation drives characteristic residue-level epistasis-not only between a protein and its own mRNA but also between a protein and the mRNA of an entirely different protein. © 2018 Wiley Periodicals, Inc.

  20. Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

    Science.gov (United States)

    Morimoto, Shimpei; Yahara, Koji

    2018-03-01

    Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

  1. Being WISE. I. Validating stellar population models and M */L ratios at 3.4 and 4.6 μm

    International Nuclear Information System (INIS)

    Norris, Mark A.; Meidt, Sharon; Van de Ven, Glenn; Schinnerer, Eva; Groves, Brent; Querejeta, Miguel

    2014-01-01

    Using data from the Wide-field Infrared Survey Explorer mission, we have measured near infra-red (NIR) photometry of a diverse sample of dust-free stellar systems (globular clusters, dwarf and giant early-type galaxies) which have metallicities that span the range -2.2 < [Fe/H] (dex) < 0.3. This dramatically increases the sample size and broadens the metallicity regime over which the 3.4 (W1) and 4.6 μm (W2) photometry of stellar populations have been examined. We find that the W1 – W2 colors of intermediate and old (>2 Gyr) stellar populations are insensitive to the age of the stellar population, but that the W1 – W2 colors become bluer with increasing metallicity, a trend not well reproduced by most stellar population synthesis (SPS) models. In common with previous studies, we attribute this behavior to the increasing strength of the CO absorption feature located in the 4.6 μm bandpass with metallicity. Having used our sample to validate the efficacy of some of the SPS models, we use these models to derive stellar mass-to-light ratios in the W1 and W2 bands. Utilizing observational data from the SAURON and ATLAS3D surveys, we demonstrate that these bands provide extremely simple, yet robust stellar mass tracers for dust free older stellar populations that are freed from many of the uncertainties common among optical estimators.

  2. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

    Science.gov (United States)

    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

    2017-10-01

    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Sequence variability is correlated with weak immunogenicity in Streptococcus pyogenes M protein.

    Science.gov (United States)

    Lannergård, Jonas; Kristensen, Bodil M; Gustafsson, Mattias C U; Persson, Jenny J; Norrby-Teglund, Anna; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

    2015-10-01

    The M protein of Streptococcus pyogenes, a major bacterial virulence factor, has an amino-terminal hypervariable region (HVR) that is a target for type-specific protective antibodies. Intriguingly, the HVR elicits a weak antibody response, indicating that it escapes host immunity by two mechanisms, sequence variability and weak immunogenicity. However, the properties influencing the immunogenicity of regions in an M protein remain poorly understood. Here, we studied the antibody response to different regions of the classical M1 and M5 proteins, in which not only the HVR but also the adjacent fibrinogen-binding B repeat region exhibits extensive sequence divergence. Analysis of antisera from S. pyogenes-infected patients, infected mice, and immunized mice showed that both the HVR and the B repeat region elicited weak antibody responses, while the conserved carboxy-terminal part was immunodominant. Thus, we identified a correlation between sequence variability and weak immunogenicity for M protein regions. A potential explanation for the weak immunogenicity was provided by the demonstration that protease digestion selectively eliminated the HVR-B part from whole M protein-expressing bacteria. These data support a coherent model, in which the entire variable HVR-B part evades antibody attack, not only by sequence variability but also by weak immunogenicity resulting from protease attack. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  4. [Identification of C(2)M interacting proteins by yeast two-hybrid screening].

    Science.gov (United States)

    Yue, Shan-shan; Xia, Lai-xin

    2015-11-01

    The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

  5. Discovery of Proteomic Code with mRNA Assisted Protein Folding

    Directory of Open Access Journals (Sweden)

    Jan C. Biro

    2008-12-01

    Full Text Available The 3x redundancy of the Genetic Code is usually explained as a necessity to increase the mutation-resistance of the genetic information. However recent bioinformatical observations indicate that the redundant Genetic Code contains more biological information than previously known and which is additional to the 64/20 definition of amino acids. It might define the physico-chemical and structural properties of amino acids, the codon boundaries, the amino acid co-locations (interactions in the coded proteins and the free folding energy of mRNAs. This additional information, which seems to be necessary to determine the 3D structure of coding nucleic acids as well as the coded proteins, is known as the Proteomic Code and mRNA Assisted Protein Folding.

  6. Ductless personalized ventilation with local air cleaning

    DEFF Research Database (Denmark)

    Dalewski, Mariusz; Vesely, Michal; Melikov, Arsen Krikor

    2012-01-01

    An experiment with 28 human subjects was performed to examine effects of using a local air cleaning device combined with ductless personalized ventilation (DPV) on perceived air quality. Experiments were performed in a test room with displacement ventilation. The DPV at one of two desks was equip......An experiment with 28 human subjects was performed to examine effects of using a local air cleaning device combined with ductless personalized ventilation (DPV) on perceived air quality. Experiments were performed in a test room with displacement ventilation. The DPV at one of two desks...... was equipped with an activated carbon filter installed at the air intake, while the DPV at the second desk was without such a filter. The air temperature in the occupied zone (1.1 m above the floor) was 29 °C. The pollution load in the room was simulated by PVC floor covering. The subjects assessed...

  7. Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance

    Directory of Open Access Journals (Sweden)

    Shimpei Morimoto

    2018-03-01

    Full Text Available Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes (ADC17 and KIN1 that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after

  8. Death of a dogma: eukaryotic mRNAs can code for more than one protein.

    Science.gov (United States)

    Mouilleron, Hélène; Delcourt, Vivian; Roucou, Xavier

    2016-01-08

    mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5' UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3' UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Immunoglobulin deposits in peripheral nerve endings detected by skin biopsy in patients with IgM M proteins and neuropathy

    DEFF Research Database (Denmark)

    Jønsson, V; Jensen, T S; Friis, M L

    1987-01-01

    biopsies provide a simple effective method of detecting immunoglobulin binding to peripheral nerves in patients suspected of having an autoimmune neuropathy. In contrast to sural nerve biopsy, skin biopsy does not cause sensory loss or pain in a denervated area and can easily be repeated.......Immunofluorescence studies of sural nerve and skin biopsies from three patients with IgM M proteins and clinical neuropathy showed that IgM M protein was bound to the nerve myelin in two patients and by the peri- and endoneurium in one. It is suggested that immunohistochemical studies of skin...

  10. Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro.

    Science.gov (United States)

    Arcangeletti, Maria-Cristina; Rodighiero, Isabella; Mirandola, Prisco; De Conto, Flora; Covan, Silvia; Germini, Diego; Razin, Sergey; Dettori, Giuseppe; Chezzi, Carlo

    2011-01-01

    The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.

  11. Selective translational repression of truncated proteins from frameshift mutation-derived mRNAs in tumors.

    Directory of Open Access Journals (Sweden)

    Kwon Tae You

    2007-05-01

    Full Text Available Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs. Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD system. However, PTCs located within 50-55 nucleotides of the last exon-exon junction are not recognized by NMD (NMD-irrelevant, and some PTC-containing mRNAs can escape from the NMD system (NMD-escape. We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression.

  12. Being WISE. I. Validating stellar population models and M {sub *}/L ratios at 3.4 and 4.6 μm

    Energy Technology Data Exchange (ETDEWEB)

    Norris, Mark A.; Meidt, Sharon; Van de Ven, Glenn; Schinnerer, Eva; Groves, Brent; Querejeta, Miguel, E-mail: norris@mpia.de [Max Planck Institut für Astronomie, Königstuhl 17, D-69117, Heidelberg (Germany)

    2014-12-10

    Using data from the Wide-field Infrared Survey Explorer mission, we have measured near infra-red (NIR) photometry of a diverse sample of dust-free stellar systems (globular clusters, dwarf and giant early-type galaxies) which have metallicities that span the range -2.2 < [Fe/H] (dex) < 0.3. This dramatically increases the sample size and broadens the metallicity regime over which the 3.4 (W1) and 4.6 μm (W2) photometry of stellar populations have been examined. We find that the W1 – W2 colors of intermediate and old (>2 Gyr) stellar populations are insensitive to the age of the stellar population, but that the W1 – W2 colors become bluer with increasing metallicity, a trend not well reproduced by most stellar population synthesis (SPS) models. In common with previous studies, we attribute this behavior to the increasing strength of the CO absorption feature located in the 4.6 μm bandpass with metallicity. Having used our sample to validate the efficacy of some of the SPS models, we use these models to derive stellar mass-to-light ratios in the W1 and W2 bands. Utilizing observational data from the SAURON and ATLAS3D surveys, we demonstrate that these bands provide extremely simple, yet robust stellar mass tracers for dust free older stellar populations that are freed from many of the uncertainties common among optical estimators.

  13. ALS Associated Mutations in Matrin 3 Alter Protein-Protein Interactions and Impede mRNA Nuclear Export.

    Science.gov (United States)

    Boehringer, Ashley; Garcia-Mansfield, Krystine; Singh, Gurkaran; Bakkar, Nadine; Pirrotte, Patrick; Bowser, Robert

    2017-11-06

    Mutations in Matrin 3 have recently been linked to ALS, though the mechanism that induces disease in these patients is unknown. To define the protein interactome of wild-type and ALS-linked MATR3 mutations, we performed immunoprecipitation followed by mass spectrometry using NSC-34 cells expressing human wild-type or mutant Matrin 3. Gene ontology analysis identified a novel role for Matrin 3 in mRNA transport centered on proteins in the TRanscription and EXport (TREX) complex, known to function in mRNA biogenesis and nuclear export. ALS-linked mutations in Matrin 3 led to its re-distribution within the nucleus, decreased co-localization with endogenous Matrin 3 and increased co-localization with specific TREX components. Expression of disease-causing Matrin 3 mutations led to nuclear mRNA export defects of both global mRNA and more specifically the mRNA of TDP-43 and FUS. Our findings identify a potential pathogenic mechanism attributable to MATR3 mutations and further link cellular transport defects to ALS.

  14. Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis.

    Science.gov (United States)

    Cai, Hui; Zhang, Yu; Lu, Mijia; Liang, Xueya; Jennings, Ryan; Niewiesk, Stefan; Li, Jianrong

    2016-08-15

    Human metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis in vivo The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus

  15. hCLP46 regulates U937 cell proliferation via Notch signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Wenzhan; Du, Jie; Chu, Qiaoyun [College of Life Science, Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Youxin [School of Public Health and Family Medicine, Capital Medical University, Beijing 100069 (China); Liu, Lixin [College of Life Science, Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Song, Manshu [School of Public Health and Family Medicine, Capital Medical University, Beijing 100069 (China); Wang, Wei, E-mail: wei6014@yahoo.com [College of Life Science, Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); School of Public Health and Family Medicine, Capital Medical University, Beijing 100069 (China)

    2011-04-29

    Highlights: {yields} Knock down of hCLP46 by RNAi impairs mammalian Notch signaling. {yields} hCLP46 affects neither cell surface Notch1 expression nor ligand-receptor binding. {yields} Knock down of hCLP46 inhibits U937 cell-growth by up-regulation of CDKN1B. -- Abstract: Human CAP10-like protein 46 kDa (hCLP46) is the homolog of Rumi, which is the first identified protein O-glucosyltransferase that modifies Notch receptor in Drosophila. Dysregulation of hCLP46 occurs in many hematologic diseases, but the role of hCLP46 remains unclear. Knockdown of hCLP46 by RNA interference resulted in decreased protein levels of endogenous Notch1, Notch intracellular domain (NICD) and Notch target gene Hes-1, suggesting the impairment of the Notch signaling. However, neither cell surface Notch expression nor ligand binding activities were affected. In addition, down-regulated expression of hCLP46 inhibited the proliferation of U937 cells, which was correlated with increased cyclin-dependent kinase inhibitor (CDKI) CDKN1B (p27) and decreased phosphorylation of retinoblastoma (RB) protein. We showed that lack of hCLP46 results in impaired ligand induced Notch activation in mammalian cell, and hCLP46 regulates the proliferation of U937 cell through CDKI-RB signaling pathway, which may be important for the pathogenesis of leukemia.

  16. Uncovering genes with divergent mRNA-protein dynamics in Streptomyces coelicolor.

    Directory of Open Access Journals (Sweden)

    Karthik P Jayapal

    2008-05-01

    Full Text Available Many biological processes are intrinsically dynamic, incurring profound changes at both molecular and physiological levels. Systems analyses of such processes incorporating large-scale transcriptome or proteome profiling can be quite revealing. Although consistency between mRNA and proteins is often implicitly assumed in many studies, examples of divergent trends are frequently observed. Here, we present a comparative transcriptome and proteome analysis of growth and stationary phase adaptation in Streptomyces coelicolor, taking the time-dynamics of process into consideration. These processes are of immense interest in microbiology as they pertain to the physiological transformations eliciting biosynthesis of many naturally occurring therapeutic agents. A shotgun proteomics approach based on mass spectrometric analysis of isobaric stable isotope labeled peptides (iTRAQ enabled identification and rapid quantification of approximately 14% of the theoretical proteome of S. coelicolor. Independent principal component analyses of this and DNA microarray-derived transcriptome data revealed that the prominent patterns in both protein and mRNA domains are surprisingly well correlated. Despite this overall correlation, by employing a systematic concordance analysis, we estimated that over 30% of the analyzed genes likely exhibited significantly divergent patterns, of which nearly one-third displayed even opposing trends. Integrating this data with biological information, we discovered that certain groups of functionally related genes exhibit mRNA-protein discordance in a similar fashion. Our observations suggest that differences between mRNA and protein synthesis/degradation mechanisms are prominent in microbes while reaffirming the plausibility of such mechanisms acting in a concerted fashion at a protein complex or sub-pathway level.

  17. Visual analysis and Schmidt rebound hammer test of Taj-ul-Masajid

    Directory of Open Access Journals (Sweden)

    A. Hussain

    2017-09-01

    Full Text Available Taj-ul-Masajid, literally, the crown among mosques is an embodiment of genius structural engineering located in the heart of Madhya Pradesh, Bhopal. A unique combination of the Mughal Architecture in complete stone masonry and modern day RCC work, it is a liaison between the past and the present of structural engineering. A wonder in its own right, the structure is often neglected by technicians and conservationalists alike, a satire on their ingenuity. Now, in a severely dilapidated condition, the structure is in pressing need of structural rehabilitation. The authors intend to perform in-situ Non-Destructive Testing & Evaluation (NDT&E of this structure and thereby suggest steps to better its present condition. As a first step, they’ve performed the visual analysis and Schmidt Rebound Hammer Test on the concrete portion of the structure which has been presented herein. The authors have also suggested a new approach for the verification of results obtained.

  18. Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity.

    Science.gov (United States)

    Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

    2015-09-01

    Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe(2+) chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods.

  19. Electrochemical detection of C-reactive protein using Copper nanoparticles and hybridization chain reaction amplifying signal.

    Science.gov (United States)

    Zhang, Junjun; Zhang, Wenjuan; Guo, Jinjin; Wang, Junchun; Zhang, Yuzhong

    2017-12-15

    In this study, a sandwich-type electrochemical immunosensor for the detection of C-reactive protein (CRP) is described. In design, Copper nanoparticles (Cu NPs) were used for signal tag and hybridization chain reaction (HCR)amplified output signal. The immunosensor fabrication involved three steps: (i) primary antibodies (Ab 1 ) were immobilized on the surface of gold nanoparticles (Au NPs); (ii) the sandwich-type structure formation contained "primary antibodies-antigen-secondary antibodies conjugated with primer (Ab 2 -S 0 )"; and (iii) long DNA concatemers intercalating amounts of Cu NPs was linked to the sandwich-type structure via hybridization reaction. Differential pulse voltammetry (DPV) was used to record the response signal of the immunosensor in phosphate-buffered saline (PBS). Under optimal conditions, the anodic peak currents of Cu NPs at the peak potential of about 0.08V(VS.SCE) were linear with the logarithm of CRP concentration in the range of 1.0 fg mL -1 to 100 ng mL -1 with a detection limit of 0.33 fg mL -1 (at signal/noise [S/N] = 3). In addition, the practical application of immunosensor was evaluated by analyzing CRP in real human serum samples, the recoveries obtained were within 95.3%-103.8%, indicating the immunosensor possessed potential application ability for practical disease diagnosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

    Science.gov (United States)

    Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo; Saito, Hirohide

    2017-07-07

    Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Endosulfan induced alteration in bacterial protein profile and RNA yield of Klebsiella sp. M3, Achromobacter sp. M6, and Rhodococcus sp. M2.

    Science.gov (United States)

    Singh, Madhu; Singh, Dileep Kumar

    2014-01-30

    Three bacterial strains identified as Klebsiella sp. M3, Achromobacter sp. M6 and Rhodococcus sp. M2 were isolated by soil enrichment with endosulfan followed by shake flask enrichment technique. They were efficiently degrading endosulfan in the NSM (non sulfur medium) broth. Degradation of endosulfan was faster with the cell free extract of bacterial cells grown in the sulfur deficient medium (NSM) supplemented with endosulfan than that of nutrient rich medium (Luria Bertani). In the cell free extract of NSM supplemented with endosulfan as sole sulfur source, a unique band was visualized on SDS-PAGE but not with magnesium sulfate as the sole sulfur source in NSM and LB with endosulfan. Expression of a unique polypeptide band was speculated to be induced by endosulfan under sulfur starved condition. These unique polypeptide bands were identified as OmpK35 protein, sulfate binding protein and outer membrane porin protein, respectively, in Klebsiella sp. M3, Achromobacter sp. M6 and Rhodococcus sp. M2. Endosulfan showed dose dependent negative effect on total RNA yield of bacterial strains in nutrient rich medium. Absence of plasmid DNA indicated the presence of endosulfan metabolizing gene on genomic DNA. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Protein deposition in pigs as influenced by sex, type andlivemass. 1 ...

    African Journals Online (AJOL)

    protein, 1% lysine and 13,46 MJ DE/kg feed on an 'as is' basis ..... birth to 136kg with two planes of nutrition from 53 to 136 kilograms ... teenoor droe voeding op die spekvark. M.Sc. (Agric.) tesis. SIEBRITS, F.K., KEMM, E.H. & RAS, M.N., 1981.

  3. Comparison of GLUT1, GLUT3, and GLUT4 mRNA and the subcellular distribution of their proteins in normal human muscle

    Science.gov (United States)

    Stuart, C. A.; Wen, G.; Gustafson, W. C.; Thompson, E. A.

    2000-01-01

    Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

  4. Nanostructured Modified Electrode for Electrocatalytic Determination of Epinephrine in the Presence of Acetaminophen

    Directory of Open Access Journals (Sweden)

    M. Mazloum-Ardakani

    2011-04-01

    Full Text Available In this paper, a nanostructured modified electrode was fabricated by incorporating of 2,2′-[1,9-nonanediylbis(nitriloethylidyne]-bis-hydroquinone (NNH as a newly synthesized modifier and TiO2 nanoparticles to the carbon paste (MTCPE and then was used for the electroanalysis of epinephrine (EP. The electrochemical studies were carried out by using cyclic voltammetry, chronoamperometry and differential pulse voltammetry (DPV techniques. It has been found that the oxidation of EP at the surface of this electrode occurs at a potential about 235 mV less positive than that of an unmodified carbon paste electrode. A dynamic range of 1.0–2000.0 μM, with a detection limit of 0.37 μM for EP, was obtained using DPV. Also, this modified electrode exhibits well separated oxidation peaks for EP and acetaminophen (AC using DPV.

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

    NARCIS (Netherlands)

    Stopar, D.

    1997-01-01

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

  6. Capacity Enhancement for Hybrid Fiber-Wireless Channels with 46.8Gbit/sWireless Multi-CAP Transmission over 50m at W-Band

    DEFF Research Database (Denmark)

    Rommel, Simon; Puerta Ramírez, Rafael; Vegas Olmos, Juan José

    2017-01-01

    Transmission of a 46.8 Gbit/s multi-band CAP signal is experimentally demonstrated over a 50 m W-band radio-over-fiber link. Bit error rates below 3.8×10-3 are achieved, employing nine CAP bands with bit and power loading.......Transmission of a 46.8 Gbit/s multi-band CAP signal is experimentally demonstrated over a 50 m W-band radio-over-fiber link. Bit error rates below 3.8×10-3 are achieved, employing nine CAP bands with bit and power loading....

  7. Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

    Directory of Open Access Journals (Sweden)

    Yuping Ren

    2017-12-01

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

  8. Improvement of reduced serum cartilage oligomeric matrix protein levels in systemic juvenile idiopathic arthritis patients treated with the anti-interleukin-6 receptor monoclonal antibody tocilizumab.

    Science.gov (United States)

    Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

    2009-01-01

    In this study, we determined serum cartilage oligomeric matrix protein (COMP) levels in systemic juvenile idiopathic arthritis (sJIA) patients during both the active and the remission phases to investigate how the growth cartilage turnover changed under tocilizumab treatment. Specimens were collected from 201 healthy children under 16 years of age with no growth impairment, and paired sera were collected from 11 sJIA patients treated with tocilizumab. Disease activity was assessed from white blood cell count, erythrocyte sedimentation rate, C-reactive protein, and ferritin, and the COMP concentration was determined by sandwich enzyme-linked immunosorbent assay. Serum COMP concentrations were found independent of age, and the mean value in healthy children was 17.74+/-5.6 U/L. The mean serum COMP in sJIA patients during the active phase was 10.75+/-3.9 U/L, lower than that of healthy children. The mean serum COMP in the remission phase (14.89+/-3.9 U/L) was significantly higher than that in the active period (P<0.05). These results suggested that in sJIA patients, a reduced serum COMP concentration is a useful marker of active disease and growth impairment, and that the growth cartilage turnover suppressed during the active phase is improved in the remission phase under tocilizumab treatment.

  9. Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis.

    Science.gov (United States)

    Tudor, Jennifer C; Davis, Emily J; Peixoto, Lucia; Wimmer, Mathieu E; van Tilborg, Erik; Park, Alan J; Poplawski, Shane G; Chung, Caroline W; Havekes, Robbert; Huang, Jiayan; Gatti, Evelina; Pierre, Philippe; Abel, Ted

    2016-04-26

    Sleep deprivation is a public health epidemic that causes wide-ranging deleterious consequences, including impaired memory and cognition. Protein synthesis in hippocampal neurons promotes memory and cognition. The kinase complex mammalian target of rapamycin complex 1 (mTORC1) stimulates protein synthesis by phosphorylating and inhibiting the eukaryotic translation initiation factor 4E-binding protein 2 (4EBP2). We investigated the involvement of the mTORC1-4EBP2 axis in the molecular mechanisms mediating the cognitive deficits caused by sleep deprivation in mice. Using an in vivo protein translation assay, we found that loss of sleep impaired protein synthesis in the hippocampus. Five hours of sleep loss attenuated both mTORC1-mediated phosphorylation of 4EBP2 and the interaction between eukaryotic initiation factor 4E (eIF4E) and eIF4G in the hippocampi of sleep-deprived mice. Increasing the abundance of 4EBP2 in hippocampal excitatory neurons before sleep deprivation increased the abundance of phosphorylated 4EBP2, restored the amount of eIF4E-eIF4G interaction and hippocampal protein synthesis to that seen in mice that were not sleep-deprived, and prevented the hippocampus-dependent memory deficits associated with sleep loss. These findings collectively demonstrate that 4EBP2-regulated protein synthesis is a critical mediator of the memory deficits caused by sleep deprivation. Copyright © 2016, American Association for the Advancement of Science.

  10. Hidden photoinduced reactivity of the blue fluorescent protein mKalama1

    DEFF Research Database (Denmark)

    Vegh, Russell B.; Bloch, Dmitry A.; Bommarius, Andreas S.

    2015-01-01

    , is largely unexplored. Here, by using transient absorption spectroscopy spanning the time scale from picoseconds to seconds, we reveal a hidden reactivity of the bright blue-light emitting protein mKalama1 previously thought to be inert. This protein shows no excited-state proton transfer during its...

  11. A role for the membrane protein M6 in the Drosophila visual system.

    Science.gov (United States)

    Zappia, María Paula; Bernabo, Guillermo; Billi, Silvia C; Frasch, Alberto C; Ceriani, María Fernanda; Brocco, Marcela Adriana

    2012-07-04

    Members of the proteolipid protein family, including the four-transmembrane glycoprotein M6a, are involved in neuronal plasticity in mammals. Results from our group previously demonstrated that M6, the only proteolipid protein expressed in Drosophila, localizes to the cell membrane in follicle cells. M6 loss triggers female sterility, which suggests a role for M6 in follicular cell remodeling. These results were the basis of the present study, which focused on the function and requirements of M6 in the fly nervous system. The present study identified two novel, tissue-regulated M6 isoforms with variable N- and C- termini, and showed that M6 is the functional fly ortholog of Gpm6a. In the adult brain, the protein was localized to several neuropils, such as the optic lobe, the central complex, and the mushroom bodies. Interestingly, although reduced M6 levels triggered a mild rough-eye phenotype, hypomorphic M6 mutants exhibited a defective response to light. Based on its ability to induce filopodium formation we propose that M6 is key in cell remodeling processes underlying visual system function. These results bring further insight into the role of M6/M6a in biological processes involving neuronal plasticity and behavior in flies and mammals.

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

    NARCIS (Netherlands)

    Sanders, J.C.

    1992-01-01

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

  13. Acute guttate psoriasis patients have positive streptococcus hemolyticus throat cultures and elevated antistreptococcal M6 protein titers.

    Science.gov (United States)

    Zhao, Guang; Feng, Xiaoling; Na, Aihua; Yongqiang, Jiang; Cai, Qing; Kong, Jian; Ma, Huijun

    2005-02-01

    To further study the role of Streptococci hemolyticus infection and streptococcal M6 protein in the pathogenesis of acute guttate psoriasis, streptococcal cultures were taken from the throats of 68 patients with acute guttate psoriasis. PCR technique was applied to detect M6 protein encoding DNA from those cultured streptococci. Pure M6 protein was obtained by Sephacry/S-200HR and Mono-Q chromatography from proliferated Streptococcus hemolyticus. Antistreptococcal M6 protein titers were measured in the serum of patients with acute guttate psoriasis, plaque psoriasis and healthy controls by ELISA. A high incidence of Streptococcus hemolyticus culture was observed in the guttate psoriatic group compared with the plaque psoriasis and control groups. Fourteen strains of Streptococcus hemolyticus were cultured from the throats of 68 acute guttate psoriasis patients. Of these, 5 strains contain DNA encoding the M6 protein gene as confirmed by PCR technique. More than 85% purification of M6 protein was obtained from Streptococcus pyogenes. Applying our pure M6 protein with the ELISA methods, we found that the titer of antistreptococcal M6 protein was significantly higher in the serum of guttate psoriasis patients than in the control or plaque psoriasis groups (P M6 protein in their sera.

  14. Mutation in WDR4 impairs tRNA m(7)G46 methylation and causes a distinct form of microcephalic primordial dwarfism.

    Science.gov (United States)

    Shaheen, Ranad; Abdel-Salam, Ghada M H; Guy, Michael P; Alomar, Rana; Abdel-Hamid, Mohamed S; Afifi, Hanan H; Ismail, Samira I; Emam, Bayoumi A; Phizicky, Eric M; Alkuraya, Fowzan S

    2015-09-28

    Primordial dwarfism is a state of extreme prenatal and postnatal growth deficiency, and is characterized by marked clinical and genetic heterogeneity. Two presumably unrelated consanguineous families presented with an apparently novel form of primordial dwarfism in which severe growth deficiency is accompanied by distinct facial dysmorphism, brain malformation (microcephaly, agenesis of corpus callosum, and simplified gyration), and severe encephalopathy with seizures. Combined autozygome/exome analysis revealed a novel missense mutation in WDR4 as the likely causal variant. WDR4 is the human ortholog of the yeast Trm82, an essential component of the Trm8/Trm82 holoenzyme that effects a highly conserved and specific (m(7)G46) methylation of tRNA. The human mutation and the corresponding yeast mutation result in a significant reduction of m(7)G46 methylation of specific tRNA species, which provides a potential mechanism for primordial dwarfism associated with this lesion, since reduced m(7)G46 modification causes a growth deficiency phenotype in yeast. Our study expands the number of biological pathways underlying primordial dwarfism and adds to a growing list of human diseases linked to abnormal tRNA modification.

  15. The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

    International Nuclear Information System (INIS)

    Zhang, Yangli; Gao, Zengqiang; Guo, Zhen; Zhang, Hongpeng; Zhang, Zhenzhen; Luo, Miao; Hou, Haifeng; Huang, Ailong; Dong, Yuhui; Wang, Deqiang

    2013-01-01

    Highlights: •We determined the first structure of human α1M with heavy electron density of the chromophore. •We proposed a new structural model of the chromophore. •We first revealed that the two conserved surface regions of α1M are proposed as putative protein–protein interface sites. -- Abstract: Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners

  16. Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation.

    Science.gov (United States)

    Suryawan, Agus; Jeyapalan, Asumthia S; Orellana, Renan A; Wilson, Fiona A; Nguyen, Hanh V; Davis, Teresa A

    2008-10-01

    Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.

  17. Structural and Biochemical Studies of LysM Proteins

    DEFF Research Database (Denmark)

    Wong, Mei Mei Jaslyn Elizabeth

    2017-01-01

    . Most of the signalling components in the Nod factor signalling pathway have been identified through genetic approaches. The current symbiosis signalling model, however, lacks components that could link Nod factor perception at the plasma membrane to downstream responses, such as calcium influx and perinuclear calcium...... involved in peptidoglycan hydrolysis; the Cell Wall Lytic enzyme associated with cell Separation (CwlS) from Bacillus subtilis, and P60_Tth from Thermus thermopiles. Biochemical studies conducted on purified CwlS showed that multiple LysM modules function cooperatively to bind N-acetylglucosamine (NAG......-induced intermolecular dimerization was observed in the co-crystal structure of P60_2LysM and NAG6. Until today, this is the only structural evidence illustrating intermolecular dimerization of LysM proteins. Intermolecular dimerization of plant LysM receptor kinases (RK) has been proposed as a mechanism...

  18. Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

    Science.gov (United States)

    Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

    2017-08-01

    Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

  19. Identification of novel RNA viruses in alfalfa (Medicago sativa): an Alphapartitivirus, a Deltapartitivirus, and a Marafivirus.

    Science.gov (United States)

    Kim, Hyein; Park, Dongbin; Hahn, Yoonsoo

    2018-01-05

    Genomic RNA molecules of plant RNA viruses are often co-isolated with the host RNAs, and their sequences can be detected in plant transcriptome datasets. Here, an alfalfa (Medicago sativa) transcriptome dataset was analyzed and three new RNA viruses were identified, which were named Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa deltapartitivirus 1 (MsDPV1), and Medicago sativa marafivirus 1 (MsMV1). The RNA-dependent RNA polymerases of MsAPV1, MsDPV1, and MsMV1 showed about 68%, 58%, and 46% amino acid sequence identity, respectively, with their closest virus species. Sequence similarity and phylogenetic analyses indicated that MsAPV1, MsDPV1, and MsMV1 were novel RNA virus species that belong to the genus Alphapartitivirus of the family Partitiviridae, the genus Deltapartitivirus of the family Partitiviridae, and the genus Marafivirus of the family Tymoviridae, respectively. The bioinformatics procedure applied in this study may facilitate the identification of novel RNA viruses from plant transcriptome data. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

    DEFF Research Database (Denmark)

    Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

    2008-01-01

    AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

  1. Reassessment of the relationship between M-protein decrement and survival in multiple myeloma.

    Science.gov (United States)

    Palmer, M; Belch, A; Hanson, J; Brox, L

    1989-01-01

    The relationship between percentage M-protein decrement and survival is assessed in 134 multiple myeloma patients. The correlation did not achieve statistical significance (P = 0.069). Multivariate analysis using the Cox proportional hazards model, including a number of previously recognised prognostic factors, showed only percentage M-protein decrement, creatinine and haemoglobin to be significantly correlated with survival. However, the R'-statistic for each of these variables was low, indicating that their prognostic power is weak. We conclude that neither the percentage M-protein decrement nor the response derived from it can be used as an accurate means of assessing the efficacy of treatment in myeloma. Mature survival data alone should be used for this purpose.

  2. Reassessment of the relationship between M-protein decrement and survival in multiple myeloma.

    Science.gov (United States)

    Palmer, M.; Belch, A.; Hanson, J.; Brox, L.

    1989-01-01

    The relationship between percentage M-protein decrement and survival is assessed in 134 multiple myeloma patients. The correlation did not achieve statistical significance (P = 0.069). Multivariate analysis using the Cox proportional hazards model, including a number of previously recognised prognostic factors, showed only percentage M-protein decrement, creatinine and haemoglobin to be significantly correlated with survival. However, the R'-statistic for each of these variables was low, indicating that their prognostic power is weak. We conclude that neither the percentage M-protein decrement nor the response derived from it can be used as an accurate means of assessing the efficacy of treatment in myeloma. Mature survival data alone should be used for this purpose. PMID:2757916

  3. Mechanism of mRNA-STAR domain interaction: Molecular dynamics simulations of Mammalian Quaking STAR protein.

    Science.gov (United States)

    Sharma, Monika; Anirudh, C R

    2017-10-03

    STAR proteins are evolutionary conserved mRNA-binding proteins that post-transcriptionally regulate gene expression at all stages of RNA metabolism. These proteins possess conserved STAR domain that recognizes identical RNA regulatory elements as YUAAY. Recently reported crystal structures show that STAR domain is composed of N-terminal QUA1, K-homology domain (KH) and C-terminal QUA2, and mRNA binding is mediated by KH-QUA2 domain. Here, we present simulation studies done to investigate binding of mRNA to STAR protein, mammalian Quaking protein (QKI). We carried out conventional MD simulations of STAR domain in presence and absence of mRNA, and studied the impact of mRNA on the stability, dynamics and underlying allosteric mechanism of STAR domain. Our unbiased simulations results show that presence of mRNA stabilizes the overall STAR domain by reducing the structural deviations, correlating the 'within-domain' motions, and maintaining the native contacts information. Absence of mRNA not only influenced the essential modes of motion of STAR domain, but also affected the connectivity of networks within STAR domain. We further explored the dissociation of mRNA from STAR domain using umbrella sampling simulations, and the results suggest that mRNA binding to STAR domain occurs in multi-step: first conformational selection of mRNA backbone conformations, followed by induced fit mechanism as nucleobases interact with STAR domain.

  4. Immunoglobulin M and G antibody responses to Plasmodium falciparum glutamate-rich protein

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Rowe, P; Bennett, S

    1993-01-01

    were measured with a recombinant fusion protein consisting of the carboxy-terminal 783 amino acids of the GLURP. Samples for the study were obtained during a longitudinal malaria morbidity survey performed in The Gambia; cross-sectional surveys were performed at the beginning of the transmission season......The aims of the present study were to describe the age-related immunoglobulin M (IgM) and IgG response to part of a 220-kDa glutamate-rich protein (GLURP) from Plasmodium falciparum and to determine possible correlations of possession of these antibodies with malaria morbidity. IgM and IgG levels...

  5. PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis.

    Directory of Open Access Journals (Sweden)

    Kathrin Thedieck

    Full Text Available TOR (Target of Rapamycin is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1 and TOR complex 2 (TORC2. In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS based proteomic strategy to identify new mammalian TOR (mTOR binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40 and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5 and was therefore named PRR5-Like (PRR5L. PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1 and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

  6. Autoimmunity related to IgM monoclonal gammopathy of undetermined significance. Peripheral neuropathy and connective tissue sensibilization caused by IgM M-proteins

    DEFF Research Database (Denmark)

    Jønsson, V; Schrøder, H D; Nolsøe, C

    1988-01-01

    of them, including two siblings with a demyelinating peripheral neuropathy, the IgM was bound to the myelin-associated glycoprotein (MAG) of peripheral nerves. One had axonal neuropathy with IgM activity against the peri- and endoneurium, while another case with post-infectious neuritis had IgM activity......In eight of 10 consecutive cases of IgM monoclonal gammopathy of undetermined significance (MGUS), the M-protein had specificity towards various tissues as estimated by direct and indirect immunofluorescence studies of skin and/or sural nerve biopsies. Five of the cases had neuropathy. In three...

  7. Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

    Science.gov (United States)

    Clemens, Michael J; Elia, Androulla; Morley, Simon J

    2013-01-01

    The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

  8. m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application.

    OpenAIRE

    Zor, T; Halifa, I; Kleinhaus, S; Chorev, M; Selinger, Z

    1995-01-01

    A novel photoaffinity label, m-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In the presence of the beta-adrenergic agonist isoprenaline, it activates turkey erythrocyte adenylate cyclase. This activation persists even when the beta-adrenergic receptor is subsequently blocked by antagonist, indicating that ...

  9. Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

    Science.gov (United States)

    Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

    2016-06-01

    Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Czech Academy of Sciences Publication Activity Database

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

    2012-01-01

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

  11. Biochemical and biological properties of the binding of human fibrinogen to M protein in group A streptococci

    International Nuclear Information System (INIS)

    Whitnack, E.; Beachey, E.H.

    1985-01-01

    Fibrinogen is known to bind to group A streptococci and precipitate with extracts containing streptococcal M protein. The authors have previously shown that the binding of fibrinogen to M-positive streptococci prevents opsonization by complement and protects that organism from phagocytosis in nonimmune blood. In the present study, they used 3 H-labeled fibrinogen, a highly purified peptide fragment of type 24 M protein (pep M24), and anti-pep M sera to show that fibrinogen binds to M-positive streptococci with high affinity; occupation of the high-affinity binding sites suffices to protect the organism from phagocytosis; proteolytic treatments that remove M protein from streptococcal cells abolish binding; binding is competitively inhibited by anti-pep M sera; pep M24 precipitates fibrinogen; and binding to type 24 cells is inhibited by pep M24. They conclude that M protein is the cell surface structure principally responsible for binding fibrinogen on the surface of M-positive streptococci and that this binding contributes to the known antiopsonic property of M proteins

  12. Heterogeneous nuclear ribonuclear protein K interacts with Sindbis virus nonstructural proteins and viral subgenomic mRNA

    International Nuclear Information System (INIS)

    Burnham, Andrew J.; Gong, Lei; Hardy, Richard W.

    2007-01-01

    Alphaviruses are a group of arthropod-borne human and animal pathogens that can cause epidemics of significant public health and economic consequence. Alphavirus RNA synthesis requires four virally encoded nonstructural proteins and probably a number of cellular proteins. Using comparative two-dimensional electrophoresis we were able to identify proteins enriched in cytoplasmic membrane fractions containing viral RNA synthetic complexes following infection with Sindbis virus. Our studies demonstrated the following: (i) the host protein hnRNP K is enriched in cytoplasmic membrane fractions following Sindbis virus infection, (ii) viral nonstructural proteins co-immunoprecipitate with hnRNP K, (iii) nsP2 and hnRNP K co-localize in the cytoplasm of Sindbis virus infected cells, (iv) Sindbis virus subgenomic mRNA, but not genomic RNA co-immunoprecipitates with hnRNP K, (v) viral RNA does not appear to be required for the interaction of hnRNP K with the nonstructural proteins. Potential functions of hnRNP K during virus replication are discussed

  13. Resource Allocation and Interference Management for D2D-Enabled DL/UL Decoupled Het-Nets

    KAUST Repository

    Celik, Abdulkadir

    2017-10-06

    In this paper, resource allocation and interference mitigation are investigated for heterogeneous networks (HetNets) where the lowest tier consists of device-to-device (D2D) cells. In order to alleviate dead-zone problem, we first consider downlink/ uplink (DL/UL) decoupling (DUDe) user association and quantify its capability on interference management and networkwide D2D performance enhancement. Secondly, we propose an UL fractional frequency reuse (FFR) scheme where subband (SB) bandwidths are adaptively determined based on: i) user equipment (UE) density, ii) e-node-B (eNB) density, and iii) ON/OFF switching frequency of smallcells. Obtained results show that the adaptive method significantly reduces the number of outage users. Thereafter, a novel concatenated bi-partite matching (CBM) method is proposed for joint SB assignment (SA) and resource block allocation (RA) of cellular UEs (CUEs). Numerical results show that the CBM provides a close performance to exhaustive solution with greatly reduced running time. The CBM is then extended to a centralized mode selection, SA, and RA for D2D cells. Alternatively, we develop offline and online semidistributed approaches where a D2D-cell can reuse white-list RBs (WRBs), which are not occupied by the adjacent smallcells. In the former, D2D-cell members are not aware of intra-cell and inter-cell interference and uniformly distribute their maximum permissible power to WRBs. In the latter, we put D2D sumrate maximization into a convex form by exploiting the proximity gain of D2D UEs (DUEs). Online distributed solution is then developed by message passing of dual variables and consistency prices. Finally, virtues and drawbacks of the developed approaches are compared and explained.

  14. Resource Allocation and Interference Management for D2D-Enabled DL/UL Decoupled Het-Nets

    KAUST Repository

    Celik, Abdulkadir; Radaydeh, Redha Mahmoud Mesleh; Al-Qahtani, Fawaz S.; Alouini, Mohamed-Slim

    2017-01-01

    In this paper, resource allocation and interference mitigation are investigated for heterogeneous networks (HetNets) where the lowest tier consists of device-to-device (D2D) cells. In order to alleviate dead-zone problem, we first consider downlink/ uplink (DL/UL) decoupling (DUDe) user association and quantify its capability on interference management and networkwide D2D performance enhancement. Secondly, we propose an UL fractional frequency reuse (FFR) scheme where subband (SB) bandwidths are adaptively determined based on: i) user equipment (UE) density, ii) e-node-B (eNB) density, and iii) ON/OFF switching frequency of smallcells. Obtained results show that the adaptive method significantly reduces the number of outage users. Thereafter, a novel concatenated bi-partite matching (CBM) method is proposed for joint SB assignment (SA) and resource block allocation (RA) of cellular UEs (CUEs). Numerical results show that the CBM provides a close performance to exhaustive solution with greatly reduced running time. The CBM is then extended to a centralized mode selection, SA, and RA for D2D cells. Alternatively, we develop offline and online semidistributed approaches where a D2D-cell can reuse white-list RBs (WRBs), which are not occupied by the adjacent smallcells. In the former, D2D-cell members are not aware of intra-cell and inter-cell interference and uniformly distribute their maximum permissible power to WRBs. In the latter, we put D2D sumrate maximization into a convex form by exploiting the proximity gain of D2D UEs (DUEs). Online distributed solution is then developed by message passing of dual variables and consistency prices. Finally, virtues and drawbacks of the developed approaches are compared and explained.

  15. Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

    Energy Technology Data Exchange (ETDEWEB)

    Reimers, Kerstin [Klinik fuer Plastische, Hand-und Wiederherstellungschirurgie, Podbielskistrasse 380, D-30659 Hannover (Germany); Buchholz, Katja [Institut fuer Medizinische Mikrobiologie, Otto-von-Guericke-Universitaet Magdeburg, Leipzigerstrasse 44, D-39120 Magdeburg (Germany); Werchau, Hermann [Institut fuer Medizinische Mikrobiologie, Otto-von-Guericke-Universitaet Magdeburg, Leipzigerstrasse 44, D-39120 Magdeburg (Germany)

    2005-01-20

    Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cells revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.

  16. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein.

    Directory of Open Access Journals (Sweden)

    Anthony J Brzoska

    Full Text Available Actin-like proteins (Alps are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments.

  17. Clofibrate-induced increases in peroxisomal proteins: effect on synthesis, degradation, and mRNA activity

    International Nuclear Information System (INIS)

    Mortensen, R.M.

    1983-01-01

    The effect of clofibrate on the polypeptide composition of peroxisomes was determined. A simple method was developed for the isolation of peroxisomes with a purity of 90-95% using sedimentation in a metrizamide gradient. The specific activities of HD did not change with clofibrate treatment so that the increases in enzyme activities are solely due to increases in protein amounts. The hepatic concentration of HD increased 63 times. The HD synthesis rate, as measured by the incorporation of [ 3 H]leucine, increased 74 times, so that the increase in the synthesis was sufficient to account for the increase in protein. Clofibrate caused no discernible change in the degradation rate of HD labeled with [ 14 C]bicarbonate. The half-life of HD was approximately 2 days. The translatable mRBA coding for HD increased 55 times. This value is not significantly different from the increase in HD protein or in HD synthesis. This observation was also true for several other peroxisomal proteins. Therefore, clofibrate causes an increase in the mRNA activity, which increases the synthesis of HD leading to an accumulation of protein and enzyme activity. The kinetics of the clofibrate-induced changes in HD synthesis rate, protein level, and enzymatic activity was analyzed using a simple model which included the half-lives of the drug, mRNA, and protein. The best fit of the model to the data gave an mRNA half-life of 10 hours and a protein half-life of 1.8 days, with no significant change by clofibrate

  18. Human metapneumovirus M2-2 protein inhibits innate immune response in monocyte-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Junping Ren

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS. Whether M2-2 regulates the innate immunity in human dendritic cells (DC, an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2 produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT, suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88, an essential adaptor for Toll-like receptors (TLRs, plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.

  19. Characterization of structural proteins of hirame rhabdovirus, HRV

    Science.gov (United States)

    Nishizawa, Toyohiko; Yoshimizu, Mamoru; Winton, James; Ahne, Winfried; Kimura, Takahisa

    1991-01-01

    Structural proteins of hirame rhabdovirus (HRV) were analyzed by SDS-polyacrylarnide gel electrophoresis, western blotting, 2-dimensional gel electrophoresis, and Triton X-100 treatment. Purified HRV virions were composed of: polymerase (L), glycoprotein (G), nucleoprotein (N), and 2 matrix proteins (M1 and M2). Based upon their relative mobilities, the estimated molecular weights of the proteins were: L, 156 KDa; G, 68 KDa; N, 46.4 KDa; M1, 26.4 KDa; and M2, 19.9 KDa. The electrophorehc pattern formed by the structural proteins of HRV was clearly different from that formed by pike fry rhabdovirus, spring viremia of carp virus, eel virus of America, and eel virus European X which belong to the Vesiculovirus genus; however, it resembled the pattern formed by structural proteins of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) which are members of the Lyssavirus genus. Among HRV, IHNV, and VHSV, differences were observed in the relative mobilities of the G, N, M1, and M2 proteins. Western blot analysis revealed that the G. N, and M2 proteins of HRV shared antigenic determinants with IHNV and VHSV, but not with any of the 4 fish vesiculoviruses tested. Cross-reactions between the M1 proteins of HRV, IHNV, or VHSV were not detected in this assay. Two-dimensional gel electrophoresis was used to show that HRV differed from IHNV or VHSV in the isoelectric point (PI) of the M1 and M2 proteins. In this system, 2 forms of the M1 protein of HRV and IHNV were observed.These subspecies of M1 had the same relative mobility but different p1 values. Treatment of purified virions with 2% Triton X-100 in Tris buffer containing NaCl removed the G, M1, and M2 proteins of IHNV, but HRV virions were more stable under these conditions.

  20. Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

    OpenAIRE

    Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

    2015-01-01

    Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we sh...

  1. Functional motifs responsible for human metapneumovirus M2-2-mediated innate immune evasion.

    Science.gov (United States)

    Chen, Yu; Deng, Xiaoling; Deng, Junfang; Zhou, Jiehua; Ren, Yuping; Liu, Shengxuan; Prusak, Deborah J; Wood, Thomas G; Bao, Xiaoyong

    2016-12-01

    Human metapneumovirus (hMPV) is a major cause of lower respiratory infection in young children. Repeated infections occur throughout life, but its immune evasion mechanisms are largely unknown. We recently found that hMPV M2-2 protein elicits immune evasion by targeting mitochondrial antiviral-signaling protein (MAVS), an antiviral signaling molecule. However, the molecular mechanisms underlying such inhibition are not known. Our mutagenesis studies revealed that PDZ-binding motifs, 29-DEMI-32 and 39-KEALSDGI-46, located in an immune inhibitory region of M2-2, are responsible for M2-2-mediated immune evasion. We also found both motifs prevent TRAF5 and TRAF6, the MAVS downstream adaptors, to be recruited to MAVS, while the motif 39-KEALSDGI-46 also blocks TRAF3 migrating to MAVS. In parallel, these TRAFs are important in activating transcription factors NF-kB and/or IRF-3 by hMPV. Our findings collectively demonstrate that M2-2 uses its PDZ motifs to launch the hMPV immune evasion through blocking the interaction of MAVS and its downstream TRAFs. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-05-18

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

  3. mTORC1-independent reduction of retinal protein synthesis in type 1 diabetes.

    Science.gov (United States)

    Fort, Patrice E; Losiewicz, Mandy K; Pennathur, Subramaniam; Jefferson, Leonard S; Kimball, Scot R; Abcouwer, Steven F; Gardner, Thomas W

    2014-09-01

    Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2(Akita) diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  4. The murine gammaherpesvirus-68 chemokine-binding protein M3 inhibits experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Millward, Jason M; Holst, Peter J; Høgh-Petersen, Mette

    2010-01-01

    M3 (AdM3) directly to the CNS to evaluate the capacity of this protein to inhibit neuroinflammation using the experimental autoimmune encephalomyelitis (EAE) model. Treatment with the AdM3 vector significantly reduced the clinical severity of EAE, attenuated CNS histopathology, and reduced numbers......Chemokines are critical mediators of immune cell entry into the central nervous system (CNS), as occurs in neuroinflammatory disease such as multiple sclerosis. Chemokines are also implicated in the immune response to viral infections. Many viruses encode proteins that mimic or block chemokine...... of immune cells infiltrating the CNS. These results suggest that M3 may represent a novel therapeutic approach to neuroinflammatory disease....

  5. Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

    International Nuclear Information System (INIS)

    Wu, William Ka Kei; Volta, Viviana; Cho, Chi Hin; Wu, Ya Chun; Li, Hai Tao; Yu, Le; Li, Zhi Jie; Sung, Joseph Jao Yiu

    2009-01-01

    Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of 35 S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.

  6. Breast-milk radioactivity after a Tc-99m DTPA aerosol/Tc-99m MAA lung study

    International Nuclear Information System (INIS)

    Mountford, P.J.; Hall, F.M.; Wells, C.P.; Coakley, A.J.

    1984-01-01

    Measurements were made of the concentration of Tc-99m activity in samples of breast milk following an administration of Tc-99m DTPA aerosol for a lung ventilation image and one of Tc-99m MAA for lung perfusion. The activity was 222 nCi/ml of milk (8.2 kBq/ml) at 2 hr after the MAA injection, and it was found to be excreted exponentially with an effective half-life of 4.6 hr. There was a small incorporation of Tc-99m into breast-milk protein. The authors conclude that the combined use of these two Tc-99m agents did not indicate the interruption of breast feeding beyond 24 hr after administration of the MAA, and that for an aerosol ventiliation study alone, breast feeding need not be interrupted for more than 4 hr after the test

  7. Evaluation of the in vivo binding of 99mTc-MDP on serum proteins

    International Nuclear Information System (INIS)

    Freitas, R.S.F.; Mattos, D.M.M.; Gomes, M.L.; Moreno, S.R.F.; Lima, E.A.C.; Dire, G.F.; Aleixo, L.C.M.; Lima Filho, G.L.; Bernardo-Filho, M.

    2002-01-01

    Aim: In the clinical application of technetium-99m-radiopharmaceuticals (99mTc) these agents are injected into the blood stream and they may bind reversibly and irreversibly to the blood proteins. To evaluate the protein binding of the radiopharmaceutical, the protein-bound 99mTc-radiopharmaceuticals must be separated from the free 99mTc-radiopharmaceuticals complexes. This has been accomplished by precipitation of the proteins. In this study, we have compared the results obtained with the evaluation of the binding of the radiopharmaceutical Tc-99m methylenediphosphonic acid (99mTc-MDP) on serum proteins using precipitation methods with TCA and AS. Material and Methods: 99mTc-MDP was administrated in Wistar rats and after 10 minutes blood was withdrawn and serum (S) isolated. Aliquots of S were precipitated with 1 ml of TCA or AS at various concentrations (0.1, 0.5, 1.0, 5.0, 10.0 and 20.0%) and soluble (SF) and insoluble fractions (IF) were separated. The samples (IF-S and SF-S) were counted in a well counter with NaI(TI)crystal. The percent of the radioactivity (%ATI) was determined in each fraction. Results: The results obtained with the comparison of the in vivo binding of 99mTc-MDP when the precipitation was performed with TCA showed that the %ATI was only statistically different (ANOVA, p<0.05) to the 20% TCA concentration. When various concentrations of AS were used no differences in the %ATI were found and the results were similar to the obtained with TCA, except to the concentration of 20%(TCA - 51.70 ± 10.19 and SA - 10.17 ± 0.98 %). Conclusion: It is possible to suggest that the different concentrations of the precipitating agents (TCA and AS) act, in general, on the same binding sites of the evaluate radiopharmaceutical to the proteins of the serum, except to concentration of 20%

  8. SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G.

    Science.gov (United States)

    Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

    2017-01-01

    The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm -positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis , respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis . The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.

  9. Reassessment of the relationship between M-protein decrement and survival in multiple myeloma.

    OpenAIRE

    Palmer, M.; Belch, A.; Hanson, J.; Brox, L.

    1989-01-01

    The relationship between percentage M-protein decrement and survival is assessed in 134 multiple myeloma patients. The correlation did not achieve statistical significance (P = 0.069). Multivariate analysis using the Cox proportional hazards model, including a number of previously recognised prognostic factors, showed only percentage M-protein decrement, creatinine and haemoglobin to be significantly correlated with survival. However, the R'-statistic for each of these variables was low, indi...

  10. Mobile platform for fish migration upstream from the discharge sill situated near Dacia bridge on Crișul Repede River

    Directory of Open Access Journals (Sweden)

    Răzvan VOICU

    2016-06-01

    Full Text Available Longitudinal connectivity represent the way in which organisms move the energy and material exchanges located throughout the water. Fragmentation the longitudinal connectivity of watercourses caused by dams or other hydrotechnical constructions represent a major impact on sediment transport, hydrological regime, downstream moving and biota migration. The hydromorphological elements (river continuity, as well as chemical, biological, physicochemical elements characterize the ecological status of rivers. Migratory fish species: nase (Chondrostoma nasus - protected by Bern Convention - Appendix III, barbel (Barbus barbus - rare species, protected Habitats Directive (Annex V, annex 4A of Low nr.462 and Red List of RBDD and Freshwater bream (Abramis brama - protected by Bern Convention (Appendix III are blocked by the hydrotechnical constructions (discharge sills, dams located across the watercourse Crișul Repede River. One of the important think of this system is the gravitational fall of water. This solution will lead to the restoration of the longitudinal connection of the Crișul Repede River in the Oradea City, near Dacia Bridge. Romania is part of the European Union and it has the obligation to implement the provisions of the Water Framework Directive 2000/60/EC, transposed into Romanian legislation by the Water Law 107/1996 as supplemented and amended (Act 310/2004.

  11. Molecularly imprinted polymer based electrochemical detection of L-cysteine at carbon paste electrode.

    Science.gov (United States)

    Aswini, K K; Vinu Mohan, A M; Biju, V M

    2014-04-01

    A methacrylic acid (MAA) based molecularly imprinted polymer (MIP) modified carbon paste electrode (CPE) was developed for electrochemical detection of L-cysteine (Cys). Characterisation of MIP was done with FTIR and the modified electrode with cyclic voltammetry (CV) and differential pulse voltammetry (DPV). CV, DPV and impedance analysis demonstrated that the modified electrode is responsive towards the target molecule. The optimum percentage composition of MIP for MIP/CPE and the effect of pH towards the electrode response for Cys were studied. The detection of Cys in the range of 2×10(-8) to 18×10(-8)M at MIP/CPE was monitored by DPV with a limit of detection of 9.6nM and R(2) of 0.9974. Also, various physiological interferents such as ascorbic acid, L-tryptophan, D-glucose, D-cysteine and L-cysteine were found to have little effect on DPV response at MIP/CPE. The utility of the electrode was proved by the effective detection of Cys from tap water and human blood plasma samples with reproducible results. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

    Science.gov (United States)

    Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

    2011-11-01

    Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

  13. 99mTc-HYNIC-derivatized ternary ligand complexes for 99mTc-labeled polypeptides with low in vivo protein binding

    International Nuclear Information System (INIS)

    Ono, Masahiro; Arano, Yasushi; Mukai, Takahiro; Fujioka, Yasushi; Ogawa, Kazuma; Uehara, Tomoya; Saga, Tsuneo; Konishi, Junji; Saji, Hideo

    2001-01-01

    6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m ( 99m Tc)-labeled polypeptides with tricine as a coligand. However, 99m Tc-HYNIC-labeled polypeptides show delayed elimination rates of the radioactivity not only from the blood but also from nontarget tissues such as the liver and kidney. In this study, a preformed chelate of tetrafluorophenol (TFP) active ester of [ 99m Tc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare 99m Tc-labeled polypeptides with higher stability against exchange reactions with proteins in plasma and lysosomes using the Fab fragment of a monoclonal antibody and galactosyl-neoglycoalbumin (NGA) as model polypeptides. When incubated in plasma, [ 99m Tc](HYNIC-Fab)(tricine)(BP) showed significant reduction of the radioactivity in high molecular weight fractions compared with [ 99m Tc](HYNIC-Fab)(tricine) 2. When injected into mice, [ 99m Tc](HYNIC-NGA)(tricine)(BP) was metabolized to [ 99m Tc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivity detected in protein-bound fractions in contrast to the observations with [ 99m Tc](HYNIC-NGA)(tricine) 2. In addition, [ 99m Tc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [ 99m Tc](HYNIC-NGA)(tricine) 2. Similar results were observed with 99m Tc-labeled Fab fragments where [ 99m Tc](HYNIC-Fab)(tricine)(BP) exhibited significantly faster elimination rates of the radioactivity not only from the blood but also from the kidney. These findings indicated that conjugation of [ 99m Tc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides accelerated elimination rates of the radioactivity from the blood and nontarget tissues due to low binding of the [ 99m Tc](HYNIC)(tricine)(BP) complex with proteins in the blood and in the lysosomes. Such characteristics would render the TFP active ester of [ 99m Tc](HYNIC)(tricine)(BP) complex

  14. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

    Science.gov (United States)

    Soheilypour, M; Mofrad, M R K

    2016-11-02

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

  15. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Maric, Martina; Haugo, Alison C. [Department of Microbiology, University of Iowa, Iowa City, IA 52242 (United States); Dauer, William [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Johnson, David [Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201 (United States); Roller, Richard J., E-mail: richard-roller@uiowa.edu [Department of Microbiology, University of Iowa, Iowa City, IA 52242 (United States)

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.

  16. Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

    Science.gov (United States)

    Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

    2017-08-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus. IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

  17. Drosophila Protein interaction Map (DPiM)

    OpenAIRE

    Guruharsha, K.G.; Obar, Robert A.; Mintseris, Julian; Aishwarya, K.; Krishnan, R.T.; VijayRaghavan, K.; Artavanis-Tsakonas, Spyros

    2012-01-01

    Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when,...

  18. Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

    Science.gov (United States)

    Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

    2015-01-01

    Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

  19. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-01-01

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

  20. Functional dissection of Streptococcus pyogenes M5 protein: the hypervariable region is essential for virulence.

    Directory of Open Access Journals (Sweden)

    Johan Waldemarsson

    Full Text Available The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region.

  1. ASSESSMENT OF MOVEMENT SKILL PERFORMANCE IN PRESCHOOL CHILDREN: CONVERGENT VALIDITY BETWEEN MOT 4-6 AND M-ABC

    Directory of Open Access Journals (Sweden)

    Wouter Cools

    2010-12-01

    Full Text Available The purpose was to determine the level of agreement between the Motoriktest für Vier- bis Sechsjährige Kinder [MOT 4-6] and the Movement Assessment Battery for Children [M-ABC]. 48 preschool children participated in the study (Mean age = 5 years, 6 months, SD = 3 months. There was high classification agreement (90% between both tests. A Kappa correlation coefficient (0.67 provided moderately strong support for convergent validity. Less agreement was shown in identification of motor difficulties (58%. This was reflected by lower correlation coefficients on the fine movement cluster and test item level. The MOT 4-6 showed values within the range of similar movement skill performance assessment protocols. Because of its specific focus it may be of meaningful value to assess movement skill competence in typically developing preschool children (ages 4 to 6.

  2. An integrative genomic and transcriptomic analysis reveals potential targets associated with cell proliferation in uterine leiomyomas.

    Directory of Open Access Journals (Sweden)

    Priscila Daniele Ramos Cirilo

    Full Text Available Uterine Leiomyomas (ULs are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs. Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs.We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC and gene expression microarrays (SAM. The CONEXIC algorithm was applied to integrate the data.The integrated analysis identified the top 30 significant genes (P<0.01, which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively and IGFBP5 (P = 0.0002 and P = 0.006, respectively were up-regulated in the tumours when compared with the adjacent normal myometrium.The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs.

  3. Prevalence of Anti Human Herpes Virus-6 IgG and its Receptor in Acute Leukemia (Membrane Cofactor Protein: MCP, CD46)

    International Nuclear Information System (INIS)

    Assem, M.M; El-Sharkawy, N.M.; Tarek, H.; Kamel, A.M.; Gad, W.H.; El-Rouby, M.N.; Ghaleb, F.M.

    2005-01-01

    CD46 is a membrane cofactor protein, which acts as a cofactor for factor I proteolytic cleavage of C3, so it protects the cells expressing it on their surface from autologous complement attack. It has been recently described as a receptor for HHV-6. Also, it has been shown to be highly expressed on malignant cells as compared to normal cells, thus playing a major role by which these cells, either cells of haematological malignancy or cells of other body cancers, can protect themselves against complement attack so they can survive and metastasize. Patients and methods: This study has been done to detect the sero prevalence of HHV-6 among 47 Egyptian adult cases of acute leukemia using the anti-HHV-6 IgG ELISA serological technique. CD46 receptor expression and immuno phenotyping technique were performed using FCM. Twenty nine of the cases were ANLL, while 18 were ALL cases. Sixteen age- and sex-matched control cases were also studied for both anti-HHV-6 IgG and CD46 receptor expression. HHV-6 IgG antibodies were encountered in 29 (100%), 14 (77.8%) and 12 (75%) of the ANLL, ALL and the control group, cases, respectively. CD46 expression was encountered in 21 (72.4%) of the ANLL cases and in 10 (55.6%) of the ALL cases. Concordance between HHV6 sero positivity and CD46 expression was encountered in 31 cases (29 positive and 2 negative). Dis concordance was encountered in 16 cases with 14 showing HHV-6 IgG sero positivity with no CD46 expression and 2 showing the reverse. The lack of significant correlation between CD46 expression and sero positivity would exclude CD46 expression as a cause of contracting HHV-6 infection in leukemic patients

  4. Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein

    DEFF Research Database (Denmark)

    Dziegiel, M; Borre, Mette; Petersen, E

    1992-01-01

    This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactos......This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta...

  5. Glucocorticoid effects on hippocampal protein synthesis

    International Nuclear Information System (INIS)

    Schlatter, L.K.

    1988-01-01

    Following subcutaneous injection of rats with 5 mg corticosterone, hippocampal slices in vitro show increased [ 35 S]-methionine labeling of a cytosolic protein with an apparent molecular weight (M r ) of 35,000 and an isoelectric point (IEP) of 6.6. This labeling is temporally consistent with a transcriptional event, and is steroid- and tissue-specific. The pear serum concentration of steroid occurs one hour or less following the injection. Maximal labeling of this protein is reached whenever serum corticosterone values are approximately 100 ng/ml. When endogenous corticosterone levels are elevated to 100 ng/ml through stressors or exogenous ACTH injections the same maximal increase in synthesis of the 35,000 M r protein is observed. Adrenalectomy prevents the observed response from occurring following stressor application or ACTH injections. Comparison of the increases observed after administration of the type 2 receptor agonist RU 28362 and aldosterone, which has a higher affinity for the type 1 receptor, shows a 50-fold greater sensitivity of the response to the type 2 receptor agonist. Synthesis of this protein following serum increases of steroid possibly correlates to the theorized function of the type 2 receptor feedback regulation. The similar protein in the liver has an IEP of 6.8 and a slightly higher M r . A second hippocampal protein with an M r of 46,000 and an IEP of 6.2 is also increased in labeling. Two additional liver proteins, one of Mr 53,000 (IEP of 6.2) and the other with an M r of 45,000 (IEP of 8.7-7.8) are increased in the liver following glucocorticoid administration

  6. An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.

    Directory of Open Access Journals (Sweden)

    Mariana Serpeloni

    Full Text Available In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II, but not RNA polymerase I (RNA pol I or Spliced Leader (SL transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and

  7. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Role of protein and mRNA oxidation in seed dormancy and germination

    Directory of Open Access Journals (Sweden)

    hayat eel-maarouf-bouteau

    2013-04-01

    Full Text Available Reactive oxygen species (ROS are key players in the regulation of seed germination and dormancy. Although their regulated accumulation is a prerequisite for germination, the cellular basis of their action remains unknown, but very challenging to elucidate due to the lack of specificity of these compounds that can potentially react with all biomolecules. Among these, nucleic acids and proteins are very prone to oxidative damage. RNA is highly sensitive to oxidation because of its single-stranded structure and the absence of a repair system. Oxidation of mRNAs induces their decay through processing bodies or results in the synthesis of aberrant proteins through altered translation. Depending on the oxidized amino acid, ROS damage of proteins can be irreversible (i.e. carbonylation thus triggering the degradation of the oxidized proteins by the cytosolic 20S proteasome or can be reversed through the action of thioredoxins, peroxiredoxins or glutaredoxins (cysteine oxidation or by methionine sulfoxide reductase (methionine oxidation. Seed dormancy alleviation in the dry state, referred to as after-ripening, requires both selective mRNA oxidation and protein carbonylation. Similarly, seed imbibition of non-dormant seeds is associated with targeted oxidation of a subset of proteins. Altogether, these specific features testify that such oxidative modifications play important role in commitment of the cellular functioning toward germination completion.

  9. M13 Bacteriophage Based Protein Sensors

    Science.gov (United States)

    Lee, Ju Hun

    Despite significant progress in biotechnology and biosensing, early detection and disease diagnosis remains a critical issue for improving patient survival rates and well-being. Many of the typical detection schemes currently used possess issues such as low sensitivity and accuracy and are also time consuming to run and expensive. In addition, multiplexed detection remains difficult to achieve. Therefore, developing advanced approaches for reliable, simple, quantitative analysis of multiple markers in solution that also are highly sensitive are still in demand. In recent years, much of the research has primarily focused on improving two key components of biosensors: the bio-recognition agent (bio-receptor) and the transducer. Particular bio-receptors that have been used include antibodies, aptamers, molecular imprinted polymers, and small affinity peptides. In terms of transducing agents, nanomaterials have been considered as attractive candidates due to their inherent nanoscale size, durability and unique chemical and physical properties. The key focus of this thesis is the design of a protein detection and identification system that is based on chemically engineered M13 bacteriophage coupled with nanomaterials. The first chapter provides an introduction of biosensors and M13 bacteriophage in general, where the advantages of each are provided. In chapter 2, an efficient and enzyme-free sensor is demonstrated from modified M13 bacteriophage to generate highly sensitive colorimetric signals from gold nanocrystals. In chapter 3, DNA conjugated M13 were used to enable facile and rapid detection of antigens in solution that also provides modalities for identification. Lastly, high DNA loadings per phage was achieved via hydrozone chemistry and these were applied in conjunction with Raman active DNA-gold/silver core/shell nanoparticles toward highly sensitive SERS sensing.

  10. Optimized enzyme-linked immunosorbent assay for detecting cytomegalovirus infections during clinical trials of recombinant vaccines.

    Science.gov (United States)

    Pagnon, Anke; Piras, Fabienne; Gimenez-Fourage, Sophie; Dubayle, Joseline; Arnaud-Barbe, Nadège; Hessler, Catherine; Caillet, Catherine

    2017-11-01

    In clinical trials of cytomegalovirus (CMV) glycoprotein B (gB) vaccines, CMV infection is detected by first depleting serum of anti-gB antibodies and then measuring anti-CMV antibodies with a commercially available enzyme-linked immunosorbent assay (ELISA) kit, with confirmation of positive findings by immunoblot. Identification of CMV immunoantigens for the development of an ELISA that detects specifically CMV infection in clinical samples from individuals immunized with gB vaccines. Sensitivity and specificity of ELISAs using antigenic regions of CMV proteins UL83/pp65, UL99/pp28, UL44/pp52, UL80a/pp38, UL57, and UL32/pp150 were measured. An IgG ELISA using a UL32/pp150 [862-1048] capture peptide was the most specific (93.7%) and sensitive (96.4%) for detecting CMV-specific antibodies in sera. The ELISA successfully detected CMV-specific antibodies in 22 of 22 sera of subjects who had been vaccinated with a gB vaccine but who had later been infected with CMV. The ELISA was linear over a wide range of CMV concentrations (57-16,814 ELISA units/mL) and was reproducible as indicated by a 5% intra-day and 7% inter-day coefficients of variation. The signal was specifically competed by UL32/pp150 [862-1048] peptide but not by CMV-gB or herpes simplex virus 2 glycoprotein D. Lipid and hemoglobin matrix did not interfere with the assay. The UL32/pp150 [862-1048] IgG ELISA can be used for the sensitive and specific detection of CMV infection in gB-vaccinated individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Association of FAS A-670G Polymorphism and Risk of Uterine Leiomyoma in a Southeast Iranian Population

    Directory of Open Access Journals (Sweden)

    Abbas Mohammadpour-Gharehbagh

    2016-10-01

    Full Text Available Background: Uterine leiomyoma (UL is a benign tumor of uterine smooth muscle that affects women in reproductive ages. FAS has an important role in initial stages of apoptosis. Previous studies have shown an association between the FAS gene and tumorigenesis. In the present study, we evaluated the relationship between FAS A-670G (rs 1800682 and UL risk. Methods: The FAS gene polymorphism of 155 women with UL and 157 healthy controls was analyzed by the polymerase chain reaction restriction fragment length polymorphism method. Results: The AA, AG, and GG genotype frequencies of the FAS A-670G polymorphism were respectively 37.4, 42.6, and 20% in women with UL, and 46, 42.6, and 11.5% in healthy controls. The risk of UL in women was 1.5-fold greater in GG-genotype women than in AA-genotype women. The G allele frequencies were 41% in women with UL and 33% in healthy controls and statistically different (P = 0.03. Conclusions: The FAS polymorphism was associated with the risk of UL in a sample of Iranian women.

  12. Association of FAS A-670G Polymorphism and Risk of Uterine Leiomyoma in a Southeast Iranian Population

    Science.gov (United States)

    Mohammadpour-Gharehbagh, Abbas; Salimi, Saeedeh; Keshavarzi, Farshid; Zakerian, Sepideh; Sajadian, Mojtaba; Mokhtari, Mojgan

    2016-01-01

    Background: Uterine leiomyoma (UL) is a benign tumor of uterine smooth muscle that affects women in reproductive ages. FAS has an important role in initial stages of apoptosis. Previous studies have shown an association between the FAS gene and tumorigenesis. In the present study, we evaluated the relationship between FAS A-670G (rs 1800682) and UL risk Methods: The FAS gene polymorphism of 155 women with UL and 157 healthy controls was analyzed by the polymerase chain reaction restriction fragment length polymorphism method Results: The AA, AG, and GG genotype frequencies of the FAS A-670G polymorphism were respectively 37.4, 42.6, and 20% in women with UL, and 46, 42.6, and 11.5% in healthy controls. The risk of UL in women was 1.5-fold greater in GG-genotype women than in AA-genotype women. The G allele frequencies were 41% in women with UL and 33% in healthy controls and statistically different (P = 0.03) Conclusion: The FAS polymorphism was associated with the risk of UL in a sample of Iranian women. PMID:28070535

  13. Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

    Directory of Open Access Journals (Sweden)

    O Rickard Nilsson

    Full Text Available Recent studies indicate that defective activity of complement factor H (FH is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

  14. Tyrosine 129 of the murine gammaherpesvirus M2 protein is critical for M2 function in vivo.

    Science.gov (United States)

    Rangaswamy, Udaya S; O'Flaherty, Brigid M; Speck, Samuel H

    2014-01-01

    A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes--predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.

  15. Tyrosine 129 of the murine gammaherpesvirus M2 protein is critical for M2 function in vivo.

    Directory of Open Access Journals (Sweden)

    Udaya S Rangaswamy

    Full Text Available A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes--predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68 infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.

  16. Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein.

    Science.gov (United States)

    Lyabin, D N; Ovchinnikov, L P

    2016-03-02

    The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors.

  17. Supercharging by m-NBA Improves ETD-Based Quantification of Hydroxyl Radical Protein Footprinting

    Science.gov (United States)

    Li, Xiaoyan; Li, Zixuan; Xie, Boer; Sharp, Joshua S.

    2015-08-01

    Hydroxyl radical protein footprinting (HRPF) is an MS-based technique for analyzing protein structure based on measuring the oxidation of amino acid side chains by hydroxyl radicals diffusing in solution. Spatial resolution of HRPF is limited by the smallest portion of the protein for which oxidation amounts can be accurately quantitated. Previous work has shown electron transfer dissociation (ETD) to be the most reliable method for quantifying the amount of oxidation of each amino acid side chain in a mixture of peptide oxidation isomers, but efficient ETD requires high peptide charge states, which limits its applicability for HRPF. Supercharging reagents have been used to enhance peptide charge state for ETD analysis, but previous work has shown supercharging reagents to enhance charge state differently for different peptides sequences; it is currently unknown if different oxidation isomers will experience different charge enhancement effects. Here, we report the effect of m-nitrobenzyl alcohol ( m-NBA) on the ETD-based quantification of peptide oxidation. The addition of m-NBA to both a defined mixture of synthetic isomeric oxidized peptides and Robo-1 protein subjected to HRPF increased the abundance of higher charge state ions, improving our ability to perform efficient ETD of the mixture. No differences in the reported quantitation by ETD were noted in the presence or absence of m-NBA, indicating that all oxidation isomers were charge-enhanced to a similar extent. These results indicate the utility of m-NBA for residue-level quantification of peptide oxidation in HRPF and other applications.

  18. Process for the separation of proteins from acid whey

    Energy Technology Data Exchange (ETDEWEB)

    Mirabel, B

    1980-01-01

    Acid whey from cheese or casein manufacture (pH less than 4.6) and containing about 5.2 g protein/l is passed through a cation exchange resin (of silica coated with a copolymer of styrene/vinyltriethoxysilane carrying SO/sub 3/H functional groups). The proteins adsorbed on the resin (alpha-lactalbumin, beta-lactoglobulin, serum albumin and immunoglobulins) are eluated with an 0.1 M ammonia solution, concentrated under vacuum and freeze-dried, obtaining a final product with 88% undenatured protein. The products are for use in the food and pharmaceutical industries and for dietetic and veterinary purposes.

  19. The cross section measurement for the reactions of 48,46Ti(n,p) 48,46Sc, 50Ti(n, α)47Ca and 58Ni (n, 2n)57Ni, 58Ni(n,p)58m+gCo

    International Nuclear Information System (INIS)

    Yuan Junqian; Wang Yongchang; Kong Xiangzhong; Yang Jingkang

    1992-01-01

    The cross sections for the 50 Ti(n, α) 47 Ca, 46 Ti(n, p) 46 Sc, 48 Ti(n, p) 48 Sc and 58 Ni(n, 2n) 57 Ni, 58 Ni(n, p) 58m+g Co reactions have been measured by using the activation method relative to the cross sections of the 27 Al(n, α) 24 Na reaction in the neutron energy range of 13.50-14.81 MeV. The neutron energies were determined by the cross section ratios of the 90 Zr(n, 2n) 89m+g Zr and 93 Nb(n, 2n) 92m Nb reactions. The results obtained are compared with the published and to be published data of several authors

  20. The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation.

    Directory of Open Access Journals (Sweden)

    Andrea M Siegel

    2008-04-01

    Full Text Available Murine gammaherpesvirus 68 (MHV68 establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV. EBV encodes an interleukin-10 (IL-10 homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25 and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis

  1. The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation.

    Science.gov (United States)

    Siegel, Andrea M; Herskowitz, Jeremy H; Speck, Samuel H

    2008-04-04

    Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis-identifying a

  2. The Mycobacterium tuberculosis Complex has a Pathway for the Biosynthesis of 4-Formamido-4,6-Dideoxy-d-Glucose.

    Science.gov (United States)

    Brown, Haley A; Vinogradov, Evgeny; Gilbert, Michel; Holden, Hazel M

    2018-05-15

    Recent studies have demonstrated that the O-antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N-formylated sugars (3-formamido-3,6-dideoxy-d-glucose or 4-formamido-4,6-dideoxy-d-glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6-dehydratase, a pyridoxal 5'-phosphate or PLP-dependent aminotransferase, and an N-formyltransferase. To date, there have been no published reports of N-formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N-formyltransferase. Given that M. tuberculosis produces l-rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6-dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N-formylated sugar in M. tuberculosis, namely a PLP-dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP-4-formamido-4,6-dideoxy-d-glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined. This article is protected by copyright. All rights reserved. © 2018 The Protein Society.

  3. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

    Science.gov (United States)

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

    2016-03-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. © 2016 Müller-McNicoll et al.; Published by Cold Spring Harbor Laboratory Press.

  4. Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

  5. The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

    Science.gov (United States)

    Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

    2016-05-21

    The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

  6. Characterization of an electrochemical mercury sensor using alternating current, cyclic, square wave and differential pulse voltammetry

    International Nuclear Information System (INIS)

    Guerreiro, Gabriela V.; Zaitouna, Anita J.; Lai, Rebecca Y.

    2014-01-01

    Graphical abstract: -- Highlights: •An electrochemical Hg(II) sensor based on T–Hg(II)–T sensing motif was fabricated. •A methylene blue-modified DNA probe was used to fabricate the sensor. •Sensor performance was evaluated using ACV, CV, SWV, and DPV. •The sensor behaves as a “signal-off” sensor in ACV and CV. •The sensor behaves as either a “signal-on” or “signal-off” sensor in SWV and DPV. -- Abstract: Here we report the characterization of an electrochemical mercury (Hg 2+ ) sensor constructed with a methylene blue (MB)-modified and thymine-containing linear DNA probe. Similar to the linear probe electrochemical DNA sensor, the resultant sensor behaved as a “signal-off” sensor in alternating current voltammetry and cyclic voltammetry. However, depending on the applied frequency or pulse width, the sensor can behave as either a “signal-off” or “signal-on” sensor in square wave voltammetry (SWV) and differential pulse voltammetry (DPV). In SWV, the sensor showed “signal-on” behavior at low frequencies and “signal-off” behavior at high frequencies. In DPV, the sensor showed “signal-off” behavior at short pulse widths and “signal-on” behavior at long pulse widths. Independent of the sensor interrogation technique, the limit of detection was found to be 10 nM, with a linear dynamic range between 10 nM and 500 nM. In addition, the sensor responded to Hg 2+ rather rapidly; majority of the signal change occurred in 2+ , which has not been previously reported. More importantly, the observed “switching” behavior in SWV and DPV is potentially generalizable and should be applicable to most sensors in this class of dynamics-based electrochemical biosensors

  7. Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs.

    Science.gov (United States)

    Manjarín, Rodrigo; Columbus, Daniel A; Suryawan, Agus; Nguyen, Hanh V; Hernandez-García, Adriana D; Hoang, Nguyet-Minh; Fiorotto, Marta L; Davis, Teresa

    2016-01-01

    Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight(-1) · day(-1)) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E · eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E · eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs.

  8. A conserved small RNA promotes silencing of the outer membrane protein YbfM

    DEFF Research Database (Denmark)

    Rasmussen, Anders Aamann; Johansen, Jesper; Nielsen, Jesper S

    2009-01-01

    important physiological role of regulatory RNA molecules in Gram-negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP-sRNA network by showing that the expression of the outer membrane protein YbfM is silenced by a conserved sRNA......In the past few years an increasing number of small non-coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the post-transcriptional level. These RNAs act under various growth and stress conditions, suggesting that one......, designated MicM (also known as RybC/SroB). The regulation is strictly dependent on the RNA chaperone Hfq, and mutational analysis indicates that MicM sequesters the ribosome binding site of ybfM mRNA by an antisense mechanism. Furthermore, we provide evidence that Hfq strongly enhances the on-rate of duplex...

  9. Nifedipine effect on the labelling of blood cells and plasma proteins with Tc-99m

    International Nuclear Information System (INIS)

    Gutfilen, B.; Boasquevisque, E.M.; Bernardo Filho, M.

    1988-01-01

    The labeling of red blood cells (RBC) with Tc-99m depends on the presence of stannous ion (Sn) that helps this radionuclide's fixation on the hemoglobin molecule. Nifedipine is an agent capable to block a specific way where calcius (Ca) ion acrosses the cellular membrane and to bind itself on plasma proteins. The effect of nifedipine in the labeling of RBC and plasma proteins with Tc-99m was studied because of similarities between Ca and Sn ions. Blood with anticoagulant was treated with nifedipine concentration of 10 -6 M for 15 min at 37 0 C. The labeling of RBC with Tc-99m was done incubating with Sn ion solution (3 uM) for different times. The % of radioactivity in RBC was determined. Samples of plasma were precipited with trichloroacetic acid and the % of radiocctivity in insoluble fraction was calculated. The same procedure was done using different nifedipine concentrations and the blood was incubated for 60 min with Sn ion. The determination of the % of Tc-99m labeled in RBC and plasma proteins showed that this drug does not have the capability to alter this incorporation because the results are similar to control. It is suggested that the Sn ions passage across RBC is not altered by nifedipine although this drug could bind to plasma protein, it does not modify the Tc-99m fixation on it. (author) [pt

  10. A novel frameshift mutation in CX46 associated with hereditary dominant cataracts in a Chinese family

    Directory of Open Access Journals (Sweden)

    Xiu-Kun Cui

    2017-05-01

    Full Text Available AIM: To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family. METHODS: A Chinese family consisting of 20 cataract patients (including 9 male and 11 female and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46 mutant. Immunoblotting was conduceted for testing the expression of CX46. RESULTS: To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3 were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400 compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression. CONCLUSION: The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.

  11. Selective translation of the measles virus nucleocapsid mRNA by La protein

    Directory of Open Access Journals (Sweden)

    Yoshihisa eInoue

    2011-08-01

    Full Text Available Measles, caused by measles virus (MeV infection, is the leading cause of death in children because of secondary infections attributable to MeV-induced immune suppression. Recently, we have shown that wild-type MeVs induce the suppression of protein synthesis in host cells (referred to as "shutoff" and that viral mRNAs are preferentially translated under shutoff conditions in infected cells. To determine the mechanism behind the preferential translation of viral mRNA, we focused on the 5 untranslated region (UTR of nucleocapsid (N mRNA. The La/SSB autoantigen (La was found to specifically bind to an N-5UTR probe. Recombinant La enhanced the translation of luciferase mRNA containing the N-5UTR (N-fLuc, and RNA interference of La suppressed N-fLuc translation. Furthermore, recombinant MeV lacking the La-binding motif in the N-5UTR displayed delayed viral protein synthesis and growth kinetics at an early phase of infection. These results suggest that La induced predominant translation of N mRNA via binding to its 5UTR under shutoff conditions. This is the first report on a cellular factor that specifically regulates paramyxovirus mRNA translation.

  12. Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

    Directory of Open Access Journals (Sweden)

    Ganesh Ambigapathy

    Full Text Available Brain-derived neurotrophic factor (BDNF has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

  13. Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

    Science.gov (United States)

    Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

    2013-01-01

    Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

  14. Prevention of Pulmonary Fibrosis via Trichostatin A (TSA) in Bleomycin Induced Rats.

    Science.gov (United States)

    Ye, Qing; Li, Yanqin; Jiang, Handong; Xiong, Jianfei; Xu, Jiabo; Qin, Hui; Liu, Bin

    2014-10-20

    To investigate the effects of non selective histone deacetylase inhibitors Trichostatin A (TSA)on bleomycin-induced pulmonary fibrosis. To investigate the effects of non selective histone deacetylase inhibitors Trichostatin A ( TSA ) on HDAC2, p-SMAD2, HDAC2 mRNA, SMAD2mRNA in pulmonary fibrosis rats and investigate impossible mechanism. 46 SPF level male SD rats were randomly divided into four groups: ten for normal control group, fourteen for model control group I, twelve for model control group II and ten for treatment group. Rat pulmonary fibrosis was induced by bleomycin(5mg/kg) via single intratracheal perfusion in the two model control groups and treatment group. Normal control mice were instilled with a corresponding volume of 0.9% saline intratracheally. Treatment group was treated by the dilution of TSA 2mg/kg DMSO 60ul and0.9% saline 1.2ml intraperitoneal injection from the next day ,once a day for three days. Model control group II was treated by the dilution of DMSO 60ul and0.9% saline 1.2ml intraperitoneal injection from the next day once a day for three days. Model control group I and normal control group were treated by 0.9% saline 1.2ml intraperitoneal injection from the next day once a day for three days. All the animals were sacrificed on the 21 day after modeling. The pathological changes were observed by hematoxylin and eosin(HE)stain and masson trichrome stain. The expression of HDAC2 mRNA,SMAD2 mRNA were measured by real-time PCR. The protein level of HDAC2 and p-SMAD2 in serum was measured by Western blot. The pulmonary fibrosis in treatment group were significantly alleviated compared to the two model control groups (P0.05). Western blot indicated that the protein level of HDAC2 and p-SMAD2 in serum increased in the two model control groups compared with normal control group(P0.05). Non selective histone deacetylase inhibitors of Trichostatin A (TSA) can reduce the bleomycin induced pulmonary fibrosis in rats. TSA attenuates pulmonary

  15. AcEST: DK962942 [AcEST

    Lifescience Database Archive (English)

    Full Text Available Swiss-Prot sp_hit_id P09287 Definition sp|P09287|UL25_VZVD Virion gene 34 protein OS=Varicella-zoster virus (strain Dumas...in 47 O... 30 8.7 >sp|P09287|UL25_VZVD Virion gene 34 protein OS=Varicella-zoster virus (strain Dumas) GN=34

  16. Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation.

    Science.gov (United States)

    Ziehr, Benjamin; Lenarcic, Erik; Vincent, Heather A; Cecil, Chad; Garcia, Benjamin; Shenk, Thomas; Moorman, Nathaniel J

    2015-06-01

    Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

    Science.gov (United States)

    Wu, Fan-Lin; Liu, Yin; Jiang, He-Wei; Luan, Yi-Zhao; Zhang, Hai-Nan; He, Xiang; Xu, Zhao-Wei; Hou, Jing-Li; Ji, Li-Yun; Xie, Zhi; Czajkowsky, Daniel M; Yan, Wei; Deng, Jiao-Yu; Bi, Li-Jun; Zhang, Xian-En; Tao, Sheng-Ce

    2017-08-01

    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Alterations of proteins in MDCK cells during acute potassium deficiency.

    Science.gov (United States)

    Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

    2016-06-01

    Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition.

    Science.gov (United States)

    Marques-Ramos, Ana; Candeias, Marco M; Menezes, Juliane; Lacerda, Rafaela; Willcocks, Margaret; Teixeira, Alexandre; Locker, Nicolas; Romão, Luísa

    2017-11-01

    The mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5' untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions. © 2017 Marques-Ramos et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  20. Long distance movement of an Arabidopsis Translationally Controlled Tumor Protein (AtTCTP2 mRNA and protein in tobacco

    Directory of Open Access Journals (Sweden)

    Roberto eToscano-Morales

    2014-12-01

    Full Text Available TCTP (Translationally Controlled Tumor Protein is an almost ubiquitous protein found in eukaryotes, fundamental for the regulation of development and general growth. The multiple functions of TCTP have been inferred from its involvement in several cell pathways, but the specific function of TCTP is still not known in detail. On the other hand, TCTP seems to respond to a plethora of external signals, and appears to be regulated at the transcriptional and/or translational levels by mechanisms yet to be determined. In the present work, we analyzed the capacity of AtTCTP2 gene products (mRNA and protein to translocate long distance through tobacco heterografts (Transgenic/WT and WT/Transgenic. The results indicate that both AtTCTP2 mRNA and protein are capable of moving long distance in both directions (stock-scion and scion-stock with a tendency for movement from source to sink tissue (stock to scion. Interestingly, aerial roots emerged only in heterografts where the protein was detected in both stock and scion, suggesting a correlation between the presence of AtTCTP2 and appearance of aerial adventitious roots. More detailed analysis showed that these adventitious aerial roots harbored the transgene and expressed both transcript and protein. In addition, the protein localization pattern in transgenic aerial and primary roots was basically the same, indicating specific nuclear destination in roots, but also in leaves. These findings provide an approach to understand the role of long-distance movement in the function of plant TCTPs, supporting the notion that some of these act in a non-cell autonomous manner, as the human counterpart, the Histamine Releasing Factor (HRF.

  1. The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity

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    Minako Ogino

    2016-05-01

    Full Text Available The large (L protein of rabies virus (RABV plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5′-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5′-triphosphorylated but not 5′-diphosphorylated RABV mRNA-start sequences, 5′-AACA(C/U, with GDP to generate the 5′-terminal cap structure G(5′ppp(5′A. The 5′-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286 in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

  2. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    DEFF Research Database (Denmark)

    Beuchert Kallehauge, Thomas; Li, Shangzhong; Pedersen, Lasse Ebdrup

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as effici......Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated...... as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated...... as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we...

  3. Targeting natural killer cell reactivity by employing antibody to NKp46: implications for type 1 diabetes.

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    Rami Yossef

    Full Text Available Natural killer (NK cells belong to the innate lymphoid cells. Their cytotoxic activity is regulated by the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that the NKp46 receptor is involved in the development of type 1 diabetes (T1D. Subsequently, we hypothesized that blocking this receptor could prevent or hinder disease development. To address this goal, we developed monoclonal antibodies for murine NKp46. One mAb, named NCR1.15, recognizes the mouse homologue protein of NKp46, named Ncr1, and was able to down-regulate the surface expression of NKp46 on primary murine NK cells following antibody injection in vivo. Additionally, NCR1.15 treatments were able to down-regulate cytotoxic activity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, non-obese diabetic mice and low-dose streptozotocin. Our results show a significantly lower incidence of diabetic mice in the NCR1.15-treated group compared to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests a novel treatment strategy for early insulitis.

  4. Characterization of protein kinase CK2 protein subunits and p53 in F9 teratocarcinoma cells in the absence and presence of cisplatin

    DEFF Research Database (Denmark)

    Küpper, M; Köster, M; Schmidt-Spaniol, I

    1994-01-01

    cell extracts treated with and without cisplatin were analyzed by ion exchange chromatography for protein kinase CK2 alpha/beta subunits and p53 distribution. The following results were obtained: (a) in crude extracts of cisplatin-treated cells, CK2 activity was sometimes reduced by as much as 50%; (b......The effect of cis-diaminedichloroplatinum(II) (cisplatin) on the induction of p53 and protein kinase CK2 activity was studied in the mouse teratocarcinoma cell line F9. Treatment of the cells with the chemotherapeutic agent cisplatin led to the detection of p53 3 h after addition of the drug. F9...... by immunostaining, we have detected, at a concentration of approximately 200 mM NaCl, a protein of approximately 46 kDa which reacted with the CK2 alpha-specific antibody. This fraction was devoid of CK2 activity; and (d) cisplatin-treated cells exhibited p53 protein, which was mostly eluting ahead but also partly...

  5. Increased Dapivirine tissue accumulation through vaginal film codelivery of dapivirine and Tenofovir.

    Science.gov (United States)

    Akil, Ayman; Devlin, Brid; Cost, Marilyn; Rohan, Lisa Cencia

    2014-05-05

    The HIV-1 replication inhibitor dapivirine (DPV) is one of the most promising drug candidates being used in topical microbicide products for prevention of HIV-1 sexual transmission. To be able to block HIV-1 replication, DPV must have access to the viral reverse transcriptase enzyme. The window for DPV to access the enzyme happens during the HIV-1 cellular infection cycle. Thus, in order for DPV to exert its anti-HIV activity, it must be present in the mucosal tissue or cells where HIV-1 infection occurs. A dosage form containing DPV must be able to deliver the drug to the tissue site of action. Polymeric films are solid dosage forms that dissolve and release their payload upon contact with fluids. Films have been used as vaginal delivery systems of topical microbicide drug candidates including DPV. For use in topical microbicide products containing DPV, polymeric films must prove their ability to deliver DPV to the target tissue site of action. Ex vivo exposure studies of human ectocervical tissue to DPV film revealed that DPV was released from the film and did diffuse into the tissue in a concentration dependent manner indicating a process of passive diffusion. Analysis of drug distribution in the tissue revealed that DPV accumulated mostly at the basal layer of the epithelium infiltrating the upper part of the stroma. Furthermore, as a combination microbicide product, codelivery of DPV and TFV from a polymeric film resulted in a significant increase in DPV tissue concentration [14.21 (single entity film) and 31.03 μg/g (combination film)], whereas no impact on TFV tissue concentration was found. In vitro release experiments showed that this observation was due to a more rapid DPV release from the combination film as compared to the single entity film. In conclusion, the findings of this study confirm the ability of polymeric films to deliver DPV and TFV to human ectocervical tissue and show that codelivery of the two agents has a significant impact on DPV

  6. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    Directory of Open Access Journals (Sweden)

    Haohan Wang

    2016-03-01

    Full Text Available Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

  7. Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

    Science.gov (United States)

    Ohnheiser, Johanna; Ferlemann, Eva; Haas, Astrid; Müller, Jan P; Werwein, Eugen; Fehler, Olesja; Biyanee, Abhiruchi; Klempnauer, Karl-Heinz

    2015-07-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. The use of supersaturation for the vaginal application of microbicides: a case study with dapivirine.

    Science.gov (United States)

    Grammen, Carolien; Plum, Jakob; Van Den Brande, Jeroen; Darville, Nicolas; Augustyns, Koen; Augustijns, Patrick; Brouwers, Joachim

    2014-11-01

    In this study, we investigated the potential of supersaturation for the formulation of the poorly water-soluble microbicide dapivirine (DPV) in an aqueous vaginal gel in order to enhance its vaginal tissue uptake. Different excipients such as hydroxypropylmethylcellulose, polyethylene glycol 1000, and cyclodextrins were evaluated for their ability to inhibit precipitation of supersaturated DPV in the formulation vehicle as such as well as in biorelevant media. In vitro permeation assessment across HEC-1A cell layers demonstrated an enhanced DPV flux from supersaturated gels compared with suspension gels. The best performing supersaturated gel containing 500 μM DPV (supersaturation degree of 4) in the presence of sulfobutyl ether-beta-cyclodextrin (2.5%) appeared to be stable for at least 3 months. In addition, the gel generated a significant increase in vaginal drug uptake in rabbits as compared with suspension gels. We conclude that supersaturation is a possible strategy to enhance the vaginal concentration of hydrophobic microbicides, thereby increasing permeation into the vaginal submucosa. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  9. Differential effects of hGH and IGF-I on body proportions.

    Science.gov (United States)

    Laron, Zvi; Silbergeld, Aviva; Kauli, Rivka

    2012-07-01

    The differential growth effects of hGH and IGF-I on the upper/lower (U/L) body segment in relation to height (Ht) were analyzed in 15 patients with isolated Growth hormone deficiency (IGHD,:7M, 8F) mean age 5.0 +/- 3.2 (SD) years treated with hGH; 21 patients with multiple pituitary hormone deficiency including growth hormone (MPHD: 14M, 7F) aged 10.0 +/- 3.8, treated with hGH; 9 patients with Laron Syndrome (LS) (4M,5F) aged 6.9 +/- 5.6 years treated with IGF-I; 9 boys with intrauterine growth retardation (IUGR) aged 6.3 +/- 1.25 years treated by hGH; and 22 boys with idiopathic short stature (ISS) aged 8.0 +/- 1.55 years treated by hGH. The dose of hGH was 33 microg/kg/day, that of IGF-I 180-200 microg/kg/day. the U/L body segment ratio in IGHD patients decreased from 2.3 +/- 0.7 to 1.1 +/- 0.7 (p <0.001), and the Ht SDS increased from -4.9 +/- 1.3 to 2.3 +/- 1 (p < 0.001) following treatment. In MPHD patients the U/L body segment decreased from 1.1 +/- 1.1 to -0.6 +/- 1.0 (p < 0.001), and the Ht SDS increased from -3.3 +/- 1.4 to -2.5 +/- 1.0 (p < 0.009). In the LS group the U/L body segment ratio did not change with IGF-I treatment but Ht improved from -6.1 +/- 1.3 to -4.6 +/- 1.2 (p < 0.001), The differential growth response of the children with IUGR and with ISS resembled that of the children with LS. hGH and IGF-I act differentially on the spine and limbs.

  10. M6 membrane protein plays an essential role in Drosophila oogenesis.

    Science.gov (United States)

    Zappia, María Paula; Brocco, Marcela Adriana; Billi, Silvia C; Frasch, Alberto C; Ceriani, María Fernanda

    2011-01-01

    We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila.

  11. Correlation between Body Composition and Walking Capacity in Severe Obesity.

    Science.gov (United States)

    Correia de Faria Santarém, G; de Cleva, R; Santo, Marco Aurélio; Bernhard, Aline Biaseto; Gadducci, Alexandre Vieira; Greve, Julia Maria D'Andrea; Silva, Paulo Roberto Santos

    2015-01-01

    Obesity is associated with mobility reduction due to mechanical factors and excessive body fat. The six-minute walk test (6MWT) has been used to assess functional capacity in severe obesity. To determine the association of BMI, total and segmental body composition with distance walked (6MWD) during the six-minute walk test (6MWT) according to gender and obesity grade. University of São Paulo Medical School, Brazil; Public Practice. Functional capacity was assessed by 6MWD and body composition (%) by bioelectrical impedance analysis in 90 patients. The mean 6MWD was 514.9 ± 50.3 m for both genders. The male group (M: 545.2 ± 46.9 m) showed a 6MWD higher (p = 0.002) than the female group (F: 505.6 ± 47.9 m). The morbid obese group (MO: 524.7 ± 44.0 m) also showed a 6MWD higher (p = 0.014) than the super obese group (SO: 494.2 ± 57.0 m). There was a positive relationship between 6MWD and fat free mass (FFM), FFM of upper limps (FFM_UL), trunk (FFM_TR) and lower limbs (FFM_LL). Female group presented a positive relationship between 6MWD and FFM, FFM_UL and FFM_LL and male group presented a positive relationship between 6MWD and FFM_TR. In morbid obese group there was a positive relationship between 6MWD with FFM, FFM_UL, FFM_TR and FFM_LL. The super obese group presented a positive relationship between 6MWD with FFM, FFM_TR and FFM_LL. Total and segmental FFM is associated with a better walking capacity than BMI.

  12. Prolonged calorie restriction downregulates skeletal muscle mTORC1 signaling independent of dietary protein intake and associated microRNA expression

    Directory of Open Access Journals (Sweden)

    Lee M Margolis

    2016-10-01

    Full Text Available Short-term (5-10 days calorie restriction (CR downregulates muscle protein synthesis, with consumption of a high protein-based diet attenuating this decline. Benefit of increase protein intake is believed to be due to maintenance of amino acid-mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1, however, there is limited evidence to support this contention. The purpose of this investigation was to determine the effects of prolonged CR and high protein diets on skeletal muscle mTORC1 signaling and expression of associated microRNA (miR. 12-wk old male Sprague Dawley rats consumed ad libitum (AL or calorie restricted (CR; 40% adequate (10%, AIN-93M or high (32% protein milk-based diets for 16 weeks. Body composition was determined using dual energy X-ray absorptiometry and muscle protein content was calculated from muscle homogenate protein concentrations expressed relative to fat-free mass to estimate protein content. Western blot and RT-qPCR were used to determine mTORC1 signaling and mRNA and miR expression in fasted mixed gastrocnemius. Independent of dietary protein intake, muscle protein content was 38% lower (P < 0.05 in CR compared to AL. Phosphorylation and total Akt, mTOR, rpS6 and p70S6K were lower (P < 0.05 in CR versus AL, and total rpS6 was associated with muscle protein content (r = 0.64, r2 = 0.36. Skeletal muscle miR expression was not altered by either energy or protein intake. This study provides evidence that chronic CR attenuates muscle protein content by downregulating mTORC1 signaling. This response is independent of skeletal muscle miR and dietary protein.

  13. Antibody classes & subclasses induced by mucosal immunization of mice with Streptococcus pyogenes M6 protein & oligodeoxynucleotides containing CpG motifs.

    Science.gov (United States)

    Teloni, R; von Hunolstein, C; Mariotti, S; Donati, S; Orefici, G; Nisini, R

    2004-05-01

    Type-specific antibodies against M protein are critical for human protection as they enhance phagocytosis and are protective. An ideal vaccine for the protection against Streptococcus pyogenes would warrant mucosal immunity, but mucosally administered M-protein has been shown to be poorly immunogenic in animals. We used a recombinant M type 6 protein to immunize mice in the presence of synthetic oligodeoxynucleotides containing CpG motifs (immunostimulatory sequences: ISS) or cholera toxin (CT) to explore its possible usage in a mucosal vaccine. Mice were immunized by intranasal (in) or intradermal (id) administration with four doses at weekly intervals of M6-protein (10 microg/mouse) with or without adjuvant (ISS, 10 microg/mouse or CT, 0,5 microg/mouse). M6 specific antibodies were measured by enzyme linked immunosorbent assay using class and subclass specific monoclonal antibodies. The use of ISS induced an impressive anti M-protein serum IgG response but when id administered was not detectable in the absence of adjuvant. When used in, M-protein in the presence of both ISS and CT induced anti M-protein IgA in the bronchoalveolar lavage, as well as specific IgG in the serum. IgG were able to react with serotype M6 strains of S. pyogenes. The level of antibodies obtained by immunizing mice in with M-protein and CT was higher in comparison to M-protein and ISS. The analysis of anti-M protein specific IgG subclasses showed high levels of IgG1, IgG2a and IgG2b, and low levels of IgG3 when ISS were used as adjuvant. Thus, in the presence of ISS, the ratio IgG2a/IgG1 and (IgG2a+IgG3)/IgG1 >1 indicated a type 1-like response obtained both in mucosally or systemically vaccinated mice. Our study offers a reproducible model of anti-M protein vaccination that could be applied to test new antigenic formulations to induce an anti-group A Streptococcus (GAS) vaccination suitable for protection against the different diseases caused by this bacterium.

  14. Ligand-free, protein-bound technetium-99m. Evidence for tumour localisation

    International Nuclear Information System (INIS)

    Jakovljevic, A.C.; Pojer, P.M.

    1984-11-01

    An hypothesis that cations accumulate in tumours independent of ligand is tested. A preparation of technetium-99m known to be ligand-free (that is, the technetium is protein bound and no other ligand is injected) has been shown to accumulate in a T-cell lymphoma

  15. Differential regulation of amyloid-β-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease

    International Nuclear Information System (INIS)

    Higgins, G.A.; Lewis, D.A.; Bahmanyar, S.; Goldgaber, D.; Gajdusek, D.C.; Young, W.G.; Morrison, J.H.; Wilson, M.C.

    1988-01-01

    The authors have mapped the neuroanatomical distribution of amyloid-β-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-β-protein mRNA appears to be expressed in all hippocampal neurons, but at different levels of abundance. In the central nervous system of monkey and normal aged human, image analysis shows that neurons of the dentate gyrus and cornu Ammonis fields contain a 2.5-times-greater hybridization signal than is present in neurons of the subiculum and entorhinal cortex. In contrast, in the Alzheimer disease hippocampal formation, the levels of amyloid-β-protein mRNA in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-β-protein gene expression may be altered in Alzheimer disease

  16. Design, Engineering and Application of an Amyloidogenic Protein, SBAFP-m1, for use in Nanotechnological Applications

    Science.gov (United States)

    Peralta, Maria del Refugio

    Nanotechnology relies on collaborations across scientific disciplines such as physics, engineering, chemistry and biology. In nanotechnology, researchers manipulate molecules on the nanometer scale for various applications, ranging from tissue engineering, nanowire synthesis, and alternative energy devices. By utilizing various biological scaffolds, namely amyloid fibrils, the work of nanometer molecular control can be achieved through the use of self-assembly systems. Here, a systematic design scheme was developed to engineer protein based amyloid fibrils and was successfully applied to the design of two, unique self-assembled monomers, SBAFP-m1 and RGAFP-m1, from naturally occurring ice binding proteins found in insects and plants. A highly idealized, in-register dimer interface was designed and experimentally synthesized and demonstrated to form micron long amyloid fibrils (Chapter 2). The strength and resistance of the designer amyloid fibrils formed by SBAFP-m1 were probed in Chapter 3. Most notably, the ultimate tensile strength of SBAFP-m1 fibrils was experimentally determined to be 2.1 +/- 1.7 GPa, on par with that of naturally occurring amyloid fibrils in literature and steel. The fibrils were found to maintain their beta-sheet structure over a wide range of temperatures, from - 80 °C to 90 °C. Fibrils were resistant to common protein denaturants like 8M urea, 2.5 M guanidine hydrochloride, 2.5 M NaCl, organic solvents (methanol, ethanol, isopropanol and acetone), and across the pH range two to 11. SBAFP-m1 was mutated to add a 5x cysteine tag to the N-terminus, allowing for gold nanoparticle conjugation along the fibril axis (Chapter 4). The gold-conjugated fibrils were then enhanced with silver to produce nanowires. Various attempts to selectively synthesize heterogeneous fibrils from SBAFP-m1 mutants were attempted in Chapter 5. An attempt to de-stabilize the homogeneous fibril assembly through unfavorable homogeneous protein interactions was not

  17. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    International Nuclear Information System (INIS)

    Keryer-Bibens, Cecile; Legagneux, Vincent; Namanda-Vanderbeken, Allen; Cosson, Bertrand; Paillard, Luc; Poncet, Didier; Osborne, H. Beverley

    2009-01-01

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  18. Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

    Directory of Open Access Journals (Sweden)

    Valentina Iadevaia

    2015-09-01

    Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

  19. Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation.

    Science.gov (United States)

    Corduan, Aurélie; Plé, Hélène; Laffont, Benoit; Wallon, Thérèse; Plante, Isabelle; Landry, Patricia; Provost, Patrick

    2015-05-01

    Platelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3'UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of proteinmRNA complexes rather than disassembly of Ago2microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation.

  20. Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling

    Directory of Open Access Journals (Sweden)

    Tzviya Zeev-Ben-Mordehai

    2015-12-01

    Full Text Available Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC, which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling.

  1. NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

    Science.gov (United States)

    Alagarsamy, Sudar; Saugstad, Julie; Warren, Lee; Mansuy, Isabelle M.; Gereau, Robert W.; Conn, P. Jeffrey

    2010-01-01

    Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor. 2005 Elsevier Ltd. All rights reserved. PMID:16005030

  2. Serum protein-binding ability of sup(99m)Tc-diethyl IDA and sup(99m)Tc-para-butyl IDA studied by electrophoresis and precipitation experiments

    International Nuclear Information System (INIS)

    Sawas-Dimopoulou, C.; Simitzis, G.; Papanicolaou, N.

    1982-01-01

    The aim of the study was to separate and to identify by electrophoresis the proteins which are able to bind 99mTc-diethyl IDA and 99mTc-p-butyl IDA when these radiopharmaceuticals are incubated in vitro with human serum. The determinations were completed by precipitation experiments. Electrophoresis showed that both radiopharmaceuticals have negative charge and move to the anode like organic anions. And it is known that organic anions can interact with cationic groups on the albumin. Moreover, it is generally accepted that the extent to which a drug is bound to a particular protein depends on the concentration of the drug. Consequently, the logical hypothesis that binding of each radiopharmaceutical to the various serum proteins could be easier demonstrated on autoradiographies and electrophoregrams by high concentrations of 99mTc-diethyl IDA and 99mTc-parabutyl IDA in the incubation medium was put to trial

  3. Planning for a Distributed Disruption: Innovative Practices for Incorporating Distributed Solar into Utility Planning

    Energy Technology Data Exchange (ETDEWEB)

    Mills, Andrew D.; Barbose, Galen L.; Seel, Joachim; Dong, Changgui; Mai, Trieu; Sigrin, Ben; Zuboy, Jarett

    2016-08-01

    The rapid growth of distributed solar photovoltaics (DPV) has critical implications for U.S. utility planning processes. This report informs utility planning through a comparative analysis of roughly 30 recent utility integrated resource plans or other generation planning studies, transmission planning studies, and distribution system plans. It reveals a spectrum of approaches to incorporating DPV across nine key planning areas, and it identifies areas where even the best current practices might be enhanced. (1) Forecasting DPV deployment: Because it explicitly captures several predictive factors, customer-adoption modeling is the most comprehensive forecasting approach. It could be combined with other forecasting methods to generate a range of potential futures. (2) Ensuring robustness of decisions to uncertain DPV quantities: using a capacity-expansion model to develop least-cost plans for various scenarios accounts for changes in net load and the generation portfolio; an innovative variation of this approach combines multiple per-scenario plans with trigger events, which indicate when conditions have changed sufficiently from the expected to trigger modifications in resource-acquisition strategy. (3) Characterizing DPV as a resource option: Today's most comprehensive plans account for all of DPV's monetary costs and benefits. An enhanced approach would address non-monetary and societal impacts as well. (4) Incorporating the non-dispatchability of DPV into planning: Rather than having a distinct innovative practice, innovation in this area is represented by evolving methods for capturing this important aspect of DPV. (5) Accounting for DPV's location-specific factors: The innovative propensity-to-adopt method employs several factors to predict future DPV locations. Another emerging utility innovation is locating DPV strategically to enhance its benefits. (6) Estimating DPV's impact on transmission and distribution investments: Innovative

  4. Validation of the FACT-B+4-UL questionnaire and exploration of its predictive value in women submitted to surgery for breast cancer.

    Science.gov (United States)

    Andrade Ortega, Juan Alfonso; Millán Gómez, Ana Pilar; Ribeiro González, Marisa; Martínez Piró, Pilar; Jiménez Anula, Juan; Sánchez Andújar, María Belén

    2017-06-21

    The early detection of upper limb complications is important in women operated on for breast cancer. The "FACT-B+4-UL" questionnaire, a specific variant of the Functional Assessment of Cancer Therapy-Breast (FACT-B) is available among others to measure the upper limb function. The Spanish version of the upper limb subscale of the FACT-B+4 was validated in a prospective cohort of 201 women operated on for breast cancer (factor analysis, internal consistency, test-retest reliability, construct validity and sensitivity to change were determined). Its predictive capacity of subsequent lymphoedema and other complications in the upper limb was explored using logistic regression. This subscale is unifactorial and has a great internal consistency (Cronbach's alpha: 0.87), its test-retest reliability and construct validity are strong (intraclass correlation coefficient: 0.986; Pearson's R with "Quick DASH": 0.81) as is its sensitivity to change. It didn't predict the onset of lymphedema. Its predictive capacity for other upper limb complications is low. FACT-B+4-UL is useful in measuring upper limb disability in women surgically treated for breast cancer; but it does not predict the onset of lymphoedema and its predictive capacity for others complications in the upper limb is low. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

  5. Levels of protein hydroperoxides and carbonyl groups in guinea pigs native of high altitudes (Huancavelica, 3660 m)

    OpenAIRE

    Huayta, Roxana; Zúñiga, Haydée; Esquerre, Cynthia; Hernández, Luz; Carranza, Elizabeth

    2014-01-01

    The influence of hypobaric hypoxia on protein oxidation in lungs, heart, liver, kidneys and testicles of high altitude native guinea pigs (Huancavelica, 3660 m) in comparison to sea level (Lima, 150 m) native guinea pigs was evaluated. The concentration of protein hydroperoxides (POOH) and carbonyl groups (GC) as markers of protein oxidation, as well as total thiols (TT) concentration, powerful reducing agents that act as live antioxidants were determined. The results showed low concentration...

  6. Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures.

    Directory of Open Access Journals (Sweden)

    Kerstin Radtke

    2010-07-01

    Full Text Available Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1 show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during

  7. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome

    Energy Technology Data Exchange (ETDEWEB)

    Heilbronn, R.; zur Hausen, H. (Deutsches Krebsforschungszentrum, Heidelberg (West Germany))

    1989-09-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of sic HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensable for SV40 DNA amplification. The results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.

  8. mPLR-Loc: an adaptive decision multi-label classifier based on penalized logistic regression for protein subcellular localization prediction.

    Science.gov (United States)

    Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

    2015-03-15

    Proteins located in appropriate cellular compartments are of paramount importance to exert their biological functions. Prediction of protein subcellular localization by computational methods is required in the post-genomic era. Recent studies have been focusing on predicting not only single-location proteins but also multi-location proteins. However, most of the existing predictors are far from effective for tackling the challenges of multi-label proteins. This article proposes an efficient multi-label predictor, namely mPLR-Loc, based on penalized logistic regression and adaptive decisions for predicting both single- and multi-location proteins. Specifically, for each query protein, mPLR-Loc exploits the information from the Gene Ontology (GO) database by using its accession number (AC) or the ACs of its homologs obtained via BLAST. The frequencies of GO occurrences are used to construct feature vectors, which are then classified by an adaptive decision-based multi-label penalized logistic regression classifier. Experimental results based on two recent stringent benchmark datasets (virus and plant) show that mPLR-Loc remarkably outperforms existing state-of-the-art multi-label predictors. In addition to being able to rapidly and accurately predict subcellular localization of single- and multi-label proteins, mPLR-Loc can also provide probabilistic confidence scores for the prediction decisions. For readers' convenience, the mPLR-Loc server is available online (http://bioinfo.eie.polyu.edu.hk/mPLRLocServer). Copyright © 2014 Elsevier Inc. All rights reserved.

  9. The Relationship Between Domestic Partner Violence and Suicidal Behaviors in an Adult Community Sample: Examining Hope Agency and Pathways as Protective Factors.

    Science.gov (United States)

    Chang, Edward C; Yu, Elizabeth A; Kahle, Emma R; Du, Yifeng; Chang, Olivia D; Jilani, Zunaira; Yu, Tina; Hirsch, Jameson K

    2017-10-01

    We examined an additive and interactive model involving domestic partner violence (DPV) and hope in accounting for suicidal behaviors in a sample of 98 community adults. Results showed that DPV accounted for a significant amount of variance in suicidal behaviors. Hope further augmented the prediction model and accounted for suicidal behaviors beyond DPV. Finally, we found that DPV significantly interacted with both dimensions of hope to further account for additional variance in suicidal behaviors above and beyond the independent effects of DPV and hope. Implications for the role of hope in the relationship between DPV and suicidal behaviors are discussed.

  10. Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.

    Science.gov (United States)

    Asenjo, Ana; Villanueva, Nieves

    2016-01-04

    The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Frequent Cross-Resistance to Dapivirine in HIV-1 Subtype C-Infected Individuals after First-Line Antiretroviral Therapy Failure in South Africa.

    Science.gov (United States)

    Penrose, Kerri J; Wallis, Carole L; Brumme, Chanson J; Hamanishi, Kristen A; Gordon, Kelley C; Viana, Raquel V; Harrigan, P Richard; Mellors, John W; Parikh, Urvi M

    2017-02-01

    A vaginal ring containing dapivirine (DPV) has shown moderate protective efficacy against HIV-1 acquisition, but the activity of DPV against efavirenz (EFV)- and nevirapine (NVP)-resistant viruses that could be transmitted is not well defined. We investigated DPV cross-resistance of subtype C HIV-1 from individuals on failing NVP- or EFV-containing antiretroviral therapy (ART) in South Africa. Plasma samples were obtained from individuals with >10,000 copies of HIV RNA/ml and with HIV-1 containing at least one non-nucleoside reverse transcriptase (NNRTI) mutation. Susceptibility to NVP, EFV, and DPV in TZM-bl cells was determined for recombinant HIV-1 LAI containing bulk-amplified, plasma-derived, full-length reverse transcriptase sequences. Fold change (FC) values were calculated compared with a composite 50% inhibitory concentration (IC 50 ) from 12 recombinant subtype C HIV-1 LAI plasma-derived viruses from treatment-naive individuals in South Africa. A total of 25/100 (25%) samples showed >500-FCs to DPV compared to treatment-naive samples with IC 50 s exceeding the maximum DPV concentration tested (132 ng/ml). A total of 66/100 (66%) samples displayed 3- to 306-FCs, with a median IC 50 of 17.6 ng/ml. Only 9/100 (9%) samples were susceptible to DPV (FC 500-fold resistance to DPV compared to samples with a ≤500-fold resistance. A total of 91% of samples with NNRTI-resistant HIV-1 from individuals on failing first-line ART in South Africa exhibited ≥3-fold cross-resistance to DPV. This level of resistance exceeds expected plasma concentrations, but very high genital tract DPV concentrations from DPV ring use could block viral replication. It is critically important to assess the frequency of transmitted and selected DPV resistance in individuals using the DPV ring. Copyright © 2017 American Society for Microbiology.

  12. Imaging diagnosis of protein-losing enteropathy by 99mTc-labeled serum albumin

    International Nuclear Information System (INIS)

    Kashiwagi, Toru; Fukui, Hiroyuki; Jyokou, Takeshi

    1990-01-01

    Abdominal scintigraphy with intravenous injection of 99m Tc-labeled serum albumin was performed in 6 patients with protein-losing enteropathy (PLE) and 3 patients with nongastrointestinal tract disorders. In 3 out of 6 patients with PLE, abnormal radioactivity was observed in the ileum region 3 hours after injection, and thereafter clear colon image was obtained. In the remaining 3 patients, the colon was visualized 24 hours after injection. On the other hand, in all patients with nongastrointestinal tract disorders, no abnormal radioactivity was observed in the abdomen until 24 hours after injection. These results indicate that gastrointestinal protein loss could be demonstrated by scintigraphy with intravenously administered 99m Tc-labeled serum albumin. In one healthy subject, 99m Tc-labeled serum albumin was administered orally and abdominal scintigraphy was performed. Gastrointestinal tract image was only observed and no other image was demonstrated until 24 hours after oral administration. This result suggests that 99m Tc excreted into the gastrointestinal tract is not reabsorbed. Therefore, abdominal scintigraphy with 99m Tc-labeled serum albumin appears to be a simple and useful method for diagnosis of PLE. (author)

  13. Simultaneous detection of mRNA and protein stem cell markers in live cells

    Directory of Open Access Journals (Sweden)

    Bao Gang

    2009-04-01

    Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

  14. Effect of low-dose irradiation on expression of mRNA and protein. Pt.1. Induction of thioredoxin as radioprotective protein in human lymphocytes

    International Nuclear Information System (INIS)

    Hoshi, Yuko; Tanooka, Hiroshi; Wakasugi, Hiro; Miyasaki, Kunihisa

    1997-01-01

    To elucidate the mechanism of hormetic effect by low-dose ionizing radiation, we studied the expression of the thioredoxin (TRX) gene in human lymphocytes after irradiation. TRX is a radioprotector and a key protein regulating cellular functions through redox reaction. The major results obtained were as follows; (1) The peaks of TRX mRNA expression and protein synthesis in human lymphocytes appeared 6-8 hr after irradiation with 25cGy. (2) At 6 hr after irradiation, the optimum dose for induction of TRX mRNA and TRX protein in human lymphocytes appeared to be 25-50cGy. (3) Induction of expression TRX mRNA had individual variations about twice. (4) Lymphocytes prepared from fresh venous blood showed the lowest TRX mRNA level in other cells such a Jurkat cells, lymphocytes stimulated for now with IL-2 and CD3 and the immortalized cell line 1G8. (5) The optimal dose and time course of induction of TRX by low-dose radiation suggest that TRX is related to the radio-adaptive response. (author)

  15. Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo.

    OpenAIRE

    Demangeat, Gerard; Hemmer, O; Reinbolt, J; Mayo, M A; Fritsch, Coralie

    1992-01-01

    The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M(r) 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a sta...

  16. The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice

    Science.gov (United States)

    You, Jae-Sung; Anderson, Garrett B.; Dooley, Matthew S.; Hornberger, Troy A.

    2015-01-01

    ABSTRACT The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate

  17. Kinetic study on ligand exchange reaction between ethylenedicysteine and 99mTc- glucoheptonate (99mTc-GH)

    International Nuclear Information System (INIS)

    Wu, C.Y.; Ji, S.R.; Lu, C.X.; Ding, S.Y.; Chen, Z.P.; Lin, X.T.

    2002-01-01

    Aim: 99m Tc-L,L-ethylenedicysteine( 99m Tc-EC)is a new type of renal imaging agent. It can be labeled very easily and efficiently at room temperature through direct labeling at pH 12. The need for direct labeling at pH 12 does not compromise the simplicity and ease of preparation of 99m Tc-EC and its practical usefulness in daily routine. On the basis of the labeling experiments, we developed a ligand exchange labeling method, in which the labeling EC with 99m Tc can be performed at pH 8. In order to provide a theoretic basis, a detailed kinetic study of ligand exchange reaction between 99m Tc- glucoheptonate( 99m Tc-GH) and EC was carried out. Materials and Methods: 99m Tc-EC is prepared as follows: 99m Tc-GH + EC → 99m Tc-EC + GH, labeling can be easily performed by adding 99m TcO 4 - (2∼6ml generator elute) to glucoheptonate solution containing SnCl 2 .2H 2 O solution to form 99m Tc-GH, then freshly prepared 99m Tc-GH is transferred to the aqueous solution of different concentrations of EC at different pH value, after being shaken, 99m Tc-EC was formed. Radiolabeling yield(RLY) and radiochemical purity(RCP) of 99m Tc-GH and 99m Tc-EC were measured by Xinhua No.1 paper with developing system of Me 2 CO/H 2 O/con.NH 3 .H 2 O=9/3/1(V/V). 99m Tc-GH(RCP must be over 98%, 80ul, 3.6∼7.4MBq) was added to 1ml of 0.5mol/L phosphate buffer(pH 12) containing different amount of EC(150, 75, 50 and 15ug), the sample was taken out at different time intervals and RCP was determined. The solution of EC(30ul, 5g/L) was added to 1ml of 0.5mol/L phosphate buffer at different pH value(pH11, 10, 9, 8, 7), after completely vortexed, 99m Tc-GH(RCP must be over 98%, 80ul, 3.6∼7.4MBq) was then added, the sample was taken out and RCP was determined as above. The rate constant(k) of ligand exchange reaction at different concentrations of EC and different reaction pH values were calculated out by integrating. Plot ln[1/(1-RLY)] vs t(time) showed a liner relationship, and the rate

  18. Features of Two New Proteins with OmpA-Like Domains Identified in the Genome Sequences of Leptospira interrogans

    Science.gov (United States)

    Teixeira, Aline F.; de Morais, Zenaide M.; Kirchgatter, Karin; Romero, Eliete C.; Vasconcellos, Silvio A.; Nascimento, Ana Lucia T. O.

    2015-01-01

    Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG)-interacting proteins, capable of generating plasmin (PLA) and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis. PMID:25849456

  19. Features of two new proteins with OmpA-like domains identified in the genome sequences of Leptospira interrogans.

    Directory of Open Access Journals (Sweden)

    Aline F Teixeira

    Full Text Available Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively. Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG-interacting proteins, capable of generating plasmin (PLA and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis.

  20. Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

    Science.gov (United States)

    Kristensen, Bodil M.; Olsen, John E.; Harris, Claire L.; Ufret-Vincenty, Rafael L.; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

    2013-01-01

    Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems. PMID:23637608

  1. Factor H binds to the hypervariable region of many Streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence.

    Directory of Open Access Journals (Sweden)

    Mattias C U Gustafsson

    Full Text Available Many pathogens express a surface protein that binds the human complement regulator factor H (FH, as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

  2. A rapid chemical method of labelling human plasma proteins with sup(99m)Tc-pertechnetate at pH 7.4

    International Nuclear Information System (INIS)

    Wong, D.W.; Mishkin, F.; Lee, T.

    1978-01-01

    A successful method for labelling human plasma proteins with sup(99m)Tc-pertechnetate by chemical means is described. The labelling methodology involves the production of Sup(99m)Tc-(Sn)citrate complex species with high protein binding capacity at pH 7.4 condition following initial chemical reduction of sodium sup(99m)Tc-pertechnetate by stannous chloride. A combined labelling efficiency range of 95-99% for sup(99m)Tc-labelled fibrinogen, immune gamma globulin and serum albumin is achieved. The actual amount of labelled protein content in the product is found to be 85-95% when assayed by ITLC and 74-85% by TCAA protein precipitation. In vitro experimental data indicate that sup(99m)Tc-fibrinogen contains an average of 85% clottable protein with an average clottability of 95%. This strongly suggests that the radioactive proteins retain much of their biological and physiological activities after the labelling process. (author)

  3. Haematological and biochemical parameters in Churra-da-Terra-Quente ewes from the northeast of Portugal Parâmetros hematológicos e bioquímicos de ovelhas da raça Churra da Terra Quente do nordeste de Portugal

    Directory of Open Access Journals (Sweden)

    I.R. Dias

    2010-04-01

    Full Text Available Hematological and biochemical parameters, including plasma electrolytes and thyroid hormones, were determined in 73 clinically healthy Churra-da-Terra-Quente ewes, a typical breed from the northeast of Portugal. The hemogram values were: erythrocytes 9.8±1.5×10(12/L; haemoglobin 118.1±19.1g/L; haematocrit 40.8±5.9%; leukocytes 5.7±1.8×10(9/L; and platelets 544.3±177.2×10(9/L. The thrombin time was 17.3±1.7 seconds. The values of biochemical parameters were: total protein 76.4±6.1g/L; glucose 2.87±0.60mmol/L; total cholesterol 1.65±0.33mmol/L; aspartate aminotransferase 155.9±49.2U/L; alanine aminotransferase 23.2±9.6U/L; γ-glutamyl transferase 48.0±18.7U/L; total alkaline phosphatase 121.6±76.1U/L; glutamate dehydrogenase 6.4±3.7U/L; urea 7.32±2.22mmol/L; creatinine 123.0±54.1μmol/L; total calcium 2.53±0.25mmol/L; phosphorus 2.10±0.46mmol/L; magnesium 1.01±0.09mmol/L; sodium 152.04±3.65mmol/L; potassium 4.7±0.4mmol/L; ionized calcium 1.32±0.07mmol/L; total thyroxine 111.75±42.29nmol/L; total triiodothyronine 1.01±0.28nmol/L; free T4 11.93±1.78pmol/L; free T3 4.22±1.33pmol/L; and thyroid-stimulating hormone 0.18±0.19μIU/mL. Although differences among the Churra-da-Terra-Quente breed and other breeds may occur, the hematological and biochemical parameters, plasma electrolytes, and thyroid hormones, for this indigenous breed, were generally situated within the reference intervals previously reported for sheep.Os valores hematológicos e bioquímicos, incluindo os eletrólitos plasmáticos e os hormônios da tireoide, foram determinados em 73 ovelhas, clinicamente saudáveis, da raça Churra da Terra Quente, raça ovina característica do nordeste de Portugal. Os valores obtidos para o hemograma foram: eritrócitos 9,8±1,5×10(12 /L; hemoglobina 118,1±19,1g/L; hematócrito 40,8±5,9%; leucócitos 5,7±1,8×10(9 /L e plaquetas 544,3±177,2×10(9/L. O tempo de trombina foi de 17,3±1,7 segundos. Os valores dos par

  4. Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo.

    Science.gov (United States)

    Demangeat, G; Hemmer, O; Reinbolt, J; Mayo, M A; Fritsch, C

    1992-07-01

    The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M(r) 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a stable 120K protein and, only in protoplasts, small amounts of a 90K protein which contains the C-terminal part of the 120K protein and the polymerase domain. The results suggest that the polymerase and the adjacent protease function in vivo largely or solely when combined in a 120K protein. The proteins derived from the RNA-2-encoded polyprotein detected by immunoblotting were 59K and 57K proteins, which reacted with antiserum to TBRV particles, and a 46K protein. In extracts of infected Nicotiana clevelandii and Chenopodium quinoa made soon after inoculation, the 59K protein was more abundant than the 57K protein; later samples contained similar quantities of each protein. The 57K protein comigrated with protein extracted from virus particles. The results of amino acid sequencing suggested that the 57K protein is derived from the 59K protein by the loss of nine C-terminal amino acids. Antiserum to a peptide adjacent to the 57K protein in the 150K polyprotein detected a 46K protein in protoplasts and plant tissue. The results support the processing scheme for TBRV polyproteins proposed after analysis of the products of in vitro translation.

  5. Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis

    DEFF Research Database (Denmark)

    Rosenstierne, Maiken W; Vinther, Jeppe; Mittler, Gerhard

    2008-01-01

    CaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy. RESULTS: The wild...... irradiation. Several proteins (including GAPDH, heterogeneous nuclear ribonucleoprotein (hnRNP) D and A/B) were identified from the MCF-7 cytoplasmic extracts that bound specifically to the CPEs. CONCLUSION: Two conserved CPEs in the p53 3'UTR regulate stability and translation of a reporter mRNA in non...

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  7. Metabotropic Glutamate Receptor I (mGluR1) Antagonism Impairs Cocaine-Induced Conditioned Place Preference via Inhibition of Protein Synthesis

    OpenAIRE

    Yu, Fei; Zhong, Peng; Liu, Xiaojie; Sun, Dalong; Gao, Hai-qing; Liu, Qing-song

    2013-01-01

    Antagonism of group I metabotropic glutamate receptors (mGluR1 and mGluR5) reduces behavioral effects of drugs of abuse, including cocaine. However, the underlying mechanisms remain poorly understood. Activation of mGluR5 increases protein synthesis at synapses. Although mGluR5-induced excessive protein synthesis has been implicated in the pathology of fragile X syndrome, it remains unknown whether group I mGluR-mediated protein synthesis is involved in any behavioral effects of drugs of abus...

  8. Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

    International Nuclear Information System (INIS)

    Han, Mingyuan; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan

    2017-01-01

    Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of 126 -LQxxLxxxGL- 135 . In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine arteritis virus.

  9. Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

    Energy Technology Data Exchange (ETDEWEB)

    Han, Mingyuan, E-mail: hanming@umich.edu; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan, E-mail: dyoo@illinois.edu

    2017-05-15

    Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of {sub 126}-LQxxLxxxGL-{sub 135}. In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine

  10. USING THE FOURNIER INDEXES IN ESTIMATING RAINFALL EROSIVITY. CASE STUDY - THE SECAŞUL MARE BASIN

    Directory of Open Access Journals (Sweden)

    M. COSTEA

    2012-03-01

    Full Text Available Using the Fournier Index in Estimating Rainfall Erosivity. Case Study - The Secaşul Mare Basin. Climatic aggressiveness is one of the most important factors in relief dynamic. Of all climatic parameters, rainfall is directly involved in versant dynamic, in the loss of soil quality and through pluvial denudation and the processes associated with it, through the erosivity of torrential rain. We analyzed rainfall aggressiveness based on monthly and annual average values through the Fournier's index (1970 and Fournier's index modified by Arnoldus (1980. They have the advantage that they can be used not only for evaluating the land susceptibility to erosion and the calculation of erodibility of land and soil losses, but also in assessing land susceptibility to sliding (Aghiruş, 2010. The literature illustrates the successful use of this index which provides a summary assessment of the probability of rainfall with significant erosive effects. The results obtained allow observation of differences in space and time of the distribution of this index.

  11. Characterization of an electrochemical mercury sensor using alternating current, cyclic, square wave and differential pulse voltammetry

    Energy Technology Data Exchange (ETDEWEB)

    Guerreiro, Gabriela V.; Zaitouna, Anita J.; Lai, Rebecca Y., E-mail: rlai2@unl.edu

    2014-01-31

    Graphical abstract: -- Highlights: •An electrochemical Hg(II) sensor based on T–Hg(II)–T sensing motif was fabricated. •A methylene blue-modified DNA probe was used to fabricate the sensor. •Sensor performance was evaluated using ACV, CV, SWV, and DPV. •The sensor behaves as a “signal-off” sensor in ACV and CV. •The sensor behaves as either a “signal-on” or “signal-off” sensor in SWV and DPV. -- Abstract: Here we report the characterization of an electrochemical mercury (Hg{sup 2+}) sensor constructed with a methylene blue (MB)-modified and thymine-containing linear DNA probe. Similar to the linear probe electrochemical DNA sensor, the resultant sensor behaved as a “signal-off” sensor in alternating current voltammetry and cyclic voltammetry. However, depending on the applied frequency or pulse width, the sensor can behave as either a “signal-off” or “signal-on” sensor in square wave voltammetry (SWV) and differential pulse voltammetry (DPV). In SWV, the sensor showed “signal-on” behavior at low frequencies and “signal-off” behavior at high frequencies. In DPV, the sensor showed “signal-off” behavior at short pulse widths and “signal-on” behavior at long pulse widths. Independent of the sensor interrogation technique, the limit of detection was found to be 10 nM, with a linear dynamic range between 10 nM and 500 nM. In addition, the sensor responded to Hg{sup 2+} rather rapidly; majority of the signal change occurred in <20 min. Overall, the sensor retains all the characteristics of this class of sensors; it is reagentless, reusable, sensitive, specific and selective. This study also highlights the feasibility of using a MB-modified probe for real-time sensing of Hg{sup 2+}, which has not been previously reported. More importantly, the observed “switching” behavior in SWV and DPV is potentially generalizable and should be applicable to most sensors in this class of dynamics-based electrochemical biosensors.

  12. Dynamic Proteomics Emphasizes the Importance of Selective mRNA Translation and Protein Turnover during Arabidopsis Seed Germination*

    Science.gov (United States)

    Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

    2014-01-01

    During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [35S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment. PMID:24198433

  13. microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein.

    Science.gov (United States)

    Pinder, Benjamin D; Smibert, Craig A

    2013-01-01

    Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

  14. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    International Nuclear Information System (INIS)

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-01-01

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

  15. The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.

    Science.gov (United States)

    Sirena, Dominique; Lilienfeld, Benjamin; Eisenhut, Markus; Kälin, Stefan; Boucke, Karin; Beerli, Roger R; Vogt, Lorenz; Ruedl, Christiane; Bachmann, Martin F; Greber, Urs F; Hemmi, Silvio

    2004-05-01

    Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.

  16. 46 CFR 114.600 - Incorporation by reference.

    Science.gov (United States)

    2010-10-01

    ... Test Method for Rate of Burning and/or Extent and Time of Burning of Plastics in a Horizontal Position... Burning Characteristics of Building Materials 116.405; 116.422; 116.423 ASTM E 648-97, Standard Test... Storage Tank Water Heaters 119.320 UL 486A-1992—Wire Connectors and Soldering Lugs For Use With Copper...

  17. Role of a reducing environment in disassembly of the herpesvirus tegument

    Energy Technology Data Exchange (ETDEWEB)

    Newcomb, William W. [Department of Microbiology Immunology and Cancer Biology, University of Virginia Health System, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave. Charlottesville, VA 22908 (United States); Jones, Lisa M. [Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130 (United States); Dee, Alexander [Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030 (United States); Chaudhry, Farid [Department of Microbiology Immunology and Cancer Biology, University of Virginia Health System, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave. Charlottesville, VA 22908 (United States); Brown, Jay C., E-mail: JCB2G@VIRGINIA.EDU [Department of Microbiology Immunology and Cancer Biology, University of Virginia Health System, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave. Charlottesville, VA 22908 (United States)

    2012-09-15

    Initiation of infection by herpes family viruses involves a step in which most of the virus tegument becomes detached from the capsid. Detachment takes place in the host cell cytosol near the virus entry site and it is followed by dispersal of tegument proteins and disappearance of the tegument as a distinct entity. Here we describe the results of experiments designed to test the idea that the reducing environment of the cytosol may contribute to tegument detachment and disassembly. Non-ionic detergent was used to remove the membrane of purified herpes simplex virus under control and reducing conditions. The effects on the tegument were then examined by SDS-PAGE and electron microscopy. Protein analysis demonstrated that most major tegument proteins were removed under both oxidizing and reducing conditions except for UL49 which required a reducing environment. It is proposed therefore that the reducing conditions in the cytosol are involved in removal of UL49 protein. Electron microscopic analysis revealed that capsids produced under oxidizing conditions contained a coating of protein that was absent in reduced virions and which correlated uniquely with the presence of UL49. This capsid-associated layer is suggested to be the location of UL49 in the extracted virion.

  18. A new method for 99mTc-labelling of proteins, leucocytes and platelets for nuclear medicine application

    International Nuclear Information System (INIS)

    Sundrehagen, E.

    1984-01-01

    A reduced state of 99mTc was obtained by concentrated hydrocloric acid treatment of the 99mTc(VII)/0.15 M NaCl eluate from 99Mo/99mTc generators. Non-acidic reduced state of 99mTc in dry NaCl deposit was obtained by vacuum evaporation of concentrated HCl and water. A monitored vacuum evaporator built for this purpose is presented, as well as methods of formation of various 99mTc-protein and 99mTc-polypeptide complexes. After careful protein precipation or anionic adsorption of pertechnetate and 99mTc-gentisic acid complexes, high radiochemical purities of labelled proteins were demonstrated by gel chromatography studies, radioimmunological methods, radioaffinity testing studies and ampholyte displacement radiochromatography. Preparative methods for 99mTc-plasmin (at pH=2), 99mTc-secretin (at pH=3) and 99mTc-IgG (at pH=4) are presented. The role and the limitations of 99mTc-plasmin for diagnosis of deep vein thrombosis were investigated in experimentally induced jugular vein thrombosis in rabbits. The in vivo distribution of intravenously injected 99mTc-secretin was found to be in correspondance with that of unlabelled secretin. Labelling of platelets and leucoytes from human blood with 99mTc was carried out at pH=7.2. Data for a remarkable high stability of the labelled cells are presented

  19. Metabotropic glutamate receptor I (mGluR1) antagonism impairs cocaine-induced conditioned place preference via inhibition of protein synthesis.

    Science.gov (United States)

    Yu, Fei; Zhong, Peng; Liu, Xiaojie; Sun, Dalong; Gao, Hai-Qing; Liu, Qing-Song

    2013-06-01

    Antagonism of group I metabotropic glutamate receptors (mGluR1 and mGluR5) reduces behavioral effects of drugs of abuse, including cocaine. However, the underlying mechanisms remain poorly understood. Activation of mGluR5 increases protein synthesis at synapses. Although mGluR5-induced excessive protein synthesis has been implicated in the pathology of fragile X syndrome, it remains unknown whether group I mGluR-mediated protein synthesis is involved in any behavioral effects of drugs of abuse. We report that group I mGluR agonist DHPG induced more pronounced initial depression of inhibitory postsynaptic currents (IPSCs) followed by modest long-term depression (I-LTD) in dopamine neurons of rat ventral tegmental area (VTA) through the activation of mGluR1. The early component of DHPG-induced depression of IPSCs was mediated by the cannabinoid CB1 receptors, while DHPG-induced I-LTD was dependent on protein synthesis. Western blotting analysis indicates that mGluR1 was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation. We also show that cocaine conditioning activated translation machinery in the VTA via an mGluR1-dependent mechanism. Furthermore, intra-VTA microinjections of mGluR1 antagonist JNJ16259685 and protein synthesis inhibitor cycloheximide significantly attenuated or blocked the acquisition of cocaine-induced conditioned place preference (CPP) and activation of translation elongation factors. Taken together, these results suggest that mGluR1 antagonism inhibits de novo protein synthesis; this effect may block the formation of cocaine-cue associations and thus provide a mechanism for the reduction in CPP to cocaine.

  20. PURIFICATION OF ANGIOTENSIN CONVERTING ENZYME INHIBITORY PEPTIDE DERIVED FROM KACANG GOAT MEAT PROTEIN HYDROLYSATE

    Directory of Open Access Journals (Sweden)

    J. Jamhari

    2014-10-01

    Full Text Available The objective of this study was to identify the Angiotensin Converting Enzyme (ACE inhibitorypeptide derived from Kacang goat meat protein hydrolysate. Kacang goat meat loin section washydrolyzed with pepsin, trypsin and chymotrypsin. Protein hydrolysate of Kacang goat meat was thentested the protein concentration and ACE inhibitory activity. ACE inhibitory peptide of the proteinhydrolysate was purified through several steps of purification by column SEP-PAK Plus C18 Cartridgeand RP-HPLC using a Cosmosil column 5PE-SM, 4.6 x 250 mm. The sequence of amino acid of ACEinhibitory peptide was identified by amino acid sequencer. The results showed that amino acidssequence of ACE inhibitory peptide derived from protein hydrolysate of Kacang goat meat was leu-thrglu-ala-pro-leu-asn-pro-lys-ala-arg- asn-glu-lys. It had a molecular weight (MW of 1581 and occurredat the position of 20th to 33rd residues of b-actin of goat meat protein (Capra hircus. The ACE inhibitoryactivity (IC50 of the peptide was 190 mg/mL or 120 mM.

  1. Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins

    International Nuclear Information System (INIS)

    Zullo, J.; Stiles, C.D.; Garcea, R.L.

    1987-01-01

    The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus. Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection. At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter. Empty virions (capsids) and recombinant VP 1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation. Virions, capsids, and recombinant VP 1 protein stimulated [ 3 H]thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma. The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition. These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for the cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins

  2. Crystallization and preliminary crystallographic studies of L30e, a ribosomal protein from Methanocaldococcus jannaschii (MJ1044)

    International Nuclear Information System (INIS)

    Rangarajan, Sarani; Jeyakanthan, Jeyaraman; Mridula, Palappetty; Sakamoto, Keiko; Kitamura, Yoshiaki; Agari, Yoshihiro; Shinkai, Akeo; Ebihara, Akio; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sekar, Kanagaraj

    2008-01-01

    The ribosomal protein (L30e) from M. jannaschii was cloned from the gene MJ1044, expressed, purified and crystallized. The crystal belongs to the primitive tetragonal space group P4 3 and diffracted to 1.9 Å resolution. In view of the biological significance of understanding the ribosomal machinery of both prokaryotes and eukaryotes, the L30e ribosomal protein from Methanocaldococcus jannaschii was cloned, overexpressed, purified and crystallized using the microbatch-under-oil method with the crystallization conditions 40% PEG 400, 0.1 M MES pH 6.0 and 5% PEG 3000 at 291 K. A diffraction-quality crystal (0.20 × 0.20 × 0.35 mm) was obtained that belonged to the primitive tetragonal space group P4 3 , with unit-cell parameters a = 46.1, b = 46.1, c = 98.5 Å, and diffracted to a resolution of 1.9 Å. Preliminary calculations reveal that the asymmetric unit contains two monomers with a Matthews coefficient (V M ) of 2.16 Å 3 Da −1

  3. Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

    International Nuclear Information System (INIS)

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon

    2006-01-01

    The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K + channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments

  4. Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

    Science.gov (United States)

    Motohashi, Hiromi; Mukudai, Yoshiki; Ito, Chihiro; Kato, Kosuke; Shimane, Toshikazu; Kondo, Seiji; Shirota, Tatsuo

    2017-05-04

    Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  5. Synthesis of 17α-[(E)-2-[125I]iodoethenyl]androsta-4-6-dien-17β-o l-3-one, an active -site-directed photoaffinity radiolabel for androgen-binding proteins

    International Nuclear Information System (INIS)

    Diaz Cruz, P.J.; Smith, H.E.; Vanderbilt Univ., Nashville, TN; Danzo, B.J.; Clanton, J.A.; Mason, N.S.

    1993-01-01

    The active-site-directed photoaffinity radiolabel for androgen-binding proteins, 17α-[(E)-2-[ 125 I]iodoethenyl]androsta-4,6-dien-17β-ol-3-one, was prepared by reaction of 17α-[(E)-2-tributyltin(IV)ethenyl]androsta-4,6-dien-17β-ol-3-one with carrier added sodium iodide-125 in the presence of hydrogen peroxide and acetic acid. Purification by HPLC gave the radiolabeled steroid in 52% radiochemical yield with a specific activity of 27 Ci/mmol and 100% radiochemical purity. (author)

  6. Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

    Directory of Open Access Journals (Sweden)

    Matej Skočaj

    Full Text Available Ostreolysin A (OlyA is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II-Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1-positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

  7. In Vitro Quantified Determination of β-Amyloid 42 Peptides, a Biomarker of Neuro-Degenerative Disorders, in PBS and Human Serum Using a Simple, Cost-Effective Thin Gold Film Biosensor.

    Science.gov (United States)

    Dai, Yifan; Molazemhosseini, Alireza; Liu, Chung Chiun

    2017-07-20

    A simple in vitro biosensor for the detection of β-amyloid 42 in phosphate-buffered saline (PBS) and undiluted human serum was fabricated and tested based on our platform sensor technology. The bio-recognition mechanism of this biosensor was based on the effect of the interaction between antibody and antigen of β-amyloid 42 to the redox couple probe of K₄Fe(CN)₆ and K₃Fe(CN)₆. Differential pulse voltammetry (DPV) served as the transduction mechanism measuring the current output derived from the redox coupling reaction. The biosensor was a three-electrode electrochemical system, and the working and counter electrodes were 50 nm thin gold film deposited by a sputtering technique. The reference electrode was a thick-film printed Ag/AgCl electrode. Laser ablation technique was used to define the size and structure of the biosensor. Cost-effective roll-to-roll manufacturing process was employed in the fabrication of the biosensor, making it simple and relatively inexpensive. Self-assembled monolayers (SAM) of 3-Mercaptopropionic acid (MPA) was employed to covalently immobilize the thiol group on the gold working electrode. A carbodiimide conjugation approach using N -(3-dimethylaminopropyl)- N '-ethylcarbodiimide hydrochloride (EDC) and N -hydroxysuccinimide (NHS) was undertaken for cross-linking antibody of β-amyloid 42 to the carboxylic groups on one end of the MPA. The antibody concentration of β-amyloid 42 used was 18.75 µg/mL. The concentration range of β-amyloid 42 in this study was from 0.0675 µg/mL to 0.5 µg/mL for both PBS and undiluted human serum. DPV measurements showed excellent response in this antigen concentration range. Interference study of this biosensor was carried out in the presence of Tau protein antigen. Excellent specificity of this β-amyloid 42 biosensor was demonstrated without interference from other species, such as T-tau protein.

  8. On the Spatial Organization of mRNA, Plasmids, and Ribosomes in a Bacterial Host Overexpressing Membrane Proteins.

    Directory of Open Access Journals (Sweden)

    Lieke A van Gijtenbeek

    2016-12-01

    Full Text Available By using fluorescence imaging, we provide a time-resolved single-cell view on coupled defects in transcription, translation, and growth during expression of heterologous membrane proteins in Lactococcus lactis. Transcripts encoding poorly produced membrane proteins accumulate in mRNA-dense bodies at the cell poles, whereas transcripts of a well-expressed homologous membrane protein show membrane-proximal localization in a translation-dependent fashion. The presence of the aberrant polar mRNA foci correlates with cessation of cell division, which is restored once these bodies are cleared. In addition, activation of the heat-shock response and a loss of nucleoid-occluded ribosomes are observed. We show that the presence of a native-like N-terminal domain is key to SRP-dependent membrane localization and successful production of membrane proteins. The work presented gives new insights and detailed understanding of aberrant membrane protein biogenesis, which can be used for strategies to optimize membrane protein production.

  9. IGF2 mRNA-binding protein 2: biological function and putative role in type 2 diabetes

    DEFF Research Database (Denmark)

    Christiansen, J.; Kolte, A.M.; Hansen, T.O.

    2009-01-01

    Recent genome-wide association (GWA) studies of type 2 diabetes (T2D) have implicated IGF2 mRNA-binding protein 2 (IMP2/IGF2BP2) as one of the several factors in the etiology of late onset diabetes. IMP2 belongs to a family of oncofetal mRNA-binding proteins implicated in RNA localization...... and T2D Udgivelsesdato: 2009/11......, stability, and translation that are essential for normal embryonic growth and development. This review provides a background to the IMP protein family with an emphasis on human IMP2, followed by a closer look at the GWA studies to evaluate the significance, if any, of the proposed correlation between IMP2...

  10. C-reactive protein inhibits survivin expression via Akt/mTOR pathway downregulation by PTEN expression in cardiac myocytes.

    Directory of Open Access Journals (Sweden)

    Beom Seob Lee

    Full Text Available C-reactive protein (CRP is one of the most important biomarkers for arteriosclerosis and cardiovascular disease. Recent studies have shown that CRP affects cell cycle and inflammatory process in cardiac myocytes. Survivin is also involved in cardiac myocytes replication and apoptosis. Reduction of survivin expression is associated with less favorable cardiac remodeling in animal models. However, the effect of CRP on survivin expression and its cellular mechanism has not yet been studied. We demonstrated that treatment of CRP resulted in a significant decrease of survivin protein expression in a concentration-dependent manner in cardiac myocytes. The upstream signaling proteins of survivin, such as Akt, mTOR and p70S6K, were also downregulated by CRP treatment. In addition, CRP increased the protein and mRNA levels of PTEN. The siRNA transfection or specific inhibitor treatment for PTEN restored the CRP-induced downregulation of Akt/mTOR/p70S6K pathway and survivin protein expression. Moreover, pretreatment with a specific p53 inhibitor decreased the CRP-induced PTEN expression. ERK-specific inhibitor also blocked the p53 phosphorylation and PTEN expression induced by CRP. Our study provides a novel insight into CRP-induced downregulation of survivin protein expression in cardiac myocytes through mechanisms that involved in downregulation of Akt/mTOR/p70S6K pathway by expression of PTEN.

  11. Study of factors that interfere in the labelling process of erythrocytes and plasma proteins with Technetium-99m

    International Nuclear Information System (INIS)

    Gutfilen, Bianca

    1989-01-01

    The labelling of red blood cells (RBC) with technetium-99m (Tc-99m) depends on several factors, as the stannous ion (Sn++) concentration, time, temperature, the presence of plasma proteins (PP) and others. However the Sn++ concentration seems to be the most important factor; probably because the uptake of this reducing agent by RBC is limited. The excess of Sn++ in extracellular medium can determine the labelling of PP. the modifications of RBC at 50 deg C described in the literature, the possibility of labelling RBC with Tc-99m at this temperature and experimental results obtained made it possible to perform spleen selective scintigraphy through a simple technique with few manipulations. The effect of gentamicin, nifedipine and verapamil in the labelling of RBC and plasma proteins with Tc-99m was studied because of similarities between Ca++ and Sn++. The results show that, under some conditions, these drugs are capable to alter this Tc-99m incorporation. The modification of the ionic distribution determined by these drugs or the blockage of Sn++ and/or Tc-99m or the fact that they bind theirselves to plasma proteins, or the possibility of the labelling of these drugs, are factors that can interfere in the labelling process of red blood cells and plasma proteins with Tc-99m. (author)

  12. Characterization of the GXXXG motif in the first transmembrane segment of Japanese encephalitis virus precursor membrane (prM protein

    Directory of Open Access Journals (Sweden)

    Wu Suh-Chin

    2010-05-01

    Full Text Available Abstract The interaction between prM and E proteins in flavivirus-infected cells is a major driving force for the assembly of flavivirus particles. We used site-directed mutagenesis to study the potential role of the transmembrane domains of the prM proteins of Japanese encephalitis virus (JEV in prM-E heterodimerization as well as subviral particle formation. Alanine insertion scanning mutagenesis within the GXXXG motif in the first transmembrane segment of JEV prM protein affected the prM-E heterodimerization; its specificity was confirmed by replacing the two glycines of the GXXXG motif with alanine, leucine and valine. The GXXXG motif was found to be conserved in the JEV serocomplex viruses but not other flavivirus groups. These mutants with alanine inserted in the two prM transmembrane segments all impaired subviral particle formation in cell cultures. The prM transmembrane domains of JEV may play importation roles in prM-E heterodimerization and viral particle assembly.

  13. Dwarfism and impaired gut development in insulin-like growth factor II mRNA-binding protein 1-deficient mice

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Hammer, Niels A; Nielsen, Jacob

    2004-01-01

    Insulin-like growth factor II mRNA-binding protein 1 (IMP1) belongs to a family of RNA-binding proteins implicated in mRNA localization, turnover, and translational control. Mouse IMP1 is expressed during early development, and an increase in expression occurs around embryonic day 12.5 (E12.5). T...

  14. Dietary protein sources differentially affect microbiota, mTOR activity and transcription of mTOR signaling pathways in the small intestine.

    Directory of Open Access Journals (Sweden)

    Soumya K Kar

    Full Text Available Dietary protein sources can have profound effects on host-microbe interactions in the gut that are critically important for immune resilience. However more knowledge is needed to assess the impact of different protein sources on gut and animal health. Thirty-six wildtype male C57BL/6J mice of 35 d age (n = 6/group; mean ± SEM body weight 21.9 ± 0.25 g were randomly assigned to groups fed for four weeks with semi synthetic diets prepared with one of the following protein sources containing (300 g/kg as fed basis: soybean meal (SBM, casein, partially delactosed whey powder, spray dried plasma protein, wheat gluten meal and yellow meal worm. At the end of the experiment, mice were sacrificed to collect ileal tissue to acquire gene expression data, and mammalian (mechanistic target of rapamycin (mTOR activity, ileal digesta to study changes in microbiota and serum to measure cytokines and chemokines. By genome-wide transcriptome analysis, we identified fourteen high level regulatory genes that are strongly affected in SBM-fed mice compared to the other experimental groups. They mostly related to the mTOR pathway. In addition, an increased (P < 0.05 concentration of granulocyte colony-stimulating factor was observed in serum of SBM-fed mice compared to other dietary groups. Moreover, by 16S rRNA sequencing, we observed that SBM-fed mice had higher (P < 0.05 abundances of Bacteroidales family S24-7, compared to the other dietary groups. We showed that measurements of genome-wide expression and microbiota composition in the mouse ileum reveal divergent responses to diets containing different protein sources, in particular for a diet based on SBM.

  15. Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

    Science.gov (United States)

    Stengel, Gudrun; Kuchta, Robert D

    2011-01-01

    The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not

  16. Drosophila protein interaction map (DPiM): a paradigm for metazoan protein complex interactions.

    Science.gov (United States)

    Guruharsha, K G; Obar, Robert A; Mintseris, Julian; Aishwarya, K; Krishnan, R T; Vijayraghavan, K; Artavanis-Tsakonas, Spyros

    2012-01-01

    Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein-especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex "map" provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.

  17. Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.

    Directory of Open Access Journals (Sweden)

    Pegine Walrad

    2009-02-01

    Full Text Available The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

  18. Optimized localization of bacterial infections with technetium-99m labelled human immunoglobulin after protein charge selection

    International Nuclear Information System (INIS)

    Welling, M.; Feitsma, H.I.J.; Calame, W.; Ensing, G.J.; Goedemans, W.; Pauwels, E.K.J.

    1994-01-01

    To improve the scintigraphic detection of bacterial infections a protein charge-purified fraction of polyclonal human immunoglobulin was applied as a radiopharmaceutical. This purification was achieved by attaching the immunoglobulin to an anion-exchanger column and by obtaining the column-bound fraction with buffer. The binding to bacteria in vitro and the target to non-target ratios of an experimental thigh infection with Staphylococcus aureus or Klebsiella pneumoniae in mice were evaluated to compare the purified and the unpurified immunoglobulin. The percentage of binding to all gram-positive and gram-negative bacteria used in this study was significantly (P 99m Tc-labelled protein charge-purified polyclonal human immunoglobulin was administered intravenously. At all time intervals the target (infected thighs) to non-target (non-infected thighs) ratios for both infections were significantly higher (P 99m Tc-labelled protein charge-purified immunoglobulin localizes both a gram-positive and a gram-negative thigh infection more intensely and faster than 99m Tc-labelled unpurified immunoglobulin. (orig.)

  19. Effect of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G in cervical cancer.

    Science.gov (United States)

    Xu, Yanhua; Leng, Junhong; Xue, Fang; Dong, Ruiqian

    2015-01-01

    Cervical cancer is one of the most common gynecologic cancers. The role of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G (APCBEC-3G) in cervical cancer has yet to be elucidated. This study intends to explore the effect of APCBEC-3G on cervical cancer cell proliferation and invasion. In vitro, the cervical cancer cell line Hela was transfected by APCBEC-3G plasmid. The mRNA and protein expression levels of APCBEC-3G were detected by Real-time PCR and Western blot, respectively. Cervical cancer cell proliferation was determined by MTT. Transwell assay was applied to measure the effect of APCBEC-3G on cell invasion. APCBEC-3G mRNA and protein increased significantly after transfection (P3G serves as a suppressor of cervical cancer cell proliferation and invasion. Our research provides theoretical basis for further investigation APOBEC-3G effect in cervical cancer occurrence and development.

  20. In situ localization of phenylpropanoid biosynthetic mRNAs and proteins in Parsley (Petroselinum crispum)

    International Nuclear Information System (INIS)

    Reinold, S.; Hahlbrock, K.

    1997-01-01

    Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together constituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence. (author)

  1. Nutrient-induced stimulation of protein synthesis in mouse skeletal muscle is limited by the mTORC1 repressor REDD1.

    Science.gov (United States)

    Gordon, Bradley S; Williamson, David L; Lang, Charles H; Jefferson, Leonard S; Kimball, Scot R

    2015-04-01

    In skeletal muscle, the nutrient-induced stimulation of protein synthesis requires signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Expression of the repressor of mTORC1 signaling, regulated in development and DNA damage 1 (REDD1), is elevated in muscle during various atrophic conditions and diminished under hypertrophic conditions. The question arises as to what extent REDD1 limits the nutrient-induced stimulation of protein synthesis. The objective was to examine the role of REDD1 in limiting the response of muscle protein synthesis and mTORC1 signaling to a nutrient stimulus. Wild type REDD1 gene (REDD1(+/+)) and disruption in the REDD1 gene (REDD1(-/-)) mice were feed deprived for 16 h and randomized to remain feed deprived or refed for 15 or 60 min. The tibialis anterior was then removed for analysis of protein synthesis and mTORC1 signaling. In feed-deprived mice, protein synthesis and mTORC1 signaling were significantly lower in REDD1(+/+) than in REDD1(-/-) mice. Thirty minutes after the start of refeeding, protein synthesis in REDD1(+/+) mice was stimulated by 28%, reaching a value similar to that observed in feed-deprived REDD1(-/-) mice, and was accompanied by increased phosphorylation of mTOR (Ser2448), p70S6K1 (Thr389), and 4E-BP1 (Ser65) by 81%, 167%, and 207%, respectively. In refed REDD1(-/-) mice, phosphorylation of mTOR (Ser2448), p70S6K1 (Thr389), and 4E-BP1 (Ser65) were significantly augmented above the values observed in refed REDD1(+/+) mice by 258%, 405%, and 401%, respectively, although protein synthesis was not coordinately increased. Seventy-five minutes after refeeding, REDD1 expression in REDD1(+/+) mice was reduced (∼15% of feed-deprived REDD1(+/+) values), and protein synthesis and mTORC1 signaling were not different between refed REDD1(+/+) mice and REDD1(-/-) mice. The results show that REDD1 expression limits protein synthesis in mouse skeletal muscle by inhibiting mTORC1 signaling during periods of feed

  2. Influence of connection tubing in modern size exclusion chromatography and its impact on the characterization of mAbs.

    Science.gov (United States)

    Fekete, Szabolcs; Guillarme, Davy

    2018-02-05

    The goal of the study was to evaluate the impact of connection tubing in modern size exclusion chromatography (SEC), since it may strongly impact the apparent column efficiency, as the compounds are not retained in SEC. For this purpose, a reference SEC column of 150×4.6mm, 1.8μm was considered, and various proteins were tested as model compounds. Different tube geometries (lengths and internal diameters) and materials (stainless steel and PEEK) were evaluated in a systematic way. Large proteins always showed larger tube dispersion vs. small molecules, especially when the residence time in the tube was long (at low flow rate). This confirms the need to drastically reduce the tube volume (using the shortest and narrowest connector tubing) to attain the full benefits of UHPSEC columns. In addition, PEEK tubing were found to be more appropriate than stainless steel tubing, since adsorption of proteins was less pronounced, and higher plate count can be obtained. Finally, after a careful system optimization, up to 40% increase of apparent column efficiency can be achieved compared to a regular UHPLC system, when using a 150×4.6mm UHPSEC columns packed with sub-3μm particles. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. CERKL, a retinal disease gene, encodes an mRNA-binding protein that localizes in compact and untranslated mRNPs associated with microtubules.

    Directory of Open Access Journals (Sweden)

    Alihamze Fathinajafabadi

    Full Text Available The function of CERKL (CERamide Kinase Like, a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases.

  4. BIOLOGICALLY ACTIVE SUBSTANCES OF SPIRIT PRODUCTION WASTE

    Directory of Open Access Journals (Sweden)

    A. S. Kayshev

    2014-01-01

    Full Text Available A content of biologically active compounds (BAC with signified pharmacological activity in distillers grains was proved. It is prospective for applications of these grains as a raw material resource of pharmaceuticals. A composition of BAC distillers grains received from wheat, corn, barley, millet at different spirit enterprises which use hydro fermentative grain processing. Considering polydispersity of distillers grains they were separated on solid and liquid phases preliminary. Physical and chemical characteristics of distillers grains' liquid base were identified. Elementary composition of distillers grains is signified by active accumulation of biogenic elements (phosphorus, potassium, magnesium, calcium, sodium, iron and low content of heavy metals. The solid phase of distillers grains accumulates carbon, hydrogen and nitrogen in high concentration. The liquid phase of distillers grains contains: proteins and amino acids (20-46%, reducing sugars (5,6%-17,5%, galacturonides (0,8-1,4%, ascorbic acid (6,2-11,4 mg%. The solid base of distillers grains contains: galacturonides (3,4-5,3%, fatty oil (8,4-11,1% with predomination of essential fatty acids, proteins and amino acids (2,1-2,5%, flavonoids (0,4-0,9%, tocopherols (3,4-7,7 mg%. A method of complex processing of distillers grains based on application of membrane filtering of liquid phase and liquid extraction by inorganic and organic solvents of solid phase, which allows almost full extraction of the sum of biologically active compounds (BAC from liquid phase (Biobardin BM and solid phase (Biobardin UL. Biobardin BM comprises the following elements: proteins and amino acids (41-69%, reducing sugars (3,5-15,6%, fatty oil (0,2-0,3%, flavonoids (0,2-0,7%, ascorbic acid (17-37 mg%. Biobardin UL includes: oligouronids (16,4-19,5%, proteins and amino acids (11-21%, fatty oil (3,2-4,9% which includes essential acids; flavonoids (0,6-1,5%, tocopherols (6,6-10,2 mg%, carotinoids (0,13-0,21 mg

  5. Protective immune response of oral rabies vaccine in stray dogs, corsacs and steppe wolves after a single immunization.

    Science.gov (United States)

    Zhugunissov, K; Bulatov, Ye; Taranov, D; Yershebulov, Z; Koshemetov, Zh; Abduraimov, Ye; Kondibayeva, Zh; Samoltyrova, A; Amanova, Zh; Khairullin, B; Sansyzbay, A

    2017-11-01

    In this study the safety and protective immunity of an oral rabies vaccine, based on the live, modified rabies virus strain VRC-RZ2, was examined in stray dogs (Canis Sp.), corsacs (Vulpes corsac) and steppe wolves (Canis lupus campestris). In the safety group (dogs, n=6; corsacs, n=3; wolves, n=3) which was vaccinated with a 10-times field dose/animal, no animals showed any signs of disease or changes in behavior or appetite during the period of clinical observation, similar to the animals in the negative control group. Saliva samples taken from animals prior and post (5 th and 10 th days) vaccination failed to demonstrate rabies virus antigen. Observations of immunogenicity in vaccinated carnivores (dogs, corsacs and wolves) during a 180 day period showed the titers of virus neutralizing antibodies (VNA) in the blood sera of vaccinated dogs to be within 0.59-1.37 IU/mL. On 14 days post vaccination (dpv), all the wild carnivores had detectable levels of neutralizing antibodies, with mean titers ranging from 0.50 ± 0.07 IU/mL (for wolves) to 0.59 ± 0.10 IU/mL (for corsacs). Weeks after vaccination, all the vaccinated wolves and corsacs had higher levels of neutralizing antibodies: 0.70 ± 0.10 - 0.71 ± 0.08 IU/mL at 30 dpv, 1.06 ± 0.08 - 1.28 ± 0.21 IU/mL at 60 dpv and 0.41 ± 0.09 - 047 ± 0.06 at 180 dpv. The highest level of VNA (˃1.0 IU/ml) was detected at 60 dpv, in all vaccinated animals. After challenge all vaccinated dogs remained healthy for 180 days. Control animals (unvaccinated dogs) developed symptoms of rabies on day 6 post administration of a virulent virus and died of rabies on days 11-13. Of note, the VNA titers in all the wild carnivores (corsacs and wolves) immunized with VRC-RZ2 were higher than 0.5 IU/ml (0.59 ± 0.11 IU/ml), even as early as 14 days post vaccination. These, presumably protective, titers of antibodies to rabies virus were present in the dogs and wild carnivores examined in this study for at

  6. M protein typing of Thai group A streptococcal isolates by PCR-Restriction fragment length polymorphism analysis

    Directory of Open Access Journals (Sweden)

    Good Michael F

    2005-10-01

    Full Text Available Abstract Background Group A streptococcal (GAS infections can lead to the development of severe post-infectious sequelae, such as rheumatic fever (RF and rheumatic heart disease (RHD. RF and RHD are a major health concern in developing countries, and in indigenous populations of developed nations. The majority of GAS isolates are M protein-nontypeable (MNT by standard serotyping. However, GAS typing is a necessary tool in the epidemiologically analysis of GAS and provides useful information for vaccine development. Although DNA sequencing is the most conclusive method for M protein typing, this is not a feasible approach especially in developing countries. To overcome this problem, we have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP-based assay for molecular typing the M protein gene (emm of GAS. Results Using one pair of primers, 13 known GAS M types showed one to four bands of PCR products and after digestion with Alu I, they gave different RFLP patterns. Of 106 GAS isolates examined from the normal Thai population and from patients with GAS-associated complications including RHD, 95 isolates gave RFLP patterns that corresponded to the 13 known M types. Only 11 isolates gave RFLP patterns that differed from the 13 known M types. These were then analyzed by DNA sequencing and six additional M types were identified. In addition, we found that M93 GAS was the most common M type in the population studied, and is consistent with a previous study of Thai GAS isolates. Conclusion PCR-RFLP analysis has the potential for the rapid screening of different GAS M types and is therefore considerably advantageous as an alternative M typing approach in developing countries in which GAS is endemic.

  7. Environmental Assessment of Lead at Camp Edwards, Massachusetts, Small Arms Ranges

    Science.gov (United States)

    2007-08-01

    hydroxyapatite [Pb5(PO4)3(OH)], and lead oxyphosphate [Pb4O(PO4)2]. Lead phosphate minerals, in particular, are so sparingly soluble, thermodynamic...M ea n XR F Le ad Pr of ile R es ul ts (m g/ kg ) 1 -1 0 in ch es bg s M ea n XR F Le ad Pr of ile R es ul ts (m g/ kg ) 1 0- 20 in...ch es bg s M ea n XR F Le ad Pr of ile R es ul ts (m g/ kg ) 2 0- 30 in ch es

  8. Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism

    International Nuclear Information System (INIS)

    Khotin, Mikhail; Turoverova, Lidia; Aksenova, Vasilisa; Barlev, Nikolai; Borutinskaite, Veronika Viktorija; Vener, Alexander; Bajenova, Olga; Magnusson, Karl-Eric; Pinaev, George P.; Tentler, Dmitri

    2010-01-01

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  9. Miosite e rabdomiólise na doença mão-pé-boca na infância Myositis and rhabdomyolysis in hand-foot-mouth disease in childhood

    Directory of Open Access Journals (Sweden)

    Maria Helena Vaisbich

    2010-03-01

    Full Text Available OBJETIVO: Relatar um caso de doença mão-pé-boca complicada por miosite, rabdomiólise e hepatite, interessante por ser a doença frequente em crianças e poder apresentar complicações graves, apesar de raras. DESCRIÇÃO DO CASO: Paciente de três anos de idade, sexo feminino, com história de febre por três dias, seguida pelo aparecimento de lesões ulceradas em mucosa oral e mialgia intensa. Após três dias, voltou a apresentar febre por mais dois dias (febre bifásica. Nesses dois dias, apresentou lesões eritematosas pelo corpo, principalmente nos pés, mãos e face, e procurou atendimento médico. Evoluiu com aumento de enzimas musculares e hepáticas (CPK com valor máximo de 345.007U/L, TGO 2041U/L, TGP 1589U/L, gama-GT 94U/L e aumento transitório da creatinina sérica, com clearance de creatinina estimado pela estatura de 73mL/minuto/1,73m2 de superfície corporal. Houve melhora progressiva, com hidratação vigorosa e alcalinização da urina, sem necessidade de diálise. COMENTÁRIOS: Trata-se de uma criança com doença mão-pé-boca, com miosite, rabdomiólise e hepatite. São enfatizados os critérios clínicos laboratoriais para o diagnóstico e a importância da monitorização das complicações da doençaOBJECTIVE: To report a hand-foot-mouth disease case, complicated by myositis, rhabdomyolisis and hepatitis, since this is a common disease in children and can result in rare but severe complications. CASE DESCRIPTION: A three-year-old girl with fever for three days, followed by the appearance of ulcerative lesions in the oral mucosa and severe muscular pain; after three days, she presented fever for two more days. At the same time, she had widespread erythematosus rash, especially in her hands, feet and face. She presented high levels of muscular and hepatic enzymes (maximum value of CPK 345.007IU/L, AST 2041IU/L, ALT 1589IU/L, GT 94IU/L, and transitory increase in serum creatinine (maximum value of 0.73mg/dL, creatinine

  10. Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

    Directory of Open Access Journals (Sweden)

    Randall Marcelo Chin

    2018-03-01

    Full Text Available Summary: Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression—the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3′ UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene. : Allotopic expression of proteins normally encoded by mtDNA is a promising therapy for mitochondrial disease. Chin et al. use an unbiased and high-content imaging-based screening platform to optimize allotopic expression. Modified mRNAs encoding for the optimized allotopic expression constructs rescued the respiration and growth of mtATP6-deficient cells. Keywords: mitochondria, mitochondrial disease, mRNA, modified mRNA, ATP6, allotopic expression, rare disease, gene therapy, screening, high content imaging

  11. Sa Rocca Ulàri Cave, near S. Pietro di Sorres, Borutta (SS: archaeological aspects.

    Directory of Open Access Journals (Sweden)

    Pier Paolo Soro

    2009-12-01

    Full Text Available The Cave of Sa Rocca Ulàri, in the municipality of Borutta (SS, is fully inserted into the dense settlement of the natural cavities of Sardinia during the prehistory and early history. Its size and the particular relation to the configuration of the land in relation to the richness of the Mejlogu landscape, allowed small groups of people to settle in the cavity that opens into the northern slope of the Hill of Sorres. Attendance could be certified without interruption from the Neolithic until the Middle Ages. The preliminary study of materials, has determined that the most popular period for residential purposes is referring to the end of the Neolithic culture of Ozieri, and the first Eneolithic Sub Ozieri. The funerary use is assumed for the subsequent cultural phases Monte Claro (Evolved Eneolithic and Bonnanaro I facies Corona Moltana (Bronze Age. In the Nuragic Era (Middle Bronze / Late / Final and early Iron Age the cavity was initially used for storing food, then for other purposes probably linked to the cult.

  12. Influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting virus replication

    International Nuclear Information System (INIS)

    Wanitchang, Asawin; Wongthida, Phonphimon; Jongkaewwattana, Anan

    2016-01-01

    The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein. -- Highlights: •Flu A M2 protein (AM2) can be functionally replaced by that of Flu B (BM2). •Both AM2 and BM2 with extended cytoplasmic tail are functional. •Compatibility between the ion channel and the cytoplasmic tail is critical for M2 function. •M2 with higher ion channel activity may augment influenza virus replication.

  13. Influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Wanitchang, Asawin; Wongthida, Phonphimon; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

    2016-11-15

    The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein. -- Highlights: •Flu A M2 protein (AM2) can be functionally replaced by that of Flu B (BM2). •Both AM2 and BM2 with extended cytoplasmic tail are functional. •Compatibility between the ion channel and the cytoplasmic tail is critical for M2 function. •M2 with higher ion channel activity may augment influenza virus replication.

  14. Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling

    NARCIS (Netherlands)

    Zeev-Ben-Mordehai, Tzviya; Weberruss, Marion; Lorenz, Michael; Cheleski, Juliana; Hellberg, Teresa; Whittle, Cathy; El Omari, Kamel; Vasishtan, Daven; Dent, Kyle C.; Harlos, Karl; Franzke, Kati; Hagen, Christoph; Klupp, Barbara G.; Antonin, Wolfram; Mettenleiter, Thomas C.; Gruenewald, Kay

    2015-01-01

    Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates

  15. HCMV Infection of Human Trophoblast Progenitor Cells of the Placenta Is Neutralized by a Human Monoclonal Antibody to Glycoprotein B and Not by Antibodies to the Pentamer Complex

    Directory of Open Access Journals (Sweden)

    Martin Zydek

    2014-03-01

    Full Text Available Human cytomegalovirus (HCMV is the major viral cause of congenital infection and birth defects. Primary maternal infection often results in virus transmission, and symptomatic babies can have permanent neurological deficiencies and deafness. Congenital infection can also lead to intrauterine growth restriction, a defect in placental transport. HCMV replicates in primary cytotrophoblasts (CTBs, the specialized cells of the placenta, and inhibits differentiation/invasion. Human trophoblast progenitor cells (TBPCs give rise to the mature cell types of the chorionic villi, CTBs and multi-nucleated syncytiotrophoblasts (STBs. Here we report that TBPCs are fully permissive for pathogenic and attenuated HCMV strains. Studies with a mutant virus lacking a functional pentamer complex (gH/gL/pUL128-131A showed that virion entry into TBPCs is independent of the pentamer. In addition, infection is blocked by a potent human neutralizing monoclonal antibody (mAb, TRL345, reactive with glycoprotein B (gB, but not mAbs to the pentamer proteins pUL130/pUL131A. Functional studies revealed that neutralization of infection preserved the capacity of TBPCs to differentiate and assemble into trophospheres composed of CTBs and STBs in vitro. Our results indicate that mAbs to gB protect trophoblast progenitors of the placenta and could be included in antibody treatments developed to suppress congenital infection and prevent disease.

  16. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    Science.gov (United States)

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  17. IFT46 plays an essential role in cilia development

    Science.gov (United States)

    Lee, Mi-Sun; Hwang, Kyu-Seok; Oh, Hyun-Woo; Ji-Ae, Kim; Kim, Hyun-Taek; Cho, Hyun-Soo; Lee, Jeong-Ju; Ko, Je Yeong; Choi, Jung-Hwa; Jeong, Yun-Mi; You, Kwan-Hee; Kim, Joon; Park, Doo-Sang; Nam, Ki-Hoan; Aizawa, Shinichi; Kiyonari, Hiroshi; Shioi, Go; Park, Jong-Hoon; Zhou, Weibin; Kim, Nam-Soon; Kim, Cheol-Hee

    2015-01-01

    Cilia are microtubule-based structures that project into the extracellular space. Ciliary defects are associated with several human diseases, including polycystic kidney disease, primary ciliary dyskinesia, left-right axis patterning, hydrocephalus and retinal degeneration. However, the genetic and cellular biological control of ciliogenesis remains poorly understood. The IFT46 is one of the highly conserved intraflagellar transport complex B proteins. In zebrafish, ift46 is expressed in various ciliated tissues such as Kupffer’s vesicle, pronephric ducts, ears and spinal cord. We show that ift46 is localized to the basal body. Knockdown of ift46 gene results in multiple phenotypes associated with various ciliopathies including kidney cysts, pericardial edema and ventral axis curvature. In ift46 morphants, cilia in kidney and spinal canal are shortened and abnormal. Similar ciliary defects are observed in otic vesicles, lateral line hair cells, olfactory pits, but not in Kupffer’s vesicle. To explore the functions of Ift46 during mouse development, we have generated Ift46 knock-out mice. The Ift46 mutants have developmental defects in brain, neural tube and heart. In particular Ift46(−/−) homozygotes displays randomization of the embryo heart looping, which is a hallmark of defective left-right (L/R) axis patterning. Taken together, our results demonstrated that IFT46 has an essential role in vertebrate ciliary development. PMID:25722189

  18. Fluorescent S-layer fusion proteins

    International Nuclear Information System (INIS)

    Kainz, B.

    2010-01-01

    This work describes the construction and characterisation of fluorescent S-layer fusion proteins used as building blocks for the fabrication of nanostructured monomolecular biocoatings on silica particles with defined fluorescence properties. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the pH-dependant cyan, green and yellow variant of the green fluorescent protein (GFP) and the red fluorescent protein mRFP1. These fluorescent S-layer fusion proteins, acting as scaffold and optical sensing element simultaneously, were able to reassemble in solution and on silica particles forming 2D nanostructures with p2 lattice symmetry (a=11 ±0.5 nm, b=14 ±0.4 nm, g=80 ±1 o ). The pH-dependant fluorescence behaviour was studied with fluorimetry, confocal microscopy and flow cytometry. These fluorescent S-layer fusion proteins can be used as pH-sensor. 50% of the fluorescence intensity decreases at their calculated pKa values (pH6 - pH5). The fluorescence intensity of the GFP variants vanished completely between pH4 and pH3 whereas the chromophore of the red protein mRFP1 was only slightly affected in acidic conditions. At the isoelectric point of the S-layer coated silica particles (pH4.6 ±0.2) an increase in particle aggregation was detected by flow cytometry. The cyan and yellow fluorescent proteins were chosen to create a bi-fluorescent S-layer tandem fusion protein with the possibility for resonance energy transfer (FRET). A transfer efficiency of 20% and a molecular distance between the donor (ECFP) and acceptor (YFP) chromophores of around 6.2 nm could be shown. This bi-fluorescent ECFP-SgsE-YFP tandem fusion protein was able to reassemble on solid surfaces. The remarkable combination of fluorescence and self-assembly and the design of bi-functional S-layer tandem fusion protein matrices makes them to a promising tool in nanobiotechnology. (author) [de

  19. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.

  20. The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1.

    Science.gov (United States)

    Kathage, Barbara; Gehlert, Sebastian; Ulbricht, Anna; Lüdecke, Laura; Tapia, Victor E; Orfanos, Zacharias; Wenzel, Daniela; Bloch, Wilhelm; Volkmer, Rudolf; Fleischmann, Bernd K; Fürst, Dieter O; Höhfeld, Jörg

    2017-01-01

    The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Stabilising the Herpes Simplex Virus capsid by DNA packaging

    Science.gov (United States)

    Wuite, Gijs; Radtke, Kerstin; Sodeik, Beate; Roos, Wouter

    2009-03-01

    Three different types of Herpes Simplex Virus type 1 (HSV-1) nuclear capsids can be distinguished, A, B and C capsids. These capsids types are, respectively, empty, contain scaffold proteins, or hold DNA. We investigate the physical properties of these three capsids by combining biochemical and nanoindentation techniques. Atomic Force Microscopy (AFM) experiments show that A and C capsids are mechanically indistinguishable whereas B capsids already break at much lower forces. By extracting the pentamers with 2.0 M GuHCl or 6.0 M Urea we demonstrate an increased flexibility of all three capsid types. Remarkably, the breaking force of the B capsids without pentamers does not change, while the modified A and C capsids show a large drop in their breaking force to approximately the value of the B capsids. This result indicates that upon DNA packaging a structural change at or near the pentamers occurs which mechanically reinforces the capsids structure. The reported binding of proteins UL17/UL25 to the pentamers of the A and C capsids seems the most likely candidate for such capsids strengthening. Finally, the data supports the view that initiation of DNA packaging triggers the maturation of HSV-1 capsids.

  2. Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mTOR-independent process.

    Science.gov (United States)

    Jeyapalan, Asumthia S; Orellana, Renan A; Suryawan, Agus; O'Connor, Pamela M J; Nguyen, Hanh V; Escobar, Jeffery; Frank, Jason W; Davis, Teresa A

    2007-08-01

    Skeletal muscle protein synthesis is elevated in neonates in part due to an enhanced response to the rise in insulin and amino acids after eating. In vitro studies suggest that glucose plays a role in protein synthesis regulation. To determine whether glucose, independently of insulin and amino acids, is involved in the postprandial rise in skeletal muscle protein synthesis, pancreatic-substrate clamps were performed in neonatal pigs. Insulin secretion was inhibited with somatostatin and insulin was infused to reproduce fasting or fed levels, while glucose and amino acids were clamped at fasting or fed levels. Fractional protein synthesis rates and translational control mechanisms were examined. Raising glucose alone increased protein synthesis in fast-twitch glycolytic muscles but not in other tissues. The response in muscle was associated with increased phosphorylation of protein kinase B (PKB) and enhanced formation of the active eIF4E.eIF4G complex but no change in phosphorylation of AMP-activated protein kinase (AMPK), tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mTOR), 4E-binding protein-1 (4E-BP1), ribosomal protein S6 kinase (S6K1), or eukaryotic elongation factor 2 (eEF2). Raising glucose, insulin, and amino acids increased protein synthesis in most tissues. The response in muscle was associated with phosphorylation of PKB, mTOR, S6K1, and 4E-BP1 and enhanced eIF4E.eIF4G formation. The results suggest that the postprandial rise in glucose, independently of insulin and amino acids, stimulates protein synthesis in neonates, and this response is specific to fast-twitch glycolytic muscle and occurs by AMPK- and mTOR-independent pathways.

  3. Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

    International Nuclear Information System (INIS)

    Zhdanov, V. P.

    2010-01-01

    Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

  4. NNLO corrections to anti B {yields} X{sub u}l anti {nu} in the shape-function region

    Energy Technology Data Exchange (ETDEWEB)

    Asatrian, H.M. [Erevanskij Fizicheskij Inst., Erevan (Armenia); Greub, C. [Bern Univ. (Switzerland). Inst. for Theoretical Physics; Pecjak, B.D. [Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany)

    2008-10-15

    The inclusive decay anti B {yields} X{sub u}l anti {nu} is of much interest because of its potential to constrain the CKM element vertical stroke V{sub ub} vertical stroke. Experimental cuts required to suppress charm background restrict measurements of this decay to the shape-function region, where the hadronic final state carries a large energy but only a moderate invariant mass. In this kinematic region, the differential decay distributions satisfy a factorization formula of the form H.J x S, where S is the non-perturbative shape function, and the object H.J is a perturbatively calculable hard-scattering kernel. In this paper we present the calculation of the hard function H at next-to-next-to-leading order (NNLO) in perturbation theory. Combined with the known NNLO result for the jet function J, this completes the perturbative part of the NNLO calculation for this process. (orig.)

  5. Interference of vaccinal antibodies on serological diagnosis of leptospirosis in vaccinated buffalo using two types of commercial vaccines Interferência de anticorpos vacinais no diagnóstico sorológico da leptospirose em búfalas vacinadas com dois tipos de vacinas comerciais

    Directory of Open Access Journals (Sweden)

    Geraldo de Nardi Júnior

    2007-06-01

    representa um dos entraves para o sorodiagnóstico da leptospirose. Raros estudos dimensionam prospectivamente esse efeito na espécie bubalina. O presente trabalho objetivou traçar o perfil de aglutininas séricas anti-Leptospira spp.em búfalas vacinadas contra leptospirose com dois tipos de vacina comercial : bacterina e de membrana externa purificada e avaliar a interferência temporal dos títulos vacinais no sorodiagnóstico. Três grupos de 11 fêmeas bubalinas adultas: G1- controle não vacinado, G2 -vacinado com bacterina contendo seis sorovares e G3- recebeu vacina com membrana externa purificada de cinco sorovares, receberam reforço 30 dias pós primo vacinação (dpv e duas revacinações semestrais nos dias 210 e 390. Foram colhidas amostras sorológicas nos dias 0, 15, 30, 45, 60 e a cada 30 dias até 540 dpv e analisadas pela reação de Soroaglutinação Microscópica-SAM frente aos sorovares presentes nas vacinas. G1 manteve-se sempre negativo. Ambas vacinas induziram resposta sorológica na SAM aos 15º dpv para todos os sorovares e revelaram títulos máximos ao redor do 45º e 60º dpv.: Pomona: G2 (1600 e G3 (3200; Hardjo: G2 e G3 (1600; Wolffi: G2 (800 e G3 (1600, Icterohaemorrhagiae: G2 e G3 (800, Grippotyphosa: G2 e G3 (200 e Canicola: G2 (NR e G3 (400. Apesar da vacina de membrana externa não possuir o sorovar Wolffi, G3 revelou resposta para este sorovar, provavelmente pelo sorovar Hardjo vacinal. Os perfis sorológicos representados graficamente pela média geométrica dos títulos de aglutininas foram semelhantes, porém em G3 mais precoces e mais elevados que G2. Na revacinação houve aumento do nível de aglutininas, porém de menor intensidade que o anterior; e ao final de seis meses da segunda revacinação (540 dpv eram quase nulos, demonstrando curta duração da interferência ao diagnóstico.

  6. Regulation of 2-5A Dependent RNase at the Level of its Phosphorylation

    Science.gov (United States)

    1991-06-26

    extract as follows: 25 ul wheat germ extract 10 ul H2O 1 ul RNasin ribonuclease inhibitor (40 u/ml) 7 ul ImM amino acid mixture 1 ul IM...diacylglycerol (DAG) 2. TPA 3. Indolactam Figure 6. Chemical structure of: 1. H-7 (A kinase inhibitor) 2. okadaic acid (A phosphatase inhibitor) Figure 7...elevating agents: Forskolin and Cholera toxin Figure 17. Down-regulation of 2-5A-depRNase by Okadaic 77 acid : A phosphatase inhibitor Figure 18

  7. Proteins in olive fruit and oil.

    Science.gov (United States)

    Montealegre, Cristina; Esteve, Clara; García, Maria Concepción; García-Ruiz, Carmen; Marina, Maria Luisa

    2014-01-01

    This paper is a comprehensive review grouping the information on the extraction, characterization, and quantitation of olive and olive oil proteins and providing a practical guide about these proteins. Most characterized olive proteins are located in the fruit, mainly in the seed, where different oleosins and storage proteins have been found. Unlike the seed, the olive pulp contains a lower protein content having been described a polypeptide of 4.6 kDa and a thaumain-like protein. Other important proteins studied in olive fruits have been enzymes which could play important roles in olives characteristics. Part of these proteins is transferred from the fruit to the oil during the manufacturing process of olive oil. In fact, the same polypeptide of 4.6 kDa found in the pulp has been described in the olive oil and, additionally, the presence of other proteins and enzymes have also been described. Protein profiles have recently been proposed as an interesting strategy for the varietal classification of olive fruits and oils. Nevertheless, there is still a lot of knowledge without being explored requiring new studies focused on the determination and characterization of these proteins.

  8. 46 CFR 46.10-65 - Construction.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Construction. 46.10-65 Section 46.10-65 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) LOAD LINES SUBDIVISION LOAD LINES FOR PASSENGER VESSELS Administration § 46.10-65 Construction. (a) The watertight subdivision of every passenger vessel...

  9. A Cuprous Oxide Thin Film Non-Enzymatic Glucose Sensor Using Differential Pulse Voltammetry and Other Voltammetry Methods and a Comparison to Different Thin Film Electrodes on the Detection of Glucose in an Alkaline Solution

    Directory of Open Access Journals (Sweden)

    Yifan Dai

    2018-01-01

    Full Text Available A cuprous oxide (Cu2O thin layer served as the base for a non-enzymatic glucose sensor in an alkaline medium, 0.1 NaOH solution, with a linear range of 50–200 mg/dL using differential pulse voltammetry (DPV measurement. An X-ray photoelectron spectroscopy (XPS study confirmed the formation of the cuprous oxide layer on the thin gold film sensor prototype. Quantitative detection of glucose in both phosphate-buffered saline (PBS and undiluted human serum was carried out. Neither ascorbic acid nor uric acid, even at a relatively high concentration level (100 mg/dL in serum, interfered with the glucose detection, demonstrating the excellent selectivity of this non-enzymatic cuprous oxide thin layer-based glucose sensor. Chronoamperometry and single potential amperometric voltammetry were used to verify the measurements obtained by DPV, and the positive results validated that the detection of glucose in a 0.1 M NaOH alkaline medium by DPV measurement was effective. Nickel, platinum, and copper are commonly used metals for non-enzymatic glucose detection. The performance of these metal-based sensors for glucose detection using DPV were also evaluated. The cuprous oxide (Cu2O thin layer-based sensor showed the best sensitivity for glucose detection among the sensors evaluated.

  10. Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

    Directory of Open Access Journals (Sweden)

    Gaëlle Gonzalez

    Full Text Available Cell microparticles (MPs released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5, and serotype 35 (HAdV35, respectively. We found that MPs derived from CHO cells (MP-donor cells constitutively expressing CAR (MP-CAR or CD46 (MP-CD46 were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

  11. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization.

    Directory of Open Access Journals (Sweden)

    Kuo-Chen Chou

    Full Text Available One of the fundamental goals in proteomics and cell biology is to identify the functions of proteins in various cellular organelles and pathways. Information of subcellular locations of proteins can provide useful insights for revealing their functions and understanding how they interact with each other in cellular network systems. Most of the existing methods in predicting plant protein subcellular localization can only cover three or four location sites, and none of them can be used to deal with multiplex plant proteins that can simultaneously exist at two, or move between, two or more different location sits. Actually, such multiplex proteins might have special biological functions worthy of particular notice. The present study was devoted to improve the existing plant protein subcellular location predictors from the aforementioned two aspects. A new predictor called "Plant-mPLoc" is developed by integrating the gene ontology information, functional domain information, and sequential evolutionary information through three different modes of pseudo amino acid composition. It can be used to identify plant proteins among the following 12 location sites: (1 cell membrane, (2 cell wall, (3 chloroplast, (4 cytoplasm, (5 endoplasmic reticulum, (6 extracellular, (7 Golgi apparatus, (8 mitochondrion, (9 nucleus, (10 peroxisome, (11 plastid, and (12 vacuole. Compared with the existing methods for predicting plant protein subcellular localization, the new predictor is much more powerful and flexible. Particularly, it also has the capacity to deal with multiple-location proteins, which is beyond the reach of any existing predictors specialized for identifying plant protein subcellular localization. As a user-friendly web-server, Plant-mPLoc is freely accessible at http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/. Moreover, for the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web-server to

  12. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development

    DEFF Research Database (Denmark)

    Nielsen, J; Christiansen, J; Lykke-Andersen, J

    1999-01-01

    Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5' untranslated regions (5' UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins.......5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5' UTR-binding proteins control IGF-II biosynthesis during late mammalian development....... and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growth-dependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 -luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12...

  13. Increased immunogenicity and protective efficacy of influenza M2e fused to a tetramerizing protein.

    Directory of Open Access Journals (Sweden)

    Anne-Marie Carola Andersson

    Full Text Available The ectodomain of the matrix 2 protein (M2e of influenza A virus represents an attractive target for developing a universal influenza A vaccine, with its sequence being highly conserved amongst human variants of this virus. With the aim of targeting conformational epitopes presumably shared by diverse influenza A viruses, a vaccine (M2e-NSP4 was constructed linking M2e (in its consensus sequence to the rotavirus fragment NSP4(98-135; due to its coiled-coil region this fragment is known to form tetramers in aqueous solution and in this manner we hoped to mimick the natural configuration of M2e as presented in membranes. M2e-NSP4 was then evaluated side-by-side with synthetic M2e peptide for its immunogenicity and protective efficacy in a murine influenza challenge model. Here we demonstrate that M2e fused to the tetramerizing protein induces an accelerated, augmented and more broadly reactive antibody response than does M2e peptide as measured in two different assays. Most importantly, vaccination with M2e-NSP4 caused a significant decrease in lung virus load early after challenge with influenza A virus and maintained its efficacy against a lethal challenge even at very low vaccine doses. Based on the results presented in this study M2e-NSP4 merits further investigation as a candidate for or as a component of a universal influenza A vaccine.

  14. Technetium-99m dextran: a promising new protein-losing enteropathy imaging agent

    International Nuclear Information System (INIS)

    Bhatnagar, A.; Singh, A.K.; Lahoti, D.; Singh, T.; Khanna, C.M.

    1996-01-01

    The purpose of this study was to evaluate technetium-99m dextran ( 99m Tc-Dx; molecular weight 81000) as a prospective protein-losing enteropathy (PLE) imaging agent. Twenty-two patients iwth diseases commonly associated with PLE and 12 healthy control subjects underwent intravenous 99m Tc-Dx scintigraphy. All of the 22 test patients showed significant radiotracer accumulation in the intestines within 3-4 h post injection. The focal, regional or generalised nature of the enteropathy and involvement of the large or small intestine could be identified in most cases. Four of the 12 apparently healthy subjects also showed minimal accumulation in the abdominal area occurring late in the study period. This could have been physiological, related to food habits or due to unsuspected intestinal worms. We attribute the high sensitivity of 99m Tc-Dx to its relatively fast blood (background) clearance. The radiotracer may have several other advantages over 99m Tc-labelled human serum albumin in imaging PLE. (orig.)

  15. Human response to ductless personalized ventilation coupled with displacement ventilation

    DEFF Research Database (Denmark)

    Dalewski, Mariusz; Veselý, Michal; Melikov, Arsen K.

    2012-01-01

    A human subject experiment was carried out to investigate the extent to which ductless personalized ventilation (DPV) in conjunction with displacement ventilation can improve perceived air quality (PAQ) and thermal comfort at elevated room air temperature in comparison with displacement ventilation...... alone. The experimental conditions comprised displacement ventilation alone (room air temperature of 23 °C, 26 °C, 29 °C) and DPV with displacement ventilation (26 °C, 29 °C), both operating at supply air temperatures 3, 5 or 6K lower than room air temperature, as well as mixing ventilation (23 °C, 3 K......). During one hour exposure participants answered questionnaires regarding PAQ and thermal comfort. PAQ was significantly better with DPV than without DPV at the same background conditions. Thermal comfort improved when DPV was used. Combining DPV with displacement ventilation showed the potential...

  16. Passively mode-locked 4.6 and 10.5 GHz quantum dot laser diodes around 1.55 μm with large operating regime

    NARCIS (Netherlands)

    Heck, M.J.R.; Renault, A.; Bente, E.A.J.M.; Oei, Y.S.; Smit, M.K.; Eikema, K.S.E.; Ubachs, W.; Anantathanasarn, S.; Nötzel, R.

    2009-01-01

    Passive mode-locking in two-section InAs/InP quantum dot laser diodes operating at wavelengths around 1.55 µm is reported. For a 4.6-GHz laser, a large operating regime of stable mode-locking, with RF-peak heights of over 40 dB, is found for injection currents of 750 mA up to 1.0 A and for values of

  17. Effect of diclofenac sodium on the level of non-steroidal anti-inflammatory drug-activated gene-1 in patients with post-ERCP pancreatitis

    Directory of Open Access Journals (Sweden)

    HU Cui

    2018-03-01

    Full Text Available ObjectiveTo investigate the effect of diclofenac sodium on the level of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1 in patients with post-ERCP pancreatitis (PEP. MethodsA total of 120 patients who underwent ERCP in The First Affiliated Hospital of Anhui Medical University from September 2012 to October 2013 were enrolled and randomly divided into diclofenac sodium group and control group, with 60 patients in each group. The patients in the diclofenac sodium group were given intramuscular injection of Olfen (containing diclofenac sodium 75 mg immediately after ERCP. Blood samples were collected before surgery and at 3 and 24 hours after surgery, and the level of amylase was measured. The incidence of abdominal pain was also observed and the incidence rate of PEP was calculated for both groups. RT-PCR and Western blot were used to measure the mRNA and protein expression of NAG-1 in plasma. An repeated-measures analysis of variance was used for comparison of continuous data, a univariate analysis of variance was used for data meeting the requirements of sphericity test, and the Greenhouse-Geisser correction method was used for data which did not meet the requirements of sphericity test. The chi-square test was used for comparison of categorical data between groups. ResultsThe diclofenac sodium group had a significantly lower incidence rate of PEP than the control group [6.67% (4/60 vs 20.00% (12/60, χ2=4.62, P=0.03]. The diclofenac sodium group had a significantly lower level of amylase than the control group at 3 and 24 hours after surgery (at 3 hours after surgery: 202.70±120.44 U/L vs 283.57±178.39 U/L, t=2.06, P<0.05, at 24 hours after surgery: 209.13±157.14 U/L vs 30597±20869 U/L, t=2.03, P<0.05. At 3 hours after ERCP, the diclofenac sodium group had significant increases in the mRNA and protein expression of NAG-1 in plasma and significantly higher mRNA and protein expression of NAG-1 than the control group

  18. The Rapamycin-Binding Domain of the Protein Kinase mTOR is a Destabilizing Domain*

    Science.gov (United States)

    Edwards, Sarah R.; Wandless, Thomas J.

    2013-01-01

    Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding domain (FRB) of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to ten-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retain the ability to inhibit mTOR, albeit with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wildtype FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems. PMID:17350953

  19. 46 CFR 46.10-60 - Control.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Control. 46.10-60 Section 46.10-60 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) LOAD LINES SUBDIVISION LOAD LINES FOR PASSENGER VESSELS Administration § 46.10-60 Control. (a) The District Director of Customs or the Coast Guard District Commander may...

  20. Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium.

    Science.gov (United States)

    Shishkov, Alexander; Bogacheva, Elena; Fedorova, Natalia; Ksenofontov, Alexander; Badun, Gennadii; Radyukhin, Victor; Lukashina, Elena; Serebryakova, Marina; Dolgov, Alexey; Chulichkov, Alexey; Dobrov, Evgeny; Baratova, Lyudmila

    2011-12-01

    The structure of the C-terminal domain of the influenza virus A matrix M1 protein, for which X-ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally-activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C-terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C-terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C-terminal domain is an almost flat layer with a three-α-helical structure. To explain the high level of tritium label incorporation into the C-terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI-TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C-terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell. © 2011 The Authors Journal compilation © 2011 FEBS.

  1. Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

    Science.gov (United States)

    Redka, Dar'ya S; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V; Ellis, John; Ernst, Oliver P; Wells, James W

    2014-08-29

    G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Voltammetric Determination of Flunixin on Molecularly Imprinted Polypyrrole Modified Glassy Carbon Electrode

    Directory of Open Access Journals (Sweden)

    Abd-Elgawad Radi

    2016-01-01

    Full Text Available A novel electrochemical sensing approach, based on electropolymerization of a molecularly imprinted polypyrrole (MIPpy film onto a glassy carbon electrode (GCE surface, was developed for the detection of flunixin (FXN. The sensing conditions and the performance of the constructed sensor were assessed by cyclic, differential pulse and (DPV square wave voltammetry (SWV. The sensor exhibited high sensitivity, with linear responses in the range of 5.0 to 50.0 µM with detection limits of 1.5 and 1.0 µM for DPV and SWV, respectively. In addition, the sensor showed high selectivity towards FXN in comparison to other interferents. The sensor was successfully utilized for the direct determination of FXN in buffalo raw milk samples.

  3. Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR.

    Science.gov (United States)

    Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

    2009-09-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.

  4. Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

    Science.gov (United States)

    Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

    2013-08-01

    Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

  5. Chromatoid Body Protein TDRD6 Supports Long 3' UTR Triggered Nonsense Mediated mRNA Decay.

    Directory of Open Access Journals (Sweden)

    Grigorios Fanourgakis

    2016-05-01

    Full Text Available Chromatoid bodies (CBs are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3' UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3' UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3' UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs.

  6. Deployment Area Selection and Land Withdrawal/Acquisition. M-X/MPS (M-X/Multiple Protective Shelter) Environmental Technical Report. Texas ROI (Region of Influence).

    Science.gov (United States)

    1981-10-02

    C-<a wa-w7-> 4 -0. C zt ə C; 4W ɜ : rZ)zcz ɜ > -(yC4 w 4 < < Z < ’w0 4 C ( (C 0Z a C-- Zaw - - W0 U )-20 :3ZZ, U- ZZCzr. a> C U a a.C. Co 0o00 0 0...zWOWI.+0L tn 0 .4 z l-0uK0WznUof ’ 00 0. 0 -0. IL 9 -- . I J-Z𔃼 0 U.J In V 1 w ul a)l-0 tun 0 In 0 0 U (x> m.~ Z V)I >1-w o 00 3In 0 w 0.-Q.- ik .0

  7. CCS mRNA transcripts and serum CCS protein as copper marker in adults suffering inflammatory processes.

    Science.gov (United States)

    Araya, Magdalena; Gutiérrez, Ricardo; Arredondo, Miguel

    2014-08-01

    The chaperone to Zn-Cu superoxide dismutase (CCS) has been postulated as a candidate copper indicator, changing in a consistent manner in induced and recovered copper deficiency, in experimental cell and animal models. In real life people have various conditions that may modify molecules acting as acute phase proteins, such as serum ceruloplasmin and copper concentration and could alter CCS responses. With the hypothesis that CCS mRNA transcripts and protein would be different in individuals suffering inflammatory processes in comparison to healthy individuals, we assessed adult individuals who, although not ill had conditions known to induce variable degrees of inflammation. Screening of 600 adults resulted in two study groups, formed on the basis of their clinical history and levels of serum C reactive protein (CRP): Group 1 (n = 61, mean (range) CRP = 0.9 (0.3-2.0 mg/dL) and Group 2 (n = 150, mean (range) CRP = 6.1 (4.3-8.7 mg/dL). Results showed that mRNA transcripts relative abundance was not different for CCS, MTIIA, TNF-alpha and Cu-Zn-SOD by group (p > 0.05, one way Anova), nor between sexes (p > 0.05, one way Anova). Distribution of CCS mRNA transcripts and CCS protein in serum did not show any differences or trends. Results disproved our hypothesis that CCS abundance of transcripts and CCS protein would be different in individuals suffering inflammatory processes, adding further support to the idea that CCS may be a copper marker.

  8. Non-visualized Thyroid Gland by Tc-{sup 99m} MIBI Scan with Normal Thyroid Scan

    Energy Technology Data Exchange (ETDEWEB)

    Koca, Gokhan; Atilgan, Hasan Ikbal; Baskin, Aylin; Demirel, Koray; Korkmaz, Meliha [Ankara Training and Research Hospital, Ankara (Turkmenistan)

    2013-09-15

    We present the case of a 21-year-old man who was referred to us for parathyroid scintigraphy with high blood levels of intact parathormone and osteoporosis. Several methods and radiopharmaceuticals, e.g., Tc-99m MIBI and Tl-201 chloride/Tc-99m pertechnetate (Tl-201/TcPO{sup -4}) subtraction, are commonly used for this purpose. We present the case of a thyroid gland that demonstrates quite normal Tc-99m pertechnetate uptake, no accumulation of Tc-99m MIBI, and very low grade Tl-201 uptake. To the best of our knowledge, no similar case has been reported previously. A 21-year-old male with osteoporosis and growth-development retardation was referred for MIBI parathyroid scan because of high blood levels of intact parathormone and bone-specific alkaline-phosphatase, which were 219.4 (15-88 pg/ml) and 355 (21-58 U/L), respectively. In his Tc-99m pertechnetate (TcPO{sup -4}) pinhole scintigraphy, bilateral clearly visualized radioactivity accumulation in the thyroid gland was seen. In both early or late images of the Tc-99m MIBI parathyroid scan, the thyroid gland was not visualized. Therefore, a Tl-201/TcPO{sub -4} subtraction scan method was used. However, the Tl-201 accumulation level in the thyroid gland was not sufficient for the subtraction method. In his thyroid ultrasonography, the thyroid gland echo was homogenous, and there was neither any solid nor cystic lesion. The physical examination of his neck was normal. Other laboratory findings were all normal as follows. TSH: 3.03 (0.35-5.6 IU/mL), free T3: 3.66 (2.5-3.9 pg/mL), free T4: 0.90 (0.59-1.3 ng/dL), Anti-TPO:0.3 (0.40 IU/mL), Anti-TG-Ab: <2.2 (0-+u/L), TSH receptor Ab: 1.0 (0.14 U/L), osteocalcine: 9.13 (1.5-15 ng/dL), growth hormone: 1.3 (0.014-5.21), calcitonin:17 (0.150 ng/mL), sedimentation:6 (0.15 mm/h). There were no significant symptoms of acute or chronic thyroiditis. The cause for discordant uptake in the thyroid gland with T1-201 and Tc-99m MIBI scan could not be provided through clinical or

  9. Uncovering SUMOylation Dynamics during Cell-Cycle Progression Reveals FoxM1 as a Key Mitotic SUMO Target Protein

    DEFF Research Database (Denmark)

    Schimmel, Joost; Eifler, Karolin; Sigurdsson, Jón Otti

    2014-01-01

    Loss of small ubiquitin-like modification (SUMOylation) in mice causes genomic instability due to the missegregation of chromosomes. Currently, little is known about the identity of relevant SUMO target proteins that are involved in this process and about global SUMOylation dynamics during cell......-cycle progression. We performed a large-scale quantitative proteomics screen to address this and identified 593 proteins to be SUMO-2 modified, including the Forkhead box transcription factor M1 (FoxM1), a key regulator of cell-cycle progression and chromosome segregation. SUMOylation of FoxM1 peaks during G2 and M...... relieving FoxM1 autorepression. Cells deficient for FoxM1 SUMOylation showed increased levels of polyploidy. Our findings contribute to understanding the role of SUMOylation during cell-cycle progression....

  10. Planning for a Distributed Disruption: Innovative Practices for Incorporating Distributed Solar into Utility Planning

    Energy Technology Data Exchange (ETDEWEB)

    Mill, Andrew [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Barbose, Galen [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Seel, Joachim [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Dong, Changgui [National Renewable Energy Lab. (NREL), Golden, CO (United States); Mai, Trieu [National Renewable Energy Lab. (NREL), Golden, CO (United States); Sigrin, Ben [National Renewable Energy Lab. (NREL), Golden, CO (United States); Zuboy, Jarrett [Independent Consultant

    2016-08-19

    The rapid growth of distributed solar photovoltaics (DPV) has critical implications for U.S. utility planning processes. This report informs utility planning through a comparative analysis of roughly 30 recent utility integrated resource plans or other generation planning studies, transmission planning studies, and distribution system plans. It reveals a spectrum of approaches to incorporating DPV across nine key planning areas, and it identifies areas where even the best current practices might be enhanced. 1) Forecasting DPV deployment: Because it explicitly captures several predictive factors, customer-adoption modeling is the most comprehensive forecasting approach. It could be combined with other forecasting methods to generate a range of potential futures. 2) Ensuring robustness of decisions to uncertain DPV quantities: using a capacity-expansion model to develop least-cost plans for various scenarios accounts for changes in net load and the generation portfolio; an innovative variation of this approach combines multiple per-scenario plans with trigger events, which indicate when conditions have changed sufficiently from the expected to trigger modifications in resource-acquisition strategy. 3) Characterizing DPV as a resource option: Today’s most comprehensive plans account for all of DPV’s monetary costs and benefits. An enhanced approach would address non-monetary and societal impacts as well. 4) Incorporating the non-dispatchability of DPV into planning: Rather than having a distinct innovative practice, innovation in this area is represented by evolving methods for capturing this important aspect of DPV. 5) Accounting for DPV’s location-specific factors: The innovative propensity-to-adopt method employs several factors to predict future DPV locations. Another emerging utility innovation is locating DPV strategically to enhance its benefits. 6) Estimating DPV’s impact on transmission and distribution investments: Innovative practices are being

  11. Avaliação Bioquímica do líquido cefalorraquidiano de cães normais e de cães jovens portadores de encefalite por cinomose

    Directory of Open Access Journals (Sweden)

    Mary Marcondes Feitosa

    1997-04-01

    Full Text Available O presente experimento teve como objetivos estabelecer padrões de referência para constituintes do líquido cefalorraquidiano, determinar os valores de cloretos, creatina fosfoquinase e aspartato aminotransferase no soro sangüíneo e dosagem de glicose plasmática, na tentativa de verificar se existe correlação entre essas variáveis no sangue e no liquor, de cães normais e de cães jovens portadores de encefalite por cinomose. Foram utilizados 40 animais, sendo 20 cães normais e 20 cães com cinomose. O exame bioquímico do liquor dos cães normais permitiu o estabelecimento de valores para concentração de glicose: 86,09 ± 17,76 mg/dl, níveis de cloretos: 117, 36 ± 21,35 mEq/L, níveis de creatina fosfoquinase; 3,77 ± 1,72 Ul/L e níveis de aspartato aminotransferase 7,43± 4,61 Ul/L. O exame bioquímico do liquor de cães com cinomose permitiu o estabelecimento de valores para concentração de glicose: 80,85 ± 14,82 mg/dl, níveis de cloretos: 118,67± 12,82 mEq/L, níveis de creatina fosfoquinase: 26,38 ± 29,27 Ul/L e níveis de aspartato aminotransferase; 65,70 ± 23,72 Ul/L. Os níveis de cloretos, creatina fosfoquinase e aspartato aminotransferase no soro, e glicose no plasma nos cães normais e nos cães com cinomose foram, respectivamente, 103,43 ± 16,00 e 103,43 ±11,99 mEq/L, 95,75 ± 22,83 e 272,50 ± 140,95 Ul/L, 23,77 ± 5,20 e 23,44 ± 4,81 Ul/L, 119,12 ± 21,46 e 106,12 ± 17,73 mg/dl. Verificou-se uma correlação positiva entre a concentração de glicose liquórica e plasmática nos animais normais e nos animais com cinomose, e entre os níveis de CPK liquórica e sérica nos animais com cinomose. Não foram constatadas diferenças estatisticamente significativas entre os dois grupos de animais com relação aos níveis de cloretos do liquor e do soro. As amostras do liquor dos cães com cinomose apresentaram valores mais elevados de CPK e AST do que as amostras dos cães normais.

  12. SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

    Science.gov (United States)

    Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A; Twiss, Jeffery L; Heise, Tilman

    2016-01-01

    The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

  13. SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

    Directory of Open Access Journals (Sweden)

    Venkatesh Kota

    Full Text Available The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO, but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP elements from the 5' untranslated regions (UTR of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1 mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

  14. Contrasting the excited-state dynamics of the photoactive yellow protein chromophore: Protein versus solvent environments

    NARCIS (Netherlands)

    Vengris, M.; Horst, M.A.; Zgrablic, G.; van Stokkum, I.H.M.; Haacke, S.; Chergui, M.; Hellingwerf, K.J.; van Grondelle, R.; Larsen, D.S.

    2004-01-01

    Wavelength- and time-resolved fluorescence experiments have been performed on the photoactive yellow protein, the E46Q mutant, the hybrids of these proteins containing a nonisomerizing "locked" chromophore, and the native and locked chromophores in aqueous solution. The ultrafast dynamics of these

  15. Extracellular matrix collagen alters cell proliferation and cell cycle progression of human uterine leiomyoma smooth muscle cells.

    Science.gov (United States)

    Koohestani, Faezeh; Braundmeier, Andrea G; Mahdian, Arash; Seo, Jane; Bi, JiaJia; Nowak, Romana A

    2013-01-01

    Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs.

  16. Deficiency of the Survival of Motor Neuron Protein Impairs mRNA Localization and Local Translation in the Growth Cone of Motor Neurons.

    Science.gov (United States)

    Fallini, Claudia; Donlin-Asp, Paul G; Rouanet, Jeremy P; Bassell, Gary J; Rossoll, Wilfried

    2016-03-30

    Spinal muscular atrophy (SMA) is a neurodegenerative disease primarily affecting spinal motor neurons. It is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays an essential role in the biogenesis of spliceosomal small nuclear ribonucleoproteins in all tissues. The etiology of the specific defects in the motor circuitry in SMA is still unclear, but SMN has also been implicated in mediating the axonal localization of mRNA-protein complexes, which may contribute to the axonal degeneration observed in SMA. Here, we report that SMN deficiency severely disrupts local protein synthesis within neuronal growth cones. We also identify the cytoskeleton-associated growth-associated protein 43 (GAP43) mRNA as a new target of SMN and show that motor neurons from SMA mouse models have reduced levels ofGAP43mRNA and protein in axons and growth cones. Importantly, overexpression of two mRNA-binding proteins, HuD and IMP1, restoresGAP43mRNA and protein levels in growth cones and rescues axon outgrowth defects in SMA neurons. These findings demonstrate that SMN plays an important role in the localization and local translation of mRNAs with important axonal functions and suggest that disruption of this function may contribute to the axonal defects observed in SMA. The motor neuron disease spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays a key role in assembling RNA/protein complexes that are essential for mRNA splicing. It remains unclear whether defects in this well characterized housekeeping function cause the specific degeneration of spinal motor neurons observed in SMA. Here, we describe an additional role of SMN in regulating the axonal localization and local translation of the mRNA encoding growth-associated protein 43 (GAP43). This study supports a model whereby SMN deficiency impedes transport and local translation of mRNAs important for neurite outgrowth and stabilization

  17. The Conservation Measures of NATURA 2000 "Someşul Rece" Site Management Plan

    Directory of Open Access Journals (Sweden)

    Marian Proorocu

    2016-11-01

    Full Text Available Natura 2000 is a European network of protected natural areas including a significant number of natural habitats and wild species for the interest of comunnity. Natura 2000 ROSCI 0233 "Someşul Rece"  Site is situated in the south-western county of Cluj, on the administrative territory of Măguri-Răcătău and Ierii Valley. It has an area of 8529 ha and is a framed area of the Apuseni Mountains Alpine bioregions. The site preserves the following natural habitats: Rough mountain beech forests Asperulo-Fagetum, beech forests of Luzulo-Fagetum, forests acidophilous Picea Abies mountain region and protect important species and active fish fauna, flora and fauna of the Apuseni Mountains. It is also home for several species (mammals, amphibians, fish and beetles like: lynx, wolf or otter. The conservation measures of Natura 2000 Somesul Rece Site, elaborated in order to protect the habitats and the species are part of the management plan. These measures were developed in close connection with the conservation status of habitats and species, but also taking into account the needs of local communities. These measures include: maintaining habitats in favorable conservation status; maintain the current habitat areas; preventing and combating poaching and overfishing;ensuring peace in areas of rock (for large mammals.

  18. Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

    Science.gov (United States)

    Zhang, Cathy X. Y.; Brooks, Brian W.; Huang, Hongsheng; Pagotto, Franco

    2016-01-01

    ABSTRACT The Gram-positive bacterium Listeria monocytogenes causes a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range of L. monocytogenes serotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of the L. monocytogenes serotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains of L. monocytogenes which are variable among other Listeria species. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46 L. monocytogenes lineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17 L. monocytogenes lineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some other Listeria species grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker of L. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples. IMPORTANCE L. monocytogenes is

  19. Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

    Science.gov (United States)

    Shenje, Lincoln T; Andersen, Peter; Halushka, Marc K; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A S; Chen, Yan; Chelko, Stephen; Crosson, Jane E; Scheel, Janet; Vricella, Luca; Craig, Brian D; Marosy, Beth A; Mohr, David W; Hetrick, Kurt N; Romm, Jane M; Scott, Alan F; Valle, David; Naggert, Jürgen K; Kwon, Chulan; Doheny, Kimberly F; Judge, Daniel P

    2014-03-04

    Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

  20. A randomized male tolerance study of dapivirine gel following multiple topical penile exposures (MTN 012/IPM 010).

    Science.gov (United States)

    Cranston, Ross D; Hoesley, Craig; Carballo-Diéguez, Alex; Hendrix, Craig W; Husnik, Marla; Levy, Lisa; Hall, Wayne; Soto-Torres, Lydia; Nel, Annalene M

    2014-02-01

    Dapivirine (DPV) is a nonnucleoside reverse transcriptase inhibitor with a favorable safety profile following vaginal application. A penile tolerance study was conducted prior to further development of DPV as a candidate vaginal microbicide. Twenty-four circumcised and 24 uncircumcised (N=48) healthy HIV-negative male participants aged 18 years or older were randomized 2:1:1 to apply DPV 0.05% gel, matched placebo gel, or universal placebo gel, respectively, to their penis once daily for 7 sequential days. The safety, acceptability, and pharmacokinetic profile of DPV 0.05% gel were assessed by the presence of Grade 2 or higher genitourinary adverse events (AEs) and systemic AEs, a behavioral questionnaire, and pharmacokinetic plasma blood draw, respectively, at the final clinic visit (FCV). There were no Grade 2 genitourinary AEs in 47 participants completing the FCV. One participant in the DPV arm failed to attend the FCV. There were 13 AEs reported; all were Grade 1 except one Grade 2 corneal laceration unrelated to study product. Participants liked the gel to a moderate extent, yet 72% reported they would be "very likely" to use a gel like the one they used in the study every time they have intercourse. DPV was detectable in plasma in all 23 DPV arm study participants at the FCV. On average, the circumcised participants' DPV concentrations were 54% of those in uncircumcised participants (p=0.07). Topical seven-day penile application of DPV 0.05% gel was locally and systemically safe, was acceptable to male participants, and resulted in systemic exposure to the drug.

  1. Alternative protein secretion: The Mam1 ABC transporter supports secretion of M-factor linked GFP in fission yeast

    International Nuclear Information System (INIS)

    Kjaerulff, Soren; Mueller, Sven; Jensen, Martin Roland

    2005-01-01

    To examine whether the fission yeast Mam1 ABC transporter can be used for secretion of heterologous proteins, thereby bypassing the classical secretion pathway, we have analyzed chimeric forms of the M-factor precursor. It was demonstrated that GFP can be exported when fused to both the amino-terminal prosequence from mfm1 and a CaaX motif. This secretion was dependent on the Mam1 transporter and not the classical secretion pathway. The secretion efficiency of GFP, however, was relatively low and most of the reporter protein was trapped in the vacuolar membranes. Our findings suggest that the Mam1 ABC protein is a promiscuous peptide transporter that can accommodate globular proteins of a relatively large size. Furthermore, our results help in defining the sequences required for processing and secretion of natural M-factor

  2. Exercise training and work task induced metabolic and stress-related mRNA and protein responses in myalgic muscles

    DEFF Research Database (Denmark)

    Sjøgaard, Gisela; Zebis, Mette Kreutzfeldt; Kiilerich, Kristian

    2013-01-01

    healthy controls. Those with myalgia performed similar to 7 hrs repetitive stressful work and were subsequently randomized to 10 weeks of specific strength training, general fitness training, or reference intervention. Muscles biopsies were taken from the trapezius muscle at baseline, after work and after...... 10 weeks intervention. The main findings are that the capacity of carbohydrate oxidation was reduced in myalgic compared with healthy muscle. Repetitive stressful work increased mRNA content for heat shock proteins and decreased levels of key regulators for growth and oxidative metabolism......The aim was to assess mRNA and/or protein levels of heat shock proteins, cytokines, growth regulating, and metabolic proteins in myalgic muscle at rest and in response to work tasks and prolonged exercise training. A randomized controlled trial included 28 females with trapezius myalgia and 16...

  3. Standard model Wilson coefficients for c → ul{sup +}l{sup -} transitions at next-to-leading order

    Energy Technology Data Exchange (ETDEWEB)

    Boer, Stefan de [TU Dortmund (Germany); Mueller, Bastian; Seidel, Dirk [Uni Siegen (Germany)

    2016-07-01

    The standard theoretical framework to deal with exclusive, weak decays of heavy mesons is the so-called weak effective Hamiltonian. It involves the short-distance Wilson coefficients, which depend on the renormalization scale μ. For specific calculations one has to evolve the Wilson coefficients down from the electroweak scale μ{sub W} to the typical mass scale of the decay under consideration. This is done by solving a renormalization group equation for the effective operator basis. In this talk the results of a consistent two-step running of the c → ul{sup +}l{sup -} Wilson coefficients are presented. This running involves the intermediate scale μ{sub b} (with μ{sub W} > μ{sub b} > μ{sub c}) where the bottom quark is integrated out. All the matching coefficients and anomalous dimensions are taken to the required order by generalizing and extending results from b → s or s → d transitions available in the literature.

  4. Fasting conditions: Influence of water intake on clinical chemistry analytes.

    Science.gov (United States)

    Benozzi, Silvia F; Unger, Gisela; Campion, Amparo; Pennacchiotti, Graciela L

    2018-02-15

    Currently available recommendations regarding fasting requirements before phlebotomy do not specify any maximum water intake volume permitted during the fasting period. The aim was to study the effects of 300 mL water intake 1 h before phlebotomy on specific analytes. Blood was collected from 20 women (median age (min-max): 24 (22 - 50) years) in basal state (T 0 ) and 1 h after 300 mL water intake (T 1 ). Glucose, total proteins (TP), urea, creatinine, cystatin C, total bilirubin (BT), total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides (Tg), uric acid (UA), high-sensitivity C-reactive protein, gamma-glutamyl transferase (GGT), aspartate-aminotransferase (AST), alanine-aminotransferase and lactate-dehydrogenase (LD) were studied. Results were analyzed using Wilcoxon test. Mean difference (%) was calculated for each analyte and was further compared with reference change value (RCV). Only mean differences (%) higher than RCV were considered clinically significant. Significant differences (median T 0 vs median T 1 , P) were observed for TP (73 vs 74 g/L, 0.001); urea (4.08 vs 4.16 mmol/L, 0.010); BT (12 vs 13 µmol/L, 0.021); total cholesterol (4.9 vs 4.9 mmol/L, 0.042); Tg (1.05 vs 1.06 mmol/L, 0.002); UA (260 vs 270 µmol/L, 0.006); GGT (12 vs 12 U/L, 0.046); AST (22 vs 24 U/L, 0.001); and LD (364 vs 386 U/L, 0.001). Although the differences observed were statistically significant, they were not indicative of clinically significant changes. A water intake of 300 mL 1 h prior to phlebotomy does not interfere with the analytes studied in the present work.

  5. microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein

    OpenAIRE

    Pinder, Benjamin D; Smibert, Craig A

    2012-01-01

    Argonaute 1 directly interacts with the RNA binding protein Smaug in Drosophila, is thereby recruited to the Smaug target nanos mRNA and is required for Smaug-mediated translational repression of the nanos mRNA.

  6. Core drilling of deep borehole OL-KR46 at Olkiluoto in Eurajoki 2007

    International Nuclear Information System (INIS)

    Toropainen, V.

    2007-09-01

    Posiva Oy submitted an application to the Finnish Government in May 1999 for the Decision in Principle to choose Olkiluoto in the municipality of Eurajoki as the site of the final disposal facility for spent nuclear fuel. A positive decision was made at the end of 2000 by the Government. The Finnish Parliament ratified the decision in May 2001. The decision makes it possible for Posiva to focus the confirming bedrock investigations at Olkiluoto, where in the next few years an underground rock characterisation facility, ONKALO, will be constructed. As a part of the investigations Suomen Malmi Oy (Smoy) core drilled 600.10 m and 45.16 m deep boreholes with a diameter of 75.7 mm at Olkiluoto in May - June 2007. The identification numbers of the boreholes are OL-KR46 and OL-KR46B, respectively. A set of monitoring measurements and samplings from the drilling and returning water was carried out during the drilling. Both the volume and the electric conductivity of the returning water, and the volume of drilling water were recorded. The drill rig was computer controlled and during drilling the computer recorded drilling parameters. The objective of all these measurements was to obtain more information about bedrock and groundwater properties. Sodium fluorescein was used as a label agent in the drilling water. The total volumes of the used drilling and flushing water were 466 m 3 and 20 m 3 in boreholes OL-KR46 and OL-KR46B, respectively. Measured volumes of the returning water were 407 m 3 in borehole OL-KR46 and 12 m 3 in borehole OL-KR46B. The deviation of the boreholes was measured with the deviation measuring instruments EMS and Maxibor. Uniaxial compressive strength, Young's Modulus and Poisson's ratio were measured from the core samples. The average uniaxial compressive strength is 116.5 MPa, the average Young's Modulus is 31.5 GPa and the average Poisson's ratio is 0.20. The main rock types are veined gneiss, tonalitic-granodioritic-granitic gneiss and pegmatite

  7. Effects of low dose radiation on expressions of ICAM-1 mRNA and protein in kidney of diabetic mice

    International Nuclear Information System (INIS)

    Zhang Chi; Li Xiaokun; Gong Shouliang; Liu Xiaoju; Zhao Xue; Liu Xiaoju; Zhao Xue; Shen Wenjie; Li Cai; Cai Lu

    2010-01-01

    Objective: To study the effects of low dose radiation (LDR) on the expressions of intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in kidney of diabetes mellitus (DM) mice and illuminate that anti-inflammation of LDR is a main mechanism for diabetic therapy. Methods: The healthy and right age C57BL/6J mice were divided into 4 groups including control, DM, LDR and DM/LDR. The mice in DM and DM/LDR groups were injected intraperitoneally with streptozocin (STZ) to set up DM models. The mice in DM/LDR and LDR groups were irradiated with 25 mGy every other day for 4 weeks. The expressions of ICAM-1 mRNA and protein in kidney were detected with RT-PCR and Western blotting 2, 4, 8, 12 and 16 weeks after irradiation. Results: The expressions of ICAM-1 mRNA and protein in kidney had no significant difference among 4 groups before LDR (P>0.05). The expressions of ICAM-1 mRNA and protein 2 weeks after irradiation with LDR were higher than those in the other 3 groups (P<0.05). The expressions of ICAM-1 mRNA and protein in the DM/LDR group 4 weeks after irradiation were also significantly higher than those in non-DM groups (P<0.05), but still significantly lower than those in DM group (P<0.05), and the significant differences were kept to 16 weeks after irradiation. But the expressions of ICAM-1 mRNA and protein in LDR group were significantly higher than those in control group (P<0.05). IHC assay showed that the glomerular and tubular in DM and DM/LDR groups were abnormal and the quantities of the positive staining cells were significantly increased compared with non-DM groups. However the damage of glomerular and tubular in DM/LDR was significantly supressed compared with DM group and the positive staining cells were also decreased. Conclusion: Under the circumstance of DM, LDR can significantly decrease the expressions of ICAM-1 mRNA and protein in mouse kidney to relief the inflammation reaction in kidney; but in normal condition, LDR can improve the immunity and

  8. Prediction of protein modification sites of pyrrolidone carboxylic acid using mRMR feature selection and analysis.

    Directory of Open Access Journals (Sweden)

    Lu-Lu Zheng

    Full Text Available Pyrrolidone carboxylic acid (PCA is formed during a common post-translational modification (PTM of extracellular and multi-pass membrane proteins. In this study, we developed a new predictor to predict the modification sites of PCA based on maximum relevance minimum redundancy (mRMR and incremental feature selection (IFS. We incorporated 727 features that belonged to 7 kinds of protein properties to predict the modification sites, including sequence conservation, residual disorder, amino acid factor, secondary structure and solvent accessibility, gain/loss of amino acid during evolution, propensity of amino acid to be conserved at protein-protein interface and protein surface, and deviation of side chain carbon atom number. Among these 727 features, 244 features were selected by mRMR and IFS as the optimized features for the prediction, with which the prediction model achieved a maximum of MCC of 0.7812. Feature analysis showed that all feature types contributed to the modification process. Further site-specific feature analysis showed that the features derived from PCA's surrounding sites contributed more to the determination of PCA sites than other sites. The detailed feature analysis in this paper might provide important clues for understanding the mechanism of the PCA formation and guide relevant experimental validations.

  9. Atorvastatin protects against ischemia-reperfusion injury in fructose-induced insulin resistant rats.

    Science.gov (United States)

    Prakash, Prem; Khanna, Vivek; Singh, Vishal; Jyoti, Anupam; Jain, Manish; Keshari, Ravi Shankar; Barthwal, Manoj Kumar; Dikshit, Madhu

    2011-08-01

    High fructose (HFr) intake is known to cause insulin resistance syndrome (IRS), however its effect against acute coronary events remains elusive. The present study was undertaken to evaluate the effect of HFr (60%) diet on myocardial ischemia-reperfusion (MI-RP) injury and its modulation by atorvastatin treatment. Wistar rats kept on HFr/chow feeding for 10 weeks, received atorvastatin (30 mg/kg, per oral) or vehicle for two additional weeks followed by MI-RP injury. MI-RP injury was significantly augmented in HFr fed rats, as evident by the increase in infarct size (IS, 65 ± 5% vs. 43 ± 7%) and activities of cardiac injury biomarkers [serum lactate dehydrogenase (LDH, 698 ± 57 vs. 444 ± 26 U/L), creatinine kinase (CK-MB, 584 ± 58 vs. 435 ± 28 U/L) and tissue myeloperoxidase (MPO, 235 ± 15 vs. 101 ± 11 μM/min/100 mg tissue)]. Insulin resistance (plasma glucose, 64 ± 5 vs. 100 ± 5 mg/dl; AUC (0-120 min), p < 0.05), MI-RP injury (IS 20 ± 5%, LDH 292 ± 28 U/L, CK-MB 257 ± 13 U/L, MPO 95 ± 5 μM/min/100 mg tissue) and triglyceride (TG) level were significantly reduced, while myocardial Akt, p-Akt, eNOS, p-eNOS and iNOS protein expression were significantly enhanced following atorvastatin treatment in comparison to HFr fed rats. Oxidative stress marker, malondialdehyde and circulating levels of inflammatory cytokines (CRP, IL-6, IFN-γ and TNF) were significantly reduced, while total nitrite content in the tissue and plasma was significantly augmented in atorvastatin treated rats. Atorvastatin also ameliorated endothelial dysfunction and significantly enhanced aortic Akt and eNOS protein expression. Atorvastatin conferred significant protection against MI-RP injury and alleviated HFr induced IRS possibly by increasing NOS expression through Akt dependent pathway.

  10. Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor.

    Science.gov (United States)

    Weidmann, Chase A; Goldstrohm, Aaron C

    2012-01-01

    Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains.

  11. M/sub r/ 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor

    International Nuclear Information System (INIS)

    Moscatelli, D.; Joseph-Silverstein, J.; Manejias, R.; Rifkin, D.B.

    1987-01-01

    A M/sub r/ 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig grain along with the typical M/sub r/ 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The M/sub r/ 25,000 form, like the M/sub r/ 18,000 form was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The M/sub r/ 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentration of 0.1-10 ngml, the same range that was effective for guinea pig and human M/sub r/ 18,000 bFGFs. The binding of human 125 I-labeled bFGF to baby hamster kidney cells is inhibited equally by the M/sub r/ 25,000 guinea pig protein and the M/sub r/ 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the M/sub r/ 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the M/sub r/ 25,000 and M/sub r/ 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the M/sub r/ 25,000 guinea pig bFGF was converted to a M/sub r/ 18,000 protein. These results show that the two molecules are closely related and suggest that the M/sub r/ 25,000 protein shares substantial homology with the M/sub r/ 18,000 bFGF

  12. Voltammetric analysis of N-containing drugs using the hanging galinstan drop electrode (HGDE).

    Science.gov (United States)

    Channaa, H; Surmann, P

    2009-03-01

    The electrochemical behaviour of several N-containing voltammetric active drugs such as 1,4-benzodiazepines (chlordiazepoxide, nitrazepam and diazepam) as well as one nitro-compound (nitrofurantoin) and one azo-compound (phenazopyridine) is described using a new kind of liquid electrode, the hanging galinstan drop electrode. Concentrations of 10(-5) - 10(-8) mol L(-1) are generally measurable. Differential pulse and adsorptive stripping voltammograms are recorded in different supporting electrolytes, like 0.1 M KNO3, acetate buffer solution pH = 4.6 and phosphate buffer solution pH = 7.0. The effects of varying the starting potentials, U(start) for DPV and accumulation times, t(acc) for AdSV are considered. Briefly, it is shown that the novel galinstan electrode is suitable for reducing several functional groups in organic substances, here presented for N-oxide-, azomethine-, nitro- and azo-groups.

  13. Glomerular cell death and inflammation with high-protein diet and diabetes.

    Science.gov (United States)

    Meek, Rick L; LeBoeuf, Renee C; Saha, Sandeep A; Alpers, Charles E; Hudkins, Kelly L; Cooney, Sheryl K; Anderberg, Robert J; Tuttle, Katherine R

    2013-07-01

    Overfeeding amino acids (AAs) increases cellular exposure to advanced glycation end-products (AGEs), a mechanism for protein intake to worsen diabetic kidney disease (DKD). This study assessed receptor for AGE (RAGE)-mediated apoptosis and inflammation in glomerular cells exposed to metabolic stressors characteristic of high-protein diets and/or diabetes in vitro with proof-of-concept appraisal in vivo. Mouse podocytes and mesangial cells were cultured under control and metabolic stressor conditions: (i) no addition; (ii) increased AAs (4-6-fold>control); (iii) high glucose (HG, 30.5 mM); (iv) AA/HG combination; (v) AGE-bovine serum albumin (AGE-BSA, 300 µg/mL); (vi) BSA (300 µg/mL). RAGE was inhibited by blocking antibody. Diabetic (streptozotocin) and nondiabetic mice (C57BL/6J) consumed diets with protein calories of 20 or 40% (high) for 20 weeks. People with DKD and controls provided 24-h urine samples. In podocytes and mesangial cells, apoptosis (caspase 3/7 activity and TUNEL) increased in all metabolic stressor conditions. Both inflammatory mediator expression (real-time reverse transcriptase-polymerase chain reaction: serum amyloid A, caspase-4, inducible nitric oxide synthase, and monocyte chemotactic protein-1) and RAGE (immunostaining) also increased. RAGE inhibition prevented apoptosis and inflammation in podocytes. Among mice fed high protein, podocyte number (WT-1 immunostaining) decreased in the diabetic group, and only these diabetic mice developed albuminuria. Protein intake (urea nitrogen) correlated with AGE excretion (carboxymethyllysine) in people with DKD and controls. High-protein diet and/or diabetes-like conditions increased glomerular cell death and inflammation, responses mediated by RAGEs in podocytes. The concept that high-protein diets exacerbate early indicators of DKD is supported by data from mice and people.

  14. Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

    International Nuclear Information System (INIS)

    Klinefelter, G.R.; Hamilton, D.W.

    1985-01-01

    We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-[35S]methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-[35S]methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB[3H]4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis

  15. Influence of biflorin on the labelling of red blood cells, plasma protein, cell protein, and lymphocytes with technetium-99m: in vitro study

    Directory of Open Access Journals (Sweden)

    Thiago M. Aquino

    Full Text Available In this paper we report the results of an in vitro study involving the influence of biflorin (an o-quinone isolated from Capraria biflora L. that has potent antimicrobial activity on the Tc-99m labeling of red blood cells, plasma protein, cells protein, and lymphocytes. Blood was withdrawn from Wistar rats and incubated with various concentrations of biflorin, and solutions of stannous chloride and Tc-99m were added. Plasma (P and red blood cells (RBC were isolated, precipitated, and centrifuged, and soluble (SF and insoluble (IF fractions were isolated. The results show that the highest concentration (100% of biflorin is able to reduce the uptake of Tc-99m (%ATI on RBC and the fixation on IF-P. To study the influence of biflorin on 99mTc lymphocyte labeling, human blood was submitted to a technique with Ficoll-Hypac and centrifuged, and white cells were isolated. Lymphocytes (2.5 mL; 1.0 x 10(6 cells/mL were obtained and a 0.2 mL solution was incubated with biflorin (0.1 mL. Solutions of stannous chloride and 99mTc were added. Lymphocytes were separated and the %ATI bound in these cells was evaluated. A reduction in %ATI (from 97.85 ± 0.99 to 88.86 ± 5 was observed for RBC and for IF-P (73.24 ± 5.51 to 20.72 ± 6.95. In this case the results showed no decrease in %ATI for the lymphocytes with biflorin.

  16. Bactericidal activity of M protein conserved region antibodies against group A streptococcal isolates from the Northern Thai population

    Directory of Open Access Journals (Sweden)

    Pruksachatkunakorn Chulabhorn

    2006-08-01

    Full Text Available Abstract Background Most group A streptococcal (GAS vaccine strategies have focused on the surface M protein, a major virulence factor of GAS. The amino-terminus of the M protein elicits antibodies, that are both opsonic and protective, but which are type specific. J14, a chimeric peptide that contains 14 amino acids from the M protein conserved C-region at the carboxy-terminus, offers the possibility of a vaccine which will elicit protective opsonic antibodies against multiple different GAS strains. In this study, we searched for J14 and J14-like sequences and the number of their repeats in the C-region of the M protein from GAS strains isolated from the Northern Thai population. Then, we examined the bactericidal activity of J14, J14.1, J14-R1 and J14-R2 antisera against multiple Thai GAS strains. Results The emm genes of GAS isolates were sequenced and grouped as 14 different J14-types. The most diversity of J14-types was found in the C1-repeat. The J14.1 type was the major sequence in the C2 and C3-repeats. We have shown that antisera raised against the M protein conserved C-repeat region peptides, J14, J14.1, J14-R1 and J14-R2, commonly found in GAS isolates from the Northern Thai population, are able to kill GAS of multiple different emm types derived from an endemic area. The mean percent of bactericidal activities for all J14 and J14-like peptide antisera against GAS isolates were more than 70%. The mean percent of bactericidal activity was highest for J14 antisera followed by J14-R2, J14.1 and J14-R1 antisera. Conclusion Our study demonstrated that antisera raised against the M protein conserved C-repeat region are able to kill multiple different strains of GAS isolated from the Northern Thai population. Therefore, the four conserved "J14" peptides have the potential to be used as GAS vaccine candidates to prevent streptococcal infections in an endemic area.

  17. The ROSCOE Manual. Volume 2-1. Sample Case.

    Science.gov (United States)

    1980-03-01

    tC _ iui)i.’:r:<RC — ü in M M M M w w u) i a: u O Ott *-OOG<i*»< O CJ p- c j UJ tu UJ IL ^ UJ o: UJ fr- 3Ct a: a u ^ i...Ul U 00 X O X X o i ul X z t- • ui O i U Ul I O UJ ox 54 M«n""w* piM >w« 1 -1 3 J>«r^«9o<H^i^7l/)«^C09O««(MHt3kn4)h-(D9O-«njr󈧑l04)M0

  18. Transpiração e condutância foliar à difusão de vapor de feijoeiro irrigado em função da temperatura da folhagem e variáveis ambientais = Transpiration and stomatal conductance of irrigated bean in relation to foliage temperature and environmental variables

    Directory of Open Access Journals (Sweden)

    Paulo Augusto Manfron

    2007-01-01

    Full Text Available Áreas com cultivo irrigado têm o déficit de saturação de vapor (DPV etemperatura do ar modificados. Sendo a resposta estomática influenciada por essas variáveis e outras como temperatura do dossel, a cultura do feijão irrigado tende a apresentar condutância estomática à difusão de vapor (Gva e transpiração, diferenciados com relação ao cultivo de sequeiro. Avaliando-se Gva e transpiração com porômetros de equilíbrio dinâmico, verificou-se que a taxa de transpiração apresentou melhor correlação em relação à temperatura da folhagem em condições de folhas ao sol, do que em relação a folhassombreadas. Relações de Gva com temperatura do ar, DPV e radiação fotossinteticamente ativa (PAR reforçam a interação dos fatores ambientais com a resposta estomática. Valores de Gva apresentaram correlação exponencial negativa tanto com temperatura do ar e DPV,para valores entre 20 e 35°C, de 0,5 à 3 KPa, respectivamente e aumento exponencial quando relacionada a PAR, mesmo com valores superiores a 2000 mmol m-2 s-1.Irrigated areas present environmental variables such as vapor pressure deficit (DPV and modified air temperature. The stomatal response is not only affected by these modified environmental conditions, but also by others such as canopy temperature. Thus, an irrigated bean crop tend to present modifications in stomatal conductance (Gva and transpiration in relation to a non irrigatedcommon bean crop. Gva and transpiration were measured with steady-state null-balance porometers. Results showed that transpiration rate correlated better with canopy temperature in conditions of sunny leaves than of shaded leaves. The relation between Gva and air temperature, and between DPV and photosynthetic active radiation (PAR reinforce the interaction of the environmental variables with stomatal response. Gva values presented negative exponential correlation with air temperature and DPV, for values between 20 and 35°C, and 0

  19. Dynamic subframe allocation for mobile broadband m-health using IEEE 802.16j mobile multihop relay networks.

    Science.gov (United States)

    Alinejad, Ali; Istepanian, R S H; Philip, N

    2012-01-01

    The concept of 4G health will be one of the key focus areas of future m-health research and enterprise activities in the coming years. WiMAX technology is one of the constituent 4G wireless technologies that provides broadband wireless access (BWA). Despite the fact that WiMAX is able to provide a high data rate in a relatively large coverage; this technology has specific limitations such as: coverage, signal attenuation problems due to shadowing or path loss, and limited available spectrum. The IEEE 802.16j mobile multihop relay (MMR) technology is a pragmatic solution designed to overcome these limitations. The aim of IEEE 802.16j MMR is to expand the IEEE 802.16e's capabilities with multihop features. In particular, the uplink (UL) and downlink (DL) subframe allocation in WiMAX network is usually fixed. However, dynamic frame allocation is a useful mechanism to optimize uplink and downlink subframe size dynamically based on the traffic conditions through real-time traffic monitoring. This particular mechanism is important for future WiMAX based m-health applications as it allows the tradeoff in both UL and DL channels. In this paper, we address the dynamic frame allocation issue in IEEE 802.16j MMR network for m-health applications. A comparative performance analysis of the proposed approach is validated using the OPNET Modeler(®). The simulation results have shown an improved performance of resource allocation and end-to-end delay performance for typical medical video streaming application.

  20. [Expression of heat shock protein 70 and its mRNA in career exposure to manganese].

    Science.gov (United States)

    Chen, Wenwen; Shao, Hua; Chi, Mingfeng; Zhang, Zhihu; Shan, Yongle; Zou, Wei

    2015-10-01

    To analyze the expression levels of heat shock protein70 (HSPs70) and HSPs70 mRNA in different exposure to manganese, and research the neuroprotective effect on the career exposure to manganese. From 2008 to 2009, with cross-sectional study design, and in a locomotive and rolling stock works, by stratified random sampling method, the exposed sample consisted of 180 welders from different welding shops and 100 unexposed in the last three years, non-welder controls with age-matched workers of similar socioeconomic status from the same industry. The control workers had not been exposed to neurotoxic chemicals. The mRNA expressions of four different metabolic enzyme were detected by SYBR Green I quantitative real-time polymerase chain reaction. The expression levels of the two enzymes mRNA in different exposure to manganese were analyzed. The expressions of HSPs70 were detected by Western blot. The concentration of air manganese was determined by GFAAS. The average concentration of 8 h time (8h-TWA) was used to express the level of individual exposure to manganese, according to the air manganese workplace occupational exposure limit (8h-TWA=0.15 mg/m3), the exposed group is divided into high exposed group (>0.15 mg/m3) and low exposure group (<0.15 mg/m3). The individuals exposed to manganese dose of exposed group ((0.25±0.31) mg/m3) was higher than the control group ((0.06±0.02) mg/m3) (t=6.15, P=0.001); individuals exposed to manganese dose of high exposure group for (0.42±0.34) mg/m3, which was higher than low exposure group (0.09±0.07) mg/m3 (t=9.80, P=0.001). HSPs70 mRNA and protein of exposure group (5.65±0.21, 3.26±0.15) were higher than the reference group (0.41±0.03, 1.32±0.12) (t=18.91, t=8.68, P=0.001). HSP70 mRNA and protein of high exposure group (6.48±0.37, 3.67±0.26) were higher than the low exposure group (5.15±0.23, 3.02±0.19) (t=3.24, t=2.01, P=0.003, P=0.043). The expression of peripheral blood lymphocytes HSPs70 level and HSPs70 m