Science.gov

Sample records for 5a ns5a protein

  1. Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A.

    PubMed

    Arima, N; Kao, C Y; Licht, T; Padmanabhan, R; Sasaguri, Y; Padmanabhan, R

    2001-04-20

    Hepatitis C virus nonstructural protein, NS5A, is a phosphoprotein produced from the processing of the viral polyprotein precursor. NS5A associates with several cellular proteins in mammalian cells, and the biological consequences of this interaction are currently unknown. To this end, five stable NS5A-expressing murine and human cell lines were established. Tetracycline-regulated NIH3T3 cells and rat liver epithelial cells as well as the constitutive, NS5A-expressing, human Chang liver, HeLa, and NIH3T3 cells all exhibited cell growth retardation compared with the control cells. Cell cycle analysis by flow cytometry indicated that the NS5A-expressing human epitheloid tumor cells had a reduced S phase and an increase in the G(2)/M phase, which could be explained by a p53-dependent induction of p21(Waf1/Cip1) protein and mRNA levels. NS5A interacts with Cdk1 in vivo and in vitro, and a significant portion of the p21(Waf1/Cip1) was found to be in a complex with Cdk2 in the NS5A-expressing human hepatic cell line. Cdk1 and cyclin B1 proteins were also reduced in human Chang liver cells consistent with the increase in G(2)/M phase. Our results suggest that the NS5A protein causes growth inhibition and cell cycle perturbations by targeting the Cdk1/2-cyclin complexes.

  2. Hepatitis C virus NS5A protein modulates IRF-7-mediated interferon-α signaling.

    PubMed

    Chowdhury, Joydip Bhanja; Kim, Hangeun; Ray, Ranjit; Ray, Ratna B

    2014-01-01

    Hepatitis C virus (HCV) establishes chronic infection in a large number of infected individuals. We have previously shown that HCV infection in hepatocytes blocks poly (I-C) or interferon (IFN)-α-mediated IRF-7 nuclear translocation (Raychoudhuri and others 2010). However, the mechanism of IRF-7 regulation by HCV remained unknown. In this study, we have observed that HCV NS5A physically associates with IRF-7. A subsequent study suggested that the HCV NS5A protein blocks IRF-7-mediated IFN-α14 promoter activation. Further analyses demonstrated that site-specific mutagenesis of the 2 basic arginine residues (amino acids Arg(216) and Arg(217)) in the NS5A is critical for IRF-7-mediated IFN-α14 promoter regulation. Together, our results suggested that the HCV NS5A protein limits the IFN-α-signaling pathway in association with IRF-7, and may, in part, be responsible for the establishment of chronic infection.

  3. Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein

    PubMed Central

    Tellinghuisen, Timothy L.; Foss, Katie L.; Treadaway, Jason

    2008-01-01

    Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to

  4. Crystal Structure of a Novel Dimeric Form of NS5A Domain I Protein from Hepatitis C Virus

    SciTech Connect

    Love, Robert A.; Brodsky, Oleg; Hickey, Michael J.; Wells, Peter A.; Cronin, Ciarán N.; Pfizer

    2009-07-10

    A new protein expression vector design utilizing an N-terminal six-histidine tag and tobacco etch virus protease cleavage site upstream of the hepatitis C virus NS5A sequence has resulted in a more straightforward purification method and improved yields of purified NS5A domain I protein. High-resolution diffracting crystals of NS5A domain I (amino acids 33 to 202) [NS5A(33-202)] were obtained by using detergent additive crystallization screens, leading to the structure of a homodimer which is organized differently from that published previously (T. L. Tellinghuisen, J. Marcotrigiano, and C. M. Rice, Nature 435:374-379, 2005) yet is consistent with a membrane association model for NS5A. The monomer-monomer interface of NS5A(33-202) features an extensive buried surface area involving the most-highly conserved face of each monomer. The two alternate structural forms of domain I now available may be indicative of the multiple roles emerging for NS5A in viral RNA replication and viral particle assembly.

  5. Genetic diversity of NS5A protein from hepatitis C virus genotype 3a and its relationship to therapy response

    PubMed Central

    2010-01-01

    Background The quasispecies nature of HCV may have important implications for viral persistence, pathogenicity and resistance to antiviral agents. The variability of one of the viral proteins, NS5A, is believed to be related to the response to IFN therapy, the standard treatment for infection. In this study we analyzed the quasispecies composition of NS5A protein in patients infected with HCV genotype 3a, before IFN therapy. Methods Viral RNA was isolated from samples of 12 patients: four sustained virological responders (SVR), four non-responders (NR), and four end-of-treatment responders (ETR). cDNA was synthesized, the NS5A region was amplified and the fragments obtained were cloned. Fifteen clones from each patient were sequenced with eight primers, generating 179 contigs. Results Higher values for substitution (either synonymous or non-synonymous) and for distance were found in the SVR group. However, the NR group showed relatively more non-synonymous mutations than the other groups, owing to the higher values of dN/dS in complete NS5A and most specific regions. Overall, NS5A protein is undergoing purifying selection, since all dN/dS ratios values are below 0.5. Conclusions Our study provides an overview of the genetic variability of complete NS5A protein in HCV genotype 3a. PMID:20178583

  6. Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A.

    PubMed

    Kim, Nari; Kim, Min-Jung; Sung, Pil Soo; Bae, Yong Chul; Shin, Eui-Cheol; Yoo, Joo-Yeon

    2016-02-12

    Hepatitis C virus (HCV) utilizes autophagy to promote its propagation. Here we show the autophagy-mediated suppression of HCV replication via the endoplasmic reticulum (ER) protein SCOTIN. SCOTIN overexpression inhibits HCV replication and infectious virion production in cells infected with cell culture-derived HCV. HCV nonstructural 5A (NS5A) protein, which is a critical factor for HCV RNA replication, interacts with the IFN-β-inducible protein SCOTIN, which transports NS5A to autophagosomes for degradation. Furthermore, the suppressive effect of SCOTIN on HCV replication is impaired in both ATG7-silenced cells and cells treated with autophagy or lysosomal inhibitors. SCOTIN does not affect the overall flow of autophagy; however, it is a substrate for autophagic degradation. The physical association between the transmembrane/proline-rich domain (TMPRD) of SCOTIN and Domain-II of NS5A is essential for autophagosomal trafficking and NS5A degradation. Altogether, our findings suggest that IFN-β-induced SCOTIN recruits the HCV NS5A protein to autophagosomes for degradation, thereby restricting HCV replication.

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

    PubMed

    Galli, Andrea; Scheel, Troels K H; Prentoe, Jannick C; Mikkelsen, Lotte S; Gottwein, Judith M; Bukh, Jens

    2013-10-01

    Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A recombinants produced infectivity titres of 10(2.5)-10(4.5) f.f.u. ml(-1). Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1-7 core-NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.

  8. The interaction between the hepatitis C proteins NS4B and NS5A is involved in viral replication.

    PubMed

    David, Naama; Yaffe, Yakey; Hagoel, Lior; Elazar, Menashe; Glenn, Jeffrey S; Hirschberg, Koret; Sklan, Ella H

    2015-01-15

    Hepatitis C virus (HCV) replicates in membrane associated, highly ordered replication complexes (RCs). These complexes include viral and host proteins necessary for viral RNA genome replication. The interaction network among viral and host proteins underlying the formation of these RCs is yet to be thoroughly characterized. Here, we investigated the association between NS4B and NS5A, two critical RC components. We characterized the interaction between these proteins using fluorescence resonance energy transfer and a mammalian two-hybrid system. Specific tryptophan residues within the C-terminal domain (CTD) of NS4B were shown to mediate this interaction. Domain I of NS5A, was sufficient to mediate its interaction with NS4B. Mutations in the NS4B CTD tryptophan residues abolished viral replication. Moreover, one of these mutations also affected NS5A hyperphosphorylation. These findings provide new insights into the importance of the NS4B-NS5A interaction and serve as a starting point for studying the complex interactions between the replicase subunits.

  9. The interaction between the Hepatitis C proteins NS4B and NS5A is involved in viral replication

    PubMed Central

    David, Naama; Yaffe, Yakey; Hagoel, Lior; Elazar, Menashe; Glenn, Jeffrey S.; Hirschberg, Koret; Sklan, Ella H.

    2015-01-01

    Hepatitis C virus (HCV) replicates in membrane associated, highly ordered replication complexes (RCs). These complexes include viral and host proteins necessary for viral RNA genome replication. The interaction network among viral and host proteins underlying the formation of these RCs is yet to be thoroughly characterized. Here, we investigated the association between NS4B and NS5A, two critical RC components. We characterized the interaction between these proteins using fluorescence resonance energy transfer and a mammalian two-hybrid system. Specific tryptophan residues within the C-terminal domain (CTD) of NS4B were shown to mediate this interaction. Domain I of NS5A, was sufficient to mediate its interaction with NS4B. Mutations in the NS4B CTD tryptophan residues abolished viral replication. Moreover, one of these mutations also affected NS5A hyperphosphorylation. These findings provide new insights into the importance of the NS4B–NS5A interaction and serve as a starting point for studying the complex interactions between the replicase subunits. PMID:25462354

  10. Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

    PubMed

    Hoffman, Brett; Li, Zhubing; Liu, Qiang

    2015-08-01

    Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

  11. Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway

    PubMed Central

    Xie, Zhengyuan; Xiao, Zhihua; Wang, Fenfen

    2017-01-01

    The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor (TNF)-α. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of NF-κB and miR-503. We found that overexpression of NS5A inhibited TNF-αinduced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the TNF-α induced Hep-mock cells was significantly less than the viability of the TNF-α induced Hep-NS5A cells, which demonstrates that NS5A inhibited TNF-α-induced HepG2 cell apoptosis. Under TNF-α treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited TNF-α-induced NF-κB activation and NF-κB regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse TNF-α-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits NF-κB activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis. PMID:28343379

  12. Synergistic Activity of Combined NS5A Inhibitors

    PubMed Central

    Nower, Peter T.; Gao, Min; Fridell, Robert; Wang, Chunfu; Hewawasam, Piyasena; Lopez, Omar; Tu, Yong; Meanwell, Nicholas A.; Belema, Makonen; Roberts, Susan B.; Cockett, Mark; Sun, Jin-Hua

    2015-01-01

    Daclatasvir (DCV) is a first-in-class hepatitis C virus (HCV) nonstructural 5A replication complex inhibitor (NS5A RCI) that is clinically effective in interferon-free combinations with direct-acting antivirals (DAAs) targeting alternate HCV proteins. Recently, we reported NS5A RCI combinations that enhance HCV inhibitory potential in vitro, defining a new class of HCV inhibitors termed NS5A synergists (J. Sun, D. R. O’Boyle II, R. A. Fridell, D. R. Langley, C. Wang, S. Roberts, P. Nower, B. M. Johnson F. Moulin, M. J. Nophsker, Y. Wang, M. Liu, K. Rigat, Y. Tu, P. Hewawasam, J. Kadow, N. A. Meanwell, M. Cockett, J. A. Lemm, M. Kramer, M. Belema, and M. Gao, Nature 527:245–248, 2015, doi:10.1038/nature15711). To extend the characterization of NS5A synergists, we tested new combinations of DCV and NS5A synergists against genotype (gt) 1 to 6 replicons and gt 1a, 2a, and 3a viruses. The kinetics of inhibition in HCV-infected cells treated with DCV, an NS5A synergist (NS5A-Syn), or a combination of DCV and NS5A-Syn were distinctive. Similar to activity observed clinically, DCV caused a multilog drop in HCV, followed by rebound due to the emergence of resistance. DCV–NS5A-Syn combinations were highly efficient at clearing cells of viruses, in line with the trend seen in replicon studies. The retreatment of resistant viruses that emerged using DCV monotherapy with DCV–NS5A-Syn resulted in a multilog drop and rebound in HCV similar to the initial decline and rebound observed with DCV alone on wild-type (WT) virus. A triple combination of DCV, NS5A-Syn, and a DAA targeting the NS3 or NS5B protein cleared the cells of viruses that are highly resistant to DCV. Our data support the observation that the cooperative interaction of DCV and NS5A-Syn potentiates both the genotype coverage and resistance barrier of DCV, offering an additional DAA option for combination therapy and tools for explorations of NS5A function. PMID:26711745

  13. Small heterodimer partner 1 directly interacts with NS5A viral protein and has a key role in HCV related liver cell transformation.

    PubMed

    Conti, Beatrice; Porcu, Cristiana; Viscomi, Carmela; Minutolo, Antonella; Costantini, Susan; Corazzari, Marco; Iannucci, Gino; Barbaro, Barbara; Balsano, Clara

    2016-12-20

    HCV life cycle is strictly correlated with the hepatocyte lipid metabolism; moreover, the progression of HCV chronic hepatitis is accelerated by the presence of liver steatosis. Among the steatogenic genes deregulated during the HCV infection one of the most attractive is the Small Heterodimer Protein 1 (SHP1; NR0B2), that is involved in a remarkable number of metabolic functions. HCV NS5A is an essential and integral component of the HCV membranous-web replicon complex (RC) and plays an essential role to transfer the viral genome from the RCs to the surface of the lipid droplets (LDs) that, in turn, play a key function during HCV life cycle.With the help of a HCV infection model, we demonstrate a functional interaction between SHP1 and HCV NS5A protein. SHP1 silencing (siSHP1) reversed the pro-oncogenic effects of HCV infection, inducing a significant decrease in liver lipid accumulation and in NS5A protein expression. Moreover, siSHP1 causes a strong modulation of some genes involved in HCV-related EMT, such as: HNF4, a central regulators of hepatocyte differentiation, E-Cadherin, SNAILs.Our data suggest that SHP1 results not only to be strictly connected to the pathogenesis of HCV-related liver steatosis, but also to its progression towards the liver transformation.

  14. TRIM14 inhibits hepatitis C virus infection by SPRY domain-dependent targeted degradation of the viral NS5A protein

    PubMed Central

    Wang, Shanshan; Chen, Yongzhi; Li, Chunfeng; Wu, Yaoxing; Guo, Lei; Peng, Changwei; Huang, Yueping; Cheng, Genhong; Qin, F. Xiao-Feng

    2016-01-01

    Tripartite motif 14 (TRIM14) was reported to function as a mitochondrial signaling adaptor in mediating innate immune responses. However, the involvement of TRIM14 in host defense against viral infection and molecular mechanisms remain unclear. Here, we demonstrated that enforced expression of TRIM14 could potently inhibit the infection and replication of HCV in hepatocytes, whereas TRIM14 knockout cells became more susceptible to HCV infection. Interestingly, further experiments revealed that such anti-HCV activity was independent of activating the NF-κB or interferon pathways but required the C-terminal SPRY domain of no signaling capacity. In searching for mechanisms how TRIM14 exerts its antiviral function we found that TRIM14 interacted with HCV encoded non-structural protein NS5A and could strongly induce its degradation dependent on the NS5A1 subdomain. Interestingly extensive domain mapping analyses revealed that NS5A degradation was mediated by the highly conserved SPRY domain of TRIM14, which might involve the K48 ubiquitination pathway. Collectively, our work uncovered a new mechanism responsible for host defense against HCV infection, and could potentially aid the development of novel anti-HCV therapeutics. PMID:27578425

  15. Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides

    PubMed Central

    Chukkapalli, Vineela; Berger, Kristi L.; Kelly, Sean M.; Thomas, Meryl; Deiters, Alexander; Randall, Glenn

    2014-01-01

    Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies are HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation. PMID:25546252

  16. Cellular immunogenicity of a multi-epitope peptide vaccine candidate based on hepatitis C virus NS5A, NS4B and core proteins in HHD-2 mice.

    PubMed

    Huang, Xiao-Jun; Lü, Xin; Lei, Ying-Feng; Yang, Jing; Yao, Min; Lan, Hai-Yun; Zhang, Jian-Min; Jia, Zhan-Sheng; Yin, Wen; Xu, Zhi-Kai

    2013-04-01

    To develop a vaccine against hepatitis C virus (HCV), a multi-epitope peptide was synthesized from nonstructural proteins containing HLA-A2 epitopes inducing mainly responses in natural infection. The engineered vaccine candidate, VAL-44, consists of multiple epitopes from the HCV NS5A, NS4B and core proteins. Immunization with the VAL-44 peptide induced higher CTL responses than those by the smaller VL-20 peptide. VAL-44 induced antigen-specific IFN-γ-producing CD4+ T cells and CD8+ T cells. VAL-44 elicited a Th1-biased immune response with secretion of high amounts of IFN-γ and IL-2, compared with VL-20. These results suggest that VAL-44 can elicit strong cellular immune responses. The VAL-44 peptide stimulated IFN-γ production from viral-specific peripheral blood mononuclear cells (PBMCs) of patients infected with HCV. These results suggest that VAL-44 could be developed as a potential HCV multi-epitope peptide vaccine.

  17. Identification of cleavage of NS5A of C-strain classical swine fever virus.

    PubMed

    Xie, Jinxin; Guo, Huancheng; Gong, Wenjie; Jiang, Daliang; Zhang, Li; Jia, Junjie; Tu, Changchun

    2017-02-01

    NS5A is a multifunctional non-structural protein of classical swine fever virus (CSFV) that plays an important role in viral replication, but how it exerts its functions is unknown. Here, we report the cleavage of NS5A of the vaccine C-strain, resulting in two truncated forms (b and c). Further experiments using calpain- and caspase-family-specific inhibitors, followed by a caspase-6-specific shRNAs and inhibitor, showed that the cleavage of C-strain NS5A to produce truncated form c is mediated by caspase-6, mapping to (272)DTTD(275), while the cleavage producing truncated form b is probably mediated by another unknown protease. shRNA-mediated downregulation of caspase-6 and blocking of enzyme activity in ST cells significantly impaired genome replication and virus production, indicating that NS5A cleavage is required for CSFV replication.

  18. HCV NS5A co-operates with PKR in modulating HCV IRES-dependent translation.

    PubMed

    Karamichali, Eirini; Foka, Pelagia; Tsitoura, Eliza; Kalliampakou, Katerina; Kazazi, Dorothea; Karayiannis, Peter; Georgopoulou, Urania; Mavromara, Penelope

    2014-08-01

    Translation initiation of the Hepatitis C virus (HCV) genome is driven by an internal ribosome entry site (IRES), located within the 5' non-coding region. Several studies have suggested that different cellular non canonical proteins or viral proteins can regulate the HCV IRES activity. However, the role of the viral proteins on HCV translation remains controversial. In this report, we confirmed previous studies showing that NS5A down-regulates IRES activity in HepG2 but not in Huh7 cells suggesting that the NS5A effect on HCV IRES is cell-type dependent. Additionally, we provide strong evidence that activated PKR up-regulates the IRES activity while silencing of endogenous PKR had the opposite effect. Furthermore, we present data indicating that the NS5A-mediated inhibitory effect on IRES-dependent translation could be linked with the PKR inactivation. Finally, we show that NS5A from GBV-C but not from GBV-B down-regulates HCV IRES activity in the absence or the presence of PKR over expression. Notably, HCV and GBV-C but not GBV-B NS5A contains a previously identified PKR interacting protein domain.

  19. GPS2 is required for the association of NS5A with VAP-A and hepatitis C virus replication.

    PubMed

    Xu, Guodong; Xin, Xiu; Zheng, Congyi

    2013-01-01

    Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of the replication complex associated with various cellular proteins. It has been reported that G protein pathway suppressor 2 (GPS2) is a potential NS5A-binding factor, as identified in a yeast two-hybrid screens of human cDNA library using viral proteins as baits [1]. In this study, we demonstrated the interaction between GPS2 and NS5A in mammalian cells by coimmunoprecipitation analysis and found that both exogenously and endogenously expressed GPS2 interacted with NS5A of genotype 1b and 2a. Mutagenesis study demonstrated that Domain I of NS5A and coiled-coil domain of GPS2 are responsible for the interaction. Knockdown of GPS2 in hepatoma cell lines suppressed the replication of HCV RNA, which can be rescued by the expression of an RNAi-resistant GPS2. Furthermore, overexpression of GPS2 enhanced the association of NS5A with a proviral cellular factor, human vesicle-associated membrane protein-associated protein A (VAP-A), while knockdown of GPS2 disrupted interaction between VAP-A and NS5A. Taken together, our results suggest that GPS2 acts as a bridge between NS5A and VAP-A and is required for efficient HCV replication.

  20. HCV RNA traffic and association with NS5A in living cells

    SciTech Connect

    Fiches, Guillaume N.; Eyre, Nicholas S.; Aloia, Amanda L.; Van Der Hoek, Kylie; Betz-Stablein, Brigit; Luciani, Fabio; Chopra, Abha; Beard, Michael R.

    2016-06-15

    The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3′-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly. - Highlights: • HCV can tolerate can bacteriophage MS2 stem-loop insertions within the 3′ UTR. • MS2 stem-loop containing HCV genomes allow for real-time imaging of HCV RNA. • HCV RNA is both static and motile and associates with NS5A and lipid droplets.

  1. Understanding the biological context of NS5A-host interactions in HCV infection: a network-based approach.

    PubMed

    Tripathi, Lokesh P; Kambara, Hiroto; Chen, Yi-An; Nishimura, Yorihiro; Moriishi, Kohji; Okamoto, Toru; Morita, Eiji; Abe, Takayuki; Mori, Yoshio; Matsuura, Yoshiharu; Mizuguchi, Kenji

    2013-06-07

    Hepatitis C virus (HCV) is a major cause of chronic liver disease. HCV NS5A protein plays an important role in HCV infection through its interactions with other HCV proteins and host factors. In an attempt to further our understanding of the biological context of protein interactions between NS5A and host factors in HCV pathogenesis, we generated an extensive physical interaction map between NS5A and cellular factors. By combining a yeast two-hybrid assay with comprehensive literature mining, we built the NS5A interactome composed of 132 human proteins that interact with NS5A. These interactions were integrated into a high-confidence human protein interactome (HPI) with the help of the TargetMine data warehouse system to infer an overall protein interaction map linking NS5A with the components of the host cellular networks. The NS5A-host interactions that were integrated with the HPI were shown to participate in compact and well-connected cellular networks. Functional analysis of the NS5A "infection" network using TargetMine highlighted cellular pathways associated with immune system, cellular signaling, cell adhesion, cellular growth and death among others, which were significantly targeted by NS5A-host interactions. In addition, cellular assays with in vitro HCV cell culture systems identified two ER-localized host proteins RTN1 and RTN3 as novel regulators of HCV propagation. Our analysis builds upon the present understanding of the role of NS5A protein in HCV pathogenesis and provides potential targets for more effective anti-HCV therapeutic intervention.

  2. Hepatitis C virus NS5A inhibitors and drug resistance mutations.

    PubMed

    Nakamoto, Shingo; Kanda, Tatsuo; Wu, Shuang; Shirasawa, Hiroshi; Yokosuka, Osamu

    2014-03-21

    Some direct-acting antiviral agents for hepatitis C virus (HCV), such as telaprevir and boceprevir have been available since 2011. It was reported that HCV NS5A is associated with interferon signaling related to HCV replication and hepatocarcinogenesis. HCV NS5A inhibitors efficiently inhibited HCV replication in vitro. Human studies showed that dual, triple and quad regimens with HCV NS5A inhibitors, such as daclatasvir and ledipasvir, in combination with other direct-acting antiviral agents against other regions of HCV with or without peginterferon/ribavirin, could efficiently inhibit HCV replication according to HCV genotypes. These combinations might be a powerful tool for "difficult-to-treat" HCV-infected patients. "First generation" HCV NS5A inhibitors such as daclatasvir, ledipasvir and ABT-267, which are now in phase III clinical trials, could result in resistance mutations. "Second generation" NS5A inhibitors such as GS-5816, ACH-3102, and MK-8742, have displayed improvements in the genetic barrier while maintaining potency. HCV NS5A inhibitors are safe at low concentrations, which make them attractive for use despite low genetic barriers, although, in fact, HCV NS5A inhibitors should be used with HCV NS3/4A inhibitors, HCV NS5B inhibitors or peginterferon plus ribavirin. This review article describes HCV NS5A inhibitor resistance mutations and recommends that HCV NS5A inhibitors be used in combination regimens potent enough to prevent the emergence of resistant variants.

  3. Progress on New Hepatitis C Virus Targets: NS2 and NS5A

    NASA Astrophysics Data System (ADS)

    Marcotrigiano, Joseph

    Hepatitis C virus (HCV) is a major global health problem, affecting about 170 million people worldwide. Chronic infection can lead to cirrhosis and liver cancer. The replication machine of HCV is a multi-subunit membrane associated complex, consisting of nonstructural proteins (NS2-5B), which replicate the viral RNA genome. The structures of NS5A and NS2 were recently determined. NS5A is an essential replicase component that also modulates numerous cellular processes ranging from innate immunity to cell growth and survival. The structure reveals a novel protein fold, a new zinc coordination motif, a disulfide bond and a dimer interface. Analysis of molecular surfaces suggests the location of the membrane interaction surface of NS5A, as well as hypothetical protein and RNA binding sites. NS2 is one of two virally encoded proteases that are required for processing the viral polyprotein into the mature nonstructural proteins. NS2 is a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer and the nucleophilic cysteine by the other. The C-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. The structure also reveals possible sites of membrane interaction, a rare cis-proline residue, and highly conserved dimer contacts. The novel features of both structures have changed the current view of HCV polyprotein replication and present new opportunities for antiviral drug design.

  4. Interferon alpha (IFNα)-induced TRIM22 interrupts HCV replication by ubiquitinating NS5A.

    PubMed

    Yang, Chen; Zhao, Xinhao; Sun, Dakang; Yang, Leilei; Chong, Chang; Pan, Yu; Chi, Xiumei; Gao, Yanhang; Wang, Moli; Shi, Xiaodong; Sun, Haibo; Lv, Juan; Gao, Yuanda; Zhong, Jin; Niu, Junqi; Sun, Bing

    2016-01-01

    TRIM22, a tripartite-motif (TRIM) protein, is upregulated upon interferon alpha (IFNα) administration to hepatitis C virus (HCV)-infected patients. However, the physiological role of TRIM22 upregulation remains unclear. Here, we describe a potential antiviral function of TRIM22's targeting of the HCV NS5A protein. NS5A is important for HCV replication and for resistance to IFNα therapy. During the first 24 h following the initiation of IFNα treatment, upregulation of TRIM22 in the peripheral blood mononuclear cells (PBMCs) of HCV patients correlated with a decrease in viral titer. This phenomenon was confirmed in the hepatocyte-derived cell line Huh-7, which is highly permissive for HCV infection. TRIM22 over-expression inhibited HCV replication, and Small interfering RNA (siRNA)-mediated knockdown of TRIM22 diminished IFNα-induced anti-HCV function. Furthermore, we determined that TRIM22 ubiquitinates NS5A in a concentration-dependent manner. In summary, our results suggest that TRIM22 upregulation is associated with HCV decline during IFNα treatment and plays an important role in controlling HCV replication in vitro.

  5. Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

    SciTech Connect

    Eyre, Nicholas S.; Hampton-Smith, Rachel J.; Aloia, Amanda L.; Eddes, James S.; Simpson, Kaylene J.; Hoffmann, Peter; Beard, Michael R.

    2016-04-15

    Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.

  6. Regulation of PKR by HCV IRES RNA: Importance of Domain II and NS5A

    PubMed Central

    Toroney, Rebecca; Nallagatla, Subba Rao; Boyer, Joshua A.; Cameron, Craig E.; Bevilacqua, Philip C.

    2010-01-01

    The protein kinase PKR is an essential component of the innate immune response. In the presence of dsRNA, PKR is autophosphorylated, which enables it to phosphorylate its substrate, eIF2α, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5′-UTR of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and non-canonical features of domain II cumulate to a total of ∼33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV non-structural protein NS5A to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection. PMID:20447405

  7. Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor

    PubMed Central

    Brasher, Bradley B.; Polemeropoulos, Alex; Rhodin, Michael H. J.; McAllister, Nicole; Wong, Kelly A.; Jones, Christopher T.; Jiang, Lijuan; Lin, Kai; Or, Yat Sun

    2016-01-01

    EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo. This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro. Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.) PMID:27503644

  8. A synthetic codon-optimized hepatitis C virus nonstructural 5A DNA vaccine primes polyfunctional CD8+ T cell responses in wild-type and NS5A-transgenic mice.

    PubMed

    Holmström, Fredrik; Pasetto, Anna; Nähr, Veronica; Brass, Anette; Kriegs, Malte; Hildt, Eberhard; Broderick, Kate E; Chen, Margaret; Ahlén, Gustaf; Frelin, Lars

    2013-02-01

    The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5A-transgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >10(4) postimmunization. With respect to CD8(+) T cell responses, the coNS5A gene primed more potent IFN-γ-producing and lytic cytotoxic T cells in wt mice compared with NS5A-Tg mice. In addition, high frequencies of NS5A-specific CD8(+) T cells were found in wt mice after a single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the coNS5A gene primed polyfunctional NS5A-specific CD8(+) T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections.

  9. Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B.

    PubMed

    Hamamoto, Itsuki; Nishimura, Yorihiro; Okamoto, Toru; Aizaki, Hideki; Liu, Minyi; Mori, Yoshio; Abe, Takayuki; Suzuki, Tetsuro; Lai, Michael M C; Miyamura, Tatsuo; Moriishi, Kohji; Matsuura, Yoshiharu

    2005-11-01

    The hepatitis C virus (HCV) nonstructural protein (NS) 5A is a phosphoprotein that associates with various cellular proteins and participates in the replication of the HCV genome. Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. To obtain more information on the NS5A protein in HCV replication, we screened human brain and liver libraries by a yeast two-hybrid system using NS5A as bait and identified VAP-B as an NS5A-binding protein. Immunoprecipitation and mutation analyses revealed that VAP-B binds to both NS5A and NS5B in mammalian cells and forms homo- and heterodimers with VAP-A. VAP-A interacts with VAP-B through the transmembrane domain. NS5A interacts with the coiled-coil domain of VAP-B via 70 residues in the N-terminal and 341 to 344 amino acids in the C-terminal polyproline cluster region. NS5A was colocalized with VAP-B in the endoplasmic reticulum and Golgi apparatus. The specific antibody to VAP-B suppressed HCV RNA replication in a cell-free assay. Overexpression of VAP-B, but not of a mutant lacking its transmembrane domain, enhanced the expression of NS5A and NS5B and the replication of HCV RNA in Huh-7 cells harboring a subgenomic replicon. In the HCV replicon cells, the knockdown of endogenous VAP-B by small interfering RNA decreased expression of NS5B, but not of NS5A. These results suggest that VAP-B, in addition to VAP-A, plays an important role in the replication of the HCV genome.

  10. Discovery of novel highly potent hepatitis C virus NS5A inhibitor (AV4025).

    PubMed

    Ivachtchenko, Alexandre V; Mitkin, Oleg D; Yamanushkin, Pavel M; Kuznetsova, Irina V; Bulanova, Elena A; Shevkun, Natalia A; Koryakova, Angela G; Karapetian, Ruben N; Bichko, Vadim V; Trifelenkov, Andrey S; Kravchenko, Dmitry V; Vostokova, Natalia V; Veselov, Mark S; Chufarova, Nina V; Ivanenkov, Yan A

    2014-09-25

    A series of next in class small-molecule hepatitis C virus (HCV) NS5A inhibitors with picomolar potency containing 2-pyrrolidin-2-yl-5-{4-[4-(2-pyrrolidin-2-yl-1H-imidazol-5-yl)buta-1,3-diynyl]phenyl}-1H-imidazole cores was designed based on the SAR studies available for the reported NS5A inhibitors. Compound 13a (AV4025), with (S,S,S,S)-stereochemistry (EC50 = 3.4 ± 0.2 pM, HCV replicon genotype 1b), was dramatically more active than were the compounds with two (S)- and two (R)-chiral centers. Human serum did not significantly reduce the antiviral activity (<4-fold). Relatively favorable pharmacokinetic features and good oral bioavailability were observed during animal studies. Compound 13a was well tolerated in rodents (in mice, LD50 = 2326 mg/kg or higher), providing a relatively high therapeutic index. During safety, pharmacology and subchronic toxicity studies in rats and dogs, it was not associated with any significant pathological or clinical findings. This compound is currently being evaluated in phase I/II clinical trials for the treatment of HCV infection.

  11. Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented in trans

    PubMed Central

    Grassmann, Claus W.; Isken, Olaf; Tautz, Norbert; Behrens, Sven-Erik

    2001-01-01

    The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5′ and 3′ untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cis or in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be cis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of

  12. Kushenin induces the apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A.

    PubMed

    Zhou, Yi; Chen, Na; Liu, Xiaojing; Lin, Shumei; Luo, Wenjuan; Liu, Min

    2016-07-01

    With the increased burden induced by HCV, there is an urgent need to develop better-tolerated agents with good safety. In this study, we evaluated the anti-HCV capability of kushenin, as well as the possible mechanism to Huh7.5-HCV cells. The results demonstrated that kushenin significantly inhibited the HCV-RNA level. Similarly, the expression of HCV-specific protein NS5A was also decreased. Molecular docking results displayed that kushenin bonded well to the active pockets of HCV NS5A, further confirming the effects of kushenin on HCV replication. Coimmunoprecipitation assay determined that kushenin suppressed the interaction between PI3K and NS5A in HCV-replicon cells. Furthermore, kushenin exerted an obviously induced function on HCV-replicon cells apoptosis by inhibiting PI3K-Akt-mTOR pathway, which could be ameliorated by the specific activator IGF-1 addition. Taken together, kushenin possesses the ability to inhibit HCV replication, and contributes to the increased apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A. Our results provide important evidence for a better understanding of the pathogenesis of HCV infection, and suggest that kushenin has the potential to treat HCV disease.

  13. Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.

    PubMed

    Reiss, Simon; Rebhan, Ilka; Backes, Perdita; Romero-Brey, Ines; Erfle, Holger; Matula, Petr; Kaderali, Lars; Poenisch, Marion; Blankenburg, Hagen; Hiet, Marie-Sophie; Longerich, Thomas; Diehl, Sarah; Ramirez, Fidel; Balla, Tamas; Rohr, Karl; Kaul, Artur; Bühler, Sandra; Pepperkok, Rainer; Lengauer, Thomas; Albrecht, Mario; Eils, Roland; Schirmacher, Peter; Lohmann, Volker; Bartenschlager, Ralf

    2011-01-20

    Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.

  14. Effects of Resistance-Associated NS5A Mutations in Hepatitis C Virus on Viral Production and Susceptibility to Antiviral Reagents

    PubMed Central

    Nitta, Sayuri; Asahina, Yasuhiro; Matsuda, Mami; Yamada, Norie; Sugiyama, Ryuichi; Masaki, Takahiro; Suzuki, Ryosuke; Kato, Nobuyuki; Watanabe, Mamoru; Wakita, Takaji; Kato, Takanobu

    2016-01-01

    Direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have potent anti-HCV effects but may provoke resistance-associated variants (RAVs). In this study, we assessed the characteristics of these RAVs and explored efficacious anti-HCV reagents using recombinant HCV with NS5A from a genotype 1b strain. We replaced the NS5A of JFH1 with that of Con1 (JFH1/5ACon1) and introduced known NS5A inhibitor resistance mutations (L31M, L31V, L31I and Y93H) individually or in combination. Susceptibilities against anti-HCV reagents were also investigated. RAVs with Y93H exhibited high extracellular core antigen levels and infectivity titers. Variants with any single mutation showed mild to moderate resistance against NS5A inhibitors, whereas variants with double mutations at both L31 and Y93 showed severe resistance. The variants with mutations exhibited similar levels of susceptibility to interferon (IFN)-α, IFN-λ1, IFN-λ3 and Ribavirin. Variants with the Y93H mutation were more sensitive to protease inhibitors compared with JFH1/5ACon1. In conclusion, the in vitro analysis indicated that the Y93H mutation enhanced infectious virus production, suggesting advantages in the propagation of RAVs with this mutation. However, these RAVs were susceptible to protease inhibitors. Thus, a therapeutic regimen that includes these reagents is a promising means to eradicate these RAVs. PMID:27703205

  15. Optimizing choice of oral interferon-free treatment for genotype 1 hepatitis C virus using testing for NS5A resistance: a cost-utility analysis from the perspective of the Italian National Health Service

    PubMed Central

    Westerhout, Kirsten Y; Bouwmeester, Walter; Duchesne, Inge; Pisini, Marta; Piena, Marjanne A; Damele, Francesco; Gueron, Beatrice; Treur, Maarten; Belsey, Jonathan

    2017-01-01

    Background Patients with genotype-1 hepatitis C virus infection who have failed to respond to standard therapy or who relapse following treatment may be considered for an interferon-free regimen incorporating a nonstructural protein 5A (NS5A) inhibitor. Sustained virologic response (SVR) with these regimens is typically >90%, but this is reduced in patients with NS5A resistance. European Association for Study of the Liver guidelines recommend simeprevir + sofosbuvir ± ribavirin (SMV+SOF±R) for re-treating patients failing an NS5A inhibitor-containing regimen. An alternative strategy would be to test for NS5A resistance prior to treatment, with therapy optimized based on the results. This study investigates the cost-effectiveness of this strategy. Materials and methods A Markov model was used to estimate disease progression for treatment-experienced genotype 1 patients with severe fibrosis or compensated cirrhosis. Targeted treatment with either SMV+SOF±R or sofosbuvir + ledipasvir ± ribavirin (SOF+LDV±R) based on pretreatment NS5A resistance testing was compared to routine SOF+LDV±R without testing. Treatment duration was 12 or 24 weeks for patients with severe fibrosis or compensated cirrhosis (Metavir F3/F4). SVR data for the treatment options were based on the results of published clinical trials. The analysis was carried out from the perspective of the Italian National Health Service. Results Optimized treatment using NS5A resistance testing yielded 0.163 additional QALYs and increased costs of €2,789 per patient versus no testing. The incremental cost-effectiveness ratio (ICER) was €17,078/QALY. Sensitivity analysis identified the SVR attributable to each of the treatment regimens as the most sensitive determinant of ICER (range: €10,055/QALY–€43,501/QALY across plausible range). Probabilistic sensitivity analysis demonstrated that, at a willingness-to-pay threshold of €30,000/QALY, the probability that NS5A-directed treatment will be cost

  16. Effect of Hepatitis C Virus Genotype 1b Core and NS5A Mutations on Response to Peginterferon Plus Ribavirin Combination Therapy.

    PubMed

    Nakamoto, Shingo; Imazeki, Fumio; Arai, Makoto; Yasui, Shin; Nakamura, Masato; Haga, Yuki; Sasaki, Reina; Kanda, Tatsuo; Shirasawa, Hiroshi; Yokosuka, Osamu

    2015-09-07

    We examined whether hepatitis C virus (HCV) genotype 1b core- and NS5A-region mutations are associated with response to peginterferon α-2b plus ribavirin combination therapy. A total of 103 patients with high HCV genotype 1b viral loads (≥ 100 KIU/mL) were treated with the combination therapy. Pretreatment mutations in the core region and interferon sensitivity determining region (ISDR) in the NS5A region were analyzed. In univariate analysis, arginine and leucine at positions 70 and 91 in the core region, defined as double wild (DW)-type, were associated with early virologic response (p = 0.002), sustained virologic response (SVR) (p = 0.004), and non-response (p = 0.005). Non-threonine at position 110 was associated with SVR (p = 0.004). Multivariate analysis showed the following pretreatment predictors of SVR: hemoglobin level ≥ 14 g/dL (odds ratio (OR) 6.2, p = 0.04); platelet count ≥ 14 × 10⁴/mm³ (OR 5.2, p = 0.04); aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio < 0.9 (OR 6.17, p = 0.009); DW-type (OR 6.8, p = 0.02); non-threonine at position 110 (OR 14.5, p = 0.03); and ≥ 2 mutations in the ISDR (OR 12.3, p = 0.02). Patients with non-DW-type, non-threonine at position 110, and < 2 ISDR mutations showed significantly lower SVR rates than others (11/45 (24.4%) vs. 27/37 (73.0%), respectively; p < 0.001). SVR can be predicted through core and NS5A region mutations and host factors like hemoglobin, platelet count, and AST/ALT ratio in HCV genotype 1b-infected patients treated with peginterferon and ribavirin combination therapy.

  17. Hepatitis C virus NS5A replication complex inhibitors. Part 6: Discovery of a novel and highly potent biarylimidazole chemotype with inhibitory activity toward genotypes 1a and 1b replicons.

    PubMed

    Belema, Makonen; Nguyen, Van N; Romine, Jeffrey L; St Laurent, Denis R; Lopez, Omar D; Goodrich, Jason T; Nower, Peter T; O'Boyle, Donald R; Lemm, Julie A; Fridell, Robert A; Gao, Min; Fang, Hua; Krause, Rudolph G; Wang, Ying-Kai; Oliver, A Jayne; Good, Andrew C; Knipe, Jay O; Meanwell, Nicholas A; Snyder, Lawrence B

    2014-03-13

    A medicinal chemistry campaign that was conducted to address a potential genotoxic liability associated with an aniline-derived scaffold in a series of HCV NS5A inhibitors with dual GT-1a/-1b inhibitory activity is described. Anilides 3b and 3c were used as vehicles to explore structural modifications that retained antiviral potency while removing the potential for metabolism-based unmasking of the embedded aniline. This effort resulted in the discovery of a highly potent biarylimidazole chemotype that established a potency benchmark in replicon assays, particularly toward HCV GT-1a, a strain with significant clinical importance. Securing potent GT-1a activity in a chemotype class lacking overt structural liabilities was a critical milestone in the effort to realize the full clinical potential of targeting the HCV NS5A protein.

  18. Priming and stimulation of hepatitis C virus-specific CD4+ and CD8+ T cells against HCV antigens NS4, NS5a or NS5b from HCV-naive individuals: implications for prophylactic vaccine.

    PubMed

    Li, Wen; Krishnadas, Deepa K; Kumar, Rakesh; Tyrrell, D Lorne J; Agrawal, Babita

    2008-01-01

    Hepatitis C virus (HCV) is a devastating human pathogen, yet there is no vaccine available for this virus. From studies with acute or chronic HCV-infected humans and chimpanzees, T-cell responses against HCV-derived conserved non-structural antigens have been correlated with viral clearance. In this study, recombinant adenoviral vectors containing HCV-derived NS4, NS5a or NS5b genes were employed to endogenously express the HCV antigens in human dendritic cells (DCs). The DCs expressing these HCV antigens exhibited normal phenotype and function. Intriguingly, we found that the DCs expressing HCV NS4, NS5a or NS5b antigens were able to significantly stimulate autologous T cells obtained from uninfected healthy individuals. These T cells produced various cytokines and proliferated in an HCV antigen-dependent manner. Evidence of both CD4(+) and CD8(+) T-cell responses generated in vitro against HCV NS4, NS5a or NS5b were obtained. HCV NS4 was much less stimulatory for CD4(+) and CD8(+) T cells than NS5. Further, in secondary assays, the CD4(+) T cells primed in vitro exhibited HCV antigen-specific proliferative responses against recombinant protein antigens. In summary, we provide conclusive evidence of in vitro stimulation of CD4(+) and CD8(+) T cells from HCV-naive individuals against HCV antigens NS4, NS5a and NS5b. The studies with naive T cells represent early events in the induction of cellular immune responses, which most likely govern the outcome of HCV infection. These studies have significant implications in designing vaccines for HCV infection in both prophylactic and therapeutic settings.

  19. Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease.

    PubMed Central

    Shimizu, Y; Yamaji, K; Masuho, Y; Yokota, T; Inoue, H; Sudo, K; Satoh, S; Shimotohno, K

    1996-01-01

    In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation. PMID:8523516

  20. Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses.

    PubMed

    Gottwein, Judith M; Jensen, Sanne B; Li, Yi-Ping; Ghanem, Lubna; Scheel, Troels K H; Serre, Stéphanie B N; Mikkelsen, Lotte; Bukh, Jens

    2013-03-01

    With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3' untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ~4 log(10) focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low

  1. The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons

    PubMed Central

    Bystol, Karin; Curry, Stephanie; McMonagle, Patricia; Xia, Ellen; Ingravallo, Paul; Chase, Robert; Liu, Rong; Black, Todd; Hazuda, Daria; Howe, Anita Y. M.; Asante-Appiah, Ernest

    2016-01-01

    The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for

  2. HCV NS5A Up-Regulates COX-2 Expression via IL-8-Mediated Activation of the ERK/JNK MAPK Pathway

    PubMed Central

    Chen, Wei-Chun; Tseng, Chin-Kai; Chen, Yen-Hsu; Lin, Chun-Kuang; Hsu, Shih-hsien; Wang, Shen-Nien; Lee, Jin-Ching

    2015-01-01

    Chronic hepatitis C virus (HCV) infection leads to intrahepatic inflammation and liver cell injury, which are considered a risk factor for virus-associated hepatitis, cirrhosis, and hepatocellular carcinoma worldwide. Inflammatory cytokines are critical components of the immune system and influence cellular signaling, and genetic imbalances. In this study, we found that cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) were significantly induced by HCV infection and HCV NS5A expression, and induction of COX-2 correlated with HCV-induced IL-8 production. We also found that the ERK and JNK signaling pathways were involved in the regulation of IL-8-mediated COX-2 induction in response to HCV infection. Using a promoter-linked reporter assay, we identified that the C/EBP regulatory element within the COX-2 promoter was the dominant factor responsible for the induction of COX-2 by HCV. Silencing C/EBP attenuated HCV-induced COX-2 expression. Our results revealed that HCV-induced inflammation promotes viral replication, providing new insights into the involvement of IL-8-mediated COX-2 induction in HCV replication. PMID:26231035

  3. Frequency of Natural Resistance within NS5a Replication Complex Domain in Hepatitis C Genotypes 1a, 1b: Possible Implication of Subtype-Specific Resistance Selection in Multiple Direct Acting Antivirals Drugs Combination Treatment

    PubMed Central

    Bagaglio, Sabrina; Andolina, Andrea; Merli, Marco; Uberti-Foppa, Caterina; Morsica, Giulia

    2016-01-01

    Different HCV subtypes may naturally harbor different resistance selection to anti-NS5a inhibitors. 2761 sequences retrieved from the Los Alamos HCV database were analyzed in the NS5a domain 1, the target of NS5a inhibitors. The NS5a resistance-associated polymorphisms (RAPs) were more frequently detected in HCV G1b compared to G1a. The prevalence of polymorphisms associated with cross-resistance to compounds in clinical use (daclatasvir, DCV, ledipasvir, LDV, ombitasvir, and OMV) or scheduled to come into clinical use in the near future (IDX719, elbasvir, and ELV) was higher in G1b compared to G1a (37/1552 (2.4%) in 1b sequences and 15/1209 (1.2%) in 1a isolates, p = 0.040). Interestingly, on the basis of the genotype-specific resistance pattern, 95 (6.1%) G1b sequences had L31M RAP to DCV/IDX719, while 6 sequences of G1a (0.5%) harbored L31M RAP, conferring resistance to DCV/LDV/IDX719/ELV (p < 0.0001). Finally, 28 (2.3%) G1a and none of G1b isolates harbored M28V RAP to OMV (p < 0.0001). In conclusion, the pattern of subtype-specific resistance selection in the naturally occurring strains may guide the treatment option in association with direct acting antivirals (DAAs) targeting different regions, particularly in patients that are difficult to cure, such as those with advanced liver disease or individuals who have failed previous DAAs. PMID:27023593

  4. Nonstructural 5A Protein of Hepatitis C Virus Interacts with Pyruvate Carboxylase and Modulates Viral Propagation

    PubMed Central

    Kim, Jong-Wook; Hwang, Soon B.

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular factors for its own propagation. By employing tandem affinity purification method, we identified pyruvate carboxylase (PC) as a cellular partner for NS5A protein. NS5A interacted with PC through the N-terminal region of NS5A and the biotin carboxylase domain of PC. PC expression was decreased in cells expressing NS5A and HCV-infected cells. Promoter activity of PC was also decreased by NS5A protein. However, FAS expression was increased in cells expressing NS5A and cell culture grown HCV (HCVcc)-infected cells. Silencing of PC promoted fatty acid synthase (FAS) expression level. These data suggest HCV may modulate PC via NS5A protein for its own propagation. PMID:23861867

  5. Strong HCV NS3/4a, NS4b, NS5a, NS5b-specific cellular immune responses induced in Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine.

    PubMed

    Latimer, Brian; Toporovski, Roberta; Yan, Jian; Pankhong, Panyupa; Morrow, Matthew P; Khan, Amir S; Sardesai, Niranjan Y; Welles, Seth L; Jacobson, Jeffrey M; Weiner, David B; Kutzler, Michele A

    2014-01-01

    Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world's population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study the immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million peripheral blood mononuclear cells (PBMCs) as measured by Interferon-γ ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD45RA- memory-like T cells

  6. Evaluation of Resistance-Associated Substitutions in NS5A Using Direct Sequence and Cycleave Method and Treatment Outcome with Daclatasvir and Asunaprevir for Chronic Hepatitis C Genotype 1

    PubMed Central

    Ishii, Kunihide; Morita, Masaru; Morita, Yasuyo; Sugiyama, Gen; Fukushima, Hirofumi; Yano, Yoichi; Noguchi, Kazunori; Nakamura, Hiroki; Hisatomi, Junjiro; Kumemura, Hiroto; Shirachi, Miki; Iwane, Shinji; Okada, Michiaki; Honma, Yuichi; Arinaga-Hino, Teruko; Miyajima, Ichiro; Ogata, Kei; Kuwahara, Reiichiro; Amano, Keisuke; Kawaguchi, Toshihiro; Kuromatsu, Ryoko; Torimura, Takuji

    2016-01-01

    Background The aim of this study was to evaluate the efficacy of daclatasvir plus asunaprevir therapy in patients infected with hepatitis C virus and determine its relevance to resistant variants. Methods A total of 629 consecutive patients infected with hepatitis C virus genotype 1 were assessed. Daclatasvir (60 mg/day) plus asunaprevir (200 mg/day) was given for 24 weeks. The virological responses and resistance-associated substitutions of hepatitis C virus mutants were examined by the direct sequence and cycleave methods were evaluated. Results Overall, 89.4% (555/621) of patients exhibited a sustained virological response (SVR). The SVR rates in the patients with wild type, mixed, and mutant type Y93 by direct sequencing were 92.5% (520/562), 70.3% (26/37), and 42.9% (9/21), respectively. The SVR rates in the patients with 100%, 90%, 80%-30%, and 20%-0% Y93 wild by the cycleave method were 93.4% (456/488), 88.2%(30/34), 56.0%(14/25), and 36.8%(7/19), respectively. In contrast, the SVR rates for the wild type and mixed/mutant type L31 by direct sequencing were 90.2% (534/592) and 72.4% (21/29), respectively. In the multivariate analyses, the wild type Y93, no history of simeprevir therapy, the wild type L31, and low HCV RNA level were independent factors of SVR. Conclusion NS5A resistance-associated substitutions, especially Y93H, were major factors predicting the SVR. Although direct sequencing can predict the SVR rate, the cycleave method is considered to be more useful for predicting the SVR when used in combination. PMID:27684567

  7. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    SciTech Connect

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  8. Screening of cellular proteins that interact with the classical swine fever virus non-structural protein 5A by yeast two-hybrid analysis.

    PubMed

    Zhang, Chengcheng; He, Lei; Kang, Kai; Chen, Heng; Xu, Lei; Zhang, Yanming

    2014-03-01

    Classical swine fever virus (CSFV), the pathogen of classical swine fever (CSF), causes severe hemorrhagic fever and vascular necrosis in domestic pigs and wild boar. A large number of evidence has proven that non-structural 5A (NS5A) is not only a very important part of viral replication complex, but also can regulate host cell's function; however, the underlying mechanisms remain poorly understood. In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database revealed 16 interactive proteins: DDX5, PSMC3, NAV1, PHF5A, GNB2L1, CSDE1, HSPA8, BRMS1, PPP2R3C, AIP, TMED10, POLR1C, TMEM70, METAP2, CHORDC1 and COPS6. These proteins are mostly related to gene transcription, protein folding, protein degradation and metabolism. The interactions detected by the Y2H system should be considered as preliminary results. Since identifying novel pathways and host targets, which play essential roles during infection, may provide potential targets for therapeutic development. The finding of proteins obtained from the SUVEC cDNA library that interact with the CSFV NS5A protein provide valuable information for better understanding the interactions between this viral protein and the host target proteins.

  9. Increased Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression and Steatosis During Hepatitis C Virus (HCV) Subgenome Replication: Role of Nonstructural Component-5A (NS5A) and CCAAT/Enhancer Binding Protein ß (C/EBPß)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated tra...

  10. Functional differences in hepatitis C virus nonstructural (NS) 3/4A- and 5A-specific T cell responses

    PubMed Central

    Holmström, Fredrik; Chen, Margaret; Balasiddaiah, Anangi; Sällberg, Matti; Ahlén, Gustaf; Frelin, Lars

    2016-01-01

    The hepatitis C virus nonstructural (NS) 3/4A and NS5A proteins are major targets for the new direct-acting antiviral compounds. Both viral proteins have been suggested as modulators of the response to the host cell. We have shown that NS3/4A- and NS5A-specific T cell receptors confer different effector functions, and that killing of NS3/4A-expressing hepatocytes is highly dependent on IFN-γ. We here characterize the functional differences in the T cell responses to NS3/4A and NS5A. NS3/4A- and NS5A-specific T cells could be induced at various frequencies in wild-type-, NS3/4A-, and NS5A-transgenic mice. Priming of NS5A-specific T cells required a high DNA dose, and was unlike NS3/4A dependent on both CD4+ and CD8+ T cells, but less influenced by CD25+/GITR+ regulatory T cells. The presence of IL-12 greatly improved specific CD8+ T cell priming by NS3/4A but not by NS5A, suggesting a less dependence of IFN-γ for NS5A. This notion was supported by the observation that NS5A-specific T cells could eliminate NS5A-expressing hepatocytes also in the absence of IFN-γ-receptor-2. This supports that NS3/4A- and NS5A-specific T cells become activated and eliminate antigen expressing, or infected hepatocytes, by distinct mechanisms, and that NS5A-specific T cells show an overall less dependence of IFN-γ. PMID:27141891

  11. Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver

    PubMed Central

    Okushin, Kazuya; Enooku, Kenichiro; Fujinaga, Hidetaka; Moriya, Kyoji; Yotsuyanagi, Hiroshi; Aizaki, Hideki; Suzuki, Tetsuro; Matsuura, Yoshiharu; Koike, Kazuhiko

    2017-01-01

    The hepatitis C virus nonstructural protein NS5A is involved in resistance to the host immune response, as well as the viral lifecycle such as replication and maturation. Here, we established transgenic mice expressing NS5A protein in the liver and examined innate immune responses against lipopolysaccharide (LPS) in vivo. Intrahepatic gene expression levels of cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly suppressed after LPS injection in the transgenic mouse liver. Induction of the C-C motif chemokine ligand 2, 4, and 5 was also suppressed. Phosphorylation of the signal transducer and activator of transcription 3, which is activated by cytokines, was also reduced, and expression levels of interferon-stimulated genes, 2’-5’ oligoadenylate synthase, interferon-inducible double-stranded RNA-activated protein kinase, and myxovirus resistance 1 were similarly suppressed. Since LPS binds to toll-like receptor 4 and stimulates the downstream pathway leading to induction of these genes, we examined the extracellular signal-regulated kinase and IκB-α. The phosphorylation levels of these molecules were reduced in transgenic mouse liver, indicating that the pathway upstream of the molecules was disrupted by NS5A. Further analyses revealed that the interaction between interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor associated factor-6 was dispersed in transgenic mice, suggesting that NS5A may interfere with this interaction via myeloid differentiation primary response gene 88, which was shown to interact with NS5A. Since the gut microbiota, a source of LPS, is known to be associated with pathological conditions in liver diseases, our results suggest the involvement of NS5A in the pathogenesis of HCV infected-liver via the suppression of innate immunity. PMID:28107512

  12. Increased phosphoenolpyruvate carboxykinase gene expression and steatosis during hepatitis C virus subgenome replication: role of nonstructural component 5A and CCAAT/enhancer-binding protein β.

    PubMed

    Qadri, Ishtiaq; Choudhury, Mahua; Rahman, Shaikh Mizanoor; Knotts, Trina A; Janssen, Rachel C; Schaack, Jerome; Iwahashi, Mieko; Puljak, Livia; Simon, Francis R; Kilic, Gordan; Fitz, J Gregory; Friedman, Jacob E

    2012-10-26

    Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated transcription factors are dramatically up-regulated in Huh.8 cells, which stably express an HCV subgenome replicon. HCV increased activation of cAMP response element-binding protein (CREB), CCAAT/enhancer-binding protein (C/EBPβ), forkhead box protein O1 (FOXO1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and involved activation of the cAMP response element in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPβ-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1α or FOXO1 levels. Notably, expression of HCV nonstructural component NS5A in Huh7 or primary hepatocytes stimulated PEPCK gene expression and glucose output in HepG2 cells, whereas a deletion in NS5A reduced PEPCK expression and lowered cellular lipids but was without effect on insulin resistance, as demonstrated by the inability of insulin to stimulate mobilization of a pool of insulin-responsive vesicles to the plasma membrane. HCV-replicating cells demonstrated increases in cellular lipids with insulin resistance at the level of the insulin receptor, increased insulin receptor substrate 1 (Ser-312), and decreased Akt (Ser-473) activation in response to insulin. C/EBPβ-RNAi normalized lipogenic genes sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and liver X receptor α but was unable to reduce accumulation of triglycerides in Huh.8 cells or reverse the increase in ApoB expression, suggesting a role for increased lipid retention in steatotic hepatocytes. Collectively, these data reveal an important role of NS5A, C/EBPβ, and pCREB in promoting HCV-induced gluconeogenic gene expression

  13. Characterization of SLCO5A1/OATP5A1, a Solute Carrier Transport Protein with Non-Classical Function

    PubMed Central

    Sebastian, Katrin; Detro-Dassen, Silvia; Rinis, Natalie; Fahrenkamp, Dirk; Müller-Newen, Gerhard; Merk, Hans F.; Schmalzing, Günther

    2013-01-01

    Organic anion transporting polypeptides (OATP/SLCO) have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (∼6.5-fold) and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of ∼105 kDa and ∼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of ∼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20) and genes implicated in developmental processes (e.g. TGM2). A single nucleotide polymorphism leading to the exchange of amino acid 33 (L→F) revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration. PMID:24376674

  14. Hypusine-containing protein eIF5A promotes translation elongation.

    PubMed

    Saini, Preeti; Eyler, Daniel E; Green, Rachel; Dever, Thomas E

    2009-05-07

    Translation elongation factors facilitate protein synthesis by the ribosome. Previous studies identified two universally conserved translation elongation factors, EF-Tu in bacteria (known as eEF1A in eukaryotes) and EF-G (eEF2), which deliver aminoacyl-tRNAs to the ribosome and promote ribosomal translocation, respectively. The factor eIF5A (encoded by HYP2 and ANB1 in Saccharomyces cerevisiae), the sole protein in eukaryotes and archaea to contain the unusual amino acid hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine), was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis-a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation. Hypusine is required for eIF5A to associate with ribosomes and to stimulate methionyl-puromycin synthesis. Because eIF5A did not stimulate earlier steps of translation initiation, and depletion of eIF5A in yeast only modestly impaired protein synthesis, it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular messenger RNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay, including the nonsense-mediated mRNA decay pathway, and to nucleocytoplasmic transport. Here we use molecular genetic and biochemical studies to show that eIF5A promotes translation elongation. Depletion or inactivation of eIF5A in the yeast S. cerevisiae resulted in the accumulation of polysomes and an increase in ribosomal transit times. Addition of recombinant eIF5A from yeast, but not a derivative lacking hypusine, enhanced the rate of tripeptide synthesis in vitro. Moreover, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin, indicating that eIF5A might function together with eEF2 to promote ribosomal translocation. Because eIF5A is a structural homologue of the bacterial

  15. Purification and characterization of Ras related protein, Rab5a from Tinospora cordifolia.

    PubMed

    Amir, Mohd; Wahiduzzaman; Dar, Mohammad Aasif; Haque, Md Anzarul; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-01-01

    Ras related protein (Rab5a) is one of the most important member of the Rab family which regulates the early endosome fusion in endocytosis, and it also helps in the regulation of the budding process. Here, for the first time we report a simple and reproducible method for the purification of the Rab5a from a medicinal plant Tinospora cordifolia. We have used weak cation-exchange (CM-Sepharose-FF) followed by gel-filtration chromatography. A purified protein of 22-kDa was observed on SDS-PAGE which was identified as Rab5a using MALDI-TOF/MS. Our purification procedure is fast and simple with high yield. The purified protein was characterized using circular dichroism for the measurement of secondary structure followed by GdmCl- and urea-induced denaturation to calculate the values of Gibbs free energy change (ΔGD), ΔGD°, midpoint of the denaturation Cm, i.e. molar GdmCl [GdmCl] and molar urea [Urea] concentration at which ΔGD=0; and m, the slope (=∂ΔGD/∂[d]) values. Furthermore, thermodynamic properties of Rab5a were also measured by differential scanning calorimeter. Here, using isothermal calorimeteric measurements we further showed that Rab5a binds with the GTP. This is a first report on the purification and biophysical characterization of Rab5a protein from T. cordifolia.

  16. Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins*

    PubMed Central

    Nguyen, Suong; Leija, Chrisopher; Kinch, Lisa; Regmi, Sandesh; Li, Qiong; Grishin, Nick V.; Phillips, Margaret A.

    2015-01-01

    The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens. PMID:26082486

  17. The ankyrin repeat containing SOCS box protein 5: a novel protein associated with arteriogenesis.

    PubMed

    Boengler, Kerstin; Pipp, Frederic; Fernandez, Borja; Richter, Alexandra; Schaper, Wolfgang; Deindl, Elisabeth

    2003-02-28

    Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbit by occlusion of the femoral artery. In order to identify and characterize genes differentially expressed during the early phase of arteriogenesis, cDNA of collateral arteries 24h after femoral ligation or sham operation was subjected to suppression subtractive hybridization. We identified the ankyrin repeat containing SOCS box protein 5 (asb5) and cloned the rabbit full-length cDNA. Asb5 was demonstrated to be a single-copy gene. We localized the asb5 protein in vivo in endothelial and smooth muscle cells of collateral arteries as well as in satellite cells. Asb5 was significantly upregulated in growing collateral arteries on mRNA and protein level. The infusion of doxorubicin in rabbit led to a significant decrease of the asb5 mRNA. In summary, our data show that asb5 is a novel protein implicated in the initiation of arteriogenesis.

  18. Agonistic and antagonistic effects of C5a-chimera bearing S19 ribosomal protein tail portion on the C5a receptor of monocytes and neutrophils, respectively.

    PubMed

    Oda, Yuuichiro; Tokita, Kazutaka; Ota, Yoshihiko; Li, Ying; Taniguchi, Keisuke; Nishino, Norikazu; Takagi, Katsumasa; Yamamoto, Tetsuro; Nishiura, Hiroshi

    2008-09-01

    C-terminus of S19 ribosomal protein (RP S19) endows the cross-linked homodimer with a dual effect on the C5a receptor in leucocyte chemoattraction; agonistic effect on the monocyte receptor, and antagonistic effect on the neutrophil receptor. C5a exhibits the uniform agonistic effect on this receptor of both cell types. We have currently prepared a recombinant C5a-chimeric protein bearing the C-terminus of RP S19 (C5a/RP S19 chimera) to be used as a substitute of the RP S19 dimer. In vitro, this chimera similarly inhibited the intracellular Ca(2+) mobilization of neutrophils induced by C5a to the RP S19 dimer did. In the guinea pig skin, 10(-7) M C5a/RP S19 chimera exhibited an inhibitory capacity to the neutrophil infiltration induced by 3 x 10(-7) M C5a without enhancing monocyte infiltration. In reverse passive Arthus reaction, the neutrophil infiltration associated with plasma extravasation was significantly reduced by the simultaneous administration of 10(-7) M C5a/RP S19 chimera with antibodies. The C5a/RP S19 chimera is a useful tool not only to examine the molecular mechanism that underlies the functional difference of the C5a receptor between monocytes and neutrophils, but also to prevent C5a-mediated hyper-response of neutrophils in acute inflammation.

  19. Gc-globulin (vitamin D-binding protein) enhances the neutrophil chemotactic activity of C5a and C5a des Arg.

    PubMed Central

    Kew, R R; Webster, R O

    1988-01-01

    Several serum proteins have been shown to be important in modulating leukocyte chemotaxis and inflammation. We investigated the possibility that the multifunctional serum protein Gc-globulin (vitamin D-binding protein) may also enhance the neutrophil chemotactic activity of complement-derived peptides. Purified Gc-globulin by itself did not induce chemotaxis of human neutrophils. However, as little as 0.01 nM Gc-globulin greatly enhanced the neutrophil chemotactic activity of C5a and its derivative, C5a des Arg over a wide concentration range. The effect was most pronounced at nonchemotactic doses of C5a (0.01 nM) and C5a des Arg (1 nM). Gc-globulin was unable to augment the neutrophil chemotactic activity of FMLP and leukotriene B4. This enhancing activity was not due to a nonspecific effect of anionic proteins since other purified serum proteins, of similar size and charge as Gc-globulin (alpha 1 acid glycoprotein, alpha 2 HS glycoprotein, alpha 2 histidine-rich glycoprotein), could not increase the chemotactic activity of C5a des Arg. Serum depleted of Gc-globulin by immunoaffinity chromatography totally lacked chemotactic enhancing activity for C5a des Arg. Gc-globulin-depleted serum activated with zymosan also had significantly less chemotactic activity than control- (sham-depleted) activated serum. Finally, radioiodinated C5a or C5a des Arg formed a 1:1 complex with purified Gc-globulin when analyzed by gel filtration chromatography. These results indicate that Gc-globulin is the major chemotactic enhancing factor in serum and may function as an up-regulator of the chemotactic activity of C5-derived peptides. PMID:3392213

  20. Hepatitis C virus infection protein network.

    PubMed

    de Chassey, B; Navratil, V; Tafforeau, L; Hiet, M S; Aublin-Gex, A; Agaugué, S; Meiffren, G; Pradezynski, F; Faria, B F; Chantier, T; Le Breton, M; Pellet, J; Davoust, N; Mangeot, P E; Chaboud, A; Penin, F; Jacob, Y; Vidalain, P O; Vidal, M; André, P; Rabourdin-Combe, C; Lotteau, V

    2008-01-01

    A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein-protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFbeta pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.

  1. Hepatitis C virus infection protein network

    PubMed Central

    de Chassey, B; Navratil, V; Tafforeau, L; Hiet, M S; Aublin-Gex, A; Agaugué, S; Meiffren, G; Pradezynski, F; Faria, B F; Chantier, T; Le Breton, M; Pellet, J; Davoust, N; Mangeot, P E; Chaboud, A; Penin, F; Jacob, Y; Vidalain, P O; Vidal, M; André, P; Rabourdin-Combe, C; Lotteau, V

    2008-01-01

    A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein–protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFβ pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins. PMID:18985028

  2. Cysteine residues of the porcine reproductive and respiratory syndrome virus ORF5a protein are not essential for virus viability.

    PubMed

    Sun, Lichang; Zhou, Yan; Liu, Runxia; Li, Yanhua; Gao, Fei; Wang, Xiaomin; Fan, Hongjie; Yuan, Shishan; Wei, Zuzhang; Tong, Guangzhi

    2015-02-02

    ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for virus viability, but all serine mutant clones produced infectious progeny viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30.

  3. HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis

    PubMed Central

    Nawkar, Ganesh M.; Kang, Chang Ho; Maibam, Punyakishore; Park, Joung Hun; Jung, Young Jun; Chae, Ho Byoung; Chi, Yong Hun; Jung, In Jung; Kim, Woe Yeon; Yun, Dae-Jin; Lee, Sang Yeol

    2017-01-01

    Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box–like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression. PMID:28167764

  4. HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis.

    PubMed

    Nawkar, Ganesh M; Kang, Chang Ho; Maibam, Punyakishore; Park, Joung Hun; Jung, Young Jun; Chae, Ho Byoung; Chi, Yong Hun; Jung, In Jung; Kim, Woe Yeon; Yun, Dae-Jin; Lee, Sang Yeol

    2017-02-21

    Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box-like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression.

  5. Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A

    PubMed Central

    Kang, Yoon-Suk; Brame, Keenan; Jetter, Jonathan; Bothner, Brian B.; Wang, Gejiao

    2016-01-01

    ABSTRACT ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCE Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR. This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate

  6. Induced gene expression of the hypusine-containing protein eukaryotic initiation factor 5A in activated human T lymphocytes.

    PubMed Central

    Bevec, D; Klier, H; Holter, W; Tschachler, E; Valent, P; Lottspeich, F; Baumruker, T; Hauber, J

    1994-01-01

    The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation. Images PMID:7971969

  7. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

    SciTech Connect

    Nemoto, Eiji; Ebe, Yukari; Kanaya, Sousuke; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. Black-Right-Pointing-Pointer Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. Black-Right-Pointing-Pointer Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. Black-Right-Pointing-Pointer Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through {beta}-catenin-dependent canonical and {beta}-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent

  8. Orphan G protein-coupled receptor GPRC5A modulates integrin β1-mediated epithelial cell adhesion.

    PubMed

    Bulanova, Daria R; Akimov, Yevhen A; Rokka, Anne; Laajala, Teemu D; Aittokallio, Tero; Kouvonen, Petri; Pellinen, Teijo; Kuznetsov, Sergey G

    2016-10-07

    G-Protein Coupled Receptor (GPCR), Class C, Group 5, Member A (GPRC5A) has been implicated in several malignancies. The underlying mechanisms, however, remain poorly understood. Using a panel of human cell lines, we demonstrate that CRISPR/Cas9-mediated knockout and RNAi-mediated depletion of GPRC5A impairs cell adhesion to integrin substrates: collagens I and IV, fibronectin, as well as to extracellular matrix proteins derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Matrigel). Consistent with the phenotype, knock-out of GPRC5A correlated with a reduced integrin β1 (ITGB1) protein expression, impaired phosphorylation of the focal adhesion kinase (FAK), and lower activity of small GTPases RhoA and Rac1. Furthermore, we provide the first evidence for a direct interaction between GPRC5A and a receptor tyrosine kinase EphA2, an upstream regulator of FAK, although its contribution to the observed adhesion phenotype is unclear. Our findings reveal an unprecedented role for GPRC5A in regulation of the ITGB1-mediated cell adhesion and it's downstream signaling, thus indicating a potential novel role for GPRC5A in human epithelial cancers.

  9. Regulation of steroid 5-{alpha} reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    SciTech Connect

    Seo, Young-Kyo; Zhu, Bing; Jeon, Tae-Il; Osborne, Timothy F.

    2009-11-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  10. KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis

    PubMed Central

    Wang, Chuandong; Wang, Jing; Li, Jiao; Hu, Guoli; Shan, Shengzhou; Li, Qingfeng; Zhang, Xiaoling

    2016-01-01

    Bone morphogenetic protein 2 (BMP2) has been used to induce bone regeneration by promoting osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs). However, its effect is attenuated in osteoporotic conditions by unknown mechanisms. In this study, we investigated the molecular mechanisms of reduced osteogenic effect of BMP2 in osteoporotic conditions. By interrogating the microarray data from osteoporosis patients, we revealed an upregulation of the epigenetic modifying protein lysine (K)-specific demethylase 5A (KDM5A) and decreased Runt-related transcription factor 2 (RUNX2) expression. Further studies were focused on the role of KDM5A in osteoporosis. We first established ovariectomized (OVX) mouse model and found that the BMP2-induced osteogenic differentiation of osteoporotic MSCs was impaired. The elevated level of KDM5A was confirmed in osteoporotic MSCs. Overexpression of KDM5A in normal MSCs inhibited BMP2-induced osteogenesis. Moreover, osteogenic differentiation of osteoporotic MSCs was restored by specific KDM5A short hairpin RNA or inhibitor. Furthermore, by chromatin immunoprecipitation assay we demonstrated that KDM5A functions as endogenous modulator of osteogenic differentiation by decreasing H3K4me3 levels on promoters of Runx2, depend on its histone methylation activity. More importantly, we found an inhibitory role of KDM5A in regulating bone formation in osteoporotic mice, and pretreatment with KDM5A inhibitor partly rescued the bone loss during osteoporosis. Our results show, for the first time, that KDM5A-mediated H3K4me3 modification participated in the etiology of osteoporosis and may provide new strategies to improve the clinical efficacy of BMP2 in osteoporotic conditions. PMID:27512956

  11. Recombinant Trichomonas vaginalis eIF-5A protein expressed from a eukaryotic system binds specifically to mammalian and putative trichomonal eIF-5A response elements (EREs).

    PubMed

    Carvajal-Gamez, Bertha Isabel; Carrillo, Laura Vázquez; Torres-Romero, Julio César; Camacho-Nuez, Minerva; Ponce-Regalado, María Dolores; Camarillo, César López; Alvarez-Sánchez, María Elizbeth

    2016-12-01

    Trichomonas vaginalis eIF-5A-like protein (TveIF-5A) belongs to the highly conserved eIF-5A family of proteins that contains a unique polyamine-derived amino acid, hypusine. Recently, we determined that the polyamine putrescine is required for tveif-5a mRNA stability, and it is necessary for stability and maturation of the TveIF-5A protein. Eukaryotic eIF-5A is known to be involved in mRNA turnover and is capable of sequence-specific RNA binding to eIF-5A response elements (EREs). These ERE sequences are present in diverse mammalian mRNAs, including human cyclooxygenase-2 (cox-2). Here, we cloned the complete coding sequence of TveIF-5A and overexpressed it in a eukaryotic system. The recombinant protein (rTveIF-5A) was purified in soluble form using size-exclusion chromatography. Because of the polyamine-dependent regulation of TvCP39 (a protease of T. vaginalis) at the protein and RNA messenger (mRNA) levels, we looked for an ERE-like structure in the 3' region of tvcp39 mRNA. In RNA gel-shift assays, rTveIF-5A bound to transcripts at the EREs of cox-2 or tvcp39 mRNAs. This work shows the eIF-5A/ERE-like interaction in T. vaginalis.

  12. Sab (SH3BP5), a novel mitochondria-localized JNK-interacting protein.

    PubMed

    Wiltshire, C; Gillespie, D A F; May, G H W

    2004-12-01

    The JNK (c-Jun N-terminal kinase) pathway is activated by diverse stresses and can have an effect on a number of different cellular processes. Protein-protein interactions are critical for efficient signalling from JNK to multiple targets; through a screen for interacting proteins, we identified a novel JNK-interacting protein, Sab (SH3BP5). Sab has previously been found to interact with the Src homology 3 domain of Bruton's tyrosine kinase; however, the interaction with JNK occurs through a mitogen-activated protein KIM (kinase interaction motif) in a region distinct from the Bruton's tyrosine kinase-binding domain. As with c-Jun, the presence of this KIM is essential for Sab to act as a JNK substrate. Interestingly, Sab is associated with the mitochondria and co-localizes with a portion of active JNK after stress treatment. The present study and previously reported work may suggest a possible role for Sab in targeting JNK to this subcellular compartment and/or mediating crosstalk between different signal-transduction pathways.

  13. The Characteristics of Rare Codon Clusters in the Genome and Proteins of Hepatitis C Virus; a Bioinformatics Look

    PubMed Central

    Fattahi, Mohammadreza; Malekpour, Abdorrasoul; Mortazavi, Mojtaba; Safarpour, Alireza; Naseri, Nasrin

    2014-01-01

    BACKGROUND Recent studies suggest that rare codon clusters are functionally important for protein activity. METHODS Here, for the first time we analyzed and reported rare codon clusters in Hepatitis C Virus (HCV) genome and then identified the location of these rare codon clusters in the structure of HCV protein. This analysis was performed using the Sherlocc program that detects statistically relevant conserved rare codon clusters. RESULTS By this program, we identified the rare codon cluster in three regions of HCV genome; NS2, NS3, and NS5A coding sequence of HCV genome. For further understanding of the role of these rare codon clusters, we studied the location of these rare codon clusters and critical residues in the structure of NS2, NS3 and NS5A proteins. We identified some critical residues near or within rare codon clusters. It should be mentioned that characteristics of these critical residues such as location and situation of side chains are important in assurance of the HCV life cycle. CONCLUSION The characteristics of these residues and their relative status showed that these rare codon clusters play an important role in proper folding of these proteins. Thus, it is likely that these rare codon clusters may have an important role in the function of HCV proteins. This information is helpful in development of new avenues for vaccine and treatment protocols. PMID:25349685

  14. MSG5, a novel protein phosphatase promotes adaptation to pheromone response in S. cerevisiae.

    PubMed Central

    Doi, K; Gartner, A; Ammerer, G; Errede, B; Shinkawa, H; Sugimoto, K; Matsumoto, K

    1994-01-01

    Pheromone-stimulated yeast cells and haploid gpa1 deletion mutants arrest their cell cycle in G1. Overexpression of a novel gene called MSG5 suppresses this inhibition of cell division. Loss of MSG5 function leads to a diminished adaptive response to pheromone. Genetic analysis indicates that MSG5 acts at a stage where the protein kinases STE7 and FUS3 function to transmit the pheromone-induced signal. Since loss of MSG5 function causes an increase in FUS3 enzyme activity but not STE7 activity, we propose that MSG5 impinges on the pathway at FUS3. Sequence analysis suggests that MSG5 encodes a protein tyrosine phosphatase. This is supported by the finding that recombinant MSG5 has phosphatase activity in vitro and is able to inactivate autophosphorylated FUS3. Thus MSG5 might stimulate recovery from pheromone by regulating the phosphorylation state of FUS3. Images PMID:8306972

  15. The Nuclear Death Domain Protein p84N5; a Candidate Breast Cancer Susceptibility Gene

    DTIC Science & Technology

    2006-05-01

    transcriptional elongation, mRNA export, breast cancer 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF PAGES 19a. NAME...Body 4 Key Research Accomplishments 16 Reportable Outcomes 17 Conclusions 18 References 19 Appendices None 3 Godwin, A.K., Ph.D. INTRODUCTION: A...the basis of its ability to encode a protein that specifically associates with the N-terminal half of p110RB ( 18 ). This study demonstrated that

  16. MAP5: a novel brain microtubule-associated protein under strong developmental regulation.

    PubMed

    Riederer, B; Cohen, R; Matus, A

    1986-12-01

    A novel microtubule-associated protein, MAP5, is described, whose chemical properties and cytological distribution distinguish it from other known microtubule-associated proteins (MAPs). Its status as a MAP is indicated by the observations that (i) it co-assembles efficiently with microtubules in vitro, (ii) it is localized on microtubules in brain sections by immunogold staining with monoclonal antibody against MAP5 and (iii) immunoaffinity purified MAP5 stimulates tubulin polymerization. Immunoperoxidase staining of brain sections showed that MAP5 is present in neurons throughout the brain and that in them it is evenly distributed throughout axons, dendrites and cell bodies. In this respect it differs from previously described MAPs (1, 2, 3 and tau) which are differentially compartmentalized in brain neurons. MAP5 is not present in axon terminals, dendritic spines or other synaptic elements. It is present at substantially higher levels in neonatal brain than adult and it is more abundant than either MAP1 or MAP2a up to postnatal day 10. The fall in amount of MAP5, from juvenile to adult levels, is completed between postnatal days 10 and 20. This suggests that MAP5 is particularly important in modulating microtubule function during the formation of neuronal processes.

  17. GPRC5A suppresses protein synthesis at the endoplasmic reticulum to prevent radiation-induced lung tumorigenesis

    PubMed Central

    Wang, Jian; Farris, Alton B.; Xu, Kaiming; Wang, Ping; Zhang, Xiangming; Duong, Duc M.; Yi, Hong; Shu, Hui-Kuo; Sun, Shi-Yong; Wang, Ya

    2016-01-01

    GPRC5A functions as a lung tumour suppressor to prevent spontaneous and environmentally induced lung carcinogenesis; however, the underlying mechanism remains unclear. Here we reveal that GPRC5A at the endoplasmic reticulum (ER) membrane suppresses synthesis of the secreted or membrane-bound proteins including a number of oncogenes, the most important one being Egfr. The ER-located GPRC5A disturbs the assembly of the eIF4F-mediated translation initiation complex on the mRNA cap through directly binding to the eIF4F complex with its two middle extracellular loops. Particularly, suppression of EGFR by GPRC5A contributes significantly to preventing ionizing radiation (IR)-induced lung tumorigenesis. Thus, GPRC5A deletion enhances IR-promoted EGFR expression through an increased translation rate, thereby significantly increasing lung tumour incidence in Gprc5a−/− mice. Our findings indicate that under-expressed GPRC5A during lung tumorigenesis enhances any transcriptional stimulation through an active translational status, which can be used to control oncogene expression and potentially the resulting related disease. PMID:27273304

  18. Discovery of G Protein-Biased Dopaminergics with a Pyrazolo[1,5-a]pyridine Substructure.

    PubMed

    Möller, Dorothee; Banerjee, Ashutosh; Uzuneser, Taygun C; Skultety, Marika; Huth, Tobias; Plouffe, Bianca; Hübner, Harald; Alzheimer, Christian; Friedland, Kristina; Müller, Christian P; Bouvier, Michel; Gmeiner, Peter

    2017-03-01

    1,4-Disubstituted aromatic piperazines are privileged structural motifs recognized by aminergic G protein-coupled receptors. Connection of a lipophilic moiety to the arylpiperazine core by an appropriate linker represents a promising concept to increase binding affinity and to fine-tune functional properties. In particular, incorporation of a pyrazolo[1,5-a]pyridine heterocyclic appendage led to a series of high affinity dopamine receptor partial agonists. Comprehensive pharmacological characterization involving BRET biosensors, binding studies, electrophysiology and complementation-based assays revealed compounds favoring activation of G proteins (preferably Go) over β-arrestin recruitment at dopamine D2 receptors. The feasibility to design G protein-biased partial agonists as putative novel therapeutics was demonstrated for the representative 2-methoxyphenylpiperazine 16c, which unequivocally displayed antipsychotic activity in vivo. Moreover, combination of the pyrazolo[1,5-a]pyridine appendage with a 5-hydroxy-N-propyl-2-aminotetraline unit led to balanced or G protein-biased dopaminergic ligands depending on the stereochemistry of the head group, illustrating the complex structure-functional selectivity relationships at dopamine D2 receptors.

  19. CDKL5, a protein associated with rett syndrome, regulates neuronal morphogenesis via Rac1 signaling.

    PubMed

    Chen, Qian; Zhu, Yong-Chuan; Yu, Jing; Miao, Sheng; Zheng, Jing; Xu, Li; Zhou, Yang; Li, Dan; Zhang, Chi; Tao, Jiong; Xiong, Zhi-Qi

    2010-09-22

    Mutations in cyclin-dependent kinase-like 5 (CDKL5), also known as serine/threonine kinase 9 (STK9), have been identified in patients with Rett syndrome (RTT) and X-linked infantile spasm. However, the function of CDKL5 in the brain remains unknown. Here, we report that CDKL5 is a critical regulator of neuronal morphogenesis. We identified a neuron-specific splicing variant of CDKL5 whose expression was markedly induced during postnatal development of the rat brain. Downregulating CDKL5 by RNA interference (RNAi) in cultured cortical neurons inhibited neurite growth and dendritic arborization, whereas overexpressing CDKL5 had opposite effects. Furthermore, knocking down CDKL5 in the rat brain by in utero electroporation resulted in delayed neuronal migration, and severely impaired dendritic arborization. In contrast to its proposed function in the nucleus, we found that CDKL5 regulated dendrite development through a cytoplasmic mechanism. In fibroblasts and in neurons, CDKL5 colocalized and formed a protein complex with Rac1, a critical regulator of actin remodeling and neuronal morphogenesis. Overexpression of Rac1 prevented the inhibition of dendrite growth caused by CDKL5 knockdown, and the growth-promoting effect of ectopically expressed CDKL5 on dendrites was abolished by coexpressing a dominant-negative form of Rac1. Moreover, CDKL5 was required for brain-derived neurotrophic factor (BDNF)-induced activation of Rac1. Together, these results demonstrate a critical role of CDKL5 in neuronal morphogenesis and identify a Rho GTPase signaling pathway which may contribute to CDKL5-related disorders.

  20. Major urinary protein 5, a scent communication protein, is regulated by dietary restriction and subsequent re-feeding in mice

    PubMed Central

    Giller, K.; Huebbe, P.; Doering, F.; Pallauf, K.; Rimbach, G.

    2013-01-01

    Major urinary proteins (Mups) are important for rodent scent communication and sexual behaviour. Recent evidence suggests that Mup1 may be regulated by fasting and re-feeding (RF). However, other Mup isoforms are poorly investigated, and data on the impact of long-term dietary restriction (DR) and ad libitum RF on Mup expression are missing. We investigated the effects of long-term 25 per cent DR and subsequent RF on Mup expression in male C57BL6 mice. DR significantly decreased Mup gene expression, hepatic and urinary protein levels compared with ad libitum (AL) fed control mice, with the greatest downregulation found for Mup5 expression. The decline in Mup expression was inverted by six months of RF. Because of inhibitory glucocorticoid response elements in the genomic sequence of the Mup5 gene, the observed inverse correlation of nuclear glucocorticoid receptor levels with Mup expression in response to DR and subsequent RF is a possible regulatory mechanism. Additionally, gene-expression-inhibiting histone deacetylation (H3K9) occurred in the region of the Mup5 gene in response to DR. We assume that Mup may act as a molecular switch linking nutritional status to sexual behaviour of mice, and thereby regulating male fertility and reproduction in response to food supply. PMID:23446533

  1. A novel ER-localized transmembrane protein, EMC6, interacts with RAB5A and regulates cell autophagy.

    PubMed

    Li, Yanjun; Zhao, Yuanbo; Hu, Jia; Xiao, Juan; Qu, Liujing; Wang, Zhenda; Ma, Dalong; Chen, Yingyu

    2013-02-01

    Autophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of the cytoplasm for delivery to the lysosome. Phosphatidylinositol 3-phosphate (PtdIns3P) produced by the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is essential for canonical autophagosome formation. RAB5A, a small GTPase localized to early endosomes, has been shown to associate with the class III PtdIns3K complex, regulate its activity and promote autophagosome formation. However, little is known about how endosome-localized RAB5A functions with the class III PtdIns3K complex. Here we identified a novel endoplasmic reticulum (ER)-localized transmembrane protein, ER membrane protein complex subunit 6 (EMC6), which interacted with both RAB5A and BECN1/Beclin 1 and colocalized with the omegasome marker ZFYVE1/DFCP1. It was shown to regulate autophagosome formation, and its deficiency caused the accumulation of autophagosomal precursor structures and impaired autophagy. Our study showed for the first time that EMC6 is a novel regulator involved in autophagy.

  2. Plk5, a Polo Box Domain-Only Protein with Specific Roles in Neuron Differentiation and Glioblastoma Suppression▿ †

    PubMed Central

    de Cárcer, Guillermo; Escobar, Beatriz; Higuero, Alonso M.; García, Laura; Ansón, Alejandra; Pérez, Gema; Mollejo, Manuela; Manning, Gerard; Meléndez, Bárbara; Abad-Rodríguez, José; Malumbres, Marcos

    2011-01-01

    Polo-like kinases (Plks) are characterized by the presence of a specific domain, known as the polo box (PBD), involved in protein-protein interactions. Plk1 to Plk4 are involved in centrosome biology as well as the regulation of mitosis, cytokinesis, and cell cycle checkpoints in response to genotoxic stress. We have analyzed here the new member of the vertebrate family, Plk5, a protein that lacks the kinase domain in humans. Plk5 does not seem to have a role in cell cycle progression; in fact, it is downregulated in proliferating cells and accumulates in quiescent cells. This protein is mostly expressed in the brain of both mice and humans, and it modulates the formation of neuritic processes upon stimulation of the brain-derived neurotrophic factor (BDNF)/nerve growth factor (NGF)-Ras pathway in neurons. The human PLK5 gene is significantly silenced in astrocytoma and glioblastoma multiforme by promoter hypermethylation, suggesting a tumor suppressor function for this gene. Indeed, overexpression of Plk5 has potent apoptotic effects in these tumor cells. Thus, Plk5 seems to have evolved as a kinase-deficient PBD-containing protein with nervous system-specific functions and tumor suppressor activity in brain cancer. PMID:21245385

  3. Expression of heterologous proteins flanked by NS3-4A cleavage sites within the hepatitis C virus polyprotein.

    PubMed

    Horwitz, Joshua A; Dorner, Marcus; Friling, Tamar; Donovan, Bridget M; Vogt, Alexander; Loureiro, Joana; Oh, Thomas; Rice, Charles M; Ploss, Alexander

    2013-04-25

    Hepatitis C virus (HCV) contributes substantially to human morbidity and mortality world-wide. The development of HCV genomes expressing heterologous proteins has enhanced the ability to study viral infection, but existing systems have drawbacks. Recombinant viruses often require adaptive mutations to compensate for reduced viral titers, or rely on an artificial genomic organization that uncouples viral protein expression from recombinant gene expression. Here, we sought to exploit the viral polyprotein processing machinery to express heterologous proteins within the context of the HCV polyprotein. We show that HCV genotypes 2a and 1b permit insertion of reporter proteins between NS5A and NS5B with minimal impact on viral fitness. Using this strategy we constructed reporter genomes exhibiting a wide dynamic range, simplifying analysis of HCV infection in primary hepatocytes. Expression of heterologous proteins within the HCV genome offers new opportunities to analyze HCV infection in experimental systems without perturbing functions of individual viral proteins.

  4. iHADAMAC: A complementary tool for sequential resonance assignment of globular and highly disordered proteins

    NASA Astrophysics Data System (ADS)

    Feuerstein, Sophie; Plevin, Michael J.; Willbold, Dieter; Brutscher, Bernhard

    2012-01-01

    An experiment, iHADAMAC, is presented that yields information on the amino-acid type of individual residues in a protein by editing the 1H- 15N correlations into seven different 2D spectra, each corresponding to a different class of amino-acid types. Amino-acid type discrimination is realized via a Hadamard encoding scheme based on four different spin manipulations as recently introduced in the context of the sequential HADAMAC experiment. Both sequential and intra-residue HADAMAC experiments yield highly complementary information that greatly facilitate resonance assignment of proteins with high frequency degeneracy, as demonstrated here for a 188-residue intrinsically disordered protein fragment of the hepatitis C virus protein NS5A.

  5. Identification of the C5a des Arg cochemotaxin. Homology with vitamin D-binding protein (group-specific component globulin).

    PubMed Central

    Perez, H D; Kelly, E; Chenoweth, D; Elfman, F

    1988-01-01

    The chemotactic activity of human C5a des Arg is enhanced significantly by an anionic polypeptide (cochemotaxin) in normal human serum and plasma. The cochemotaxin attaches to sialic acid residues within the oligosaccharide chain of native C5a des Arg to form a complex with potent chemotactic activity for human PMN. We investigated the nature of the cochemotaxin and found that vitamin D-binding protein is the putative cochemotaxin. Vitamin D-binding protein enhanced the chemotactic activity of native C5a des Arg, but had no effect on the chemotactic activity of either native C5a or FMLP. Sialic acid prevented both enhancement by vitamin D-binding protein of the chemotactic activity of native C5a des Arg and formation of C5a des Arg-vitamin D-binding protein complexes, detected by molecular sieve chromatography. Furthermore, vitamin D-binding protein and cochemotaxin exhibited identical molecular weights, isoelectric points, antigenic reactivity, and amino acid composition. Images PMID:3392212

  6. Heterotrimeric G protein-dependent WNT-5A signaling to ERK1/2 mediates distinct aspects of microglia proinflammatory transformation

    PubMed Central

    2012-01-01

    Background WNT-5A signaling in the central nervous system is important for morphogenesis, neurogenesis and establishment of functional connectivity; the source of WNT-5A and its importance for cellular communication in the adult brain, however, are mainly unknown. We have previously investigated the inflammatory effects of WNT/β-catenin signaling in microglia in Alzheimer's disease. WNT-5A, however, generally recruits β-catenin-independent signaling. Thus, we aim here to characterize the role of WNT-5A and downstream signaling pathways for the inflammatory transformation of the brain's macrophages, the microglia. Methods Mouse brain sections were used for immunohistochemistry. Primary isolated microglia and astrocytes were employed to characterize the WNT-induced inflammatory transformation and underlying intracellular signaling pathways by immunoblotting, quantitative mRNA analysis, proliferation and invasion assays. Further, measurements of G protein activation by [γ-35 S]GTP binding, examination of calcium fluxes and cyclic AMP production were used to define intracellular signaling pathways. Results Astrocytes in the adult mouse brain express high levels of WNT-5A, which could serve as a novel astroglia-microglia communication pathway. The WNT-5A-induced proinflammatory microglia response is characterized by increased expression of inducible nitric oxide synthase, cyclooxygenase-2, cytokines, chemokines, enhanced invasive capacity and proliferation. Mapping of intracellular transduction pathways reveals that WNT-5A activates heterotrimeric Gi/o proteins to reduce cyclic AMP levels and to activate a Gi/o protein/phospholipase C/calcium-dependent protein kinase/extracellular signal-regulated kinase 1/2 (ERK1/2) axis. We show further that WNT-5A-induced ERK1/2 signaling is responsible for distinct aspects of the proinflammatory transformation, such as matrix metalloprotease 9/13 expression, invasion and proliferation. Conclusions Thus, WNT-5A-induced and G

  7. Wnt5a is required for endothelial differentiation of embryonic stem cells and vascularization via pathways involving both Wnt/beta-catenin and protein kinase Calpha.

    PubMed

    Yang, Dong-Hwa; Yoon, Ju-Young; Lee, Soung-Hoon; Bryja, Vitezslav; Andersson, Emma R; Arenas, Ernest; Kwon, Young-Guen; Choi, Kang-Yell

    2009-02-13

    In this study, we examined the signaling pathways activated by Wnt5a in endothelial differentiation of embryonic stem (ES) cells and the function of Wnt5a during vascular development. We first found that Wnt5a(-/-) mouse embryonic stem (mES) cells exhibited a defect in endothelial differentiation, which was rescued by addition of Wnt5a, suggesting that Wnt5a is required for endothelial differentiation of ES cells. Involvement of both beta-catenin and protein kinase (PK)Calpha pathways in endothelial differentiation of mES cells requiring Wnt5a was indicated by activation of both beta-catenin and PKCalpha in Wnt5a(+/-) but not in Wnt5a(-/-) mES cells. We also found that beta-catenin or PKCalpha knockdowns inhibited the Wnt5a-induced endothelial differentiation of ES cells. Moreover, the lack of endothelial differentiation of Wnt5a(-/-) mES cells was rescued only by transfection of both beta-catenin and PKCalpha, indicating that both genes are required for Wnt5a-mediated endothelial differentiation. Wnt5a was also found to be essential for the differentiation of mES cells into immature endothelial progenitor cells, which are known to play a role in repair of damaged endothelium. Furthermore, a defect in the vascularization of the neural tissue was detected at embryonic day 14.5 in Wnt5a(-/-) mice, implicating Wnt5a in vascular development in vivo. Thus, we conclude that Wnt5a is involved in the endothelial differentiation of ES cells via both Wnt/beta-catenin and PKC signaling pathways and regulates embryonic vascular development.

  8. Signal transducer and activator of transcription 5a inhibited by pimozide may regulate survival of goat mammary gland epithelial cells by regulating parathyroid hormone-related protein.

    PubMed

    Li, Hui; Zheng, Huiling; Sun, Yongsen; Yu, Qian; Li, Lihui

    2014-11-10

    The signal transducer and activator of transcription 5a (Stat5a) modulates genes involved in proliferation and survival and plays pivotal roles in regulating the function of the mammary gland during pregnancy, lactation, and involution. However, there is little information about the effects of Stat5a on apoptosis of goat mammary gland epithelial cells (GMECs). In addition, parathyroid hormone-related protein (PTHrP) is a key regulator in cellular calcium transport, mammary gland development and breast tumor biology. This study aimed to explore the interaction of Stat5a and PTHrP in GMEC apoptosis. Quantitative real time PCR (qRT-PCR) suggested that Stat5a was predominantly expressed in the mammary gland, lung, liver and spleen of goats. Treating the GMECs with pimozide, an inhibitor of Stat5a that decreases Stat5a tyrosine phosphorylation, increased PTHrP levels in GMECs in a dose-dependent manner and simultaneously promoted apoptosis of the GMECs. We also demonstrated that PTHrP inhibition induced GMEC apoptosis and restrained cell proliferation. In contrast, PTHrP overexpression protected GMECs from pimozide- and calcium-induced apoptosis, and promoted cell proliferation. Furthermore, pimozide and CaCl2 downregulated the antiapoptotic protein Bcl-2 mRNA expression, respectively, and these effects were protected by PTHrP overexpression. Interestingly, we also found that Stat5a suppressed the expression of matrix metalloproteinase 9 (MMP-9) which can induce goat mammary epithelial cell migration, but PTHrP increased MMP-9 mRNA level. Thus, Stat5a may regulate GMEC survival by regulating the expression of PTHrP.

  9. Inflammatory responses induced by lipopolysaccharide are amplified in primary human monocytes but suppressed in macrophages by complement protein C5a.

    PubMed

    Seow, Vernon; Lim, Junxian; Iyer, Abishek; Suen, Jacky Y; Ariffin, Juliana K; Hohenhaus, Daniel M; Sweet, Matthew J; Fairlie, David P

    2013-10-15

    Monocytes and macrophages are important innate immune cells equipped with danger-sensing receptors, including complement and Toll-like receptors. Complement protein C5a, acting via C5aR, is shown in this study to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. Whereas C5a enhanced secretion of LPS-induced IL-6 and TNF from primary human monocytes, C5a inhibited these responses while increasing IL-10 secretion in donor-matched human monocyte-derived macrophages differentiated by GM-CSF or M-CSF. Gαi/c-Raf/MEK/ERK signaling induced by C5a was amplified in macrophages but not in monocytes by LPS. Accordingly, the Gαi inhibitor pertussis toxin and MEK inhibitor U0126 blocked C5a inhibition of LPS-induced IL-6 and TNF production from macrophages. This synergy was independent of IL-10, PI3K, p38, JNK, and the differentiating agent. Furthermore, C5a did not inhibit IL-6 production from macrophages induced by other TLR agonists that are selective for Toll/IL-1R domain-containing adapter inducing IFN-β (polyinosinic-polycytidylic acid) or MyD88 (imiquimod), demonstrating selectivity for C5a regulation of LPS responses. Finally, suppression of proinflammatory cytokines IL-6 and TNF in macrophages did not compromise antimicrobial activity; instead, C5a enhanced clearance of the Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium from macrophages. C5aR is thus a regulatory switch that modulates TLR4 signaling via the Gαi/c-Raf/MEK/ERK signaling axis in human macrophages but not monocytes. The differential effects of C5a are consistent with amplifying monocyte proinflammatory responses to systemic danger signals, but attenuating macrophage cytokine responses (without compromising microbicidal activity), thereby restraining inflammatory responses to localized infections.

  10. Cofactor Regulation of C5a Chemotactic Activity in Physiological Fluids. Requirement for the Vitamin D Binding Protein, Thrombospondin-1 and its Receptors

    PubMed Central

    Trujillo, Glenda; Zhang, Jianhua; Habiel, David M.; Ge, Lingyin; Ramadass, Mahalakshmi; Ghebrehiwet, Berhane; Kew, Richard R.

    2011-01-01

    Factors in physiological fluids that regulate the chemotactic activity of complement activation peptides C5a and C5a des Arg are not well understood. The vitamin D binding protein (DBP) has been shown to significantly enhance chemotaxis to C5a/C5a des Arg. More recently, platelet-derived thrombospondin-1 (TSP-1) has been shown to facilitate the augmentation of C5a-induced chemotaxis by DBP. The objective of this study was to better characterize these chemotactic cofactors and investigate the role that cell surface TSP-1 receptors CD36 and CD47 may play in this process. The chemotactic activity in C-activated normal serum, citrated plasma, DBP-depleted serum or C5 depleted serum was determined for both normal human neutrophils and U937 cell line transfected with the C5a receptor (U937-C5aR). In addition, levels of C5a des Arg, DBP and TSP-1 in these fluids were measured by RIA or ELISA. Results show that there is a clear hierarchy with C5a being the essential primary signal (DBP or TSP-1 will not function in the absence of C5a), DBP the necessary cofactor and TSP-1 a dependent tertiary factor, since it cannot function to enhance chemotaxis to C5a without DBP. Measurement of the C5a-induced intracellular calcium flux confirmed the same hierarchy observed with chemotaxis. Moreover, analysis of bronchoalveolar lavage fluid (BALF) from patients with the adult respiratory distress syndrome (ARDS) demonstrated that C5a-dependent chemotactic activity is significantly decreased after anti-DBP treatment. Finally, results show that TSP-1 utilizes cell surface receptors CD36 and CD47 to augment chemotaxis, but DBP does not bind to TSP-1, CD36 or CD47. The results clearly demonstrate that C5a/C5a des Arg needs both DBP and TSP-1 for maximal chemotactic activity and suggest that the regulation of C5a chemotactic activity in physiological fluids is more complex than previously thought. PMID:22014686

  11. Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

    PubMed Central

    Li, Lian-Feng; Yu, Jiahui; Li, Yongfeng; Wang, Jinghan; Li, Su; Zhang, Lingkai; Xia, Shui-Li; Yang, Qian; Wang, Xiao; Yu, Shaoxiong; Luo, Yuzi; Sun, Yuan; Zhu, Yan; Munir, Muhammad

    2016-01-01

    ABSTRACT Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity. IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV

  12. Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

    SciTech Connect

    Suhadolnik, R.J.; Kariko, K.; Sobol, R.W. Jr.; Li, S.W.; Reichenbach, N.L.; Haley, B.E.

    1988-11-29

    The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from (..gamma..-/sup 32/P)2-azidoATP and (..cap alpha..-/sup 32/P)8-azidoAPT by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p/sub 3/A/sub 4/(/sup 32/P)pCp from RNase L with affinity equivalent to p/sub 3/A/sub 3/. The 8-azido photoprobe also activates RNase L to hydrolyze poly(U)(/sup 32/P)pCp 50% at 7 /times/ 10/sup /minus/9/ M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p/sub 3/A/sub 3/ activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10/sup /minus/9/ M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The (..gamma..-/sup 32/P)2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the (..cap alpha..-/sup 32/P)8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p/sub 3/A/sub 3/ allosteric binding site was obtained.

  13. Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin.

    PubMed

    Otto, Edgar A; Loeys, Bart; Khanna, Hemant; Hellemans, Jan; Sudbrak, Ralf; Fan, Shuling; Muerb, Ulla; O'Toole, John F; Helou, Juliana; Attanasio, Massimo; Utsch, Boris; Sayer, John A; Lillo, Concepcion; Jimeno, David; Coucke, Paul; De Paepe, Anne; Reinhardt, Richard; Klages, Sven; Tsuda, Motoyuki; Kawakami, Isao; Kusakabe, Takehiro; Omran, Heymut; Imm, Anita; Tippens, Melissa; Raymond, Pamela A; Hill, Jo; Beales, Phil; He, Shirley; Kispert, Andreas; Margolis, Benjamin; Williams, David S; Swaroop, Anand; Hildebrandt, Friedhelm

    2005-03-01

    Nephronophthisis (NPHP) is the most frequent genetic cause of chronic renal failure in children. Identification of four genes mutated in NPHP subtypes 1-4 (refs. 4-9) has linked the pathogenesis of NPHP to ciliary functions. Ten percent of affected individuals have retinitis pigmentosa, constituting the renal-retinal Senior-Loken syndrome (SLSN). Here we identify, by positional cloning, mutations in an evolutionarily conserved gene, IQCB1 (also called NPHP5), as the most frequent cause of SLSN. IQCB1 encodes an IQ-domain protein, nephrocystin-5. All individuals with IQCB1 mutations have retinitis pigmentosa. Hence, we examined the interaction of nephrocystin-5 with RPGR (retinitis pigmentosa GTPase regulator), which is expressed in photoreceptor cilia and associated with 10-20% of retinitis pigmentosa. We show that nephrocystin-5, RPGR and calmodulin can be coimmunoprecipitated from retinal extracts, and that these proteins localize to connecting cilia of photoreceptors and to primary cilia of renal epithelial cells. Our studies emphasize the central role of ciliary dysfunction in the pathogenesis of SLSN.

  14. Decreased activity of the Na+/H+ exchanger by phosphodiesterase 5A inhibition is attributed to an increase in protein phosphatase activity.

    PubMed

    Yeves, Alejandra M; Garciarena, Carolina D; Nolly, Mariela B; Chiappe de Cingolani, Gladys E; Cingolani, Horacio E; Ennis, Irene L

    2010-10-01

    The beneficial effect of phosphodiesterase 5A inhibition in ischemia/reperfusion injury and cardiac hypertrophy is well established. Inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1) exerts beneficial effects on these same conditions, and a possible link between these therapeutic strategies was suggested. Experiments were performed in isolated cat cardiomyocytes to gain insight into the intracellular pathway involved in the reduction of NHE-1 activity by phosphodiesterase 5A inhibition. NHE-1 activity was assessed by the rate of intracellular pH recovery from a sustained acidic load in the absence of bicarbonate. Phosphodiesterase 5A inhibition with sildenafil (1 μmol/L) did not affect basal intracellular pH; yet, it did decrease proton efflux (J(H); in millimoles per liter per minute) after the acidic load (proton efflux: 6.97±0.43 in control versus 3.31±0.58 with sildenafil; P<0.05). The blockade of both protein phosphatase 1 and 2A with 100 nmol/L of okadaic acid reverted the sildenafil effect (proton efflux: 6.77±0.82). In contrast, selective inhibition of protein phosphatase 2A (1 nmol/L of okadaic acid or 100 μmol/L of endothall) did not (3.86±1.0 and 2.61±1.2), suggesting that only protein phosphatase 1 was involved in sildenafil-induced NHE-1 inhibition. Moreover, sildenafil prevented the acidosis-induced increase in NHE-1 phosphorylation without affecting activation of the extracellular signal-regulated kinase 1/2-p90(RSK) pathway. Our results suggest that phosphodiesterase 5A inhibition decreases NHE-1 activity, during intracellular pH recovery after an acidic load, by a protein phosphatase 1-dependent reduction in NHE-1 phosphorylation.

  15. A vaccine composed of a hypothetical protein and the eukaryotic initiation factor 5a from Leishmania braziliensis cross-protection against Leishmania amazonensis infection.

    PubMed

    Duarte, Mariana Costa; Lage, Daniela Pagliara; Martins, Vívian Tamietti; Costa, Lourena Emanuele; Carvalho, Ana Maria Ravena Severino; Ludolf, Fernanda; Santos, Thaís Teodoro de Oliveira; Vale, Danniele Luciana; Roatt, Bruno Mendes; Menezes-Souza, Daniel; Fernandes, Ana Paula; Tavares, Carlos Alberto Pereira; Coelho, Eduardo Antonio Ferraz

    2017-02-01

    In the present study, two proteins cloned from Leishmania braziliensis species, a hypothetical protein (LbHyp) and the eukaryotic initiation factor 5a (EiF5a), were evaluated to protect BALB/c mice against L. amazonensis infection. The animals were immunized with the antigens, either separately or in combination, using saponin as an immune adjuvant in both cases. Spleen cells from vaccinated and later infected mice produced significantly higher levels of protein and parasite-specific IFN-γ, IL-12, and GM-CSF, in addition to low levels of IL-4 and IL-10. Evaluating the parasite load by means of a limiting dilution technique and quantitative Real-Time PCR, vaccinated animals presented significant reductions in the parasite load in both infected tissues and organs, as well as lower footpad swelling, when compared to the control (saline and saponin) groups. The best results regarding the protection of the animals were achieved when the combined vaccine was administered into the animals. Protection was associated with an IFN-γ production against parasite antigens, which was mediated by both CD4(+) and CD8(+) T cells and correlated with antileishmanial nitrite production. In conclusion, data from the present study show that this polyprotein vaccine, which combines two L. braziliensis proteins, can induce protection against L. amazonensis infection.

  16. The wingless-related integration site-5a/secreted frizzled-related protein-5 system is dysregulated in human sepsis.

    PubMed

    Schulte, D M; Kragelund, D; Müller, N; Hagen, I; Elke, G; Titz, A; Schädler, D; Schumacher, J; Weiler, N; Bewig, B; Schreiber, S; Laudes, M

    2015-04-01

    Sepsis and type 2 diabetes exhibit insulin resistance as a common phenotype. In type 2 diabetes we and others have recently provided evidence that alterations of the proinflammatory wingless-related integration site (wnt)-5a/anti-inflammatory secreted frizzled-related protein (sFRP)-5 system are involved in the pathogenesis of insulin resistance. The aim of the present study was to investigate whether this novel cytokine system is dysregulated in human sepsis, which may indicate a potential mechanism linking inflammation to metabolism. In this single-centre prospective observational study, critically ill adult septic patients were examined and proinflammatory wnt5a and wnt5a inhibitor sFRP5 were measured in serum samples by enzyme-linked immunosorbent assay (ELISA) at admission to the intensive care unit (ICU) and 5 days later. Sixty sepsis patients were included, and 30 healthy individuals served as controls. Wnt5a levels were found to be increased significantly in septic patients compared to healthy controls (2·21 ± 0·33 versus 0·32 ± 0·03 ng/ml, P < 0·0001). In contrast, sFRP5 was not altered significantly in septic patients (19·72 ± 3·06 versus 17·48 ± 6·38 ng/ml, P = 0·07). On admission to the ICU, wnt5a levels exhibited a significant positive correlation with the leucocyte count (rs  = 0·3797, P = 0·004). Interestingly, in patients recovering from sepsis, wnt5a levels declined significantly within 5 days (2·17 ± 0·38-1·03 ± 0·28 ng/ml, P < 0·01). In contrast, if sepsis was worsening, wnt5a levels increased in the same time-period by trend (2·34 ± 0·59-3·25 ± 1·02 ng/ml, P > 0·05). sFRP5 levels did not change significantly throughout the study period. The wnt5a/sFRP5 system is altered in human sepsis and might therefore be of interest for future studies on molecular pathophysiology of this common human disease.

  17. p300/β-Catenin Interactions Regulate Adult Progenitor Cell Differentiation Downstream of WNT5a/Protein Kinase C (PKC)*

    PubMed Central

    Rieger, Megan E.; Zhou, Beiyun; Solomon, Nicola; Sunohara, Mitsuhiro; Li, Changgong; Nguyen, Cu; Liu, Yixin; Pan, Jie-hong; Minoo, Parviz; Crandall, Edward D.; Brody, Steven L.; Kahn, Michael; Borok, Zea

    2016-01-01

    Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of β-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential β-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific β-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/β-catenin but not CBP/β-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/β-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/β-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/β-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting β-catenin to modulate adult progenitor cell

  18. A Critical Role of Lyst-Interacting Protein5, a Positive Regulator of Multivesicular Body Biogenesis, in Plant Responses to Heat and Salt Stresses1

    PubMed Central

    Wang, Fei; Yang, Yan; Wang, Zhe; Zhou, Jie; Fan, Baofang; Chen, Zhixiang

    2015-01-01

    Multivesicular bodies (MVBs) are unique endosomes containing vesicles in the lumen and play critical roles in many cellular processes. We have recently shown that Arabidopsis (Arabidopsis thaliana) Lyst-Interacting Protein5 (LIP5), a positive regulator of the Suppressor of K+ Transport Growth Defect1 (SKD1) AAA ATPase in MVB biogenesis, is a critical target of the mitogen-activated protein kinases MPK3 and MPK6 and plays an important role in the plant immune system. In this study, we report that the LIP5-regulated MVB pathway also plays a critical role in plant responses to abiotic stresses. Disruption of LIP5 causes compromised tolerance to both heat and salt stresses. The critical role of LIP5 in plant tolerance to abiotic stresses is dependent on its ability to interact with Suppressor of K+ Transport Growth Defect1. When compared with wild-type plants, lip5 mutants accumulate increased levels of ubiquitinated protein aggregates and NaCl under heat and salt stresses, respectively. Further analysis using fluorescent dye and MVB markers reveals that abiotic stress increases the formation of endocytic vesicles and MVBs in a largely LIP5-dependent manner. LIP5 is also required for the salt-induced increase of intracellular reactive oxygen species, which have been implicated in signaling of salt stress responses. Basal levels of LIP5 phosphorylation by MPKs and the stability of LIP5 are elevated by salt stress, and mutation of MPK phosphorylation sites in LIP5 reduces the stability and compromises the ability to complement the lip5 salt-sensitive mutant phenotype. These results collectively indicate that the MVB pathway is positively regulated by pathogen/stress-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in broad plant responses to biotic and abiotic stresses. PMID:26229051

  19. Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental disorder.

    PubMed

    Sekiguchi, Mari; Katayama, Syouichi; Hatano, Naoya; Shigeri, Yasushi; Sueyoshi, Noriyuki; Kameshita, Isamu

    2013-07-15

    Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in brain and mutations of its gene are known to be associated with neurodevelopmental disorders such as X-linked West syndrome and Rett syndrome. However, the physiological substrates of CDKL5 that are directly linked to these neurodevelopmental disorders are currently unknown. In this study, we explored endogenous substrates for CDKL5 in mouse brain extracts fractionated by a liquid-phase isoelectric focusing. In conjunction with CDKL5 phosphorylation assay, this approach detected a protein band with an apparent molecular mass of 120kDa that is remarkably phosphorylated by CDKL5. This 120-kDa protein was identified as amphiphysin 1 (Amph1) by LC-MS/MS analysis, and the site of phosphorylation by CDKL5 was determined to be Ser-293. The phosphorylation mimic mutants, Amph1(S293E) and Amph1(S293D), showed significantly reduced affinity for endophilin, a protein involved in synaptic vesicle endocytosis. Introduction of point mutations in the catalytic domain of CDKL5, which are disease-causing missense mutations found in Rett patients, resulted in the impairment of kinase activity toward Amph1. These results suggest that Amph1 is the cytoplasmic substrate for CDKL5 and that its phosphorylation may play crucial roles in the neuronal development.

  20. Approach for in vivo protein binding of 5-n-butyl-pyrazolo[1,5-a]pyrimidine bioactivated in chimeric mice with humanized liver by two-dimensional electrophoresis with accelerator mass spectrometry.

    PubMed

    Yamazaki, Hiroshi; Kuribayashi, Shunji; Inoue, Tae; Tateno, Chise; Nishikura, Yasufumi; Oofusa, Ken; Harada, Daisuke; Naito, Shinsaku; Horie, Toru; Ohta, Shigeru

    2010-01-01

    Drug development of a potential analgesic agent 5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine was withdrawn because of its limited hepatotoxic effects in humans that could not be predicted from regulatory animal or in vitro studies. In vivo formation of glutathione conjugates and covalent binding of a model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine were investigated in the present study after intravenous administration to chimeric mice with a human or rat liver because of an interesting capability of human cytochrome P450 1A2 in forming a covalently bound metabolite in vitro. Rapid distribution and elimination of radiolabeled 5-n-butyl-pyrazolo[1,5-a]pyrimidine in plasma or liver fractions were seen in chimeric mice after intravenous administration. However, similar covalent binding in liver was detected over 0.17-24 h after intravenous administration. Radio-LC analyses revealed that the chimeric mice with humanized liver preferentially gave the 3-hydroxylated metabolite and its glutathione conjugate in the plasma and liver. On the contrary, chimeric mice with a rat liver had some rat-specific metabolites in vivo. Analyses by electrophoresis with accelerator mass spectrometry of in vivo radiolabeled liver proteins in chimeric mice revealed that bioactivated 5-n-butyl-pyrazolo[1,5-a]pyrimidine bound nonspecifically to a variety of microsomal proteins including human P450 1A2 as well as cytosolic proteins in the livers from chimeric mice with humanized liver. These results suggest that the hepatotoxic model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine was activated by human liver microsomal P450 1A2 to reactive intermediate(s) in vivo in humanized chimeric mice and could relatively nonspecifically bind to biomolecules such as P450 1A2 and other proteins.

  1. Molecular and immunological characterization of β'-component (Onc k 5), a major IgE-binding protein in chum salmon roe.

    PubMed

    Shimizu, Yutaka; Kishimura, Hideki; Kanno, Gaku; Nakamura, Atsushi; Adachi, Reiko; Akiyama, Hiroshi; Watanabe, Kazuhiko; Hara, Akihiko; Ebisawa, Motohiro; Saeki, Hiroki

    2014-03-01

    Salmon roe has a high allergic potency and often causes anaphylaxis in Japan. The major allergic protein of salmon roe is β'-component, which is a 35kDa vitellogenin fragment consisting of two subunits. To elucidate structural information and immunological characteristics, β'-component and the subunit components were purified from chum salmon (Onchorhincus keta) roe and vitellogenin-encoding mRNA was used to prepare β'-component subunit-encoding cDNA. This was PCR-amplified, cloned and sequenced and the deduced amino acid sequence compared with partial sequences of β'-component obtained by peptide mapping. The recombinant β'-component subunit was produced by bacterial expression in Escherichia coli and its IgE-binding ability was measured by ELISA using the sera of a patient allergic to salmon roe. This was then compared with that of the native β'-component with and without carboxymethylation. Following successful cloning of the cDNA encoding the β'-component subunit, 170 amino acid residues were deduced and matched with the amino acid sequences of 121 and 88 residues in the 16kDa and 18kDa subunits, respectively. The sequences of both β'-component subunits were almost identical, and the predicted secondary structure of the β'-component showed a high content of β-pleated sheets and no α-helices. There was no difference in IgE-binding ability between the native and recombinant β'-component subunits at the same protein concentration, regardless of carboxymethylation. In conclusion, β'-component is a homodimer protein composed of two isoform subunits having the same level of IgE-binding ability and, therefore, allergenic identity.

  2. TsAg5, a Taenia solium cysticercus protein with a marginal trypsin-like activity in the diagnosis of human neurocysticercosis.

    PubMed

    Rueda, Analiz; Sifuentes, Cecilia; Gilman, Robert H; Gutiérrez, Andrés H; Piña, Ruby; Chile, Nancy; Carrasco, Sebastián; Larson, Sandra; Mayta, Holger; Verástegui, Manuela; Rodriguez, Silvia; Gutiérrez-Correa, Marcel; García, Héctor H; Sheen, Patricia; Zimic, Mirko

    2011-12-01

    Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection. We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis.

  3. PRMT5, a novel TRAIL receptor-binding protein, inhibits TRAIL-induced apoptosis via nuclear factor-kappaB activation.

    PubMed

    Tanaka, Hiroshi; Hoshikawa, Yutaka; Oh-hara, Tomoko; Koike, Sumie; Naito, Mikihiko; Noda, Tetsuo; Arai, Hiroyuki; Tsuruo, Takashi; Fujita, Naoya

    2009-04-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has selective antitumor activity. Although TNF-alpha-induced intracellular signaling pathways have been well studied, TRAIL signaling is not fully understood. Here, we identified a novel TRAIL receptor-binding protein, protein arginine methyltransferase 5 (PRMT5), as a result of proteomic screening. PRMT5 selectively interacted with death receptor 4 and death receptor 5 but not with TNF receptor 1 or Fas. PRMT5 gene silencing sensitized various cancer cells to TRAIL without affecting TRAIL resistance in nontransformed cells. PRMT5 contributed to TRAIL-induced activation of inhibitor of kappaB kinase (IKK) and nuclear factor-kappaB (NF-kappaB), leading to induction of several NF-kappaB target genes. Although IKK inhibition increased sensitivity to both TRAIL and TNF-alpha, PRMT5 knockdown potentiated TRAIL-mediated cytotoxicity alone. PRMT5 had no effect on TNF-alpha-mediated NF-kappaB signaling. These results show the selectivity of PRMT5 for TRAIL signaling. The PRMT5 small interfering RNA-mediated susceptibility to TRAIL was rescued by ectopic expression of active IKKbeta, confirming the involvement of PRMT5 in TRAIL resistance by activating the NF-kappaB pathway. Collectively, our findings suggest the therapeutic potential of PRMT5 in TRAIL-based cancer treatments

  4. The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity

    PubMed Central

    Teyssier, Catherine; Déméné, Hélène; Carvalho, João E.; Bird, Louise E.; Lebedev, Andrey; Fattori, Juliana; Schubert, Michael; Dumas, Christian; Bourguet, William; le Maire, Albane

    2016-01-01

    Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism. PMID:27812132

  5. Expression of hepatitis C virus proteins in epithelial intestinal cells in vivo

    PubMed Central

    Deforges, Séverine; Evlashev, Alexey; Perret, Magali; Sodoyer, Mireille; Pouzol, Stéphane; Scoazec, Jean-Yves; Bonnaud, Bertrand; Diaz, Olivier; Paranhos-Baccalà, Glaucia; Lotteau, Vincent; André, Patrice

    2004-01-01

    Previous work on hepatitis C virus (HCV) led to the discovery of a new form of viral particles associating viral and lipoprotein elements. These hybrid particles (LVP for lipo-viro-particles) are enriched in triglycerides and contain at least apolipoprotein B (apoB), HCV RNA and core protein. These findings suggest that LVP synthesis could occur in liver and intestine, the two main organs specialized in the production of apoB containing lipoprotein. To precise the site of LVP production, we studied the genetic diversity and phylogenetic relationship of HCV quasispecies from purified LVP, whole serum and liver biopsies from chronically infected patients. HCV quasispecies from LVP and liver differed significantly suggesting that LVP were not predominantly synthetized in the liver but that they might also originate from the intestine. We thus searched for presence of HCV in the small intestine. Paraffin embedded intestinal biopsies from ten HCV chronically infected patients and from twelwe HCV RNA negative controls (10 anti-HCV antibody negative and 2 anti-HCV antibody positive patients) were tested for HCV protein expression. HCV NS3 and NS5A proteins were stained in small intestine epithelial cells in 4 out of 10 chronically infected patients and not in controls. Cells expressing HCV proteins were apoB producing enterocytes but not mucus secreting cells. These data indicate that small intestine can be infected by HCV and identify this organ as a potential reservoir and replication site. This further emphasizes the interaction between lipoprotein metabolism and HCV, and opens new insights in hepatitis C infection and pathophysiology. PMID:15302945

  6. MYO5A — EDRN Public Portal

    Cancer.gov

    The MYO5A gene is one of three myosin V heavy-chain genes, belonging to the myosin gene superfamily. The MYO5A protein functions as a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament. MYO5A is also involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. Different isoforms, encoded by alternatively spliced transcript variants, have been reported.

  7. Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

    PubMed

    Stewart, Emerson V; Lloyd, S Julie-Ann; Burg, John S; Nwosu, Christine C; Lintner, Robert E; Daza, Riza; Russ, Carsten; Ponchner, Karen; Nusbaum, Chad; Espenshade, Peter J

    2012-01-02

    Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.

  8. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

    PubMed Central

    Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves

    2016-01-01

    ABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. PMID:27803188

  9. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment.

    PubMed

    Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves; Einav, Shirit

    2016-11-01

    Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses.

  10. Emergence of occult minority genotype 2b hepatitis C infection in an HIV-1-co-infected patient treated for genotype 5a HCV infection with 48 weeks of pegylated-interferon-alpha 2b and ribavirin.

    PubMed

    Buckton, A J; Kulasegaram, R; Ngui, S L; Fisher, M; James, R; Rangarajan, S; Teo, C G

    2007-09-01

    An HIV-1/hepatitis C virus (HCV) co-infected patient with haemophilia received a 48-week course of pegylated interferon-alpha-2b and ribavirin therapy for genotype 5a HCV infection. Virological response was achieved at week 24. At the end of treatment, HCV RNA in serum was detected and identified to belong to genotype 2b, rather than genotype 5a. A sensitive method for identifying minority HCV genotypes in pre-treatment serum showed genotype 2b HCV carriage prior to treatment. Sequencing the interferon sensitivity-determining region of the HCV NS5A gene obtained from pre-, intra- and post-treatment sera revealed emergence of quasispecies bearing R-->K and M-->A/T mutations at codons 2222 and 2223, respectively. Occult presence of minority HCV subpopulations and their acquisition of mutations following therapy can result in poor treatment outcome.

  11. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  12. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    SciTech Connect

    Kramer, J.M. . Dept. of Zoology)

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs.

  13. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  14. Protein

    MedlinePlus

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  15. Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation

    PubMed Central

    Wong, Mun-Teng

    2016-01-01

    ABSTRACT Hepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-α (hCKα) plays in HCV replication remains elusive. Here, we first showed that hCKα activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKα colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKα localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKα on the ER, and hCKα activity was required for effective trafficking of hCKα and NS5A to the ER. Coimmunoprecipitation showed that hCKα was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCKα silencing or treatment with CK37, an hCKα activity inhibitor, abolished HCV-induced MW formation. In addition, hCKα depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKα activity was not essential for the binding of NS5A to hCKα or NS5B. These findings demonstrate that hCKα forms a complex with NS5A and that hCKα activity enhances the targeting of the complex to the ER, where hCKα protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication. IMPORTANCE HCV infection reorganizes the cellular membrane to create an active viral replication site named the membranous web (MW). Here, we report that human choline kinase-α (hCKα) acts as an essential host factor for HCV RNA replication. A fraction of hCKα colocalizes with the viral replication complex (RC) on the endoplasmic reticulum

  16. NnHSP17.5, a cytosolic class II small heat shock protein gene from Nelumbo nucifera, contributes to seed germination vigor and seedling thermotolerance in transgenic Arabidopsis.

    PubMed

    Zhou, Yuliang; Chen, Huhui; Chu, Pu; Li, Yin; Tan, Bin; Ding, Yu; Tsang, Edward W T; Jiang, Liwen; Wu, Keqiang; Huang, Shangzhi

    2012-02-01

    In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.

  17. Structure based design of novel 6,5 heterobicyclic mitogen-activated protein kinase kinase (MEK) inhibitors leading to the discovery of imidazo[1,5-a] pyrazine G-479.

    PubMed

    Robarge, Kirk D; Lee, Wendy; Eigenbrot, Charles; Ultsch, Mark; Wiesmann, Christian; Heald, Robert; Price, Steve; Hewitt, Joanne; Jackson, Philip; Savy, Pascal; Burton, Brenda; Choo, Edna F; Pang, Jodie; Boggs, Jason; Yang, April; Yang, Xioaye; Baumgardner, Matthew

    2014-10-01

    Use of the tools of SBDD including crystallography led to the discovery of novel and potent 6,5 heterobicyclic MEKi's [J. Med. Chem.2012, 55, 4594]. The core change from a 5,6 heterobicycle to a 6,5 heterobicycle was driven by the desire for increased structural diversity and aided by the co-crystal structure of G-925 [J. Med. Chem.2012, 55, 4594]. The key design feature was the shift of the attachment of the five-membered heterocyclic ring towards the B ring while maintaining the key hydroxamate and anilino pharamcophoric elements in a remarkably similar position as in G-925. From modelling, changing the connection point of the five membered ring heterocycle placed the H-bond accepting nitrogen within a good distance and angle to the Ser212 [J. Med. Chem.2012, 55, 4594]. The resulting novel 6,5 benzoisothiazole MEKi G-155 exhibited improved potency versus aza-benzofurans G-925 and G-963 but was a potent inhibitor of cytochrome P450's 2C9 and 2C19. Lowering the logD by switching to the more polar imidazo[1,5-a] pyridine core significantly diminished 2C9/2C19 inhibition while retaining potency. The imidazo[1,5-a] pyridine G-868 exhibited increased potency versus the starting point for this work (aza-benzofuran G-925) leading to deprioritization of the azabenzofurans. The 6,5-imidazo[1,5-a] pyridine scaffold was further diversified by incorporating a nitrogen at the 7 position to give the imidazo[1,5-a] pyrazine scaffold. The introduction of the C7 nitrogen was driven by the desire to improve metabolic stability by blocking metabolism at the C7 and C8 positions (particularly the HLM stability). It was found that improving on G-868 (later renamed GDC-0623) required combining C7 nitrogen with a diol hydroxamate to give G-479. G-479 with polarity distributed throughout the molecule was improved over G-868 in many aspects.

  18. Eukaryotic translation initiation factor 5A (eIF5A) is essential for HIF-1α activation in hypoxia.

    PubMed

    Tariq, Mohammad; Ito, Akihiro; Ishfaq, Muhammad; Bradshaw, Elliot; Yoshida, Minoru

    2016-02-05

    The eukaryotic initiation factor 5A (eIF5A) is an essential protein involved in translation elongation and cell proliferation. eIF5A undergoes several post-translational modifications including hypusination and acetylation. Hypusination is indispensable for the function of eIF5A. On the other hand, the precise function of acetylation remains unknown, but it may render the protein inactive since hypusination blocks acetylation. Here, we report that acetylation of eIF5A increases under hypoxia. During extended hypoxic periods an increase in the level of eIF5A acetylation correlated with a decrease in HIF-1α, suggesting involvement of eIF5A activity in HIF-1α expression under hypoxia. Indeed, suppression of eIF5A by siRNA oligo-mediated knockdown or treatment with GC7, a deoxyhypusine synthase inhibitor, led to significant reduction of HIF-1α activity. Furthermore, knockdown of eIF5A or GC7 treatment reduced tumor spheroid formation with a concomitant decrease in HIF-1α expression. Our results suggest that functional, hypusinated eIF5A is necessary for HIF-1α expression during hypoxia and that eIF5A is an attractive target for cancer therapy.

  19. A nuclear protein tyrosine phosphatase TC-PTP is a potential negative regulator of the PRL-mediated signaling pathway: dephosphorylation and deactivation of signal transducer and activator of transcription 5a and 5b by TC-PTP in nucleus.

    PubMed

    Aoki, Naohito; Matsuda, Tsukasa

    2002-01-01

    In the present study we examined involvement of nuclear protein tyrosine phosphatase TC-PTP in PRL-mediated signaling. TC-PTP could dephosphorylate signal transducer and activator of transcription 5a (STAT5a) and STAT5b, but the apparent dephosphorylation activity of TC-PTP was weaker than that of cytosolic PTP1B 30 min after PRL stimulation in transfected COS-7 cells, whereas both STAT5a and STAT5b were dephosphorylated to the same extent by recombinant TC-PTP and PTP1B in vitro. Tyrosine-phosphorylated STAT5 was coimmunoprecipitated with substrate trapping mutants of TC-PTP, suggesting that STAT5 is a specific substrate of TC-PTP. These observations were further extended in mammary epithelial COMMA-1D cells stably expressing TC-PTP. A time-course study revealed that dephosphorylation of STAT5 by TC-PTP was delayed compared with that by cytosolic PTP1B due to nuclear localization of TC-PTP throughout PRL stimulation in mammary epithelial cells. Endogenous beta-casein gene expression and beta-casein gene promoter activation in COS-7 cells were largely suppressed by TC-PTP wild type as well as catalytically inactive mutants, suggesting that stable complexes formed between STAT5 and TC-PTP in the nucleus. Taken together, we conclude that TC-PTP is catalytically competent with respect to dephosphorylation and deactivation of PRL-activated STAT5 in the nucleus.

  20. eIF5A promotes translation of polyproline motifs.

    PubMed

    Gutierrez, Erik; Shin, Byung-Sik; Woolstenhulme, Christopher J; Kim, Joo-Ran; Saini, Preeti; Buskirk, Allen R; Dever, Thomas E

    2013-07-11

    Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.

  1. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  2. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  3. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  4. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  5. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  6. Identification and characterization of human LL5A gene and mouse Ll5a gene in silico.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2003-11-01

    ARCN1, KIAA0638, TREH, DDX6, BLR1, BCL9L, UPK2, DLNB13, DLNB14, RPS25, SBDN, G6PT1, HYOU1, VPS11, HMBS, H2AFX, DPAGT1, KIAA0285, MIZF, ABCG4, NOD9, PDZK2, CBL, MCAM, RNF26, C1QTNF5, MFRP, USP2, THY1, and PVRL1 genes are located within the commonly deleted region of neuroblastoma at human chromosome 11q23.3. Here, we characterized the KIAA0638 gene within the 11q23.3 region by using bioinformatics. Because human KIAA0638 gene was homologous to human LL5B gene encoding LL5beta protein, KIAA0638 was designated LL5A gene encoding LL5alpha protein. LL5A isoform 1 (FLJ00141 type) consists of exons 1-12, 14-21 and 23, while LL5A isoform 2 (KIAA0638 type) consists of exon 1-23. LL5A isoform 1 was the major transcript among LL5A isoforms generated due to alternative splicing. Nucleotide sequence of mouse Ll5a cDNA was determined by assembling CB522359 EST and 5'-truncated mKIAA0638 cDNA. Human LL5alpha isoform 2 showed 94.4 and 35.9% total-amino-acid identity with mouse Ll5alpha and human LL5beta, respectively. LL5alpha proteins consist of Forkhead associated (FHA) domain, bipartite nuclear localization signal (NLS), Chromosome segregation ATPases (SMC) domain, and Pleckstrin homology (PH) domain. LL5alpha proteins were homologous to PtdIns(3,4,5)P3 sensor protein LL5beta in the SMC and PH domains, and were also homologous to KIF1A, KIF1B, KIF13A, KIF13B, KIF14, and SNX23 proteins in the FHA domain. LL5alpha protein might be a transducer of PtdIns(3,4,5)P3 levels to the intracellular trafficking system.

  7. Wnt5a Signaling in Cancer

    PubMed Central

    Asem, Marwa S.; Buechler, Steven; Wates, Rebecca Burkhalter; Miller, Daniel L.; Stack, M. Sharon

    2016-01-01

    Wnt5a is involved in activating several non-canonical WNT signaling pathways, through binding to different members of the Frizzled- and Ror-family receptors. Wnt5a signaling is critical for regulating normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However, the aberrant activation or inhibition of Wnt5a signaling is emerging as an important event in cancer progression, exerting both oncogenic and tumor suppressive effects. Recent studies show the involvement of Wnt5a in regulating cancer cell invasion, metastasis, metabolism and inflammation. In this article, we review findings regarding the molecular mechanisms and roles of Wnt5a signaling in various cancer types, and highlight Wnt5a in ovarian cancer. PMID:27571105

  8. ISG12a Restricts Hepatitis C Virus Infection through the Ubiquitination-Dependent Degradation Pathway

    PubMed Central

    Xue, Binbin; Yang, Darong; Wang, Jingjing; Xu, Yan; Wang, Xiaohong; Qin, Yuwen; Tian, Renyun; Chen, Shengwen; Xie, Qinya; Liu, Nianli

    2016-01-01

    ABSTRACT Interferons (IFNs) restrict various kinds of viral infection via induction of hundreds of IFN-stimulated genes (ISGs), while the functions of the majority of ISGs are broadly unclear. Here, we show that a high-IFN-inducible gene, ISG12a (also known as IFI27), exhibits a nonapoptotic antiviral effect on hepatitis C virus (HCV) infection. Viral NS5A protein is targeted specifically by ISG12a, which mediates NS5A degradation via a ubiquitination-dependent proteasomal pathway. K374R mutation in NS5A domain III abrogates ISG12a-induced ubiquitination and degradation of NS5A. S-phase kinase-associated protein 2 (SKP2) is identified as an ubiquitin E3 ligase for NS5A. ISG12a functions as a crucial adaptor that promotes SKP2 to interact with and degrade viral protein. Moreover, the antiviral effect of ISG12a is dependent on the E3 ligase activity of SKP2. These findings uncover an intriguing mechanism by which ISG12a restricts viral infection and provide clues for understanding the actions of innate immunity. IMPORTANCE Upon virus invasion, IFNs induce numerous ISGs to control viral spread, while the functions of the majority of ISGs are broadly unclear. The present study shows a novel antiviral mechanism of ISGs and elucidated that ISG12a recruits an E3 ligase, SKP2, for ubiquitination and degradation of viral protein and restricts viral infection. These findings provide important insights into exploring the working principles of innate immunity. PMID:27194766

  9. Putrescine is required for the expression of eif-5a in Trichomonas vaginalis.

    PubMed

    Carvajal-Gamez, Bertha Isabel; Arroyo, Rossana; Camacho-Nuez, Minerva; Lira, Rosalia; Martínez-Benitez, Máximo; Alvarez-Sánchez, María Elizbeth

    2011-11-01

    Recently, we found that Trichomonas vaginalis contains a eukaryotic translation initiation factor 5A (TveIF-5A) with unknown function in this parasite. eIF-5A is the only cellular protein dependent of polyamines to form a hypusine residue, an unusual basic amino acid that is post-translationally formed by modification of a single specific lysine residue in an eIF-5A precursor protein. The purpose of this study was to determine the effect of a putrescine analogue, 1,4-diamino-2-butanone (DAB), on tveif-5a mRNA and TveIF-5A protein expression. TveIF-5A protein expression was reduced by inhibition of putrescine biosynthesis, and tveif-5a mRNA levels were reduced ∼90%, as shown by western blot and immunofluorescence assays. Cycloheximide treatment reduced the amount of mature TveIF-5A protein at 4h and decreased the tveif-5a transcript level at 2h, according to western blot, RT-PCR and qRT-PCR analyses. Actinomycin D treatment showed that the tveif-5a mRNA had half-life of ∼2.5h in DAB-treated parasites. The half-life of tveif-5a mRNA was ∼4.5h under exogenous putrescine conditions. These results suggest that putrescine is required for tveif-5a mRNA stability, and it is necessary for the expression, stability and maturation of TveIF-5A protein.

  10. Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses

    SciTech Connect

    Costa-Neto, Claudio M. . E-mail: claudio@fmrp.usp.br; Parreiras-e-Silva, Lucas T.; Ruller, Roberto; Oliveira, Eduardo B.; Miranda, Antonio; Oliveira, Laerte; Ward, Richard J.

    2006-09-01

    The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.

  11. Zeolite 5A Catalyzed Etherification of Diphenylmethanol

    ERIC Educational Resources Information Center

    Cooke, Jason; Henderson, Eric J.; Lightbody, Owen C.

    2009-01-01

    An experiment for the synthetic undergraduate laboratory is described in which zeolite 5A catalyzes the room temperature dehydration of diphenylmethanol, (C[subscript 6]H[subscript 5])[subscript 2]CHOH, producing 1,1,1',1'-tetraphenyldimethyl ether, (C[subscript 6]H[subscript 5])[subscript 2]CHOCH(C[subscript 6]H[subscript 5])[subscript 2]. The…

  12. Complement C5a Generation by Staphylococcal Biofilms

    PubMed Central

    Satorius, Ashley E.; Szafranski, Jacob; Pyne, Derek; Ghanesan, Mahesh; Solomon, Michael J.; Newton, Duane W.; Bortz, David M.

    2013-01-01

    Biofilms production is a central feature of nosocomial infection of catheters and other medical devices used in resuscitation and critical care. However, the very effective biofilm forming pathogen Staphylococcus epidermidis often produces a modest host inflammatory response and few of the signs and symptoms associated with more virulent pathogens. To examine the impact of bacterial biofilm formation on provocation of an innate immune response, we studied the elaboration of the major complement anaphylatoxin C5a by human serum upon contact with S. epidermidis biofilms. Wildtype S. epidermidis and mutants of sarA (a regulatory protein that promotes synthesis of the biofilm-forming polysaccharide intercellular adhesin, PIA) and icaB (responsible for post-export processing of PIA) were studied. C5a release, as a function of exposed biofilm surface area, was on the order of 1 fmol cm−2 sec−1 and was dependent on the presence of PIA. Experimental results were used to inform a physiologically-based pharmacokinetic model of C5a release by an infected central venous catheter, one of S. epidermidis' primary means of causing human disease. These simulations revealed that the magnitude of C5a release on a superior vena cava catheter completely covered with S. epidermidis would be lower than necessary to alert circulating leukocytes. Combined, the experimental and computational results are highly consistent with clinical observations in which the clinical signs of central line associated bloodstream infection are often muted in association with this important pathogen. PMID:23459111

  13. 5 A, version modifée

    PubMed Central

    Vallis, Michael; Piccinini-Vallis, Helena; Sharma, Arya M.; Freedhoff, Yoni

    2013-01-01

    Objectif Adapter le modèle 5 A pour offrir aux professionnels des soins primaires un cadre de counseling sur l'obésité. Sources des données Une recension systématique des ouvrages spécialisés a été effectuée dans MEDLINE à l'aide des expressions de recherche 5 A's (49 articles recensés, tous pertinents) et 5 A's et primary care (8 articles recensés, tous redondants). On a aussi fait une recherche dans les sites web du National Institute of Health et de l'Organisation mondiale de la Santé. Message principal L'approche 5 A (autorisation, analyse, avis, accord et aide ou ask, assess, advise, agree et assist en anglais), élaborée pour la cessation du tabagisme, peut être adaptée au counseling sur l'obésité. Demander l'autorisation de discuter du poids; ne pas porter de jugement et explorer la volonté du patient de changer. Analyser l'indice de masse corporelle, la circonférence de la taille et le stade de l'obésité; explorer les déclencheurs et les complications de l'excès de poids. Donner son avis sur les risques de l'obésité pour la santé, les bienfaits d'une modeste perte de poids, la nécessité d'une stratégie à long terme et les options de traitements. Se mettre d'accord sur des attentes, des objectifs et des changements comportementaux réalistes pour perdre du poids et sur les détails précis du plan de traitement. Assister dans l'identifcation et l'atténuation des obstacles; offrir des ressources, aider à trouver et à consulter les services appropriés et organiser un suivi périodique. Conclusion Les 5 A représentent une stratégie d'intervention comportementale fondée sur des données probantes qui a le potentiel d'améliorer le taux de réussite de la gestion du poids en soins primaires.

  14. WNT5A promotes stemness characteristics in nasopharyngeal carcinoma cells leading to metastasis and tumorigenesis.

    PubMed

    Qin, Li; Yin, Yan-Tao; Zheng, Fang-Jing; Peng, Li-Xia; Yang, Chang-Fu; Bao, Ying-Na; Liang, Ying-Ying; Li, Xin-Jian; Xiang, Yan-Qun; Sun, Rui; Li, An-Hua; Zou, Ru-Hai; Pei, Xiao-Qing; Huang, Bi-Jun; Kang, Tie-Bang; Liao, Duan-Fang; Zeng, Yi-Xin; Williams, Bart O; Qian, Chao-Nan

    2015-04-30

    Nasopharyngeal carcinoma (NPC) has the highest metastasis rate among head and neck cancers with unclear mechanism. WNT5A belongs to the WNT family of cysteine-rich secreted glycoproteins. Our previous high-throughput gene expression profiling revealed that WNT5A was up-regulated in highly metastatic cells. In the present study, we first confirmed the elevated expression of WNT5A in metastatic NPC tissues at both the mRNA and protein levels. We then found that WNT5A promoted epithelial-mesenchymal transition (EMT) in NPC cells, induced the accumulation of CD24-CD44+ cells and side population, which are believed to be cancer stem cell characteristics. Moreover, WNT5A promoted the migration and invasion of NPC cells in vitro, while in vivo treatment with recombinant WNT5A promoted lung metastasis. Knocking down WNT5A diminished NPC tumorigenesis in vivo. When elevated expression of WNT5A coincided with the elevated expression of vimentin in the primary NPC, the patients had a poorer prognosis. Among major signaling pathways, protein kinase C (PKC) signaling was activated by WNT5A in NPC cells. A positive feedback loop between WNT5A and phospho-PKC to promote EMT was also revealed. Taken together, these data suggest that WNT5A is an important molecule in promoting stem cell characteristics in NPC, leading to tumorigenesis and metastasis.

  15. RAB5A — EDRN Public Portal

    Cancer.gov

    From UniProtKB/Swiss-Prot: The small GTPases Rab are key regulators of intracellular membrane trafficking, from the formation of transport vesicles to their fusion with membranes. Rabs cycle between an inactive GDP-bound form and an active GTP-bound form that is able to recruit to membranes different sets of downstream effectors directly responsible for vesicle formation, movement, tethering and fusion. RAB5A is required for the fusion of plasma membranes and early endosomes. Contributes to the regulation of filopodia extension.

  16. Calcium and cargoes as regulators of myosin 5a activity

    SciTech Connect

    Sellers, James R. Thirumurugan, Kavitha; Sakamoto, Takeshi; Hammer, John A.; Knight, Peter J.

    2008-04-25

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.

  17. Functional identification of the stable transfection C5aR cell line Molt-4.

    PubMed

    Zhang, Chunmei; Xu, Ruonan; Wang, Jianan; Han, Gencheng; Chen, Guojiang; Wang, Renxi; Wei, Huawei; Shen, Beifen; Ma, Yuanfang; Li, Yan

    2007-12-01

    The complement C5 anaphylatoxin receptor is a member of the seven transmembrane-spanning G protein-coupled receptor superfamily that signals through Galphai and Galpha16. C5aR is mostly expressed on neutrophils, macrophages and endothelial cells. C5a and C5aR interaction plays an important role in numerous biological effects such as in vivo cytokine storm which results in inflammatory damage. Considering the limitation of collection of human peripheral blood neutrophils and their short half life, the stably transfected cell line for studying the biological effects of C5aR is needed. In this study, we transfected C5aR gene into Molt-4 cell line and examined the function of ectopic C5aR. Our results showed stable expression of the C5aR in Molt-4 cell line and their interaction with human C5a induced ERK1/2 phosphorylation, Ca++ influx. This stable transfected cell line may provide a useful tool for studying signal pathways related to C5a and C5aR interplay and antibody development specific for C5aR.

  18. WNT5A inhibits human dental papilla cell proliferation and migration

    SciTech Connect

    Peng, L.; Ye, L.; Dong, G.; Ren, L.B.; Wang, C.L.; Xu, P.; Zhou, X.D.

    2009-12-18

    WNT proteins are a large family of cysteine-rich secreted molecules that are linked to both canonical and non-canonical signal pathways, and have been implicated in oncogenesis and tissue development. Canonical WNT proteins have been proven to play critical roles in tooth development, while little is known about the role of non-canonical WNT proteins such as WNT5A. In this study, WNT5A was localized to human dental papilla tissue and human dental papilla cells (HDPCs) cultured in vitro, using immunochemistry and RT-PCR. Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of WNT5A on HDPCs. The BrdU incorporation assay, the MTT assay and flow cytometric analysis showed that over-expression of Wnt5a strongly inhibited the proliferation of HDPCs in vitro. Wound healing and transwell migration assays indicated that over-expression of WNT5A reduced migration of HDPCs. In conclusion, our results showed that WNT5A negatively regulates both proliferation and migration of HDPCs, suggesting its important role in odontogenesis via controlling the HDPCs.

  19. A Sequence of the CIS Gene Promoter Interacts Preferentially with Two Associated STAT5A Dimers: a Distinct Biochemical Difference between STAT5A and STAT5B

    PubMed Central

    Verdier, Frédérique; Rabionet, Raquel; Gouilleux, Fabrice; Beisenherz-Huss, Christian; Varlet, Paule; Muller, Odile; Mayeux, Patrick; Lacombe, Catherine; Gisselbrecht, Sylvie; Chretien, Stany

    1998-01-01

    Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5. PMID:9742102

  20. Autophagy enhances the replication of classical swine fever virus in vitro.

    PubMed

    Pei, Jingjing; Zhao, Mingqiu; Ye, Zuodong; Gou, Hongchao; Wang, Jiaying; Yi, Lin; Dong, Xiaoying; Liu, Wenjun; Luo, Yongwen; Liao, Ming; Chen, Jinding

    2014-01-01

    Autophagy plays an important role in cellular responses to pathogens. However, the impact of the autophagy machinery on classical swine fever virus (CSFV) infection is not yet confirmed. In this study, we showed that CSFV infection significantly increases the number of autophagy-like vesicles in the cytoplasm of host cells at the ultrastructural level. We also found the formation of 2 ubiquitin-like conjugation systems upon virus infection, including LC3-I/LC3-II conversion and ATG12-ATG5 conjugation, which are considered important indicators of autophagy. Meanwhile, high expression of ATG5 and BECN1 was detected in CSFV-infected cells; conversely, degradation of SQSTM1 was observed by immunoblotting, suggesting that CSFV infection triggered a complete autophagic response, most likely by the NS5A protein. Furthermore, by confocal immunofluorescence analysis, we discovered that both envelope protein E2 and nonstructural protein NS5A colocalized with LC3 and CD63 during CSFV infection. Examination by immunoelectron microscopy further confirmed the colocalization of both E2 and NS5A proteins with autophagosome-like vesicles, indicating that CSFV utilizes the membranes of these vesicles for replication. Finally, we demonstrated that alteration of cellular autophagy by autophagy regulators and shRNAs affects progeny virus production. Collectively, these findings provide strong evidence that CSFV infection needs an autophagy pathway to enhance viral replication and maturity in host cells.

  1. DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate

    PubMed Central

    Ge, Rongbin; Wang, Zongwei; Bechis, Seth K.; Otsetov, Alexander G.; Hua, Shengyu; Wu, Shulin; Wu, Chin-Lee; Tabatabaei, Shahin; Olumi, Aria F.

    2016-01-01

    5-α Reductase type 2 (SRD5A2) is a critical enzyme for prostatic development and growth. Inhibition of SRD5A2 by finasteride is used commonly for the management of urinary obstruction caused by benign prostatic hyperplasia. Contrary to common belief, we have found that expression of SRD5A2 is variable and absent in one third of benign adult prostates. In human samples, absent SRD5A2 expression is associated with hypermethylation of the SRD5A2 promoter, and in vitro SRD5A2 promoter activity is suppressed by methylation. We show that methylation of SRD5A2 is regulated by DNA methyltransferase 1, and inflammatory mediators such as tumor necrosis factor α, NF-κB, and IL-6 regulate DNA methyltransferase 1 expression and thereby affect SRD5A2 promoter methylation and gene expression. Furthermore, we show that increasing age in mice and humans is associated with increased methylation of the SRD5A2 promoter and concomitantly decreased protein expression. Artificial induction of inflammation in prostate primary epithelial cells leads to hypermethylation of the SRD5A2 promoter and silencing of SRD5A2, whereas inhibition with tumor necrosis factor α inhibitor reactivates SRD5A2 expression. Therefore, expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases. PMID:25700986

  2. Rab5a-mediated autophagy regulates the phenotype and behavior of vascular smooth muscle cells

    PubMed Central

    Tan, Jin-Yun; Jia, Luo-Qi; Shi, Wei-Hao; He, Qing; Zhu, Lei; Yu, Bo

    2016-01-01

    Rab5a, a key member of the Rab family of GTPases, was determined to be a regulator of vascular smooth muscle cell (VSMC) proliferation and migration. However, the exact regulatory mechanism remains unclear. As Rab5a has been shown to be associated with autophagy, which is essential for the conversion of VSMCs from a contractile to a synthetic phenotype in order to prevent cell death due to oxidative stress. The present study hypothesized that autophagy may be responsible for the proliferation and migration of VSMCs via the Rab5a protein. The aim of the present study was to evaluate the effect of Rab5a on autophagy in VSMCs. The human aorta vascular smooth muscle cell line, T/G HA-VSMCs, was treated with small interfering (si)RNA against Rab5a and/or platelet-derived growth factor (PDGF). Following treatment, the phenotype transition of the VSMCs was evaluated by detecting the mRNA and protien expression levels of VSMC molecular markers using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. In addition, autophagy in VSMCs was evaluated by western blotting for autophagy-associated proteins, flow cytometry of acidic vesicular organelles, punctate fluorescence of microtubule associated protein light chain 3 and transmission electron microscopy of typical scattered double-membrane vacuolar structures. Additionally, the proliferation, migration, cell cycle and apoptotic response of VSMCs were detected by sulforhodamine B assay, transwell assay and flow cytometry, respectively. The results revealed that transfection with siRNA against Rab5a led to a significant decrease in Rab5a protein expression, while the reduced expression trend of Rab5a was rescued by intervention with PDGF. Furthermore, cells transfected with siRNA against Rab5a inhibited the autophagy of VSMCs. Downregulated Rab5a inhibited the phenotype transition of VSMCs. Additionally, downregulated Rab5a led to slowed cell growth, decreased numbers of migrated

  3. Real-Time Imaging of Interactions of Neutrophils with Cryptococcus neoformans Demonstrates a Crucial Role of Complement C5a-C5aR Signaling.

    PubMed

    Sun, Donglei; Zhang, Mingshun; Liu, Gongguan; Wu, Hui; Zhu, Xiaoping; Zhou, Hong; Shi, Meiqing

    2015-10-26

    Neutrophils have been shown to efficiently kill Cryptococcus neoformans, a causative agent of meningoencephalitis. Here, using live-cell imaging, we characterize the dynamic interactions of neutrophils with C. neoformans and the underlying mechanisms in real time. Neutrophils were directly seen to chase C. neoformans cells and then rapidly internalize them. Complement C5a-C5aR signaling guided neutrophils to migrate to the yeast cells, resulting in optimal phagocytosis and subsequent killing of the organisms. The addition of recombinant complement C5a enhanced neutrophil movement but did not induce chemotaxis, suggesting that the C5a gradient is crucial. Incubation with C. neoformans resulted in enhanced activation of Erk and p38 mitogen-activated protein (MAP) kinases (MAPKs) in neutrophils. Inhibition of the p38 MAPK pathway, but not the Erk pathway, significantly impaired neutrophil migration and its subsequent killing of C. neoformans. Deficiency of CD11b or blocking of CD11b did not affect the migration of neutrophils toward C. neoformans but almost completely abolished phagocytosis and killing of the organisms by neutrophils. C5a-C5aR signaling induced enhanced surface expression of CD11b. Interestingly, the original surface expression of CD11b was essential and sufficient for neutrophils to attach to C. neoformans but was unable to mediate phagocytosis. In contrast, the enhanced surface expression of CD11b induced by C5a-C5aR signaling was essential for neutrophil phagocytosis and subsequent killing of yeast cells. Collectively, this is the first report of the dynamic interactions of neutrophils with C. neoformans, demonstrating a crucial role of C5a-C5aR signaling in neutrophil killing of C. neoformans in real time.

  4. Real-Time Imaging of Interactions of Neutrophils with Cryptococcus neoformans Demonstrates a Crucial Role of Complement C5a-C5aR Signaling

    PubMed Central

    Sun, Donglei; Zhang, Mingshun; Liu, Gongguan; Wu, Hui; Zhu, Xiaoping; Zhou, Hong

    2015-01-01

    Neutrophils have been shown to efficiently kill Cryptococcus neoformans, a causative agent of meningoencephalitis. Here, using live-cell imaging, we characterize the dynamic interactions of neutrophils with C. neoformans and the underlying mechanisms in real time. Neutrophils were directly seen to chase C. neoformans cells and then rapidly internalize them. Complement C5a-C5aR signaling guided neutrophils to migrate to the yeast cells, resulting in optimal phagocytosis and subsequent killing of the organisms. The addition of recombinant complement C5a enhanced neutrophil movement but did not induce chemotaxis, suggesting that the C5a gradient is crucial. Incubation with C. neoformans resulted in enhanced activation of Erk and p38 mitogen-activated protein (MAP) kinases (MAPKs) in neutrophils. Inhibition of the p38 MAPK pathway, but not the Erk pathway, significantly impaired neutrophil migration and its subsequent killing of C. neoformans. Deficiency of CD11b or blocking of CD11b did not affect the migration of neutrophils toward C. neoformans but almost completely abolished phagocytosis and killing of the organisms by neutrophils. C5a-C5aR signaling induced enhanced surface expression of CD11b. Interestingly, the original surface expression of CD11b was essential and sufficient for neutrophils to attach to C. neoformans but was unable to mediate phagocytosis. In contrast, the enhanced surface expression of CD11b induced by C5a-C5aR signaling was essential for neutrophil phagocytosis and subsequent killing of yeast cells. Collectively, this is the first report of the dynamic interactions of neutrophils with C. neoformans, demonstrating a crucial role of C5a-C5aR signaling in neutrophil killing of C. neoformans in real time. PMID:26502909

  5. Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice.

    PubMed

    Zhang, Jia-xiang; Zha, Wan-sheng; Ye, Liang-ping; Wang, Feng; Wang, Hui; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

    2016-02-01

    We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals.

  6. Wnt-5a/Frizzled9 Receptor Signaling through the Gαo-Gβγ Complex Regulates Dendritic Spine Formation.

    PubMed

    Ramírez, Valerie T; Ramos-Fernández, Eva; Henríquez, Juan Pablo; Lorenzo, Alfredo; Inestrosa, Nibaldo C

    2016-09-02

    Wnt ligands play crucial roles in the development and regulation of synapse structure and function. Specifically, Wnt-5a acts as a secreted growth factor that regulates dendritic spine formation in rodent hippocampal neurons, resulting in postsynaptic development that promotes the clustering of the PSD-95 (postsynaptic density protein 95). Here, we focused on the early events occurring after the interaction between Wnt-5a and its Frizzled receptor at the neuronal cell surface. Additionally, we studied the role of heterotrimeric G proteins in Wnt-5a-dependent synaptic development. We report that FZD9 (Frizzled9), a Wnt receptor related to Williams syndrome, is localized in the postsynaptic region, where it interacts with Wnt-5a. Functionally, FZD9 is required for the Wnt-5a-mediated increase in dendritic spine density. FZD9 forms a precoupled complex with Gαo under basal conditions that dissociates after Wnt-5a stimulation. Accordingly, we found that G protein inhibition abrogates the Wnt-5a-dependent pathway in hippocampal neurons. In particular, the activation of Gαo appears to be a key factor controlling the Wnt-5a-induced dendritic spine density. In addition, we found that Gβγ is required for the Wnt-5a-mediated increase in cytosolic calcium levels and spinogenesis. Our findings reveal that FZD9 and heterotrimeric G proteins regulate Wnt-5a signaling and dendritic spines in cultured hippocampal neurons.

  7. Wnt5a: a potential factor linking psoriasis to metabolic complications.

    PubMed

    Gerdes, Sascha; Laudes, Matthias; Neumann, Katrin; Baurecht, Hansjoerg; Mrowietz, Ulrich

    2014-06-01

    Psoriasis is associated with comorbidity including obesity, insulin resistance and diabetes mellitus type 2. In obesity, the protein wingless-type MMTV integration site Family, Member 5a (wnt5a) is released from adipose tissue macrophages and was shown to be of importance in the development of insulin resistance. As wnt5a was also shown to be upregulated in psoriatic skin lesions, we investigated whether wnt5a and its counterpart secreted frizzled-related protein 5 are altered in the circulation of lean and obese patients with psoriasis compared with lean and obese healthy volunteers by measuring serum concentrations of both proteins. Our results showed that wnt5a was significantly higher in lean patients with psoriasis (0.096 ng/ml; SD 0.12) compared with lean healthy controls (0.020 ng/ml; SD 0.04; P ≤ 0.01) as well as in obese patients (0.177 ng/ml; SD 0.14) compared with obese healthy controls (0.011 ng/ml; SD 0.03; P ≤ 0.001). Therefore, we suggest that in psoriasis, an increase in wnt5a may contribute to the development of metabolic comorbidity.

  8. The hypusine-containing translation factor eIF5A.

    PubMed

    Dever, Thomas E; Gutierrez, Erik; Shin, Byung-Sik

    2014-01-01

    In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A (eIF5A) and its bacterial ortholog elongation factor P (EF-P) were initially characterized based on their ability to stimulate methionyl-puromycin (Met-Pmn) synthesis, a model assay for protein synthesis; however, the function of these factors in cellular protein synthesis has been difficult to resolve. Interestingly, a conserved lysine residue in eIF5A is post-translationally modified to hypusine and the corresponding lysine residue in EF-P from at least some bacteria is modified by the addition of a β-lysine moiety. In this review, we provide a summary of recent data that have identified a novel role for the translation factor eIF5A and its hypusine modification in the elongation phase of protein synthesis and more specifically in stimulating the production of proteins containing runs of consecutive proline residues.

  9. Crystal Structure of Hypusine-Containing Translation Factor eIF5A Bound to a Rotated Eukaryotic Ribosome.

    PubMed

    Melnikov, Sergey; Mailliot, Justine; Shin, Byung-Sik; Rigger, Lukas; Yusupova, Gulnara; Micura, Ronald; Dever, Thomas E; Yusupov, Marat

    2016-09-11

    Eukaryotic translation initiation factor eIF5A promotes protein synthesis by resolving polyproline-induced ribosomal stalling. Here, we report a 3.25-Å resolution crystal structure of eIF5A bound to the yeast 80S ribosome. The structure reveals a previously unseen conformation of an eIF5A-ribosome complex and highlights a possible functional link between conformational changes of the ribosome during protein synthesis and the eIF5A-ribosome association.

  10. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development

    PubMed Central

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W.; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B.

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  11. Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

    SciTech Connect

    Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S. )

    1991-02-01

    The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.

  12. Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

    PubMed Central

    Nausch, Henrik; Mischofsky, Heike; Koslowski, Roswitha; Meyer, Udo; Broer, Inge; Huckauf, Jana

    2012-01-01

    We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T0 transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T2 generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active. PMID:23285250

  13. A Dynamic View of Hepatitis C Virus Replication Complexes▿ ‡

    PubMed Central

    Wölk, Benno; Büchele, Benjamin; Moradpour, Darius; Rice, Charles M.

    2008-01-01

    Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex (RC). Specific membrane alterations, designated membranous webs, represent predominant sites of HCV RNA replication. The principles governing HCV RC and membranous web formation are poorly understood. Here, we used replicons harboring a green fluorescent protein (GFP) insertion in nonstructural protein 5A (NS5A) to study HCV RCs in live cells. Two distinct patterns of NS5A-GFP were observed. (i) Large structures, representing membranous webs, showed restricted motility, were stable over many hours, were partitioned among daughter cells during cell division, and displayed a static internal architecture without detectable exchange of NS5A-GFP. (ii) In contrast, small structures, presumably representing small RCs, showed fast, saltatory movements over long distances. Both populations were associated with endoplasmic reticulum (ER) tubules, but only small RCs showed ER-independent, microtubule (MT)-dependent transport. We suggest that this MT-dependent transport sustains two distinct RC populations, which are both required during the HCV life cycle. PMID:18715913

  14. A neuronal C5a receptor and an associated apoptotic signal transduction pathway.

    PubMed

    Farkas, I; Baranyi, L; Takahashi, M; Fukuda, A; Liposits, Z; Yamamoto, T; Okada, H

    1998-03-15

    1. We report the first experimental evidence of a neuronal C5a receptor (nC5aR) in human cells of neuronal origin. Expression of nC5aR mRNA was demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) in TGW human neuroblastoma cells. 2. Expression of a functional C5aR was supported by the finding that C5a evoked a transient increase in the intracellular calcium level as measured by flow cytometry (FACS). 3. To analyse the function of the nC5aR, an antisense peptide fragment of the C5aR was used. Previous data showed that a C5aR fragment (a peptide termed PR226) has C5aR agonist and antagonist effects in U-937 cells depending on the concentration of the peptide. We found that a multiple antigenic peptide (MAP) form of the same peptide (termed PR226-MAP) induced rapid elevation of nuclear c-fos immunoreactivity and resulted in DNA fragmentation, a characteristic sign of apoptosis, in TGW cells. 4. Early electrophysiological events characteristic of apoptosis were also detected: intermittent calcium current pulses were recorded within 1-2 min of peptide administration. C5a pretreatment delayed the onset of this calcium influx. 5. We also demonstrated that the apoptotic pathway is linked to nC5aR via pertussis toxin-sensitive G-proteins. 6. Although the function of C5a and its receptor on neurons is unknown, these results suggest that an abnormal activation of this signal transduction pathway can result in apoptosis and, subsequently, in neurodegeneration.

  15. C5a, but not C5a-des Arg, induces upregulation of heteromer formation between complement C5a receptors C5aR and C5L2.

    PubMed

    Croker, Daniel E; Halai, Reena; Fairlie, David P; Cooper, Matthew A

    2013-01-01

    Receptors for C5a have an important role in innate immunity and inflammation where their expression and activation is tightly regulated. There are two known receptors for C5a: the C5a receptor (C5aR) and the C5a receptor like-2 (C5L2) receptor. Here we hypothesized that activation of C5aR might lead to heteromer formation with C5L2, as a downregulatory mechanism for C5aR signaling. To investigate this experimentally, bioluminescent resonance energy transfer (BRET) was implemented and supported by wide-field microscopy to analyze receptor localization in transfected HEK293 cells and human monocyte-derived macrophages (HMDM). BRET experiments indicated the presence of constitutive C5aR-C5L2 heteromers, where C5a, but not C5a-des Arg, was able to induce further heteromer formation, which was inhibited by a C5aR-specific antagonist. The data obtained suggest that C5aR-C5L2 can form heteromers in a process enhanced by C5a, but not by C5a-des Arg. There was also a significant difference in the levels of the anti-inflammatory cytokine IL-10 detected in HMDM following exposure to C5a compared with that seen for C5a-des Arg but no differences in the pro-inflammatory cytokines TNFα and IL-6. These subtle differences in C5a and C5a-des Arg induced receptor function may be of benefit in understanding the regulation of C5a in acute inflammation.

  16. Optimization, validation, and identification of two reliable antibodies for immunodetection of WNT5A.

    PubMed

    Prgomet, Z; Andersson, T; Lindberg, P

    2017-01-01

    WNT5A is a secreted, noncanonical WNT signaling protein that has been reported to promote progression of several types of cancer, including oral squamous cell carcinoma. Many WNT5A antibodies are available commercially for immunohistochemistry (IHC) and western blot analysis. Validation of the primary antibodies, however, is often neglected. We characterized antibodies for detecting WNT5A by IHC and western blot analysis. We evaluated one polyclonal and three monoclonal commercially available WNT5A antibodies. After optimization of the IHC assay, all four antibodies showed cytoplasmic WNT5A expression in tissue samples; in contrast, only one antibody detected WNT5A in western blots. A pre-absorption test with recombinant WNT5A showed that AF645 and 3A4 antibodies specifically detected WNT5A in different assays. We suggest that the monoclonal 3A4 antibody is the most appropriate for use with IHC, while the polyclonal AF645 antibody is the best for western blot analysis.

  17. Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation.

    PubMed

    Schulte, Gunnar; Bryja, Vítezslav; Rawal, Nina; Castelo-Branco, Goncalo; Sousa, Kyle M; Arenas, Ernest

    2005-03-01

    The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.

  18. Cloning, sequencing, and expression of a Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) with novel carbohydrate-binding modules, and properties of Cel5A.

    PubMed

    Yoda, Kazutoyo; Toyoda, Atsushi; Mukoyama, Yoshihiro; Nakamura, Yutaka; Minato, Hajime

    2005-10-01

    A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.

  19. Spatial regulation of cell cohesion by Wnt5a during second heart field progenitor deployment.

    PubMed

    Li, Ding; Sinha, Tanvi; Ajima, Rieko; Seo, Hwa-Seon; Yamaguchi, Terry P; Wang, Jianbo

    2016-04-01

    Wnt5a, a non-canonical Wnt ligand critical for outflow tract (OFT) morphogenesis, is expressed specifically in second heart field (SHF) progenitors in the caudal splanchnic mesoderm (SpM) near the inflow tract (IFT). Using a conditional Wnt5a gain of function (GOF) allele and Islet1-Cre, we broadly over-expressed Wnt5a throughout the SHF lineage, including the entire SpM between the IFT and OFT. Wnt5a over-expression in Wnt5a null mutants can rescue the cell polarity and actin polymerization defects as well as severe SpM shortening, but fails to rescue OFT shortening. Moreover, Wnt5a over-expression in wild-type background is able to cause OFT shortening. We find that Wnt5a over-expression does not perturb SHF cell proliferation, apoptosis or differentiation, but affects the deployment of SHF cells by causing them to accumulate into a large bulge at the rostral SpM and fail to enter the OFT. Our immunostaining analyses suggest an inverse correlation between cell cohesion and Wnt5a level in the wild-type SpM. Ectopic Wnt5a expression in the rostral SpM of Wn5a-GOF mutants diminishes the upregulation of adherens junction; whereas loss of Wnt5a in Wnt5a null mutants causes premature increase in adherens junction level in the caudal SpM. Over-expression of mouse Wnt5a in Xenopus animal cap cells also reduces C-cadherin distribution on the plasma membrane without affecting its overall protein level, suggesting that Wnt5a may play an evolutionarily conserved role in controlling the cell surface level of cadherin to modulate cell cohesion during tissue morphogenesis. Collectively, our data indicate that restricted expression of Wnt5a in the caudal SpM is essential for normal OFT morphogenesis, and uncover a novel function of spatially regulated cell cohesion by Wnt5a in driving the deployment of SHF cells from the SpM into the OFT.

  20. Wnt5a Increases Properties of Lung Cancer Stem Cells and Resistance to Cisplatin through Activation of Wnt5a/PKC Signaling Pathway

    PubMed Central

    Yang, Jiali; Zhang, Kangjian; Wu, Jing; Shi, Juan; Xue, Jing; Li, Jing; Zhu, Yongzhao; Wei, Jun

    2016-01-01

    The development of chemoresistance to cisplatin regimens causes a poor prognosis in patients with advanced NSCLC. The role of noncanonical Wnt signaling in the regulation of properties of lung cancer stem cells and chemoresistance was interrogated, by accessing capacities of cell proliferation, migration, invasion, and clonogenicity as well as the apoptosis in A549 cell lines and cisplatin-resistant A549 cells treated with Wnt5a conditional medium or protein kinase C (PKC) inhibitor GF109203X. Results showed that the noncanonical Wnt signaling ligand, Wnt5a, could promote the proliferation, migration, invasion, and colony formation in A549 lung adenocarcinoma cells and cisplatin-resistant A549/DDP cells and increase the fraction of ALDH-positive cell in A549/DDP cells. An exposure of cells to Wnt5a led to a significant reduction of A549/DDP cell apoptosis but not A549 cells. An addition of GF109203X could both strikingly increase the baseline apoptosis and resensitize the Wnt5a-inhibited cell apoptosis. Interestingly, an inhibition of Wnt/PKC signaling pathway could reduce properties of lung cancer stem cells, promote cell apoptosis, and resensitize cisplatin-resistant cells to cisplatin via a caspase/AIF-dependent pathway. These data thus suggested that the Wnt5a could promote lung cancer cell mobility and cisplatin-resistance through a Wnt/PKC signaling pathway and a blockage of this signaling may be an alternative therapeutic strategy for NSCLC patients with resistance to chemotherapies. PMID:27895670

  1. CNS-specific expression of C3a and C5a exacerbate demyelination severity in the cuprizone model.

    PubMed

    Ingersoll, Sarah A; Martin, Carol B; Barnum, Scott R; Martin, Brian K

    2010-01-01

    Demyelination in the central nervous system (CNS) is known to involve several immune effector mechanisms, including complement proteins. Local production of complement by glial cells in the brain can be both harmful and protective. To investigate the roles of C3a and C5a in demyelination and remyelination pathology we utilized the cuprizone model. Transgenic mice expressing C3a or C5a under the control of the glial fibrillary acidic protein (GFAP) promoter had exacerbated demyelination and slightly delayed remyelination in the corpus callosum compared to WT mice. C3a and C5a transgenic mice had increased cellularity in the corpus callosum due to increase activation and/or migration of microglia. Oligodendrocytes migrated to the corpus callosum in higher numbers during early remyelination events in C3a and C5a transgenic mice, thus enabling these mice to remyelinate as effectively as WT mice by the end of the 10 week study. To determine the effects of C3a and/or C5a on individual glial subsets, we created murine recombinant C3a and C5a proteins. When microglia and mixed glial cultures were stimulated with C3a and/or C5a, we observed an increase in the production of proinflammatory cytokines and chemokines. In contrast, astrocytes had decreased cytokine and chemokine production in the presence of C3a and/or C5a. We also found that the MAPK pathway proteins JNK and ERK1/2 were activated in glia upon stimulation with C3a and C5a. Overall, our findings show that although C3a and C5a production in the brain play a negative role during demyelination, these proteins may aid in remyelination.

  2. Induced Wnt5a expression perturbs embryonic outgrowth and intestinal elongation, but is well-tolerated in adult mice.

    PubMed

    Bakker, Elvira R M; Raghoebir, Lalini; Franken, Patrick F; Helvensteijn, Werner; van Gurp, Léon; Meijlink, Frits; van der Valk, Martin A; Rottier, Robbert J; Kuipers, Ernst J; van Veelen, Wendy; Smits, Ron

    2012-09-01

    Wnt5a is essential during embryonic development, as indicated by mouse Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5 dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in Wnt5a knockout mice. However, Wnt5a induction from 13.5 dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5 dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages.

  3. High prevalence of GPRC5A germline mutations in BRCA1-mutant breast cancer patients.

    PubMed

    Sokolenko, Anna P; Bulanova, Daria R; Iyevleva, Aglaya G; Aleksakhina, Svetlana N; Preobrazhenskaya, Elena V; Ivantsov, Alexandr O; Kuligina, Ekatherina Sh; Mitiushkina, Natalia V; Suspitsin, Evgeny N; Yanus, Grigoriy A; Zaitseva, Olga A; Yatsuk, Olga S; Togo, Alexandr V; Kota, Poojitha; Dixon, J Michael; Larionov, Alexey A; Kuznetsov, Sergey G; Imyanitov, Evgeny N

    2014-05-15

    In a search for new breast cancer (BC) predisposing genes, we performed a whole exome sequencing analysis using six patient samples of familial BC and identified a germline inactivating mutation c.183delG [p. Arg61fs] in an orphan G protein-coupled receptor GPRC5A. An extended case-control study revealed a tenfold enrichment for this mutation in BC patients carrying the 5382insC allele of BRCA1, the major founder mutation in the Russian population, compared to wild-type BRCA1 BC cases [6/117 (5.1%) vs. 8/1578 (0.5%), p = 0.0002]. In mammary tumors (n = 60), the mRNA expression of GPRC5A significantly correlated with that of BRCA1 (p = 0.00018). In addition, the amount of GPRC5A transcript was significantly lower in BC obtained from BRCA1 mutation carriers (n = 17) compared to noncarriers (n = 93) (p = 0.026). Accordingly, a siRNA-mediated knockdown of either BRCA1 or GPRC5A in the MDA-MB-231 human BC cell line reduced expression of GPRC5A or BRCA1, respectively. Knockdown of GPRC5A also attenuated radiation-induced BRCA1- and RAD51-containing nuclear DNA repair foci. Taken together, these data suggest that GPRC5A is a modifier of BC risk in BRCA1 mutation carriers and reveals a functional interaction of these genes.

  4. Wnt5a inhibits K(+) currents in hippocampal synapses through nitric oxide production.

    PubMed

    Parodi, Jorge; Montecinos-Oliva, Carla; Varas, Rodrigo; Alfaro, Iván E; Serrano, Felipe G; Varas-Godoy, Manuel; Muñoz, Francisco J; Cerpa, Waldo; Godoy, Juan A; Inestrosa, Nibaldo C

    2015-09-01

    Hippocampal synapses play a key role in memory and learning processes by inducing long-term potentiation and depression. Wnt signaling is essential in the development and maintenance of synapses via several mechanisms. We have previously found that Wnt5a induces the production of nitric oxide (NO), which modulates NMDA receptor expression in the postsynaptic regions of hippocampal neurons. Here, we report that Wnt5a selectively inhibits a voltage-gated K(+) current (Kv current) and increases synaptic activity in hippocampal slices. Further supporting a specific role for Wnt5a, the soluble Frizzled receptor protein (sFRP-2; a functional Wnt antagonist) fully inhibits the effects of Wnt5a. We additionally show that these responses to Wnt5a are mediated by activation of a ROR2 receptor and increased NO production because they are suppressed by the shRNA-mediated knockdown of ROR2 and by 7-nitroindazole, a specific inhibitor of neuronal NOS. Together, our results show that Wnt5a increases NO production by acting on ROR2 receptors, which in turn inhibit Kv currents. These results reveal a novel mechanism by which Wnt5a may regulate the excitability of hippocampal neurons.

  5. Activity of purified hepatitis C virus protease NS3 on peptide substrates.

    PubMed Central

    Steinkühler, C; Urbani, A; Tomei, L; Biasiol, G; Sardana, M; Bianchi, E; Pessi, A; De Francesco, R

    1996-01-01

    The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation. PMID:8794305

  6. Complement C5a receptors in the pituitary gland: expression and function.

    PubMed

    Francis, Karen; Lewis, B Mary; Monk, Peter N; Ham, Jack

    2008-12-01

    Communication between the immune and endocrine system is important for the control of inflammation that is primarily mediated through the hypothalamic-pituitary-adrenal axis. The innate immune system rapidly responds to pathogens by releasing complement proteins that include the anaphylatoxins C3a and C5a. We previously reported the existence of C3a receptors in the anterior pituitary gland and now describe the presence of C5a receptors in the gland. C5a and its less active derivative (C5adR) can bind to its own receptor and to another receptor called C5L2. Using RT-PCR and immunocytochemistry, C5a receptors and C5L2 were demonstrated in the rat anterior pituitary gland and in several rodent anterior pituitary cell lines. Western blotting analysis showed that C5a stimulated the phosphorylation of MAPK and AKT but not p38; C5adR on the other hand, had no effect on any of the signal molecules investigated. The effects of C5a and C5adR on the secretion of the inflammatory molecule, macrophage migration inhibitory factor (MIF) were investigated by ELISA. Both compounds showed a dose-dependent inhibition of MIF release, 30-40% inhibition at around 35-70 nM agonist with IC50 values of around 20 nM. C5a and C5adR also stimulated ACTH secretion (up to 25%) from AtT-20DV16 cells. These data show that functional C5a receptors (C5a and C5L2) are present in the anterior pituitary gland and they may play a role in dampening down inflammation by inhibiting the release of MIF and stimulating the release of ACTH.

  7. Daclatasvir plus Asunaprevir Treatment for Real-World HCV Genotype 1-Infected Patients in Japan

    PubMed Central

    Kanda, Tatsuo; Yasui, Shin; Nakamura, Masato; Suzuki, Eiichiro; Arai, Makoto; Haga, Yuki; Sasaki, Reina; Wu, Shuang; Nakamoto, Shingo; Imazeki, Fumio; Yokosuka, Osamu

    2016-01-01

    Background. All-oral combination of direct-acting antivirals could lead to higher sustained virologic response (SVR) in hepatitis C virus (HCV)-infected patients. In the present study, we examined the efficacy and safety of the dual oral treatment with HCV nonstructural protein (NS) 5A inhibitor daclatasvir (DCV) plus HCV NS3/4A inhibitor asunaprevir (ASV) for 24 weeks in real-world HCV genotype 1-infected Japanese individuals. Methods. After screening for HCV NS5A resistance-associated variants (RAVs) by PCR invader assay, a total of 54 Japanese patients infected with HCV genotype 1 treated with DCV plus ASV were retrospectively analyzed. SVR12 was used for evaluation of the virologic response. Results. Of the total 54 patients, 46 patients (85.2%) were treated with DCV plus ASV for 24 weeks and achieved SVR12. The other 8 patients (14.8%) discontinued this treatment before 24 weeks due to adverse events. Of these 8 patients, 5 and 3 patients did and did not achieve SVR12, respectively. Finally, 51 of 54 (94.4%) patients achieved SVR12. Conclusion. Treatment with DCV and ASV after screening for HCV NS5A RAVs by PCR invader assay is effective and safe in the treatment of real-world HCV genotype 1-infected patients in Japan. PMID:27279790

  8. Daclatasvir plus Asunaprevir Treatment for Real-World HCV Genotype 1-Infected Patients in Japan.

    PubMed

    Kanda, Tatsuo; Yasui, Shin; Nakamura, Masato; Suzuki, Eiichiro; Arai, Makoto; Haga, Yuki; Sasaki, Reina; Wu, Shuang; Nakamoto, Shingo; Imazeki, Fumio; Yokosuka, Osamu

    2016-01-01

    Background. All-oral combination of direct-acting antivirals could lead to higher sustained virologic response (SVR) in hepatitis C virus (HCV)-infected patients. In the present study, we examined the efficacy and safety of the dual oral treatment with HCV nonstructural protein (NS) 5A inhibitor daclatasvir (DCV) plus HCV NS3/4A inhibitor asunaprevir (ASV) for 24 weeks in real-world HCV genotype 1-infected Japanese individuals. Methods. After screening for HCV NS5A resistance-associated variants (RAVs) by PCR invader assay, a total of 54 Japanese patients infected with HCV genotype 1 treated with DCV plus ASV were retrospectively analyzed. SVR12 was used for evaluation of the virologic response. Results. Of the total 54 patients, 46 patients (85.2%) were treated with DCV plus ASV for 24 weeks and achieved SVR12. The other 8 patients (14.8%) discontinued this treatment before 24 weeks due to adverse events. Of these 8 patients, 5 and 3 patients did and did not achieve SVR12, respectively. Finally, 51 of 54 (94.4%) patients achieved SVR12. Conclusion. Treatment with DCV and ASV after screening for HCV NS5A RAVs by PCR invader assay is effective and safe in the treatment of real-world HCV genotype 1-infected patients in Japan.

  9. The Nuclear Death Domain Protein p84N5; a Candidate Breast Cancer Susceptibility Gene

    DTIC Science & Technology

    2005-05-01

    p84N5. Cell proliferation and apoptosis (data not shown) was examined using Guava ViaCount and Nexin assays, respectively. Plotted is the number of...apoptosis was increased expression of p84N5 is evident comparing breast cancer examined using Guava ViaCount and Nexin assays, respectively as previously...complex was shown to be intimately involved not shown) was examined using Guava ViaCount and Nexin assays, respectively. Plotted is the number of viable

  10. The Nuclear Death Domain Protein p84N5; A Candidate Breast Cancer Susceptibility Gene

    DTIC Science & Technology

    2007-05-01

    proliferation following abrogation of hTREX84. Cell proliferation and apoptosis (data not shown) were examined using Guava ViaCount and Nexin assays...oligofectamine (mock) or transfected using a control siRNA. Cell proliferation and apoptosis was examined using Guava ViaCount and Nexin assays, respectively as...following abrogation of TREX84. Cell proliferation and apoptosis (data not shown) was examined using Guava ViaCount and Nexin assays, respectively. Plotted

  11. Exendin-4 promotes pancreatic β-cell proliferation via inhibiting the expression of Wnt5a.

    PubMed

    Wu, Xinger; Liang, Weiwei; Guan, Hongyu; Liu, Juan; Liu, Liehua; Li, Hai; He, Xiaoying; Zheng, Jing; Chen, Jie; Cao, Xiaopei; Li, Yanbing

    2017-02-01

    Exendin-4, a glucagon-like peptide-1 receptor agonist, is currently regarded as an effective therapeutic strategy for type-2 diabetes. Previous studies indicated that exendin-4 promoted β cell proliferation. However, the underlying mechanisms remain largely unknown. Recently it was reported that exendin-4 promoted pancreatic β cell proliferation by regulating the expression level of Wnt4. The present study was designed to investigate whether other Wnt isoforms take part in accommodation of β-cell proliferation. We found that exendin-4 promotes the proliferation and suppresses the expression of Wnt5a in INS-1 cell line and C57Bl/6 mouse pancreatic β-cells. Further mechanistic study demonstrated that exendin-4 promoted INS-1 cell proliferation partly through down-regulating the expression of Wnt5a. Furthermore, Wnt5a could induce the activation of calmodulin-dependent protein kinase II in INS-1 cells, thereby decreasing the cellular stable β-catenin and its nuclear translocation, and finally reduce the expression of cyclin D1. In addition, we also found that both of the receptors (Frz-2 and Ror-2) mediated the effect of Wnt5a on β cell line INS-1 proliferation. Taken together, this study suggests that Wnt5a plays a critical role in exendin-4-induced β-cell proliferation, indicating that Wnt5a might be a novel regulator in counterbalance of β cell mass.

  12. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Definition of Underserved Rural Community. 5a.3 Section 5a.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL... Professions Shortage Area, (under section 332(a)(1)(A) of the Public Health Service Act) or (2)...

  13. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  14. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Definition of Underserved Rural Community. 5a.3 Section 5a.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL... Professions Shortage Area, (under section 332(a)(1)(A) of the Public Health Service Act) or (2)...

  15. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Definition of Underserved Rural Community. 5a.3 Section 5a.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL... Professions Shortage Area, (under section 332(a)(1)(A) of the Public Health Service Act) or (2)...

  16. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Definition of Underserved Rural Community. 5a.3 Section 5a.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL... Professions Shortage Area, (under section 332(a)(1)(A) of the Public Health Service Act) or (2)...

  17. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  18. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  19. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  20. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  1. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Definition of Underserved Rural Community. 5a.3 Section 5a.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL... Professions Shortage Area, (under section 332(a)(1)(A) of the Public Health Service Act) or (2)...

  2. A review of the Model 5A uranium hexafluoride cylinder

    SciTech Connect

    Dorning, R.E. II

    1989-05-23

    Both the Model 5A (Monel 400) and 5A (Monel 400) Modified five-inch cylinders have been used at the Portsmouth GDP to withdraw, store, and ship highly enriched uranium hexafluoride. As a result of a generic cracking problem with Monel 400 valve-boss material, a cylinder modification was implemented in the mid 1970s. This modification resulted in the violation of the ASME ''Code'' stamp status of the Model 5A Modified cylinder. Hydrostatic testing-to- rupture data indicated that the Model 5A Modified cylinders had ruptured strengths equivalent to that of the original Model 5A cylinders. An independent consultant reviewed the available information and confirmed that the Model 5A Modified cylinders ''will with proper maintenance continue to perform satisfactorily for many additional years of service.'' Based on the test data and consultant's review, DOE approved continued use of the 5A Modified cylinder and also requested procurement of replacement 5B cylinders be expedited. Currently, the 5A modified cylinders are in the production, storage, shipment cycle, and a sufficient number of 5B cylinders has been ordered to accommodate the projected product shipping requirements for the Navy flow. 3 tabs.

  3. Relevance of the Axis Spermidine/eIF5A for Plant Growth and Development.

    PubMed

    Belda-Palazón, Borja; Almendáriz, Carla; Martí, Esmeralda; Carbonell, Juan; Ferrando, Alejandro

    2016-01-01

    One key role of the essential polyamine spermidine in eukaryotes is to provide the 4-aminobutyl moiety group destined to the post-translational modification of a lysine in the highly conserved translation factor eIF5A. This modification is catalyzed by two sequential enzymatic steps leading to the activation of eIF5A by the conversion of one conserved lysine to the unusual amino acid hypusine. The active translation factor facilitates the sequence-specific translation of polyproline sequences that otherwise cause ribosome stalling. In spite of the well-characterized involvement of active eIF5A in the translation of proline repeat-rich proteins, its biological role has been recently elucidated only in mammals, and it is poorly described at the functional level in plants. Here we describe the alterations in plant growth and development caused by RNAi-mediated conditional genetic inactivation of the hypusination pathway in Arabidopsis thaliana by knocking-down the enzyme deoxyhypusine synthase. We have uncovered that spermidine-mediated activation of eIF5A by hypusination is involved in several aspects of plant biology such as the control of flowering time, the aerial and root architecture, and root hair growth. In addition this pathway is required for adaptation to challenging growth conditions such as high salt and high glucose medium and to elevated concentrations of the plant hormone ABA. We have also performed a bioinformatic analysis of polyproline-rich containing proteins as putative eIF5A targets to uncover their organization in clusters of protein networks to find molecular culprits for the disclosed phenotypes. This study represents a first attempt to provide a holistic view of the biological relevance of the spermidine-dependent hypusination pathway for plant growth and development.

  4. Relevance of the Axis Spermidine/eIF5A for Plant Growth and Development

    PubMed Central

    Belda-Palazón, Borja; Almendáriz, Carla; Martí, Esmeralda; Carbonell, Juan; Ferrando, Alejandro

    2016-01-01

    One key role of the essential polyamine spermidine in eukaryotes is to provide the 4-aminobutyl moiety group destined to the post-translational modification of a lysine in the highly conserved translation factor eIF5A. This modification is catalyzed by two sequential enzymatic steps leading to the activation of eIF5A by the conversion of one conserved lysine to the unusual amino acid hypusine. The active translation factor facilitates the sequence-specific translation of polyproline sequences that otherwise cause ribosome stalling. In spite of the well-characterized involvement of active eIF5A in the translation of proline repeat-rich proteins, its biological role has been recently elucidated only in mammals, and it is poorly described at the functional level in plants. Here we describe the alterations in plant growth and development caused by RNAi-mediated conditional genetic inactivation of the hypusination pathway in Arabidopsis thaliana by knocking-down the enzyme deoxyhypusine synthase. We have uncovered that spermidine-mediated activation of eIF5A by hypusination is involved in several aspects of plant biology such as the control of flowering time, the aerial and root architecture, and root hair growth. In addition this pathway is required for adaptation to challenging growth conditions such as high salt and high glucose medium and to elevated concentrations of the plant hormone ABA. We have also performed a bioinformatic analysis of polyproline-rich containing proteins as putative eIF5A targets to uncover their organization in clusters of protein networks to find molecular culprits for the disclosed phenotypes. This study represents a first attempt to provide a holistic view of the biological relevance of the spermidine-dependent hypusination pathway for plant growth and development. PMID:26973686

  5. Cloning, expression, cellular distribution, and role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in a nonmammalian species

    USGS Publications Warehouse

    Boshra, Hani; Li, Jun; Peters, Rodney; Hansen, John; Matlapudi, Anjan; Sunyer, J. Oriol

    2004-01-01

    C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.

  6. Viral Phosphodiesterases That Antagonize Double-Stranded RNA Signaling to RNase L by Degrading 2-5A

    PubMed Central

    2014-01-01

    The host interferon (IFN) antiviral response involves a myriad of diverse biochemical pathways that disrupt virus replication cycles at many different levels. As a result, viruses have acquired and evolved genes that antagonize the host antiviral proteins. IFNs inhibit viral infections in part through the 2′,5′-oligoadenylate (2-5A) synthetase (OAS)/RNase L pathway. OAS proteins are pathogen recognition receptors that exist at different basal levels in different cell types and that are IFN inducible. Upon activation by the pathogen-associated molecular pattern viral double-stranded RNA, certain OAS proteins synthesize 2-5A from ATP. 2-5A binds to the antiviral enzyme RNase L causing its dimerization and activation. Recently, disparate RNA viruses, group 2a betacoronaviruses, and group A rotaviruses, have been shown to produce proteins with 2′,5′-phosphodiesterase (PDE) activities that eliminate 2-5A thereby evading the antiviral activity of the OAS/RNase L pathway. These viral proteins are members of the eukaryotic-viral LigT-like group of 2H phosphoesterases, so named for the presence of 2 conserved catalytic histidine residues. Here, we will review the biochemistry, biology, and implications of viral and cellular 2′,5′-PDEs that degrade 2-5A. In addition, we discuss alternative viral and cellular strategies for limiting the activity of OAS/RNase L. PMID:24905202

  7. C5a alters blood-brain barrier integrity in experimental lupus.

    PubMed

    Jacob, Alexander; Hack, Bradley; Chiang, Eddie; Garcia, Joe G N; Quigg, Richard J; Alexander, Jessy J

    2010-06-01

    The blood-brain barrier (BBB) is a crucial anatomic location in the brain. Its dysfunction complicates many neurodegenerative diseases, from acute conditions, such as sepsis, to chronic diseases, such as systemic lupus erythematosus (SLE). Several studies suggest an altered BBB in lupus, but the underlying mechanism remains unknown. In the current study, we observed a definite loss of BBB integrity in MRL/MpJ-Tnfrsf6(lpr) (MRL/lpr) lupus mice by IgG infiltration into brain parenchyma. In line with this result, we examined the role of complement activation, a key event in this setting, in maintenance of BBB integrity. Complement activation generates C5a, a molecule with multiple functions. Because the expression of the C5a receptor (C5aR) is significantly increased in brain endothelial cells treated with lupus serum, the study focused on the role of C5a signaling through its G-protein-coupled receptor C5aR in brain endothelial cells, in a lupus setting. Reactive oxygen species production increased significantly in endothelial cells, in both primary cells and the bEnd3 cell line treated with lupus serum from MRL/lpr mice, compared with those treated with control serum from MRL(+/+) mice. In addition, increased permeability monitored by changes in transendothelial electrical resistance, cytoskeletal remodeling caused by actin fiber rearrangement, and increased iNOS mRNA expression were observed in bEnd3 cells. These disruptive effects were alleviated by pretreating cells with a C5a receptor antagonist (C5aRant) or a C5a antibody. Furthermore, the structural integrity of the vasculature in MRL/lpr brain was maintained by C5aR inhibition. These results demonstrate the regulation of BBB integrity by the complement system in a neuroinflammatory setting. For the first time, a novel role of C5a in the maintenance of BBB integrity is identified and the potential of C5a/C5aR blockade highlighted as a promising therapeutic strategy in SLE and other neurodegenerative diseases.

  8. Peptidyl Arginine Deiminase from Porphyromonas gingivalis Abolishes Anaphylatoxin C5a Activity*

    PubMed Central

    Bielecka, Ewa; Scavenius, Carsten; Kantyka, Tomasz; Jusko, Monika; Mizgalska, Danuta; Szmigielski, Borys; Potempa, Barbara; Enghild, Jan J.; Prossnitz, Eric R.; Blom, Anna M.; Potempa, Jan

    2014-01-01

    Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis. PMID:25324545

  9. WNT5A Inhibits Hepatocyte Proliferation and Concludes β-Catenin Signaling in Liver Regeneration

    PubMed Central

    Yang, Jing; Cusimano, Antonella; Monga, Jappmann K.; Preziosi, Morgan E.; Pullara, Filippo; Calero, Guillermo; Lang, Richard; Yamaguchi, Terry P.; Nejak-Bowen, Kari N.; Monga, Satdarshan P.

    2016-01-01

    Activation of Wnt/β-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased β-catenin–T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in β-catenin–T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses β-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process. PMID:26100214

  10. WNT5A inhibits hepatocyte proliferation and concludes β-catenin signaling in liver regeneration.

    PubMed

    Yang, Jing; Cusimano, Antonella; Monga, Jappmann K; Preziosi, Morgan E; Pullara, Filippo; Calero, Guillermo; Lang, Richard; Yamaguchi, Terry P; Nejak-Bowen, Kari N; Monga, Satdarshan P

    2015-08-01

    Activation of Wnt/β-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased β-catenin-T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in β-catenin-T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses β-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.

  11. Peptidyl arginine deiminase from Porphyromonas gingivalis abolishes anaphylatoxin C5a activity.

    PubMed

    Bielecka, Ewa; Scavenius, Carsten; Kantyka, Tomasz; Jusko, Monika; Mizgalska, Danuta; Szmigielski, Borys; Potempa, Barbara; Enghild, Jan J; Prossnitz, Eric R; Blom, Anna M; Potempa, Jan

    2014-11-21

    Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis.

  12. Epithelial defect in prostates of Stat5a-null mice.

    PubMed

    Nevalainen, M T; Ahonen, T J; Yamashita, H; Chandrashekar, V; Bartke, A; Grimley, P M; Robinson, G W; Hennighausen, L; Rui, H

    2000-07-01

    The transcription factor Stat5a critically mediates prolactin (PRL)-induced mammary gland development and lactogenesis. PRL also stimulates growth and differentiation of prostate tissue. Specifically, hyperprolactinemia gives rise to prostate hyperplasia, and prostate size is reduced in PRL-deficient mice. We therefore investigated the importance of Stat5a for prostate development and function by examining Stat5a-null mice. The absence of Stat5a in mice was associated with a distinct prostate morphology characterized by an increased prevalence of local disorganization within acinar epithelium of ventral prostates. Affected acini were typically filled with desquamated, granular epithelial cells that had become embedded in dense, coagulated secretory material. These features were reminiscent of acinar cyst formation and degeneration frequently observed in human benign prostate hyperplasia, however, cystic changes in prostate acini of Stat5a-deficient mice were not associated with increased prostate size or morphologic hallmarks of epithelial hyperplasia. Instead, immunohistochemistry of the prostate-specific secretory marker, probasin, suggested that hypersecretory function of the epithelium could underlie local congestion and cyst formation in prostates of Stat5a-null mice. Serum testosterone and PRL levels were normal in Stat5a knockout mice, but prostate PRL receptor expression was reduced as determined by immunohistochemistry. Expression levels or activation states of other PRL signal transduction proteins, including Stat5b, Stat3, Stat1, ERK1, and ERK2 were not altered. The present study offers the first evidence for a direct role of Stat5a in the maintenance of normal tissue architecture and function of the mouse prostate.

  13. An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat

    PubMed Central

    Ru, Yan-Fei; Xue, Hai-Min; Ni, Zi-Mei; Xia, Dong; Zhou, Yu-Chuan; Zhang, Yong-Lian

    2015-01-01

    Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo. Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus-mediated siRNA in the CES5A-expressing region of the rat epididymis, Ces5a-knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility. PMID:25475668

  14. Wnt5a Regulates Midbrain Dopaminergic Axon Growth and Guidance

    PubMed Central

    Blakely, Brette D.; Bye, Christopher R.; Fernando, Chathurini V.; Horne, Malcolm K.; Macheda, Maria L.; Stacker, Steven A.; Arenas, Ernest; Parish, Clare L.

    2011-01-01

    During development, precise temporal and spatial gradients are responsible for guiding axons to their appropriate targets. Within the developing ventral midbrain (VM) the cues that guide dopaminergic (DA) axons to their forebrain targets remain to be fully elucidated. Wnts are morphogens that have been identified as axon guidance molecules. Several Wnts are expressed in the VM where they regulate the birth of DA neurons. Here, we describe that a precise temporo-spatial expression of Wnt5a accompanies the development of nigrostriatal projections by VM DA neurons. In mice at E11.5, Wnt5a is expressed in the VM where it was found to promote DA neurite and axonal growth in VM primary cultures. By E14.5, when DA axons are approaching their striatal target, Wnt5a causes DA neurite retraction in primary cultures. Co-culture of VM explants with Wnt5a-overexpressing cell aggregates revealed that Wnt5a is capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a are mediated by the Frizzled receptors and by the small GTPase, Rac1 (a component of the non-canonical Wnt planar cell polarity pathway). Moreover, the effects were specific as they could be blocked by Wnt5a antibody, sFRPs and RYK-Fc. The importance of Wnt5a in DA axon morphogenesis was further verified in Wnt5a−/− mice, where fasciculation of the medial forebrain bundle (MFB) as well as the density of DA neurites in the MFB and striatal terminals were disrupted. Thus, our results identify a novel role of Wnt5a in DA axon growth and guidance. PMID:21483795

  15. Wingless-type family member 5A (Wnt-5a) stimulates synaptic differentiation and function of glutamatergic synapses

    PubMed Central

    Varela-Nallar, Lorena; Alfaro, Iván E.; Serrano, Felipe G.; Parodi, Jorge; Inestrosa, Nibaldo C.

    2010-01-01

    Growing evidence indicates that Wingless-type (Wnt) signaling plays an important role in the maturation of the central nervous system. We report here that Wingless-type family member 5A (Wnt-5a) is expressed early in development and stimulates dendrite spine morphogenesis, inducing de novo formation of spines and increasing the size of the preexisting ones in hippocampal neurons. Wnt-5a increased intracellular calcium concentration in dendritic processes and the amplitude of NMDA spontaneous miniature currents. Acute application of Wnt-5a increased the amplitude of field excitatory postsynaptic potentials (fEPSP) in hippocampal slices, an effect that was prevented by calcium-channel blockers. The physiological relevance of our findings is supported by studies showing that Wnt scavengers decreased spine density, miniature excitatory postsynaptic currents, and fEPSP amplitude. We conclude that Wnt-5a stimulates different aspects of synaptic differentiation and plasticity in the mammalian central nervous system. PMID:21084636

  16. TXNDC5, a newly discovered disulfide isomerase with a key role in cell physiology and pathology.

    PubMed

    Horna-Terrón, Elena; Pradilla-Dieste, Alberto; Sánchez-de-Diego, Cristina; Osada, Jesús

    2014-12-17

    Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum under not fully characterized conditions As a result, TXNDC5 interacts with many cell proteins, contributing to their proper folding and correct formation of disulfide bonds through its thioredoxin domains. Moreover, it can also work as an electron transfer reaction, recovering the functional isoform of other protein disulfide isomerases, replacing reduced glutathione in its role. Finally, it also acts as a cellular adapter, interacting with the N-terminal domain of adiponectin receptor. As can be inferred from all these functions, TXNDC5 plays an important role in cell physiology; therefore, dysregulation of its expression is associated with oxidative stress, cell ageing and a large range of pathologies such as arthritis, cancer, diabetes, neurodegenerative diseases, vitiligo and virus infections. Its implication in all these important diseases has made TXNDC5 a susceptible biomarker or even a potential pharmacological target.

  17. 5. A VIEW LOOKING NORTHWEST FROM THE SOUTHEAST BANK, SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. A VIEW LOOKING NORTHWEST FROM THE SOUTHEAST BANK, SHOWING THE EAST ABUTMENT, THE BRIDGE STRUCTURE AND BOTH BANKS. - Wells County Bridge No. 74, Spanning Rock Creek Ditch at County Road 400, Bluffton, Wells County, IN

  18. 5. A PHOTO IMAGE FROM THE WEST SIDEWALK LOOKING EAST ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. A PHOTO IMAGE FROM THE WEST SIDEWALK LOOKING EAST TOWARD THE ENTRY GATES AND PORTIONS OF RILEY PARK - Delphi Bridge on U.S. Route 421, Spanning Deer Creek at U.S. Route 421, Delphi, Carroll County, IN

  19. 5. A VIEW LOOKING WEST, TOWARD THE UPSTREAM SIDE OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. A VIEW LOOKING WEST, TOWARD THE UPSTREAM SIDE OF THE PIER, SHOWING THE DETERIORATED SHEARWATER EDGE, THE NORTHEAST ABUTMENT AND WING WALL. - Cement Plant Road Bridge, Spanning Leatherwood Creek on County Road 50 South, Bedford, Lawrence County, IN

  20. Operation JANGLE. Particle Studies. Projects 2.5a-1, 2.5a-2, 2.5a-3, 2. 8,

    DTIC Science & Technology

    1979-10-01

    destroy regularity of the size separation. For a similar reason, the housing packing must be kept well oiled to pre- vent leakage through the bearings. To...chemical composition and physical properties become more and more necessary as research in contamination-decontauination %easuft and inhalation ...magnification of approximately 520X was obtained usii.g a 431 B and L objective and a filer micrometer ocular. - 26 - PROJECT 2.5a-2 Th us. of oil innersion type

  1. Mechanisms Underlying the Breast Cancer Susceptibility Locus Mcs5a

    DTIC Science & Technology

    2010-07-01

    Mcs5a2) down regulates the expression of the Fbxo10 gene in the T cells and that this reduced expression is associated with reduced mammary tumor...in primary T cells is conserved between rat and human. We demonstrate that the function of the non-coding Mcs5a locus likely is repressive gene ...regulation. We present a model that begins to explain how the Fbxo10 gene could be regulated in T cells. 15. SUBJECT TERMS mammary carcinogenesis

  2. Amino acid limitation induces down-regulation of WNT5a at transcriptional level

    SciTech Connect

    Wang Zuguang; Chen Hong

    2009-01-23

    An aberrant WNT signaling contributes to the development and progression of multiple cancers. WNT5a is one of the WNT signaling molecules. This study was designed to test the hypothesis that amino acid deprivation induces changes in the WNT signaling pathway in colon cancer cells. Results showed that targets of the amino acid response pathway, ATF3 and p21, were induced in the human colon cancer cell line SW480 during amino acid limitation. There was a significant decrease in the WNT5a mRNA level following amino acid deprivation. The down-regulation of WNT5a mRNA by amino acid deprivation is not due to mRNA destabilization. There is a reduction of nuclear {beta}-catenin protein level by amino acid limitation. Under amino acid limitation, phosphorylation of ERK1/2 was increased and the blockage of ERK1/2 by the inhibitor U0126 partially restored WNT5a mRNA level. In conclusion, amino acid limitation in colon cancer cells induces phosphorylation of ERK1/2, which then down-regulates WNT5a expression.

  3. Adipocytes WNT5a mediated dedifferentiation: a possible target in pancreatic cancer microenvironment

    PubMed Central

    Zoico, Elena; Darra, Elena; Rizzatti, Vanni; Budui, Simona; Franceschetti, Guido; Mazzali, Gloria; Rossi, Andrea P; Fantin, Francesco; Menegazzi, Marta; Cinti, Saverio; Zamboni, Mauro

    2016-01-01

    A significant epidemiological association between obesity and pancreatic ductal adenocarcinoma (PDAC) has previously been described, as well as a correlation between the degree of pancreatic steatosis, PDAC risk and prognosis. The underlying mechanisms are still not completely known. After co-culture of 3T3-L1 adipocytes and MiaPaCa2 with an in vitro transwell system we observed the appearance of fibroblast-like cells, along with a decrease in number and size of remaining adipocytes. RT-PCR analyses of 3T3-L1 adipocytes in co-culture showed a decrease in gene expression of typical markers of mature adipocytes, in parallel with an increased expression of fibroblast-specific and reprogramming genes. We found an increased WNT5a gene and protein expression early in MiaPaCa2 cells in co-culture. Additionally, EMSA of c-Jun and AP1 in 3T3-L1 demonstrated an increased activation in adipocytes after co-culture. Treatment with WNT5a neutralizing antibody completely reverted the activation of c-Jun and AP1 observed in co-cultured adipocytes. Increasing doses of recombinant SFRP-5, a competitive inhibitor for WNT5a receptor, added to the co-culture medium, were able to block the dedifferentiation of adipocytes in co-culture. These data support a WNT5a-mediated dedifferentiation process with adipocytes reprogramming toward fibroblast-like cells that might profoundly influence cancer microenvironment. PMID:26958939

  4. Wnt-5a/JNK Signaling Promotes the Clustering of PSD-95 in Hippocampal Neurons*

    PubMed Central

    Farías, Ginny G.; Alfaro, Iván E.; Cerpa, Waldo; Grabowski, Catalina P.; Godoy, Juan A.; Bonansco, Christian; Inestrosa, Nibaldo C.

    2009-01-01

    During the formation of synapses, specific regions of pre- and postsynaptic cells associate to form a single functional transmission unit. In this process, synaptogenic factors are necessary to modulate pre- and postsynaptic differentiation. In mammals, different Wnt ligands operate through canonical and non-canonical Wnt pathways, and their precise functions to coordinate synapse structure and function in the mature central nervous system are still largely unknown. Here, we studied the effect of different Wnt ligands on postsynaptic organization. We found that Wnt-5a induces short term changes in the clustering of PSD-95, without affecting its total levels. Wnt-5a promotes the recruitment of PSD-95 from a diffuse dendritic cytoplasmic pool to form new PSD-95 clusters in dendritic spines. Moreover, Wnt-5a acting as a non-canonical ligand regulates PSD-95 distribution through a JNK-dependent signaling pathway, as demonstrated by using the TAT-TI-JIP peptide in mature hippocampal neurons. Finally, using adult rat hippocampal slices, we found that Wnt-5a modulates glutamatergic synaptic transmission through a postsynaptic mechanism. Our studies indicate that the Wnt-5a/JNK pathway modulates the postsynaptic region of mammalian synapse directing the clustering and distribution of the physiologically relevant scaffold protein, PSD-95. PMID:19332546

  5. Crystal structures of the bifunctional tRNA methyltransferase Trm5a

    PubMed Central

    Wang, Caiyan; Jia, Qian; Chen, Ran; Wei, Yuming; Li, Juntao; Ma, Jie; Xie, Wei

    2016-01-01

    tRNA methyltransferase Trm5 catalyses the transfer of a methyl group from S-adenosyl-L-methionine to G37 in eukaryotes and archaea. The N1-methylated guanosine is the product of the initial step of the wyosine hypermodification, which is essential for the maintenance of the reading frame during translation. As a unique member of this enzyme family, Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4-demethylwyosine at position 37 to produce isowyosine, but the mechanism for the double methylation is poorly understood. Here we report four crystal structures of PaTrm5a ranging from 1.7- to 2.3-Å, in the apo form or in complex with various SAM analogues. These structures reveal that Asp243 specifically recognises the base moiety of SAM at the active site. Interestingly, the protein in our structures all displays an extended conformation, quite different from the well-folded conformation of Trm5b from Methanocaldococcus jannaschii reported previously, despite their similar overall architectures. To rule out the possibilities of crystallisation artefacts, we conducted the fluorescence resonance energy transfer (FRET) experiments. The FRET data suggested that PaTrm5a adopts a naturally extended conformation in solution, and therefore the open conformation is a genuine state of PaTrm5a. PMID:27629654

  6. Wnt-5a increases NO and modulates NMDA receptor in rat hippocampal neurons.

    PubMed

    Muñoz, Francisco J; Godoy, Juan A; Cerpa, Waldo; Poblete, Inés M; Huidobro-Toro, Juan Pablo; Inestrosa, Nibaldo C

    2014-02-07

    Wnt signaling has a crucial role in synaptic function at the central nervous system. Here we evaluate whether Wnts affect nitric oxide (NO) generation in hippocampal neurons. We found that non-canonical Wnt-5a triggers NO production; however, Wnt-3a a canonical ligand did not exert the same effect. Co-administration of Wnt-5a with the soluble Frizzled related protein-2 (sFRP-2) a Wnt antagonist blocked the NO production. Wnt-5a activates the non-canonical Wnt/Ca(2+) signaling through a mechanism that depends on Ca(2+) release from Ryanodine-sensitive internal stores. The increase in NO levels evoked by Wnt-5a promotes the insertion of the GluN2B subunit of the NMDA receptor (NMDAR) into the neuronal cell surface. To the best of our knowledge, this is the first time that Wnt-5a signaling is related to NO production, which in turn increases NMDARs trafficking to the cell surface.

  7. Separate and distinctive roles for Wnt5a in tongue, lingual tissue and taste papilla development

    PubMed Central

    Liu, Hong-Xiang; Grosse, Ann S.; Iwatsuki, Ken; Mishina, Yuji; Gumucio, Deborah L.; Mistretta, Charlotte M.

    2012-01-01

    Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor. PMID:22024319

  8. EGF-reduced Wnt5a transcription induces epithelial-mesenchymal transition via Arf6-ERK signaling in gastric cancer cells

    PubMed Central

    Zhang, Yujie; Du, Jun; Zheng, Jianchao; Liu, Jiaojing; Xu, Rui; Shen, Tian; Zhu, Yichao; Chang, Jun; Wang, Hong; Zhang, Zhihong; Meng, Fanqing; Wang, Yan; Chen, Yongchang; Xu, Yong; Gu, Luo

    2015-01-01

    Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells. PMID:25779663

  9. PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers.

    PubMed

    Alquézar, Carolina; Esteras, Noemí; de la Encarnación, Ana; Alzualde, Ainhoa; Moreno, Fermín; López de Munain, Adolfo; Martín-Requero, Angeles

    2014-04-01

    Loss-of-function progranulin (PGRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Previously, we reported cell cycle-related alterations in lymphoblasts from FTLD-TDP patients, carrying the c.709-1G>A null PGRN mutation, suggesting aberrant cell cycle activation in affected neurons. Here we report that PGRN haploinsufficiency activates the extracellular signal-regulated protein kinases 1 and 2 pathway in a Ca(2+), protein kinase C-dependent, and pertussis toxin-sensitive manner. Addition of exogenous PGRN or conditioned medium from control cells normalized the response of PGRN-deficient lymphoblasts to serum activation. Our data indicated that noncanonical Wnt5a signaling might be overactivated by PGRN deficiency. We detected increased cellular and secreted levels of Wnt5a in PGRN-deficient lymphoblasts associated with enhanced phosphorylated calmodulin kinase II. Moreover, treatment of control cells with exogenous Wingless-type 5a (Wnt5a)-activated Ca(2+)/calmodulin kinase II (CaMKII), increased extracellular signal-regulated protein kinases 1 and 2 activity and cell proliferation up to the levels found in c.709-1G>A carrier cells. PGRN knockdown SH-SY5Y neuroblastoma cells also show enhanced Wnt5a content and signaling. Taken together, our results revealed an important role of Wnt signaling in FTLD-TDP pathology and suggest a novel target for therapeutic intervention.

  10. A de novo microdeletion of SEMA5A in a boy with autism spectrum disorder and intellectual disability

    PubMed Central

    Mosca-Boidron, Anne-Laure; Gueneau, Lucie; Huguet, Guillaume; Goldenberg, Alice; Henry, Céline; Gigot, Nadège; Pallesi-Pocachard, Emilie; Falace, Antonio; Duplomb, Laurence; Thevenon, Julien; Duffourd, Yannis; ST-Onge, Judith; Chambon, Pascal; Rivière, Jean-Baptiste; Thauvin-Robinet, Christel; Callier, Patrick; Marle, Nathalie; Payet, Muriel; Ragon, Clemence; Goubran Botros, Hany; Buratti, Julien; Calderari, Sophie; Dumas, Guillaume; Delorme, Richard; Lagarde, Nathalie; Pinoit, Jean-Michel; Rosier, Antoine; Masurel-Paulet, Alice; Cardoso, Carlos; Mugneret, Francine; Saugier-Veber, Pascale; Campion, Dominique; Faivre, Laurence; Bourgeron, Thomas

    2016-01-01

    Semaphorins are a large family of secreted and membrane-associated proteins necessary for wiring of the brain. Semaphorin 5A (SEMA5A) acts as a bifunctional guidance cue, exerting both attractive and inhibitory effects on developing axons. Previous studies have suggested that SEMA5A could be a susceptibility gene for autism spectrum disorders (ASDs). We first identified a de novo translocation t(5;22)(p15.3;q11.21) in a patient with ASD and intellectual disability (ID). At the translocation breakpoint on chromosome 5, we observed a 861-kb deletion encompassing the end of the SEMA5A gene. We delineated the breakpoint by NGS and observed that no gene was disrupted on chromosome 22. We then used Sanger sequencing to search for deleterious variants affecting SEMA5A in 142 patients with ASD. We also identified two independent heterozygous variants located in a conserved functional domain of the protein. Both variants were maternally inherited and predicted as deleterious. Our genetic screens identified the first case of a de novo SEMA5A microdeletion in a patient with ASD and ID. Although our study alone cannot formally associate SEMA5A with susceptibility to ASD, it provides additional evidence that Semaphorin dysfunction could lead to ASD and ID. Further studies on Semaphorins are warranted to better understand the role of this family of genes in susceptibility to neurodevelopmental disorders. PMID:26395558

  11. A de novo microdeletion of SEMA5A in a boy with autism spectrum disorder and intellectual disability.

    PubMed

    Mosca-Boidron, Anne-Laure; Gueneau, Lucie; Huguet, Guillaume; Goldenberg, Alice; Henry, Céline; Gigot, Nadège; Pallesi-Pocachard, Emilie; Falace, Antonio; Duplomb, Laurence; Thevenon, Julien; Duffourd, Yannis; St-Onge, Judith; Chambon, Pascal; Rivière, Jean-Baptiste; Thauvin-Robinet, Christel; Callier, Patrick; Marle, Nathalie; Payet, Muriel; Ragon, Clemence; Goubran Botros, Hany; Buratti, Julien; Calderari, Sophie; Dumas, Guillaume; Delorme, Richard; Lagarde, Nathalie; Pinoit, Jean-Michel; Rosier, Antoine; Masurel-Paulet, Alice; Cardoso, Carlos; Mugneret, Francine; Saugier-Veber, Pascale; Campion, Dominique; Faivre, Laurence; Bourgeron, Thomas

    2016-06-01

    Semaphorins are a large family of secreted and membrane-associated proteins necessary for wiring of the brain. Semaphorin 5A (SEMA5A) acts as a bifunctional guidance cue, exerting both attractive and inhibitory effects on developing axons. Previous studies have suggested that SEMA5A could be a susceptibility gene for autism spectrum disorders (ASDs). We first identified a de novo translocation t(5;22)(p15.3;q11.21) in a patient with ASD and intellectual disability (ID). At the translocation breakpoint on chromosome 5, we observed a 861-kb deletion encompassing the end of the SEMA5A gene. We delineated the breakpoint by NGS and observed that no gene was disrupted on chromosome 22. We then used Sanger sequencing to search for deleterious variants affecting SEMA5A in 142 patients with ASD. We also identified two independent heterozygous variants located in a conserved functional domain of the protein. Both variants were maternally inherited and predicted as deleterious. Our genetic screens identified the first case of a de novo SEMA5A microdeletion in a patient with ASD and ID. Although our study alone cannot formally associate SEMA5A with susceptibility to ASD, it provides additional evidence that Semaphorin dysfunction could lead to ASD and ID. Further studies on Semaphorins are warranted to better understand the role of this family of genes in susceptibility to neurodevelopmental disorders.

  12. Semaphorin 5A mediated cellular navigation: connecting nervous system and cancer

    PubMed Central

    Purohit, Abhilasha; Sadanandam, Anguraj; Myneni, Pavan; Singh, Rakesh K.

    2014-01-01

    The ultraprecise wiring of neurons banks on the instructions provided by guidance cue proteins that steer them to their appropriate target tissue during neuronal development. Semaphorins are one such family of proteins. Semaphorins are known to play major physiological roles during the development of various organs including nervous system, cardiovascular, and immune systems. Their role in different pathologies including cancer remains an intense area of investigation. This review focuses on a novel member of this family of proteins, semaphorin 5A, which is much less explored in comparison to its other affiliates. Recent reports suggest that semaphorins play important roles in the pathology of cancer by affecting angiogenesis, tumor growth and metastasis. We will firstly give a general overview of the semaphorin family and its receptors. Next, we discuss their roles in cellular movements and how that makes them a connecting link between nervous system and cancer. Finally, we focus our discussion on semaphorin 5A to summarize the prevailing knowledge for this molecule in developmental biology and carcinogenesis. PMID:25263940

  13. Semaphorin 5A mediated cellular navigation: connecting nervous system and cancer.

    PubMed

    Purohit, Abhilasha; Sadanandam, Anguraj; Myneni, Pavan; Singh, Rakesh K

    2014-12-01

    The ultraprecise wiring of neurons banks on the instructions provided by guidance cue proteins that steer them to their appropriate target tissue during neuronal development. Semaphorins are one such family of proteins. Semaphorins are known to play major physiological roles during the development of various organs including the nervous, cardiovascular, and immune systems. Their role in different pathologies including cancer remains an intense area of investigation. This review focuses on a novel member of this family of proteins, semaphorin 5A, which is much less explored in comparison to its other affiliates. Recent reports suggest that semaphorins play important roles in the pathology of cancer by affecting angiogenesis, tumor growth and metastasis. We will firstly give a general overview of the semaphorin family and its receptors. Next, we discuss their roles in cellular movements and how that makes them a connecting link between the nervous system and cancer. Finally, we focus our discussion on semaphorin 5A to summarize the prevailing knowledge for this molecule in developmental biology and carcinogenesis.

  14. Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

    PubMed Central

    Garvey, Megan; Fischer, Rainer; Commandeur, Ulrich

    2014-01-01

    Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems. PMID:24962636

  15. Historical review of C-5A lift distribution control systems

    NASA Technical Reports Server (NTRS)

    Disney, T. E.; Eckholdt, D. C.

    1976-01-01

    Analytical and experimental development work on various load alleviation systems for the C-5A is reviewed to trace the development of the technical and hardware concepts to the present time. Variations in system objectives, means of implementation and effects on loads and airplane performance, stability and control are discussed.

  16. C-5a-substituted validamine type glycosidase inhibitors.

    PubMed

    Schalli, Michael; Wolfsgruber, Andreas; Gonzalez Santana, Andres; Tysoe, Christina; Fischer, Roland; Stütz, Arnold E; Thonhofer, Martin; Withers, Stephen G

    2017-02-22

    A series of N-alkyl derivatives of the D-galactosidase inhibitor 1,4-di-epi-validamine featuring lipophilic substituents at position C-5a was prepared and screened for their glycosidase inhibitory properties. Products turned out selective for β-galactosidases as well as β-glucosidases.

  17. Wnt5a induces renal AQP2 expression by activating calcineurin signalling pathway

    PubMed Central

    Ando, Fumiaki; Sohara, Eisei; Morimoto, Tetsuji; Yui, Naofumi; Nomura, Naohiro; Kikuchi, Eriko; Takahashi, Daiei; Mori, Takayasu; Vandewalle, Alain; Rai, Tatemitsu; Sasaki, Sei; Kondo, Yoshiaki; Uchida, Shinichi

    2016-01-01

    Heritable nephrogenic diabetes insipidus (NDI) is characterized by defective urine concentration mechanisms in the kidney, which are mainly caused by loss-of-function mutations in the vasopressin type 2 receptor. For the treatment of heritable NDI, novel strategies that bypass the defective vasopressin type 2 receptor are required to activate the aquaporin-2 (AQP2) water channel. Here we show that Wnt5a regulates AQP2 protein expression, phosphorylation and trafficking, suggesting that Wnt5a is an endogenous ligand that can regulate AQP2 without the activation of the classic vasopressin/cAMP signalling pathway. Wnt5a successfully increases the apical membrane localization of AQP2 and urine osmolality in an NDI mouse model. We also demonstrate that calcineurin is a key regulator of Wnt5a-induced AQP2 activation without affecting intracellular cAMP level and PKA activity. The importance of calcineurin is further confirmed with its activator, arachidonic acid, which shows vasopressin-like effects underlining that calcineurin activators may be potential therapeutic targets for heritable NDI. PMID:27892464

  18. Cutting edge: identification of novel T cell epitopes in Lol p5a by computational prediction.

    PubMed

    de Lalla, C; Sturniolo, T; Abbruzzese, L; Hammer, J; Sidoli, A; Sinigaglia, F; Panina-Bordignon, P

    1999-08-15

    Although atopic allergy affects protein structure and immunogenic activity of the allergens is still largely unknown. We observed that group 5 grass allergens are characterized by repeated structural motifs. Using a new algorithm, TEPITOPE, we predicted promiscuous HLA-DR ligands within the repeated motifs of the Lol p5a allergen from rye grass. In vitro binding studies confirmed the promiscuous binding characteristics of these peptides. Moreover, most of the predicted ligands were novel T cell epitopes that were able to stimulate T cells from atopic patients. We generated a panel of Lol p5a-specific T cell clones, the majority of which recognized the peptides in a cross-reactive fashion. The computational prediction of DR ligands might thus allow the design of T cell epitopes with potential useful application in novel immunotherapy strategies.

  19. Expression of genes encoding the rice translation initiation factor, eIF5A, is involved in developmental and environmental responses.

    PubMed

    Chou, Wan-Chi; Huang, Ya-Wen; Tsay, Wen-Su; Chiang, Tzen-Yuh; Huang, Dinq-Ding; Huang, Hao-Jen

    2004-05-01

    Eukaryotic translation initiation factor 5 A (eIF5A) is the only cellular protein known to contain the unusual amino acid hypusine. However, the precise cellular function of eIF5A is to date unknown. In the present study, we report on the characterization of two cDNA clones encoding eIF5A in rice (Oryza sativa). Sequence analysis revealed that the two cDNAs share 93% amino acid sequence identity. Phylogenetic analysis of the eIF5A genes revealed paraphyly of OseIF5A-1 and OseIF5A-2. Analysis at the mRNA level has shown that OseIF5A-1 and OseIF5A-2 are expressed in rice leaves and panicles and high relative amounts of both genes were detected in old leaves. In addition, both OseIF5A-1 and OseIF5A-2 were spatially regulated during rice leaf development. In suspension-cultured cells, the transcripts of OseIF5A genes were strongly reduced after sugar starvation. Abiotic stresses, salt and heavy metal, induce the accumulation of OseIF5A-1 and OseIF5A-2 mRNAs in rice cells. These results suggested that both OseIF5A genes might be regulated by plant development and environmental stresses.

  20. Modulation of protein synthesis by polyamines.

    PubMed

    Igarashi, Kazuei; Kashiwagi, Keiko

    2015-03-01

    Polyamines are ubiquitous small basic molecules that play important roles in cell growth and viability. Since polyamines mainly exist as a polyamine-RNA complex, we looked for proteins whose synthesis is preferentially stimulated by polyamines at the level of translation, and thus far identified 17 proteins in Escherichia coli and 6 proteins in eukaryotes. The mechanisms of polyamine stimulation of synthesis of these proteins were investigated. In addition, the role of eIF5A, containing hypusine formed from spermidine, on protein synthesis is described. These results clearly indicate that polyamines and eIF5A contribute to cell growth and viability through modulation of protein synthesis.

  1. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  2. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  3. GPRC5A is a potential oncogene in pancreatic ductal adenocarcinoma cells that is upregulated by gemcitabine with help from HuR

    PubMed Central

    Zhou, H; Telonis, A G; Jing, Y; Xia, N L; Biederman, L; Jimbo, M; Blanco, F; Londin, E; Brody, J R; Rigoutsos, I

    2016-01-01

    GPRC5A is an orphan G-protein coupled receptor with an intriguing dual behavior, acting as an oncogene in some cancers and as a tumor suppressor in other cancers. In the pancreatic cancer context, very little is known about GPRC5A. By analyzing messenger RNA (mRNA) expression data from 675 human cancer cell lines and 10 609 samples from The Cancer Genome Atlas (TCGA) we found that GPRC5A's abundance in pancreatic cancer is highest (cell lines) or second highest (TCGA) among all tissues and cancer types. Further analyses of an independent set of 252 pancreatic normal and cancer samples showed GPRC5A mRNA to be more than twofold upregulated in primary tumor samples compared with normal pancreas (P-value<10−5), and even further upregulated in pancreatic cancer metastases to various organs (P-value=0.0021). Immunostaining of 208 cores (103 samples) of a tissue microarray showed generally low expression of GPRC5A protein in normal pancreatic ductal cells; on the other hand, in primary and metastatic samples, GPRC5A protein levels were dramatically increased in pancreatic ductal cells. In vitro studies of multiple pancreatic cancer cell lines showed that an increase in GPRC5A protein levels promoted pancreatic cancer cell growth and migration. Unexpectedly, when we treated pancreatic cancer cell lines with gemcitabine (2′,2′-difluorodeoxycytidine), we observed an increase in GPRC5A protein abundance. On the other hand, when we knocked down GPRC5A we sensitized pancreatic cancer cells to gemcitabine. Through further experimentation we showed that the monotonic increase in GPRC5A protein levels that we observe for the first 18 h following gemcitabine treatment results from interactions between GPRC5A's mRNA and the RNA-binding protein HuR, which is an established key mediator of gemcitabine's efficacy in cancer cells. As we discovered, the interaction between GPRC5A and HuR is mediated by at least one HuR-binding site in GPRC5A's mRNA. Our findings indicate that

  4. Mag-5: a magnificent approach to upper and midfacial "magic".

    PubMed

    Flowers, Robert S; Ceydeli, Adil

    2008-10-01

    MAG-5 is an operation with five surgical components: (1) a lateral emphasis frontal lift with (2) corrugator resection, (3) lower blepharoplasty, (4) extended two-layered canthopexy, and (5) a full (or partial) subperiosteal malar release and midcheek lift assisted by an absorbable suture securing the malar tuft periosteum and fibrous tissue to the orbital rim. The procedure rejuvenates and restores the face or transforms it when, and if, transformation is the desire.

  5. Rab5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial cells.

    PubMed Central

    Bucci, C; Wandinger-Ness, A; Lütcke, A; Chiariello, M; Bruni, C B; Zerial, M

    1994-01-01

    In nonpolarized cells, the small GTPase Rab5a is localized to the plasma membrane, clathrin-coated vesicles, and early endosomes. Rab5a is required for early endosome fusion in vitro and regulates transport between the plasma membrane and early endosomes, in vivo. In polarized epithelial cells endocytosis occurs from separate apical and basolateral plasma membrane domains. Internalized molecules are initially delivered to distinct apical or basolateral early endosomes. In vitro, apical early endosomes can readily fuse with one another but not with the basolateral endosomes and vice versa, thereby indicating that the apical and basolateral early endocytic pathways are controlled by distinct machineries. Here, we have investigated the localization and function of Rab5a in polarized epithelial cells. Confocal immunofluorescence microscopy on mouse kidney sections revealed association of the protein with the apical and basolateral plasma membrane domains and underlying structures. In polarized Madin-Darby canine kidney I cells, endogenous and overexpressed Rab5a have the same distribution. Moreover, overexpression of the protein causes a 2-fold increase in fluid-phase uptake from both domains of the cell, thus showing that Rab5a functions in apical and basolateral endocytosis. Our data indicate that the apical and basolateral endocytic machineries of epithelial cells share common regulatory components and that Rab5a per se is not sufficient to target endocytic vesicles to apical or basolateral early endosomes. Images PMID:8197185

  6. Drusen complement components C3a and C5a promote choroidal neovascularization

    PubMed Central

    Nozaki, Miho; Raisler, Brian J.; Sakurai, Eiji; Sarma, J. Vidya; Barnum, Scott R.; Lambris, John D.; Chen, Yali; Zhang, Kang; Ambati, Balamurali K.; Baffi, Judit Z.; Ambati, Jayakrishna

    2006-01-01

    Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30–50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD. PMID:16452172

  7. NLRC5: a newly discovered MHC class I transactivator (CITA).

    PubMed

    Meissner, Torsten B; Li, Amy; Kobayashi, Koichi S

    2012-06-01

    Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator), is a master regulator of MHC class II gene expression as well as of some of the genes involved in MHC class II antigen presentation. It has recently been discovered that another member of the NLR protein family, NLRC5, transcriptionally activates MHC class I genes, and thus acts as "CITA" (MHC class I transactivator), a counterpart to CIITA. In addition to MHC class I genes, NLRC5 can induce the expression of β2M, TAP1 and LMP2, essential components of MHC class I antigen presentation. These findings indicate that NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and MHC class II pathways, respectively.

  8. The NPG 7120.5A Electronic Review Process

    NASA Technical Reports Server (NTRS)

    McBrayer, Robert; Ives, Mark

    1998-01-01

    The use of electronics to review a document is well within the technical realm of today's state-of-the-art workplace. File servers and web site interaction are common tools for many NASA employees. The electronic comment processing described here was developed for the NPG 7120.5A review to augment the existing NASA Online Directives Information System (NODIS). The NODIS system is NASA's official system for formal review, approval and storage of NASA Directives. The electronic review process worked so well that NASA and other agencies may want to consider it as one of our "best practices." It was participatory decision making at its very best, a process that attracted dozens of very good ideas to improve the document as well as the way we can be managing projects far more effectively. The revision of NPG 7120.5A has significant implications for the way all elements of the Agency accomplish program and project management. Therefore, the review of NPG 7120.5A was an Agencywide effort with high visibility, heavy participation and a short schedule. The level of involvement created interest in supplementing the formal NODIS system with a system to collect comments efficiently and to allow the Centers and Codes to review and consolidate their comments into the official system in a short period of time. In addition, the Program Management Council Working Group (PMCWG), responsible for the revision of the document and the disposition of official comments, needed an electronic system to manage the disposition of comments, obtain PMCWG consensus on each disposition, and coordinate the disposition with the appropriate Headquarters Code that had submitted the official comment. The combined NASA and contractor talents and resources provided a system that supplemented the NODIS system and its operating personnel to produce a thorough review and approval of NPG 7120.5A on April 3, 1998, 7.5 months from the start of the process. The original six-month schedule is indicated. All

  9. Increased mitochondrial function downstream from KDM5A histone demethylase rescues differentiation in pRB-deficient cells

    PubMed Central

    Váraljai, Renáta; Islam, Abul B.M.M.K.; Beshiri, Michael L.; Rehman, Jalees; Lopez-Bigas, Nuria; Benevolenskaya, Elizaveta V.

    2015-01-01

    The retinoblastoma tumor suppressor protein pRb restricts cell growth through inhibition of cell cycle progression. Increasing evidence suggests that pRb also promotes differentiation, but the mechanisms are poorly understood, and the key question remains as to how differentiation in tumor cells can be enhanced in order to diminish their aggressive potential. Previously, we identified the histone demethylase KDM5A (lysine [K]-specific demethylase 5A), which demethylates histone H3 on Lys4 (H3K4), as a pRB-interacting protein counteracting pRB's role in promoting differentiation. Here we show that loss of Kdm5a restores differentiation through increasing mitochondrial respiration. This metabolic effect is both necessary and sufficient to induce the expression of a network of cell type-specific signaling and structural genes. Importantly, the regulatory functions of pRB in the cell cycle and differentiation are distinct because although restoring differentiation requires intact mitochondrial function, it does not necessitate cell cycle exit. Cells lacking Rb1 exhibit defective mitochondria and decreased oxygen consumption. Kdm5a is a direct repressor of metabolic regulatory genes, thus explaining the compensatory role of Kdm5a deletion in restoring mitochondrial function and differentiation. Significantly, activation of mitochondrial function by the mitochondrial biogenesis regulator Pgc-1α (peroxisome proliferator-activated receptor γ-coactivator 1α; also called PPARGC1A) a coactivator of the Kdm5a target genes, is sufficient to override the differentiation block. Overexpression of Pgc-1α, like KDM5A deletion, inhibits cell growth in RB-negative human cancer cell lines. The rescue of differentiation by loss of KDM5A or by activation of mitochondrial biogenesis reveals the switch to oxidative phosphorylation as an essential step in restoring differentiation and a less aggressive cancer phenotype. PMID:26314709

  10. The role of mutations in the SCN5A gene in cardiomyopathies.

    PubMed

    Zaklyazminskaya, Elena; Dzemeshkevich, Sergei

    2016-07-01

    The SCN5A gene encodes the alpha-subunit of the Nav1.5 ion channel protein, which is responsible for the sodium inward current (INa). Since 1995 several hundred mutations in this gene have been found to be causative for inherited arrhythmias including Long QT syndrome, Brugada syndrome, cardiac conduction disease, sudden infant death syndrome, etc. As expected these syndromes are primarily electrical heart diseases leading to life-threatening arrhythmias with an "apparently normal heart". In 2003 a new form of dilated cardiomyopathy was identified associated with mutations in the SCN5A gene. Recently mutations have been also found in patients with arrhythmogenic right ventricular cardiomyopathy and atrial standstill. The purpose of this review is to outline and analyze the following four topics: 1) SCN5A genetic variants linked to different cardiomyopathies; 2) clinical manifestations of the known mutations; 3) possible molecular mechanisms of myocardial remodeling; and 4) the potential implications of gene-specific treatment for those disorders. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  11. Wnt-5a occludes Aβ oligomer-induced depression of glutamatergic transmission in hippocampal neurons

    PubMed Central

    2010-01-01

    Background Soluble amyloid-β (Aβ;) oligomers have been recognized to be early and key intermediates in Alzheimer's disease (AD)-related synaptic dysfunction. Aβ oligomers block hippocampal long-term potentiation (LTP) and impair rodent spatial memory. Wnt signaling plays an important role in neural development, including synaptic differentiation. Results We report here that the Wnt signaling activation prevents the synaptic damage triggered by Aβ oligomers. Electrophysiological analysis of Schaffer collaterals-CA1 glutamatergic synaptic transmission in hippocampal slices indicates that Wnt-5a increases the amplitude of field excitatory postsynaptic potentials (fEPSP) and both AMPA and NMDA components of the excitatory postsynaptic currents (EPSCs), without modifying the paired pulse facilitation (PPF). Conversely, in the presence of Aβ oligomers the fEPSP and EPSCs amplitude decreased without modification of the PPF, while the postsynaptic scaffold protein (PSD-95) decreased as well. Co-perfusion of hippocampal slices with Wnt-5a and Aβ oligomers occludes against the synaptic depression of EPSCs as well as the reduction of PSD-95 clusters induced by Aβ oligomers in neuronal cultures. Taken together these results indicate that Wnt-5a and Aβ oligomers inversely modulate postsynaptic components. Conclusion These results indicate that post-synaptic damage induced by Aβ oligomers in hippocampal neurons is prevented by non-canonical Wnt pathway activation. PMID:20205789

  12. Insight into the molecular switch mechanism of human Rab5a from molecular dynamics simulations

    SciTech Connect

    Wang, Jing-Fang; Chou, Kuo-Chen

    2009-12-18

    Rab5a is currently a most interesting target because it is responsible for regulating the early endosome fusion in endocytosis and possibly the budding process. We utilized longtime-scale molecular dynamics simulations to investigate the internal motion of the wild-type Rab5a and its A30P mutant. It was observed that, after binding with GTP, the global flexibility of the two proteins is increasing, while the local flexibility in their sensitive sites (P-loop, switch I and II regions) is decreasing. Also, the mutation of Ala30 to Pro30 can cause notable flexibility variations in the sensitive sites. However, this kind of variations is dramatically reduced after binding with GTP. Such a remarkable feature is mainly caused by the water network rearrangements in the sensitive sites. These findings might be of use for revealing the profound mechanism of the displacements of Rab5a switch regions, as well as the mechanism of the GDP dissociation and GTP association.

  13. Peroxisome proliferator-activated receptor gamma regulates expression of signal transducer and activator of transcription 5A

    SciTech Connect

    Olsen, Hanne; Haldosen, Lars-Arne . E-mail: Lars-Arne.Haldosen@mednut.ki.se

    2006-05-01

    Signal transducer and activator of transcription 5A (STAT5A) has been shown to be important for terminal differentiation of mammary epithelial cells. In order to understand regulation of expression of STAT5A, the 5' end of the mouse Stat5a gene was isolated. Putative regulatory elements was searched for and several peroxisome proliferator response elements (PPREs) were found, one with high (12/13 nucleotides) and three with less (8-10/13) similarity to the reported consensus sequence. Mouse mammary epithelial HC11 cells were treated with peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligand, the thiazolidinedione (TZD) troglitazone, and an increase in STAT5A protein expression was seen. The 5' flank of Stat5a gene was cloned in a luciferase reporter vector. A concentration dependent activation of the STAT5A-luciferase reporter was detected, when transiently transfected HC11 cells were treated with TZD. The activation could be inhibited by treatment with a PPAR{gamma} antagonist. It has earlier been shown that epidermal growth factor (EGF) induces MAPK phosphorylation of PPAR{gamma} resulting in a less transcriptionally active receptor. In HC11 cells, EGF inhibited TZD induced STAT5A-reporter activity suggesting that our previously reported EGF-mediated suppression of STAT5A expression is mediated in all or partly through inhibition of PPAR{gamma} activity. Furthermore, the MEK inhibitor PD98059 inhibited the EGF effect. All together, data presented suggest that PPAR{gamma} participates in regulation of STAT5A expression.

  14. Identification of two novel KIF5A mutations in hereditary spastic paraplegia associated with mild peripheral neuropathy.

    PubMed

    López, Eva; Casasnovas, Carlos; Giménez, Javier; Santamaría, Raúl; Terrazas, Jesús M; Volpini, Víctor

    2015-11-15

    Spastic paraplegia type 10 (SPG10) is a rare form of autosomal dominant hereditary spastic paraplegia (AD-HSP) due to mutations in KIF5A, a gene encoding the neuronal kinesin heavy-chain involved in axonal transport. KIF5A mutations have been associated with a wide clinical spectrum, ranging from pure HSP to isolated peripheral nerve involvement or complicated HSP phenotypes. Most KIF5A mutations are clustered in the motor domain of the protein that is necessary for microtubule interaction. Here we describe two Spanish families with an adult onset complicated AD-HSP in which neurological studies revealed a mild sensory neuropathy. Intention tremor was also present in both families. Molecular genetic analysis identified two novel mutations c.773 C>T and c.833 C>T in the KIF5A gene resulting in the P258L and P278L substitutions respectively. Both were located in the highly conserved kinesin motor domain of the protein which has previously been identified as a hot spot for KIF5A mutations. This study adds to the evidence associating the known occurrence of mild peripheral neuropathy in the adult onset SPG10 type of AD-HSP.

  15. Serotonin (5-HT) receptor 5A sequence variants affect human plasma triglyceride levels

    PubMed Central

    Zhang, Y.; Smith, E. M.; Baye, T. M.; Eckert, J. V.; Abraham, L. J.; Moses, E. K.; Kissebah, A. H.; Martin, L. J.

    2010-01-01

    Neurotransmitters such as serotonin (5-hydroxytryptamine, 5-HT) work closely with leptin and insulin to fine-tune the metabolic and neuroendocrine responses to dietary intake. Losing the sensitivity to excess food intake can lead to obesity, diabetes, and a multitude of behavioral disorders. It is largely unclear how different serotonin receptor subtypes respond to and integrate metabolic signals and which genetic variations in these receptor genes lead to individual differences in susceptibility to metabolic disorders. In an obese cohort of families of Northern European descent (n = 2,209), the serotonin type 5A receptor gene, HTR5A, was identified as a prominent factor affecting plasma levels of triglycerides (TG), supported by our data from both genome-wide linkage and targeted association analyses using 28 publicly available and 12 newly discovered single nucleotide polymorphisms (SNPs), of which 3 were strongly associated with plasma TG levels (P < 0.00125). Bayesian quantitative trait nucleotide (BQTN) analysis identified a putative causal promoter SNP (rs3734967) with substantial posterior probability (P = 0.59). Functional analysis of rs3734967 by electrophoretic mobility shift assay (EMSA) showed distinct binding patterns of the two alleles of this SNP with nuclear proteins from glioma cell lines. In conclusion, sequence variants in HTR5A are strongly associated with high plasma levels of TG in a Northern European population, suggesting a novel role of the serotonin receptor system in humans. This suggests a potential brain-specific regulation of plasma TG levels, possibly by alteration of the expression of HTR5A. PMID:20388841

  16. Editing CCR5: a novel approach to HIV gene therapy.

    PubMed

    Cornu, Tatjana I; Mussolino, Claudio; Bloom, Kristie; Cathomen, Toni

    2015-01-01

    Acquired immunodeficiency syndrome (AIDS) is a life-threatening disorder caused by infection of individuals with the human immunodeficiency virus (HIV). Entry of HIV-1 into target cells depends on the presence of two surface proteins on the cell membrane: CD4, which serves as the main receptor, and either CCR5 or CXCR4 as a co-receptor. A limited number of people harbor a genomic 32-bp deletion in the CCR5 gene (CCR5∆32), leading to expression of a truncated gene product that provides resistance to HIV-1 infection in individuals homozygous for this mutation. Moreover, allogeneic hematopoietic stem cell (HSC) transplantation with CCR5∆32 donor cells seems to confer HIV-1 resistance to the recipient as well. However, since Δ32 donors are scarce and allogeneic HSC transplantation is not exempt from risks, the development of gene editing tools to knockout CCR5 in the genome of autologous cells is highly warranted. Targeted gene editing can be accomplished with designer nucleases, which essentially are engineered restriction enzymes that can be designed to cleave DNA at specific sites. During repair of these breaks, the cellular repair pathway often introduces small mutations at the break site, which makes it possible to disrupt the ability of the targeted locus to express a functional protein, in this case CCR5. Here, we review the current promise and limitations of CCR5 gene editing with engineered nucleases, including factors affecting the efficiency of gene disruption and potential off-target effects.

  17. Evaluation of Recharge Potential at Crater U5a (WISHBONE)

    SciTech Connect

    Richard H. French; Samuel L. Hokett

    1998-11-01

    Radionuclides are present both below and above the water table at the Nevada Test Site (NTS), as the result of underground nuclear testing. Mobilization and transport of radionuclides from the vadose zone is a complex process that is influenced by the solubility and sorption characteristics of the individual radionuclides, as well as the soil water flux. On the NTS, subsidence craters resulting from testing underground nuclear weapons are numerous, and many intercept surface water flows. Because craters collect surface water above the sub-surface point of device detonation, these craters may provide a mechanism for surface water to recharge the groundwater aquifer system underlying the NTS. Given this situation, there is a potential for the captured water to introduce contaminants into the groundwater system. Crater U5a (WISHBONE), located in Frenchman Flat, was selected for study because of its potentially large drainage area, and significant erosional features, which suggested that it has captured more runoff than other craters in the Frenchman Flat area. Recharge conditions were studied in subsidence crater U5a by first drilling boreholes and analyzing the collected soil cores to determine the soil properties and moisture conditions. This information, coupled with a 32-year precipitation record, was used to conduct surface and vaodse zone modeling. Surface water modeling predicted that approximately 13 ponding events had occurred during the life of the crater. Vadose zone modeling indicated that since the crater's formation approximately 5,900 m3 of water were captured by the crater. Of this total, approximately 5,200 m3 of potential recahrge may have occurred, and the best estimates of annual average potential recharge rates lie between 36 and 188 cm of water per year. The term potential is used here to indicate that the water is not technically recharged because it has not yet reached the water table.

  18. Silencing of Wnt5a prevents interleukin-1β-induced collagen type II degradation in rat chondrocytes

    PubMed Central

    Shi, Shiping; Man, Zhentao; Li, Wei; Sun, Shui; Zhang, Wei

    2016-01-01

    Osteoarthritis (OA) is a joint disease, and few treatments to date have been able to delay OA progression. The degradation of collagen type II (COL2) in the cartilage matrix is an important initiating factor for OA progression; the upregulation of Wnt5a protein activates COL2 degradation. In the present study, small interfering RNA of Wnt-5a was delivered by a lentiviral vector (LV-Wnt5a-RNAi) to silence Wnt-5a mRNA and prevent COL2 degradation. To determine the function of LV-Wnt5a-RNAi, the OA chondrocyte model (OA-like chondrocytes) were constructed using interleukin (IL)-1β. Detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Wnt-5a mRNA in the OA-like chondrocytes were upregulated in a time-dependent manner, indicating that OA-like chondrocytes were successfully constructed. The bioactivity of OA-like chondrocytes was determined using Live-Dead staining, and the result illustrated that the OA-like chondrocytes stimulated with IL-1β for 6 h remained viable, and these were used in Wnt5a silencing. The OA-like chondrocytes were divided into three groups: Group I, cultivated with common medium; group II, cultivated with common medium supplemented with empty lentiviral vector; group III, cultivated with common medium supplemented with LV-Wnt5a-RNAi. The efficiency of LV-Wnt5a-RNAi transfection was determined using fluorescence microscopy, the result of which indicated that LV-Wnt5a-RNAi could efficiently be transfected into the OA-like chondrocytes. The LV-Wnt5a-RNAi efficiency for the Wnt5a mRNA silencing was determined using RT-qPCR. The result illustrated that the mRNA of Wnt5a in group III was significantly lower in group I compared with that in group II (P<0.05), indicating that the LV-Wnt5a-RNAi could successfully silence Wnt5a mRNA. To further verify whether the silencing of Wnt5a mRNA could prevent COL2 degradation, western blotting and immunohistochemical analyses were performed. The results demonstrated that COL2 in

  19. Syntrophin mutation associated with long QT syndrome through activation of the nNOS-SCN5A macromolecular complex.

    PubMed

    Ueda, Kazuo; Valdivia, Carmen; Medeiros-Domingo, Argelia; Tester, David J; Vatta, Matteo; Farrugia, Gianrico; Ackerman, Michael J; Makielski, Jonathan C

    2008-07-08

    Mutations in 11 genes that encode ion channels or their associated proteins cause inherited long QT syndrome (LQTS) and account for approximately 75-80% of cases (LQT1-11). Direct sequencing of SNTA1, the gene encoding alpha1-syntrophin, was performed in a cohort of LQTS patients that were negative for mutations in the 11 known LQTS-susceptibility genes. A missense mutation (A390V-SNTA1) was found in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms). SNTA1 links neuronal nitric oxide synthase (nNOS) to the nNOS inhibitor plasma membrane Ca-ATPase subtype 4b (PMCA4b); SNTA1 also is known to associate with the cardiac sodium channel SCN5A. By using a GST-fusion protein of the C terminus of SCN5A, we showed that WT-SNTA1 interacted with SCN5A, nNOS, and PMCA4b. In contrast, A390V-SNTA1 selectively disrupted association of PMCA4b with this complex and increased direct nitrosylation of SCN5A. A390V-SNTA1 expressed with SCN5A, nNOS, and PMCA4b in heterologous cells increased peak and late sodium current compared with WT-SNTA1, and the increase was partially inhibited by NOS blockers. Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current. We conclude that the A390V mutation disrupted binding with PMCA4b, released inhibition of nNOS, caused S-nitrosylation of SCN5A, and was associated with increased late sodium current, which is the characteristic biophysical dysfunction for sodium-channel-mediated LQTS (LQT3). These results establish an SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current and suggest that SNTA1 be considered a rare LQTS-susceptibility gene.

  20. NDR proteins

    PubMed Central

    Jones, Alan M

    2010-01-01

    N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

  1. CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo.

    PubMed

    Teng, Ooiean; Chen, Szu-Ting; Hsu, Tsui-Ling; Sia, Sin Fun; Cole, Suzanne; Valkenburg, Sophie A; Hsu, Tzu-Yun; Zheng, Jian Teddy; Tu, Wenwei; Bruzzone, Roberto; Peiris, Joseph Sriyal Malik; Hsieh, Shie-Liang; Yen, Hui-Ling

    2017-01-01

    Human infections with influenza viruses exhibit mild to severe clinical outcomes as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin domain family 5 member A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of proinflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced levels of tumor necrosis factor alpha (TNF-α) and IP-10 but elevated alpha interferon (IFN-α) compared to those of wild-type mice. The heightened type I IFN response in the macrophages of CLEC5A-deficient mice was associated with upregulated TLR3 mRNA after treatment with double-stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of proinflammatory cytokines, decreased immune cell infiltration in the lungs, and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections.

  2. On Hepatitis C Virus Evolution: The Interaction between Virus and Host towards Treatment Outcome

    PubMed Central

    Bittar, Cíntia; Jardim, Ana Carolina Gomes; Yamasaki, Lilian Hiromi Tomonari; Carareto, Claudia Márcia Aparecida; Pinho, João Renato Rebello; Lemey, Philippe; de Carvalho-Mello, Isabel Maria Vicente Guedes; Rahal, Paula

    2013-01-01

    Background Hepatitis C is a disease spread throughout the world. Hepatitis C virus (HCV), the etiological agent of this disease, is a single-stranded positive RNA virus. Its genome encodes a single precursor protein that yields ten proteins after processing. NS5A, one of the non-structural viral proteins, is most associated with interferon-based therapy response, the approved treatment for hepatitis C in Brazil. HCV has a high mutation rate and therefore high variability, which may be important for evading the immune system and response to therapy. The aim of this study was to analyze the evolution of NS5A quasispecies before, during, and after treatment in patients infected with HCV genotype 3a who presented different therapy responses. Methods Viral RNA was extracted, cDNA was synthesized, the NS5A region was amplified and cloned, and 15 clones from each time-point were sequenced. The sequences were analyzed for evolutionary history, genetic diversity and selection. Results This analysis shows that the viral population that persists after treatment for most non-responder patients is present in before-treatment samples, suggesting it is adapted to evade treatment. In contrast, the population found in before treatment samples from most end-of-treatment responder patients either are selected out or appears in low frequency after relapse, therefore changing the population structure. The exceptions illustrate the uniqueness of the evolutionary process, and therefore the treatment resistance process, in each patient. Conclusion Although evolutionary behavior throughout treatment showed that each patient presented different population dynamics unrelated to therapy outcome, it seems that the viral population from non-responders that resists the treatment already had strains that could evade therapy before it started. PMID:23638063

  3. Differential regulation of C5a receptor 1 in innate immune cells during the allergic asthma effector phase

    PubMed Central

    Schmudde, Inken; Sun, Jing; Vollbrandt, Tillman; König, Peter; Köhl, Jörg

    2017-01-01

    C5a drives airway constriction and inflammation during the effector phase of allergic asthma, mainly through the activation of C5a receptor 1 (C5aR1). Yet, C5aR1 expression on myeloid and lymphoid cells during the allergic effector phase is ill-defined. Recently, we generated and characterized a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we used this reporter strain to monitor C5aR1 expression in airway, pulmonary and lymph node cells during the effector phase of OVA-driven allergic asthma. C5aR1 reporter and wildtype mice developed a similar allergic phenotype with comparable airway resistance, mucus production, eosinophilic/neutrophilic airway inflammation and Th2/Th17 cytokine production. During the allergic effector phase, C5aR1 expression increased in lung tissue eosinophils but decreased in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs). Surprisingly, expression in neutrophils was not affected. Of note, moDCs but not CD11b+ cDCs from mediastinal lymph nodes (mLN) expressed less C5aR1 than DCs residing in the lung after OVA challenge. Finally, neither CD103+ cDCs nor cells of the lymphoid lineage such as Th2 or Th17-differentiated CD4+ T cells, B cells or type 2 innate lymphoid cells (ILC2) expressed C5aR1 under allergic conditions. Our findings demonstrate a complex regulation pattern of C5aR1 in the airways, lung tissue and mLN of mice, suggesting that the C5a/C5aR1 axis controls airway constriction and inflammation through activation of myeloid cells in all three compartments in an experimental model of allergic asthma. PMID:28231307

  4. Proteins (image)

    MedlinePlus

    ... is an important nutrient that builds muscles and bones and provides energy. Protein can help with weight control because it helps you feel full and satisfied from your meals. The healthiest proteins are the leanest. This means ...

  5. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  6. Increased Phosphoenolpyruvate Carboxykinase Gene Expression and Steatosis during Hepatitis C Virus Subgenome Replication

    PubMed Central

    Qadri, Ishtiaq; Choudhury, Mahua; Rahman, Shaikh Mizanoor; Knotts, Trina A.; Janssen, Rachel C.; Schaack, Jerome; Iwahashi, Mieko; Puljak, Livia; Simon, Francis R.; Kilic, Gordan; Fitz, J. Gregory; Friedman, Jacob E.

    2012-01-01

    Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated transcription factors are dramatically up-regulated in Huh.8 cells, which stably express an HCV subgenome replicon. HCV increased activation of cAMP response element-binding protein (CREB), CCAAT/enhancer-binding protein (C/EBPβ), forkhead box protein O1 (FOXO1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and involved activation of the cAMP response element in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPβ-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1α or FOXO1 levels. Notably, expression of HCV nonstructural component NS5A in Huh7 or primary hepatocytes stimulated PEPCK gene expression and glucose output in HepG2 cells, whereas a deletion in NS5A reduced PEPCK expression and lowered cellular lipids but was without effect on insulin resistance, as demonstrated by the inability of insulin to stimulate mobilization of a pool of insulin-responsive vesicles to the plasma membrane. HCV-replicating cells demonstrated increases in cellular lipids with insulin resistance at the level of the insulin receptor, increased insulin receptor substrate 1 (Ser-312), and decreased Akt (Ser-473) activation in response to insulin. C/EBPβ-RNAi normalized lipogenic genes sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and liver X receptor α but was unable to reduce accumulation of triglycerides in Huh.8 cells or reverse the increase in ApoB expression, suggesting a role for increased lipid retention in steatotic hepatocytes. Collectively, these data reveal an important role of NS5A, C/EBPβ, and pCREB in promoting HCV-induced gluconeogenic gene expression

  7. Ledipasvir and Sofosbuvir

    MedlinePlus

    ... inhibitors. It works by decreasing the amount of hepatitis C virus (HCV) in the body. Ledipasvir is in a ... HCV NS5A inhibitors. It works by stopping the virus that causes hepatitis C from spreading inside the body.

  8. Elbasvir and Grazoprevir

    MedlinePlus

    ... treat a certain type of chronic (long-term) hepatitis C infection (swelling of the liver caused by a ... is in a class of antiviral medications called HCV NS5A inhibitors. It works by stopping the virus ...

  9. Daclatasvir

    MedlinePlus

    ... Solvadi]) to treat a certain type of chronic hepatitis C (an ongoing viral infection that damages the liver). ... is in a class of antiviral medications called hepatitis C virus (HCV) NS5A inhibitors. It works by stopping ...

  10. Identification and validation of AIB1 and EIF5A2 for noninvasive detection of bladder cancer in urine samples.

    PubMed

    Zhou, Bang-Fen; Wei, Jin-Huan; Chen, Zhen-Hua; Dong, Pei; Lai, Ying-Rong; Fang, Yong; Jiang, Hui-Ming; Lu, Jun; Zhou, Fang-Jian; Xie, Dan; Luo, Jun-Hang; Chen, Wei

    2016-07-05

    We previously demonstrated that amplified in breast cancer 1 (AIB1) and eukaryotic initiation factor 2 (EIF5A2) overexpression was an independent predictor of poor clinical outcomes for patients with bladder cancer (BCa). In this study, we evaluated the usefulness of AIB1 and EIF5A2 alone and in combination with nuclear matrix protein 22 (NMP22) as noninvasive diagnostic tests for BCa. Using urine samples from 135 patients (training set, controls [n = 50] and BCa [n = 85]), we detected the AIB1, EIF5A2, and NMP22 concentrations using enzyme-linked immunosorbent assay. We applied multivariate logistic regression analysis to build a model based on the three biomarkers for BCa diagnosis. The diagnostic accuracy of the three biomarkers and the model were assessed and compared by the area under the curve (AUC) of the receiver operating characteristic. We validated the diagnostic accuracy of these biomarkers and the model in an independent validation cohort of 210 patients. In the training set, urinary concentrations of AIB1, EIF5A2, and NMP22 were significantly elevated in BCa. The AUCs of AIB1, EIF5A2, NMP22, and the model were 0.846, 0.761, 0.794, and 0.919, respectively. The model had the highest diagnostic accuracy when compared with AIB1, EIF5A2, or NMP22 (p < 0.05 for all). The model had 92% sensitivity and 92% specificity. We obtained similar results in the independent validation cohort. AIB1 and EIF5A2 show promise for the noninvasive detection of BCa. The model based on AIB1, EIF5A2, and NMP22 outperformed each of the three individual biomarkers for detecting BCa.

  11. Unique Function of Kinesin Kif5A in Localization of Mitochondria in Axons

    PubMed Central

    Campbell, Philip D.; Shen, Kimberle; Sapio, Matthew R.; Glenn, Thomas D.; Talbot, William S.

    2014-01-01

    Mutations in Kinesin proteins (Kifs) are linked to various neurological diseases, but the specific and redundant functions of the vertebrate Kifs are incompletely understood. For example, Kif5A, but not other Kinesin-1 heavy-chain family members, is implicated in Charcot-Marie-Tooth disease (CMT) and Hereditary Spastic Paraplegia (HSP), but the mechanism of its involvement in the progressive axonal degeneration characteristic of these diseases is not well understood. We report that zebrafish kif5Aa mutants exhibit hyperexcitability, peripheral polyneuropathy, and axonal degeneration reminiscent of CMT and HSP. Strikingly, although kif5 genes are thought to act largely redundantly in other contexts, and zebrafish peripheral neurons express five kif5 genes, kif5Aa mutant peripheral sensory axons lack mitochondria and degenerate. We show that this Kif5Aa-specific function is cell autonomous and is mediated by its C-terminal tail, as only Kif5Aa and chimeric motors containing the Kif5Aa C-tail can rescue deficits. Finally, concurrent loss of the kinesin-3, kif1b, or its adaptor kbp, exacerbates axonal degeneration via a nonmitochondrial cargo common to Kif5Aa. Our results shed light on Kinesin complexity and reveal determinants of specific Kif5A functions in mitochondrial transport, adaptor binding, and axonal maintenance. PMID:25355224

  12. eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury identified by a proteomics approach

    PubMed Central

    Liu, Wei; Shang, Fei-Fei; Xu, Yang; Belegu, Visar; Xia, Lei; Zhao, Wei; Liu, Ran; Wang, Wei; Liu, Jin; Li, Chen-Yun; Wang, Ting-Hua

    2015-01-01

    Spinal cord injury (SCI) is frequently accompanied by a degree of spontaneous functional recovery. The underlying mechanisms through which such recovery is generated remain elusive. In this study, we observed a significant spontaneous motor function recovery 14 to 28 days after spinal cord transection (SCT) in rats. Using a comparative proteomics approach, caudal to the injury, we detected difference in 20 proteins. Two of these proteins, are eukaryotic translation initiation factor 5A1 (eIF5A1) that is involved in cell survival and proliferation, and Rho GDP dissociation inhibitor alpha (RhoGDIα), a member of Rho GDI family that is involved in cytoskeletal reorganization. After confirming the changes in expression levels of these two proteins following SCT, we showed that in vivo eIF5A1 up-regulation and down-regulation significantly increased and decreased, respectively, motor function recovery. In vitro, eIF5A1 overexpression in primary neurons increased cell survival and elongated neurite length while eIF5A1 knockdown reversed these results. We found that RhoGDIα up-regulation and down-regulation rescues the effect of eIF5A1 down-regulation and up-regulation both in vivo and in vitro. Therefore, we have identified eIF5A1/RhoGDIα pathway as a new therapeutic target for treatment of spinal cord injured patients. PMID:26593060

  13. Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells.

    PubMed

    Eadon, Michael T; Jacob, Alexander; Cunningham, Patrick N; Quigg, Richard J; Garcia, Joe G N; Alexander, Jessy J

    2014-11-01

    Blood-brain barrier (BBB) disturbance is a crucial occurrence in many neurological diseases, including systemic lupus erythematosus (SLE). Our previous studies showed that experimental lupus serum altered the integrity of the mouse brain endothelial layer, an important constituent of the BBB. Complement activation occurs in lupus with increased circulating complement components. Using a genomics approach, we identified the microRNA (miRNA) altered in mouse brain endothelial cells (bEnd3) by lupus serum and the complement protein, C5a. Of the 318 miRNA evaluated, 23 miRNAs were altered by lupus serum and 32 were altered by C5a alone compared with controls. Seven miRNAs (P < 0 · 05) were differentially expressed by both treatments: mmu-miR-133a*, mmu-miR-193*, mmu-miR-26b, mmu-miR-28*, mmu-miR-320a, mmu-miR-423-3p and mmu-miR-509-5p. The microarray results were validated by quantitative RT-PCR. In line with the in vitro results, expression of miR-26b and miR-28* were also significantly up-regulated in lupus mouse brain which was reduced by C5a receptor inhibition. Target prediction analysis revealed miR gene targets encoding components involved in inflammation, matrix arrangement, and apoptosis, pathways known to play important roles in central nervous system lupus. Our findings suggest that the miRNAs reported in this study may represent novel therapeutic targets in central nervous system lupus and other similar neuroinflammatory settings.

  14. Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells

    PubMed Central

    Eadon, Michael T; Jacob, Alexander; Cunningham, Patrick N; Quigg, Richard J; Garcia, Joe G N; Alexander, Jessy J

    2014-01-01

    Blood–brain barrier (BBB) disturbance is a crucial occurrence in many neurological diseases, including systemic lupus erythematosus (SLE). Our previous studies showed that experimental lupus serum altered the integrity of the mouse brain endothelial layer, an important constituent of the BBB. Complement activation occurs in lupus with increased circulating complement components. Using a genomics approach, we identified the microRNA (miRNA) altered in mouse brain endothelial cells (bEnd3) by lupus serum and the complement protein, C5a. Of the 318 miRNA evaluated, 23 miRNAs were altered by lupus serum and 32 were altered by C5a alone compared with controls. Seven miRNAs (P < 0·05) were differentially expressed by both treatments: mmu-miR-133a*, mmu-miR-193*, mmu-miR-26b, mmu-miR-28*, mmu-miR-320a, mmu-miR-423-3p and mmu-miR-509-5p. The microarray results were validated by quantitative RT-PCR. In line with the in vitro results, expression of miR-26b and miR-28* were also significantly up-regulated in lupus mouse brain which was reduced by C5a receptor inhibition. Target prediction analysis revealed miR gene targets encoding components involved in inflammation, matrix arrangement, and apoptosis, pathways known to play important roles in central nervous system lupus. Our findings suggest that the miRNAs reported in this study may represent novel therapeutic targets in central nervous system lupus and other similar neuroinflammatory settings. PMID:24801999

  15. Therapeutic proteins.

    PubMed

    Dimitrov, Dimiter S

    2012-01-01

    Protein-based therapeutics are highly successful in clinic and currently enjoy unprecedented recognition of their potential. More than 100 genuine and similar number of modified therapeutic proteins are approved for clinical use in the European Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for almost half (48%) of the sales. Based on their pharmacological activity, they can be divided into five groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins can also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. They can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Most protein therapeutics currently on the market are recombinant and hundreds of them are in clinical trials for therapy of cancers, immune disorders, infections, and other diseases. New engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics, are currently under development. However, in the last several decades, there are no conceptually new methodological developments comparable, e.g., to genetic engineering leading to the development of recombinant therapeutic proteins. It appears that a paradigm change in methodologies and understanding of mechanisms is needed to overcome major

  16. WNT5A expression is regulated by the status of its promoter methylation in leukaemia and can inhibit leukemic cell malignant proliferation.

    PubMed

    Deng, Gang; Li, Zhao Quan; Zhao, Chen; Yuan, Yuan; Niu, Chang Chun; Zhao, Chen; Pan, Jing; Si, Wei Ke

    2011-02-01

    Although down-regulation of WNT5A expression has been reported in some types of leukaemias, the level of WNT5A expression has not been assessed in leukaemia complete remission (CR) cases, the relationship among WNT5A expression level, the status of its promoter methylation, and the curative effect of leukaemia has not been reported, and the effect of WNT5A on cell proliferation has not been assessed. In this study, we analyzed WNT5A expression in various kinds of leukaemia cases, leukaemia CR cases, non-malignant hematopoietic (NMH) cases, as well as in leukemic cell lines and CD34+ cells. The methylation status of the WNT5A promoter and the levels of the Wnt5a protein were also studied. We also investigated the effect of Wnt5a on leukemic cell proliferation. WNT5A expression level was higher in NMH but lower in leukaemia cases compared to that in CR-cases (P<0.01), and was expressed at low level in leukemic cell lines K562, U937 and Jurkat. Wnt5a protein was positive in NMH, CR cases and CD34+, but negative in leukaemia cases. WNT5A promoter was methylated in leukaemia cases and all leukemic cell lines, but not in NMH and CR cases. WNT5A expression was up-regulated after exposure to the demethylating agent 5-Aza-2'-deoxycytidine (Aza) in the K562, U937, Jurkat leukemic cell lines and in 83.3% (10/12) of CR patients after cure, respectively. The increased Wnt5a protein can inhibit K562 malignant proliferation and arrest cell cycle at the G2/M phase after exposure to Aza. These results indicate that WNT5A expression was restored in complete remission cases due to demethylation, and Wnt5a can inhibit leukaemic cell proliferation. We propose that WNT5A can act as a suppressor factor in leukemogenesis and can be used as a potential marker for curative effect assessment in leukaemia.

  17. Crystal structure of human annexin I at 2.5 A resolution.

    PubMed Central

    Weng, X.; Luecke, H.; Song, I. S.; Kang, D. S.; Kim, S. H.; Huber, R.

    1993-01-01

    cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I). PMID:8453382

  18. MADANALYSIS 5, a user-friendly framework for collider phenomenology

    NASA Astrophysics Data System (ADS)

    Conte, Eric; Fuks, Benjamin; Serret, Guillaume

    2013-01-01

    We present MADANALYSIS 5, a new framework for phenomenological investigations at particle colliders. Based on a C++ kernel, this program allows us to efficiently perform, in a straightforward and user-friendly fashion, sophisticated physics analyses of event files such as those generated by a large class of Monte Carlo event generators. MADANALYSIS 5 comes with two modes of running. The first one, easier to handle, uses the strengths of a powerful PYTHON interface in order to implement physics analyses by means of a set of intuitive commands. The second one requires one to implement the analyses in the C++ programming language, directly within the core of the analysis framework. This opens unlimited possibilities concerning the level of complexity which can be reached, being only limited by the programming skills and the originality of the user. Program summaryProgram title: MadAnalysis 5 Catalogue identifier: AENO_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENO_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Permission to use, copy, modify and distribute this program is granted under the terms of the GNU General Public License. No. of lines in distributed program, including test data, etc.: 31087 No. of bytes in distributed program, including test data, etc.: 399105 Distribution format: tar.gz Programming language: PYTHON, C++. Computer: All platforms on which Python version 2.7, Root version 5.27 and the g++ compiler are available. Compatibility with newer versions of these programs is also ensured. However, the Python version must be below version 3.0. Operating system: Unix, Linux and Mac OS operating systems on which the above-mentioned versions of Python and Root, as well as g++, are available. Classification: 11.1. External routines: ROOT (http://root.cern.ch/drupal/) Nature of problem: Implementing sophisticated phenomenological analyses in high-energy physics through a

  19. An eukaryotic translation initiation factor, AteIF5A-2, affects cadmium accumulation and sensitivity in Arabidopsis.

    PubMed

    Xu, Xiao-Yan; Ding, Zhong-Jie; Chen, Lei; Yan, Jin-Ying; Li, Gui-Xin; Zheng, Shao-Jian

    2015-10-01

    Cadmium (Cd) is one of the most toxic elements and can be accumulated in plants easily; meanwhile, eIF5A is a highly conserved protein in all eukaryotic organisms. The present work tried to investigate whether eIF5A is involved in Cd accumulation and sensitivity in Arabidopsis (Arabidopsis thaliana L.) by comparing the wild-type Columbia-0 (Col-0) with a knockdown mutant of AteIF5A-2, fbr12-3 under Cd stress conditions. The results showed that the mutant fbr12-3 accumulated more Cd in roots and shoots and had significantly lower chlorophyll content, shorter root length, and smaller biomass, suggesting that downregulation of AteIF5A-2 makes the mutant more Cd sensitive. Real-time polymerase chain reaction revealed that the expressions of metal transporters involved in Cd uptake and translocation including IRT1, ZIP1, AtNramp3, and AtHMA4 were significantly increased but the expressions of PCS1 and PCS2 related to Cd detoxification were decreased notably in fbr12-3 compared with Col-0. As a result, an increase in MDA and H2 O2 content but decrease in root trolox, glutathione and proline content under Cd stress was observed, indicating that a severer oxidative stress occurs in the mutant. All these results demonstrated for the first time that AteIF5A influences Cd sensitivity by affecting Cd uptake, accumulation, and detoxification in Arabidopsis.

  20. Histone demethylase KDM5A is regulated by its reader domain through a positive-feedback mechanism

    PubMed Central

    Torres, Idelisse Ortiz; Kuchenbecker, Kristopher M.; Nnadi, Chimno I.; Fletterick, Robert J.; Kelly, Mark J.S.; Fujimori, Danica Galonić

    2016-01-01

    The retinoblastoma binding protein KDM5A removes methyl marks from lysine 4 of histone H3 (H3K4). Misregulation of KDM5A contributes to the pathogenesis of lung and gastric cancers. In addition to its catalytic jumonji C domain, KDM5A contains three PHD reader domains, commonly recognized as chromatin recruitment modules. It is unknown whether any of these domains in KDM5A have functions beyond recruitment and whether they regulate the catalytic activity of the demethylase. Here using biochemical and nuclear magnetic resonance (NMR)-based structural studies, we show that the PHD1 preferentially recognizes unmethylated H3K4 histone tail, product of KDM5A-mediated demethylation of tri-methylated H3K4 (H3K4me3). Binding of unmodified H3 peptide to the PHD1 stimulates catalytic domain-mediated removal of methyl marks from H3K4me3 peptide and nucleosome substrates. This positive-feedback mechanism—enabled by the functional coupling between a reader and a catalytic domain in KDM5A—suggests a model for the spread of demethylation on chromatin. PMID:25686748

  1. Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy

    PubMed Central

    Freyermuth, Fernande; Rau, Frédérique; Kokunai, Yosuke; Linke, Thomas; Sellier, Chantal; Nakamori, Masayuki; Kino, Yoshihiro; Arandel, Ludovic; Jollet, Arnaud; Thibault, Christelle; Philipps, Muriel; Vicaire, Serge; Jost, Bernard; Udd, Bjarne; Day, John W.; Duboc, Denis; Wahbi, Karim; Matsumura, Tsuyoshi; Fujimura, Harutoshi; Mochizuki, Hideki; Deryckere, François; Kimura, Takashi; Nukina, Nobuyuki; Ishiura, Shoichi; Lacroix, Vincent; Campan-Fournier, Amandine; Navratil, Vincent; Chautard, Emilie; Auboeuf, Didier; Horie, Minoru; Imoto, Keiji; Lee, Kuang-Yung; Swanson, Maurice S.; de Munain, Adolfo Lopez; Inada, Shin; Itoh, Hideki; Nakazawa, Kazuo; Ashihara, Takashi; Wang, Eric; Zimmer, Thomas; Furling, Denis; Takahashi, Masanori P.; Charlet-Berguerand, Nicolas

    2016-01-01

    Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy. PMID:27063795

  2. Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy.

    PubMed

    Freyermuth, Fernande; Rau, Frédérique; Kokunai, Yosuke; Linke, Thomas; Sellier, Chantal; Nakamori, Masayuki; Kino, Yoshihiro; Arandel, Ludovic; Jollet, Arnaud; Thibault, Christelle; Philipps, Muriel; Vicaire, Serge; Jost, Bernard; Udd, Bjarne; Day, John W; Duboc, Denis; Wahbi, Karim; Matsumura, Tsuyoshi; Fujimura, Harutoshi; Mochizuki, Hideki; Deryckere, François; Kimura, Takashi; Nukina, Nobuyuki; Ishiura, Shoichi; Lacroix, Vincent; Campan-Fournier, Amandine; Navratil, Vincent; Chautard, Emilie; Auboeuf, Didier; Horie, Minoru; Imoto, Keiji; Lee, Kuang-Yung; Swanson, Maurice S; Lopez de Munain, Adolfo; Inada, Shin; Itoh, Hideki; Nakazawa, Kazuo; Ashihara, Takashi; Wang, Eric; Zimmer, Thomas; Furling, Denis; Takahashi, Masanori P; Charlet-Berguerand, Nicolas

    2016-04-11

    Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.

  3. Histone demethylase KDM5A is regulated by its reader domain through a positive-feedback mechanism

    NASA Astrophysics Data System (ADS)

    Torres, Idelisse Ortiz; Kuchenbecker, Kristopher M.; Nnadi, Chimno I.; Fletterick, Robert J.; Kelly, Mark J. S.; Fujimori, Danica Galonić

    2015-02-01

    The retinoblastoma binding protein KDM5A removes methyl marks from lysine 4 of histone H3 (H3K4). Misregulation of KDM5A contributes to the pathogenesis of lung and gastric cancers. In addition to its catalytic jumonji C domain, KDM5A contains three PHD reader domains, commonly recognized as chromatin recruitment modules. It is unknown whether any of these domains in KDM5A have functions beyond recruitment and whether they regulate the catalytic activity of the demethylase. Here using biochemical and nuclear magnetic resonance (NMR)-based structural studies, we show that the PHD1 preferentially recognizes unmethylated H3K4 histone tail, product of KDM5A-mediated demethylation of tri-methylated H3K4 (H3K4me3). Binding of unmodified H3 peptide to the PHD1 stimulates catalytic domain-mediated removal of methyl marks from H3K4me3 peptide and nucleosome substrates. This positive-feedback mechanism—enabled by the functional coupling between a reader and a catalytic domain in KDM5A—suggests a model for the spread of demethylation on chromatin.

  4. C5a alters blood-brain barrier integrity in a human in vitro model of systemic lupus erythematosus.

    PubMed

    Mahajan, Supriya D; Parikh, Neil U; Woodruff, Trent M; Jarvis, James N; Lopez, Molly; Hennon, Teresa; Cunningham, Patrick; Quigg, Richard J; Schwartz, Stanley A; Alexander, Jessy J

    2015-09-01

    The blood-brain barrier (BBB) plays a crucial role in brain homeostasis, thereby maintaining the brain environment precise for optimal neuronal function. Its dysfunction is an intriguing complication of systemic lupus erythematosus (SLE). SLE is a systemic autoimmune disorder where neurological complications occur in 5-50% of cases and is associated with impaired BBB integrity. Complement activation occurs in SLE and is an important part of the clinical profile. Our earlier studies demonstrated that C5a generated by complement activation caused the loss of brain endothelial layer integrity in rodents. The goal of the current study was to determine the translational potential of these studies to a human system. To assess this, we used a two dimensional in vitro BBB model constructed using primary human brain microvascular endothelial cells and astroglial cells, which closely emulates the in vivo BBB allowing the assessment of BBB integrity. Increased permeability monitored by changes in transendothelial electrical resistance and cytoskeletal remodelling caused by actin fiber rearrangement were observed when the cells were exposed to lupus serum and C5a, similar to the observations in mice. In addition, our data show that C5a/C5aR1 signalling alters nuclear factor-κB translocation into nucleus and regulates the expression of the tight junction proteins, claudin-5 and zonula occludens 1 in this setting. Our results demonstrate for the first time that C5a regulates BBB integrity in a neuroinflammatory setting where it affects both endothelial and astroglial cells. In addition, we also demonstrate that our previous findings in a mouse model, were emulated in human cells in vitro, bringing the studies one step closer to understanding the translational potential of C5a/C5aR1 blockade as a promising therapeutic strategy in SLE and other neurodegenerative diseases.

  5. The Globular Tail Domain of Myosin-5a Functions as a Dimer in Regulating the Motor Activity.

    PubMed

    Zhang, Wen-Bo; Yao, Lin-Lin; Li, Xiang-Dong

    2016-06-24

    Myosin-5a contains two heavy chains, which are dimerized via the coiled-coil regions. Thus, myosin-5a comprises two heads and two globular tail domains (GTDs). The GTD is the inhibitory domain that binds to the head and inhibits its motor function. Although the two-headed structure is essential for the processive movement of myosin-5a along actin filaments, little is known about the role of GTD dimerization. Here, we investigated the effect of GTD dimerization on its inhibitory activity. We found that the potent inhibitory activity of the GTD is dependent on its dimerization by the preceding coiled-coil regions, indicating synergistic interactions between the two GTDs and the two heads of myosin-5a. Moreover, we found that alanine mutations of the two conserved basic residues at N-terminal extension of the GTD not only weaken the inhibitory activity of the GTD but also enhance the activation of myosin-5a by its cargo-binding protein melanophilin (Mlph). These results are consistent with the GTD forming a head to head dimer, in which the N-terminal extension of the GTD interacts with the Mlph-binding site in the counterpart GTD. The Mlph-binding site at the GTD-GTD interface must be exposed prior to the binding of Mlph. We therefore propose that the inhibited Myo5a is equilibrated between the folded state, in which the Mlph-binding site is buried, and the preactivated state, in which the Mlph-binding site is exposed, and that Mlph is able to bind to the Myo5a in preactivated state and activates its motor function.

  6. eIF5A2 is an alternative pathway for cell proliferation in cetuximab-treated epithelial hepatocellular carcinoma

    PubMed Central

    Xue, Fei; Liu, Yanhui; Chu, Haoyuan; Wen, Yu; Yan, Lei; Tang, Qiang; Xiao, Erhui; Zhang, Dongyi; Zhang, Hongwei

    2016-01-01

    Heaptocellular carcinoma (HCC) is still a great health problem around the world. Recently, the cetuximab has been implicated to have therapeutic values for HCC. However, cetuximab-resistance has also been synchronously reported pertaining to HCC treatment. This study aimed to evaluate the role of eIF5A2 in cetuximab-treated HCC cell proliferation, and whether eIF5A2 specific inhibitor GC7 has any effects on cetuximab-mediated proliferation inhibition in HCC cell lines. It was observed that GC7 significantly inhibited cell proliferation in HCC cell lines. GC7 synergized cetuximab to inhibit the proliferation in epithelial HCC cell lines HepG2, Huh7 and Hep3B, but not in mesenchymal cell lines SNU387 and SNU449. Knockdown of eIF5A-2 by specific siRNA exhibited the similar effects as GC7 did. In cetuximab-treated cells, cetuximab decreased the protein level of EGFR and phosphorylated STAT3 and unexpectedly up-regulated the expression level of eIF5A2, indicating the activation of eIF5A2 pathway. In turn, cetuximab also synergized GC7 to inhibit cell proliferation in epithelial cell lines. GC7 also suppressed hypoxia-induced cell proliferation in epithelial cell lines. These data suggest that eIF5A2 is an alternative pathway for cell proliferation in epithelial HCC cells escaping from the cytotoxicity of cetuximab. The eIF5A inhibitor GC7 might be a potent agent that promotes the cytotoxicity of cetuximab on epithelial HCC cells. PMID:27904670

  7. Blocking eIF5A Modification in Cervical Cancer Cells Alters the Expression of Cancer-Related Genes and Suppresses Cell Proliferation

    PubMed Central

    Mémin, Elisabeth; Hoque, Mainul; Jain, Mohit R.; Heller, Debra S.; Li, Hong; Cracchiolo, Bernadette; Hanauske-Abel, Hartmut M.; Pe’ery, Tsafi; Mathews, Michael B.

    2016-01-01

    Cancer etiology is influenced by alterations in protein synthesis that are not fully understood. In this study, we took a novel approach to investigate the role of the eukaryotic translation initiation factor eIF5A in human cervical cancers, where it is widely overexpressed. eIF5A contains the distinctive amino acid hypusine, which is formed by a posttranslational modification event requiring deoxyhypusine hydroxylase (DOHH), an enzyme that can be inhibited by the drugs ciclopirox and deferiprone. We found that proliferation of cervical cancer cells can be blocked by DOHH inhibition with either of these pharmacologic agents, as well as by RNA interference–mediated silencing of eIF5A, DOHH, or another enzyme in the hypusine pathway. Proteomic and RNA analyses in HeLa cervical cancer cells identified two groups of proteins in addition to eIF5A that were coordinately affected by ciclopirox and deferiprone. Group 1 proteins (Hsp27, NM23, and DJ-1) were downregulated at the translational level, whereas group 2 proteins (TrpRS and PRDX2) were upregulated at the mRNA level. Further investigations confirmed that eIF5A and DOHH are required for Hsp27 expression in cervical cancer cells and for regulation of its key target IκB and hence NF-κB. Our results argue that mature eIF5A controls a translational network of cancer-driving genes, termed the eIF5A regulon, at the levels of mRNA abundance and translation. In coordinating cell proliferation, the eIF5A regulon can be modulated by drugs such as ciclopirox or deferiprone, which might be repositioned to control cancer cell growth. PMID:24220243

  8. Whey Protein

    MedlinePlus

    ... inflammation (polymyalgia rheumatica). Taking whey protein in a dairy product twice daily for 8 weeks does not improve muscle function, walking speed, or other movement tests in people with polymyalgia rheumatica. Other conditions. More evidence is needed to rate whey protein for these uses.

  9. Expression and modulation of C5a receptor (CD88) on skin dendritic cells. Chemotactic effect of C5a on skin migratory dendritic cells.

    PubMed Central

    Morelli, A; Larregina, A; Chuluyán, I; Kolkowski, E; Fainboim, L

    1996-01-01

    Although it is known that dendritic cells (DC) migrate in response to inflammatory stimuli. There is little information about the expression of receptors for chemotactic factors on DC. The present study has demonstrated by double immunostaining and flow cytometry of Langerhan's cell (LC)-enriched epidermal cell suspensions that a small subpopulation (5-6%) of epidermal resident DC (rLC) expresses receptors for C5a (C5aR). Epidermal rLC positive for C5aR show a round-shape morphology, were located next to the basement membrane and express HLA-DR molecules higher than C5aR negative rLC. These observations suggest that rLC would express C5aR as part of their process of maturation during tissue trafficking. To investigate whether epidermal LC up-regulate C5aR along their differentiation pathway. LC were differentiated in vitro after culture in epidermal cell suspensions supplemented with granulocyte macrophage colony-stimulating factor (GM-CSF). As a result, in vitro differentiated LC increased the expression of C5aR up to 69% of the DC population. In accordance with this observation, interdigitating DC of secondary lymphoid organs (lymph node and tonsil) also expressed (5aR. Migratory CD1a positive DC that spontaneously migrated out of dermal or split-skin organ explants were also positive for C5aR and were used for chemotaxis and chemokinesis assays in response to human recombinant C5a (rC5a). Optimum migration to rC5a was observed at 10(-8)M with a sigmoidal dose response curve. Checkboard analysis demonstrated that locomotion in response to rC5a was chemotaxis and not chemokinesis. Images Figure 3 Figure 6 Figure 7 Figure 10 Figure 11 PMID:8911150

  10. Study of PKRBD in HCV genotype 3a infected patients in response to interferon therapy in Pakistani population

    PubMed Central

    2013-01-01

    Background Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma and infects about 3% world population. Response to interferon therapy depends upon the genotype of the virus and factors associated with the host. Despite a good response to interferon therapy, a considerable number of genotype 3a infected patients remains unalleviated. Results In total forty-nine patients including twenty-five non-responders (non-SVR) and twenty-four responders (SVR) were recruited. Patients were tested for viral status at different intervals and the isolated RNA was sequenced for the NS5A region in both groups. The comparison of PKRBD of HCV between the SVR and non-SVR patients did not confirm any significant difference in the number of mutations. However, when the sequence downstream to the PKRBD of NS5A was compared, two important statistically significant mutations were observed; at positions 2309 (Ala to Ser) and 2326 (Gly to Ala). These mutations were then analysed for tertiary protein structure and important structural changes were observed. Statistically significant difference was also observed when age groups of patients were compared; younger patients showed better response than the older ones. Conclusions The region between PKRBD and IRRDR may be important for prediction of response to IFN therapy for genotype 3a. ISDR and PKRBD have not shown any involvement in treatment response. Further functional analyses of these findings can help in understanding the involvement of the NS5A region in interferon treatment of HCV-3a infected patients. PMID:24321105

  11. CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo

    PubMed Central

    Teng, Ooiean; Chen, Szu-Ting; Hsu, Tsui-Ling; Sia, Sin Fun; Cole, Suzanne; Valkenburg, Sophie A.; Hsu, Tzu-Yun; Zheng, Jian Teddy; Tu, Wenwei; Bruzzone, Roberto; Peiris, Joseph Sriyal Malik

    2016-01-01

    ABSTRACT Human infections with influenza viruses exhibit mild to severe clinical outcomes as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin domain family 5 member A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of proinflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced levels of tumor necrosis factor alpha (TNF-α) and IP-10 but elevated alpha interferon (IFN-α) compared to those of wild-type mice. The heightened type I IFN response in the macrophages of CLEC5A-deficient mice was associated with upregulated TLR3 mRNA after treatment with double-stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of proinflammatory cytokines, decreased immune cell infiltration in the lungs, and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections. IMPORTANCE Multiple

  12. Hypoxic tumor kinase signaling mediated by STAT5A in development of castration-resistant prostate cancer.

    PubMed

    Røe, Kathrine; Bratland, Åse; Vlatkovic, Ljiljana; Ragnum, Harald Bull; Saelen, Marie Grøn; Olsen, Dag Rune; Marignol, Laure; Ree, Anne Hansen

    2013-01-01

    In this study, we hypothesized that androgen-deprivation therapy (ADT) in prostate cancer, although initially efficient, induces changes in the tumor kinome, which subsequently promote development of castration-resistant (CR) disease. Recognizing the correlation between tumor hypoxia and poor prognosis in prostate cancer, we further hypothesized that such changes might be influenced by hypoxia. Microarrays with 144 kinase peptide substrates were applied to analyze CWR22 prostate carcinoma xenograft samples from ADT-naïve, androgen-deprived (AD), long-term AD (ADL), and CR disease stages. The impact of hypoxia was assessed by matching the xenograft kinase activity profiles with those acquired from hypoxic and normoxic prostate carcinoma cell cultures, whereas the clinical relevance was evaluated by analyzing prostatectomy tumor samples from patients with locally advanced disease, either in ADT-naïve or early CR disease stages. By using this novel peptide substrate microarray method we revealed high kinase activity mediated by signal transducer and activator of transcription 5A (STAT5A) in CR prostate cancer. Additionally, we uncovered high STAT5A kinase activity already in regressing ADL xenografts, before renewed CR growth was evidenced. Finally, since increased STAT5A kinase activity also was detected after exposing prostate carcinoma cells to hypoxia, we propose long-term ADT to induce tumor hypoxia and stimulate STAT5A kinase activity, subsequently leading to renewed CR tumor growth. Hence, the study detected STAT5A as a candidate to be further investigated for its potential as marker of advanced prostate cancer and as possible therapeutic target protein.

  13. Nucleation and growth control in protein crystallization

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Nyce, Thomas A.; Meehan, Edward J.; Sowers, Jennifer W.; Monaco, Lisa A.

    1990-01-01

    The five topics summarized in this final report are as follows: (1) a technique for the expedient, semi-automated determination of protein solubilities as a function of temperature and application of this technique to proteins other than lysozyme; (2) a small solution cell with adjustable temperature gradients for the growth of proteins at a predetermined location through temperature programming; (3) a microscopy system with image storage and processing capability for high resolution optical studies of temperature controlled protein growth and etching kinetics; (4) growth experiments with lysozyme in thermosyphon flow ; and (5) a mathematical model for the evolution of evaporation/diffusion induced concentration gradients in the hanging drop protein crystallization technique.

  14. Non-canonical WNT5A signaling up-regulates the expression of the tumor suppressor 15-PGDH and induces differentiation of colon cancer cells.

    PubMed

    Mehdawi, Lubna M; Prasad, Chandra Prakash; Ehrnström, Roy; Andersson, Tommy; Sjölander, Anita

    2016-11-01

    The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in prostaglandin E2 catabolism and is down-regulated in colorectal cancer (CRC) tissue. Canonical Wnt signaling is frequently elevated in colon cancers and has been shown to down-regulate 15-PGDH expression. Therefore, we have in the current study investigated if the non-canonical ligand WNT5A relates to increased expression of 15-PGDH in colon cancer cells. In the same cohort of patients, we demonstrated a parallel and significant loss of 15-PGDH and WNT5A protein expression in CRC tissues compared with matched normal colon tissues. Furthermore, patients with low 15-PGDH/WNT5A expression in their tumors showed reduced survival compared with patients with high 15-PGDH/WNT5A expression. To investigate if WNT5A signaling directly affects 15-PGDH expression, we performed in vitro analyses of colon cancer cells (HT-29 and Caco-2). Both cell lines, when treated with recombinant WNT5A (rWNT5A) or Foxy-5, a WNT5A-mimicking peptide, responded by increasing their expression of 15-PGDH mRNA and protein. Our investigations showed that rWNT5A and Foxy-5 induced this increased expression of 15-PGDH through reduced β-catenin signaling as well as increased JNK/AP-1 signaling in colon cancer cells. WNT5A signaling also induced increased 15-PGDH expression in a breast cancer cell line both in vitro and in vivo. In agreement, WNT5A signaling also increased the expression of the differentiation markers sucrose-isomaltase and mucin-2 in colon cancer cells. Our results show that WNT5A signaling regulates 15-PGDH expression, thus uncovering a novel mechanism by which WNT5A acts as a tumor suppressor and suggests that increased 15-PGDH expression could be used as an indicator of a positive response to Foxy-5 in patients treated with this WNT5A agonist.

  15. Total protein

    MedlinePlus

    ... 2016:chap 215. Read More Agammaglobulinemia Albumin - blood (serum) test Amino acids Antibody Burns Chronic Congenital nephrotic syndrome Fibrinogen blood test Glomerulonephritis Hemoglobin Liver disease Malabsorption Multiple myeloma Polycythemia vera Protein in diet ...

  16. Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells.

    PubMed

    Andersson, Emma R; Saltó, Carmen; Villaescusa, J Carlos; Cajanek, Lukas; Yang, Shanzheng; Bryjova, Lenka; Nagy, Irina I; Vainio, Seppo J; Ramirez, Carmen; Bryja, Vitezslav; Arenas, Ernest

    2013-02-12

    Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1(-/-) mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1(-/-);Wnt5a(-/-) mice revealed a greater loss of Nurr1(+) cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson's disease.

  17. Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia and failure to thrive.

    PubMed

    Baertling, Fabian; Al-Murshedi, Fathiya; Sánchez-Caballero, Laura; Al-Senaidi, Khalfan; Joshi, Niranjan P; Venselaar, Hanka; van den Brand, Mariël Am; Nijtmans, Leo Gj; Rodenburg, Richard Jt

    2017-03-01

    COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts. This article is protected by copyright. All rights reserved.

  18. Critical role of the C5a-activated neutrophils in high-fat diet-induced vascular inflammation

    PubMed Central

    Osaka, Mizuko; Ito, Shunsuke; Honda, Masaki; Inomata, Yukihiro; Egashira, Kensuke; Yoshida, Masayuki

    2016-01-01

    Exceed and chronic high-fat diet (HFD) contributes to the diagnosis and development of atherosclerosis, obesity, and metabolic syndrome. However, the key molecular component(s) triggered by HFD responsible for initiating vascular inflammation remain unknown. We observed that feeding HFD for 4 weeks is sufficient to induce leukocyte recruitment in the femoral artery of wild-type mice. Neutrophil- and monocyte-depletion analyses confirmed the preferential recruitment of neutrophils in these mice. Protein analysis of sera from HFD-fed mice revealed a marked elevation of complement component C5a levels. Exogenous C5a alone induced leukocyte recruitment, which was abolished by a C5a-receptor antagonist. We also examined the role of neutrophil-derived MCP-1 in accumulation of leukocytes in the artery. These results demonstrated a previously unrecognized role for C5a and neutrophils in the early onset of HFD-induced vascular inflammation. Further study may help in elucidating a novel regulatory pathway to control diet-induced inflammation such as that in case of atherosclerosis. PMID:26893238

  19. Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon induced antiviral activity

    PubMed Central

    Huang, Hao; Zeqiraj, Elton; Dong, Beihua; Jha, Babal Kant; Duffy, Nicole; Orlicky, Stephen; Thevakumaran, Neroshan; Talukdar, Manisha; Pillon, Monica C.; Ceccarelli, Derek F.; Wan, Leo; Juang, Yu-Chi; Mao, Daniel Y.L.; Gaughan, Christina; Brinton, Margo A.; Perelygin, Andrey A.; Kourinov, Igor; Guarné, Alba; Silverman, Robert H.; Sicheri, Frank

    2014-01-01

    Summary RNase L is an ankyrin repeat domain containing dual endoribonuclease-pseudokinase that is activated by unusual 2′,5′-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here, we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small angle x-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the non-hydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain together with nucleotide binding, impose a rigid intertwined dimer configuration that is essential for RNase catalytic and anti-viral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold. PMID:24462203

  20. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling.

    PubMed

    Abedini, Atefeh; Zamberlam, Gustavo; Lapointe, Evelyne; Tourigny, Catherine; Boyer, Alexandre; Paquet, Marilène; Hayashi, Kanako; Honda, Hiroaki; Kikuchi, Akira; Price, Christopher; Boerboom, Derek

    2016-04-01

    Whereas the roles of the canonical wingless-type MMTV (mouse mammary tumor virus) integration site family (WNT) signaling pathway in the regulation of ovarian follicle growth and steroidogenesis are now established, noncanonical WNT signaling in the ovary has been largely overlooked. Noncanonical WNTs, including WNT5a and WNT11, are expressed in granulosa cells (GCs) and are differentially regulated throughout follicle development, but their physiologic roles remain unknown. Using conditional gene targeting, we found that GC-specific inactivation ofWnt5a(but notWnt11) results in the female subfertility associated with increased follicular atresia and decreased rates of ovulation. Microarray analyses have revealed that WNT5a acts to down-regulate the expression of FSH-responsive genesin vitro, and corresponding increases in the expression of these genes have been found in the GCs of conditional knockout mice. Unexpectedly, we found that WNT5a regulates its target genes not by signalingviathe WNT/Ca(2+)or planar cell polarity pathways, but rather by inhibiting the canonical pathway, causing both β-catenin (CTNNB1) and cAMP responsive element binding (CREB) protein levels to decreaseviaa glycogen synthase kinase-3β-dependent mechanism. We further found that WNT5a prevents follicle-stimulating hormone and luteinizing protein from up-regulating the CTNNB1 and CREB proteins and their target genes, indicating that WNT5a functions as a physiologic inhibitor of gonadotropin signaling. Together, these findings identify WNT5a as a key regulator of follicle development and gonadotropin responsiveness.-Abedini, A., Zamberlam, G., Lapointe, E., Tourigny, C., Boyer, A., Paquet, M., Hayashi, K., Honda, H., Kikuchi, A., Price, C., Boerboom, D. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling.

  1. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling

    PubMed Central

    Abedini, Atefeh; Zamberlam, Gustavo; Lapointe, Evelyne; Tourigny, Catherine; Boyer, Alexandre; Paquet, Marilène; Hayashi, Kanako; Honda, Hiroaki; Kikuchi, Akira; Price, Christopher; Boerboom, Derek

    2015-01-01

    Whereas the roles of the canonical wingless-type MMTV (mouse mammary tumor virus) integration site family (WNT) signaling pathway in the regulation of ovarian follicle growth and steroidogenesis are now established, noncanonical WNT signaling in the ovary has been largely overlooked. Noncanonical WNTs, including WNT5a and WNT11, are expressed in granulosa cells (GCs) and are differentially regulated throughout follicle development, but their physiologic roles remain unknown. Using conditional gene targeting, we found that GC-specific inactivation of Wnt5a (but not Wnt11) results in the female subfertility associated with increased follicular atresia and decreased rates of ovulation. Microarray analyses have revealed that WNT5a acts to down-regulate the expression of FSH-responsive genes in vitro, and corresponding increases in the expression of these genes have been found in the GCs of conditional knockout mice. Unexpectedly, we found that WNT5a regulates its target genes not by signaling via the WNT/Ca2+ or planar cell polarity pathways, but rather by inhibiting the canonical pathway, causing both β-catenin (CTNNB1) and cAMP responsive element binding (CREB) protein levels to decrease via a glycogen synthase kinase-3β-dependent mechanism. We further found that WNT5a prevents follicle-stimulating hormone and luteinizing protein from up-regulating the CTNNB1 and CREB proteins and their target genes, indicating that WNT5a functions as a physiologic inhibitor of gonadotropin signaling. Together, these findings identify WNT5a as a key regulator of follicle development and gonadotropin responsiveness.—Abedini, A., Zamberlam, G., Lapointe, E., Tourigny, C., Boyer, A., Paquet, M., Hayashi, K., Honda, H., Kikuchi, A., Price, C., Boerboom, D. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling. PMID:26667040

  2. Role of complement C5a in mechanical inflammatory hypernociception: potential use of C5a receptor antagonists to control inflammatory pain

    PubMed Central

    Ting, E; Guerrero, A T G; Cunha, T M; Verri, W A; Taylor, S M; Woodruff, T M; Cunha, F Q; Ferreira, S H

    2007-01-01

    Background and purpose: C5a, a complement activation product, exhibits a broad spectrum of inflammatory activities particularly neutrophil chemoattraction. Herein, the role of C5a in the genesis of inflammatory hypernociception was investigated in rats and mice using the specific C5a receptor antagonist PMX53 (AcF-[OP(D-Cha)WR]). Experimental approach: Mechanical hypernociception was evaluated with a modification of the Randall–Selitto test in rats and electronic pressure meter paw test in mice. Cytokines were measured by ELISA and neutrophil migration was determined by myeloperoxidase activity. Key results: Local pretreatment of rats with PMX53 (60–180 μg per paw) inhibited zymosan-, carrageenan-, lipopolysaccharide (LPS)- and antigen-induced hypernociception. These effects were associated with C5a receptor blockade since PMX53 also inhibited the hypernociception induced by zymosan-activated serum and C5a but not by the direct-acting hypernociceptive mediators, prostaglandin E2 and dopamine. Underlying the C5a hypernociceptive mechanisms, PMX53 did not alter the cytokine release induced by inflammatory stimuli. However, PMX53 inhibited cytokine-induced hypernociception. PMX53 also inhibited the recruitment of neutrophils induced by zymosan but not by carrageenan or LPS, indicating an involvement of neutrophils in the hypernociceptive effect of C5a. Furthermore, the C5a-induced hypernociception was reduced in neutrophil-depleted rats. Extending these findings in rats, blocking C5a receptors also reduced zymosan-induced joint hypernociception in mice. Conclusions and implications: These results suggest that C5a is an important inflammatory hypernociceptive mediator, acting by a mechanism independent of hypernociceptive cytokine release, but dependent on the presence of neutrophils. Therefore, we suggest that inhibiting the action of C5a has therapeutic potential in the control of inflammatory pain. PMID:18084313

  3. Cloning and characterization of SOX5, a new member of the human SOX gene family

    SciTech Connect

    Wunderle, V.M.; Critcher, R.; Goodfellow, P.N.; Ashworth, A.

    1996-09-01

    The mammalian Y-linked testis determining gene, SRY, encodes a protein with a DNA binding motif known as the HMG box. A large family of genes sharing a high similarity with the SRY HMG box and named Sox (Sry-related HMG box) in mouse and SOX in human has been identified from various organisms. We have cloned SOX5, a new member of the human SOX gene family. SOX5 cDNAs isolated from a human adult testis cDNA library show a high similarity with the mouse Sox5 transcript over a large region identical in all the human cDNAs. However, comparison of the 5{prime} unique sequences of the cDNAs suggests that the SOX5 gene is subject to alternative splicing. Genomic analysis identified a SOX5 pseudogene located on 8q21.1, whereas the SOX5 gene itself, which contains a minimum of five introns, maps to 12p12.1. In contrast to the mouse gene, the human SOX5 gene is expressed in a variety of human tissues, and different size transcripts are observed in adult testis and fetal brain. 19 refs., 5 figs.

  4. Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption.

    PubMed

    Ghosal, Abhisek; Lambrecht, Nils; Subramanya, Sandeep B; Kapadia, Rubina; Said, Hamid M

    2013-01-01

    The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

  5. Elevated expression and potential roles of human Sp5, a member of Sp transcription factor family, in human cancers

    SciTech Connect

    Chen Yongxin; Guo Yingqiu; Ge Xijin; Itoh, Hirotaka; Watanabe, Akira; Fujiwara, Takeshi; Kodama, Tatsuhiko; Aburatani, Hiroyuki . E-mail: haburata-tky@umin.ac.jp

    2006-02-17

    In this report, we describe the expression and function of human Sp5, a member of the Sp family of zinc finger transcription factors. Like other family members, the Sp5 protein contains a Cys2His2 zinc finger DNA binding domain at the C-terminus. Our experiments employing Gal4-Sp5 fusion proteins reveal multiple transcriptional domains, including a N-terminal activity domain, an intrinsic repressive element, and a C-terminal synergistic domain. Elevated expression of Sp5 was noted in several human tumors including hepatocellular carcinoma, gastric cancer, and colon cancer. To study the effects of the Sp5 protein on growth properties of human cancer cells and facilitate the identification of its downstream genes, we combined an inducible gene expression system with microarray analysis to screen for its transcriptional targets. Transfer of Sp5 into MCF-7 cells that expressed no detectable endogenous Sp5 protein elicited significant growth promotion activity. Several of the constitutively deregulated genes have been associated with tumorigenesis (CDC25C, CEACAM6, TMPRSS2, XBP1, MYBL1, ABHD2, and CXCL12) and Wnt/{beta}-Catenin signaling pathways (BAMBI, SIX1, IGFBP5, AES, and p21{sup WAF1}). This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against human cancers.

  6. Role of spinal 5-HT5A, and 5-HT1A/1B/1D, receptors in neuropathic pain induced by spinal nerve ligation in rats.

    PubMed

    Avila-Rojas, Sabino Hazael; Velázquez-Lagunas, Isabel; Salinas-Abarca, Ana Belen; Barragán-Iglesias, Paulino; Pineda-Farias, Jorge Baruch; Granados-Soto, Vinicio

    2015-10-05

    Serotonin (5-HT) participates in pain modulation by interacting with different 5-HT receptors. The role of 5-HT5A receptor in neuropathic pain has not previously studied. The purpose of this study was to investigate: A) the role of 5-HT5A receptors in rats subjected to spinal nerve injury; B) the expression of 5-HT5A receptors in dorsal spinal cord and dorsal root ganglia (DRG). Neuropathic pain was induced by L5/L6 spinal nerve ligation. Tactile allodynia in neuropathic rats was assessed with von Frey filaments. Western blot methodology was used to determine 5-HT5A receptor protein expression. Intrathecal administration (on day 14th) of 5-HT (10-100 nmol) or 5-carboxamidotryptamine (5-CT, 0.03-0.3 nmol) reversed nerve injury-induced tactile allodynia. Intrathecal non-selective (methiothepin, 0.1-0.8 nmol) and selective (SB-699551, 1-10 nmol) 5-HT5A receptor antagonists reduced, by ~60% and ~25%, respectively, the antiallodynic effect of 5-HT (100 nmol) or 5-CT (0.3 nmol). Moreover, both selective 5-HT1A and 5-HT1B/1D receptor antagonists, WAY-100635 (0.3-1 nmol) and GR-127935 (0.3-1 nmol), respectively, partially diminished the antiallodynic effect of 5-HT or 5-CT by about 30%. Injection of antagonists, by themselves, did not affect allodynia. 5-HT5A receptors were expressed in the ipsilateral dorsal lumbar spinal cord and DRG and L5/L6 spinal nerve ligation did not modify 5-HT5A receptor protein expression in those sites. Results suggest that 5-HT5A receptors reduce pain processing in the spinal cord and that 5-HT and 5-CT reduce neuropathic pain through activation of 5-HT5A and 5-HT1A/1B/1D receptors. These receptors could be an important part of the descending pain inhibitory system.

  7. Activation of Nrf2 by the dengue virus causes an increase in CLEC5A, which enhances TNF-α production by mononuclear phagocytes.

    PubMed

    Cheng, Yi-Lin; Lin, Yee-Shin; Chen, Chia-Ling; Tsai, Tsung-Ting; Tsai, Cheng-Chieh; Wu, Yan-Wei; Ou, Yi-Dan; Chu, Yu-Yi; Wang, Ju-Ming; Yu, Chia-Yi; Lin, Chiou-Feng

    2016-08-26

    Infection by the dengue virus (DENV) threatens global public health due to its high prevalence and the lack of effective treatments. Host factors may contribute to the pathogenesis of DENV; herein, we investigated the role of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is activated by DENV in mononuclear phagocytes. DENV infection selectively activates Nrf2 following nuclear translocation. Following endoplasmic reticular (ER) stress, protein kinase R-like ER kinase (PERK) facilitated Nrf2-mediated transcriptional activation of C-type lectin domain family 5, member A (CLEC5A) to increase CLEC5A expression. Signaling downstream of the Nrf2-CLEC5A interaction enhances Toll-like receptor 3 (TLR3)-independent tumor necrosis factor (TNF)-α production following DENV infection. Forced expression of the NS2B3 viral protein induces Nrf2 nuclear translocation/activation and CLEC5A expression which increases DENV-induced TNF-α production. Animal studies confirmed Nrf2-induced CLEC5A and TNF-α in brains of DENV-infected mice. These results demonstrate that DENV infection causes Nrf2-regulated TNF-α production by increasing levels of CLEC5A.

  8. Protein Crystallizability.

    PubMed

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

  9. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  10. Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

    PubMed Central

    Kolykhalov, A A; Agapov, E V; Rice, C M

    1994-01-01

    Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B

  11. Angiogenesis Induced by Signal Transducer and Activator of Transcription 5A (STAT5A) Is Dependent on Autocrine Activity of Proliferin*

    PubMed Central

    Yang, Xinhai; Qiao, Dianhua; Meyer, Kristy; Pier, Thomas; Keles, Sunduz; Friedl, Andreas

    2012-01-01

    Multiple secreted factors induce the formation of new blood vessels (angiogenesis). The signal transduction events that orchestrate the numerous cellular activities required for angiogenesis remain incompletely understood. We have shown previously that STAT5 plays a pivotal role in angiogenesis induced by FGF2 and FGF8b. To delineate the signaling pathway downstream of STAT5, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in mouse brain endothelial cells (EC). We found that the conditioned medium from CA-STAT5A but not from dominant-negative STAT5A overexpressing EC is sufficient to induce EC invasion and tube formation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. Conversely, CA-STAT5A-induced conditioned medium had no effect on EC proliferation. Using a comparative genome-wide transcription array screen, we identified the prolactin family member proliferin (PLF1 and PLF4) as a candidate autocrine factor. The CA-STAT5A-dependent transcription and secretion of PLF by EC was confirmed by quantitative RT-PCR and Western blotting, respectively. CA-STAT5A binds to the PLF1 promoter region, suggesting a direct transcriptional regulation. Knockdown of PLF expression by shRNA or by blocking of PLF activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. Similarly, the ability of concentrated conditioned medium from CA-STAT5A transfected EC to induce angiogenesis in Matrigel plugs in vivo was abolished when PLF was depleted from the medium. These observations demonstrate a FGF/STAT5/PLF signaling cascade in EC and implicate PLF as autocrine regulator of EC invasion and tube formation. PMID:22199350

  12. Expression of cytokines by human astrocytomas following stimulation by C3a and C5a anaphylatoxins: specific increase in interleukin-6 mRNA expression.

    PubMed

    Sayah, S; Ischenko, A M; Zhakhov, A; Bonnard, A S; Fontaine, M

    1999-06-01

    C3a and C5a anaphylatoxins are two proinflammatory peptides generated during complement activation that act through distinct Gi protein-coupled receptors named C3aR and C5aR, respectively. We have demonstrated previously that human astrocytes expressed C3aR and C5aR constitutively and were able to produce a functional complement. In this study, we examined the effect of an anaphylatoxin stimulation on cytokine expression by human astrocyte cell lines. Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA expression was studied by quantitative RT-PCR. Whereas IL-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA levels remained unchanged, stimulation of astrocytoma cells (T98G, CB193, U118MG) by C3a, C5a, and peptidic C3aR and C5aR agonists induced an increase in the IL-6 mRNA level. The amount of IL-6 was markedly increased at 3 and 6 h and returned to the basal level at 9 h of stimulation. This response was specific, because pretreatment of cells with pertussis toxin or with polyclonal anti-C3aR or anti-C5aR antibodies completely blocked the IL-6 mRNA increase. The IL-6 response was also investigated at the protein level, but IL-6 protein was detected neither in cell lysates nor in supernatants of stimulated cells. The anaphylatoxin-mediated transcriptional activation of IL-6 gene suggests that C3a and C5a could play a role in priming glial cells during the inflammatory process in the brain.

  13. Cadmium induces cytotoxicity in human bronchial epithelial cells through upregulation of eIF5A1 and NF-kappaB

    SciTech Connect

    Chen, De-Ju; Xu, Yan-Ming; Du, Ji-Ying; Huang, Dong-Yang; Lau, Andy T.Y.

    2014-02-28

    Highlights: • Normal human bronchial epithelial cells (BEAS-2B) were dosed with cadmium (Cd). • A low level (2 μM) of Cd treatment for 36 h elicited negligible cytotoxicity. • High levels (20 or 30 μM) of Cd treatment for 36 h induced cell death. • High levels of Cd can upregulate the protein levels of eIF5A1 and NF-κB p65. • We suggest that eIF5A1 level is possibly modulated by NF-κB. - Abstract: Cadmium (Cd) and Cd compounds are widely-distributed in the environment and well-known carcinogens. Here, we report that in CdCl{sub 2}-exposed human bronchial epithelial cells (BEAS-2B), the level of p53 is dramatically decreased in a time- and dose-dependent manner, suggesting that the observed Cd-induced cytotoxicity is not likely due to the pro-apoptotic function of p53. Therefore, this prompted us to further study the responsive pro-apoptotic factors by proteomic approaches. Interestingly, we identified that high levels (20 or 30 μM) of Cd can significantly upregulate the protein levels of eukaryotic translation initiation factor 5A1 (eIF5A1) and redox-sensitive transcription factor NF-κB p65. Moreover, there is an enhanced NF-κB nuclear translocation as well as chromatin-binding in Cd-treated BEAS-2B cells. We also show that small interfering RNA-specific knockdown of eIF5A1 in Cd-exposed cells attenuated the Cd cytotoxicity, indicating the potential role of eIF5A1 in Cd cytotoxicity. As eIF5A1 is reported to be related with cell apoptosis but little is known about its transcriptional control, we hypothesize that NF-κB might likely modulate eIF5A1 gene expression. Notably, by bioinformatic analysis, several potential NF-κB binding sites on the upstream promoter region of eIF5A1 gene can be found. Subsequent chromatin immunoprecipitation assay revealed that indeed there is enhanced NF-κB binding on eIF5A1 promoter region of Cd-treated BEAS-2B cells. Taken together, our findings suggest for the first time a regulatory mechanism for the pro

  14. CROI 2014: Viral hepatitis and complications of HIV disease and antiretroviral therapy.

    PubMed

    Luetkemeyer, Anne F; Havlir, Diane V; Currier, Judith S

    2014-05-01

    The remarkable advances in interferon-sparing, all-oral hepatitis C virus (HCV) treatment were a highlight of the 2014 Conference on Retroviruses and Opportunistic Infections (CROI). The backbone of the nucleotide inhibitor sofosbuvir and the nonstructural protein 5A (NS5A) inhibitor ledipasvir with an additional third agent (HCV protease inhibitor or HCV nonnucleoside reverse transcriptase inhibitor) led to a sustained virologic response (SVR) rate 12 weeks after cessation of treatment of 95% to 100% after only 6 weeks of treatment. These results demonstrate the potential of combination directacting antiviral (DAA) therapy for abbreviated, well-tolerated, and highly effective HCV treatment. Two triple-drug regimens that comprised 12 weeks of an NS5A inhibitor, an HCV protease inhibitor, and a nonnucleoside inhibitor also resulted in SVRs of more than 90% in patients with HCV genotype 1. HIV coinfection does not appear to negatively impact response to DAA-based HCV therapy, as evidenced by similar response rates in HIV/HCV-coinfected patients compared with HCV-monoinfected patients receiving interferonsparing or -containing regimens. There was continued emphasis at CROI 2014 on non-AIDS complications of HIV infection, specifically cardiovascular disease, renal insufficiency, and bone and endocrine disorders that persist among patients with treated HIV disease and contribute to morbidity and mortality. Finally, new data on novel drugs and combinations for treatment of tuberculosis (TB), patient outcomes using new rapid TB diagnostics, and a short-course TB prevention strategy were presented.

  15. GEC-derived SFRP5 inhibits Wnt5a-induced macrophage chemotaxis and activation.

    PubMed

    Zhao, Chenghai; Bu, Xianmin; Wang, Wei; Ma, Tingxian; Ma, Haiying

    2014-01-01

    Aberrant macrophage infiltration and activation has been implicated in gastric inflammation and carcinogenesis. Overexpression of Wnt5a and downregulation of SFRP5, a Wnt5a antagonist, were both observed in gastric cancers recently. This study attempted to explore whether Wnt5a/SFRP5 axis was involved in macrophage chemotaxis and activation. It was found that both Wnt5a transfection and recombinant Wnt5a (rWnt5a) treatment upregulated CCL2 expression in macrophages, involving JNK and NFκB signals. Conditioned medium from Wnt5a-treated macrophages promoted macrophage chemotaxis mainly dependent on CCL2. SFRP5 from gastric epithelial cells (GECs) inhibited Wnt5a-induced CCL2 expression and macrophage chemotaxis. In addition, Wnt5a treatment stimulated macrophages to produce inflammatory cytokines and COX-2/PGE2, which was also suppressed by SFRP5 from GECs. These results demonstrate that Wnt5a induces macrophage chemotaxis and activation, which can be blocked by GEC-derived SFRP5, suggesting that Wnt5a overproduction and SFRP5 deficiency in gastric mucosa may together play an important role in gastric inflammation and carcinogenesis.

  16. 12 CFR 226.5a - Credit and charge card applications and solicitations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Credit and charge card applications and solicitations. 226.5a Section 226.5a Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM TRUTH IN LENDING (REGULATION Z) Open-End Credit § 226.5a Credit and...

  17. 12 CFR 226.5a - Credit and charge card applications and solicitations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... solicitations. 226.5a Section 226.5a Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM TRUTH IN LENDING (REGULATION Z) Open-End Credit § 226.5a Credit and charge... consumers may obtain on the Web site information about shopping for and using credit cards. (c) Direct...

  18. 12 CFR 226.5a - Credit and charge card applications and solicitations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... solicitations. 226.5a Section 226.5a Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM (CONTINUED) TRUTH IN LENDING (REGULATION Z) Open-End Credit § 226.5a Credit... consumers may obtain on the Web site information about shopping for and using credit cards. (c) Direct...

  19. 12 CFR 226.5a - Credit and charge card applications and solicitations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... solicitations. 226.5a Section 226.5a Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM (CONTINUED) TRUTH IN LENDING (REGULATION Z) Open-End Credit § 226.5a Credit... consumers may obtain on the Web site information about shopping for and using credit cards. (c) Direct...

  20. Wnt5a Directs Polarized Calcium Gradients by Recruiting Cortical Endoplasmic Reticulum to the Cell Trailing Edge

    PubMed Central

    Witze, Eric S.; Connacher, Mary Katherine; Houel, Stephane; Schwartz, Michael P.; Morphew, Mary K.; Reid, Leah; Sacks, David B.; Anseth, Kristi S.; Ahn, Natalie G.

    2013-01-01

    SUMMARY Wnt5a directs the assembly of “Wnt-receptor-actin-myosin-polarity (WRAMP)” structure, which integrates cell adhesion receptors with F-actin and myosin to form a microfilament array associated with multivesicular bodies. The WRAMP structure is polarized to the cell posterior, where it directs tail-end membrane retraction, driving forward translocation of the cell body. Here, we define constituents of the WRAMP proteome, including regulators of microfilament and microtubule dynamics, protein interactions, and enzymatic activity. IQGAP1, a scaffold for F-actin nucleation and crosslinking, is necessary for WRAMP structure formation, potentially bridging microfilaments and MVBs. Vesicle coat proteins, including coatomer-I subunits, localize to and are required for the WRAMP structure. Electron microscopy and live imaging demonstrate movement of ER to the WRAMP structure and plasma membrane, followed by elevation of intracellular Ca2+. Thus, Wnt5a controls directional movement by recruiting cortical ER to mobilize a rear-directed, localized Ca2+ signal, activating actomyosin contraction and adhesion disassembly for membrane retraction. PMID:24091015

  1. Surface Relaxation in Protein Crystals

    NASA Technical Reports Server (NTRS)

    Boutet, S.; Robinson, I. K.; Hu, Z. W.; Thomas, B. R.; Chernov, A. A.

    2002-01-01

    Surface X-ray diffraction measurements were performed on (111) growth faces of crystals of the Cellular iron-storage protein horse spleen ferritin. Crystal Trunkation Rods (CTR) were measured. A fit of the measured profile of the CTR revealed a surface roughness of 48 +/- 4.5 A and a top layer spacing contraction of 3.9 +/- 1.5%. In addition to the peak from the CTR, the rocking curves of the crystals displayed unexpected extra peaks. Multiple-scattering is demonstrated to account for them. Future applications of the method could allow the exploration of hydration effects on the growth of protein crystals.

  2. TRE5-A retrotransposition profiling reveals putative RNA polymerase III transcription complex binding sites on the Dictyostelium extrachromosomal rDNA element.

    PubMed

    Spaller, Thomas; Groth, Marco; Glöckner, Gernot; Winckler, Thomas

    2017-01-01

    The amoeba Dictyostelium discoideum has a haploid genome in which two thirds of the DNA encodes proteins. Consequently, the space available for selfish mobile elements to expand without excess damage to the host genome is limited. The non-long terminal repeat retrotransposon TRE5-A maintains an active population in the D. discoideum genome and apparently adapted to this gene-dense environment by targeting positions ~47 bp upstream of tRNA genes that are devoid of protein-coding regions. Because only ~24% of tRNA genes are associated with a TRE5-A element in the reference genome, we evaluated whether TRE5-A retrotransposition is limited to this subset of tRNA genes. We determined that a tagged TRE5-A element (TRE5-Absr) integrated at 384 of 405 tRNA genes, suggesting that expansion of the current natural TRE5-A population is not limited by the availability of targets. We further observed that TRE5-Absr targets the ribosomal 5S gene on the multicopy extrachromosomal DNA element that carries the ribosomal RNA genes, indicating that TRE5-A integration may extend to the entire RNA polymerase III (Pol III) transcriptome. We determined that both natural TRE5-A and cloned TRE5-Absr retrotranspose to locations on the extrachromosomal rDNA element that contain tRNA gene-typical A/B box promoter motifs without displaying any other tRNA gene context. Based on previous data suggesting that TRE5-A targets tRNA genes by locating Pol III transcription complexes, we propose that A/B box loci reflect Pol III transcription complex assembly sites that possess a function in the biology of the extrachromosomal rDNA element.

  3. Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

    PubMed Central

    Seow, Vernon; Lim, Junxian; Cotterell, Adam J.; Yau, Mei-Kwan; Xu, Weijun; Lohman, Rink-Jan; Kok, W. Mei; Stoermer, Martin J.; Sweet, Matthew J.; Reid, Robert C.; Suen, Jacky Y.; Fairlie, David P.

    2016-01-01

    Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1–3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents. PMID:27094554

  4. A novel mutation in the SCN5A gene contributes to arrhythmogenic characteristics of early repolarization syndrome

    PubMed Central

    GUO, QI; REN, LAN; CHEN, XUHUA; HOU, CUIHONG; CHU, JIANMIN; PU, JIELIN; ZHANG, SHU

    2016-01-01

    Several genetic variants have been associated with early repolarization syndrome (ERS). However, the lack of functional validations of the mutant effects has limited the interpretation of genetic tests. In the present study, we identified and characterized a novel sodium channel, voltage gated, type V alpha subunit (SCN5A) mutation that was associated with ERS. A 67-year-old male proband suffering from recurrent syncope underwent a documented electrocardiogram (ECG) for polymorphic ventricular tachycardia (VT). It was noted that baseline 12-lead ECG exhibited a predominantly elevated ST-segment which mimicked acute myocardial ischemia in lead V2–V6, and the ECG also demonstrated J waves in lead II, III, aVF and V2–V6. Using genetic analysis, we noted that the proband carried a novel heterozygous missense mutation of A1055G in the SCN5A gene. Whole-cell configuration of patch-clamp analysis revealed that the mutation significantly decreased peak sodium current (INa) density and shifted the steady-state inactivation curve of INa to a more negative potential. Confocal imaging suggested that in the mutant channel a defect of protein expression both on the cell membrane and in cytoplasm was present. The present study demonstrated that a novel heterozygous missense mutation of A1055G in SCN5A led to 'loss-of function' of the sodium channels, and we suggest that it accounts for the arrhythmogenic characteristics of ERS. PMID:26820605

  5. Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

    NASA Astrophysics Data System (ADS)

    Seow, Vernon; Lim, Junxian; Cotterell, Adam J.; Yau, Mei-Kwan; Xu, Weijun; Lohman, Rink-Jan; Kok, W. Mei; Stoermer, Martin J.; Sweet, Matthew J.; Reid, Robert C.; Suen, Jacky Y.; Fairlie, David P.

    2016-04-01

    Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1–3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents.

  6. Fosb gene products contribute to excitotoxic microglial activation by regulating the expression of complement C5a receptors in microglia

    PubMed Central

    Nomaru, Hiroko; Sakumi, Kunihiko; Katogi, Atsuhisa; Ohnishi, Yoshinori N; Kajitani, Kosuke; Tsuchimoto, Daisuke; Nestler, Eric J.; Nakabeppu, Yusaku

    2014-01-01

    The Fosb gene encodes subunits of the activator protein-1 transcription factor complex. Two mature mRNAs, Fosb and ΔFosb, encoding full-length FOSB and ΔFOSB proteins respectively, are formed by alternative splicing of Fosb mRNA. Fosb products are expressed in several brain regions. Moreover, Fosb-null mice exhibit depressive-like behaviors and adult-onset spontaneous epilepsy, demonstrating important roles in neurological and psychiatric disorders. Study of Fosb products has focused almost exclusively on neurons; their function in glial cells remains to be explored. In this study, we found that microglia express equivalent levels of Fosb and ΔFosb mRNAs to hippocampal neurons and, using microarray analysis, we identified six microglial genes whose expression is dependent on Fosb products. Of these genes, we focused on C5ar1 and C5ar2, which encode receptors for complement C5a. In isolated Fosb-null microglia, chemotactic responsiveness toward the truncated form of C5a was significantly lower than that in wild-type cells. Fosb-null mice were significantly resistant to kainate-induced seizures compared with wild-type mice. C5ar1 mRNA levels and C5aR1 immunoreactivity were increased in wild-type hippocampus 24 hours after kainate administration; however, such induction was significantly reduced in Fosb-null hippocampus. Furthermore, microglial activation after kainate administration was significantly diminished in Fosb-null hippocampus, as shown by significant reductions in CD68 immunoreactivity, morphological change and reduced levels of Il6 and Tnf mRNAs, although no change in the number of Iba-1-positive cells was observed. These findings demonstrate that, under excitotoxicity, Fosb products contribute to a neuroinflammatory response in the hippocampus through regulation of microglial C5ar1 and C5ar2 expression. PMID:24771617

  7. Pharmacological and biochemical characterization of the mouse 5HT5A serotonin receptor heterologously produced in the yeast Saccharomyces cerevisiae.

    PubMed

    Bach, M; Sander, P; Haase, W; Reiländer, H

    1996-01-01

    The cDNA for the mouse 5HT5A receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. The NH2-terminal end of the receptor gene was fused to the Bacillus macerans (1-3, 1-4)-beta-glucanase signal sequence to ensure proper membrane insertion and to the c-myc epitope to permit immunological detection of the heterologously expressed protein. In the resulting episomal yeast expression plasmid pCNNmm5HT5A the modified 5HT5A gene is under the transcriptional control of the endopeptidase B gene promoter (PRB1). After transformation of the vector into the protease deficient S. cerevisiae strain cI3-ABYS-86, recombinant clones were examined for the presence of functional receptor by radioligand binding using [3H]LSD. Whole cells as well as crude membrane preparations of recombinant clones showed saturable binding of the receptor with a KD of approximately 2.2 nM. The pharmacological properties for the heterologous expressed receptor, estimated by ligand-displacement studies using certain serotonin agonists and antagonists, were comparable to those reported for the receptor expressed in mammalian systems. Western blot analysis of membranes prepared from a recombinant clone using the monoclonal antibody 9E10, directed against the c-myc epitope of the modified receptor, revealed an apparent molecular mass of about 43 kDa for the receptor expressed in S. cerevisiae. Glycosylation of the receptor was analysed by EndoH digestion. A heat shock of recombinant yeast significantly increased the number of specific binding sites per cell and also improved the affinity of the receptor. Immunogold electron microscopy was used to study the localization of the heterologously expressed protein within the yeast cells.

  8. Sumoylated NHR-25/NR5A Regulates Cell Fate during C. elegans Vulval Development

    PubMed Central

    Bernal, Teresita; Ashrafi, Kaveh; Asahina, Masako; Yamamoto, Keith R.

    2013-01-01

    Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here, we examine how a single regulatory input, sumoylation, differentially modulates the activity of a conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on at least four lysines. Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1, demonstrating that sumoylation has been strongly conserved within the NR5A family. We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription, and that NHR-25 activity was enhanced in vivo upon loss of sumoylation. Knockdown of smo-1 mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells (VPCs) during development. Importantly, however, overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate. Moreover, SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs. That is, accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development, but as cells began dividing, a smo-1-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs, intermediate levels in 2° fated VPCs, and low levels in 3° fated VPCs. We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner. PMID:24348269

  9. Genomic organization of the human SCN5A gene encoding the cardiac sodium channel

    SciTech Connect

    Wang, Qing; Li, Zhizhong; Shen, Jiaxiang; Keating, M.T.

    1996-05-15

    The voltage-gated cardiac sodium channel, SCN5A, is responsible for the initial upstroke of the action potential. Mutations in the human SCN5A gene cause susceptibility to cardiac arrhythmias and sudden death in the long QT syndrome (LQT). In this report we characterize the genomic structure of SCN5A. SCN5A consists of 28 exons spanning approximately 80 kb on chromosome 3p21. We describe the sequences of all intron/exon boundaries and a dinucleotide repeat polymorphism in intron 16. Oligonucleotide primers based on exon-flanking sequences amplify all SCN5A exons by PCR. This work establishes the complete genomic organization of SCN5A and will enable high-resolution analyses of this locus for mutations associated with LQT and other phenotypes for which SCN5A may be a candidate gene. 40 refs., 4 figs., 2 tabs.

  10. Biological functions of macrophage-derived Wnt5a, and its roles in human diseases

    PubMed Central

    Shao, Yue; Zheng, Qianqian; Wang, Wei; Xin, Na; Song, Xiaowen; Zhao, Chenghai

    2016-01-01

    Wnt5a is implicated in development and tissue homeostasis by activating β-catenin-independent pathway. Excessive production of Wnt5a is related to some human diseases. Macrophage recruitment is a character of inflammation and cancer, therefore macrophage-derived Wnt5a is supposed to be a player in these conditions. Actually, macrophage-derived Wnt5a maintains macrophage immune function, stimulates pro-inflammatory cytokine release, and induces angiogenesis and lymphangiogenesis. Furthermore, macrophage-derived Wnt5a is involved in insulin resistance, atherosclerosis and cancer. These findings indicate that macrophage-derived Wnt5a may be a target in the treatment of these diseases. Notably, unlike macrophages, the exact role of macrophage-derived Wnt5a in bacterial infection remains largely unknown. PMID:27608847

  11. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  12. Complex Pattern of Resistance-Associated Substitutions of Hepatitis C Virus after Daclatasvir/Asunaprevir Treatment Failure

    PubMed Central

    Hasebe, Chitomi; Osaki, Yukio; Joko, Kouji; Yagisawa, Hitoshi; Sakita, Shinya; Okushin, Hiroaki; Satou, Takashi; Hisai, Hiroyuki; Abe, Takehiko; Tsuji, Keiji; Tamada, Takashi; Kobashi, Haruhiko; Mitsuda, Akeri; Ide, Yasushi; Ogawa, Chikara; Tsuruta, Syotaro; Takaguchi, Kouichi; Murakawa, Miyako; Asahina, Yasuhiro; Enomoto, Nobuyuki; Izumi, Namiki

    2016-01-01

    Backgrounds & Aims We aimed to clarify the characteristics of resistance-associated substitutions (RASs) after treatment failure with NS5A inhibitor, daclatasvir (DCV) in combination with NS3/4A inhibitor, asunaprevir (ASV), in patients with chronic hepatitis C virus genotype 1b infection. Methods This is a nationwide multicenter study conducted by the Japanese Red Cross Liver Study Group. The sera were obtained from 68 patients with virological failure after 24 weeks of DCV/ASV treatment. RASs in NS5A and NS3 were determined by population sequencing. Results The frequency of signature RASs at position D168 of NS3 was 68%, and at positions L31 and Y93 of NS5A was 79 and 76%, respectively. The frequency of dual signature RASs in NS5A (L31-RAS and Y93-RAS) was 63%. RASs at L28, R30, P32, Q54, P58, and A92 in addition to dual signature RAS were detected in 5, 5, 1, 22, 2, and 0 patients, respectively. In total, triple, quadruple, and quintuple RASs in combination with dual signature RAS were detected in 35, 10, and 1.5% patients, respectively. These RASs were detected in patients without baseline RASs or who prematurely discontinued therapy. Co-existence of D168 RAS in NS3 and L31 and/or Y93 RAS in NS5A was observed in 62% of patients. Conclusion Treatment-emergent RASs after failure with DCV/ASV combination therapy are highly complex in more than 50% of the patients. The identification of complex RAS patterns, which may indicate high levels of resistance to NS5A inhibitors, highlights the need for RAS sequencing when considering re-treatment with regimens including NS5A inhibitors. PMID:27776192

  13. Clinical and molecular characterization of 40 patients with classic Ehlers–Danlos syndrome: identification of 18 COL5A1 and 2 COL5A2 novel mutations

    PubMed Central

    2013-01-01

    Background Classic Ehlers–Danlos syndrome (cEDS) is a rare autosomal dominant connective tissue disorder that is primarily characterized by skin hyperextensibility, abnormal wound healing/atrophic scars, and joint hypermobility. A recent study demonstrated that more than 90% of patients who satisfy all of these major criteria harbor a type V collagen (COLLV) defect. Methods This cohort included 40 patients with cEDS who were clinically diagnosed according to the Villefranche nosology. The flowchart that was adopted for mutation detection consisted of sequencing the COL5A1 gene and, if no mutation was detected, COL5A2 analysis. In the negative patients the presence of large genomic rearrangements in COL5A1 was investigated using MLPA, and positive results were confirmed via SNP-array analysis. Results We report the clinical and molecular characterization of 40 patients from 28 families, consisting of 14 pediatric patients and 26 adults. A family history of cEDS was present in 9 patients. The majority of the patients fulfilled all the major diagnostic criteria for cEDS; atrophic scars were absent in 2 females, skin hyperextensibility was not detected in a male and joint hypermobility was negative in 8 patients (20% of the entire cohort). Wide inter- and intra-familial phenotypic heterogeneity was observed. We identified causal mutations with a detection rate of approximately 93%. In 25/28 probands, COL5A1 or COL5A2 mutations were detected. Twenty-one mutations were in the COL5A1 gene, 18 of which were novel (2 recurrent). Of these, 16 mutations led to nonsense-mediated mRNA decay (NMD) and to COLLV haploinsufficiency and 5 mutations were structural. Two novel COL5A2 splice mutations were detected in patients with the most severe phenotypes. The known p. (Arg312Cys) mutation in the COL1A1 gene was identified in one patient with vascular-like cEDS. Conclusions Our findings highlight that the three major criteria for cEDS are useful and sufficient for cEDS clinical

  14. WNT5A signaling contributes to Aβ-induced neuroinflammation and neurotoxicity.

    PubMed

    Li, Bei; Zhong, Ling; Yang, Xiangling; Andersson, Tommy; Huang, Min; Tang, Shao-Jun

    2011-01-01

    Neurodegenration is a pathological hallmark of Alzheimer's disease (AD), but the underlying molecular mechanism remains elusive. Here, we present evidence that reveals a crucial role of Wnt5a signaling in this process. We showed that Wnt5a and its receptor Frizzled-5 (Fz5) were up-regulated in the AD mouse brain, and that beta-amyloid peptide (Aβ), a major constituent of amyloid plaques, stimulated Wnt5a and Fz5 expression in primary cortical cultures; these observations indicate that Wnt5a signaling could be aberrantly activated during AD pathogenesis. In support of such a possibility, we observed that inhibition of Wnt5a signaling attenuated while activation of Wnt5a signaling enhanced Aβ-evoked neurotoxicity, suggesting a role of Wnt5a signaling in AD-related neurodegeneration. Furthermore, we also demonstrated that Aβ-induced neurotoxicity depends on inflammatory processes, and that activation of Wnt5a signaling elicited the expression of proinflammatory cytokines IL-1β and TNF-α whereas inhibition of Wnt5a signaling attenuated the Aβ-induced expression of the cytokines in cortical cultures. Our findings collectively suggest that aberrantly up-regulated Wnt5a signaling is a crucial pathological step that contributes to AD-related neurodegeneration by regulating neuroinflammation.

  15. Drosophila SLC5A11 Mediates Hunger by Regulating K(+) Channel Activity.

    PubMed

    Park, Jin-Yong; Dus, Monica; Kim, Seonil; Abu, Farhan; Kanai, Makoto I; Rudy, Bernardo; Suh, Greg S B

    2016-08-08

    Hunger is a powerful drive that stimulates food intake. Yet, the mechanism that determines how the energy deficits that result in hunger are represented in the brain and promote feeding is not well understood. We previously described SLC5A11-a sodium/solute co-transporter-like-(or cupcake) in Drosophila melanogaster, which is required for the fly to select a nutritive sugar over a sweeter nonnutritive sugar after periods of food deprivation. SLC5A11 acts on approximately 12 pairs of ellipsoid body (EB) R4 neurons to trigger the selection of nutritive sugars, but the underlying mechanism is not understood. Here, we report that the excitability of SLC5A11-expressing EB R4 neurons increases dramatically during starvation and that this increase is abolished in the SLC5A11 mutation. Artificial activation of SLC5A11-expresssing neurons is sufficient to promote feeding and hunger-driven behaviors; silencing these neurons has the opposite effect. Notably, SLC5A11 transcript levels in the brain increase significantly when flies are starved and decrease shortly after starved flies are refed. Furthermore, expression of SLC5A11 is sufficient for promoting hunger-driven behaviors and enhancing the excitability of SLC5A11-expressing neurons. SLC5A11 inhibits the function of the Drosophila KCNQ potassium channel in a heterologous expression system. Accordingly, a knockdown of dKCNQ expression in SLC5A11-expressing neurons produces hunger-driven behaviors even in fed flies, mimicking the overexpression of SLC5A11. We propose that starvation increases SLC5A11 expression, which enhances the excitability of SLC5A11-expressing neurons by suppressing dKCNQ channels, thereby conferring the hunger state.

  16. Stat5a increases lactation of dairy cow mammary gland epithelial cells cultured in vitro.

    PubMed

    Liu, Xiao Fei; Li, Meng; Li, Qing Zhang; Lu, Li Min; Tong, Hui Li; Gao, Xue Jun

    2012-10-01

    Signal transducer and activator of transcription 5a (Stat5a) transduces signals of extracellular cytokines and growth factors to the nucleus of mammary gland epithelial cells and thereby regulates gene transcription during pregnancy, lactation, and weaning. However, its function on the milk production of dairy cows needs further investigation. In this experiment, the effects of Stat5a on lactation ability of dairy cow mammary gland epithelial cells (DCMECs) were analyzed. Eukaryotic expression vector pcDNA3.1+-stat5a-αS1 was constructed by inserting stat5a gene into the plasmid vector pcDNA3.1+ and replacing CMV promoter with α-S1-casein 5' flanking sequence. The recombinant vector was stably transfected into DCMECs after geneticin (G418) selection. The proliferation and viability of DCMECs, expression of β-casein and stat5a gene, and the content of lactose were detected. The results showed that stat5a gene in eukaryotic expression vector pcDNA3.1+-stat5a-αS1 was highly expressed in DCMECs and could increase the lactation ability of DCMECs. The associativity of Stat5a with nutrients on the lactation ability of DCMECs was also evaluated. Lysine (Lys), methionine (Met), sodium acetate, β-sodium hydroxybutyrate, and glucose all had more positive effects on the lactation function of DCMECs after pcDNA3.1+-stat5a-αS1 transfection. The proliferation and viability of DCMECs, expression of β-casein and stat5a gene, and contents of lactose and triglyceride were detected. The results revealed that nutrients could promote expression of Stat5a gene to increase lactation of DCMECs. These data help to clarify the function of stat5 gene on lactation and gene regulatory networks linking stat5a.

  17. Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway

    PubMed Central

    McDonald, Chloë R.; Cahill, Lindsay S.; Ho, Keith T.; Yang, Jimmy; Kim, Hani; Silver, Karlee L.; Ward, Peter A.; Mount, Howard T.; Liles, W. Conrad; Sled, John G.; Kain, Kevin C.

    2015-01-01

    The in utero environment profoundly impacts childhood neurodevelopment and behaviour. A substantial proportion of pregnancies in Africa are at risk of malaria in pregnancy (MIP) however the impact of in utero exposure to MIP on fetal neurodevelopment is unknown. Complement activation, in particular C5a, may contribute to neuropathology and adverse outcomes during MIP. We used an experimental model of MIP and standardized neurocognitive testing, MRI, micro-CT and HPLC analysis of neurotransmitter levels, to test the hypothesis that in utero exposure to malaria alters neurodevelopment through a C5a-C5aR dependent pathway. We show that malaria-exposed offspring have persistent neurocognitive deficits in memory and affective-like behaviour compared to unexposed controls. These deficits were associated with reduced regional brain levels of major biogenic amines and BDNF that were rescued by disruption of C5a-C5aR signaling using genetic and functional approaches. Our results demonstrate that experimental MIP induces neurocognitive deficits in offspring and suggest novel targets for intervention. PMID:26402732

  18. Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

    SciTech Connect

    Briolay, A.; Lencel, P.; Caverzasio, J.; Buchet, R.; Magne, D.

    2013-01-18

    Highlights: ► Ankylosing spondylitis (AS) leads to bone fusions and ankylosis. ► TNF-α stimulates osteoblasts through growth factors in AS. ► We compare the involvement of canonical vs non-canonical Wnt signaling. ► Canonical Wnt signaling is not involved in TNF-α effects in differentiating hMSCs. ► TNF-α stimulates osteoblasts through Wnt5a autocrine secretion in hMSCs. -- Abstract: Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased

  19. Proteomics profiling of Madin-Darby canine kidney plasma membranes reveals Wnt-5a involvement during oncogenic H-Ras/TGF-beta-mediated epithelial-mesenchymal transition.

    PubMed

    Chen, Yuan-Shou; Mathias, Rommel A; Mathivanan, Suresh; Kapp, Eugene A; Moritz, Robert L; Zhu, Hong-Jian; Simpson, Richard J

    2011-02-01

    Epithelial-mesenchymal transition (EMT) describes a process whereby polarized epithelial cells with restricted migration transform into elongated spindle-shaped mesenchymal cells with enhanced motility and invasiveness. Although there are some molecular markers for this process, including the down-regulation of E-cadherin, our understanding of plasma membrane (PM) and associated proteins involved in EMT is limited. To specifically explore molecular alterations occurring at the PM, we used the cationic colloidal silica isolation technique to purify PM fractions from epithelial Madin-Darby canine kidney cells during Ras/TGF-β-mediated EMT. Proteins in the isolated membrane fractions were separated by one-dimensional SDS-PAGE and subjected to nano-LC-MS/MS-based protein identification. In this study, the first membrane protein analysis of an EMT model, we identified 805 proteins and determined their differential expression using label-free spectral counting. These data reveal that Madin-Darby canine kidney cells switch from cadherin-mediated to integrin-mediated adhesion following Ras/TGF-β-mediated EMT. Thus, during the EMT process, E-cadherin, claudin 4, desmoplakin, desmoglein-2, and junctional adhesion molecule A were down-regulated, whereas integrins α6β1, α3β1, α2β1, α5β1, αVβ1, and αVβ3 along with their extracellular ligands collagens I and V and fibronectin had increased expression levels. Conspicuously, Wnt-5a expression was elevated in cells undergoing EMT, and transient Wnt-5a siRNA silencing attenuated both cell migration and invasion in these cells. Furthermore, Wnt-5a expression suppressed canonical Wnt signaling induced by Wnt-3a. Wnt-5a may act through the planar cell polarity pathway of the non-canonical Wnt signaling pathway as several of the components and modulators (Wnt-5a, -5b, frizzled 6, collagen triple helix repeat-containing protein 1, tyrosine-protein kinase 7, RhoA, Rac, and JNK) were found to be up-regulated during Ras

  20. Protein inference: A protein quantification perspective.

    PubMed

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/.

  1. Major involvement of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) in uptake of biotin and pantothenic acid by human brain capillary endothelial cells.

    PubMed

    Uchida, Yasuo; Ito, Katsuaki; Ohtsuki, Sumio; Kubo, Yoshiyuki; Suzuki, Takashi; Terasaki, Tetsuya

    2015-07-01

    The purpose of this study was to clarify the expression of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) and its contribution to the supply of biotin and pantothenic acid to the human brain via the blood-brain barrier. DNA microarray and immunohistochemical analyses confirmed that SLC5A6 is expressed in microvessels of human brain. The absolute expression levels of SLC5A6 protein in isolated human and monkey brain microvessels were 1.19 and 0.597 fmol/μg protein, respectively, as determined by a quantitative targeted absolute proteomics technique. Using an antibody-free method established by Kubo et al. (2015), we found that SLC5A6 was preferentially localized at the luminal membrane of brain capillary endothelium. Knock-down analysis using SLC5A6 siRNA showed that SLC5A6 accounts for 88.7% and 98.6% of total [(3) H]biotin and [(3) H]pantothenic acid uptakes, respectively, by human cerebral microvascular endothelial cell line hCMEC/D3. SLC5A6-mediated transport in hCMEC/D3 was markedly inhibited not only by biotin and pantothenic acid, but also by prostaglandin E2, lipoic acid, docosahexaenoic acid, indomethacin, ketoprofen, diclofenac, ibuprofen, phenylbutazone, and flurbiprofen. This study is the first to confirm expression of SLC5A6 in human brain microvessels and to provide evidence that SLC5A6 is a major contributor to luminal uptake of biotin and pantothenic acid at the human blood-brain barrier. In humans, it was unclear (not concluded) about what transport system at the blood-brain barrier (BBB) is responsible for the brain uptakes of two vitamins, biotin and pantothenic acid, which are necessary for brain proper function. This study clarified for the first time that the solute carrier 5A6/Na(+) -dependent multivitamin transporter SLC5A6/SMVT is responsible for the supplies of biotin and pantothenic acid into brain across the BBB in humans. DHA, docosahexaenoic acid; NSAID, non-steroidal anti-inflammatory drug; PGE2, prostaglandin E2.

  2. Research on Superplasticity of 5A90-O Aluminum Alloy Sheet

    NASA Astrophysics Data System (ADS)

    Li, Zhi-qiang; LU, Wen-lin; Zhang, Feng; Hu, Fengjiao; Wang, Yong

    2017-03-01

    The influence of temperature and strain rate on the 5A90-O had been researched by costant strain-rate tensile method. The result shows that, although 5A90-O is not an ideal superplastic material, under certain conditions, with a large extension rate, the maximum elongation is193.6%. The strain rate has a significant impact on the flow stress and tensile strength under T=375°C∼500°C. With the decrease of strain rate, the load is reduced. Another important factor that affects the flow stress of 5A90 is temperature. Under the same strain rate, the flow stress of 5A90 is lower with the temperature increase. We choose the Backofen law as the constitutive equation of 5A90, the best condition of 5A90 surperplastic deformation is T=400°C, ε = 0.005s‑1.

  3. 46 CFR 30.10-5a - Cargo area-TB/ALL.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Cargo area-TB/ALL. 30.10-5a Section 30.10-5a Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS GENERAL PROVISIONS Definitions § 30.10-5a Cargo area—TB/ALL. The term cargo area means that part of a vessel that includes the cargo tanks...

  4. 46 CFR 30.10-5a - Cargo area-TB/ALL.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Cargo area-TB/ALL. 30.10-5a Section 30.10-5a Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS GENERAL PROVISIONS Definitions § 30.10-5a Cargo area—TB/ALL. The term cargo area means that part of a vessel that includes the cargo tanks...

  5. C5a Induces the Synthesis of IL-6 and TNF-α in Rat Glomerular Mesangial Cells through MAPK Signaling Pathways

    PubMed Central

    Ji, Mingde; Lu, Yanlai; Zhao, Chenhui; Gao, Wenxing; He, Fengxia; Zhang, Jing; Zhao, Dan; Qiu, Wen; Wang, Yingwei

    2016-01-01

    Inflammatory response has been reported to contribute to the renal lesions in rat Thy-1 nephritis (Thy-1N) as an animal model of human mesangioproliferative glomerulonephritis (MsPGN). Besides C5b-9 complex, C5a is also a potent pro-inflammatory mediator and correlated to severity of various nephritic diseases. However, the role of C5a in mediating pro-inflammatory cytokine production in rats with Thy-1N is poorly defined. In the present studies, the levels of C5a, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were first determined in the renal tissues of rats with Thy-1N. Then, the expression of IL-6 and TNF-α was detected in rat glomerular mesangial cells (GMC) stimulated with our recombinant rat C5a in vitro. Subsequently, the activation of mitogen-activated protein kinase (MAPK) signaling pathways (p38 MAPK, ERK1/2 and JNK) and their roles in the regulation of IL-6 and TNF-α production were examined in the GMC induced by C5a. The results showed that the levels of C5a, IL-6 and TNF-α were markedly increased in the renal tissues of Thy-1N rats. Rat C5a stimulation in vitro could up-regulate the expression of IL-6 and TNF-α in rat GMC, and the activation of MAPK signaling pathways was involved in the induction of IL-6 and TNF-α. Mechanically, p38 MAPK activation promoted IL-6 production, while either ERK1/2 or JNK activation promoted TNF-α production in the GMC with exposure to C5a. Taken together, these data implicate that C5a induces the synthesis of IL-6 and TNF-α in rat GMC through the activation of MAPK signaling pathways. PMID:27583546

  6. Wnt5a-Dopamine D2 Receptor Interactions Regulate Dopamine Neuron Development via Extracellular Signal-regulated Kinase (ERK) Activation*

    PubMed Central

    Yoon, Sehyoun; Choi, Mi-hyun; Chang, Min Seok; Baik, Ja-Hyun

    2011-01-01

    The dopamine D2 receptor (D2R) plays an important role in mesencephalic dopaminergic neuronal development, particularly coupled with extracellular signal-regulated kinase (ERK) activation. Wnt5a protein is known to regulate the development of dopaminergic neurons. We analyzed the effect of Wnt5a on dopaminergic neuron development in mesencephalic primary cultures from wild-type (WT) and D2R knock-out (D2R−/−) mice. Treatment with Wnt5a increased the number and neuritic length of dopamine neurons in primary mesencephalic neuronal cultures from WT mice, but not from D2R−/− mice. The effect of Wnt5a was completely blocked by treatment with D2R antagonist or inhibitors of MAPK or EGFR. Wnt5a-mediated ERK activation in mesencephalic neuronal cultures was inhibited by treatment of D2R antagonist and EGFR inhibitors in WT mice. However, these regulations were not observed for D2R−/− mice. Co-immunoprecipitation and displacement of [3H]spiperone from D2R by Wnt5a demonstrated that Wnt5a could bind with D2R. This interaction was confirmed by GST pulldown assays demonstrating that the domain including transmembrane domain 4, second extracellular loop, and transmembrane domain 5 of D2R binds to Wnt5a. These results suggest that the interaction between D2R and Wnt5a has an important role in dopamine neuron development in association with EGFR and the ERK pathway. PMID:21454669

  7. Grazoprevir/elbasvir combination therapy for HCV infection

    PubMed Central

    Vallet-Pichard, Anaïs; Pol, Stanislas

    2016-01-01

    Interferon-free regimens combine different second-wave direct-acting antiviral agents (DAAs), which target the main viral proteins involved in the replication cycle of hepatitis C virus (HCV): NS3/4A protease inhibitors (simeprevir or paritaprevir boosted by ritonavir), NS5B nucleos(t)idic (sofosbuvir) and nonnucleos(t)idic (dasabuvir) polymerase inhibitors, NS5A replication complex inhibitors (daclatasvir, ledipasvir, elbasvir, velpatasvir). Combinations of two or three DAAs, given for 8–24 weeks reach sustained virology response (SVR) rates greater than 90% with good tolerance. SVR rates and safety are similar in clinical trials and in real life, usually higher than 95% in the per-protocol analysis. Next-generation DAAs are now expected. To be competitive, these new combinations need to prove their added value regarding the pill burden, the reduced duration of treatment, the drug–drug interaction profile and safety. Zepatier is a fixed-dose combination product coformulating MK-5172 [grazoprevir (GZR), 100 mg QD] and MK-8742 [elbasvir or (EBR) 50 mg QD]: it combines highly potent inhibitors of the HCV NS3/4A protease and NS5A replication complex, respectively. This review provides a summary of the main evidence available for the use of GZR/EBR and highlights the strength of this combination. PMID:28286567

  8. C5A Protects Macaques from Vaginal Simian-Human Immunodeficiency Virus Challenge.

    PubMed

    Veazey, Ronald S; Chatterji, Udayan; Bobardt, Michael; Russell-Lodrigue, Kasi E; Li, Jian; Wang, Xiaolei; Gallay, Philippe A

    2015-11-09

    A safe and effective vaginal microbicide could decrease human immunodeficiency virus (HIV) transmission in women. Here, we evaluated the safety and microbicidal efficacy of a short amphipathic peptide, C5A, in a rhesus macaque model. We found that a vaginal application of C5A protects 89% of the macaques from a simian-human immunodeficiency virus (SHIV-162P3) challenge. We observed no signs of lesions or inflammation in animals vaginally treated with repeated C5A applications. With its noncellular cytotoxic activity and rare mechanism of action, C5A represents an attractive microbicidal candidate.

  9. A novel C-terminal truncating NR5A1 mutation in dizygotic twins.

    PubMed

    Hattori, Atsushi; Zukeran, Hiroaki; Igarashi, Maki; Toguchi, Suzuka; Toubaru, Yuji; Inoue, Takanobu; Katoh-Fukui, Yuko; Fukami, Maki

    2017-01-01

    Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is a nuclear receptor involved in gonadal and adrenal development. We identified a novel C-terminally truncating NR5A1 mutation, p.Leu423Trpfs*7, in dizygotic twins with 46,XY disorders of sex development. Our results highlight the functional importance of C-terminal region of NR5A1 and indicate that NR5A1 mutations can be associated with intrafamilial phenotypic variations, progressive testicular dysfunction, hypogonadotropic hypogonadism, and borderline adrenal dysfunction.

  10. Reduced expression of C5a receptors on neutrophils from cord blood

    PubMed Central

    Nybo, M.; Sorensen, O.; Leslie, R; Wang, P.

    1998-01-01

    AIM—To describe further functional deficiencies of neonatal neutrophils by measuring the expression of C5a receptors.
METHODS—C5a uptake was measured using flow cytometry with fluorescein isothiocynate labelled recombinant C5a. The response of neutrophils to stimulation with C5a and fMLP was tested by measuring migration and exocytosis of myeloperoxidase and lactoferrin.
RESULTS—C5a mean fluorescence on neutrophils from neonates was significantly lower (22.4 (SD 3.5)) than in adult controls (31.5 (3.1)). Neutrophils from neonates migrated poorly towards both C5a and fMLP compared with those from adult controls. Exocytosis of myeloperoxidase, but not lactoferrin from neonatal neutrophils stimulated with C5a, was significantly lower than in adult controls. fMLP stimulation, on the other hand, resulted in significantly higher exocytosis in neonates.
CONCLUSION—The lower expression of C5a receptors on neutrophils from neonates could be related to reduced C5a mediated exocytosis of myeloperoxidase.

 PMID:9577284

  11. Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

    PubMed Central

    Graf, Michael; Blaby, Ian K.; Makkay, Andrea M.; Starosta, Agata L.; Papke, R. Thane; Oshima, Tairo; Wilson, Daniel N.

    2016-01-01

    Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in Haloferax volcanii in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification in Hfx. volcanii that was experimentally tested. Analysis of aIF5A from Hfx. volcanii by LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene (HVO_1958) in agmatine synthesis. The agmatinase-like gene (HVO_2299) was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) from S. cerevisiae was shown to transfer 4-aminobutyl moiety from spermidine to aIF5A from Hfx. volcanii in vitro. However, at least under conditions tested, this transfer was not observed with the Hfx. volcanii DHS. Furthermore, the growth of Hfx. volcanii was not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis in Hfx. volcanii that differs from the canonical eukaryotic pathway, paving the way for further studies. PMID:28053595

  12. A novel C-terminal truncating NR5A1 mutation in dizygotic twins

    PubMed Central

    Hattori, Atsushi; Zukeran, Hiroaki; Igarashi, Maki; Toguchi, Suzuka; Toubaru, Yuji; Inoue, Takanobu; Katoh-Fukui, Yuko; Fukami, Maki

    2017-01-01

    Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is a nuclear receptor involved in gonadal and adrenal development. We identified a novel C-terminally truncating NR5A1 mutation, p.Leu423Trpfs*7, in dizygotic twins with 46,XY disorders of sex development. Our results highlight the functional importance of C-terminal region of NR5A1 and indicate that NR5A1 mutations can be associated with intrafamilial phenotypic variations, progressive testicular dysfunction, hypogonadotropic hypogonadism, and borderline adrenal dysfunction. PMID:28326187

  13. Loss Of Klotho During Melanoma Progression Leads To Increased Filamin Cleavage, Increased Wnt5A Expression and Enhanced Melanoma Cell Motility

    PubMed Central

    Camilli, Tura C.; Xu, Mai; O'Connell, Michael P.; Chien, Bonnie; Frank, Brittany P.; Subaran, Sarah; Indig, Fred E.; Morin, Patrice J.; Hewitt, Stephen M.; Weeraratna, Ashani T.

    2010-01-01

    Summary We have previously shown that Wnt5A-mediated signaling can promote melanoma metastasis. It has been shown that Wnt signaling is antagonized by the protein Klotho, which has been implicated in aging. We show here that in melanoma cells, expressions of Wnt5A and Klotho are inversely correlated. In the presence of recombinant Klotho (rKlotho) we show that Wnt5A internalization and signaling is decreased in high Wnt5A expressing cells. Moreover, in the presence of rKlotho, we observe an increase in Wnt5A remaining in the medium, coincident with an increase in sialidase activity and decrease in syndecan expression. These effects can be inhibited using a sialidase inhibitor. In addition to its effects on Wnt5A internalization, we also demonstrate that Klotho decreases melanoma cell invasive potential by a second mechanism, that involves the inhibition of calpain and a resultant decrease in filamin cleavage, which we demonstrate is critical for melanoma cell motility. PMID:20955350

  14. Expression of Wnt5a and its receptor Fzd2 is changed in the spinal cord of adult amyotrophic lateral sclerosis transgenic mice

    PubMed Central

    Li, Xiaojin; Guan, Yingjun; Chen, Yanchun; Zhang, Caixia; Shi, Caixing; Zhou, Fenghua; Yu, Li; Juan, Juan; Wang, Xin

    2013-01-01

    Wnt5a, a member of the Wnt gene family, encodes a cysteine-rich growth factor involved in signal transduction during growth and differentiation. The Fzd2 gene codes for a cell membrane receptor called Frizzled-2 have a structure similar to G protein coupled receptors. The extracellular N-terminal of the Fzd2 receptor has a cysteine-rich domain (CRD) that binds Wnt ligands and thus primes the Wnt signal pathway. Downregulation of the Wnt signal pathway occurs in neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS). However, little is known about Wnt5a/Fzd2 signaling in mammalian nerve cells, and it is not clear whether Wnt5a or Fzd2 functioning are changed in ALS. The influence of Wnt5a and Fzd2 signal transduction pathway on ALS was investigated in adult SOD1G93A transgenic mice. Changes in Wnt5a and Fzd2 expression in the spinal cord of SOD1G93A transgenic mice (ALS), SOD1G93A transfected NSC-34 cells, and primary cultures of astrocytes from SOD1G93A transgenic mice were detected by immunofluorescent staining, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting. The results provide further insight into the role of Wnt5a and Fzd2 in the pathogenesis of ALS transgenic mice, which provides evidence that should help in the search for treatments of ALS. PMID:23826406

  15. Intracellular Protein Delivery for Treating Breast Cancer

    DTIC Science & Technology

    2014-08-01

    nanocapsules with specific cancer cell targeting ligands; Task 3. Preparing and testing of MMP activatable cell penetrating peptides (ACCPs)-coupled...AD_________________ Award Number: W81XWH-11-1-0371 TITLE: Intracellular Protein Delivery for Treating Breast Cancer PRINCIPAL INVESTIGATOR: Dr...SUBTITLE Intracellular Protein Delivery for Treating Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-11-1-0371 5c. PROGRAM ELEMENT NUMBER 6

  16. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 5

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. Detail drawings of several assemblies and subassemblies are given. This is the fifth book of volume 4.

  17. Semaphorin 5A inhibits synaptogenesis in early postnatal- and adult-born hippocampal dentate granule cells

    PubMed Central

    Duan, Yuntao; Wang, Shih-Hsiu; Song, Juan; Mironova, Yevgeniya; Ming, Guo-li; Kolodkin, Alex L; Giger, Roman J

    2014-01-01

    Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A−/− GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A−/− mutants, PlexinA2−/− mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A−/− mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders. DOI: http://dx.doi.org/10.7554/eLife.04390.001 PMID:25313870

  18. Carbon-Carbon Bond Cleavage Reaction: Synthesis of Multisubstituted Pyrazolo[1,5-a]pyrimidines.

    PubMed

    Saikia, Pallabi; Gogoi, Sanjib; Boruah, Romesh C

    2015-07-02

    A new carbon-carbon bond cleavage reaction was developed for the efficient synthesis of multisubstituted pyrazolo[1,5-a]pyrimidines. This base induced reaction of 1,3,5-trisubstituted pentane-1,5-diones and substituted pyrazoles afforded good yields of the pyrazolo[1,5-a]pyrimidines.

  19. A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma

    PubMed Central

    Köhl, Jörg; Baelder, Ralf; Lewkowich, Ian P.; Pandey, Manoj K.; Hawlisch, Heiko; Wang, Lihua; Best, Jennifer; Herman, Nancy S.; Sproles, Alyssa A.; Zwirner, Jörg; Whitsett, Jeffrey A.; Gerard, Craig; Sfyroera, Georgia; Lambris, John D.; Wills-Karp, Marsha

    2006-01-01

    Complement component 5 (C5) has been described as either promoting or protecting against airway hyperresponsiveness (AHR) in experimental allergic asthma, suggesting pleomorphic effects of C5. Here we report that local pharmacological targeting of the C5a receptor (C5aR) prior to initial allergen sensitization in murine models of inhalation tolerance or allergic asthma resulted in either induction or marked enhancement of Th2-polarized immune responses, airway inflammation, and AHR. Importantly, C5aR-deficient mice exhibited a similar, increased allergic phenotype. Pulmonary allergen exposure in C5aR-targeted mice resulted in increased sensitization and accumulation of CD4+CD69+ T cells associated with a marked increase in pulmonary myeloid, but not plasmacytoid, DC numbers. Pulmonary DCs from C5aR-targeted mice produced large amounts of CC chemokine ligand 17 (CCL17) and CCL22 ex vivo, suggesting a negative impact of C5aR signaling on pulmonary homing of Th2 cells. In contrast, C5aR targeting in sensitized mice led to suppressed airway inflammation and AHR but was still associated with enhanced production of Th2 effector cytokines. These data suggest a dual role for C5a in allergic asthma, i.e., protection from the development of maladaptive type 2 immune responses during allergen sensitization at the DC/T cell interface but enhancement of airway inflammation and AHR in an established inflammatory environment. PMID:16511606

  20. C5a Increases the Injury to Primary Neurons Elicited by Fibrillar Amyloid Beta

    PubMed Central

    Hernandez, Michael X.; Namiranian, Pouya; Nguyen, Eric; Fonseca, Maria I.

    2017-01-01

    C5aR1, the proinflammatory receptor for C5a, is expressed in the central nervous system on microglia, endothelial cells, and neurons. Previous work demonstrated that the C5aR1 antagonist, PMX205, decreased amyloid pathology and suppressed cognitive deficits in two Alzheimer's Disease (AD) mouse models. However, the cellular mechanisms of this protection have not been definitively demonstrated. Here, primary cultured mouse neurons treated with exogenous C5a show reproducible loss of MAP-2 staining in a dose-dependent manner within 24 hr of treatment, indicative of injury to neurons. This injury is prevented by the C5aR1 antagonist PMX53, a close analog of PMX205. Furthermore, primary neurons derived from C5aR1 null mice exhibited no MAP-2 loss after exposure to the highest concentration of C5a tested. Primary mouse neurons treated with both 100 nM C5a and 5 µM fibrillar amyloid beta (fAβ), to model what occurs in the AD brain, showed increased MAP-2 loss relative to either C5a or fAβ alone. Blocking C5aR1 with PMX53 (100 nM) blocked the loss of MAP2 in these primary neurons to the level seen with fAβ alone. Similar experiments with primary neurons derived from C5aR1 null mice showed a loss of MAP-2 due to fAβ treatment. However, the addition of C5a to the cultures did not enhance the loss of MAP-2 and the addition of PMX53 to the cultures did not change the MAP-2 loss in response to fAβ. Thus, at least part of the beneficial effects of C5aR1 antagonist in AD mouse models may be due to protection of neurons from the toxic effects of C5a. PMID:28078911

  1. Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

    SciTech Connect

    Sakisaka, Yukihiko; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi; Nemoto, Eiji

    2015-08-01

    Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression.

  2. Learning about Proteins

    MedlinePlus

    ... What Happens in the Operating Room? Learning About Proteins KidsHealth > For Kids > Learning About Proteins A A ... the foods you eat. continue Different Kinds of Protein Protein from animal sources, such as meat and ...

  3. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    PubMed

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

  4. Improved Activity of a Thermophilic Cellulase, Cel5A, from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass

    PubMed Central

    Chen, Zhiwei; Pereira, Jose H.; Liu, Hanbin; Tran, Huu M.; Hsu, Nathan S. Y.; Dibble, Dean; Singh, Seema; Adams, Paul D.; Sapra, Rajat; Hadi, Masood Z.; Simmons, Blake A.; Sale, Kenneth L.

    2013-01-01

    Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25–42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding. PMID:24244549

  5. Protein Microarray Technology

    PubMed Central

    Hall, David A.; Ptacek, Jason

    2007-01-01

    Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays. PMID:17126887

  6. Spatiotemporal Dynamics of Complement C5a Production within Bacterial Extracellular Polymeric Substance

    PubMed Central

    Conrad, Erin C.; Hsu, Yueh-Ya; Bortz, David M.; Younger, John G.

    2013-01-01

    Opsonization and anaphylatoxin production are early events in innate response to bacterial pathogens. Opsonization alone is frequently not lethal and production of anaphylatoxins, especially C5a, allows for recruitment of cellular defenses. Complement biochemistry is extensively studied and computational models have been previously reported. However, a critical feature of complement mediated attack is its spatial dependence: diffusion of mediators into and away from a bacterium is central to understanding C5a generation. Spatial dependence is especially important in biofilms, where diffusion limitation is crucial to bacterial counterdefense. Here we develop a model of opsonization and C5a production in the presence of a common blood borne pathogen, Staphylococcus epidermidis. Our results indicate that when complement attacks a single cell, diffusion into the extracellular polymeric substance (EPS) is complete within 10 msec and that production of C5a peaks over the next 15 minutes. When longer diffusion lengths (as in an EPS-rich biofilm) are incorporated, diffusion limitation appears such that the intensity and duration of C5a production is increased. However, the amount of C5a produced under several likely clinical scenarios where single cells or sparse biofilms are present is below the kD of the C5a receptor suggesting that complement activation by a single bacterium may be difficult to detect when diffusion is taken into account. PMID:23328643

  7. Association of MMP-3 (-1612 5A/6A) polymorphism with knee osteoarthritis in Thai population.

    PubMed

    Honsawek, Sittisak; Malila, Somkiat; Yuktanandana, Pongsak; Tanavalee, Aree; Deepaisarnsakul, Benjamad; Parvizi, Javad

    2013-02-01

    Osteoarthritis (OA) is a degenerative joint disorder resulting in destruction of articular cartilage, osteophyte formation, and subchondral bone sclerosis. In recent years, numerous genetic factors have been identified and implicated in causing osteoarthritis. One such genetic defect is a single nucleotide polymorphism at position -1612 of matrix metalloproteinase-3 (MMP-3) promoter region, known to lead to three possible genotypes, 5A/5A, 6A/6A, and 5A/6A. The purpose of this study was to investigate the association of MMP-3 -1612 5A/6A gene polymorphism with knee osteoarthritis in Thai population. Genotype distributions and allelic frequencies of MMP-3 -1612 5A/6A polymorphism were investigated in 200 participants (100 patients with knee osteoarthritis and 100 healthy controls). Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism. There was no statistically significant difference between the groups with respect to genotype distribution (P > 0.05). The 5A allele frequency was indicated as 15.5 %, and 6A allele was as 84.5 % in OA patients, whereas it was 10-90 % in the control group. Accordingly, the present study has indicated that the -1612 5A/6A polymorphism genotypes of MMP-3 gene promoter do not play a role in the development of osteoarthritis in the Thai population.

  8. Crystallization and preliminary X-ray analysis of RsbS from Moorella thermoacetica at 2.5 A resolution.

    PubMed

    Quin, Maureen; Newman, Joseph; Firbank, Susan; Lewis, Richard J; Marles-Wright, Jon

    2008-03-01

    The thermophilic bacterium Moorella thermoacetica possesses an rsb operon that is related to the genetic locus common to many Gram-positive bacteria that regulates the activity of the stress-responsive sigma factor sigma(B). One of the gene products of this operon is RsbS, a single STAS-domain protein that is a component of higher order assemblies in Bacillus subtilis known as 'stressosomes'. It is expected that similar complexes are found in M. thermoacetica, but in this instance regulating the biosynthesis of cyclic di-GMP, a ubiquitous secondary messenger. Selenomethionine-labelled MtRsbS protein was crystallized at room temperature using the hanging-drop vapour-diffusion method. Crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 51.07, b = 60.52, c = 89.28 A, diffracted to 2.5 A resolution on beamline I04 of the Diamond Light Source. The selenium substructure was solved using SHELX and it is believed that this represents the first reported ab initio crystal structure to be solved using diffraction data collected at DLS.

  9. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  10. Synthesis of 5a-carba-hexopyranoses and hexopyranosylamines, as well as 5a,5a'-dicarbadisaccharides, from 3,8-dioxatricyclo[4.2.1.0(2,4)]nonan-9-ol: glycosidase inhibitory activity of N-substituted 5a-carba-beta-gluco- and beta-galactopyranosylamines, and derivatives thereof.

    PubMed

    Ogawa, Seiichiro; Funayama, Sho; Okazaki, Kensuke; Ishizuka, Fumito; Sakata, Yoko; Doi, Fuminao

    2004-10-18

    Since glycosidase and glycosyltransferase inhibitors, composed of carba-sugars, have recently attracted much attention, it is desirable to develop effective preparative routes for provision of new carba-sugar derivatives of potential biological interest. 1,2:3,6-Dianhydro-5a-carba-alpha-glucopyranose was here chosen for study of synthetic utility, and demonstrated to be a promising intermediate for supplying several carba-beta-glycosylamines and N-linked dicarba-oligosaccharides. An N-linked 5a,5a'-dicarbalactose derivative obtained here was found to be a strong alpha-galactosidase inhibitor (IC50 1.2 microM, green coffee beans).

  11. Cloning of three novel neuronal Cdk5 activator binding proteins.

    PubMed

    Ching, Y P; Qi, Z; Wang, J H

    2000-01-25

    Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana.

  12. Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

    PubMed Central

    Velázquez-Moctezuma, Rodrigo; Bukh, Jens

    2017-01-01

    Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3–4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope. By analyzing the genotype 1a envelope proteins (E1/E2) of recovered Core-NS2 recombinant H77/JFH1ΔHVR1 and performing reverse genetic studies we found that resistance to AR5A was caused by substitution L665W, also conferring resistance to the parental H77/JFH1. The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we introduced into J6/JFH1 and J6/JFH1ΔHVR1. I345V increased fitness but had no effect on AR5A resistance. L665S impaired fitness and decreased AR5A sensitivity, while S680T combined with L665S compensated for fitness loss and decreased AR5A sensitivity even further. Interestingly, S680T alone had no fitness effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2–6 parental and HVR1-deleted variants (not available for genotype 4a) we observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1

  13. VIEW OF SOUTH FRONT WITH BUILDING 5A ATTACHED TO LEFT, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF SOUTH FRONT WITH BUILDING 5A ATTACHED TO LEFT, FACING NORTHWEST. - Douglas Aircraft Company Long Beach Plant, Aircraft Parts Receiving & Storage Building, 3855 Lakewood Boulevard, Long Beach, Los Angeles County, CA

  14. Screening the SPO11 and EIF5A2 genes in a population of infertile men.

    PubMed

    Christensen, Greg L; Ivanov, Ivaylo P; Atkins, John F; Mielnik, Anna; Schlegel, Peter N; Carrell, Douglas T

    2005-09-01

    Populations of infertile and fertile men were screened for mutations in SPO11 and EIF5A2, two infertility candidate genes. Three heterozygous amino acid changes that might contribute to infertility were identified in the infertile group.

  15. Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a

    PubMed Central

    McCaffary, David; Iqbal, Asif Jilani; Greaves, David R.

    2016-01-01

    Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of

  16. Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a.

    PubMed

    Taylor, Lewis; Brodermann, Maximillian Hugo; McCaffary, David; Iqbal, Asif Jilani; Greaves, David R

    2016-01-01

    Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of

  17. Wide spectrum of NR5A1-related phenotypes in 46,XY and 46,XX individuals.

    PubMed

    Domenice, Sorahia; Zamboni Machado, Aline; Moraes Ferreira, Frederico; Ferraz-de-Souza, Bruno; Marcondes Lerario, Antonio; Lin, Lin; Yumie Nishi, Mirian; Lisboa Gomes, Nathalia; Evelin da Silva, Thatiana; Barbosa Silva, Rosana; Vieira Correa, Rafaela; Ribeiro Montenegro, Luciana; Narciso, Amanda; Maria Frade Costa, Elaine; C Achermann, John; Bilharinho Mendonca, Berenice

    2016-12-01

    Steroidogenic factor 1 (NR5A1, SF-1, Ad4BP) is a transcriptional regulator of genes involved in adrenal and gonadal development and function. Mutations in NR5A1 have been among the most frequently identified genetic causes of gonadal development disorders and are associated with a wide phenotypic spectrum. In 46,XY individuals, NR5A1-related phenotypes may range from disorders of sex development (DSD) to oligo/azoospermia, and in 46,XX individuals, from 46,XX ovotesticular and testicular DSD to primary ovarian insufficiency (POI). The most common 46,XY phenotype is atypical or female external genitalia with clitoromegaly, palpable gonads, and absence of Müllerian derivatives. Notably, an undervirilized external genitalia is frequently seen at birth, while spontaneous virilization may occur later, at puberty. In 46,XX individuals, NR5A1 mutations are a rare genetic cause of POI, manifesting as primary or secondary amenorrhea, infertility, hypoestrogenism, and elevated gonadotropin levels. Mothers and sisters of 46,XY DSD patients carrying heterozygous NR5A1 mutations may develop POI, and therefore require appropriate counseling. Moreover, the recurrent heterozygous p.Arg92Trp NR5A1 mutation is associated with variable degrees of testis development in 46,XX patients. A clear genotype-phenotype correlation is not seen in patients bearing NR5A1 mutations, suggesting that genetic modifiers, such as pathogenic variants in other testis/ovarian-determining genes, may contribute to the phenotypic expression. Here, we review the published literature on NR5A1-related disease, and discuss our findings at a single tertiary center in Brazil, including ten novel NR5A1 mutations identified in 46,XY DSD patients. The ever-expanding phenotypic range associated with NR5A1 variants in XY and XX individuals confirms its pivotal role in reproductive biology, and should alert clinicians to the possibility of NR5A1 defects in a variety of phenotypes presenting with gonadal dysfunction

  18. A Wnt5a signaling pathway in the pathogenesis of HIV-1 gp120-induced pain

    PubMed Central

    Yuan, Su-Bo; Ji, Guangchen; Li, Bei; Andersson, Tommy; Neugebauer, Volker; Tang, Shao-Jun

    2015-01-01

    Pathological pain is one of the most common neurological complications in HIV-1/AIDS patients. However, the pathogenic process is unclear. Our recent studies show that Wnt5a is up-regulated in the spinal cord dorsal horn of the HIV patients who develop pain and that HIV-1 gp120, a potential causal factor of the HIV-associated pain, rapidly up-regulates Wnt5a in the mouse SDH. Using a mouse model, we show here that a specific Wnt5a antagonist, Box-5, attenuated gp120-induced mechanical allodynia. Conversely, a Wnt5a agonist, Foxy5, facilitated the allodynia. To elucidate the molecular mechanism by which Wnt5a regulates gp120-induced allodynia, we tested the role of the JNK/TNF-α pathway. We observed that the JNK-specific inhibitor SP600125 blocked either gp120- or Foxy5-induced allodynia. Similarly, the TNF-α-specific antagonist Enbrel also reversed either gp120- or Foxy5-induced allodynia. These data suggest that JNK and TNF-α mediate the biological effects of Wnt5a in regulating gp120-induced allodynia. To investigate the cellular mechanism, we performed extracellular single-unit recording from SDH neurons in anesthetized mice. Both Box5 and SP600125 negated gp120-induced potentiation of SDH neuron spiking evoked by mechanical stimulation of the hindpaw. Furthermore, while Foxy5 potentiated spike frequency of SDH neurons, either SP600125 or Enbrel blocked the potentiation. The data indicate that Wnt5a potentiates the activity of SDH neurons via the JNK-TNF-α pathway. Collectively, our findings suggest that Wnt5a regulates the pathogenesis of gp120-induced pain, likely by sensitizing pain-processing SDH neurons via JNK/TNF-α signaling. PMID:25840108

  19. Epidural Analgesia with Ropivacaine during Labour in a Patient with a SCN5A Gene Mutation

    PubMed Central

    Duvekot, J. J.; Roos-Hesselink, J. W.; Gonzalez Candel, A.; van der Marel, C. D.; Adriaens, V. F. R.

    2016-01-01

    SCN5A gene mutations can lead to ion channel defects which can cause cardiac conduction disturbances. In the presence of specific ECG characteristics, this mutation is called Brugada syndrome. Many drugs are associated with adverse events, making anesthesia in patients with SCN5A gene mutations or Brugada syndrome challenging. In this case report, we describe a pregnant patient with this mutation who received epidural analgesia using low dose ropivacaine and sufentanil during labour. PMID:27668095

  20. A Wnt5a signaling pathway in the pathogenesis of HIV-1 gp120-induced pain.

    PubMed

    Yuan, Su-Bo; Ji, Guangchen; Li, Bei; Andersson, Tommy; Neugebauer, Volker; Tang, Shao-Jun

    2015-07-01

    Pathological pain is one of the most common neurological complications in patients with HIV-1/AIDS. However, the pathogenic process is unclear. Our recent studies show that Wnt5a is upregulated in the spinal cord dorsal horn (SDH) of the patients with HIV who develop pain and that HIV-1 gp120, a potential causal factor of the HIV-associated pain, rapidly upregulates Wnt5a in the mouse SDH. Using a mouse model, we show here that a specific Wnt5a antagonist, Box-5, attenuated gp120-induced mechanical allodynia. Conversely, a Wnt5a agonist, Foxy5, facilitated the allodynia. To elucidate the molecular mechanism by which Wnt5a regulates gp120-induced allodynia, we tested the role of the JNK/TNF-α pathway. We observed that the JNK-specific inhibitor SP600125 blocked either gp120- or Foxy5-induced allodynia. Similarly, the TNF-α-specific antagonist Enbrel also reversed either gp120- or Foxy5-induced allodynia. These data suggest that JNK and TNF-α mediate the biological effects of Wnt5a in regulating gp120-induced allodynia. To investigate the cellular mechanism, we performed extracellular single-unit recording from SDH neurons in anesthetized mice. Both Box-5 and SP600125 negated gp120-induced potentiation of SDH neuron spiking evoked by mechanical stimulation of the hind paw. Furthermore, while Foxy5 potentiated spike frequency of SDH neurons, either SP600125 or Enbrel blocked the potentiation. The data indicate that Wnt5a potentiates the activity of SDH neurons through the JNK-TNF-α pathway. Collectively, our findings suggest that Wnt5a regulates the pathogenesis of gp120-induced pain, likely by sensitizing pain-processing SDH neurons through JNK/TNF-α signaling.

  1. Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation

    SciTech Connect

    Jensen, Taylor J.; Wozniak, Ryan J.; Eblin, Kylee E.; Wnek, Sean M.; Gandolfi, A. Jay; Futscher, Bernard W.

    2009-02-15

    Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modifications and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation.

  2. Wnt5a attenuates hypoxia-induced pulmonary arteriolar remodeling and right ventricular hypertrophy in mice

    PubMed Central

    Jin, Yuling; Wang, Wang; Chai, Sanbao; Liu, Jie

    2015-01-01

    Hypoxic pulmonary hypertension (HPH), which is characterized by pulmonary arteriolar remodeling and right ventricular hypertrophy, is still a life-threatening disease with the current treatment strategies. The underlying molecular mechanisms of HPH remain unclear. Our previously published study showed that Wnt5a, one of the ligands in the Wnt family, was critically involved in the inhibition of hypoxia-induced pulmonary arterial smooth muscle cell proliferation by downregulation of β-catenin/cyclin D1 in vitro. In this study, we investigated the possible functions and mechanisms of Wnt5a in HPH in vivo. Recombinant mouse Wnt5a (rmWnt5a) or phosphate buffered saline (PBS) was administered to male C57/BL6 mice weekly from the first day to the end of the two or four weeks after exposed to hypoxia (10% O2). Hypoxia-induced pulmonary hypertension was associated with a marked increase in β-catenin/cyclin D1 expression in lungs. Right ventricular systolic pressure and right ventricular hypertrophy index were reduced in animals treated with rmWnt5a compared with PBS. Histology showed less pulmonary vascular remodeling and right ventricular hypertrophy in the group treated with rmWnt5a than with PBS. Treatment with rmWnt5a resulted in a concomitant reduction in β-catenin/cyclin D1 levels in lungs. These data demonstrate that Wnt5a exerts its beneficial effects on HPH by regulating pulmonary vascular remodeling and right ventricular hypertrophy in a manner that is associated with reduction in β-catenin/cyclin D1 signaling. A therapy targeting the β-catenin/cyclin D1 signaling pathway might be a potential strategy for HPH treatment. PMID:25956683

  3. Epigenetic Mediated Transcriptional Activation of WNT5A Participates in Arsenical-Associated Malignant Transformation

    PubMed Central

    Jensen, Taylor J.; Wozniak, Ryan J.; Eblin, Kylee E.; Wnek, Sean M.; Gandolfi, A. Jay; Futscher, Bernard W.

    2015-01-01

    Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggests that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modifications and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicate that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation. PMID:19061910

  4. Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation.

    PubMed

    Jensen, Taylor J; Wozniak, Ryan J; Eblin, Kylee E; Wnek, Sean M; Gandolfi, A Jay; Futscher, Bernard W

    2009-02-15

    Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modifications and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation.

  5. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  6. Imaging cardiac SCN5A using the novel F-18 radiotracer radiocaine

    PubMed Central

    Hooker, Jacob M.; Strebl, Martin G.; Schroeder, Frederick A.; Wey, Hsiao-Ying; Ambardekar, Amrut V.; McKinsey, Timothy A.; Schoenberger, Matthias

    2017-01-01

    The key function of the heart, a well-orchestrated series of contractions, is controlled by cardiac action potentials. These action potentials are initiated and propagated by a single isoform of voltage gated sodium channels – SCN5A. However, linking changes in SCN5A expression levels to human disease in vivo has not yet been possible. Radiocaine, an F-18 radiotracer for positron emission tomography (PET), is the first SCN5A imaging agent in the heart. Explants from healthy and failing human hearts were compared using radiocaine autoradiography to determine that the failing heart has ~30% lower SCN5A levels - the first evidence of changes in SCN5A expression in humans as a function of disease. Paving the way for translational imaging, radiocaine proved to exhibit high in vivo specific binding to the myocardium of non-human primates. We envision that SCN5A measurements using PET imaging may serve as a novel diagnostic tool to stratify arrhythmia risk and assess for progression of heart failure in patients with a broad spectrum of cardiovascular diseases. PMID:28205593

  7. MOD-5A wind turbine generator program design report: Volume 1: Executive Summary

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator covering work performed between July 1980 and June 1984 is discussed. The report is divided into four volumes: Volume 1 summarizes the entire MOD-5A program, Volume 2 discusses the conceptual and preliminary design phases, Volume 3 describes the final design of the MOD-5A, and Volume 4 contains the drawings and specifications developed for the final design. Volume 1, the Executive Summary, summarizes all phases of the MOD-5A program. The performance and cost of energy generated by the MOD-5A are presented. Each subsystem - the rotor, drivetrain, nacelle, tower and foundation, power generation, and control and instrumentation subsystems - is described briefly. The early phases of the MOD-5A program, during which the design was analyzed and optimized, and new technologies and materials were developed, are discussed. Manufacturing, quality assurance, and safety plans are presented. The volume concludes with an index of volumes 2 and 3.

  8. Noncanonical WNT-5A signaling impairs endogenous lung repair in COPD.

    PubMed

    Baarsma, Hoeke A; Skronska-Wasek, Wioletta; Mutze, Kathrin; Ciolek, Florian; Wagner, Darcy E; John-Schuster, Gerrit; Heinzelmann, Katharina; Günther, Andreas; Bracke, Ken R; Dagouassat, Maylis; Boczkowski, Jorge; Brusselle, Guy G; Smits, Ron; Eickelberg, Oliver; Yildirim, Ali Ö; Königshoff, Melanie

    2017-01-01

    Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. One main pathological feature of COPD is the loss of functional alveolar tissue without adequate repair (emphysema), yet the underlying mechanisms are poorly defined. Reduced WNT-β-catenin signaling is linked to impaired lung repair in COPD; however, the factors responsible for attenuating this pathway remain to be elucidated. Here, we identify a canonical to noncanonical WNT signaling shift contributing to COPD pathogenesis. We demonstrate enhanced expression of noncanonical WNT-5A in two experimental models of COPD and increased posttranslationally modified WNT-5A in human COPD tissue specimens. WNT-5A was increased in primary lung fibroblasts from COPD patients and induced by COPD-related stimuli, such as TGF-β, cigarette smoke (CS), and cellular senescence. Functionally, mature WNT-5A attenuated canonical WNT-driven alveolar epithelial cell wound healing and transdifferentiation in vitro. Lung-specific WNT-5A overexpression exacerbated airspace enlargement in elastase-induced emphysema in vivo. Accordingly, inhibition of WNT-5A in vivo attenuated lung tissue destruction, improved lung function, and restored expression of β-catenin-driven target genes and alveolar epithelial cell markers in the elastase, as well as in CS-induced models of COPD. We thus identify a novel essential mechanism involved in impaired mesenchymal-epithelial cross talk in COPD pathogenesis, which is amenable to therapy.

  9. Wnt5a induces Ryk-dependent and -independent effects on callosal axon and dendrite growth.

    PubMed

    Clark, Charlotte E J; Richards, Linda J; Stacker, Steven A; Cooper, Helen M

    2014-02-01

    The non-canonical Wnt receptor, Ryk, promotes chemorepulsive axon guidance in the developing mouse brain and spinal cord in response to Wnt5a. Ryk has also been identified as a major suppressor of axonal regrowth after spinal cord injury. Thus, a comprehensive understanding of how growing axons and dendrites respond to Wnt5a-mediated Ryk activation is required if we are to overcome this detrimental activity. Here we undertook a detailed analysis of the effect of Wnt5a/Ryk interactions on axonal and dendritic growth in dissociated embryonic mouse cortical neuron cultures, focusing on callosal neurons known to be responsive to Ryk-induced chemorepulsion. We show that Ryk inhibits axonal growth in response to Wnt5a. We also show that Wnt5a inhibits dendrite growth independently of Ryk. However, this inhibition is relieved when Ryk is present. Therefore, Wnt5a-mediated Ryk activation triggers divergent responses in callosal axons and dendrites in the in vitro context.

  10. Posttranslational hypusination of the eukaryotic translation initiation factor-5A regulates Fusarium graminearum virulence

    PubMed Central

    Martinez-Rocha, Ana Lilia; Woriedh, Mayada; Chemnitz, Jan; Willingmann, Peter; Kröger, Cathrin; Hadeler, Birgit; Hauber, Joachim; Schäfer, Wilhelm

    2016-01-01

    Activation of eukaryotic translation initiation factor eIF5A requires a posttranslational modification, forming the unique amino acid hypusine. This activation is mediated by two enzymes, deoxyhypusine synthase, DHS, and deoxyhypusine hydroxylase, DOHH. The impact of this enzymatic complex on the life cycle of a fungal pathogen is unknown. Plant pathogenic ascomycetes possess a single copy of the eIF5A activated by hypusination. We evaluated the importance of imbalances in eIF5A hypusination in Fusarium graminearum, a devastating fungal pathogen of cereals. Overexpression of DHS leads to increased virulence in wheat, elevated production of the mycotoxin deoxynivalenol, more infection structures, faster wheat tissue invasion in plants and increases vegetatively produced conidia. In contrast, overexpression of DOHH completely prevents infection structure formation, pathogenicity in wheat and maize, leads to overproduction of ROS, reduced DON production and increased sexual reproduction. Simultaneous overexpression of both genes restores wild type-like phenotypes. Analysis of eIF5A posttranslational modification displayed strongly increased hypusinated eIF5A in DOHH overexpression mutant in comparison to wild type, and the DHS overexpression mutants. These are the first results pointing to different functions of differently modified eIF5A. PMID:27098988

  11. The role of C5a in acute lung injury induced by highly pathogenic viral infections

    PubMed Central

    Wang, Renxi; Xiao, He; Guo, Renfeng; Li, Yan; Shen, Beifen

    2015-01-01

    The complement system, an important part of innate immunity, plays a critical role in pathogen clearance. Unregulated complement activation is likely to play a crucial role in the pathogenesis of acute lung injury (ALI) induced by highly pathogenic virus including influenza A viruses H5N1, H7N9, and severe acute respiratory syndrome (SARS) coronavirus. In highly pathogenic virus-induced acute lung diseases, high levels of chemotactic and anaphylatoxic C5a were produced as a result of excessive complement activaiton. Overproduced C5a displays powerful biological activities in activation of phagocytic cells, generation of oxidants, and inflammatory sequelae named “cytokine storm”, and so on. Blockade of C5a signaling have been implicated in the treatment of ALI induced by highly pathogenic virus. Herein, we review the literature that links C5a and ALI, and review our understanding of the mechanisms by which C5a affects ALI during highly pathogenic viral infection. In particular, we discuss the potential of the blockade of C5a signaling to treat ALI induced by highly pathogenic viruses. PMID:26060601

  12. Paracrine WNT5A Signaling Inhibits Expansion of Tumor-Initiating Cells.

    PubMed

    Borcherding, Nicholas; Kusner, David; Kolb, Ryan; Xie, Qing; Li, Wei; Yuan, Fang; Velez, Gabriel; Askeland, Ryan; Weigel, Ronald J; Zhang, Weizhou

    2015-05-15

    It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ-SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal cross-talk in normal mammary tissue.

  13. Paracrine WNT5A signaling inhibits expansion of tumor-initiating cells

    PubMed Central

    Borcherding, Nicholas; Kusner, David; Kolb, Ryan; Xie, Qing; Li, Wei; Yuan, Fang; Velez, Gabriel; Askeland, Ryan; Weigel, Ronald J.; Zhang, Weizhou

    2015-01-01

    It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared to the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of TGFβ/SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal crosstalk in normal mammary tissue. PMID:25769722

  14. C5a Regulates IL-1β Production and Leukocyte Recruitment in a Murine Model of Monosodium Urate Crystal-Induced Peritonitis

    PubMed Central

    Khameneh, Hanif J.; Ho, Adrian W. S.; Laudisi, Federica; Derks, Heidi; Kandasamy, Matheswaran; Sivasankar, Baalasubramanian; Teng, Gim Gee; Mortellaro, Alessandra

    2017-01-01

    Gouty arthritis results from the generation of monosodium urate (MSU) crystals within joints. These MSU crystals elicit acute inflammation characterized by massive infiltration of neutrophils and monocytes that are mobilized by the pro-inflammatory cytokine IL-1β. MSU crystals also activate the complement system, which regulates the inflammatory response; however, it is unclear whether or how MSU-mediated complement activation is linked to IL-1β release in vivo, and the various roles that might be played by individual components of the complement cascade. Here we show that exposure to MSU crystals in vivo triggers the complement cascade, leading to the generation of the biologically active complement proteins C3a and C5a. C5a, but not C3a, potentiated IL-1β and IL-1α release from LPS–primed MSU-exposed peritoneal macrophages and human monocytic cells in vitro; while in vivo MSU–induced C5a mediated murine neutrophil recruitment as well as IL-1β production at the site of inflammation. These effects were significantly ameliorated by treatment of mice with a C5a receptor antagonist. Mechanistic studies revealed that C5a most likely increased NLRP3 inflammasome activation via production of reactive oxygen species (ROS), and not through increased transcription of inflammasome components. Therefore we conclude that C5a generated upon MSU-induced complement activation increases neutrophil recruitment in vivo by promoting IL-1 production via the generation of ROS, which activate the NLRP3 inflammasome. Identification of the C5a receptor as a key determinant of IL-1-mediated recruitment of inflammatory cells provides a novel potential target for therapeutic intervention to mitigate gouty arthritis. PMID:28167912

  15. C5a Regulates IL-1β Production and Leukocyte Recruitment in a Murine Model of Monosodium Urate Crystal-Induced Peritonitis.

    PubMed

    Khameneh, Hanif J; Ho, Adrian W S; Laudisi, Federica; Derks, Heidi; Kandasamy, Matheswaran; Sivasankar, Baalasubramanian; Teng, Gim Gee; Mortellaro, Alessandra

    2017-01-01

    Gouty arthritis results from the generation of monosodium urate (MSU) crystals within joints. These MSU crystals elicit acute inflammation characterized by massive infiltration of neutrophils and monocytes that are mobilized by the pro-inflammatory cytokine IL-1β. MSU crystals also activate the complement system, which regulates the inflammatory response; however, it is unclear whether or how MSU-mediated complement activation is linked to IL-1β release in vivo, and the various roles that might be played by individual components of the complement cascade. Here we show that exposure to MSU crystals in vivo triggers the complement cascade, leading to the generation of the biologically active complement proteins C3a and C5a. C5a, but not C3a, potentiated IL-1β and IL-1α release from LPS-primed MSU-exposed peritoneal macrophages and human monocytic cells in vitro; while in vivo MSU-induced C5a mediated murine neutrophil recruitment as well as IL-1β production at the site of inflammation. These effects were significantly ameliorated by treatment of mice with a C5a receptor antagonist. Mechanistic studies revealed that C5a most likely increased NLRP3 inflammasome activation via production of reactive oxygen species (ROS), and not through increased transcription of inflammasome components. Therefore we conclude that C5a generated upon MSU-induced complement activation increases neutrophil recruitment in vivo by promoting IL-1 production via the generation of ROS, which activate the NLRP3 inflammasome. Identification of the C5a receptor as a key determinant of IL-1-mediated recruitment of inflammatory cells provides a novel potential target for therapeutic intervention to mitigate gouty arthritis.

  16. The Autophagic Process Occurs in Human Bone Metastasis and Implicates Molecular Mechanisms Differently Affected by Rab5a in the Early and Late Stages

    PubMed Central

    Maroni, Paola; Bendinelli, Paola; Resnati, Massimo; Matteucci, Emanuela; Milan, Enrico; Desiderio, Maria Alfonsina

    2016-01-01

    Autophagy favours metastatic growth through fuelling energy and nutrients and resistance to anoikis, typical of disseminated-tumour cells. The autophagic process, mediated by a unique organelle, the autophagosome, which fuses with lysosomes, is divided into three steps. Several stages, especially early omegasome formation and isolation-membrane initiation, remain controversial; molecular mechanisms involve the small-GTPase Rab5a, which regulates vesicle traffic for autophagosome formation. We examined Rab5a involvement in the function of key members of ubiquitin-conjugation systems, Atg7 and LC3-lipidated, interacting with the scaffold-protein p62. Immunohistochemistry of Rab5a was performed in human specimens of bone metastasis and pair-matched breast carcinoma; the autophagic-molecular mechanisms affected by Rab5a were evaluated in human 1833 bone metastatic cells, derived from breast-carcinoma MDA-MB231 cells. To clarify the role of Rab5a, 1833 cells were transfected transiently with Rab5a-dominant negative, and/or stably with the short-hairpin RNA Atg7, were exposed to two inhibitors of autolysosome function, and LC3II and p62 expression was measured. We showed basal autophagy in bone-metastatic cells and the pivotal role of Rab5a together with Beclin 1 between the early stages, elongation of isolation membrane/closed autophagosome mediated by Atg7, and the late-degradative stages. This regulatory network might occur in bone-metastasis and in high-grade dysplastic lesions, preceding invasive-breast carcinoma and conferring phenotypic characteristics for dissemination. PMID:27023526

  17. Conformational analysis of brominated pA2'-5'A2'-5'A analogs. An NMR and model-building study.

    PubMed

    van den Hoogen, Y T; Hilgersom, C M; Brozda, D; Lesiak, K; Torrence, P F; Altona, C

    1989-07-01

    NMR and model-building studies were carried out on pA2'-5'A2'-5'A and analogs in which one or more of the A residues were replaced by 8-bromoadenosine. Chemical shifts, coupling constants and NOE data were used to obtain structural information. The N/S equilibrium constant of the ribose rings as well as the phase angles and puckering amplitudes were determined from the experimental coupling constants with the aid of an improved version of the PSEUROT program. Chemical shifts in combination with NOE data were used to monitor base-base interactions and the orientation of the bases (syn or anti). The combined data suggest that different types of stacking interactions are present in the various compounds. Bromination of the first or second residue in the trimers results in a preference for N-type sugar and syn orientation of the base in these residues. When A(3) is brominated, an S-type sugar conformation together with a syn orientation of the base is favoured at the 2' terminus. Energy-minimized models of the different stacking interactions are presented which fit the present collection of data. The possible correlation between biological activity of these compounds and their conformation is briefly discussed.

  18. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  19. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  20. Frequent up-regulation of WNT5A mRNA in primary gastric cancer.

    PubMed

    Saitoh, Tetsuroh; Mine, Tetsuya; Katoh, Masaru

    2002-05-01

    WNT signal is transduced to the beta-catenin - TCF pathway, the JNK pathway, or the Ca2+-releasing pathway through seven-transmembrane-type WNT receptors encoded by Frizzled genes (FZD1-FZD10). We have previously cloned and characterized human WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, and WNT14B/WNT15 by using bioinformatics, cDNA-library screening, and cDNA-PCR. Here, we investigated expression of human WNT5A mRNA in various normal tissues, 66 primary tumors derived from various tissues, and 15 human cancer cell lines. WNT5A mRNA was relatively highly expressed in salivary gland, bladder, uterus, placenta, and fetal kidney. Up-regulation of WNT5A mRNA was detected in 5 out of 8 cases of primary gastric cancer, 5 out of 18 cases of primary colorectal tumors, and in 2 out of 7 cases of primary uterus tumors by using matched tumor/normal expression array analysis. Up-regulation of WNT5A mRNA was also detected in 7 out of 10 other cases of primary gastric cancer by using cDNA-PCR. Although low-level expression of WNT5A mRNA was detected in gastric cancer cell line MKN45, WNT5A mRNA was almost undetectable in gastric cancer cell lines OKAJIMA, TMK1, MKN7, MKN28, MKN74, and KATO-III. Compared with frequent up-regulation of WNT5A mRNA in primary gastric cancer, expression levels of WNT5A mRNA in 7 gastric cancer cell lines were significantly lower than that in normal stomach. Frequent up-regulation of WNT5A mRNA in human primary gastric cancer might be due to cancer-stromal interaction.

  1. Wnt5a is a crucial regulator of neurogenesis during cerebellum development

    PubMed Central

    Subashini, Chandramohan; Dhanesh, Sivadasan Bindu; Chen, Chih-Ming; Riya, Paul Ann; Meera, Vadakkath; Divya, Thulasi Sheela; Kuruvilla, Rejji; Buttler, Kerstin; James, Jackson

    2017-01-01

    The role of Wnt5a has been extensively explored in various aspects of development but its role in cerebellar development remains elusive. Here, for the first time we unravel the expression pattern and functional significance of Wnt5a in cerebellar development using Wnt5a−/− and Nestin-Cre mediated conditional knockout mouse models. We demonstrate that loss of Wnt5a results in cerebellar hypoplasia and depletion of GABAergic and glutamatergic neurons. Besides, Purkinje cells of the mutants displayed stunted, poorly branched dendritic arbors. Furthermore, we show that the overall reduction is due to decreased radial glial and granule neuron progenitor cell proliferation. At molecular level we provide evidence for non-canonical mode of action of Wnt5a and its regulation over genes associated with progenitor proliferation. Altogether our findings imply that Wnt5a signaling is a crucial regulator of cerebellar development and would aid in better understanding of cerebellar disease pathogenesis caused due to deregulation of Wnt signaling. PMID:28205531

  2. Wnt5a Suppresses β-catenin Signaling during Hair Follicle Regeneration

    PubMed Central

    Xing, Yizhan; Ma, Xiaogen; Guo, Haiying; Deng, Fang; Yang, Jin; Li, Yuhong

    2016-01-01

    Hair follicles display periodic growth. Wnt signaling is a critical regulator for hair follicle regeneration. Previously, we reported that Wnt5a inhibits the telogen-to-anagen transition of hair follicles, but the mechanism by which this process occurs has not yet been reported. Here, we determined the expression patterns of Wnt signaling pathway molecules by quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry and found that β-catenin signaling was suppressed by Wnt5a. We then compared the phenotypes and expression patterns following β-catenin knockdown and Wnt5a overexpression during hair follicle regeneration induced by hair depilation and observed similar patterns. In addition, we performed a rescue experiment in the JB6 cell line and found that the inhibitory effect of Wnt5a on cell proliferation could be rescued by the addition of Wnt3a. Our data reveal that Wnt5a suppresses the activation of β-catenin signaling during hair follicle regeneration. PMID:27499692

  3. Synthesis, crystal structure, characterization and antifungal activity of pyrazolo[1,5-a]pyrimidines derivatives

    NASA Astrophysics Data System (ADS)

    Zhang, Jin; Peng, Ju-Fang; Wang, Tao; Wang, Ping; Zhang, Zun-Ting

    2016-09-01

    Under microwave radiation, isomers 2-(pyrazolo[1,5-a]pyrimidin-5-yl)phenols (3) and 2-(pyrazolo[1,5-a]pyrimidin-7-yl)phenols (4) were simultaneously obtained by the condensation of chromones and 3-aminopyrazoles. These two isomers were fully characterized by IR, 1H NMR, 13C NMR and HRMS. In addition, a representative product 5-chloro-2-(2-methyl-pyrazolo[1,5-a] pyrimidin-5-yl)phenol (3e) was further conformed by the single crystal X-ray diffraction. The antifungal abilities of the obtained products 3 and 4 were evaluated against five phytopathogenic fungi (Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria solani and Fusarium solani). The results revealed that 2-(pyrazolo[1,5-a]pyrimidin-5-yl)phenol (3a) and 4-chloro-2-(2-methylpyrazolo[1,5-a]pyrimidin-7-yl)phenol (4e) exhibited good antifungal abilities against Colletotrichum gloeosporioides with the IC50 values of 24.90 and 28.28 μg/mL, respectively.

  4. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

    PubMed Central

    Lu, Yingying; Dong, Shichen; Hao, Baixia; Li, Chang; Zhu, Kaiyuan; Guo, Wenjing; Wang, Qian; Cheung, King-Ho; Wong, Connie WM; Wu, Wu-Tian; Markus, Huss; Yue, Jianbo

    2014-01-01

    Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca2+ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy. PMID:25483964

  5. Wnt5a and Wnt11 are essential for second heart field progenitor development

    PubMed Central

    Cohen, Ethan David; Miller, Mayumi F.; Wang, Zichao; Moon, Randall T.; Morrisey, Edward E.

    2012-01-01

    Wnt/β-catenin has a biphasic effect on cardiogenesis, promoting the induction of cardiac progenitors but later inhibiting their differentiation. Second heart field progenitors and expression of the second heart field transcription factor Islet1 are inhibited by the loss of β-catenin, indicating that Wnt/β-catenin signaling is necessary for second heart field development. However, expressing a constitutively active β-catenin with Islet1-Cre also inhibits endogenous Islet1 expression, reflecting the inhibitory effect of prolonged Wnt/β-catenin signaling on second heart field development. We show that two non-canonical Wnt ligands, Wnt5a and Wnt11, are co-required to regulate second heart field development in mice. Loss of Wnt5a and Wnt11 leads to a dramatic loss of second heart field progenitors in the developing heart. Importantly, this loss of Wnt5a and Wnt11 is accompanied by an increase in Wnt/β-catenin signaling, and ectopic Wnt5a/Wnt11 inhibits β-catenin signaling and promotes cardiac progenitor development in differentiating embryonic stem cells. These data show that Wnt5a and Wnt11 are essential regulators of the response of second heart field progenitors to Wnt/β-catenin signaling and that they act by restraining Wnt/β-catenin signaling during cardiac development. PMID:22569553

  6. C5a receptor signaling prevents folate deficiency-induced neural tube defects in mice.

    PubMed

    Denny, Kerina J; Coulthard, Liam G; Jeanes, Angela; Lisgo, Steven; Simmons, David G; Callaway, Leonie K; Wlodarczyk, Bogdan; Finnell, Richard H; Woodruff, Trent M; Taylor, Stephen M

    2013-04-01

    The complement system is involved in a range of diverse developmental processes, including cell survival, growth, differentiation, and regeneration. However, little is known about the role of complement in embryogenesis. In this study, we demonstrate a novel role for the canonical complement 5a receptor (C5aR) in the development of the mammalian neural tube under conditions of maternal dietary folic acid deficiency. Specifically, we found C5aR and C5 to be expressed throughout the period of neurulation in wild-type mice and localized the expression to the cephalic regions of the developing neural tube. C5aR was also found to be expressed in the neuroepithelium of early human embryos. Ablation of the C5ar1 gene or the administration of a specific C5aR peptide antagonist to folic acid-deficient pregnant mice resulted in a high prevalence of severe anterior neural tube defect-associated congenital malformations. These findings provide a new and compelling insight into the role of the complement system during mammalian embryonic development.

  7. Nuclear receptor NR5A2 controls neural stem cell fate decisions during development

    PubMed Central

    Stergiopoulos, Athanasios; Politis, Panagiotis K.

    2016-01-01

    The enormous complexity of mammalian central nervous system (CNS) is generated by highly synchronized actions of diverse factors and signalling molecules in neural stem/progenitor cells (NSCs). However, the molecular mechanisms that integrate extrinsic and intrinsic signals to control proliferation versus differentiation decisions of NSCs are not well-understood. Here we identify nuclear receptor NR5A2 as a central node in these regulatory networks and key player in neural development. Overexpression and loss-of-function experiments in primary NSCs and mouse embryos suggest that NR5A2 synchronizes cell-cycle exit with induction of neurogenesis and inhibition of astrogliogenesis by direct regulatory effects on Ink4/Arf locus, Prox1, a downstream target of proneural genes, as well as Notch1 and JAK/STAT signalling pathways. Upstream of NR5a2, proneural genes, as well as Notch1 and JAK/STAT pathways control NR5a2 endogenous expression. Collectively, these observations render NR5A2 a critical regulator of neural development and target gene for NSC-based treatments of CNS-related diseases. PMID:27447294

  8. Protein-losing enteropathy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  9. Protein in diet

    MedlinePlus

    ... basic structure of protein is a chain of amino acids. You need protein in your diet to help ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a number ...

  10. Dual Variation in SCN5A and CACNB2b Underlies the Development of Cardiac Conduction Disease without Brugada Syndrome

    PubMed Central

    Hu, Dan; Barajas-Martinez, Hector; Nesterenko, Vladislav V.; Pfeiffer, Ryan; Guerchicoff, Alejandra; Cordeiro, Jonathan M.; Curtis, Anne B.; Pollevick, Guido D.; Wu, Yuesheng; Burashnikov, Elena; Antzelevitch, Charles

    2010-01-01

    Background Inherited loss of function mutations in SCN5A have been linked to overlapping syndromes including cardiac conduction disease and Brugada syndrome (BrS). The mechanisms responsible for the development of one without the other are poorly understood. Methods Direct sequencing was performed in a family with cardiac conduction disease. Wild-type (WT) and mutant channels were expressed in TSA201 cells for electrophysiological study. Green fluorescent protein (GFP)-fused WT or mutant genes were used to assess channel trafficking. Results A novel SCN5A mutation, P1008S, was identified in all family members displaying 1st degree atrioventricular block, but not in unaffected family members nor in 430 reference alleles. Peak P1008S current was 11.77% of WT (p<0.001). Confocal microscopy showed that WT channels tagged with GFP were localized on the cell surface, whereas GFP-tagged P1008S channels remained trapped in intracellular organelles. Trafficking could be rescued by incubation at room temperature, but not by incubation with mexiletine (300µM) at 37°C. We also identified a novel polymorphism (D601E) in CACNB2b that slowed inactivation of L-type calcium current (ICa), significantly increased total charge. Using the Luo-Rudy action potential model, we show that the reduction in INa can cause loss of the right ventricular epicardial action potential (AP) dome in the absence but not in the presence of the slowed inactivation of ICa. Slowed conduction was present in both cases. Conclusions Our results suggest genetic variations leading to a loss-of-function in INa coupled with a gain of function in ICa may underlie the development of cardiac conduction disease without BrS. PMID:20025708

  11. Mod-5A Wind Turbine Generator Program Design Report. Volume 4: Drawings and Specifications, Book 1

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. Volume 4 contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This is the first of five books of volume four. It contains structural design criteria, generator step-up transformer specs, specs for design, fabrication and testing of the system, specs for the ground control enclosure, systems specs, slip ring specs, and control system specs.

  12. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 2

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This is the second book of volume four. Some of the items it contains are specs for the emergency shutdown panel, specs for the simulator software, simulator hardware specs, site operator terminal requirements, control data system requirements, software project management plan, elastomeric teeter bearing requirement specs, specs for the controls electronic cabinet, and specs for bolt pretensioning.

  13. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 4

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator are documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This volume contains 5 books of which this is the fourth, providing drawings 47A380128 through 47A387125. In addition to the parts listing and where-used list, the logic design of the controller software and the code listing of the controller software are provided. Also given are the aerodynamic profile coordinates.

  14. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 2

    NASA Astrophysics Data System (ADS)

    1984-08-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This is the second book of volume four. Some of the items it contains are specs for the emergency shutdown panel, specs for the simulator software, simulator hardware specs, site operator terminal requirements, control data system requirements, software project management plan, elastomeric teeter bearing requirement specs, specs for the controls electronic cabinet, and specs for bolt pretensioning.

  15. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  16. ADME studies and preliminary safety pharmacology of LDT5, a lead compound for the treatment of benign prostatic hyperplasia

    PubMed Central

    Noël, F.; Nascimento-Viana, J.B.; Romeiro, L.A.S.; Silva, R.O.; Lemes, L.F.N.; Oliveira, A.S.; Giorno, T.B.S.; Fernandes, P.D.; Silva, C.L.M.

    2016-01-01

    This study aimed to estimate the absorption, distribution, metabolism and excretion (ADME) properties and safety of LDT5, a lead compound for oral treatment of benign prostatic hyperplasia that has previously been characterized as a multi-target antagonist of α1A-, α1D-adrenoceptors and 5-HT1A receptors. The preclinical characterization of this compound comprised the evaluation of its in vitro properties, including plasma, microsomal and hepatocytes stability, cytochrome P450 metabolism and inhibition, plasma protein binding, and permeability using MDCK-MDR1 cells. De-risking and preliminary safety pharmacology assays were performed through screening of 44 off-target receptors and in vivo tests in mice (rota-rod and single dose toxicity). LDT5 is stable in rat and human plasma, human liver microsomes and hepatocytes, but unstable in rat liver microsomes and hepatocytes (half-life of 11 min). LDT5 is highly permeable across the MDCK-MDR1 monolayer (Papp ∼32×10-6 cm/s), indicating good intestinal absorption and putative brain penetration. LDT5 is not extensively protein-bound and is a substrate of human CYP2D6 and CYP2C19 but not of CYP3A4 (half-life >60 min), and did not significantly influence the activities of any of the human cytochrome P450 isoforms screened. LDT5 was considered safe albeit new studies are necessary to rule out putative central adverse effects through D2, 5-HT1A and 5-HT2B receptors, after chronic use. This work highlights the drug-likeness properties of LDT5 and supports its further preclinical development. PMID:27901175

  17. Transcriptomic characterization of two major Fusarium resistance quantitative trait loci (QTLs), Fhb1 and Qfhs.ifa-5A, identifies novel candidate genes

    PubMed Central

    Schweiger, Wolfgang; Steiner, Barbara; Ametz, Christian; Siegwart, Gerald; Wiesenberger, Gerlinde; Berthiller, Franz; Lemmens, Marc; Jia, Haiyan; Adam, Gerhard; Muehlbauer, Gary J; Kreil, David P; Buerstmayr, Hermann

    2013-01-01

    Fusarium head blight, caused by Fusarium graminearum, is a devastating disease of wheat. We developed near-isogenic lines (NILs) differing in the two strongest known F. graminearum resistance quantitative trait loci (QTLs), Qfhs.ndsu-3BS (also known as resistance gene Fhb1) and Qfhs.ifa-5A, which are located on the short arm of chromosome 3B and on chromosome 5A, respectively. These NILs showing different levels of resistance were used to identify transcripts that are changed significantly in a QTL-specific manner in response to the pathogen and between mock-inoculated samples. After inoculation with F. graminearum spores, 16 transcripts showed a significantly different response for Fhb1 and 352 for Qfhs.ifa-5A. Notably, we identified a lipid transfer protein which is constitutively at least 50-fold more abundant in plants carrying the resistant allele of Qfhs.ifa-5A. In addition to this candidate gene associated with Qfhs.ifa-5A, we identified a uridine diphosphate (UDP)-glycosyltransferase gene, designated TaUGT12887, exhibiting a positive difference in response to the pathogen in lines harbouring both QTLs relative to lines carrying only the Qfhs.ifa-5A resistance allele, suggesting Fhb1 dependence of this transcript. Yet, this dependence was observed only in the NIL with already higher basal resistance. The complete cDNA of TaUGT12887 was reconstituted from available wheat genomic sequences, and a synthetic recoded gene was expressed in a toxin-sensitive strain of Saccharomyces cerevisiae. This gene conferred deoxynivalenol resistance, albeit much weaker than that observed with the previously characterized barley HvUGT13248. PMID:23738863

  18. Targeted Disruption of the Gene Encoding the Murine Small Subunit of Carboxypeptidase N (CPN1) Causes Susceptibility to C5a Anaphylatoxin-Mediated Shock1

    PubMed Central

    Mueller-Ortiz, Stacey L.; Wang, Dachun; Morales, John E.; Li, Li; Chang, Jui-Yoa; Wetsel, Rick A.

    2015-01-01

    Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. Historically, CPN has been implicated as a major regulator of inflammation by its enzymatic cleavage of functionally important arginine and lysine amino acids from potent phlogistic molecules, such as the complement anaphylatoxins C3a and C5a. Because of no known complete CPN deficiencies, the biological impact of CPN in vivo has been difficult to evaluate. Here, we report the generation of a mouse with complete CPN deficiency by targeted disruption of the CPN1 gene. CPN1−/− mice were hypersensitive to lethal anaphylactic shock due to acute complement activation by cobra venom factor. This hypersensitivity was completely resolved in CPN1−/−/C5aR−/− but not in CPN1−/−/C3aR−/− mice. Moreover, CPN1−/− mice given C5a i.v., but not C3a, experienced 100% mortality. This C5a-induced mortality was reduced to 20% when CPN1−/− mice were treated with an antihistamine before C5a challenge. These studies describe for the first time a complete deficiency of CPN and demonstrate 1) that CPN plays a requisite role in regulating the lethal effects of anaphylatoxin-mediated shock, 2) that these lethal effects are mediated predominantly by C5a-induced histamine release, and 3) that C3a does not contribute significantly to shock following acute complement activation. PMID:19414808

  19. Pro-Inflammatory wnt5a and Anti-Inflammatory sFRP5 Are Differentially Regulated by Nutritional Factors in Obese Human Subjects

    PubMed Central

    Schulte, Dominik M.; Müller, Nike; Neumann, Katrin; Oberhäuser, Frank; Faust, Michael; Güdelhöfer, Heike; Brandt, Burkhard; Krone, Wilhelm; Laudes, Matthias

    2012-01-01

    Background Obesity is associated with macrophage infiltration of adipose tissue. These inflammatory cells affect adipocytes not only by classical cytokines but also by the secreted glycopeptide wnt5a. Healthy adipocytes are able to release the wnt5a inhibitor sFRP5. This protective effect, however, was found to be diminished in obesity. The aim of the present study was to examine (1) whether obese human subjects exhibit increased serum concentrations of wnt5a and (2) whether wnt5a and/or sFRP5 serum concentrations in obese subjects can be influenced by caloric restriction. Methodology 23 obese human subjects (BMI 44.1±1.1 kg/m2) and 12 age- and sex-matched lean controls (BMI 22.3±0.4 kg/m2) were included in the study. Obese subjects were treated with a very low-calorie diet (approximately 800 kcal/d) for 12 weeks. Body composition was assessed by impedance analysis, insulin sensitivity was estimated by HOMA-IR and the leptin-to-adiponectin ratio and wnt5a and sFRP5 serum concentrations were measured by ELISA. sFRP5 expression in human adipose tissue biopsies was further determined on protein level by immunohistology. Principal Findings Pro-inflammatory wnt5a was not measurable in any serum sample of lean control subjects. In patients with obesity, however, wnt5a became significantly detectable consistent with low grade inflammation in such subjects. Caloric restriction resulted in a weight loss from 131.9±4.0 to 112.3±3.2 kg in the obese patients group. This was accompanied by a significant decrease of HOMA-IR and leptin-to-adiponectin ratio, indicating improved insulin sensitivity. Interestingly, these metabolic improvements were associated with a significant increase in serum concentrations of the anti-inflammatory factor and wnt5a-inhibitor sFRP5. Conclusions/Significance Obesity is associated with elevated serum levels of pro-inflammatory wnt5a in humans. Furthermore, caloric restriction beneficially affects serum concentrations of anti-inflammatory sFRP5

  20. Microglial cell migration stimulated by ATP and C5a involve distinct molecular mechanisms

    PubMed Central

    Miller, Aaron M.; Stella, Nephi

    2009-01-01

    Microglial cells, the macrophages of the brain, play an essential role in the propagation of neuroinflammation. Increased microglial cell migration in response to specific chemoattractants has been documented, but less is known about the differences between these stimuli and the signal transduction pathways that mediate their effects. Current methods to measure cell migration are often labor-intensive and rely on the manual counting of cell number, so more efficient and objective methods are needed. Here we present an improved and higher-throughput Boyden Chamber technique that measures microglial cell migration by using DRAQ5, a nuclear dye that emits in the near-infrared. Out of a panel of chemoattractants tested, we found that ATP and C5a potently stimulate the migration of mouse primary microglial cells. The stimulatory effects of ATP and C5a displayed significant additivity, suggesting that each chemoattractant stimulated migration through independent molecular mechanisms. Accordingly, we found key differences in these responses: ATP stimulated a combination of both chemokinesis and chemotaxis, and this response was mediated by the ROCK signaling pathway; whereas C5a stimulated only chemotaxis and this response was mediated by the Rac1 signaling pathway. Finally, we found that functional PI3-kinase is only required for random basal microglial cell migration. Thus, our results show that distinct non-overlapping signal transduction pathways control different modes of microglial cell migration and suggest that the targeting of these distinct molecular mechanisms should modulate different aspects of neuroinflammation propagation. PMID:19053059

  1. Role of 5-HT5A receptors in the consolidation of memory.

    PubMed

    Gonzalez, Roberto; Chávez-Pascacio, Karla; Meneses, Alfredo

    2013-09-01

    5-HT5 receptor occurs in brain areas implicated in learning and memory. Hence, the effects (0.01-3.0 mg/kg) of SB-6995516 (a 5-HT5A receptor antagonist) in the associative learning task of autoshaping were studied. The results showed that post-training injection of SB-699551 decreased conditioned responses (CR) during short-term (STM; 1.5h; at 0.1mg/kg) and long-term memory (LTM; 24 h; at 3.0 mg/kg) relative to the vehicle animals. Moreover, considering that there are no selective 5-HT5A receptor agonists, next, diverse doses of the serotonin precursor l-tryptophan were studied during STM and LTM, showing that l-tryptophan (5-100mg/kg) facilitated performance, particularly at 50mg/kg. In interactions experiments, l-tryptophan (50 mg/kg) attenuated the impairment effect induced by SB-699551 (either 0.3 or 3.0 mg/kg). All together this evidence suggests that the blockade of 5-HT5A receptor appear to be able to impair STM and LTM (24 h), while its stimulation might facilitate it. Of course further investigation is necessary, meanly with selective 5-HT5A compounds are necessary.

  2. Investigation of Northrop F-5A wing buffet intensity in transonic flight

    NASA Technical Reports Server (NTRS)

    Chintsun, H.; Pi, W. S.

    1974-01-01

    A flight test and data processing program utilizing a Northrop F-5A aircraft instrumented to acquire buffet pressures and response data during transonic maneuvers is discussed. The data are presented in real-time format followed by spectral and statistical analyses. Also covered is a comparison of the aircraft response data with computed responses based on the measured buffet pressures.

  3. The 5 A day Virtual Classroom: an on-line strategy to promote healthful eating.

    PubMed

    DiSogra, L; Glanz, K

    2000-03-01

    Communications technology can help stimulate youth to become involved in health promotion. This article reports on an innovative, Internet-based nutrition program that encouraged children to be advocates for policies that promote eating more fruits and vegetables, the 5 A Day Virtual Classroom. Through this program, students from across the United States discussed the recommendation of 5 A Day at the same time in a classroom without walls. In September 1997 children were asked, "If you were President Clinton, how would you get kids across the country to eat 5 A Day?" Based on content analysis of responses, this article suggests strategies that policymakers could use to encourage children to consume more fruits and vegetables. Approximately 2,600 students participated; 635 entries and 910 suggestions were received. The suggestion categories cited most often were mass media (19.8%), economic issues (15.4%), and social influence (13.8%). The most frequently mentioned specific ideas were to reward children for eating fruits and vegetables and to use presidential authority. Some regional, age, and gender patterns were found. Findings support the potential impact on health education of the 5 A Day Virtual Classroom and of interventions based on communications technology.

  4. Conceptual design of the 6 MW Mod-5A wind turbine generator

    NASA Technical Reports Server (NTRS)

    Barton, R. S.; Lucas, W. C.

    1982-01-01

    The General Electric Company, Advanced Energy Programs Department, is designing under DOE/NASA sponsorship the MOD-5A wind turbine system which must generate electricity for 3.75 cent/KWH (1980) or less. During the Conceptual Design Phase, completed in March, 1981, the MOD-5A WTG system size and features were established as a result of tradeoff and optimization studies driven by minimizing the system cost of energy (COE). This led to a 400' rotor diameter size. The MOD-5A system which resulted is defined in this paper along with the operational and environmental factors that drive various portions of the design. Development of weight and cost estimating relationships (WCER's) and their use in optimizing the MOD-5A are discussed. The results of major tradeoff studies are also presented. Subsystem COE contributions for the 100th unit are shown along with the method of computation. Detailed descriptions of the major subsystems are given, in order that the results of the various trade and optimization studies can be more readily visualized.

  5. Whole-exome sequencing identifies Y1495X of SCN5A to be associated with familial conduction disease and sudden death

    PubMed Central

    Tan, Zhi-Ping; Xie, Li; Deng, Yao; Chen, Jin-Lan; Zhang, Wei-Zhi; Wang, Jian; Yang, Jin-Fu; Yang, Yi-Feng

    2014-01-01

    SCN5A mutations have been reported to underlie a variety of inherited arrhythmias, while the complex overlapping phenotype, especially with congenital heart disease (CHD), is rarely reported. The 48-year-old proband underwent a recent syncope during rest. A CHD (tetralogy of Fallot) and conduction disease was revealed by echocardiogram and ultrasonic cardiogram examination. We combined whole-exome sequencing (WES) and bioinformatics strategies to identify the pathogenic gene for this autosomal-dominant cardiac conduction disease (CCD) in a multi-generation pedigree. We examined four members of this family, including three affected and one unaffected. A novel nonsense mutation (Y1495X) in SCN5A was identified in the affected family members. This mutation is predicted to generate a truncated SCN5A protein, which could result in the loss of sodium current, a defined mechanism of SCN5A related arrhythmias. Our study provides evidence that WES is a highly effective approach for genetic analyses of rare clinical phenotypes. Our study also offers accurate genetic testing information for those yet clinically negative relatives. PMID:25010007

  6. Thermoluminescence solid-state nanodosimetry--the peak 5A/5 dosemeter.

    PubMed

    Fuks, E; Horowitz, Y S; Horowitz, A; Oster, L; Marino, S; Rainer, M; Rosenfeld, A; Datz, H

    2011-02-01

    The shape of composite peak 5 in the glow curve of LiF:Mg,Ti (TLD-100) following (90)Sr/(90)Y beta irradiation, previously demonstrated to be dependent on the cooling rate used in the 400°C pre-irradiation anneal, is shown to be dependent on ionisation density in both naturally cooled and slow-cooled samples. Following heavy-charged particle high-ionisation density (HID) irradiation, the temperature of composite peak 5 decreases by ∼5°C and the peak becomes broader. This behaviour is attributed to an increase in the relative intensity of peak 5a (a low-temperature satellite of peak 5). The relative intensity of peak 5a is estimated using a computerised glow curve deconvolution code based on first-order kinetics. The analysis uses kinetic parameters for peaks 4 and 5 determined from ancillary measurements resulting in nearly 'single-glow peak' curves for both the peaks. In the slow-cooled samples, owing to the increased relative intensity of peak 5a compared with the naturally cooled samples, the precision of the measurement of the 5a/5 intensity ratio is found to be ∼15% (1 SD) compared with ∼25% for the naturally cooled samples. The ratio of peak 5a/5 in the slow-cooled samples is found to increase systematically and gradually through a variety of radiation fields from a minimum value of 0.13±0.02 for (90)Sr/(90)Y low-ionisation density irradiations to a maximum value of ∼0.8 for 20 MeV Cu and I ion HID irradiations. Irradiation by low-energy electrons of energy 0.1-1.5 keV results in values between 1.27 and 0.95, respectively. The increasing values of the ratio of peak 5a/5 with increasing ionisation density demonstrate the viability of the concept of the peak 5a/5 nanodosemeter and its potential in the measurement of average ionisation density in a 'nanoscopic' mass containing the trapping centre/luminescent centre spatially correlated molecule giving rise to composite peak 5.

  7. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  8. Wnt-5a Ligand Modulates Mitochondrial Fission-Fusion in Rat Hippocampal Neurons*

    PubMed Central

    Godoy, Juan A.; Arrázola, Macarena S.; Ordenes, Daniela; Silva-Alvarez, Carmen; Braidy, Nady; Inestrosa, Nibaldo C.

    2014-01-01

    The Wnt signaling pathway plays an important role in developmental processes, including embryonic patterning, cell specification, and cell polarity. Wnt components participate in the development of the central nervous system, and growing evidence indicates that this pathway also regulates the function of the adult nervous system. In this study, we report that Wnt-5a, a noncanonical Wnt ligand, is a potent activator of mitochondrial dynamics and induces acute fission and fusion events in the mitochondria of rat hippocampal neurons. The effect of Wnt-5a was inhibited in the presence of sFRP, a Wnt scavenger. Similarly, the canonical Wnt-3a ligand had no effect on mitochondrial fission-fusion events, suggesting that this effect is specific for Wnt-5a alone. We also show that the Wnt-5a effects on mitochondrial dynamics occur with an increase in both intracellular and mitochondrial calcium (Ca2+), which was correlated with an increased phosphorylation of Drp1(Ser-616) and a decrease of Ser-637 phosphorylation, both indicators of mitochondrial dynamics. Electron microscope analysis of hippocampal tissues in the CA1 region showed an increase in the number of mitochondria present in the postsynaptic region, and this finding correlated with a change in mitochondrial morphology. We conclude that Wnt-5a/Ca2+ signaling regulates the mitochondrial fission-fusion process in hippocampal neurons, a feature that might help to further understand the role of Wnt-related pathologies, including neurodegenerative diseases associated with mitochondrial dysfunction, and represents a potentially important link between impaired metabolic function and degenerative disorders. PMID:25336659

  9. Wnt-5a ligand modulates mitochondrial fission-fusion in rat hippocampal neurons.

    PubMed

    Godoy, Juan A; Arrázola, Macarena S; Ordenes, Daniela; Silva-Alvarez, Carmen; Braidy, Nady; Inestrosa, Nibaldo C

    2014-12-26

    The Wnt signaling pathway plays an important role in developmental processes, including embryonic patterning, cell specification, and cell polarity. Wnt components participate in the development of the central nervous system, and growing evidence indicates that this pathway also regulates the function of the adult nervous system. In this study, we report that Wnt-5a, a noncanonical Wnt ligand, is a potent activator of mitochondrial dynamics and induces acute fission and fusion events in the mitochondria of rat hippocampal neurons. The effect of Wnt-5a was inhibited in the presence of sFRP, a Wnt scavenger. Similarly, the canonical Wnt-3a ligand had no effect on mitochondrial fission-fusion events, suggesting that this effect is specific for Wnt-5a alone. We also show that the Wnt-5a effects on mitochondrial dynamics occur with an increase in both intracellular and mitochondrial calcium (Ca(2+)), which was correlated with an increased phosphorylation of Drp1(Ser-616) and a decrease of Ser-637 phosphorylation, both indicators of mitochondrial dynamics. Electron microscope analysis of hippocampal tissues in the CA1 region showed an increase in the number of mitochondria present in the postsynaptic region, and this finding correlated with a change in mitochondrial morphology. We conclude that Wnt-5a/Ca(2+) signaling regulates the mitochondrial fission-fusion process in hippocampal neurons, a feature that might help to further understand the role of Wnt-related pathologies, including neurodegenerative diseases associated with mitochondrial dysfunction, and represents a potentially important link between impaired metabolic function and degenerative disorders.

  10. IL-4 Causes Hyperpermeability of Vascular Endothelial Cells through Wnt5A Signaling

    PubMed Central

    Skaria, Tom; Burgener, Julia; Bachli, Esther; Schoedon, Gabriele

    2016-01-01

    Microvascular leakage due to endothelial barrier dysfunction is a prominent feature of T helper 2 (Th2) cytokine mediated allergic inflammation. Interleukin-4 (IL-4) is a potent Th2 cytokine, known to impair the barrier function of endothelial cells. However, the effectors mediating IL-4 induced cytoskeleton remodeling and consequent endothelial barrier dysfunction remain poorly defined. Here we have used whole genome transcriptome profiling and gene ontology analyses to identify the genes and processes regulated by IL-4 signaling in human coronary artery endothelial cells (HCAEC). The study revealed Wnt5A as an effector that can mediate actin cytoskeleton remodeling in IL-4 activated HCAEC through the regulation of LIM kinase (LIMK) and Cofilin (CFL). Following IL-4 treatment, LIMK and CFL were phosphorylated, thereby indicating the possibility of actin stress fiber formation. Imaging of actin showed the formation of stress fibers in IL-4 treated live HCAEC. Stress fiber formation was notably decreased in the presence of Wnt inhibitory factor 1 (WIF1). Non-invasive impedance measurements demonstrated that IL-4 increased the permeability and impaired the barrier function of HCAEC monolayers. Silencing Wnt5A significantly reduced permeability and improved the barrier function of HCAEC monolayers upon IL-4 treatment. Our study identifies Wnt5A as a novel marker of IL-4 activated vascular endothelium and demonstrates a critical role for Wnt5A in mediating IL-4 induced endothelial barrier dysfunction. Wnt5A could be a potential therapeutic target for reducing microvascular leakage and edema formation in Th2 driven inflammatory diseases. PMID:27214384

  11. Protein sequence comparison and protein evolution

    SciTech Connect

    Pearson, W.R.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  12. Human VAP-C negatively regulates hepatitis C virus propagation.

    PubMed

    Kukihara, Hiroshi; Moriishi, Kohji; Taguwa, Shuhei; Tani, Hideki; Abe, Takayuki; Mori, Yoshio; Suzuki, Tetsuro; Fukuhara, Takasuke; Taketomi, Akinobu; Maehara, Yoshihiko; Matsuura, Yoshiharu

    2009-08-01

    Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) and subtype B (VAP-B) are involved in the regulation of membrane trafficking, lipid transport and metabolism, and the unfolded protein response. VAP-A and VAP-B consist of the major sperm protein (MSP) domain, the coiled-coil motif, and the C-terminal transmembrane anchor and form homo- and heterodimers through the transmembrane domain. VAP-A and VAP-B interact with NS5B and NS5A of hepatitis C virus (HCV) through the MSP domain and the coiled-coil motif, respectively, and participate in the replication of HCV. VAP-C is a splicing variant of VAP-B consisting of the N-terminal half of the MSP domain of VAP-B followed by the subtype-specific frameshift sequences, and its biological function has not been well characterized. In this study, we have examined the biological functions of VAP-C in the propagation of HCV. VAP-C interacted with NS5B but not with VAP-A, VAP-B, or NS5A in immunoprecipitation analyses, and the expression of VAP-C inhibited the interaction of NS5B with VAP-A or VAP-B. Overexpression of VAP-C impaired the RNA replication of the HCV replicon and the propagation of the HCV JFH1 strain, whereas overexpression of VAP-A and VAP-B enhanced the replication. Furthermore, the expression of VAP-C was observed in various tissues, whereas it was barely detected in the liver. These results suggest that VAP-C acts as a negative regulator of HCV propagation and that the expression of VAP-C may participate in the determination of tissue tropism of HCV propagation.

  13. Toxicology studies with recombinant staphylokinase and with SY 161-P5, a polyethylene glycol-derivatized cysteine-substitution mutant.

    PubMed

    Moons, L; Vanlinthout, I; Roelants, I; Moreadith, R; Collen, D; Rapold, H J

    2001-01-01

    SY 161-P5, a polyethylene glycol derivatized (PEGylated) mutant of the recombinant Staphylokinase (rSak) variant SakSTAR, exhibiting reduced antigenicity is in clinical development for treatment of acute myocardial infarction as a single bolus injection. A series of safety studies were performed in vivo as a routine toxicology program with SY 161-P5 (PEG-rSakSTAR) and with the recombinant Staphylokinase variant Sak42D (rSak42D). For both compounds, intravenous single bolus injections of up to 100-fold therapeutic equivalent, as well as repeated injections during 7 to 28 days revealed no significant pathological findings in mice, rats or hamsters. However, New Zealand white rabbits developed clinically silent, multifocal myocarditis following single or repeat doses of SY 161-P5 or of Sak42D. These findings were dose-independent and reversible. A similar species-specific cardiotoxic effect has previously been described for other proteolytic proteins, including the approved drugs Streptokinase and Acetylated Plasminogen Streptokinase Complex (APSAC). The large experience with these drugs, as well as the clinical data accumulated both with PEGylated and non-PEGylated rSak variants to date, do not indicate cardiotoxic hazards associated with the use of these drugs in humans.

  14. Newcastle disease virus employs macropinocytosis and Rab5a-dependent intracellular trafficking to infect DF-1 cells

    PubMed Central

    Zhan, Yuan; Yuan, Yanmei; Sun, Yingjie; Qiu, Xusheng; Meng, Chunchun; Song, Cuiping; Liao, Ying; Ding, Chan

    2016-01-01

    Oncolytic Newcastle disease virus (NDV) reportedly employs direct fusion of the viral envelope with the plasma membrane and caveolae-dependent endocytosis to enter cells. Here, we show that macropinocytosis and clathrin-mediated endocytosis are involved in NDV entry into a galline embryonic fibroblast cell line. Upon specific inhibition of clathrin assembly, GTPase dynamin, Na+/H+ exchangers, Ras-related C3 botulinum toxin substrate 1, p21 activated kinase 1 or protein kinase C, entry of NDV and its propagation were suppressed. NDV entry into cells triggers Rac1-Pak1 signaling and elicits actin rearrangement and plasma membrane ruffling. Moreover, NDV internalization within macropinosomes and trafficking involve Rab5a-positive vesicles. This is the first report demonstrating that NDV utilizes clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter cells. These findings shed new light on the molecular mechanisms underlying NDV entry into cells, and provide potential targets for NDV-mediated therapy in cancer. PMID:27861142

  15. Chronic vasodilation increases renal medullary PDE5A and α-ENaC through independent renin-angiotensin-aldosterone system pathways.

    PubMed

    West, Crystal A; Shaw, Stefan; Sasser, Jennifer M; Fekete, Andrea; Alexander, Tyler; Cunningham, Mark W; Masilamani, Shyama M E; Baylis, Chris

    2013-11-15

    We have previously observed that many of the renal and hemodynamic adaptations seen in normal pregnancy can be induced in virgin female rats by chronic systemic vasodilation. Fourteen-day vasodilation with sodium nitrite or nifedipine (NIF) produced plasma volume expansion (PVE), hemodilution, and increased renal medullary phosphodiesterase 5A (PDE5A) protein. The present study examined the role of the renin-angiotensin-aldosterone system (RAAS) in this mechanism. Virgin females were treated for 14 days with NIF (10 mg·kg(-1)·day(-1) via diet), NIF with spironolactone [SPR; mineralocorticoid receptor (MR) blocker, 200-300 mg·kg(-1)·day(-1) via diet], NIF with losartan [LOS; angiotensin type 1 (AT1) receptor blocker, 20 mg·kg(-1)·day(-1) via diet], enalapril (ENAL; angiotensin-converting enzyme inhibitor, 62.5 mg/l via water), or vehicle (CON). Mean arterial pressure (MAP) was reduced 7.4 ± 0.5% with NIF, 6.33 ± 0.5% with NIF + SPR, 13.3 ± 0.9% with NIF + LOS, and 12.0 ± 0.4% with ENAL vs. baseline MAP. Compared with CON (3.6 ± 0.3%), plasma volume factored for body weight was increased by NIF (5.2 ± 0.4%) treatment but not by NIF + SPR (4.3 ± 0.3%), NIF + LOS (3.6 ± 0.1%), or ENAL (4.0 ± 0.3%). NIF increased PDE5A protein abundance in the renal inner medulla, and SPR did not prevent this increase (188 ± 16 and 204 ± 22% of CON, respectively). NIF increased the α-subunit of the epithelial sodium channel (α-ENaC) protein in renal outer (365 ± 44%) and inner (526 ± 83%) medulla, and SPR prevented these changes. There was no change in either PDE5A or α-ENaC abundance vs. CON in rats treated with NIF + LOS or ENAL. These data indicate that the PVE and renal medullary adaptations in response to chronic vasodilation result from RAAS signaling, with increases in PDE5A mediated through AT1 receptor and α-ENaC through the MR.

  16. Whey protein fractionation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concentrated whey protein products from cheese whey, such as whey protein concentrate (WPC) and whey protein isolate (WPI), contain more than seven different types of proteins: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin ...

  17. A Novel Role for the Receptor of the Complement Cleavage Fragment C5a, C5aR1, in CCR5-Mediated Entry of HIV into Macrophages.

    PubMed

    Moreno-Fernandez, Maria E; Aliberti, Julio; Groeneweg, Sander; Köhl, Jörg; Chougnet, Claire A

    2016-04-01

    The complement system is an ancient pattern recognition system that becomes activated during all stages of HIV infection. Previous studies have shown that C5a can enhance the infection of monocyte-derived macrophages and T cells indirectly through the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and the attraction of dendritic cells. C5a exerts its multiple biologic functions mainly through activation of C5a receptor 1 (C5aR1). Here, we assessed the role of C5aR1 as an enhancer of CCR5-mediated HIV infection. We determined CCR5 and C5aR1 heterodimer formation in myeloid cells and the impact of C5aR1 blockade on HIV entry and genomic integration. C5aR1/CCR5 heterodimer formation was identified by immunoprecipitation and western blotting. THP-1 cells and monocyte-derived macrophages (MDM) were infected by R5 laboratory strains or HIV pseudotyped for the vesicular stomatitis virus (VSV) envelope. Levels of integrated HIV were measured by quantitative PCR after targeting of C5aR1 by a C5aR antagonist, neutralizing C5aR1 monoclonal antibody (mAb) or hC5a. C5aR1 was also silenced by specific siRNA prior to viral entry. We found that C5aR1 forms heterodimers with the HIV coreceptor CCR5 in myeloid cells. Targeting C5aR1 significantly decreased integration by R5 viruses but not by VSV-pseudotyped viruses, suggesting that C5aR1 is critical for viral entry. The level of inhibition achieved with C5aR1-blocking reagents was comparable to that of CCR5 antagonists. Mechanistically, C5aR1 targeting decreased CCR5 expression. MDM from CCR5Δ32 homozygous subjects expressed levels of C5aR1 similar to CCR5 WT individuals, suggesting that mere C5aR1 expression is not sufficient for HIV infection. HIV appeared to preferentially enter THP-1 cells expressing high levels of both C5aR1 and CCR5. Targeted reduction of C5aR1 expression in such cells reduced HIV infection by ~50%. Our data thus suggest that C5aR1 acts as an enhancer of CCR5-mediated HIV entry into

  18. Sequencing of SCN5A identifies rare and common variants associated with cardiac conduction

    PubMed Central

    Magnani, Jared W.; Brody, Jennifer A.; Prins, Bram P.; Arking, Dan E.; Lin, Honghuang; Yin, Xiaoyan; Liu, Ching-Ti; Morrison, Alanna C.; Zhang, Feng; Spector, Tim D.; Alonso, Alvaro; Bis, Joshua C.; Heckbert, Susan R.; Lumley, Thomas; Sitlani, Colleen M.; Cupples, L. Adrienne; Lubitz, Steven A.; Soliman, Elsayed Z.; Pulit, Sara L.; Newton-Cheh, Christopher; O'Donnell, Christopher J.; Ellinor, Patrick T.; Benjamin, Emelia J.; Muzny, Donna M.; Gibbs, Richard A.; Santibanez, Jireh; Taylor, Herman A.; Rotter, Jerome I.; Lange, Leslie A.; Psaty, Bruce M.; Jackson, Rebecca; Rich, Stephen S.; Boerwinkle, Eric; Jamshidi, Yalda; Sotoodehnia, Nona

    2014-01-01

    Background The cardiac sodium channel SCN5A regulates atrioventricular and ventricular conduction. Genetic variants in this gene are associated with PR and QRS intervals. We sought to further characterize the contribution of rare and common coding variation in SCN5A to cardiac conduction. Methods and Results In the Cohorts for Heart and Aging Research in Genomic Epidemiology Targeted Sequencing Study (CHARGE), we performed targeted exonic sequencing of SCN5A (n=3699, European-ancestry individuals) and identified 4 common (minor allele frequency >1%) and 157 rare variants. Common and rare SCN5A coding variants were examined for association with PR and QRS intervals through meta-analysis of European ancestry participants from CHARGE, NHLBI’s Exome Sequencing Project (ESP, n=607) and the UK10K (n=1275) and by examining ESP African-ancestry participants (N=972). Rare coding SCN5A variants in aggregate were associated with PR interval in European and African-ancestry participants (P=1.3×10−3). Three common variants were associated with PR and/or QRS interval duration among European-ancestry participants and one among African-ancestry participants. These included two well-known missense variants; rs1805124 (H558R) was associated with PR and QRS shortening in European-ancestry participants (P=6.25×10−4 and P=5.2×10−3 respectively) and rs7626962 (S1102Y) was associated with PR shortening in those of African ancestry (P=2.82×10−3). Among European-ancestry participants, two novel synonymous variants, rs1805126 and rs6599230, were associated with cardiac conduction. Our top signal, rs1805126 was associated with PR and QRS lengthening (P=3.35×10−7 and P=2.69×10−4 respectively), and rs6599230 was associated with PR shortening (P=2.67×10−5). Conclusions By sequencing SCN5A, we identified novel common and rare coding variants associated with cardiac conduction. PMID:24951663

  19. Isolation and Characterization of Pep5, a Gene Essential for Vacuolar Biogenesis in Saccharomyces Cerevisiae

    PubMed Central

    Woolford, C. A.; Dixon, C. K.; Manolson, M. F.; Wright, R.; Jones, E. W.

    1990-01-01

    pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37°. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursor forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein. PMID:2204580

  20. Cloning and characterization of Kin5, a novel Tetrahymena ciliary kinesin II.

    PubMed

    Awan, Aashir; Bernstein, Mitchell; Hamasaki, Toshikazu; Satir, Peter

    2004-05-01

    Two Tetrahymena kinesin-like proteins (klps) of the kinesin II subfamily, Kin1 and Kin2, were first identified by Brown et al. [1999: Mol Biol Cell 10: 3081-3096] and shown to be involved in ciliary morphogenesis probably as molecular motors in intraciliary transport (ICT). Using Tetrahymena genomic DNA as a template, we cloned Kin5, another kinesin II subfamily member. Kin5 is upregulated upon deciliation, suggesting that Kin5 is a ciliary protein. Kin5 is most closely related to Osm3, a Caenorhabditis elegans kinesin II; Osm3 and Kin5 have a 56% identity, which rises to 60.4% in the motor domain and a 45% identity in a 60 amino acid region of the C-terminal FERM (4.1, Ezrin, Radixin, Moesin) domain, not present in Kin1 or Kin2, which we hypothesize to be a critical domain either for dimerization or for cargo recognition in ICT. An antibody to a peptide sequence from the tail region of Kin5 localizes in a punctate pattern along the ciliary axoneme, colocalizing with an antibody to the raft protein IFT139. These findings suggest that Kin5 is an ICT motor like Osm3. Osm3 orthologs apparently transport membrane proteins and Kin5 may be the homodimeric kinesin II that performs this function in Tetrahymena cilia.

  1. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  2. Utility of 5A molecular sieves to measure carbon isotope ratios in lipid biomarkers.

    PubMed

    Tolosa, Imma; Ogrinc, Nives

    2007-09-21

    A procedure using 5A zeolite sorption to separate cyclic/branched organic compounds from the linear ones was developed and carbon isotopic fractionation effects were investigated in different families of compounds, e.g. within the hydrocarbon and alcohol compounds. The 5A sieve has a pore size such that only linear components can be incorporated into the pores whereas the cyclic/branched compounds are remaining free in the organic solution. The sorbed compounds were released from the molecular sieve with HF and solvent extracted with hexane. The method enables the isolation of linear saturated classes, such as n-alkanes and n-fatty alcohols from branched/cyclic compounds without isotopic fractionation for compound-specific isotope analysis (CSIA) of delta(13)C. However, alkene hydrocarbons, sterols and some aromatics were completely or partly degraded with the molecular sieve.

  3. Identification of a common protein association region in the neuronal Cdk5 activator.

    PubMed

    Wang, X; Ching, Y P; Lam, W H; Qi, Z; Zhang, M; Wang, J H

    2000-10-13

    Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity.

  4. Effects of sex steroids on indices of protein turnover in rainbow trout (Oncorhynchus mykiss) white muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Effects of 17-estradiol (E2), testosterone, and 5a-dihydrotestosterone (DHT) on protein turnover and proteolytic gene expression were determined in rainbow trout (Oncorhynchus mykiss) primary myocytes and white muscle tissue. E2 reduced rates of protein synthesis and increased rates of protein degr...

  5. Mod-5A wind turbine generator program design report. Volu