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Sample records for autographa californica gp64

  1. Autographa californica multiple nucleopolyhedrovirus GP64 protein: Analysis of domain I and V amino acid interactions and membrane fusion activity

    SciTech Connect

    Yu, Qianlong; Blissard, Gary W.; Liu, Tong-Xian; Li, Zhaofei

    2016-01-15

    The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, but no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. - Highlights: • The baculovirus envelope glycoprotein GP64 is a class III viral fusion protein. • The detailed mechanism of conformational change of GP64 is unknown. • We analyzed 24 positions that might stabilize the post-fusion structure of GP64. • We identified 4 residues in domain V that were critical for membrane fusion. • Two residues are critical for formation of the pre-fusion conformation of GP64.

  2. A single amino acid substitution modulates low-pH-triggered membrane fusion of GP64 protein in Autographa californica and Bombyx mori nucleopolyhedroviruses

    SciTech Connect

    Katou, Yasuhiro; Yamada, Hayato; Ikeda, Motoko; Kobayashi, Michihiro

    2010-09-01

    We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.

  3. Proteomics of the Autographa californica nucleopolyhedrovirus budded virions.

    PubMed

    Wang, Ranran; Deng, Fei; Hou, Dianhai; Zhao, Yong; Guo, Lin; Wang, Hualin; Hu, Zhihong

    2010-07-01

    Baculoviruses produce two progeny phenotypes during their replication cycles. The occlusion-derived virus (ODV) is responsible for initiating primary infection in the larval midgut, and the budded virus (BV) phenotype is responsible for the secondary infection. The proteomics of several baculovirus ODVs have been revealed, but so far, no extensive analysis of BV-associated proteins has been conducted. In this study, the protein composition of the BV of Autographa californica nucleopolyhedrovirus (AcMNPV), the type species of baculoviruses, was analyzed by various mass spectrometry (MS) techniques, including liquid chromatography-triple quadrupole linear ion trap (LC-Qtrap), liquid chromatography-quadrupole time of flight (LC-Q-TOF), and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). SDS-PAGE and MALDI-TOF analyses showed that the three most abundant proteins of the AcMNPV BV were GP64, VP39, and P6.9. A total of 34 viral proteins associated with the AcMNPV BV were identified by the indicated methods. Thirteen of these proteins, PP31, AC58/59, AC66, IAP-2, AC73, AC74, AC114, AC124, chitinase, polyhedron envelope protein (PEP), AC132, ODV-E18, and ODV-E56, were identified for the first time to be BV-associated proteins. Western blot analyses showed that ODV-E18 and ODV-E25, which were previously thought to be ODV-specific proteins, were also present in the envelop fraction of BV. In addition, 11 cellular proteins were found to be associated with the AcMNPV BV by both LC-Qtrap and LC-Q-TOF analyses. Interestingly, seven of these proteins were also identified in other enveloped viruses, suggesting that many enveloped viruses may commonly utilize certain conserved cellular pathways.

  4. Genetic diversity among isolates of Autographa californica multiple nucleopolyhedrovirus

    USDA-ARS?s Scientific Manuscript database

    Our knowledge of genetic variation at the nucleotide sequence level of Autographa californica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus) derives from complete genome sequences of the C6 clonal isolate of AcMNPV and the R1 and CL3 clonal isolates of AcMNPV variants Rachip...

  5. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

    SciTech Connect

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi . E-mail: hiroshi.takaku@it-chiba.ac.jp

    2006-05-05

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

  6. Mapping the conformational epitope of a neutralizing antibody (AcV1) directed against the AcMNPV GP64 protein

    SciTech Connect

    Zhou Jian; Blissard, Gary W. . E-mail: gwb1@cornell.edu

    2006-09-01

    The envelope glycoprotein GP64 of Autographa californica nucleopolyhedrovirus (AcMNPV) is necessary and sufficient for the acid-induced membrane fusion activity that is required for fusion of the budded virus (BV) envelope and the endosome membrane during virus entry. Infectivity of the budded virus (BV) is neutralized by AcV1, a monoclonal antibody (MAb) directed against GP64. Prior studies indicated that AcV1 recognizes a conformational epitope and does not inhibit virus attachment to the cell, but instead inhibits entry at a step following virus attachment. We found that AcV1 recognition of GP64 was lost upon exposure of GP64 to low pH (pH 4.5) and restored by returning GP64 to pH 6.2. In addition, the AcV1 epitope was lost upon denaturation of GP64 in SDS, but the AcV1 epitope was restored by refolding the protein in the absence of SDS. Using truncated GP64 proteins expressed in insect cells, we mapped the AcV1 epitope to a 24 amino acid region in the central variable domain of GP64. When sequences within the mapped AcV1 epitope were substituted with a c-Myc epitope and the resulting construct was used to replace wt GP64 in recombinant AcMNPV viruses, the modified GP64 protein appeared to function normally. However, an anti-c-Myc monoclonal antibody did not neutralize infectivity of those viruses. Because binding of the c-Myc MAb to the same site in the GP64 sequence did not result in neutralization, these studies suggest that AcV1 neutralization may result from a specific structural constraint caused by AcV1 binding and not simply by steric hindrance caused by antibody binding at this position in GP64.

  7. Nuclear Translocation Sequence and Region in Autographa californica Multiple Nucleopolyhedrovirus ME53 That Are Important for Optimal Baculovirus Production

    PubMed Central

    Liu, Yang; de Jong, Jondavid; Nagy, Éva; Theilmann, David A.

    2016-01-01

    ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is in the family Baculoviridae, genus Alphabaculovirus. AcMNPV me53 is a highly conserved immediate early gene in all lepidopteran baculoviruses that have been sequenced and is transcribed up to late times postinfection. Although me53 is not essential for viral DNA synthesis, infectious budded virus (BV) production is greatly attenuated when it is deleted. ME53 associates with the nucleocapsid on both budded virus and occlusion-derived virus, but not with the virus envelope. ME53 colocalizes in plasma membrane foci with the envelope glycoprotein GP64 in a GP64-dependent manner. ME53 localizes in the cytoplasm early postinfection, and despite the lack of a reported nuclear localization signal (NLS), ME53 translocates to the nucleus at late times postinfection. To map determinants of ME53 that facilitate its nuclear translocation, recombinant AcMNPV bacmids containing a series of ME53 truncations, internal deletions, and peptides fused with hemagglutinin (HA) or green fluorescent protein (GFP) tags were constructed. Intracellular-localization studies identified residues within amino acids 109 to 137 at the N terminus of ME53 that acted as the nuclear translocation sequence (NTS), facilitating its nuclear transport at late times postinfection. The first 100 N-terminal amino acids and the last 50 C-terminal amino acids of ME53 are dispensable for high levels of budded virus production. The region within amino acids 101 to 398, which also contains the NTS, is critical for optimal levels of budded virus production. IMPORTANCE Baculovirus me53 is a conserved immediate early gene found in all sequenced lepidopteran alpha- and betabaculoviruses. We first identified residues within amino acids 109 to 137 at the N terminus that act as the ME53 nuclear translocation sequence (NTS) to facilitate its nuclear translocation and defined an internal region within amino acids 101 to 398, which includes the NTS, as

  8. The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene.

    PubMed

    Durantel, D; Croizier, L; Ayres, M D; Croizier, G; Possee, R D; López-Ferber, M

    1998-03-01

    Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

  9. Identification of genes involved in DNA replication of the Autographa californica baculovirus.

    PubMed Central

    Kool, M; Ahrens, C H; Goldbach, R W; Rohrmann, G F; Vlak, J M

    1994-01-01

    By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa californica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2, and PE38 stimulate DNA replication. No stimulation by the AcMNPV pcna gene, encoding a protein with sequence homology to proliferating-cell nuclear antigen, was observed. A pattern of amino acids found in a number of single-stranded-DNA-binding proteins was identified in the carboxyl-terminal region of IE-1. Images PMID:7972036

  10. A soluble form of P74 can act as a per os infectivity factor to the autographa californica multiple nucleopolyhedrovirus

    USDA-ARS?s Scientific Manuscript database

    The baculovirus occlusion-derived virion (ODV) is required to spread virus infection among insect hosts via the per os route. The Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV) P74 protein is an ODV envelope protein that is essential for ODVs to be infectious. P74 is anchored in ...

  11. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

    SciTech Connect

    Xiao Wei; Yang Yi; Weng Qingbei; Lin Tiehao; Yuan Meijin; Yang Kai; Pang Yi

    2009-08-15

    Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

  12. BV/ODV-E26: a palmitoylated, multifunctional structural protein of Autographa californica nucleopolyhedrovirus.

    PubMed

    Burks, Jared K; Summers, Max D; Braunagel, Sharon C

    2007-04-25

    Autographa californica nucleopolyhedrovirus Ac16 is 1 of 17 genes conserved within Type 1 nucleopolyhedroviruses. This report demonstrates that multiple isoforms of the protein encoded by Ac16, BV/ODV-E26 (E26), are present in the infected cell. One form of E26 associates with viral DNA or DNA-binding proteins, while a second form associates with intracellular membranes and this is likely due to palmitoylation. The different forms of E26 present unique epitopes that can be discriminated by antiserum produced to bacterially or virally produced antigen. A summation of the data now available on E26 suggests that it is a multifunctional protein and the functional states assume unique conformations that can be discriminated by differing antisera.

  13. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    SciTech Connect

    Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  14. The mosaic structure of the polyhedrin gene of the Autographa californica nucleopolyhedrovirus (AcMNPV).

    PubMed

    Jehle, Johannes A

    2004-08-01

    The polyhedrin (polh) gene of nucleopolyhedroviruses (NPVs) encodes for the matrix protein of the virus occlusion body and is one of the most conserved baculovirus genes. Previous analyses of different NPV genes and polh genes provided conflicting results indicating that the Autographa californica nucleopolyhedrovirus (AcMNPV) is generally a member of the so-called group I NPVs and is most closely related to Rachiplusia ou (Ro) NPV, whereas the AcMNPV polh is more similar to the polh of the group II NPVs. A comparative analysis of the AcMNPV polh and its closest neighbours within group I and group II NPV, the RoMNPV and the Thysanoplusia orichalcea (Thor) NPV, was performed using Hidden Markov Models for detecting recombination. The result provided strong evidence that the AcMNPV polh is a chimerical gene which consists of a mosaic of group I and group II NPV specific sequences.

  15. Functional characterization of Autographa californica multiple nucleopolyhedrovirus gp16 (ac130)

    SciTech Connect

    Yang, Ming; Huang, Cui; Qian, Duo-Duo; Li, Lu-Lin

    2014-09-15

    To investigate the function of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp16, multiple gp16-knockout and repair mutants were constructed and characterized. No obvious difference in productivity of budded virus, DNA synthesis, late gene expression and morphogenesis was observed between gp16-knockout and repair viruses, but gp16 deletion resulted in six hours of lengthening in ST{sub 50} to the third instar Spodoptera exigua larvae in bioassays. GP16 was fractionated mainly in the light membrane fraction, by subcellular fractionation. A GP16-EGFP fusion protein was predominantly localized close around the nuclear membrane in infected cells, being coincident with formation of the vesicles associated with the nuclear membrane, which hosted nucleocapsids released from the nucleus. These data suggest that gp16 is not required for viral replication, but may be involved in membrane trafficking associated with the envelopment/de-envelopment of budded viruses when they cross over the nuclear membrane and pass through cytoplasm. - Highlights: • gp16 knockout and repair mutants of AcMNPV were constructed and characterized. • AcMNPV gp16 is not essential to virus replication. • Deletion of gp16 resulted in time lengthening to kill S. exigua larvae. • GP16 was localized close around the nuclear membrane of infected cells. • GP16 was fractionated in the light membrane fraction in subcellular fractionation.

  16. ac18 is not essential for the propagation of Autographa californica multiple nucleopolyhedrovirus

    SciTech Connect

    Wang Yanjie; Wu Wenbi; Li Zhaofei; Yuan Meijin; Feng Guozhong; Yu Qian; Yang Kai Pang Yi

    2007-10-10

    orf18 (ac18) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, an ac18 knockout AcMNPV bacmid was generated to determine the role of ac18 in baculovirus life cycle. After transfection of Sf-9 cells, the ac18-null mutant showed similar infection pattern to the parent virus and the ac18 repair virus with respect to the production of infectious budded virus, occlusion bodies, or the formation of nucleocapsids as visualized by electron microscopy. The deletion mutant did not reduce AcMNPV infectivity for Trichoplusia ni in LD{sub 50} bioassay; however, it did take 24 h longer for deleted mutant to kill T. ni larvae than wild-type virus in LT{sub 50} bioassay. Our results demonstrate that ac18 is not essential for viral propagation both in vitro and in vivo, but it may play a role in efficient virus infection in T. ni larvae.

  17. Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly

    SciTech Connect

    Liu Chao; Li Zhaofei Wu Wenbi; Li Lingling; Yuan Meijin; Pan Lijing; Yang Kai Pang Yi

    2008-12-05

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf53 (ac53) is a highly conserved gene existing in all sequenced Lepidoptera and Hymenoptera baculoviruses, but its function remains unknown. To investigate its role in the baculovirus life cycle, an ac53 deletion virus (vAc{sup ac53KO-PH-GFP}) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis revealed that vAc{sup ac53KO-PH-GFP} could not produce infectious budded virus in infected Sf9 cells. Real-time PCR demonstrated that the ac53 deletion did not affect the levels of viral DNA replication. Electron microscopy showed that many lucent tubular shells devoid of the nucleoprotein core are present in the virogenic stroma and ring zone, indicating that the ac53 knockout affected nucleocapsid assembly. With a recombinant virus expressing an Ac53-GFP fusion protein, we observed that Ac53 was distributed within the cytoplasm and nucleus at 24 h post-infection, but afterwards accumulated predominantly near the nucleus-cytoplasm boundary. These data demonstrate that ac53 is involved in nucleocapsid assembly and is an essential gene for virus production.

  18. Nuclear localization of actin requires AC102 in Autographa californica multiple nucleopolyhedrovirus-infected cells

    PubMed Central

    Ohkawa, Taro; Volkman, Loy E.

    2012-01-01

    Autographa californica multiple nucleopolyhedrovirus requires nuclear actin for progeny virus production and thereby encodes viral products that ensure actin’s translocation to and retention within the nucleus. Current evidence suggests that the ie0–ie1 gene complex along with five nuclear localization of actin (NLA) genes are sufficient for NLA in transient transfection experiments. Here we report that, during infection, only one of the five NLA genes, Ac102, was essential for NLA, and that AC102 had at least one other activity critical for budded virus (BV) production. Viral deletion mutants in the other four NLA genes were viable, with only two having replication phenotypes different from that of the wild type. Infection with AcΔpe38 revealed a delay in both BV production and NLA. Infection with AcΔ152 revealed a delay in BV production, but no corresponding delay in NLA. Infection with either AcΔpe38 or AcΔ152 resulted in slightly reduced BV titres. Deletion of Ac004 or he65 had no impact on actin translocation kinetics, timing of BV production or BV titres. These results implicate AC102 as a key player in baculovirus manipulation of actin. PMID:22592260

  19. Chitinase from Autographa californica multiple nucleopolyhedrovirus: rapid purification from Sf-9 medium and mode of action.

    PubMed

    Fukamizo, Tamo; Sato, Hirokazu; Mizuhara, Mamiko; Ohnuma, Takayuki; Gotoh, Takeshi; Hiwatashi, Kazuyuki; Takahashi, Saori

    2011-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase is involved in the final liquefaction of infected host larvae. We purified the chitinase rapidly to homogeneity from Sf-9 cells infected with AcMNPV by a simple procedure using a pepstatin-aminohexyl-Sepharose column. In past studies, a recombinant AcMNPV chitinase was found to exhibit both exo- and endo-chitinase activities by analysis using artificial substrates with a fluorescent probe. In this study, however, we obtained more accurate information on the mode of action of the chitinase by HPLC analysis of the enzymatic products using natural oligosaccharide and polysaccharide substrates. The AcMNPV chitinase hydrolyzed the second β-1,4 glycosidic linkage from the non-reducing end of the chitin oligosaccharide substrates [(GlcNAc)(n), n=4, 5, and 6], producing the β-anomer of (GlcNAc)₂. The mode of action was similar to that of Serratia marcescens chitinase A (SmChiA), the amino acid sequence of which is 60.5% homologous to that of the AcMNPV enzyme. The enzyme also hydrolyzed solid β-chitin, producing only (GlcNAc)₂. The AcMNPV chitinase processively hydrolyzes solid β-chitin in a manner similar to SmChiA. The processive mechanism of the enzyme appears to be advantageous in liquefaction of infected host larvae.

  20. Autographa californica multiple nucleopolyhedrovirus odv-e66 is an essential gene required for oral infectivity.

    PubMed

    Xiang, Xingwei; Chen, Lin; Hu, Xiaolong; Yu, Shaofang; Yang, Rui; Wu, Xiaofeng

    2011-06-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e66 is a core gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E66. The N-terminal 23 amino acid of the envelope protein ODV-E66 are sufficient to direct native and fusion proteins to induced membrane microvesicles and the viral envelope during infection with AcMNPV. In this study, an odv-e66-knockout bacmid can not express N-terminal hydrophobic domains was constructed via homologous recombination in Escherichia coli. The odv-e66 deletion had no effect on budded virus (BV) production and viral DNA replication in infected Sf9 cells. Larval bioassays demonstrated that injection of odv-e66 deletion BV into the hemocoel could kill P. xylostella larvae as efficiently as repaired and control viruses; however, odv-e66 deletion mutant resulted in a 50% lethal dose that was 10(3) higher than that of the repaired and control viruses when inoculated per os. These results indicated that ODV-E66 envelope protein most likely played an important role in the oral infectivity of AcMNPV, but is not essential for virus replication.

  1. The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.

    PubMed

    Belyavskyi, M; Braunagel, S C; Summers, M D

    1998-09-15

    Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box. Immunoprecipitation results using antibodies to -EC27 show that -EC27 can associate with either cdc2 or cdk6 resulting in active kinase complexes that can phosphorylate histone H1 and retinoblastoma protein in vitro. The cdk6-EC27 complex also associates with proliferating cell nuclear antigen (PCNA) and we demonstrate that PCNA is a structural protein of both the budded virus and the occlusion-derived virus. These results suggest that -EC27 can function as a multifunctional cyclin: when associated with cdc2, it exhibits cyclin B-like activity; when associated with cdk6, the complex possesses cyclin D-like activity and binds PCNA. The possible roles of such a multifunctional cyclin during the life cycle of baculovirus are discussed, along with potential implications relative to the expression of functionally authentic recombinant proteins by using baculovirus-infected cells.

  2. Semipermissive replication of a nuclear polyhedrosis virus of Autographa californica in a gypsy moth cell line

    SciTech Connect

    McClintock, J.T.; Dougherty, E.M.; Weiner, R.M.

    1986-01-01

    Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of Autographa californica (AcMNPV). In this report, the authors demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with (/sup 35/S)methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early ..cap alpha.. proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.

  3. A microRNA encoded by Autographa californica nucleopolyhedrovirus regulates expression of viral gene ODV-E25.

    PubMed

    Zhu, Mengxiao; Wang, Jinwen; Deng, Riqiang; Xiong, Peiwen; Liang, Hai; Wang, Xunzhang

    2013-12-01

    Baculovirus-encoded microRNAs (miRNAs) have been described in Bombyx mori nucleopolyhedrovirus; however, most of their functions remain unclear. Here we report the identification and characterization of an miRNA encoded by Autographa californica nucleopolyhedrovirus. The identified miRNA, AcMNPV-miR-1, perfectly matched a segment in the coding sequence of the viral gene ODV-E25 and downregulated ODV-E25 mRNA expression, which likely resulted in a reduction of infectious budded virions and accelerated the formation of occlusion-derived virions.

  4. Selection and Characterization of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Mutations

    PubMed Central

    Feng, Guozhong; Thumbi, David K.; de Jong, Jondavid; Hodgson, Jeffrey J.; Arif, Basil M.; Doucet, Daniel

    2012-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) is essential for viral DNA replication. AcMNPV mutants resistant to aphidicolin, a selective inhibitor of viral DNA replication, and abacavir, an efficacious nucleoside analogue with inhibitory activity against reverse transcriptase, were selected by the serial passage of the parental AcMNPV in the presence of increasing concentrations of aphidicolin or abacavir. These drug-resistant mutants had either a single (C543R) (aphidicolin) or a double (C543R and S611T) (abacavir) point mutation within conserved regions II and III. To confirm the role of these point mutations in AcMNPV DNA polymerase, a dnapol knockout virus was first generated, and several repair viruses were constructed by transposing the dnapol wild-type gene or ones containing a single or double point mutation into the polyhedrin locus of the dnapol knockout bacmid. The single C543R or double C543R/S611T mutation showed increased resistance to both aphidicolin and abacavir and, even in the absence of drug, decreased levels of virus and viral DNA replication compared to the wild-type repair virus. Surprisingly, the dnapol mutant repair viruses led to the generation of occlusion-derived viruses with mostly single and only a few multiple nucleocapsids in the ring zone and within polyhedra. Thus, these point mutations in AcMNPV DNA polymerase increased drug resistance, slightly compromised virus and viral DNA replication, and influenced the viral morphogenesis of occlusion-derived virus. PMID:23035236

  5. Functional characterization of the ubiquitin variant encoded by the baculovirus Autographa californica.

    PubMed

    Haas, A L; Katzung, D J; Reback, P M; Guarino, L A

    1996-04-30

    The marked evolutionary conservation of ubiquitin is assumed to arise from constraints imposed by folding, stability, and interaction of the polypeptide with various components of the ATP, ubiquitin-dependent degradative pathway. The present studies characterize the most divergent (75% identity) of the species-specific ubiquitin isoforms encoded as a late gene product of the baculovirus Autographa californica [Guarino, L. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 409-413]. Viral ubiquitin supports 40% of the rate of ATP-dependent degradation exhibited by eukaryotic ubiquitin. Inhibition of proteolysis correlated with a lower steady-state concentration of ubiquitin-conjugated degradative intermediates. Rate studies revealed that viral ubiquitin exerts its effect at the step of isopeptide ligase-catalyzed (E3) ubiquitin conjugation since viral and eukaryotic polypeptides are identical in their abilities to support ATP-coupled activation by E1 and transthiolation to E2 carrier proteins. Other studies demonstrated viral ubiquitin severely attenuated the rate of K48-linked multiubiquitin chain formation in E3-independent conjugation catalyzed by recombination yeast CDC34 or rabbit reticulocyte E232K but not chain elongation of alternate linkages formed by yeast RAD6 or human E2EPF. The latter observations suggest nonconserved positions on viral ubiquitin constitute recognition signals for K48-linked chain formation. Sequence comparison of species-specific ubiquitin isoforms indicates that nonconserved positions localized to a defined region on the polypeptide surface distinct from the basic face required for E1 binding. These results suggest this novel ubiquitin isoform may function in baculoviral replication to block destruction of a short-lived protein(s) by the host degradative pathway, targeted through either E2-catalyzed K48-linked multibiquitin chain formation or general E3-mediated conjugation.

  6. A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus

    USGS Publications Warehouse

    Leisy, D.J.; Rasmussen, C.; Owusu, E.O.; Rohrmann, G.F.

    1997-01-01

    The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with Bg/II sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (ple-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1- specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.

  7. Multiple nucleocapsid packaging of Autographa californica nucleopolyhedrovirus accelerates the onset of systemic infection in Trichoplusia ni.

    PubMed

    Washburn, J O; Lyons, E H; Haas-Stapleton, E J; Volkman, L E

    1999-01-01

    Among the nucleopolyhedroviruses (Baculoviridae), the occlusion-derived virus (ODV), which initiates infection in host insects, may contain only a single nucleocapsid per virion (the SNPVs) or one to many nucleocapsids per virion (the MNPVs), but the significance of this difference is unclear. To gain insight into the biological relevance of these different packaging strategies, we compared pathogenesis induced by ODV fractions enriched for multiple nucleocapsids (ODV-M) or single nucleocapsids (ODV-S) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) containing a beta-galactosidase reporter gene. In time course experiments wherein newly molted fourth-instar Trichoplusia ni were challenged with doses of ODV-S or ODV-M that yielded the same final mortality ( approximately 70%), we characterized viral foci as either being restricted to the midgut or involving tracheal cells (the secondary target tissue, indicative of systemic infection). We found that while the timing of primary infection by ODV-S and ODV-M was similar, ODV-S established significantly more primary midgut cell foci than ODV-M, but ODV-M infected tracheal cells at twice the rate of ODV-S. The more efficient establishment of tracheal infections by ODV-M decreased the probability that infections were lost by midgut cell sloughing, explaining why higher numbers of primary infections established by ODV-S within larvae were needed to achieve the same final mortality. These results showed that the multiple nucleocapsid packaging strategy of AcMNPV accelerates the onset of irreversible systemic infections and may indicate why MNPVs have wider individual host ranges than SNPVs.

  8. Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

    SciTech Connect

    Mikhailov, Victor S. Vanarsdall, Adam L.; Rohrmann, George F.

    2008-01-20

    DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His{sub 6}-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA and that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.

  9. Expression and mutational analysis of Autographa californica nucleopolyhedrovirus HCF-1: functional requirements for cysteine residues.

    PubMed

    Wilson, Joyce A; Forney, Scott D; Ricci, Alessondra M; Allen, Emily G; Hefferon, Kathleen L; Miller, Lois K

    2005-11-01

    The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica multiple nucleopolyhedrovirus is required for efficient virus growth in TN368 cells but is dispensable for virus replication in SF21 cells. However, the mechanism of action of hcf-1 is unknown. To begin to understand its function in virus replication we have investigated the expression and localization pattern of HCF-1 in infected cells. Analysis of virus-infected TN368 cells showed that hcf-1 is expressed at an early time in the virus life cycle, between 2 and 12 h postinfection, and localized the protein to punctate nuclear foci. Through coprecipitation experiments we have confirmed that HCF-1 self-associates into dimers or higher-order structures. We also found that overexpression of HCF-1 repressed expression from the hcf-1 promoter in transient reporter assays. Mutagenesis of cysteine residues within a putative RING finger domain in the amino acid sequence of HCF-1 abolished self-association activity and suggests that the RING domain may be involved in this protein-protein interaction. A different but overlapping set of cysteine residues were required for efficient gene repression activity. Functional analysis of HCF-1 mutants showed that the cysteine amino acids required for both self-association and gene repression activities of HCF-1 were also required for efficient late-gene expression and occlusion body formation in TN368 cells. Mutational analysis also identified essential charged and hydrophobic amino acids located between two of the essential cysteine residues. We propose that HCF-1 is a RING finger-containing protein whose activity requires HCF-1 self-association and gene repression activity.

  10. A novel third chromosomal locus controls susceptibility to Autographa californica multiple nucleopolyhedrovirus in the silkworm, Bombyx mori.

    PubMed

    Xu, Jian; Kusakabe, Takahiro; Yamamoto, Kimiko; Suetsugu, Yoshitaka; Mon, Hiroaki; Li, Zhiqing; Zhu, Li; Iiyama, Kazuhiro; Banno, Yutaka; Yoshimura, Kaito; Lee, Jae Man

    2014-04-01

    Baculovirus demonstrates specific infection spectrums and thus one certain host exhibits particular response to single baculovirus isolate. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is considered to be not an innate pathogen to Bombyx mori, but some silkworm strains have been identified to be permissive to AcMNPV, indicating the positive or negative involvement of certain host factors in baculovirus replications in vivo. To provide a fundamental knowledge of this process, we performed large-scale screening to investigate the responses of 448 silkworm strains against recombinant AcMNPV inoculation. By genetic analysis between permissive and resistant strains identified, we further confirmed that a potential corresponding locus on chromosome 3 regulates host responses to AcMNPV in silkworm. Additionally, we found that it is available for AcMNPV-silkworm baculovirus expression vector system to produce proteins of interest.

  11. Genetic variation and virulence of Autographa californica multiple nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus isolates

    USDA-ARS?s Scientific Manuscript database

    To determine the genetic diversity within the baculovirus species Autographa calfornica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus), a PCR-based method was used to identify and classify baculoviruses found in virus samples from the lepidopteran host species A. californi...

  12. Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

    PubMed Central

    Biswas, Siddhartha; Blissard, Gary W.

    2016-01-01

    ABSTRACT The mechanism by which nucleocapsids of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with β-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with the Drosophila melanogaster kinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteran Trichoplusia ni KLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated with T. ni KLC. Direct analysis of the role of T. ni kinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation. IMPORTANCE In two key processes of the replication cycle of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are

  13. Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment

    SciTech Connect

    McCarthy, Christina B.; Da, Xiaojiang; Donly, Cam; Theilmann, David A.

    2008-03-15

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac142 is a baculovirus core gene and encodes a protein previously shown to associate with occlusion-derived virus (ODV). To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac142 deletion virus (AcBAC{sup ac142KO-PH-GFP}). Fluorescence and light microscopy revealed that AcBAC{sup ac142KO-PH-GFP} exhibits a single-cell infection phenotype. Titration assays and Western blot confirmed that AcBAC{sup ac142KO-PH-GFP} is unable to produce budded virus (BV). However, viral DNA replication is unaffected and the development of occlusion bodies in AcBAC{sup ac142KO-PH-GFP}-transfected cells evidenced progression to very late phases of the viral infection. Western blot analysis showed that AC142 is expressed in the cytoplasm and nucleus throughout infection and that it is a structural component of BV and ODV which localizes to nucleocapsids. Electron microscopy indicates that ac142 is required for nucleocapsid envelopment to form ODV and their subsequent occlusion, a fundamental process to all baculoviruses.

  14. Autographa californica multiple nucleopolyhedrovirus and Choristoneura fumiferana multiple nucleopolyhedrovirus v-cath genes are expressed as pre-proenzymes.

    PubMed

    Hodgson, Jeffrey J; Arif, Basil M; Krell, Peter J

    2009-04-01

    Intracellular processing and trafficking of the baculovirus v-cath expressed cathepsin (V-CATH), which lacks canonical targeting signals, are poorly understood. The cathepsins of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) and most other alphabaculovirus group I nucleopolyhedroviruses have well-conserved N-termini containing overlapping chymotrypsin-cleavage (Y(11)) and myristoylation (G(12)) motifs, which are suggestive of proteolytic signal-peptide cleavage to generate proV-CATH and subsequent acylation. To determine proteolytic N-terminal processing of V-CATH, haemagglutinin epitope-coding tags were fused to the 5' and/or 3' ends of AcMNPV and CfMNPV v-cath. Immunoblot analysis suggested that a small N-terminal peptide is cleaved for both viruses, indicating that v-cath is expressed as a pre-proenzyme. The two viral homologues undergo similar proteolytic processing, but have different glycosylation or other post-translational modifications. An AcMNPV V-CATH-DsRED fusion protein co-localized to the endoplasmic reticulum with an HDEL motif-containing green fluorescent protein. Based on these findings, pre-proV-CATH processing and trafficking mechanisms are postulated.

  15. Mapping of Early and Late Transcripts Encoded by the Autographa californica Nuclear Polyhedrosis Virus Genome: Is Viral RNA Spliced?

    PubMed Central

    Lübbert, Hermann; Doerfler, Walter

    1984-01-01

    Early and late transcripts were mapped on the Autographa californica nuclear polyhedrosis virus genome by Northern blotting and hybridization with the cloned viral EcoRI fragments. At least 11 early and about 90 late RNAs were compared with over 32 polypeptides synthesized by in vitro translation of hybrid-selected RNA. The latter method, of course, had its limitations also and did not guarantee that all viral RNAs could be detected in this way. A comparison of cytoplasmic and total cellular RNAs showed no clear-cut differences in their size distributions. We found that there were more RNA classes than corresponding proteins encoded by them and mapped by in vitro translation. By using the Berk-Sharp method and analyses of DNA-RNA hybrids by one-dimensional or two-dimensional neutral and alkaline gel electrophoreses, we were unable to adduce evidence for RNA splicing in this viral system. Minor splices, particularly at sites close to the termini of RNA molecules, could not be excluded. Images PMID:16789249

  16. A soluble form of P74 can act as a per os infectivity factor to the Autographa californica multiple nucleopolyhedrovirus.

    PubMed

    Slack, Jeffrey M; Lawrence, Susan D; Krell, Peter J; Arif, Basil M

    2010-04-01

    The baculovirus occlusion-derived virion (ODV) is required to spread virus infection among insect hosts via the per os route. The Autographa californica multicapsid nucleopolyhedrovirus P74 protein is an ODV envelope protein that is essential for ODVs to be infectious. P74 is anchored in the ODV envelope by a C-terminal transmembrane anchor domain and is N-terminally exposed on the ODV surface. In the present study, a series of N-terminal and C-terminal truncation mutants of P74 were evaluated for their ability to rescue per os infectivity of the P74-null virus, AcLP4. It was discovered that a P74 truncation mutant lacking the C-terminal transmembrane anchor domain of P74 was able to rescue per os infection. This result shows that a soluble form of P74 retains per os infectivity factor function and suggests that P74 may be complexed with other proteins in the ODV envelope.

  17. Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment.

    PubMed

    McCarthy, Christina B; Dai, Xiaojiang; Donly, Cam; Theilmann, David A

    2008-03-15

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac142 is a baculovirus core gene and encodes a protein previously shown to associate with occlusion-derived virus (ODV). To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac142 deletion virus (AcBAC(ac142KO-PH-GFP)). Fluorescence and light microscopy revealed that AcBAC(ac142KO-PH-GFP) exhibits a single-cell infection phenotype. Titration assays and Western blot confirmed that AcBAC(ac142KO-PH-GFP) is unable to produce budded virus (BV). However, viral DNA replication is unaffected and the development of occlusion bodies in AcBAC(ac142KO-PH-GFP)-transfected cells evidenced progression to very late phases of the viral infection. Western blot analysis showed that AC142 is expressed in the cytoplasm and nucleus throughout infection and that it is a structural component of BV and ODV which localizes to nucleocapsids. Electron microscopy indicates that ac142 is required for nucleocapsid envelopment to form ODV and their subsequent occlusion, a fundamental process to all baculoviruses.

  18. A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag.

    PubMed

    Slack, J M; Dougherty, E M; Lawrence, S D

    2001-09-01

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74-GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74-GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74-GFP chimeras in recombinant baculoviruses. When C-terminal region S580-F645 was deleted from p74, p74-GFP chimera localization became non-specific and chimeras became soluble. p74 region S580-F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.

  19. Identification of fragments from Autographa californica polyhedrin protein essential for self-aggregation and exogenous protein incorporation.

    PubMed

    Sampieri, Alicia; Luz-Madrigal, Agustín; Zepeda, Jesus; Vaca, Luis

    2015-02-04

    Baculoviruses are widely used for the production of recombinant proteins, biopesticides and as gene delivery systems. One of the viral forms called polyhedra has been recently exploited as a scaffold system to incorporate or encapsulate foreign proteins or peptide fragments. However, an efficient strategy for foreign protein incorporation has not been thoroughly studied. Based on the crystal structure of polyhedrin, we conducted an in silico analysis of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) polyhedrin protein to select the minimum fragments of polyhedrin that could be incorporated into polyhedra. Using confocal and transmission electron microscopy we analyzed the expression and cellular localization of the different polyhedrin fragments fused to the green fluorescent protein (EGFP) used as reporter. The amino fragment 1-110 contains two repeats formed each of two β sheets followed by a α helix (amino acids 1-58 and 58-110) that are important for the formation and stability of polyhedra. These fragments 1-58, 58-110 and 1-110 could be incorporated into polyhedra. However, only fragments 1-110 and 58-110 can self-aggregate. These results demonstrate that 58-110 is the minimum fragment that contributes to the assembly of the recombinant polyhedra via self-aggregation. This is the minimum sequence that can be used to efficiently incorporate foreign proteins into polyhedra.

  20. Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection.

    PubMed

    Wei, Wenqiang; Wang, Hongju; Li, Xiaoya; Fang, Na; Yang, Shili; Liu, Hongyan; Kang, Xiaonan; Sun, Xiulian; Ji, Shaoping

    2016-05-07

    At present, the details of lamina alterations after baculovirus infection remain elusive. In this study, a lamin gene in the Sf9 cell line of Spodoptera frugiperda was cloned. The open reading frame (orf) of the Sf9 lamin was 1860 bp and encoded a protein with a molecular weight of 70 kDa. A transfection assay with a red fluorescence protein (rfp)-lamin fusion protein indicated that Sf9 lamin was localized in the nuclear rim. Transmission electron microscopy observations indicated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsids may pass through the nuclear envelope. Immunofluorescence assay indicated that the lamina showed a ruffled staining pattern with the formation of invaginations in the Sf9 cells infected with AcMNPV, while it was evenly distributed at the nuclear periphery of mock-infected cells. Western blotting results indicated that the total amount of lamin in the baculovirus-infected Sf9 cells was significantly decreased compared with the mock-infected cells. These results imply that AcMNPV infection induces structural and biochemical rearrangements of lamina of Sf9 cells.

  1. Tightly Regulated Expression of Autographa californica Multicapsid Nucleopolyhedrovirus Immediate Early Genes Emerges from Their Interactions and Possible Collective Behaviors

    PubMed Central

    Taka, Hitomi; Asano, Shin-ichiro; Matsuura, Yoshiharu; Bando, Hisanori

    2015-01-01

    To infect their hosts, DNA viruses must successfully initiate the expression of viral genes that control subsequent viral gene expression and manipulate the host environment. Viral genes that are immediately expressed upon infection play critical roles in the early infection process. In this study, we investigated the expression and regulation of five canonical regulatory immediate-early (IE) genes of Autographa californica multicapsid nucleopolyhedrovirus: ie0, ie1, ie2, me53, and pe38. A systematic transient gene-expression analysis revealed that these IE genes are generally transactivators, suggesting the existence of a highly interactive regulatory network. A genetic analysis using gene knockout viruses demonstrated that the expression of these IE genes was tolerant to the single deletions of activator IE genes in the early stage of infection. A network graph analysis on the regulatory relationships observed in the transient expression analysis suggested that the robustness of IE gene expression is due to the organization of the IE gene regulatory network and how each IE gene is activated. However, some regulatory relationships detected by the genetic analysis were contradictory to those observed in the transient expression analysis, especially for IE0-mediated regulation. Statistical modeling, combined with genetic analysis using knockout alleles for ie0 and ie1, showed that the repressor function of ie0 was due to the interaction between ie0 and ie1, not ie0 itself. Taken together, these systematic approaches provided insight into the topology and nature of the IE gene regulatory network. PMID:25816136

  2. Cellular VPS4 Is Required for Efficient Entry and Egress of Budded Virions of Autographa californica Multiple Nucleopolyhedrovirus

    PubMed Central

    Li, Zhaofei

    2012-01-01

    Membrane budding is essential for the egress of many enveloped viruses, and this process shares similarities with the biogenesis of multivesicular bodies (MVBs). In eukaryotic cells, the budding of intraluminal vesicles (IVLs) is mediated by the endosomal sorting complex required for transport (ESCRT) machinery and some viruses require ESCRT machinery components or functions to bud from host cells. Baculoviruses, such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), enter host cells by clathrin-mediated endocytosis. Viral DNA replication and nucleocapsid assembly occur within the nucleus. Some progeny nucleocapsids are subsequently trafficked to, and bud from, the plasma membrane, forming budded virions (BV). To determine whether the host ESCRT machinery is important or necessary for AcMNPV replication, we cloned a cDNA of Spodoptera frugiperda VPS4, a key regulator for disassembly and recycling of ESCRT III. We then examined viral infection and budding in the presence of wild-type (WT) or dominant negative (DN) forms of VPS4. First, we used a viral complementation system, in combination with fluorescent tags, to examine the effects of transiently expressed WT or DN VPS4 on viral entry. We found that dominant negative VPS4 substantially inhibited virus entry. Entering virus was observed within aberrant compartments containing the DN VPS4 protein. We next used recombinant bacmids expressing WT or DN VPS4 proteins to examine virus egress. We found that production of infectious AcMNPV BV was substantially reduced by expression of DN VPS4 but not by WT VPS4. Together, these results indicate that a functional VPS4 is necessary for efficient AcMNPV BV entry into, and egress from, insect cells. PMID:22072775

  3. Identification of a novel regulatory sequence of actin nucleation promoting factor encoded by Autographa californica multiple nucleopolyhedrovirus.

    PubMed

    Wang, Yun; Zhang, Yongli; Han, Shili; Hu, Xue; Zhou, Yuan; Mu, Jingfang; Pei, Rongjuan; Wu, Chunchen; Chen, Xinwen

    2015-04-10

    Actin polymerization induced by nucleation promoting factors (NPFs) is one of the most fundamental biological processes in eukaryotic cells. NPFs contain a conserved output domain (VCA domain) near the C terminus, which interacts with and activates the cellular actin-related protein 2/3 complex (Arp2/3) to induce actin polymerization and a diverse regulatory domain near the N terminus. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid protein P78/83 is a virus-encoded NPF that contains a C-terminal VCA domain and induces actin polymerization in virus-infected cells. However, there is no similarity between the N terminus of P78/83 and that of other identified NPFs, suggesting that P78/83 may possess a unique regulatory mechanism. In this study, we identified a multifunctional regulatory sequence (MRS) located near the N terminus of P78/83 and determined that one of its functions is to serve as a degron to mediate P78/83 degradation in a proteasome-dependent manner. In AcMNPV-infected cells, the MRS also binds to another nucleocapsid protein, BV/ODV-C42, which stabilizes P78/83 and modulates the P78/83-Arp2/3 interaction to orchestrate actin polymerization. In addition, the MRS is also essential for the incorporation of P78/83 into the nucleocapsid, ensuring virion mobility powered by P78/83-induced actin polymerization. The triple functions of the MRS enable P78/83 to serve as an essential viral protein in the AcMNPV replication cycle, and the possible roles of the MRS in orchestrating the virus-induced actin polymerization and viral genome decapsidation are discussed.

  4. Effects of Early or Overexpression of the Autographa californica Multiple Nucleopolyhedrovirus orf94 (ODV-e25) on Virus Replication.

    PubMed

    Luo, Xiao-Chun; Wang, Shan-Shan; Zhang, Jie; Qian, Duo-Duo; Wang, Si-Min; Li, Lu-Lin

    2013-01-01

    odv-e25(e25) is one of the core genes of baculoviruses. To investigate how it functions in the replication cycle of a baculovirus, a number of Autographa californica multiple nucleopolyhedrovirus recombinants with e25 under control of the promoter of immediate early gene ie1, or the promoter of the very late hyperexpressed gene p10, were constructed using a bacmid system, and the effects of early expression or overexpression of e25 on replication of the virus were evaluated. Microscopy and titration assays demonstrated that bacmids with e25 under control of ie1 promoter were unable to produce budded viruses; and that the recombinant viruses with e25 under control of p10 promoter generated budded virus normally, but formation of occlusion bodies were dramatically reduced and delayed in the infected cells. Electron microscopy showed that there were no mature virions or intact nucleocapsids present in the cells transfected with a recombinant bacmid with e25 under control of ie1 promoter. Quantitative real-time PCR analysis demonstrated that alteration of the e25 promoter did not affect viral DNA synthesis. The reporter gene expression from the promoter of the major capsid protein gene vp39 was reduced 63% by early expression of e25. Confocal microscopy revealed that E25 was predominantly localized in nuclei by 24 hours post infection with wild-type virus, but it remained in the cytoplasm in the cells transfected with a recombinant bacmid with e25 under control of the ie1 promoter, suggesting that the transport of E25 into nuclei was regulated in a specific and strict time dependent manner.

  5. Viral and host cellular transcription in Autographa californica nuclear polyhedrosis virus-infected gypsy moth cell lines.

    PubMed Central

    Guzo, D; Rathburn, H; Guthrie, K; Dougherty, E

    1992-01-01

    Infection of two gypsy moth cell lines (IPLB-Ld652Y and IPLB-LdFB) by the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) is characterized by extremely attenuated viral protein synthesis followed by a total arrest of all viral and cellular protein production. In this study, AcMNPV- and host cell-specific transcription were examined. Overall levels of viral RNAs in infected gypsy moth cells were, at most measured times, comparable to RNA levels from an infected cell line (TN-368) permissive for AcMNPV replication. Northern blot (RNA) analyses using viral and host gene-specific probes revealed predominantly normal-length virus- and cell-specific transcripts postinfection. Transport of viral RNAs from the nucleus to the cytoplasm and transcript stability in infected gypsy moth cells also appeared normal compared with similar parameters for AcMNPV-infected TN-368 cells. Host cellular and viral mRNAs extracted from gypsy moth and TN-368 cells at various times postinfection and translated in vitro yielded similar spectra of host and viral proteins. Treatment of infected gypsy moth cells with the DNA synthesis inhibitor aphidicolin eliminated the total protein synthesis shutoff in infected IPLB-LdFB cells but had no effect on protein synthesis inhibition in infected IPLB-Ld652Y cells. The apparent selective block in the translation of viral transcripts early in infection and the absence of normal translation or transcription of host cellular genes at later times is discussed. Images PMID:1560533

  6. Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes

    PubMed Central

    Garry, Courtney E; Garry, Robert F

    2008-01-01

    Background Members of the Baculoviridae encode two types of proteins that mediate virus:cell membrane fusion and penetration into the host cell. Alignments of primary amino acid sequences indicate that baculovirus fusion proteins of group I nucleopolyhedroviruses (NPV) form the GP64 superfamily. The structure of these viral penetrenes has not been determined. The GP64 superfamily includes the glycoprotein (GP) encoded by members of the Thogotovirus genus of the Orthomyxoviridae. The entry proteins of other baculoviruses, group II NPV and granuloviruses, are class I penetrenes. Results Class III penetrenes encoded by members of the Rhabdoviridae and Herpesviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Similar sequences and structural/functional motifs that characterize class III penetrenes are located collinearly in GP64 of group I baculoviruses and related glycoproteins encoded by thogotoviruses. Structural models based on a prototypic class III penetrene, vesicular stomatitis virus glycoprotein (VSV G), were established for Thogoto virus (THOV) GP and Autographa california multiple NPV (AcMNPV) GP64 demonstrating feasible cysteine linkages. Glycosylation sites in THOV GP and AcMNPV GP64 appear in similar model locations to the two glycosylation sites of VSV G. Conclusion These results suggest that proteins in the GP64 superfamily are class III penetrenes. PMID:18282283

  7. A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

    PubMed Central

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R.; Sampieri, Alicia

    2013-01-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV. PMID:23986592

  8. A cholesterol recognition amino acid consensus domain in GP64 fusion protein facilitates anchoring of baculovirus to mammalian cells.

    PubMed

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R; Sampieri, Alicia; Vaca, Luis

    2013-11-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.

  9. The autographa californica multiple nucleopolyhedrovirus ODV-E56 envelope protein is required for oral infectivity and can be functionally substituted by rachiplusia ou multiple nucleopolyhedrovirus ODV-E56

    USDA-ARS?s Scientific Manuscript database

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. In a previous analysis, the odv-e56 gene was found to be under positive selection pressure, suggesting that it may be a determinant of viral ho...

  10. Autographa californica multiple nucleopolyhedrovirus ODV-E56 is a per os infectivity factor, but is not essential for binding and fusion of occlusion-derived virus to the host midgut

    USDA-ARS?s Scientific Manuscript database

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) envelope protein ODV-E56 is essential for oral infection of neonate Heliothis virescens larvae. Here, we present a more detailed study of ODV-E56 function. Bioassays with recombinant clones of AcMNPV lack...

  11. The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis-Acting Element That Is Essential for Nucleocapsid Assembly.

    PubMed

    Huang, Zhihong; Pan, Mengjia; Zhu, Silei; Zhang, Hao; Wu, Wenbi; Yuan, Meijin; Yang, Kai

    2017-03-01

    Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83-encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis-acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis-acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis-acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process.IMPORTANCE Virus nucleocapsid assembly usually requires specific cis-acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome

  12. Autographa californica multiple nucleopolyhedrovirus nucleocapsid protein BV/ODV-C42 mediates the nuclear entry of P78/83.

    PubMed

    Wang, Yun; Wang, Qian; Liang, Changyong; Song, Jianhua; Li, Ni; Shi, Hui; Chen, Xinwen

    2008-05-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-c42 (orf101; c42), which encodes a 41.5-kDa viral nucleocapsid protein with a putative nuclear localization signal (NLS) motif at the C terminus, is a highly conserved gene among members of the Baculoviridae family. C42 is demonstrated to be essential for AcMNPV propagation and can bind to nucleocapsid protein P78/83, a viral activator for the actin-related protein 2/3 (ARP2/3) complex to initiate nuclear actin polymerization, which is essential for viral nucleocapsid morphogenesis during AcMNPV infection. Here, we report the identification of a novel pathway through which c42 functions in nucleocapsid assembly. Cotransfection of Sf9 cells with c42 and p78/83 plasmids demonstrated that C42 was capable of recruiting P78/83 to the nuclei of uninfected cells and that the NLS motif of C42 was essential for this process. To validate this nuclear relocation mode in bacmid-transfected cells, a c42-disrupted bacmid (vAc(c42ko-gfp)) and rescued bacmids with wild-type c42 (vAc(c42res-gfp)) or with NLS coding sequence-mutated c42 (vAc(c42nls-gfp)) were prepared. By immuno-staining, P78/83 was found to be localized in the cytoplasm of either vAc(c42ko-gfp)- or vAc(c42nls-gfp)-transfected cells, whereas P78/83 was relocated to the nuclei of vAc(c42res-gfp)-transfected cells. Furthermore, F-actin-specific staining confirmed that there was no actin polymerization activity in the nuclei of either vAc(c42ko-gfp)- or vAc(c42nls-gfp)-transfected cells, which might be attributed to the absence of nuclear P78/83, an activator of the ARP2/3 complex to initiate nuclear actin polymerization. We therefore hypothesize a mode of action where C42 binds to P78/83 in the cytoplasm to form a protein complex and cotransports to the nucleus under the direction of the NLS motif in C42 during AcMNPV infection.

  13. Three-dimensional visualization of the Autographa californica multiple nucleopolyhedrovirus occlusion-derived virion envelopment process gives new clues as to its mechanism.

    PubMed

    Shi, Yang; Li, Kunpeng; Tang, Peiping; Li, Yinyin; Zhou, Qiang; Yang, Kai; Zhang, Qinfen

    2015-02-01

    Baculoviruses produce two virion phenotypes, occlusion-derived virion (ODV) and budded virion (BV). ODV envelopment occurs in the nucleus. Morphogenesis of the ODV has been studied extensively; however, the mechanisms underlying microvesicle formation and ODV envelopment in nuclei remain unclear. In this study, we used electron tomography (ET) together with the conventional electron microscopy to study the envelopment of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ODV. Our results demonstrate that not only the inner but also the outer nuclear membrane can invaginate and vesiculate into microvesicles and that intranuclear microvesicles are the direct source of the ODV membrane. Five main events in the ODV envelopment process are summarized, from which we propose a model to explain this process.

  14. Autographa californica Multiple Nucleopolyhedrovirus orf132 Encodes a Nucleocapsid-Associated Protein Required for Budded-Virus and Multiply Enveloped Occlusion-Derived Virus Production

    PubMed Central

    Yang, Ming; Wang, Shuo; Yue, Xiu-Li

    2014-01-01

    ABSTRACT Autographa californica multiple nucleopolyhedrovirus orf132 (named ac132) has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in the viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kDa, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 h postinfection with the virus. It formed a ring zone at the periphery of the nucleus by 24 h postinfection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132 knockout bacmid, infection was restricted to single cells, and the titer of infectious budded virus was reduced to an undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under the control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132 knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsids were reduced, and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids. IMPORTANCE To our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. This article reveals unique phenotypic changes induced by ac132

  15. Functional Regulation of an Autographa californica Nucleopolyhedrovirus-Encoded MicroRNA, AcMNPV-miR-1, in Baculovirus Replication

    PubMed Central

    Zhu, Mengxiao; Deng, Riqiang

    2016-01-01

    ABSTRACT An Autographa californica nucleopolyhedrovirus-encoded microRNA (miRNA), AcMNPV-miR-1, downregulates the ac94 gene, reducing the production of infectious budded virions and accelerating the formation of occlusion-derived virions. In the current study, four viruses that constitutively overexpress AcMNPV-miR-1 were constructed to further explore the function of the miRNA. In addition to the ac94 gene, two new viral gene targets (ac18 and ac95) of AcMNPV-miR-1 were identified, and the possible interacting proteins were verified and tested. In the context of AcMNPV-miR-1 overexpression, ac18 was slightly upregulated, and ac95 was downregulated. Several interacting proteins were identified, and a functional pathway for AcMNPV-miR-1 was deduced. AcMNPV-miR-1 overexpression decreased budded virus infectivity, reduced viral DNA replication, accelerated polyhedron formation, and promoted viral infection efficiency in Trichoplusia ni larvae, suggesting that AcMNPV-miR-1 restrains virus infection of cells but facilitates virus infection of larvae. IMPORTANCE Recently, microRNAs (miRNAs) have been widely reported as moderators or regulators of mammalian cellular processes, especially disease-related pathways in humans. However, the roles played by miRNAs encoded by baculoviruses, which infect numerous beneficial insects and agricultural pests, have rarely been described. To explore the actions of virus-encoded miRNAs, we investigated an miRNA encoded by Autographa californica nucleopolyhedrovirus (AcMNPV-miR-1). We previously identified this miRNA through the exogenous addition of AcMNPV-miR-1 mimics. In the current study, we constitutively overexpressed AcMNPV-miR-1 and analyzed the resultant effects to more comprehensively assess what is indeed the function of this miRNA during viral infection. In addition, we widely explored the target genes for the miRNA in the viral and host genomes and proposed a possible functional network for AcMNPV-miR-1, which provides a

  16. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment.

    PubMed

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Vaca, Luis; Laclette, Juan Pedro; Carrero, Julio César

    2015-01-01

    In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.

  17. Autographa californica multiple nucleopolyhedrovirus ac66 is required for the efficient egress of nucleocapsids from the nucleus, general synthesis of preoccluded virions and occlusion body formation

    SciTech Connect

    Ke Jianhao Wang Jinwen; Deng Riqiang; Wang Xunzhang

    2008-05-10

    Although orf66 (ac66) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in all sequenced lepidopteran baculovirus genomes, its function is not known. This paper describes generation of an ac66 knockout AcMNPV bacmid mutant and analyses of the influence of ac66 deletion on the virus replication in Sf-9 cells so as to determine the role of ac66 in the viral life cycle. Results indicated that budded virus (BV) yields were reduced over 99% in ac66-null mutant infected cells in comparison to that in wild-type virus infected cells. Optical microscopy revealed that occlusion body synthesis was significantly reduced in the ac66 knockout bacmid-transfected cells. In addition, ac66 deletion interrupted preoccluded virion synthesis. The mutant phenotype was rescued by an ac66 repair bacmid. On the other hand, real-time PCR analysis indicated that ac66 deletion did not affect the levels of viral DNA replication. Electron microscopy revealed that ac66 is not essential for nucleocapsid assembly, but for the efficient transport of nucleocapsids from the nucleus to the cytoplasm. These results suggested that ac66 plays an important role for the efficient exit of nucleocapsids from the nucleus to the cytoplasm for BV synthesis as well as for preoccluded virion and occlusion synthesis.

  18. Autographa californica Multiple Nucleopolyhedrovirus Ac34 Protein Retains Cellular Actin-Related Protein 2/3 Complex in the Nucleus by Subversion of CRM1-Dependent Nuclear Export

    PubMed Central

    Mu, Jingfang; Zhang, Yongli; Hu, Yangyang; Hu, Xue; Zhou, Yuan; Pei, Rongjuan; Wu, Chunchen; Chen, Jizheng; van Oers, Monique M.; Chen, Xinwen; Wang, Yun

    2016-01-01

    Actin, nucleation-promoting factors (NPFs), and the actin-related protein 2/3 complex (Arp2/3) are key elements of the cellular actin polymerization machinery. With nuclear actin polymerization implicated in ever-expanding biological processes and the discovery of the nuclear import mechanisms of actin and NPFs, determining Arp2/3 nucleo-cytoplasmic shuttling mechanism is important for understanding the function of nuclear actin. A unique feature of alphabaculovirus infection of insect cells is the robust nuclear accumulation of Arp2/3, which induces actin polymerization in the nucleus to assist in virus replication. We found that Ac34, a viral late gene product encoded by the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is involved in Arp2/3 nuclear accumulation during virus infection. Further assays revealed that the subcellular distribution of Arp2/3 under steady-state conditions is controlled by chromosomal maintenance 1 (CRM1)-dependent nuclear export. Upon AcMNPV infection, Ac34 inhibits CRM1 pathway and leads to Arp2/3 retention in the nucleus. PMID:27802336

  19. Three-dimensional visualization of the Autographa californica multiple nucleopolyhedrovirus occlusion-derived virion envelopment process gives new clues as to its mechanism

    SciTech Connect

    Shi, Yang; Li, Kunpeng; Tang, Peiping; Li, Yinyin; Zhou, Qiang; Yang, Kai; Zhang, Qinfen

    2015-02-15

    Baculoviruses produce two virion phenotypes, occlusion-derived virion (ODV) and budded virion (BV). ODV envelopment occurs in the nucleus. Morphogenesis of the ODV has been studied extensively; however, the mechanisms underlying microvesicle formation and ODV envelopment in nuclei remain unclear. In this study, we used electron tomography (ET) together with the conventional electron microscopy to study the envelopment of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ODV. Our results demonstrate that not only the inner but also the outer nuclear membrane can invaginate and vesiculate into microvesicles and that intranuclear microvesicles are the direct source of the ODV membrane. Five main events in the ODV envelopment process are summarized, from which we propose a model to explain this process. - Highlights: • Both the inner and outer nuclear membranes could invaginate. • Both the inner and outer nuclear membranes could vesiculate into microvesicles. • Five main events in the ODV envelopment process are summarized. • A model is proposed to explain this ODV envelopment.

  20. A peptide with similarity to baculovirus ODV-E66 binds the gut epithelium of Heliothis virescens and impedes infection with Autographa californica multiple nucleopolyhedrovirus.

    PubMed

    Sparks, Wendy O; Rohlfing, Amy; Bonning, Bryony C

    2011-05-01

    Baculoviruses infect their lepidopteran hosts via the midgut epithelium through binding of occlusion-derived virus (ODV) and fusion between the virus envelope and microvillar membranes. To identify genes and sequences that are involved in this process, a random phage display library was screened for peptides that bound to brush border membrane vesicles (BBMV) derived from the midgut epithelium of Heliothis virescens. Seventeen peptides that bound to BBMV were recovered. Two of these, HV1 and HV2, had sequence similarity to the ODV envelope protein ODV-E66 that is found in five species of alphabaculoviruses. Chemically synthesized versions of HV1 and HV2, and two peptides (AcE66A and AcE66B) derived from similar sequences of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ODV-E66, bound to unfixed cryosections of whole midgut tissues. AcE66A, but not HV1, bound to H. virescens gut BBMV proteins on a far-Western blot. Competition assays with HV1 and purified AcMNPV ODV resulted in decreased mortality of H. virescens larvae at a dose of 1 LD(50), and a significant increase in survival time at higher virus concentrations. These results suggest a role for ODV-E66 in baculovirus infection of lepidopteran larval midgut epithelium.

  1. Identification and analysis of an Autographa californica nuclear polyhedrosis virus structural protein of the occlusion-derived virus envelope: ODV-E56.

    PubMed

    Braunagel, S C; Elton, D M; Ma, H; Summers, M D

    1996-03-01

    An Autographa californica nuclear polyhedrosis virus gene encoding an occlusion-derived virus (ODV) envelope protein of 56 kDa was identified and sequenced. Transcription initiates from a conserved baculovirus late motif (ATAAG) with transcripts detected from 16 through 72 hr p.i. The protein is detected in infected cell extracts from 36 hr p.i. Western blot assay of ODV, BV, viral envelope, and nucleocapsid preparations coupled with immunoelectron microscopy reveal that this protein localizes to the ODV envelope. This protein is named ODV-E56 to identify its viral origin, envelope location, and apparent molecular weight. ODV-E56 is enriched in viral induced intranuclear microvesicles as determined by immunogold labeling. A mutant was constructed with the C-terminal portion of the protein replaced with beta-galactosidase. The fusion protein, E56-beta-gal, locates to the viral nucleocapsids and not to the ODV envelope or intranuclear microvesicles. This suggests that the signals necessary for transport and/or retention into these structures lies within the C-terminal portion of ODV-E56. Additionally, both ODV-E56 and E56-beta-gal are enriched in electron dense regions that cluster around the inner nuclear membrane and within the nucleoplasm.

  2. Identification of Autographa californica nucleopolyhedrovirus ac93 as a core gene and its requirement for intranuclear microvesicle formation and nuclear egress of nucleocapsids.

    PubMed

    Yuan, Meijin; Huang, Zhenqiu; Wei, Denghui; Hu, Zhaoyang; Yang, Kai; Pang, Yi

    2011-11-01

    Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusion-derived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.

  3. Autographa californica multiple nucleopolyhedrovirus odv-e25 (Ac94) is required for budded virus infectivity and occlusion-derived virus formation.

    PubMed

    Chen, Lin; Hu, Xiaolong; Xiang, Xingwei; Yu, Shaofang; Yang, Rui; Wu, Xiaofeng

    2012-04-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e25 is a core gene found in all lepidopteran baculoviruses, but its function is unknown. In this study, we generated an odv-e25-knockout AcMNPV and investigated the roles of ODV-E25 in the baculovirus life cycle. The odv-e25 knockout was subsequently rescued by reinserting the odv-e25 gene into the same virus genome. Fluorescence microscopy showed that transfection with the odv-e25-null bacmid vAcBac(KO) was insufficient for propagation in cell culture, whereas the 'repair' virus vAcBac(RE) was able to function in a manner similar to that of the control vAcBac. We found that odv-e25 was not essential for the release of budded viruses (BVs) into culture medium, although the absence of odv-e25 resulted in a 100-fold lower viral titer at 24 h post-transfection (p.t.). Analysis of viral DNA replication in the absence of odv-e25 showed that viral DNA replication was unaffected in the first 24 h p.t. Furthermore, electron microscopy revealed that polyhedra were found in the nucleus, while mature occlusion-derived viruses (ODVs) were not found in the nucleus or polyhedra in odv-e25 null transfected cells, which indicated that ODV-E25 was required for the formation of ODV.

  4. P74 mediates specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens Larvae.

    PubMed

    Haas-Stapleton, Eric J; Washburn, Jan O; Volkman, Loy E

    2004-07-01

    P74, an envelope protein of the occlusion-derived virus (ODV) of Autographa californica M nucleopolyhedrovirus (AcMNPV), is critical for oral infection of Trichoplusia ni larvae. The role of P74 during primary infection, however, is unknown. Here we provide evidence that P74 facilitates binding of AcMNPV ODV to a specific receptor within the larval midgut epithelia of another host species, Heliothis virescens. We adapted a fluorescence dequenching assay to compare binding, fusion, and competition of wild-type AcMNPV ODV in vivo with itself and with the ODV of a p74-deficient AcMNPV mutant. We found that relative to wild-type ODV, binding and fusion of ODV deficient in P74 were both qualitatively and quantitatively different. Unlike wild-type ODV, an excess of P74-deficient ODV failed to compete effectively with wild-type ODV binding, and the overall binding level of the mutant ODV was one-third that of the wild type. These results implicated P74 as an ODV attachment protein that binds to a specific receptor on primary target cells within the midgut.

  5. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment

    PubMed Central

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Laclette, Juan Pedro; Carrero, Julio César

    2015-01-01

    In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4. PMID:26090442

  6. Expression of the Cydia pomonella granulovirus matrix metalloprotease enhances Autographa californica multiple nucleopolyhedrovirus virulence and can partially substitute for viral cathepsin

    PubMed Central

    Ishimwe, Egide; Hodgson, Jeffrey J.; Passarelli, A. Lorena

    2015-01-01

    The Cydia pomonella granulovirus open reading frame 46 (CpGV-ORF46) contains predicted domains found in matrix metalloproteases (MMPs), a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. We showed that CpGV-MMP was active in vitro. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing CpGV-ORF46 replicated similarly to a control virus lacking CpGV-ORF46 in cultured cells. The effects of AcMNPV expressing CpGV-MMP on virus infection in cultured cells and Trichoplusia ni larvae in the presence or absence of other viral degradative enzymes, cathepsin and chitinase, were evaluated. In the absence of cathepsin and chitinase or cathepsin alone, larval time of death was significantly delayed. This delay was compensated by the expression of CpGV-MMP. CpGV-MMP was also able to promote larvae melanization in the absence of cathepsin and chitinase. In addition, CpGV-MMP partially substituted for cathepsin in larvae liquefaction when chitinase, which is usually retained in the endoplasmic reticulum, was engineered to be secreted. PMID:25795312

  7. Autographa californica Nucleopolyhedrovirus Ac76: a Dimeric Type II Integral Membrane Protein That Contains an Inner Nuclear Membrane-Sorting Motif

    PubMed Central

    Wei, Denghui; Wang, Yan; Zhang, Xiaomei; Hu, Zhaoyang; Yang, Kai

    2014-01-01

    Our previous study showed that the Autographa californica Nucleopolyhedrovirus (AcMNPV) ac76 gene is essential for both budded virion (BV) and occlusion-derived virion (ODV) development. More importantly, deletion of ac76 affects intranuclear microvesicle formation. However, the exact role by which ac76 affects virion morphogenesis remains unknown. In this report, we characterized the expression, distribution, and topology of Ac76 to further understand the functional role of Ac76 in virion morphogenesis. Ac76 contains an α-helical transmembrane domain, and phase separation showed that it was an integral membrane protein. In AcMNPV-infected cells, Ac76 was detected as a stable dimer that was resistant to SDS and thermal denaturation, and only a trace amount of monomer was detected. A coimmunoprecipitation assay demonstrated the dimerization of Ac76 by high-affinity self-association. Western blot analyses of purified virions and their nucleocapsid and envelope fractions showed that Ac76 was associated with the envelope fractions of both BVs and ODVs. Immunoelectron microscopy revealed that Ac76 was localized to the plasma membrane, endoplasmic reticulum (ER), nuclear membrane, intranuclear microvesicles, and ODV envelope. Amino acids 15 to 48 of Ac76 were identified as an atypical inner nuclear membrane-sorting motif because it was sufficient to target fusion proteins to the ER and nuclear membrane in the absence of viral infection and to the intranuclear microvesicles and ODV envelope during infection. Topology analysis of Ac76 by selective permeabilization showed that Ac76 was a type II integral membrane protein with an N terminus exposed to the cytosol and a C terminus hidden in the ER lumen. PMID:24198428

  8. The putative pocket protein binding site of Autographa californica nucleopolyhedrovirus BV/ODV-C42 is required for virus-induced nuclear actin polymerization.

    PubMed

    Li, Kun; Wang, Yun; Bai, Huimin; Wang, Qian; Song, Jianhua; Zhou, Yuan; Wu, Chunchen; Chen, Xinwen

    2010-08-01

    Nuclear filamentous actin (F-actin) is essential for nucleocapsid morphogenesis of lepidopteran nucleopolyhedroviruses. Previously, we had demonstrated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-C42 (C42) is involved in nuclear actin polymerization by recruiting P78/83, an AcMNPV orf9-encoded N-WASP homology protein that is capable of activating an actin-related-protein 2/3 (Arp2/3) complex to initiate actin polymerization, to the nucleus. To further investigate the role of C42 in virus-induced actin polymerization, the recombinant bacmid vAc(p78/83nls-gfp), with a c42 knockout, p78/83 tagged with a nuclear localization signal coding sequence, and egfp as a reporter gene under the control of the Pp10 promoter, was constructed and transfected to Sf9 cells. In the nuclei of vAc(p78/83nls-gfp)-transfected cells, polymerized F-actin filaments were absent, whereas other actin polymerization elements (i.e., P78/83, G-actin, and Arp2/3 complex) were present. This in vivo evidence indicated that C42 actively participates in the nuclear actin polymerization process as a key element, besides its role in recruiting P78/83 to the nucleus. In order to collect in vitro evidence for the participation of C42 in actin polymerization, an anti-C42 antibody was used to neutralize the viral nucleocapsid, which is capable of initiating actin polymerization in vitro. Both the kinetics of pyrene-actin polymerization and F-actin-specific staining by phalloidin indicated that anti-C42 can significantly attenuate the efficiency of F-actin formation compared to that with control antibodies. Furthermore, we have identified the putative pocket protein binding sequence (PPBS) on C42 that is essential for C42 to exert its function in nuclear actin polymerization.

  9. Role of Interactions between Autographa californica Multiple Nucleopolyhedrovirus Procathepsin and Chitinase Chitin-Binding or Active-Site Domains in Viral Cathepsin Processing

    PubMed Central

    Hodgson, Jeffrey J.; Arif, Basil M.

    2013-01-01

    The binding of Autographa californica multiple nucleopolyhedrovirus chitinase (CHIA) to viral cathepsin protease progenitor (proV-CATH) governs cellular/endoplasmic reticulum (ER) coretention of CHIA and proV-CATH, thus coordinating simultaneous cellular release of both host tissue-degrading enzymes upon host cell death. CHIA is a proposed proV-CATH folding chaperone because insertional inactivation of chiA causes production of proV-CATH aggregates that are incompetent for proteolytic maturation into active V-CATH enzyme. We wanted to determine whether the N-terminal chitin-binding domain (CBD, 149 residues) and C-terminal CHIA active-site domain (ASD, 402 residues) of CHIA bind to proV-CATH independently of one another and whether either domain is dispensable for CHIA's putative proV-CATH folding chaperone activity. We demonstrate that N-terminally green fluorescent protein (GFP)-fused CHIA, ASD, and CBD each colocalize with proV-CATH-RFP in ER-like patterns and that both ASD and CBD independently associate with proV-CATH in vivo using bimolecular fluorescence complementation (BiFC) and in vitro using reciprocal nickel-histidine pulldown assays. Altogether, the data from colocalization, BiFC, and reciprocal copurification analyses suggest specific and independent interactions between proV-CATH and both domains of CHIA. These data also demonstrate that either CHIA domain is dispensable for normal proV-CATH processing. Furthermore, in contrast to prior evidence suggesting that a lack of chiA expression causes proV-CATH to become aggregated, insoluble, and unable to mature into V-CATH, a chiA deletion bacmid virus we engineered to express just v-cath produced soluble proV-CATH that was prematurely secreted from cells and proteolytically matured into active V-CATH enzyme. PMID:23302896

  10. Spodoptera frugiperda resistance to oral infection by Autographa californica multiple nucleopolyhedrovirus linked to aberrant occlusion-derived virus binding in the midgut.

    PubMed

    Haas-Stapleton, Eric J; Washburn, Jan O; Volkman, Loy E

    2005-05-01

    Spodoptera frugiperda larvae are highly resistant to oral infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) (LD(50), approximately 9200 occlusions), but extremely susceptible to budded virus within the haemocoel (LD(50), <1 p.f.u.). The inability of AcMNPV occlusion-derived virus (ODV) to establish primary infections readily within midgut cells accounts for a major proportion of oral resistance. To determine whether inappropriate binding of AcMNPV ODV to S. frugiperda midgut cells contributes to lack of oral infectivity, the binding and fusion properties of AcMNPV ODV were compared with those of the ODV of a new isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) obtained from a field-collected larva (oral LD(50), 12 occlusions). By using a fluorescence-dequenching assay conducted in vivo, it was found that AcMNPV ODV bound to the midgut epithelia of S. frugiperda larvae at approximately 15 % of the level of SfMNPV ODV, but that, once bound, the efficiencies of fusion for the two ODVs were similar: 60 % for AcMNPV and 53 % for SfMNPV. Whilst the difference in binding efficiencies was significant, it could not account entirely for the observed differences in infectivity. Competition experiments, however, revealed that, in S. frugiperda larvae, SfMNPV ODV bound to a midgut cell receptor that was not bound by AcMNPV ODV, indicating that ODV interaction with a specific receptor(s) was necessary for productive infection of midgut columnar epithelial cells. Fusion in the absence of this ligand-receptor interaction did not result in productive infections.

  11. Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27.

    PubMed

    Braunagel, S C; He, H; Ramamurthy, P; Summers, M D

    1996-08-01

    This paper identifies two structural proteins of the occluded derived viral envelope of Autographa californica nuclear polyhedrosis virus (AcMNPV): ODV-E18 and ODV-E35. In addition, we identify a protein, ODV-EC27, that is incorporated into the capsid of occluded virus, which is not detected in budded virus. The genes for these proteins reside within the IE0 intron. The intron was sequenced, and five open reading frames (ORF) were identified. ORF 3 (genomic ORF 143) codes for the ODV envelope protein, ODV-E18. ORF 4 (genomic ORF 144) codes for ODV-EC27, and Western blot analyses locate this protein to both the ODV capsid and envelope. Transcripts for both ODV-E18 and ODV-EC27 initiate from conserved TAAG motifs, and transcripts are detected from 16 through 72 hr p.i. Antiserum to ODV-E18 recognizes a band of 18 kDa on Western blots of extracts from infected cells and bands of 18 and 35 kDa on Western blots of proteins from purified ODV envelope. N-terminal amino acid sequencing reveals that both ODV-E18 and ODV-E35 contain the same N-terminus. Antiserum to ODV-EC27 recognizes a protein of 27 kDa on Western blots of extracts from infected cells and bands of 27 and 35 kDa on Western blots of proteins from purified ODV. Using immunogold labeling techniques, ODV-E18 and/or ODV-E35 are detected in viral induced intranuclear microvesicles and are not detected in the plasma membrane, cytoplasmic membranes, or the nuclear envelope. Immunogold labeling using antisera to ODV-EC27 detects this protein on both the ODV envelope and capsid.

  12. Autographa californica nuclear polyhedrosis virus: subcellular localization and protein trafficking of BV/ODV-E26 to intranuclear membranes and viral envelopes.

    PubMed

    Beniya, H; Braunagel, S C; Summers, M D

    1998-01-05

    The Autographa californica nuclear polyhedrosis virus da26 gene codes for an envelope protein of both budded virus (BV) and occlusion derived virus (ODV). Western blot and temporal analysis of infected cell extracts detected a protein of 26 kDa by 4 h postinfection (p.i.). The amount of protein increased by 16 h p.i. and remained at high levels throughout infection. By 36 h p.i. several additional immunoreactive proteins were detected which migrated at approximately 18 kDa and remained through 96 h p.i. Western blot analysis of purified virus envelope and nucleocapsid preparations revealed that both the 26- and 18-kDa proteins are structural proteins of the envelope of BV and ODV. Immunoelectron microscopy performed at a time when only the 26-kDa species of the protein was present confirmed that the protein located to ODV envelope. The protein was named BV/ODV-E26 to designate incorporation into viral progeny, envelope location, and apparent molecular weight. Studies designed to follow localization of BV/ODV-E26 demonstrated that early in infection, the protein was incorporated into cytoplasmic vesicles and by 16 h p.i., BV/ODV-E26 was detected in the nucleus associated with virus-induced intranuclear microvesicles and ODV envelope. Coimmunoprecipitation and yeast two-hybrid assays showed that BV/ODV-E26 and FP25K were capable of interacting with each other to form a complex and coimmunoprecipitation assays indicated that cellular actin was a third component of this complex. Together, these data suggest that FP25K and cellular actin may participate in the regulation, or movement through the cell, of baculovirus proteins and/or virus nucleocapsids.

  13. Autographa californica multicapsid nucleopolyhedrovirus efficiently infects Sf9 cells and transduces mammalian cells via direct fusion with the plasma membrane at low pH.

    PubMed

    Dong, Sicong; Wang, Manli; Qiu, Zhijuan; Deng, Fei; Vlak, Just M; Hu, Zhihong; Wang, Hualin

    2010-05-01

    The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue the infectivity of AcMNPV in the presence of endocytosis pathway inhibitors. A colocalization assay of the major capsid protein VP39 with the early endosome marker EEA1 showed that at low pH, AcMNPV entered Sf9 cells via an endosome-independent pathway. Using a fluorescent probe (R18), we showed that at low pH, the viral nucleocapsid entered Sf9 cells via direct fusion at the cell surface. By using the myosin-specific inhibitor 2,3-butanedione monoxime (BDM) and the microtubule inhibitor nocodazole, the low pH-triggered direct fusion was demonstrated to be dependent on myosin-like proteins and independent of microtubules. The reverse transcription-PCR of the IE1 gene as a marker for viral entry showed that the kinetics of AcMNPV in cells triggered by low pH was similar to that of the normal entry via endocytosis. The low pH-mediated infection assay and VP39 and EEA1 colocalization assay also demonstrated that AcMNPV could efficiently transduce mammalian cells via direct membrane fusion at the cell surface. More importantly, we found that a low-pH trigger could significantly improve the transduction efficiency of AcMNPV in mammalian cells, leading to the potential application of this method when using baculovirus as a vector for heterologous gene expression and for gene therapy.

  14. Cloning and characterization of a Dim1-like mitosis gene of Spodoptera frugiperda cells (Sf9) induced by Autographa californica multiple nucleopolyhedrovirus.

    PubMed

    Mehrabadi, Mohammad; Hussain, Mazhar; Asgari, Sassan

    2013-06-01

    Dim1 proteins are evolutionarily highly conserved throughout the eukaryotes and are present in numerous species. These proteins are essential for mitosis and pre-mRNA splicing. In this study, the full-length cDNA of Dim1-like gene from Spodoptera frugiperda cells (Sf9) was obtained. S. frugiperda Dim1 (SfDim1) cDNA is comprised of 975 bp including a 429 bp open reading frame (ORF), 225 bp 5' untranslated region (5' UTR), and 321 bp 3' untranslated region (3' UTR) with a poly A tail. The predicted polypeptide encoded by this gene is 142 aa with a molecular weight of 16.76 kDa and a PI of 5.53. Sequence alignment and phylogenetic analysis showed high similarities with Dim1 of other species. The evolutionary conserved site of Dim1 proteins ((35)Asp-Pro-Thr-Cys(38)) is also present in SfDim1. Silencing of SfDim1 gene decreased cell proliferation at 72 h post-treatment in comparison to mock and control transfected cells. Using RT-PCR, we found relatively higher SfDim1 transcript levels following Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection compared to mock-infected cells from 4h post-infection (hpi) up until 24 hpi. The expression level diminished dramatically at 36 hpi up to 120 hpi with no expression detected at 144 hpi. Silencing of SfDim1 resulted in lower levels of virus DNA production in comparison to mock-infected cells, which suggested that SfDim1 might benefit the virus and facilitate viral replication. Overall, the results showed that SfDim1 protein is involved in cell proliferation as well as cell-virus interaction.

  15. Reduced expression of the immediate-early protein IE0 enables efficient replication of Autographa californica multiple nucleopolyhedrovirus in poorly permissive Spodoptera littoralis cells.

    PubMed

    Lu, Liqun; Du, Quansheng; Chejanovsky, Nor

    2003-01-01

    Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.

  16. Abortive infection of the baculovirus Autographa californica nuclear polyhedrosis virus in Sf-9 cells after mutation of the putative DNA helicase gene.

    PubMed Central

    Kamita, S G; Maeda, S

    1996-01-01

    Homologous recombination between the Autographa californica nuclear polyhedrosis virus (AcNPV) genome and a 0.6-kbp-long DNA fragment derived from the putative DNA helicase gene of Bombyx mori nuclear polyhedrosis virus generates eh2-AcNPV, an expanded-host-range AcNPV mutant (S. Maeda, S.G. Kamita, and A. Kondo, J. Virol. 67:6234-6238, 1993). After inoculation at a high multiplicity of infection (MOI), eh2-AcNPV replicates efficiently in both the Sf-9 (AcNPV-permissive) and BmN (non-AcNPV-permissive) cell lines. In this study, we found that after the inoculation of Sf-9 cells at a low MOI (i.e., 1 and 0.1 PFU per cell), the release of eh2-AcNPV virions was dramatically reduced (approximately 900- and 10,000-fold, respectively, at 72 h postinoculation) compared with that of wild-type AcNPV. In addition, the titer of eh2-AcNPV determined by plaque assay on Sf-9 cells was approximately 200-fold lower than that determined by plaque assay on BmN cells. Analyses of gene expression and viral DNA replication after low-MOI eh2-AcNPV inoculation of Sf-9 cells indicated that viral early genes were expressed normally. However, DNA replication and late-gene expression were significantly reduced. These findings suggested that abortive infection occurred at the stage of viral DNA replication in nearly all low-MOI eh2-AcNPV-infected Sf-9 cells. In the larvae of Spodoptera frugiperda, the organism from which Sf-9 cells are derived, the infectivity of eh2-AcNPV was lower than that of AcNPV; however, abortive infection was not found. PMID:8709251

  17. Mutations within the Autographa californica nucleopolyhedrovirus FP25K gene decrease the accumulation of ODV-E66 and alter its intranuclear transport.

    PubMed

    Braunagel, S C; Burks, J K; Rosas-Acosta, G; Harrison, R L; Ma, H; Summers, M D

    1999-10-01

    Previous reports indicate that mutations within the Autographa californica nucleopolyhedrosis virus FP25K gene (open reading frame 61) significantly reduce incorporation of enveloped nucleocapsids into viral occlusions. We report that FP25K is a nucleocapsid protein of both the budded virus (BV) and occluded virus (ODV), and we describe the effects of two FP25K mutations (480-1 [N-terminal truncation] and FP-betagal [C-terminal fusion]) on the expression and cellular localization of ODV-E66 and ODV-E25. Significantly decreased amounts of ODV-E66 are detected in cells infected with 480-1 or FP-betagal viral mutants, even though during FP-betagal infection, steady-state levels of ODV-E66 transcripts remain unchanged. While ODV-E66 is normally detected in intranuclear microvesicles and ODV envelopes by 24 h postinfection (p.i.), ODV-E66 remains cytosolic throughout infection in cells infected with 480-1 virus (up to 96 h p.i.), and its intranuclear localization is not detected until 96 h p.i. in cells infected with the FP-betagal mutant virus. The nuclear localization of ODV-E25 is not affected during infection by the FP-betagal mutant; however, its trafficking is significantly delayed during infection by the 480-1 mutant. Temporal Western blot analyses of cell lysates show that both 480-1 and FP-betagal mutant virus infections result in altered accumulation patterns of several structural proteins, including gp67, BV/ODV-E26, and the major capsid protein p39. In addition to BV/ODV-E26, ODV-E66 and gp67 may interact with FP25K, and ODV-E25 and p39 may also be components of a protein complex containing ODV-E66 and FP25K. Together, these data suggest that FP25K and its associated protein complex(es) may play an important role in the targeting and intracellular transport of viral proteins during infection.

  18. The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

    PubMed Central

    Guan, Zhanwen; Zhong, Ling; Li, Chunyan; Wu, Wenbi; Yuan, Meijin

    2016-01-01

    ABSTRACT Baculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate the ac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma, ac54 ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasin d-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected by ac54 deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly. IMPORTANCE Baculoviruses are large DNA viruses whose replication occurs within the host nucleus. The localization of

  19. The Trichoplusia ni single nucleopolyhedrovirus tn79 gene encodes a functional sulfhydryl oxidase enzyme that is able to support the replication of Autographa californica multiple nucleopolyhedrovirus lacking the sulfhydryl oxidase ac92 gene

    PubMed Central

    Clem, Stian A.; Wu, Wenbi; Lorena Passarelli, A.

    2014-01-01

    The Autographa californica multiple nucleopolyhedrovirus ac92 is a conserved baculovirus gene with homology to flavin adenine dinucleotide-linked sulfhydryl oxidases. Its product, Ac92, is a functional sulfhydryl oxidase. Deletion of ac92 results in almost negligible levels of budded virus (BV) production, defects in occlusion-derived virus (ODV) co-envelopment and their inefficient incorporation into occlusion bodies. To determine the role of sulfhydryl oxidation in the production of BV, envelopment of nucleocapsids, and nucleocapsid incorporation into occlusion bodies, the Trichoplusia ni single nucleopolyhedrovirus ortholog, Tn79, was substituted for ac92. Tn79 was found to be an active sulfhydryl oxidase that substituted for Ac92, resulting in the production of infectious BV, albeit about 10-fold less than an ac92-containing virus. Tn79 rescued defects in ODV morphogenesis caused by a lack of ac92. Active Tn79 sulfhydryl oxidase activity is required for efficient BV production, ODV envelopment, and their subsequent incorporation into occlusion bodies in the absence of ac92. PMID:25010286

  20. Autographa californica multiple nucleopolyhedrovirus ODV-E56 is a per os infectivity factor, but is not essential for binding and fusion of occlusion-derived virus to the host midgut

    SciTech Connect

    Sparks, Wendy O.; Harrison, Robert L.; Bonning, Bryony C.

    2011-01-05

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) envelope protein ODV-E56 is essential for oral infection of larvae of Heliothis virescens. Bioassays with recombinant clones of AcMNPV lacking a functional odv-e56 gene showed that ODV-E56 was required for infectivity of both polyhedra and to a lesser extent, purified ODV. However, binding and fusion assays showed that ODV lacking ODV-E56 bound and fused to midgut cells at levels similar to ODV of wild-type virus. Fluorescence microscopy of midguts from larvae inoculated with ODV-E56-positive and -negative viruses that express GFP indicated that ODV-E56 was required for infection of the midgut epithelium. Purified ODV-E56 bound to several proteins in midgut-derived brush border membrane vesicles, but failed to rescue infectivity of ODV-E56-negative viruses in trans. These results indicate that ODV-E56 is a per os infectivity factor (pif-5) required for primary midgut infection at a point before or after virion binding and fusion.

  1. Characterization of the interaction between P143 and LEF-3 from two different baculovirus species: Choristoneura fumiferana nucleopolyhedrovirus LEF-3 can complement Autographa californica nucleopolyhedrovirus LEF-3 in supporting DNA replication.

    PubMed

    Chen, Tricia; Sahri, Daniela; Carstens, Eric B

    2004-01-01

    The baculovirus protein P143 is essential for viral DNA replication in vivo, likely as a DNA helicase. We have demonstrated that another viral protein, LEF-3, first described as a single-stranded DNA binding protein, is required for transporting P143 into the nuclei of insect cells. Both of these proteins, along with several other early viral proteins, are also essential for DNA replication in transient assays. We now describe the identification, nucleotide sequences, and transcription patterns of the Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) homologues of p143 and lef-3 and demonstrate that CfMNPV LEF-3 is also responsible for P143 localization to the nucleus. We predicted that the interaction between P143 and LEF-3 might be critical for cross-species complementation of DNA replication. Support for this hypothesis was generated by substitution of heterologous P143 and LEF-3 between two different baculovirus species, Autographa californica nucleopolyhedrovirus and CfMNPV, in transient DNA replication assays. The results suggest that the P143-LEF-3 complex is an important baculovirus replication factor.

  2. Identification of BV/ODV-C42, an Autographa californica nucleopolyhedrovirus orf101-encoded structural protein detected in infected-cell complexes with ODV-EC27 and p78/83.

    PubMed

    Braunagel, S C; Guidry, P A; Rosas-Acosta, G; Engelking, L; Summers, M D

    2001-12-01

    orf101 is a late gene of Autographa californica nucleopolyhedrovirus (AcMNPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of orf101 was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 (1629K) and ODV-EC27 (orf144). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.

  3. Autographa californica multiple nucleopolyhedrovirus ODV-E56 is a per os infectivity factor, but is not essential for binding and fusion of occlusion-derived virus to the host midgut.

    PubMed

    Sparks, Wendy O; Harrison, Robert L; Bonning, Bryony C

    2011-01-05

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) envelope protein ODV-E56 is essential for oral infection of larvae of Heliothis virescens. Bioassays with recombinant clones of AcMNPV lacking a functional odv-e56 gene showed that ODV-E56 was required for infectivity of both polyhedra and to a lesser extent, purified ODV. However, binding and fusion assays showed that ODV lacking ODV-E56 bound and fused to midgut cells at levels similar to ODV of wild-type virus. Fluorescence microscopy of midguts from larvae inoculated with ODV-E56-positive and -negative viruses that express GFP indicated that ODV-E56 was required for infection of the midgut epithelium. Purified ODV-E56 bound to several proteins in midgut-derived brush border membrane vesicles, but failed to rescue infectivity of ODV-E56-negative viruses in trans. These results indicate that ODV-E56 is a per os infectivity factor (pif-5) required for primary midgut infection at a point before or after virion binding and fusion.

  4. Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115.

    PubMed

    Ohkawa, Taro; Washburn, Jan O; Sitapara, Ronika; Sid, Eric; Volkman, Loy E

    2005-12-01

    Per os infectivity factors PIF1 (Ac119) and PIF2 (Ac022), like P74, are essential for oral infection of lepidopteran larval hosts of Autographa californica M nucleopolyhedrovirus (AcMNPV). Here we show that Ac115 also is a PIF (PIF3) and that, unlike PIF1 and PIF2, it does not mediate specific binding of AcMNPV occlusion-derived virus (ODV) to midgut target cells. We used an improved in vivo fluorescence dequenching assay to compare binding, fusion, and competition among control AcMNPV ODV and the ODVs of AcMNPV PIF1, PIF2, and PIF3 deletion mutants. Our results showed that binding and fusion of PIF1 and PIF2 mutants, but not the PIF3 mutant, were both qualitatively and quantitatively different from those of control ODV. Unlike control and PIF3-deficient ODV, an excess of PIF1- or PIF2-deficient ODV failed to compete effectively with control ODV's binding to specific receptors on midgut epithelial cells. Moreover, the levels of PIF1- and PIF2-deficient ODV binding were depressed threefold compared to control levels. Binding, fusion, and competition by PIF3-deficient ODV, however, were all indistinguishable from those of control ODV. These results implicated PIF1 and PIF2 as ODV envelope attachment proteins that mediate specific binding to primary target cells within the midgut. In contrast, PIF3 mediates another unidentified, but critical, early event during primary infection.

  5. N-terminal sequences from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus.

    PubMed

    Hong, T; Summers, M D; Braunagel, S C

    1997-04-15

    Baculovirus occlusion-derived virus (ODV) derives its envelope from an intranuclear membrane source. N-terminal amino acid sequences of the Autographa californica nuclear polyhedrosis virus (AcMNPV) envelope proteins, ODV-E66 and ODV-E25 (23 and 24 amino acids, respectively) are highly hydrophobic. Recombinant viruses that express the two N-terminal amino acid sequences fused to green fluorescent protein (23GFP or 24GFP) provided visual markers to follow protein transport and localization within the nucleus during infection. Autoflourescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized as foci to discrete locations within the nucleus. Immunoelectron microscopy confirmed that these foci predominantly contained intranuclear microvesicles and the reporter fusion proteins were also detected in cytoplasmic membranes near the nucleus, and the outer and inner nuclear membrane. Therefore, these defined hydrophobic domains are sufficient to direct native and fusion proteins to induced membrane microvesicles within a baculovirus-infected cell nucleus and the viral envelope. In addition, these data suggest that movement of these proteins into the nuclear envelope may initiate through cytoplasmic membranes, such as endoplasmic reticulum, and that transport into the nucleus may be mediated through the outer and inner nuclear membrane.

  6. Reduction of polyhedrin mRNA and protein expression levels in Sf9 and Hi5 cell lines, but not in Sf21 cells, infected with Autographa californica multiple nucleopolyhedrovirus fp25k mutants.

    PubMed

    Cheng, Xin-Hua; Hillman, Christopher C; Zhang, Chuan-Xi; Cheng, Xiao-Wen

    2013-01-01

    During cell infection, the fp25k gene of baculoviruses frequently mutates, producing the few polyhedra (FP) per cell phenotype with reduced polyhedrin (polh) expression levels compared with wild-type baculoviruses. Here we report that the fp25k gene of the model baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), contains two hypermutable seven-adenine (A7) mononucleotide repeats (MNRs) that were mutated to A8 MNRs and a TTAA site that had host DNA insertions, producing fp25k mutants during Sf21 cell infection. The FP phenotype in Sf9 and Hi5 cells was more pronounced than in Sf21 cells. AcMNPV fp25k mutants produced similar levels of polyhedra or enhanced GFP, which were both under the control of the AcMNPV polh promoter for expression, in Sf21 cells but lower levels in Sf9 and Hi5 cells compared with AcMNPV with an intact fp25k gene. This correlated with the polh mRNA levels detected in each cell line. The majority of Sf21 cells infected with fp25 mutants showed high polh promoter-mediated GFP expression levels. Two cell lines subcloned from Sf21 cells that were infected with fp25k mutants showed different GFP expression levels. Furthermore, a small proportion of Hi5 cells infected with fp25k mutants showed higher production of polyhedra and GFP expression than the rest, and the latter was not correlated with increased m.o.i. Therefore, these data suggest that AcMNPV polh promoter-mediated gene expression activities differ in the three cell lines and are influenced by different cells within the cell line.

  7. Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses.

    PubMed Central

    Godeau, F; Saucier, C; Kourilsky, P

    1992-01-01

    The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation. Images PMID:1335569

  8. A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein

    PubMed Central

    Ardisson-Araújo, Daniel M. P.; Melo, Fernando L.; Clem, Rollie J.; Wolff, José L. C.

    2015-01-01

    The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage. PMID:26537678

  9. Interaction of Autographa californica Multiple Nucleopolyhedrovirus Cathepsin Protease Progenitor (proV-CATH) with Insect Baculovirus Chitinase as a Mechanism for proV-CATH Cellular Retention▿†

    PubMed Central

    Hodgson, Jeffrey J.; Arif, Basil M.; Krell, Peter J.

    2011-01-01

    The insect baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, enhancing the dissemination of progeny virions away from the host cadavers. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures that liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not yet been determined. Signal peptide cleavage occurs upon cotranslational ER import of the v-cath-expressed protein, and ER-resident CHIA is needed for the folding of proV-CATH. Although this implies that CHIA and proV-CATH bind each other in the ER, the putative CHIA–proV-CATH interaction has not been experimentally verified. We demonstrate that the amino-terminal 22 amino acids (aa) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) preproV-CATH are responsible for the entry of proV-CATH into the ER. Furthermore, the CHIA–green fluorescent protein (GFP) and proV-CATH-red fluorescent protein (RFP) fusion proteins colocalize in the ER. Using monomeric RFP (mRFP)-based bimolecular fluorescence complementation (BiFC), we determined that CHIA and proV-CATH interact directly with each other in the ER during virus replication. Moreover, reciprocal Ni/His pulldowns of His-tagged proteins confirmed the CHIA–proV-CATH interaction biochemically. The reciprocal copurification of CHIA and proV-CATH suggests a specific CHIA–proV-CATH interaction and corroborates our BiFC data. Deletion of the CHIA KDEL motif allowed for premature CHIA secretion from cells, and proV-CATH was similarly prematurely secreted from cells along with ΔKDEL-CHIA. These data suggest that CHIA and proV-CATH interact directly with each other and that this interaction aids the cellular retention of proV-CATH. PMID:21289117

  10. Autographa californica Multiple Nucleopolyhedrovirus AC83 is a Per Os Infectivity Factor (PIF) Protein Required for Occlusion-Derived Virus (ODV) and Budded Virus Nucleocapsid Assembly as well as Assembly of the PIF Complex in ODV Envelopes.

    PubMed

    Javed, Muhammad Afzal; Biswas, Siddhartha; Willis, Leslie G; Harris, Stephanie; Pritchard, Caitlin; van Oers, Monique M; Donly, B Cameron; Erlandson, Martin A; Hegedus, Dwayne D; Theilmann, David A

    2017-03-01

    Baculovirus occlusion-derived virus (ODV) initiates infection of lepidopteran larval hosts by binding to the midgut epithelia, which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins, of which PIF1 to PIF4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif gene results in ODV being unable to bind or enter midgut cells. AC83 also associates with the PIF complex, and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes, but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding domain but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 (ac110) is reported as the 38th baculovirus core gene.IMPORTANCE ODV is essential for the per os infectivity of the baculovirus AcMNPV. To initiate infection, ODV binds to microvilli of lepidopteran midgut cells, a process which requires a group of seven virion envelope proteins called PIFs. In this study, we reexamined the function of AC83, a protein that copurifies with the ODV PIFs, to determine its role in the oral infection process. A zinc finger domain was identified and

  11. Autographa californica multiple nucleopolyhedrovirus ODV-E56 envelope protein is required for oral infectivity and can be substituted functionally by Rachiplusia ou multiple nucleopolyhedrovirus ODV-E56.

    PubMed

    Harrison, Robert L; Sparks, Wendy O; Bonning, Bryony C

    2010-05-01

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. In a previous analysis, the odv-e56 gene was found to be under positive selection pressure, suggesting that it may be a determinant of virus host range. To assess the role of ODV-E56 in oral infectivity and host range, we constructed recombinant AcMNPV clones (Ac69GFP-e56lacZ and AcIEGFP-e56lacZ) in which ODV-E56 protein synthesis was eliminated by inserting a beta-galactosidase (lacZ) expression cassette into the odv-e56 open reading frame. We also constructed a recombinant virus, Ac69GFP-Roe56, in which the native AcMNPV odv-e56 coding sequence was replaced with that of Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV), a closely related virus that is significantly more virulent towards some host species than AcMNPV. The odv-e56 recombinant viruses exhibited no alterations in polyhedron production and morphogenesis or in the production of infectious budded virus in cell culture. In bioassays using three lepidopteran host species, the oral infectivities of the odv-e56 mutant viruses Ac69GFP-e56lacZ and AcIEGFP-e56lacZ were profoundly impaired compared with those of wild-type and control recombinant viruses. Oral infectivity was restored fully by marker rescue of the odv-e56 mutant viruses with either the AcMNPV or the RoMNPV odv-e56 gene. In bioassays using two host species that are more susceptible to RoMNPV than to AcMNPV, Ac69GFP-Roe56 killed larvae with LC50 values similar to those of recombinant viruses expressing AcMNPV ODV-E56. This result indicated that replacement of the AcMNPV odv-e56 gene with the RoMNPV orthologue did not increase virulence against these two species.

  12. Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

    PubMed Central

    Oakland, Mayumi; Maury, Wendy; McCray, Paul B; Sinn, Patrick L

    2013-01-01

    Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN) responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV) in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction. PMID:23360952

  13. Promoter analysis of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum.

    PubMed

    Takaoka, N; Fukuzawa, M; Saito, T; Sakaitani, T; Ochiai, H

    1999-10-28

    We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position -47 to -37 and to an initiator (Inr, PyPyCAPyPyPyPy) at position -3 to +5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the beta-galactosidase reporter gene in transformed cells; also the difference in activity between growth conditions was compared. The results indicated that there are two positive vegetative regulatory elements extending between -187 and -62 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium.

  14. Autographa caljfornica nuclear polyhedrosis virus replication in non-permissive Lymantria dispar cell lines

    Treesearch

    Edward M. Dougherty; David Guzo; Kathleen S. Shields; Dwight E. Lynn; Susan K. Braun

    1991-01-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) the prototypic group A baculovirus, has the widest reported host range of the baculoviruses and is considered to be one of the most virulent baculoviruses studied. The gypsy moth Lymantria dispar is not considered a natural host of AcNPV, however. To determine the factors...

  15. A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells.

    PubMed

    Wu, Chunxiao; Wang, Shu

    2012-01-01

    Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.

  16. A MicroRNA-regulated and GP64-pseudotyped Lentiviral Vector Mediates Stable Expression of FVIII in a Murine Model of Hemophilia A

    PubMed Central

    Matsui, Hideto; Hegadorn, Carol; Ozelo, Margareth; Burnett, Erin; Tuttle, Angie; Labelle, Andrea; McCray, Paul B; Naldini, Luigi; Brown, Brian; Hough, Christine; Lillicrap, David

    2011-01-01

    The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences. In contrast to outcomes using this strategy to deliver FIX, this modified delivery strategy was in and of itself insufficient to prevent an anti-FVIII immune response in treated hemophilia A mice. However, pseudotyping the lentivirus with the GP64 envelope glycoprotein, in conjunction with a liver-restricted promoter and a miRNA-regulated FVIII transgene resulted in sustained, therapeutic levels of FVIII. These modifications to the lentiviral delivery system effectively restricted FVIII transgene expression to the liver. Plasma levels of FVIII could be increased to around 9% that of normal levels when macrophages were depleted prior to treating the hemophilia A mice with the modified lentiviral FVIII delivery system. PMID:21285959

  17. Autographa californica multiple nucleopolyhedrovirus PK-1 is essential for nucleocapsid assembly

    SciTech Connect

    Liang, Changyong; Li, Min; Dai, Xuejuan; Zhao, Shuling; Hou, Yanling; Zhang, Yongli; Lan, Dandan; Wang, Yun; Chen, Xinwen

    2013-09-01

    PK-1 (Ac10) is a baculovirus-encoded serine/threonine kinase and its function is unclear. Our results showed that a pk-1 knockout AcMNPV failed to produce infectious progeny, while the pk-1 repair virus could rescue this defect. qPCR analysis demonstrated that pk-1 deletion did not affect viral DNA replication. Analysis of the repaired recombinants with truncated pk-1 mutants demonstrated that the catalytic domain of protein kinases of PK-1 was essential to viral infectivity. Moreover, those PK-1 mutants that could rescue the infectious BV production defect exhibited kinase activity in vitro. Therefore, it is suggested that the kinase activity of PK-1 is essential in regulating viral propagation. Electron microscopy revealed that pk-1 deletion affected the formation of normal nucleocapsids. Masses of electron-lucent tubular structures were present in cell transfected with pk-1 knockout bacmid. Therefore, PK-1 appears to phosphorylate some viral or cellular proteins that are essential for DNA packaging to regulate nucleocapsid assembly. - Highlights: • A pk-1 knockout AcMNPV failed to produce infectious progeny. • The pk-1 deletion did not affect viral DNA replication. • The catalytic domain of protein kinases (PKc) of PK-1 was essential to viral infectivity. • The kinase activity of PK-1 is essential in regulating viral propagation. • PK-1 appears to phosphorylate some viral proteins that are essential for DNA packaging to regulate nucleocapsid assembly.

  18. Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells.

    PubMed

    Lyupina, Yulia V; Abaturova, Svetlana B; Erokhov, Pavel A; Orlova, Olga V; Beljelarskaya, Svetlana N; Mikhailov, Victor S

    2013-02-05

    Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24-48 hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

  19. Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells

    SciTech Connect

    Lyupina, Yulia V.; Abaturova, Svetlana B.; Erokhov, Pavel A.; Orlova, Olga V.; Beljelarskaya, Svetlana N.; Mikhailov, Victor S.

    2013-02-05

    Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24-48 hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

  20. Susceptibility of Autographa californica multiple nucleopolyhedrovirus to inhibitors of DNA replication.

    PubMed

    Thumbi, David K; Arif, Basil M; Krell, Peter J

    2007-12-01

    The objectives of this study were to develop methods to evaluate the susceptibility of the type baculovirus AcMNPV to various antiviral compounds and to select potential inhibitors for investigating baculovirus DNA replication. In concert with the classical cytopathic effects (CPE) and cytotoxicity inhibition assays, two approaches, which could be amenable for high throughput application for evaluating several classes of known antiviral compounds were developed. (i) An indirect approach based on spectrofluorimetric analysis of EGFP expression in Sf21 cells infected with a recombinant AcMNPV (AcEGFP) and (ii) a direct DNA quantitative assay based on quantitative real time PCR (qPCR). Initial CPE results suggested that of 21 compounds tested, aphidicolin, abacavir, camptothecin, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), l-mimosine, hydroxyurea and phosphonoacetic acid (PAA) were selective inhibitors of AcMNPV replication. Consistent with the CPE results, the EGFP fluorescence and the qPCR of viral DNA accumulation exhibited a dose dependent depression of EGFP expression and DNA accumulation, respectively, in infected cells exposed to them. The inhibitory effects of aphidicolin, abacavir, l-mimosine and hydroxyurea on AcMNPV DNA replication were reversible. Taken together, both spectrofluorimetric and qPCR assays are suitable and rapid quantitative approaches to investigate inhibitors of baculovirus DNA replication in infected cells.

  1. Developmental transcriptome of Aplysia californica.

    PubMed

    Heyland, Andreas; Vue, Zer; Voolstra, Christian R; Medina, Mónica; Moroz, Leonid L

    2011-03-15

    Genome-wide transcriptional changes in development provide important insight into mechanisms underlying growth, differentiation, and patterning. However, such large-scale developmental studies have been limited to a few representatives of Ecdysozoans and Chordates. Here, we characterize transcriptomes of embryonic, larval, and metamorphic development in the marine mollusc Aplysia californica and reveal novel molecular components associated with life history transitions. Specifically, we identify more than 20 signal peptides, putative hormones, and transcription factors in association with early development and metamorphic stages-many of which seem to be evolutionarily conserved elements of signal transduction pathways. We also characterize genes related to biomineralization-a critical process of molluscan development. In summary, our experiment provides the first large-scale survey of gene expression in mollusc development, and complements previous studies on the regulatory mechanisms underlying body plan patterning and the formation of larval and juvenile structures. This study serves as a resource for further functional annotation of transcripts and genes in Aplysia, specifically and molluscs in general. A comparison of the Aplysia developmental transcriptome with similar studies in the zebra fish Danio rerio, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and other studies on molluscs suggests an overall highly divergent pattern of gene regulatory mechanisms that are likely a consequence of the different developmental modes of these organisms.

  2. Developmental Transcriptome of Aplysia californica

    PubMed Central

    HEYLAND, ANDREAS; VUE, ZER; VOOLSTRA, CHRISTIAN R.; MEDINA, MÓNICA; MOROZ, LEONID L.

    2014-01-01

    Genome-wide transcriptional changes in development provide important insight into mechanisms underlying growth, differentiation, and patterning. However, such large-scale developmental studies have been limited to a few representatives of Ecdysozoans and Chordates. Here, we characterize transcriptomes of embryonic, larval, and metamorphic development in the marine mollusc Aplysia californica and reveal novel molecular components associated with life history transitions. Specifically, we identify more than 20 signal peptides, putative hormones, and transcription factors in association with early development and metamorphic stages—many of which seem to be evolutionarily conserved elements of signal transduction pathways. We also characterize genes related to biomineralization—a critical process of molluscan development. In summary, our experiment provides the first large-scale survey of gene expression in mollusc development, and complements previous studies on the regulatory mechanisms underlying body plan patterning and the formation of larval and juvenile structures. This study serves as a resource for further functional annotation of transcripts and genes in Aplysia, specifically and molluscs in general. A comparison of the Aplysia developmental transcriptome with similar studies in the zebra fish Danio rerio, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and other studies on molluscs suggests an overall highly divergent pattern of gene regulatory mechanisms that are likely a consequence of the different developmental modes of these organisms. PMID:21328528

  3. Midgut transcriptomic response of the gypsy moth, lymantria dispar, to infection with l. dispar and Autographa californica multiple nucleopolyhedroviruses

    USDA-ARS?s Scientific Manuscript database

    Developmental resistance of gypsy moth (Lymantria dispar) larvae to baculovirus infection consists of both midgut-based and systemic components. To characterize the midgut response of larvae to baculovirus infection and identify larval host genes putatively involved in the midgut component of devel...

  4. Effects of deletion of the ac109 gene of Autographa californica nucleopolyhedrovirus on interactions with mammalian cells.

    PubMed

    Alfonso, Victoria; Amalfi, Sabrina; López, María Gabriela; Taboga, Oscar

    2017-03-01

    Baculoviruses are able to enter into mammalian cells, where they can express a transgene that is placed under an appropriate promoter, without producing infectious progeny. ORF109 encodes an essential baculovirus protein that participates in the interaction of the baculovirus with mammalian cells. To date, the mechanisms underlying this interaction are not yet known. We demonstrated that although a Ac109 knock out virus maintained its ability to enter into BHK-21 cells, there was a marked reduction in the expression efficiency of the nuclear transgene. Moreover, the amount of free cytoplasmic viral DNA, which was detected by transcription of a reporter gene, was severely diminished. These results suggest Ac109 could be involved in maintaining the integrity of the viral nucleic acid.

  5. Protein synthesis in cells infected by Autographa californica nuclear polyhedrosis virus (Ac-NPV): the effect of cytosine arabinoside.

    PubMed

    Dobos, P; Cochran, M A

    1980-06-01

    Twenty-two virion polypeptides (VP) were detected reproducibly when the occluded form (OV) of [35S]methionine-labeled Ac-NPV, purified from polyhedral inclusion bodies (PIB), was analyzed by polyacrylamide gel electrophoresis and autoradiography. Ten of the VPs and the polyhedral protein (PP) were phosphorylated and 6 VPs were glycoproteins. Purified, nonoccluded virus (NOV) revealed 25 polypeptides. During a single cycle of virus replication, the synthesis of 33 infected cell polypeptides (1CP) was detected in different relative proportions at different times. They were arbitrarily designated as early (0-12hr), middle (12-18 hr), and late 18-24 hr) polypeptides. Most of the middle and late proteins seemed to be viral structural proteins. Rapid post-translational cleavage of IPCs was not observed; however, pulse-chase experiments revealed post-translational modifications of at least four polypeptides. Inhibition of DNA synthesis at the time of infection did not prevent the synthesis of ICPs, VPs, or the formation of PIBs. Progeny PIB from both control and cytosine arabinoside (Ara C)-treated cells that were infected with radiochemically pure [3H]thymidine-labeled NOV contained parental virus DNA. Electron micrographs of thin sections showed that, whereas PIBs from control cultures contained complete virions, those from Ara (C-treated cells contained both full and empty virus.

  6. Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones.

    PubMed

    Lyupina, Yulia V; Orlova, Olga V; Abaturova, Svetlana B; Beljelarskaya, Svetlana N; Lavrov, Andrey N; Mikhailov, Victor S

    2014-11-04

    The induction of heat shock proteins in baculovirus infected cells is well documented. However a role of these chaperones in infection cycle remains unknown. The observation that HSP70s are associated with virions of different baculoviruses reported by several researchers suggests that HSPs might be structural components of viruses or involved in virion assembly. These hypotheses were examined by using a novel inhibitor of the ATPase and chaperoning activity of HSP/HSC70s, VER-155008. When VER-155008 was added early in infection, the synthesis of viral proteins, genome replication and the production of budded virions (BV) were markedly inhibited indicating the dependence of virus reproduction on host chaperones. However, BV production was unaffected when VER-155008 was added in the mid-replication phase which is after accumulation of products required for completion of the viral DNA replication. These results suggest that the final stages in assembly of BV and their egress from cells do not depend on chaperone activity of host HSP/HSC70s.

  7. Characterization of Sleep in Aplysia californica

    PubMed Central

    Vorster, Albrecht P.A.; Krishnan, Harini C.; Cirelli, Chiara; Lyons, Lisa C.

    2014-01-01

    Study Objective: To characterize sleep in the marine mollusk, Aplysia californica. Design: Animal behavior and activity were assessed using video recordings to measure activity, resting posture, resting place preference, and behavior after rest deprivation. Latencies for behavioral responses were measured for appetitive and aversive stimuli for animals in the wake and rest states. Setting: Circadian research laboratory for Aplysia. Patients or Participants: A. californica from the Pacific Ocean. Interventions: N/A. Measurements and Results: Aplysia rest almost exclusively during the night in a semi-contracted body position with preferential resting locations in the upper corners of their tank. Resting animals demonstrate longer latencies in head orientation and biting in response to a seaweed stimulus and less frequent escape response steps following an aversive salt stimulus applied to the tail compared to awake animals at the same time point. Aplysia exhibit rebound rest the day following rest deprivation during the night, but not after similar handling stimulation during the day. Conclusions: Resting behavior in Aplysia fulfills all invertebrate characteristics of sleep including: (1) a specific sleep body posture, (2) preferred resting location, (3) reversible behavioral quiescence, (4) elevated arousal thresholds for sensory stimuli during sleep, and (5) compensatory sleep rebound after sleep deprivation. Citation: Vorster AP, Krishnan HC, Cirelli C, Lyons LC. Characterization of sleep in Aplysia californica. SLEEP 2014;37(9):1453-1463. PMID:25142567

  8. Antibacterial activity of native California medicinal plant extracts isolated from Rhamnus californica and Umbellularia californica.

    PubMed

    Carranza, Maria G; Sevigny, Mary B; Banerjee, Debashree; Fox-Cubley, Lacie

    2015-05-23

    Antimicrobial resistance (AMR) is a major threat to global public health. Medicinal plants have long been used as remedies for infectious diseases by native cultures around the world and have the potential for providing effective treatments for antibiotic-resistant infections. Rhamnus californica (Rhamnaceae) and Umbellularia californica (Lauraceae) are two indigenous California plant species historically used by Native Americans to treat skin, respiratory and gastrointestinal infections. This study aimed to assess the in vitro antimicrobial activity of methanolic extracts of leaves and bark of R. and U. californica against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive and Gram-negative bacteria. Methanolic extracts of leaves and bark of R. and U. californica were prepared by soxhlet extraction and evaluated for their antimicrobial activity against Bacillus cereus, Streptococcus pyogenes, Mycobacterium smegmatis, Staphylococcus aureus, MRSA, Escherichia coli and Pseudomonas aeruginosa using disc diffusion and minimal inhibitory concentration (MIC) assays. Chemical profiling of the extracts was performed using standard methods. All extracts inhibited the growth of MRSA and other Gram-positive bacteria with MICs of 3.3-6.0 mg/ml. Gram-negative organisms were unaffected by these extracts. U. californica extracts (leaves and bark) had the lowest MIC values. Chemical profiling detected the presence of quinones, alkaloids, flavonoids, cardenolides, tannins and saponins in these extracts. Our study is the first to report the antimicrobial properties of R. and U. californica and illustrates their promising anti-MRSA potential. Our results give scientific credence to the traditional medicinal uses of these plants by the indigenous peoples of California. Further investigation of the secondary metabolites responsible for the antimicrobial activity of these extracts against MRSA is warranted.

  9. Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer.

    PubMed

    Sinn, Patrick L; Burnight, Erin R; Hickey, Melissa A; Blissard, Gary W; McCray, Paul B

    2005-10-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

  10. Metamorphosis of Aplysia californica in Laboratory Culture

    PubMed Central

    Kriegstein, Arnold R.; Castellucci, Vincent; Kandel, Eric R.

    1974-01-01

    To utilize the advantages offered by the large identified nerve cells of the marine mollusc Aplysia californica for cellular biological studies of development, we have devised simple techniques for growing this species in the laboratory in large number with a generation time as short as nineteen weeks. We have used the cultured animals to study the life cycle from fertilized egg to reproductive adult. The major developmental and behavioral changes occur at metamorphosis, when the larvae settle on the seaweed Laurencia pacifica and the locomotor and feeding behaviors are transformed into their adult forms. We have examined the timetable for the abandonment of larval behaviors and the emergence of adult ones and found that the transition from swimming to crawling occurs first and marks the onset of metamorphosis. The change from ciliary feeding to radular feeding occurs later and signals the end of metamorphosis. Other adult behaviors, such as the reflex responses and fixed-action patterns of the mantle organs, appear after metamorphosis. PMID:4530325

  11. Irreversible thermal denaturation of Torpedo californica acetylcholinesterase.

    PubMed Central

    Kreimer, D. I.; Shnyrov, V. L.; Villar, E.; Silman, I.; Weiner, L.

    1995-01-01

    Thermal denaturation of Torpedo californica acetylcholinesterase, a disulfide-linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the two-state kinetic scheme N-->D, with activation energy 131 +/- 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 +/- 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol-specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state. PMID:8563632

  12. Aplysia californica neurons express microinjected neuropeptide genes.

    PubMed Central

    DesGroseillers, L; Cowan, D; Miles, M; Sweet, A; Scheller, R H

    1987-01-01

    Neuropeptide genes are expressed in specific subsets of large polyploid neurons in Aplysia californica. We have defined the transcription initiation sites of three of these neuropeptide genes (the R14, L11, and ELH genes) and determined the nucleotide sequence of the promoter regions. The genes contain the usual eucaryotic promoter signals as well as other structures of potential regulatory importance, including inverted and direct repeats. The L11 and ELH genes, which are otherwise unrelated, have homology in the promoter regions, while the R14 promoter was distinct. When cloned plasmids were microinjected into Aplysia neurons in organ culture, transitions between supercoiled, relaxed circular, and linear DNAs occurred along with ligation into high-molecular-weight species. About 20% of the microinjected neurons expressed the genes. The promoter region of the R14 gene functioned in expression of the microinjected DNA in all cells studied. When both additional 5' and 3' sequences were included, the gene was specifically expressed only in R14, suggesting that the specificity of expression is generated by a multicomponent repression system. Finally, the R14 peptide could be expressed in L11, demonstrating that it is possible to alter the transmitter phenotype of these neurons by introduction of cloned genes. Images PMID:3670293

  13. Dietary metal toxicity to the marine sea hare, Aplysia californica.

    PubMed

    Jarvis, Tayler A; Capo, Thomas R; Bielmyer-Fraser, Gretchen K

    2015-01-01

    Metal pollution from anthropogenic inputs is a concern in many marine environments. Metals accumulate in tissue and in excess cause toxicity in marine organisms. This study investigated the accumulation and effects of dietary metals in a macroinvertebrate. The green seaweed, Ulva lactuca and the red seaweed, Agardhiella subulata were each concurrently exposed to two concentrations (100 or 1000 μg/L) of five metals (Cu, Ni, Pb, Cd, and Zn). Additionally, U. lactuca was exposed to 10 μg/L of the metal mixture as well as 10 or 100 μg/L of each metal individually for 48 h. The seaweeds were then used as food for the sea hare, Aplysia californica for two to three weeks depending on the exposure concentration. Body mass of A. californica was measured weekly, and at the end of the exposure duration, metal concentrations were quantified in dissected organs (mouth, esophagus, crop, gizzard, ovotestis, heart, hepatopancreas, gill, and the carcass). Metal distribution and accumulation in the organs of A. californica varied with the metal. A. californica fed the metal-exposed diets had significantly reduced body weight by the end of the exposure periods, as compared to controls; however, differences were observed in the extent of growth reductions, dependent on exposure concentration, duration, and exposure regime (metal mixture versus individual metal-exposed diet). Metal mixture diets decreased A. californica growth more so than comparable individual metal diets, despite more metal accumulating in the individual metal diets. Additionally, Zn- and Cu-contaminated algal diets decreased control-normalized growth of A. californica significantly more than comparable Cd-, Pb-, or Ni-contaminated diets. The seaweed diets in this study contained environmentally relevant tissue metal burdens. Therefore, these results have implications for metals in marine systems.

  14. Sodium-phosphate symport by Aplysia californica gut.

    PubMed

    Gerencser, George A; Levin, Randy; Zhang, Jianliang

    2002-02-01

    Phosphate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Phosphate transport across apical membranes of vertebrate gastrointestinal epithelia requires sodium; whereas, its transport across the basolateral membrane requires antiport processes involving primarily chloride or bicarbonate. To decipher the phosphate transport mechanism in the foregut apical membrane of the mollusc, Aplysia californica, in vitro short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and phosphate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na+ was enhanced by the presence of phosphate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of phosphate was dependent upon the presence of Na+ and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetande, added to either side, had no effect on transepithelial difference or short-circuit current in the Aplysia gut bathed in a Na2HPO4 seawater medium. However, mucosal arsenate inhibited the net mucosal-to-serosal fluxes of both phosphate and Na+ and the arsenate-sensitive Na+ flux to that of phosphate was 2:1. These results suggest the presence of a Na-PO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.

  15. Effects of Hypergravity on Statocyst Development in Embryonic Aplysia californica

    NASA Technical Reports Server (NTRS)

    Pedrozo, Hugo A.; Wiederhold, Michael L.

    1994-01-01

    Aplysia californica is a marine gastropod mollusc with bilaterally paired statocysts as gravity-reccptor organs. Data from three experiments in which embryonic Aplysia californica were exposed to 2 x g arc discussed. The experimental groups were exposed to excess gravity until hatching (9-12 day), whereas control groups were maintained at normal gravity. Body diameter was measured before exposure to 2 x g. Statocyst, statolith and body diameter were each determined for samples of 20 embryos from each group on successive days. Exposure to excess gravity led to an increase in body size. Statocyst size was not affected by exposure to 2 x g. Statolith size decreased with treatment as indicated by smaller statolith-to-body ratios observed in the 2 x g group in all three experiments. Mean statolith diameter was significantly smaller for the 2 x g group in Experiment 1 but not in Experiments 2 and 3. Defective statocysts, characterized by very small or no statoliths, were found in the 2 x g group in Experiments 1 and 2.

  16. Abortive replication of Bombyx mori nucleopolyhedrovirus in Sf9 and High Five cells: Defective nuclear transport of the virions

    SciTech Connect

    Katou, Yasuhiro; Ikeda, Motoko; Kobayashi, Michihiro . E-mail: michihir@agr.nagoya-u.ac.jp

    2006-04-10

    Despite close genetic relationship, Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) display a distinct host range property. Here, BmNPV replication was examined in Sf9 and High Five cells that were nonproductive for BmNPV infection but supported high titers of AcMNPV replication. Recombinant BmNPV, vBm/gfp/lac, containing bm-ie1 promoter-driven egfp showed that few Sf9 and High Five cells infected with vBm/gfp/lac expressed EGFP, while large proportion of EGFP-expressing cells was observed when transfected with vBm/gfp/lac DNA. Immunocytochemical analysis showed that BmNPV was not imported into the nucleus of these two cell lines, while recombinant BmNPV, vBm{delta}64/ac-gp64 possessing AcMNPV gp64 was imported into the nucleus, yielding progeny virions in High Five cells, but not Sf9 cells. These results indicate that the defective nuclear import of infected virions due to insufficient BmNPV GP64 function is involved in the restricted BmNPV replication in Sf9 and High Five cells.

  17. Abortive replication of Bombyx mori nucleopolyhedrovirus in Sf9 and High Five cells: defective nuclear transport of the virions.

    PubMed

    Katou, Yasuhiro; Ikeda, Motoko; Kobayashi, Michihiro

    2006-04-10

    Despite close genetic relationship, Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) display a distinct host range property. Here, BmNPV replication was examined in Sf9 and High Five cells that were nonproductive for BmNPV infection but supported high titers of AcMNPV replication. Recombinant BmNPV, vBm/gfp/lac, containing bm-ie1 promoter-driven egfp showed that few Sf9 and High Five cells infected with vBm/gfp/lac expressed EGFP, while large proportion of EGFP-expressing cells was observed when transfected with vBm/gfp/lac DNA. Immunocytochemical analysis showed that BmNPV was not imported into the nucleus of these two cell lines, while recombinant BmNPV, vBmDelta64/ac-gp64 possessing AcMNPV gp64 was imported into the nucleus, yielding progeny virions in High Five cells, but not Sf9 cells. These results indicate that the defective nuclear import of infected virions due to insufficient BmNPV GP64 function is involved in the restricted BmNPV replication in Sf9 and High Five cells.

  18. Use of Cobra Lily (Darlingtonia californica) & Drosophila for Investigating Predator-Prey Relationships.

    ERIC Educational Resources Information Center

    Pratt, Carl R.

    1994-01-01

    Describes an experiment that uses the cobra lily (Darlingtonia californica) and fruit flies (Drosophila virilis) to investigate predator-prey relationships in a classroom laboratory. Suggestions for classroom extension of this experimental system are provided. (ZWH)

  19. First case of synophthalmia and albinism in the Pacific angel shark Squatina californica.

    PubMed

    Escobar-Sánchez, O; Moreno-Sánchez, X G; Aguilar-Cruz, C A; Abitia-Cárdenas, L A

    2014-08-01

    The first record in Mexican waters of albinism and synophthalmia (partial cyclopia) in the Pacific angel shark, Squatina californica is presented. Albinism is not lethal, but synophthalmia may cause the death of the individual immediately after birth.

  20. Use of Cobra Lily (Darlingtonia californica) & Drosophila for Investigating Predator-Prey Relationships.

    ERIC Educational Resources Information Center

    Pratt, Carl R.

    1994-01-01

    Describes an experiment that uses the cobra lily (Darlingtonia californica) and fruit flies (Drosophila virilis) to investigate predator-prey relationships in a classroom laboratory. Suggestions for classroom extension of this experimental system are provided. (ZWH)

  1. Bacteria facilitate prey retention by the pitcher plant Darlingtonia californica.

    PubMed

    Armitage, David W

    2016-11-01

    Bacteria are hypothesized to provide a variety of beneficial functions to plants. Many carnivorous pitcher plants, for example, rely on bacteria for digestion of captured prey. This bacterial community may also be responsible for the low surface tensions commonly observed in pitcher plant digestive fluids, which might facilitate prey capture. I tested this hypothesis by comparing the physical properties of natural pitcher fluid from the pitcher plant Darlingtonia californica and cultured 'artificial' pitcher fluids and tested these fluids' prey retention capabilities. I found that cultures of pitcher leaves' bacterial communities had similar physical properties to raw pitcher fluids. These properties facilitated the retention of insects by both fluids and hint at a previously undescribed class of plant-microbe interaction.

  2. Metal exposure in a benthic macroinvertebrate, Hydropsyche californica, related to mine drainage in the Sacramento River

    USGS Publications Warehouse

    Cain, D.J.; Carter, J.L.; Fend, S.V.; Luoma, S.N.; Alpers, C.N.; Taylor, H.E.

    2000-01-01

    A biomonitoring technique was employed to complement studies of metal transport in the upper Sacramento River affected by acid mine drainage. Metals (Al, Cd, Cu, Fe, Hg, Pb, and Zn) were determined in a resident invertebrate, Hydropsyche californica (Insecta: Trichoptera), and streambed sediments (<62 ??m) to assess metal contamination within a 111-km section of the river downstream of the mining area. Metals in H. californica also were interpreted to be broadly indicative of metal exposure in fish. Total Hg was determined in the whole body of the insect, whereas Al, Cd, Cu, Fe, Pb, and Zn were additionally separated into operationally defined cytosolic (used as an indicator of exposure to bioavailable metal) and particulate fractions. Total concentrations of Cd, Cu, Hg, Pb, and Zn in sediments were consistent with documented upstream sources of acid mine drainage. Metal distribution patterns in H. californica and sediments were generally consistent for Cd, Cu, and Pb but inconsistent for Hg and Zn. Concentrations in H. californica indicated that bioavailable Cd, Cu, Pb, and Zn was transported at least 120 km downstream of the mine sources. Zinc in H. californica was elevated, but unlike sediments, did not decrease downstream. Mercury in H. californica was not elevated.

  3. Regulation of statoconia mineralization in Aplysia californica in vitro

    NASA Technical Reports Server (NTRS)

    Pedrozo, H. A.; Schwartz, Z.; Dean, D. D.; Wiederhold, M. L.; Boyan, B. D.

    1996-01-01

    Statoconia are calcium carbonate inclusions in the lumen of the gravity-sensing organ, the statocyst, of Aplysia californica. The aim of the present study was to examine the role of carbonic anhydrase and urease in statoconia mineralization in vitro. The experiments were performed using a previously described culture system (Pedrozo et al., J. Comp. Physiol. (A) 177:415-425). Inhibition of carbonic anhydrase by acetazolamide decreased statoconia production and volume, while inhibition of urease by acetohydroxamic acid reduced total statoconia number, but had no affect on statoconia volume. Inhibition of carbonic anhydrase initially increased and then decreased the statocyst pH, whereas inhibition of urease decreased statocyst pH at all times examined; simultaneous addition of both inhibitors also decreased pH. These effects were dose and time dependent. The results show that carbonic anhydrase and urease are required for statoconia formation and homeostasis, and for regulation of statocyst pH. This suggests that these two enzymes regulate mineralization at least partially through regulation of statocyst pH.

  4. Regulation of statoconia mineralization in Aplysia californica in vitro

    NASA Technical Reports Server (NTRS)

    Pedrozo, H. A.; Schwartz, Z.; Dean, D. D.; Wiederhold, M. L.; Boyan, B. D.

    1996-01-01

    Statoconia are calcium carbonate inclusions in the lumen of the gravity-sensing organ, the statocyst, of Aplysia californica. The aim of the present study was to examine the role of carbonic anhydrase and urease in statoconia mineralization in vitro. The experiments were performed using a previously described culture system (Pedrozo et al., J. Comp. Physiol. (A) 177:415-425). Inhibition of carbonic anhydrase by acetazolamide decreased statoconia production and volume, while inhibition of urease by acetohydroxamic acid reduced total statoconia number, but had no affect on statoconia volume. Inhibition of carbonic anhydrase initially increased and then decreased the statocyst pH, whereas inhibition of urease decreased statocyst pH at all times examined; simultaneous addition of both inhibitors also decreased pH. These effects were dose and time dependent. The results show that carbonic anhydrase and urease are required for statoconia formation and homeostasis, and for regulation of statocyst pH. This suggests that these two enzymes regulate mineralization at least partially through regulation of statocyst pH.

  5. Population trends of the common murre (Uria aalge californica)

    USGS Publications Warehouse

    Carter, Harry R.; Wilson, Ulrich W.; Lowe, Roy W.; Rodway, M.S.; Manuwal, David Allen; Takekawa, Jean E.; Yee, Julie L.; Manuwal, David Allen; Carter, Harry R.; Zimmerman, Tara S.; Orthmeyer, Dennis L.

    2001-01-01

    Population trends for the common murre (Uria aalge californica) were determined from available whole-colony counts of murres in California, Oregon, Washington, and British Columbia from 1800 to 1995.From 1800 to 1978, historical counts were sporadic and not standardized. From 1979 to 1995, standardized whole-colony counts from aerial photographs were conducted in many years in California, Oregon, and Washington. In contrast, no aerial photographs of murre colonies in British Columbia have been taken and only a few other whole-colony counts have been conducted. Direct comparisons and statistical treatment of whole-colony counts were conducted using 1979-95 data. Complete data for all colonies were available only in 1988-89 when the breeding murre population was estimated to be 1.1 million, about 5-8% of the world population and 13-28% of the Pacific Ocean population. A summary of various natural and anthropogenic factors affecting murre populations in western North America since 1800, and particularly in 1979-95, also is provided.

  6. Fatal systemic toxoplasmosis in Valley quail (Callipepla californica)

    PubMed Central

    Casagrande, Renata A.; Pena, Hilda F.J.; Cabral, Aline D.; Rolim, Veronica M.; de Oliveira, Luiz G.S.; Boabaid, Fabiana M.; Wouters, Angelica T.B.; Wouters, Flademir; Cruz, Cláudio E.F.; Driemeier, David

    2015-01-01

    An adult, captive raised male Valley quail (Callipepla californica) acquired by a southern Brazilian aviary suddenly showed severe apathy, dyspnea and diarrhea, and died 18 hours after the onset of illness. At necropsy, pale muscles and whitish areas in the heart, splenomegaly, hepatomegaly, and consolidated red lungs were observed. Histological findings were mainly mononuclear inflammation with necrosis of liver, heart, spleen, bone marrow and lung. There were large numbers of Toxoplasma gondii tachyzoitesorganisms in the liver, heart, spleen, bone marrow, lungs, trachea, kidneys, adrenal glands, testes, intestines, and pancreas. These organisms were seen free in the organs' stroma or within macrophages and stained positively with polyclonal antiserum to T. gondii. Genomic DNA was extracted from the tissues and PCR was used to target the B1 gene of T. gondii. The genotypic characterization by PCR-RFLP with 11 markers (SAG1, SAG2 and alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3) revealed the ToxoDB-PCR-RFLP #87 genotype, the same as previously identified in a backyard chicken (TgCkBr156) in Rio Grande do Sul, Brazil. PMID:26101744

  7. Digestive Physiology and Nutritional Responses of Autographa gamma (L.) (Lepidoptera: Noctuidae) on Different Sugar Beet Cultivars

    PubMed Central

    Naseri, Bahram; Golikhajeh, Neshat; Rahimi Namin, Foroogh

    2016-01-01

    Digestive enzymatic activity and nutritional responses of Autographa gamma (L.) (Lepidoptera: Noctuidae), an important insect pest of sugar beet, on nine sugar beet cultivars (Peritra, Karolina, Paolita, Lenzier, Tiller, Ardabili, Persia, Rozier, and Dorothea) were studied. The highest proteolytic activity of fourth and fifth instar of A. gamma was in larvae fed on cultivar Persia. The highest amylolytic activity of fourth and fifth instar was observed in larvae fed on cultivars Rozier and Dorothea, respectively. The lowest proteolytic and amylolytic activities in fourth instar were observed on cultivar Tiller; whereas the lowest activities in fifth instar were detected on cultivars Karolina and Tiller, respectively. Larval weight in both larval instars (fourth and fifth) was the heaviest on cultivar Persia and the lightest on cultivar Karolina. Furthermore, weight gain of larvae was the highest on cultivar Persia and the lowest on cultivar Karolina. The results of this study suggest that cultivar Tiller was the most unsuitable host plant for feeding of A. gamma. PMID:27324581

  8. Comparative investigation of Umbellularia californica and Laurus nobilis Leaf essential oils and identification of constituents active against Aedes aegypti

    USDA-ARS?s Scientific Manuscript database

    Umbellularia californica (California Bay Laurel) is a native species from California and its leaves are commonly used as spice and insect repellent. The leaves of U. californica may be mistaken or used as a substitute for Mediterranean bay laurel (Laurus nobilis) on the market. The essential oils fr...

  9. [First report in Panama on Nerocila californica Schioedte and Meinert 1881 (Isopoda: Cymothoidea) in Sciaenops ocellatus (L) (Pisces: Sciaenidae)].

    PubMed

    Garcés, H A

    1993-01-01

    One juvenile specimen of the isopod Nerocila californica (acuminata form) was found on the skin of a cage-raised red drum, Sciaenops ocellatus, in Aguadulce, Cocle Province. This finding is the first report of the occurrence of Nerocila californica as ectoparasitic of fishes on the Pacific side of the Republic of Panama.

  10. Acetylcholine receptors and cholinergic ligands: biochemical and genetic aspects in Torpedo californica and Drosophila melanogaster

    SciTech Connect

    Rosenthal, L.S.

    1987-01-01

    This study evaluates the biochemical and genetic aspects of the acetylcholine receptor proteins and cholinergic ligands in Drosophila melanogaster and Torpedo californica. Included are (1) a comparative study of nicotinic ligand-induced cation release from acetylcholine receptors isolated from Torpedo californica and from Drosophila melanogaster, (2) solution studies of the cholinergic ligands, nikethamide and ethamivan, aimed at measuring internal molecular rotational barriers in solvents of different polarity; and (3) the isolation and characterization of the gene(s) for the acetylcholine receptor in Drosophila melasogaster. Acetylcholine receptor proteins isolated from Drosphila melanogaster heads were found to behave kinetically similar (with regards to cholinergic ligand-induced /sup 155/Eu:/sup 3 +/ displacement from prelabeled proteins) to receptor proteins isolated from Torpedo californica electric tissue, providing additional biochemical evidence for the existence of a Drosophila acetylcholine receptor.

  11. Functional Analysis of the Putative Fusion Domain of the Baculovirus Envelope Fusion Protein F

    PubMed Central

    Westenberg, Marcel; Veenman, Frank; Roode, Els C.; Goldbach, Rob W.; Vlak, Just M.; Zuidema, Douwe

    2004-01-01

    Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F1. The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect. PMID:15194771

  12. Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen.

    PubMed

    Weiner, Lev; Roth, Esther; Silman, Israel

    2011-01-01

    The photosensitizer, methylene blue (MB), is a strong reversible inhibitor of Torpedo californica acetylcholinesterase (AChE) in the dark. Under illumination it causes irreversible inactivation. Loss of fluorescence of the singlet oxygen ((1)O(2)) trap, 9,10-dimethylanthracene, was retarded in the presence of AChE, and the rate of photo-inactivation was increased in the presence of D(2)O, indicating that inactivation was due to (1)O(2) generated by the photosensitizer. CD revealed slightly reduced far-UV ellipticity, and slightly enhanced binding of an amphiphilic probe, indicating limited unfolding of the photo-oxidized AChE. However, both near-UV ellipticity and intrinsic fluorescence were markedly reduced, suggesting photo-oxidative damage to tryptophans, (Trp) supported by appearance of novel emission peaks ascribed to N'-formylkynurenine and/or kynurenine. Like other partially unfolded forms, the photo-oxidized AChE was sensitive to proteolysis. Photosensitized inactivation produced exclusively chemically cross-linked dimers, whereas irradiation of a partially unfolded state generated higher-order oligomers. The active-site gorge of AChE contains Trp in inhibitor-binding sites that might be targets for photo-oxidation. Indeed, reversible inhibitors retard photo-inactivation, and photo-inactivation destroys their binding sites. An excess of AChE protects paraoxonase from photo-inactivation by sequestering the photosensitizer. Affinity photo-oxidation of AChE by MB thus provides a valuable model for studying site-specific photo-inactivation of enzymes in both fundamental and clinical contexts. © 2010 The Authors. Photochemistry and Photobiology © 2010 The American Society of Photobiology.

  13. Modulation of antioxidant defense system after long term arsenic exposure in Zantedeschia aethiopica and Anemopsis californica.

    PubMed

    Del-Toro-Sánchez, Carmen Lizette; Zurita, Florentina; Gutiérrez-Lomelí, Melesio; Solis-Sánchez, Brenda; Wence-Chávez, Laura; Rodríguez-Sahagún, Araceli; Castellanos-Hernández, Osvaldo A; Vázquez-Armenta, Gabriela; Siller-López, Fernando

    2013-08-01

    Zantedeschia aethiopica (calla lily) and Anemopsis californica (yerba mansa) are plant species capable of accumulating arsenic (As) and therefore proposed as phytoremediation for removal of As from drinking water. The effects of a continuous 6 month As exposure (34±11 μg/L) from local contaminated groundwater on the antioxidant response of Z. aethiopica and A. californica were evaluated in leaves and stems of the plants bimonthly in a subsurface flow constructed wetland. As increased the activities of the antioxidant enzymes ascorbate peroxidase, glutathione reductase and catalase where higher levels were observed in Z. aethiopica than A. californica. No significant differences were detected on lipid peroxidation levels or antioxidant capacity evaluated by ORAC and DPPH assays or total phenol contents in any part of the plant, although in general the leaves of both plants showed the best antioxidant defense against the metal. In conclusion, Z. aethiopica and A. californica were able to cope to As through induction of a more sensitive enzymatic antioxidant response mechanism.

  14. Composition of Pteryxia terebinthina var. californica (Coult. and Rose) Mathias essential oils

    USGS Publications Warehouse

    Beauchamp, Philip E.; Dev, Vasu; Munevar-Mendoza, Elsa; Moore, Peggy E.

    2000-01-01

    β-Pinene (35.0%, 53.8%) was the major component of both the aerial parts and the root oils of Pteryxia terebinthina var. californica, respectively. β-Phellandrene (12.2%) was the other most abundant component of the oil from aeial parts while δ-3-carene (14.2%) was the second abundant component of the root oil.

  15. Modulation of CYPs, P-gp, and PXR by Eschscholzia californica (California poppy) and its alkaloids

    USDA-ARS?s Scientific Manuscript database

    Eschscholzia californica Cham., a native US plant, is traditionally used as a sedative, analgesic and anxiolytic herb. With the rapid rise in the use of herbal supplements together with over the counter (OTC) and prescription drugs, the risk for potential herb-drug interactions is also increasing. M...

  16. Variation in susceptibility of Umbellularia californica (Bay Laurel) to Phytophthora ramorum

    Treesearch

    Matthew Meshriy; Daniel Hüberli; Tamar Harnik; Lori Miles; Keefe Reuther; Matteo Garbelotto

    2006-01-01

    Bay laurel (Umbellularia californica) is an important foliar host in terms of spore production and transmission of disease. We designed a bioassay to screen for variation in susceptibility to Phytophthora ramorum among populations of bay laurel collected along the coast of California to southern Oregon and also from Yosemite....

  17. Hybridization of cultivated Vitis vinifera with wild V. californica and V. girdiana in California

    USDA-ARS?s Scientific Manuscript database

    The native wild grape species of northern California, Vitis californica Benth. (California wild grape), and V. girdiana Munson (desert wild grape) in southern California are under increasing pressure from loss of habitat and from interbreeding with the domesticated grapevine, V. vinifera L. For its...

  18. Effect of aluminum chloride and zinc sulfate on Autographa california nuclear polyhedrosis virus (ACNPV) replication in cell culture.

    PubMed

    Weiss, S A; Smith, G C; Vaughn, J L; Dougherty, E M; Tompkins, G J

    1982-11-01

    When IPL-SF-21AE III continuous insect cell line was grown and maintained in IPL-41 insect cell culture medium supplemented with 16 microM of AlCl3 or 0.24 microM of ZnSO4 . 7H2O, or both metallic salts, and then infected with Autographa california nuclear polyhedrosis virus, virus replication was increased significantly. The yield of polyhedral inclusion bodies (PIB) was enhanced up to 121%. Synthesis of cell-free nonoccluded virus was increased to 365% when infectivity was assayed by the plaque method. Newly applied electron microscopic quantitation and stereological techniques also revealed a significant increase in virus particles (VP) and in amount and size of PIB as well as number of VP per PIB.

  19. Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Ojala, Kirsi; Michel, Patrik O; Vuento, Matti; Oker-Blom, Christian

    2002-12-01

    Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89 +/- 0.74) x 10(-8) cm2s(-1) and an apparent hydrodynamic radius of 83.35 +/- 21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.57 x 10(-7) cm2s(-1)), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

  20. Modulation of CYPs, P-gp, and PXR by Eschscholzia californica (California Poppy) and Its Alkaloids.

    PubMed

    Manda, Vamshi K; Ibrahim, Mohamed A; Dale, Olivia R; Kumarihamy, Mallika; Cutler, Stephen J; Khan, Ikhlas A; Walker, Larry A; Muhammad, Ilias; Khan, Shabana I

    2016-04-01

    Eschscholzia californica, a native US plant, is traditionally used as a sedative, analgesic, and anxiolytic herb. With the rapid rise in the use of herbal supplements together with over-the-counter and prescription drugs, the risk for potential herb-drug interactions is also increasing. Most of the clinically relevant pharmacokinetic drug interactions occur due to modulation of cytochrome P450 enzymes (CYPs), P-glycoprotein, and the pregnane X receptor by concomitantly used herbs. This study aimed to determine the effects of an EtOH extract, aqueous extract (tea), basic CHCl3 fractions, and isolated major alkaloids, namely protopine (1), escholtzine (2), allocryptopine (3), and californidine (4), of E. californica on the activity of cytochrome P450s, P-glycoprotein and the pregnane X receptor. The EtOH extract and fractions showed strong time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19, and reversible inhibition of CYP 2D6. Among the alkaloids, escholtzine (2) and allocryptopine (3) exhibited time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19 (IC50 shift ratio > 2), while protopine (1) and allocryptopine (3) showed reversible inhibition of CYP 2D6 enzyme. A significant activation of the pregnane X receptor (> 2-fold) was observed with the EtOH extract, basic CHCl3 fraction, and alkaloids (except protopine), which resulted into an increased expression of mRNA and the activity of CYP 3A4 and CYP 1A2. The expression of P-glycoprotein was unaffected. However, aqueous extract (tea) and its main alkaloid californidine (4) did not affect cytochrome P450s, P-glycoprotein, or the pregnane X receptor. This data suggests that EtOH extract of E. californica and its major alkaloids have a potential of causing interactions with drugs that are metabolized by cytochrome P450s, while the tea seems to be safer.

  1. Localization and functional characterization of a novel adipokinetic hormone in the mollusk, Aplysia californica.

    PubMed

    Johnson, Joshua I; Kavanaugh, Scott I; Nguyen, Cindy; Tsai, Pei-San

    2014-01-01

    Increasing evidence suggests that gonadotropin-releasing hormone (GnRH), corazonin, adipokinetic hormone (AKH), and red pigment-concentrating hormone all share common ancestry to form a GnRH superfamily. Despite the wide presence of these peptides in protostomes, their biological effects remain poorly characterized in many taxa. This study had three goals. First, we cloned the full-length sequence of a novel AKH, termed Aplysia-AKH, and examined its distribution in an opisthobranch mollusk, Aplysia californica. Second, we investigated in vivo biological effects of Aplysia-AKH. Lastly, we compared the effects of Aplysia-AKH to a related A. californica peptide, Aplysia-GnRH. Results suggest that Aplysia-AKH mRNA and peptide are localized exclusively in central tissues, with abdominal, cerebral, and pleural ganglia being the primary sites of Aplysia-AKH production. However, Aplysia-AKH-positive fibers were found in all central ganglia, suggesting diverse neuromodulatory roles. Injections of A. californica with Aplysia-AKH significantly inhibited feeding, reduced body mass, increased excretion of feces, and reduced gonadal mass and oocyte diameter. The in vivo effects of Aplysia-AKH differed substantially from Aplysia-GnRH. Overall, the distribution and biological effects of Aplysia-AKH suggest it has diverged functionally from Aplysia-GnRH over the course of evolution. Further, that both Aplysia-AKH and Aplysia-GnRH failed to activate reproduction suggest the critical role of GnRH as a reproductive activator may be a phenomenon unique to vertebrates.

  2. A water-borne adhesive modeled after the sandcastle glue of P. californica.

    PubMed

    Shao, Hui; Bachus, Kent N; Stewart, Russell J

    2009-05-13

    Polyacrylate glue protein analogs of the glue secreted by Phragmatopoma californica, a marine polycheate, were synthesized with phosphate, primary amine, and catechol sidechains with molar ratios similar to the natural glue proteins. Aqueous mixtures of the mimetic polyelectrolytes condensed into liquid complex coacervates around neutral pH. Wet cortical bone specimens bonded with the coacervates, oxidatively crosslinked through catechol sidechains, had bond strengths nearly 40% of the strength of a commercial cyanoacrylate. The unique material properties of complex coacervates may be ideal for development of clinically useful adhesives and other biomaterials.

  3. Antimutagenicity of Methanolic Extracts from Anemopsis californica in Relation to Their Antioxidant Activity.

    PubMed

    Del-Toro-Sánchez, Carmen Lizette; Bautista-Bautista, Nereyda; Blasco-Cabal, José Luis; Gonzalez-Ávila, Marisela; Gutiérrez-Lomelí, Melesio; Arriaga-Alba, Myriam

    2014-01-01

    Anemopsis californica has been used empirically to treat infectious diseases. However, there are no antimutagenic evaluation reports on this plant. The present study evaluated the antioxidant activity in relation to the mutagenic and antimutagenic activity properties of leaf (LME) and stem (SME) methanolic extracts of A. californica collected in the central Mexican state of Querétaro. Antioxidant properties and total phenols of extracts were evaluated using DPPH (1,1-diphenyl-2-picrylhydrazyl) and Folin-Ciocalteu methods, respectively. Mutagenicity was evaluated using the Ames test employing Salmonella enterica serovar Typhimurium strains (TA98, TA100, and TA102), with and without an aroclor 1254 (S9 mixture). Antimutagenesis was performed against mutations induced on the Ames test with MNNG, 2AA, or 4NQO. SME presented the highest antioxidant capacity and total phenolic content. None of the extracts exhibited mutagenicity in the Ames test. The extracts produced a significant reduction in 2AA-induced mutations in S. typhimurium TA98. In both extracts, mutagenesis induced by 4NQO or methyl-N'-nitro-N-nitrosoguanidine (MNNG) was reduced only if the exposure of strains was <10 μg/Petri dish. A. californca antioxidant properties and its capacity to reduce point mutations render it suitable to enhance medical cancer treatments. The significant effect against antimutagenic 2AA suggests that their consumption would provide protection against carcinogenic polycyclic aromatic compounds.

  4. Antimutagenicity of Methanolic Extracts from Anemopsis californica in Relation to Their Antioxidant Activity

    PubMed Central

    Del-Toro-Sánchez, Carmen Lizette; Bautista-Bautista, Nereyda; Blasco-Cabal, José Luis; Gonzalez-Ávila, Marisela; Gutiérrez-Lomelí, Melesio; Arriaga-Alba, Myriam

    2014-01-01

    Anemopsis californica has been used empirically to treat infectious diseases. However, there are no antimutagenic evaluation reports on this plant. The present study evaluated the antioxidant activity in relation to the mutagenic and antimutagenic activity properties of leaf (LME) and stem (SME) methanolic extracts of A. californica collected in the central Mexican state of Querétaro. Antioxidant properties and total phenols of extracts were evaluated using DPPH (1,1-diphenyl-2-picrylhydrazyl) and Folin-Ciocalteu methods, respectively. Mutagenicity was evaluated using the Ames test employing Salmonella enterica serovar Typhimurium strains (TA98, TA100, and TA102), with and without an aroclor 1254 (S9 mixture). Antimutagenesis was performed against mutations induced on the Ames test with MNNG, 2AA, or 4NQO. SME presented the highest antioxidant capacity and total phenolic content. None of the extracts exhibited mutagenicity in the Ames test. The extracts produced a significant reduction in 2AA-induced mutations in S. typhimurium TA98. In both extracts, mutagenesis induced by 4NQO or methyl-N′-nitro-N-nitrosoguanidine (MNNG) was reduced only if the exposure of strains was <10 μg/Petri dish. A. californca antioxidant properties and its capacity to reduce point mutations render it suitable to enhance medical cancer treatments. The significant effect against antimutagenic 2AA suggests that their consumption would provide protection against carcinogenic polycyclic aromatic compounds. PMID:25152760

  5. Selective prey avoidance learning in the predatory sea slug Pleurobranchaea californica.

    PubMed

    Noboa, Vanessa; Gillette, Rhanor

    2013-09-01

    Predator-prey interactions involving aposematic signaling, where predators learn the warning cues of well-defended prey, are clear examples of cost-benefit decisions in foraging animals. However, knowledge of the selectivity of predator learning and the natural conditions where it occurs is lacking for those foragers simpler in brain and body plan. We pursued the question in the sea slug Pleurobranchaea californica, a generalist forager of marked simplicity of body form, nervous system and behavior. This predator exploits many different types of prey, some of which are costly to attack. When offered Flabellina iodinea, an aeolid nudibranch with a stinging defense, biting attack was followed by rapid rejection and aversive turns. The predatory sea slug rapidly learned avoidance. Notable exceptions were animals with extremely high or low feeding thresholds that either ignored F. iodinea or completely consumed it, respectively. Experienced slugs showed strong avoidance of F. iodinea for days after exposure. Aposematic odor learning was selective: avoidance was not linked to change in feeding thresholds, and trained animals readily attacked and consumed a related aeolid, Hermissenda crassicornis. For P. californica, aposematic learning is a cognitive adaptation in which sensation, motivation and memory are integrated to direct cost-benefit choice, and thereby lend flexibility to the generalist's foraging strategy.

  6. Behavioural effects of the American traditional plant Eschscholzia californica: sedative and anxiolytic properties.

    PubMed

    Rolland, A; Fleurentin, J; Lanhers, M C; Younos, C; Misslin, R; Mortier, F; Pelt, J M

    1991-06-01

    Eschsholzia californica Cham. is a traditional medicinal plant of the Indians used by the rural population of California for its analgesic and sedative properties. Our study on the aqueous extract shows that this plant reduced the behavioural parameters measured in a familiar environment test in mice (novelty preference, locomotion and rearings in two compartments test) at doses above 100 mg/kg and in non-familiar environment tests (staircase test) at doses above 200 mg/kg. This finding validates its traditional sedative properties confirmed by the sleeping induction at doses above 100 mg/kg. Furthermore, when administered at a dose a of 25 mg/kg, E. californica appeared to also have an anxiolytic action since it produced an increase of the number of steps climbed by mice in the staircase test (anticonflict effect) and that of the time spent by animals in the lit box when they were confronted with the light/dark choice situation. Before evaluation of the behavioural effects, it was verified that our aqueous extract did not induce any toxic effect when administered i.p. and p.o.

  7. EF-1α DNA Sequences Indicate Multiple Origins of Introduced Populations of Essigella californica (Hemiptera: Aphididae).

    PubMed

    Théry, Thomas; Brockerhoff, Eckehard G; Carnegie, Angus J; Chen, Rui; Elms, Stephen R; Hullé, Maurice; Glatz, Richard; Ortego, Jaime; Qiao, Ge-Xia; Turpeau, Évelyne; Favret, Colin

    2017-06-01

    Aphids in the pine-feeding Nearctic genus Essigella (Sternorrhyncha, Aphididae, Lachninae) have been introduced in Europe, North Africa, Oceania, and South America. Mitochondrial, nuclear, and endosymbiont DNA sequences of 12 introduced populations from three continents confirm they all belong to Essigella californica (Essig, 1909). Intron sequence variation of the nuclear gene EF-1α has revealed the existence of four distinct groups. Group I gathers one population from China, where the species is newly reported, and several from Europe (France and Italy); Group II is represented by one population from Argentina; Group III includes two populations from Southern Australia with one from New Zealand; and Group IV corresponds to five populations from Eastern and South-Eastern Australia. These results indicate that introduced populations of E. californica have at least four source populations. They also show that intron variation of EF-1α can be a method to discriminate populations of asexually reproducing aphids. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Deviating from the Norm: Peculiarities of Aplysia cf. californica Early Cleavage Compared to Traditional Spiralian Models.

    PubMed

    Chávez-Viteri, Yolanda E; Brown, Federico D; Pérez, Oscar D

    2017-01-01

    Spiralia represents one of the main clades of bilaterally symmetrical metazoans (Bilateria). This group is of particular interest due to the remarkable conservation of its early developmental pattern despite of the high diversity of larval and adult body plans. Variations during embryogenesis are considered powerful tools to determine ancestral and derived characters under a phylogenetic framework. By direct observation of embryos cultured in vitro, we analyzed the early cleavage of the euopisthobranchs Aplysia cf. californica. We used tubulin immunocytochemistry to stain mitotic spindles during early cleavages, and followed each division with the aid of an autofluorescent compound inside yolk platelets, which differed from the characteristic pink-brownish pigment of the vegetal cytoplasm in zygotes and early embryos. We found that this species exhibits an unequal cleavage characterized by ooplasmic segregation, oblique inclination of mitotic spindles, and differences in size and positioning of the asters in relation to the cellular cortex. Furthermore, we detected asynchrony in cleavage timing between the two large macromeres C and D, which increases the number of cleavage rounds required to reach a particular cell stage in comparison to other spiralians. Here, we report the presence of a transient and previously undescribed U-shaped embryo in this species. The present detailed description of A. californica early development deviates considerably from stereotypical patterns described in other spiralians. Our observations demonstrate that early spiralian development can be more plastic than previously thought.

  9. Comparative investigation of Umbellularia californica and Laurus nobilis leaf essential oils and identification of constituents active against Aedes aegypti.

    PubMed

    Tabanca, Nurhayat; Avonto, Cristina; Wang, Mei; Parcher, Jon F; Ali, Abbas; Demirci, Betul; Raman, Vijayasankar; Khan, Ikhlas A

    2013-12-18

    Umbellularia californica (California bay laurel) and Laurus nobilis (Mediterranean bay laurel) leaves may be mistaken or used as a substitute on the market due to their morphological similarity. In this study, a comparison of anatomical and chemical features and biological activity of both plants is presented. L. nobilis essential oil biting deterrent and larvicidal activity were negligible. On the other hand, U. californica leaf oil showed biting deterrent activity against Aedes aegypti . The identified active repellents was thymol, along with (-)-umbellulone, 1,8-cineole, and (-)-α-terpineol. U. californica essential oil also demonstrated good larvicidal activity against 1-day-old Ae. aegypti larvae with a LD50 value of 52.6 ppm. Thymol (LD50 = 17.6 ppm), p-cymene, (-)-umbellulone, and methyleugenol were the primary larvicidal in this oil. Umbellulone was found as the principal compound (37%) of U. californica essential oil, but was not present in L. nobilis essential oil. Umbellulone mosquito activity is here reported for the first time.

  10. Development of Screening Trials to Rank Pinus radiata Genotypes for Resistance to Defoliation by Monterey Pine Aphid (Essigella californica)

    Treesearch

    Stephen Elms; Peter Ades; Nick Collet

    2012-01-01

    The Monterey pine aphid (Essigella californica) is a recent arrival in Australia, having first been detected in 1998. It quickly spread throughout the national radiata pine (Pinus radiata D. Don) plantation estate, causing seasonal defoliation and compromising tree growth in many areas. Selection of resistant radiata...

  11. Factors influencing Phytophthora ramorum infectivity on Umbellularia californica and testing of a defoliation-based control method

    Treesearch

    Christine Windsor Colijn; Michael Cohen; Steve Johnston; Whalen Dillon; Nathan Rank

    2013-01-01

    The primary foliar host for Phytophthora ramorum is California bay laurel, Umbellularia californica (Hook. & Arn.) Nutt., a main reservoir for the pathogen in California woodlands. We investigated environmental and pathogen-mediated influences on incidence and severity of P. ramorum infection of

  12. Mercury Concentrations in Pacific Angel Sharks (Squatina californica) and Prey Fishes from Southern Gulf of California, Mexico.

    PubMed

    Escobar-Sánchez, O; Ruelas-Inzunza, J; Moreno-Sánchez, X G; Romo-Piñera, A K; Frías-Espericueta, M G

    2016-01-01

    Concentrations of mercury (Hg) were quantified in muscle tissues of the Pacific angel shark, Squatina californica sampled from Southern Gulf of California, Mexico, considering total length, sex, diet and the dietary risk assessment. High Hg levels are typically associated with carnivorous fishes, however S. californica showed low Hg concentrations (<1.0 µg g(-1)) in muscle (0.24 ± 0.27 µg g(-1) wet weight; n = 94). No effect of sex, total length and weight on Hg concentrations were observed in the shark (p > 0.05). Hg concentrations were highest in the darkedge mishipman: Porichthys analis (0.14 ± 0.08 µg g(-1)) and red-eye round herring Etrumeus teres (0.13 ± 0.05 µg g(-1)) relative to other prey species, which could suggest that Hg concentrations in S. californica were influenced by these species. Given the relatively low concentration of Hg across age-classes and sex, consumption of S. californica's muscle tissue poses limited risk to humans.

  13. Comparative transcriptomics among floral organs of the basal eudicot Eschscholzia californica as reference for floral evolutionary developmental studies

    PubMed Central

    2010-01-01

    Background Molecular genetic studies of floral development have concentrated on several core eudicots and grasses (monocots), which have canalized floral forms. Basal eudicots possess a wider range of floral morphologies than the core eudicots and grasses and can serve as an evolutionary link between core eudicots and monocots, and provide a reference for studies of other basal angiosperms. Recent advances in genomics have enabled researchers to profile gene activities during floral development, primarily in the eudicot Arabidopsis thaliana and the monocots rice and maize. However, our understanding of floral developmental processes among the basal eudicots remains limited. Results Using a recently generated expressed sequence tag (EST) set, we have designed an oligonucleotide microarray for the basal eudicot Eschscholzia californica (California poppy). We performed microarray experiments with an interwoven-loop design in order to characterize the E. californica floral transcriptome and to identify differentially expressed genes in flower buds with pre-meiotic and meiotic cells, four floral organs at pre-anthesis stages (sepals, petals, stamens and carpels), developing fruits, and leaves. Conclusions Our results provide a foundation for comparative gene expression studies between eudicots and basal angiosperms. We identified whorl-specific gene expression patterns in E. californica and examined the floral expression of several gene families. Interestingly, most E. californica homologs of Arabidopsis genes important for flower development, except for genes encoding MADS-box transcription factors, show different expression patterns between the two species. Our comparative transcriptomics study highlights the unique evolutionary position of E. californica compared with basal angiosperms and core eudicots. PMID:20950453

  14. Biosynthesis of the Torpedo californica Acetylcholine Receptor α Subunit in Yeast

    NASA Astrophysics Data System (ADS)

    Fujita, Norihisa; Nelson, Nathan; Fox, Thomas D.; Claudio, Toni; Lindstrom, Jon; Riezman, Howard; Hess, George P.

    1986-03-01

    Yeast cells were transformed with a plasmid containing complementary DNA encoding the α subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the α subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The α subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.

  15. Biochemical isolation and physiological identification of the egg- laying hormone in Aplysia californica

    PubMed Central

    1976-01-01

    It has been determined that the bag cells of Aplysia californica produce two polypeptide species that comigrate on electrophoretic gels containing sodium dodecyl sulfate. By this separation procedure both species can be assigned a molecular weight of approximately 6,000. One of these molecules has an Rf of 0.65 on alkaline discontinuous electrophoresis gels, an isoelectric point at pH 4.8, a gel filtration molecular weight of approximately 12,000, and has no known biological function. The other does not enter alkaline disk gels, has an isoelectric point at approximately pH 9.3, shows a gel filtration molecular weight consistent with that determined by SDS gel electrophoresis, and is the egg-laying hormone. PMID:956770

  16. Aging in Sensory and Motor Neurons Results in Learning Failure in Aplysia californica.

    PubMed

    Kempsell, Andrew T; Fieber, Lynne A

    2015-01-01

    The physiological and molecular mechanisms of age-related memory loss are complicated by the complexity of vertebrate nervous systems. This study takes advantage of a simple neural model to investigate nervous system aging, focusing on changes in learning and memory in the form of behavioral sensitization in vivo and synaptic facilitation in vitro. The effect of aging on the tail withdrawal reflex (TWR) was studied in Aplysia californica at maturity and late in the annual lifecycle. We found that short-term sensitization in TWR was absent in aged Aplysia. This implied that the neuronal machinery governing nonassociative learning was compromised during aging. Synaptic plasticity in the form of short-term facilitation between tail sensory and motor neurons decreased during aging whether the sensitizing stimulus was tail shock or the heterosynaptic modulator serotonin (5-HT). Together, these results suggest that the cellular mechanisms governing behavioral sensitization are compromised during aging, thereby nearly eliminating sensitization in aged Aplysia.

  17. Isolation of sensory neurons of Aplysia californica for patch clamp recordings of glutamatergic currents.

    PubMed

    Fieber, Lynne A; Carlson, Stephen L; Kempsell, Andrew T; Greer, Justin B; Schmale, Michael C

    2013-07-10

    The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

  18. Synthesis, accumulation, and release of D-aspartate in the Aplysia californica central nervous system

    PubMed Central

    Scanlan, Cory; Shi, Ting; Hatcher, Nathan G.; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2010-01-01

    D-aspartate (D-Asp) is an endogenous molecule that is often detected in central nervous system and endocrine tissues. Using capillary electrophoresis and a variety of radionuclide detection techniques, we examine the synthesis, release, and uptake/accumulation of D-Asp in the central nervous system of the marine mollusk Aplysia californica. We observe the preferential synthesis and accumulation of D-Asp over L-aspartate (L-Asp) in neuron-containing ganglia compared to surrounding sheath tissues. Little conversion of D-Asp to L-Asp is detected. The Ca2+ ionophore ionomycin and elevated extracellular potassium stimulates release of D-Asp from the cerebral ganglia. Lastly, radioactive D-Asp in the extracellular media is efficiently taken up and accumulated by individual F-cluster neurons. These observations point to a role for D-Asp in cell-to-cell signaling with many characteristics similar to classical transmitters. PMID:20874765

  19. No evidence of temporal preferences in caching by Western scrub-jays (Aphelocoma californica)☆

    PubMed Central

    Thom, James M.; Clayton, Nicola S.

    2014-01-01

    Humans and other animals often favour immediate gratification over long-term gain. Primates, including humans, appear more willing to wait for rewards than other animals, such as rats or pigeons. Another group displaying impressive patience are the corvids, which possess large brains and show sophisticated cognitive abilities. Here, we assess intertemporal choice in one corvid species, the Western scrub-jay (Aphelocoma californica). These birds cache food for future consumption and respond flexibly to future needs. Cache-theft and cache-degradation are time-dependent processes in scrub-jay ecology that might necessitate sensitivity to delays between caching and retrieval. We adopt a caching paradigm with delays of up to 49 h. Across two experiments we find no evidence of a preference for earlier recovery. We highlight the possibility that, although scrub-jays can discriminate between the present and the future, they may not understand how far into the future an event will occur. PMID:24378212

  20. Number of conspecifics and reproduction in the invasive plant Eschscholzia californica (Papaveraceae): is there a pollinator-mediated Allee effect?

    PubMed

    Anic, V; Henríquez, C A; Abades, S R; Bustamante, R O

    2015-05-01

    The component Allee effect has been defined as 'a positive relationship between any measure of individual fitness and the number or density of conspecifics'. Larger plant populations or large patches have shown a higher pollinator visitation rate, which may give rise to an Allee effect in reproduction of the plants. We experimentally tested the effect of number of conspecifics on reproduction and pollinator visitation in Eschscholzia californica Cham., an invasive plant in Chile. We then built patches with two, eight and 16 flowering individuals of E. californica (11 replicates per treatment) in an area characterised by dominance of the study species. We found that E. californica exhibits a component Allee effect, as the number of individuals of this species has a positive effect on individual seed set. However, individual fruit production was not affected by the number of plants examined. Pollinator visitation rate was also independent of the number of plants, so this factor would not explain the Allee effect. This rate was positively correlated with the total number of flowers in the patches. We also found that the number of plants did not affect the seed mass or proportion of germinated seeds in the patches. Higher pollen availability in patches with 16 plants and pollination by wind could explain the Allee effect. The component Allee effect identified could lead to a weak demographic Allee effect that might reduce the rate of spread of E. californica. Knowledge of this would be useful for management of this invasive plant in Chile. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  1. Climatic Niche Conservatism and Biogeographical Non-Equilibrium in Eschscholzia californica (Papaveraceae), an Invasive Plant in the Chilean Mediterranean Region

    PubMed Central

    Peña-Gómez, Francisco T.; Guerrero, Pablo C.; Bizama, Gustavo; Duarte, Milén; Bustamante, Ramiro O.

    2014-01-01

    Species climate requirements are useful for predicting their geographic distribution. It is often assumed that the niche requirements for invasive plants are conserved during invasion, especially when the invaded regions share similar climate conditions. California and central Chile have a remarkable degree of convergence in their vegetation structure, and a similar Mediterranean climate. Such similarities make these geographic areas an interesting natural experiment for testing climatic niche dynamics and the equilibrium of invasive species in a new environment. We tested to see if the climatic niche of Eschscholzia californica is conserved in the invaded range (central Chile), and we assessed whether the invasion process has reached a biogeographical equilibrium, i.e., occupy all the suitable geographic locations that have suitable conditions under native niche requirements. We compared the climatic niche in the native and invaded ranges as well as the projected potential geographic distribution in the invaded range. In order to compare climatic niches, we conducted a Principal Component Analysis (PCA) and Species Distribution Models (SDMs), to estimate E. californica's potential geographic distribution. We also used SDMs to predict altitudinal distribution limits in central Chile. Our results indicated that the climatic niche occupied by E. californica in the invaded range is firmly conserved, occupying a subset of the native climatic niche but leaving a substantial fraction of it unfilled. Comparisons of projected SDMs for central Chile indicate a similarity, yet the projection from native range predicted a larger geographic distribution in central Chile compared to the prediction of the model constructed for central Chile. The projected niche occupancy profile from California predicted a higher mean elevation than that projected from central Chile. We concluded that the invasion process of E. californica in central Chile is consistent with climatic niche

  2. Disassembly of structurally modified viral nanoparticles: characterization by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Gilbert, Leona; Michel, Patrik; White, Daniel; Vuento, Matti; Oker-Blom, Christian

    2005-12-01

    Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS, fluorescent subunits with diffusion times of 0.2 ms were observed. Additional treatment with DTT caused a drop in the diffusion time to 0.1 ms. Changes in the amplitude of the autocorrelation function suggested a 3-fold increase in fluorescent particle number when viral particles were treated with SDS, and a further 1.5-fold increase with additional treatment with DTT. Thus, the data showed that an average of 4.5 molecules of gp64-GFP was incorporated in the membrane of the modified baculovirus. Further, this suggests that each fluorescent gp64 trimer carries on average 1.5 fluorescent units. Similar experiments were carried out with two non-enveloped fluorescent virus-like particles (fVLPs) that displayed enhanced green fluorescent protein (EGFP). These, fVLPs of canine and human B19 parvoviruses were treated with 6 M urea and 5 mM SDS, respectively. Correspondingly, the original hydrodynamic radii of 17 and 14 nm were reduced to 9 and 5 nm after treatment. Here, the change in the amplitude of the autocorrelation curve suggested a 10-fold increase in particle number when viral particles of CPV were treated with 6 M urea at 50 degrees C for 10 min. For EGFP-B19, there was a decrease in the amplitude, accompanied by a 9-fold increase in the number of fluorescent units with SDS treatment

  3. Botrytis californica, a new cryptic species in the B. cinerea species complex causing gray mold in blueberries and table grapes.

    PubMed

    Saito, S; Margosan, D; Michailides, T J; Xiao, C L

    2016-01-01

    The Botrytis cinerea species complex comprises two cryptic species, originally referred to Group I and Group II based on Bc-hch gene RFLP haplotyping. Group I was described as a new cryptic species B. pseudocinerea During a survey of Botrytis spp. causing gray mold in blueberries and table grapes in the Central Valley of California, six isolates, three from blueberries and three from table grapes, were placed in Group I but had a distinct morphological character with conidiophores significantly longer than those of B. cinerea and B. pseudocinerea We compared these with B. cinerea and B. pseudocinerea by examining morphological and physiological characters, sensitivity to fenhexamid and phylogenetic analysis inferred from sequences of three nuclear genes. Phylogenetic analysis with the three partial gene sequences encoding glyceraldehyde-3-phosate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60) and DNA-dependent RNA polymerase subunit II (RPB2) supported the proposal of a new Botrytis species, B. californica, which is closely related genetically to B. cinerea, B. pseudocinerea and B. sinoviticola, all known as causal agents of gray mold of grapes. Botrytis californica caused decay on blueberry and table grape fruit inoculated with the fungus. This study suggests that B. californica is a cryptic species sympatric with B. cinerea on blueberries and table grapes in California.

  4. Microbiomes of Muricea californica and M. fruticosa: Comparative Analyses of Two Co-occurring Eastern Pacific Octocorals

    PubMed Central

    Holm, Johanna B.; Heidelberg, Karla B.

    2016-01-01

    Octocorals are sources of novel but understudied microbial diversity. Conversely, scleractinian or reef-building coral microbiomes have been heavily examined in light of the threats of climate change. Muricea californica and Muricea fruticosa are two co-occurring species of gorgonian octocoral abundantly found in the kelp forests of southern California, and thus provide an excellent basis to determine if octocoral microbiomes are host specific. Using Illumina MiSeq amplicon sequencing and replicate samples, we evaluated the microbiomes collected from multiple colonies of both species of Muricea to measure both inter- and intra-colony microbiome variabilities. In addition, microbiomes from overlying sea water and nearby zoanthids (another benthic invertebrate) were also included in the analysis to evaluate whether bacterial taxa specifically associate with octocorals. This is also the first report of microbiomes from these species of Muricea. We show that microbiomes isolated from each sample type are distinct, and specifically, that octocoral species type had the greatest effect on predicting the composition of the Muricea microbiome. Bacterial taxa contributing to compositional differences include distinct strains of Mycoplasma associated with either M. californica or M. fruticosa, an abundance of Spirochaetes observed on M. californica, and a greater diversity of γ-Proteobacteria associated with M. fruticosa. Many of the bacterial taxa contributing to these differences are known for their presence in photosymbiont-containing invertebrate microbiomes. PMID:27445997

  5. Microbiomes of Muricea californica and M. fruticosa: Comparative Analyses of Two Co-occurring Eastern Pacific Octocorals.

    PubMed

    Holm, Johanna B; Heidelberg, Karla B

    2016-01-01

    Octocorals are sources of novel but understudied microbial diversity. Conversely, scleractinian or reef-building coral microbiomes have been heavily examined in light of the threats of climate change. Muricea californica and Muricea fruticosa are two co-occurring species of gorgonian octocoral abundantly found in the kelp forests of southern California, and thus provide an excellent basis to determine if octocoral microbiomes are host specific. Using Illumina MiSeq amplicon sequencing and replicate samples, we evaluated the microbiomes collected from multiple colonies of both species of Muricea to measure both inter- and intra-colony microbiome variabilities. In addition, microbiomes from overlying sea water and nearby zoanthids (another benthic invertebrate) were also included in the analysis to evaluate whether bacterial taxa specifically associate with octocorals. This is also the first report of microbiomes from these species of Muricea. We show that microbiomes isolated from each sample type are distinct, and specifically, that octocoral species type had the greatest effect on predicting the composition of the Muricea microbiome. Bacterial taxa contributing to compositional differences include distinct strains of Mycoplasma associated with either M. californica or M. fruticosa, an abundance of Spirochaetes observed on M. californica, and a greater diversity of γ-Proteobacteria associated with M. fruticosa. Many of the bacterial taxa contributing to these differences are known for their presence in photosymbiont-containing invertebrate microbiomes.

  6. Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica.

    PubMed

    Johnson, Paul M; Kicklighter, Cynthia E; Schmidt, Manfred; Kamio, Michiya; Yang, Hsiuchin; Elkin, Dimitry; Michel, William C; Tai, Phang C; Derby, Charles D

    2006-01-01

    Sea hares protect themselves from predatory attacks with several modes of chemical defenses. One of these is inking, which is an active release of a protective fluid upon predatory attack. In many sea hares including Aplysia californica and A. dactylomela, this fluid is a mixture of two secretions from two separate glands, usually co-released: ink, a purple fluid from the ink gland; and opaline, a white viscous secretion from the opaline gland. These two secretions are mixed in the mantle cavity and directed toward the attacking predator. Some of the chemicals in these secretions and their mechanism of action have been identified. In our study, we used western blots, immunocytochemistry, amino acid analysis, and bioassays to examine the distribution of these components: (1) an L-amino acid oxidase called escapin for A. californica and dactylomelin-P for A. dactylomela, which has antimicrobial activity but we believe its main function is in defending sea hares against predators that evoke its release; and (2) escapin's major amino acid substrates--L-lysine and L-arginine. Escapin is exclusively produced in the ink gland and is not present in any other tissues or secretions. Furthermore, escapin is only sequestered in the amber vesicles of the ink glandand not in the red-purple vesicles, which contain algal-derived chromophores that give ink its distinctive purple color. The concentration of escapin and dactylomelin-P in ink, both in the gland and after its release, is as high as 2 mg ml(-1), or 30 micromol ml(-1), which is well above its antimicrobial threshold. Lysine and arginine (and other amino acids) are packaged into vesicles in the ink and opaline glands, but arginine is present in ink and opaline at <1 mmol l(-1) and lysine is present in ink at <1 mmol l(-1) but in opaline at 65 mmol l(-1). Our previous results showed that both lysine and arginine mediate escapin's bacteriostatic effects, but only lysine mediates its bactericidal effects. Given that escapin

  7. Characterization of the rapid transcriptional response to long-term sensitization training in Aplysia californica

    PubMed Central

    Herdegen, Samantha; Holmes, Geraldine; Cyriac, Ashly; Calin-Jageman, Irina E.; Calin-Jageman, Robert J.

    2014-01-01

    We used a custom-designed microarray and quantitative PCR to characterize the rapid transcriptional response to long-term sensitization training in the marine mollusk Aplysia californica. Aplysia were exposed to repeated noxious shocks to one side of the body, a procedure known to induce a longlasting, transcription-dependent increase in reflex responsiveness that is restricted to the side of training. One hour after training, pleural ganglia from the trained and untrained sides of the body were harvested; these ganglia contain the sensory nociceptors which help mediate the expression of longterm sensitization memory. Microarray analysis from 8 biological replicates suggests that long-term sensitization training rapidly regulates at least 81 transcripts. We used qPCR to test a subset of these transcripts and found that 83% were confirmed in the same samples, and 86% of these were again confirmed in an independent sample. Thus, our new microarray design shows strong convergent and predictive validity for analyzing the transcriptional correlates of memory in Aplysia. Fully validated transcripts include some previously identified as regulated in this paradigm (ApC/EBP and ApEgr) but also include novel findings. Specifically, we show that long-term sensitization training rapidly upregulates the expression of transcripts which may encode Aplysia homologs of a C/EBPγ transcription factor, a glycine transporter (GlyT2), and a vacuolar-protein-sorting-associated protein (VPS36). PMID:25117657

  8. Transcriptional Changes following Long-Term Sensitization Training and In Vivo Serotonin Exposure in Aplysia californica

    PubMed Central

    Bonnick, Kristine; Bayas, Karla; Belchenko, Dmitry; Cyriac, Ashly; Dove, Michael; Lass, Jamie; McBride, Benora; Calin-Jageman, Irina E.; Calin-Jageman, Robert J.

    2012-01-01

    We used Aplysia californica to compare the transcriptional changes evoked by long-term sensitization training and by a treatment meant to mimic this training, in vivo exposure to serotonin. We focused on 5 candidate plasticity genes which are rapidly up-regulated in the Aplysia genus by in vivo serotonin treatment, but which have not yet been tested for regulation during sensitization: CREB1, matrilin, antistasin, eIF3e, and BAT1 homolog. CREB1 was rapidly up-regulated by both treatments, but the regulation following training was transient, falling back to control levels 24 hours after training. This suggests some caution in interpreting the proposed role of CREB1 in consolidating long-term sensitization memory. Both matrilin and eIF3e were up-regulated by in vivo serotonin but not by long-term sensitization training. This suggests that in vivo serotonin may produce generalized transcriptional effects that are not specific to long-term sensitization learning. Finally, neither treatment produced regulation of antistasin or BAT1 homolog, transcripts regulated by in vivo serotonin in the closely related Aplysia kurodai. This suggests either that these transcripts are not regulated by experience, or that transcriptional mechanisms of memory may vary within the Aplysia genus. PMID:23056638

  9. Transcriptional analysis of a whole-body form of long-term habituation in Aplysia californica.

    PubMed

    Holmes, Geraldine; Herdegen, Samantha; Schuon, Jonathan; Cyriac, Ashly; Lass, Jamie; Conte, Catherine; Calin-Jageman, Irina E; Calin-Jageman, Robert J

    2014-01-01

    Habituation is the simplest form of learning, but we know little about the transcriptional mechanisms that encode long-term habituation memory. A key obstacle is that habituation is relatively stimulus-specific and is thus encoded in small sets of neurons, providing poor signal/noise ratios for transcriptional analysis. To overcome this obstacle, we have developed a protocol for producing whole-body long-term habituation of the siphon-withdrawal reflex (SWR) of Aplysia californica. Specifically, we constructed a computer-controlled brushing apparatus to apply low-intensity tactile stimulation over the entire dorsal surface of Aplysia at regular intervals. We found that 3 d of training (10 rounds of stimulation/day; each round = 15 min brushing at a 10-sec ISI; 15-min rest between rounds) produces habituation with several characteristics favorable for mechanistic investigation. First, habituation is widespread, with SWR durations reduced whether the reflex is evoked by tactile stimulation to the head, tail, or the siphon. Second, long-term habituation is sensitive to the pattern of training, occurring only when brushing sessions are spaced out over 3 d rather than massed into a single session. Using a custom-designed microarray and quantitative PCR, we show that long-term habituation produces long-term up-regulation of an apparent Aplysia homolog of cornichon, a protein important for glutamate receptor trafficking. Our training paradigm provides a promising starting point for characterizing the transcriptional mechanisms of long-term habituation memory.

  10. Transcriptional changes following long-term sensitization training and in vivo serotonin exposure in Aplysia californica.

    PubMed

    Bonnick, Kristine; Bayas, Karla; Belchenko, Dmitry; Cyriac, Ashly; Dove, Michael; Lass, Jamie; McBride, Benora; Calin-Jageman, Irina E; Calin-Jageman, Robert J

    2012-01-01

    We used Aplysia californica to compare the transcriptional changes evoked by long-term sensitization training and by a treatment meant to mimic this training, in vivo exposure to serotonin. We focused on 5 candidate plasticity genes which are rapidly up-regulated in the Aplysia genus by in vivo serotonin treatment, but which have not yet been tested for regulation during sensitization: CREB1, matrilin, antistasin, eIF3e, and BAT1 homolog. CREB1 was rapidly up-regulated by both treatments, but the regulation following training was transient, falling back to control levels 24 hours after training. This suggests some caution in interpreting the proposed role of CREB1 in consolidating long-term sensitization memory. Both matrilin and eIF3e were up-regulated by in vivo serotonin but not by long-term sensitization training. This suggests that in vivo serotonin may produce generalized transcriptional effects that are not specific to long-term sensitization learning. Finally, neither treatment produced regulation of antistasin or BAT1 homolog, transcripts regulated by in vivo serotonin in the closely related Aplysia kurodai. This suggests either that these transcripts are not regulated by experience, or that transcriptional mechanisms of memory may vary within the Aplysia genus.

  11. Neurogenesis in Aplysia californica resembles nervous system formation in vertebrates. [Sponges

    SciTech Connect

    Jacob, M.H.

    1984-05-01

    The pattern of neurogenesis of the central nervous system of Aplysia californica was investigated by (/sup 3/H)thymidine autoradiography. Large numbers of animals at a series of early developmental stages were labeled with (/sup 3/H)thymidine for 24 or 48 hr and were subsequently sampled at specific intervals throughout the life cycle. I found that proliferative zones, consisting of columnar and placodal ectodermal cells, are established in regions of the body wall adjacent to underlying mesodermal cells. Mitosis in the proliferative zones generates a population of cells which leave the surface and migrate inward to join the nearby forming ganglia. Tracing specific (/sup 3/H)thymidine-labeled cells from the body wall to a particular ganglion and within the ganglion over time suggests that the final genomic replication of the neuronal precursors occurs before the cells join the ganglion while glial cell precursors and differentiating glial cells continue to divide within the ganglion for some time. Ultrastructural examination of the morphological features of the few mitosing cells observed within the Aplysia central nervous system supports this interpretation. The pattern of neurogenesis in the Aplysia central nervous system resembles the proliferation of cells in the neural tube and the migration of neural crest and ectodermal placode cells in the vertebrate nervous system but differs from the pattern described for other invertebrates.

  12. The transcriptome of the early life history stages of the California Sea Hare Aplysia californica.

    PubMed

    Fiedler, T J; Hudder, A; McKay, S J; Shivkumar, S; Capo, T R; Schmale, M C; Walsh, P J

    2010-06-01

    Aplysia californica is a marine opisthobranch mollusc used as a model organism in neurobiology for cellular analyses of learning and behavior because it possesses a comparatively small number of neurons of large size. The mollusca comprise the second largest animal phylum, yet detailed genetic and genomic information is only recently beginning to accrue. Thus developmental and comparative evolutionary biology as well as biomedical research would benefit from additional information on DNA sequences of Aplysia. Therefore, we have constructed a series of unidirectional cDNA libraries from different life stages of Aplysia. These include whole organisms from the egg, veliger, metamorphic, and juvenile stages as well as adult neural tissue for reference. Individual clones were randomly picked, and high-throughput, single pass sequence analysis was performed to generate 7971 sequences. Of these, there were 5507 quality-filtered ESTs that clustered into 1988 unigenes, which are annotated and deposited into GenBank. A significant number (497) of ESTs did not match existing Aplysia ESTs and are thus potentially novel sequences for Aplysia. GO and KEGG analyses of these novel sequences indicated that a large number were involved in protein binding and translation, consistent with the predominant biosynthetic role in development and the presence of stage-specific protein isoforms.

  13. Carbonic Anhydrase is Required for Statoconia Homeostasis in Organ Cultures of Statocysts from Aplysia californica

    NASA Technical Reports Server (NTRS)

    Pedrozo, H. A.; Schwartz, Z.; Nakaya, H.; Harrison, J. L.; Dean, D. D.; Wiederhold, M. L.; Boyan, B. D.

    1995-01-01

    A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (LI5) medium supplemented with salts and Aplysia haemolymph for four days at 17 C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 x 10(exp -4) M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially, via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.

  14. Characterization of the rapid transcriptional response to long-term sensitization training in Aplysia californica.

    PubMed

    Herdegen, Samantha; Holmes, Geraldine; Cyriac, Ashly; Calin-Jageman, Irina E; Calin-Jageman, Robert J

    2014-12-01

    We used a custom-designed microarray and quantitative PCR to characterize the rapid transcriptional response to long-term sensitization training in the marine mollusk Aplysia californica. Aplysia were exposed to repeated noxious shocks to one side of the body, a procedure known to induce a long-lasting, transcription-dependent increase in reflex responsiveness that is restricted to the side of training. One hour after training, pleural ganglia from the trained and untrained sides of the body were harvested; these ganglia contain the sensory nociceptors which help mediate the expression of long-term sensitization memory. Microarray analysis from 8 biological replicates suggests that long-term sensitization training rapidly regulates at least 81 transcripts. We used qPCR to test a subset of these transcripts and found that 83% were confirmed in the same samples, and 86% of these were again confirmed in an independent sample. Thus, our new microarray design shows strong convergent and predictive validity for analyzing the transcriptional correlates of memory in Aplysia. Fully validated transcripts include some previously identified as regulated in this paradigm (ApC/EBP and ApEgr) but also include novel findings. Specifically, we show that long-term sensitization training rapidly up-regulates the expression of transcripts which may encode Aplysia homologs of a C/EBPγ transcription factor, a glycine transporter (GlyT2), and a vacuolar-protein-sorting-associated protein (VPS36). Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Molecular recognition of thiaclopride by Aplysia californica AChBP: new insights from a computational investigation

    NASA Astrophysics Data System (ADS)

    Alamiddine, Zakaria; Selvam, Balaji; Cerón-Carrasco, José P.; Mathé-Allainmat, Monique; Lebreton, Jacques; Thany, Steeve H.; Laurent, Adèle D.; Graton, Jérôme; Le Questel, Jean-Yves

    2015-12-01

    The binding of thiaclopride (THI), a neonicotinoid insecticide, with Aplysia californica acetylcholine binding protein ( Ac-AChBP), the surrogate of the extracellular domain of insects nicotinic acetylcholine receptors, has been studied with a QM/QM' hybrid methodology using the ONIOM approach (M06-2X/6-311G(d):PM6). The contributions of Ac-AChBP key residues for THI binding are accurately quantified from a structural and energetic point of view. The importance of water mediated hydrogen-bond (H-bond) interactions involving two water molecules and Tyr55 and Ser189 residues in the vicinity of the THI nitrile group, is specially highlighted. A larger stabilization energy is obtained with the THI- Ac-AChBP complex compared to imidacloprid (IMI), the forerunner of neonicotinoid insecticides. Pairwise interaction energy calculations rationalize this result with, in particular, a significantly more important contribution of the pivotal aromatic residues Trp147 and Tyr188 with THI through CH···π/CH···O and π-π stacking interactions, respectively. These trends are confirmed through a complementary non-covalent interaction (NCI) analysis of selected THI- Ac-AChBP amino acid pairs.

  16. Photosynthetic, hydraulic and biomechanical responses of Juglans californica shoots to wildfire.

    PubMed

    Utsumi, Yasuhiro; Bobich, Edward G; Ewers, Frank W

    2010-10-01

    Leaf gas exchange and stem xylem hydraulic and mechanical properties were studied for unburned adults and resprouting burned Juglans californica (southern California black walnut) trees 1 year after a fire to explore possible trade-offs between mechanical and hydraulic properties of plants. The CO(2) uptake rates and stomatal conductance were 2-3 times greater for resprouting trees than for unburned adults. Both predawn and midday water potentials were more negative for unburned adult trees, indicating that the stems were experiencing greater water stress than the stems of resprouting trees. In addition, the xylem specific conductivity was similar in the two growth forms, even though the stems of resprouting trees were less vulnerable to water-stress-induced embolism than similar diameter, but older, stems of adult trees. The reduced vulnerability may have been due to less cavitation fatigue in stems of resprouts. The modulus of elasticity, modulus of rupture and xylem density were all greater for resprouts, indicating that resprouts have greater mechanical strength than do adult trees. The data suggest that there is no trade-off between stem mechanical strength and shoot hydraulic and photosynthetic efficiency in resprouts, which may have implications for the success of this species in the fire-prone plant communities of southern California.

  17. Proteomic characterization of the abdominal ganglion of Aplysia californica-a protein resource for neuroscience.

    PubMed

    Birner-Gruenberger, Ruth; Darnhofer, Barbara; Chen, Wei-Qiang; Monje, Francisco J; Lubec, Gert

    2012-08-01

    Aplysia californica (AC) is a widely used model for testing learning and memory. Although ESTs have been generated, proteomics studies on AC proteins are limited. Studies at the protein level, however, are mandatory, not only due to the fact that studies at the nucleic acid level are not allowing conclusions about PTMs. A gel-based proteomics method was therefore applied to carry out protein profiling in abdominal ganglia from AC. Abdominal ganglia were extirpated, proteins extracted and run on 2DE with subsequent in-gel digestion with trypsin, chymotrypsin, and partially by subtilisin. Peptides were identified using a nano-LC-ESI-LTQ-FT-mass spectrometer. MS/MS data were analyzed by searching the NCBI nonredundant public AC EST database and the NCBI nonredundant public AC protein database. A total of 477 different proteins represented by 363 protein spots were detected and were assigned to different protein pathways as for instance signaling (receptors, protein kinases, and phosphatases), metabolism, protein synthesis, handling and degradation, cytoskeleton and structural, oxido-redox, heat shock and chaperone, hypothetical, predicted and unnamed proteins. The generation of a protein map of soluble proteins shows the existence of so far hypothetical and predicted proteins and is allowing and challenging further work at the protein level, in particular in the field of neuroscience.

  18. Structure and dynamics of the fibronectin-III domains of Aplysia californica cell adhesion molecules.

    PubMed

    Kelly, Catherine M; Muzard, Julien; Brooks, Bernard R; Lee, Gil U; Buchete, Nicolae-Viorel

    2015-04-21

    Due to their homophilic and heterophilic binding properties, cell adhesion molecules (CAMs) such as integrin, cadherin and the immunoglobulin superfamily CAMs are of primary importance in cell-cell and cell-substrate interactions, signalling pathways and other crucial biological processes. We study the molecular structures and conformational dynamics of the two fibronectin type III (Fn-III) extracellular domains of the Aplysia californica CAM (apCAM) protein, by constructing and probing an atomically-detailed structural model based on apCAM's homology with other CAMs. The stability and dynamic properties of the Fn-III domains, individually and in tandem, are probed and analysed using all-atom explicit-solvent molecular dynamics (MD) simulations and normal mode analysis of their corresponding elastic network models. The refined structural model of the Fn-III tandem of apCAM reveals a specific pattern of amino acid interactions that controls the stability of the β-sheet rich structure and could affect apCAM's response to physical or chemical changes of its environment. It also exposes the important role of several specific charged residues in modulating the structural properties of the linker segment connecting the two Fn-III domains, as well as of the inter-domain interface.

  19. Efficient expression of acetylcholine-binding protein from Aplysia californica in Bac-to-Bac system.

    PubMed

    Lin, Bo; Meng, Hailing; Bing, Hui; Zhangsun, Dongting; Luo, Sulan

    2014-01-01

    The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 10(6) cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

  20. Gonadotropin-releasing hormone in protostomes: insights from functional studies on Aplysia californica.

    PubMed

    Sun, Biao; Kavanaugh, Scott I; Tsai, Pei-San

    2012-05-01

    Several protostomian molecules that structurally resemble chordate gonadotropin-releasing hormone (GnRH) have been identified through cloning, biochemical purification or data mining. These molecules share considerable sequence and structural similarities with chordate GnRH, leading to the current belief that protostomian and chordate forms of GnRH share a common ancestor. However, the physiological significance of these protostomian GnRH-like molecules remains poorly understood. This knowledge gap hampers our understanding of how GnRH has evolved functionally over time. This review provides a summary of our recent functional characterization of a GnRH-like molecule (ap-GnRH) in a gastropod mollusk, Aplysia californica, and presents preliminary proof for a cognate ap-GnRH receptor (ap-GnRHR). Our data reveal that ap-GnRH is a general neural regulator capable of exerting diverse central and motor effects, but plays little or no role in reproductive activation. This notion is supported by the abundance of a putative ap-GnRHR transcript in the central nervous system and the foot. Comparing these results to the available functional data from a cephalopod mollusk, Octopus vulgaris, we surmise that protostomian GnRH-like molecules are likely to assume a wide range of physiological roles, and reproductive activation is not an evolutionarily conserved role of these molecules. Future functional studies using suitable protostomian models are required to identify functional changes in protostomian GnRH-like molecules that accompany major taxa-level transitions.

  1. Molecular recognition of thiaclopride by Aplysia californica AChBP: new insights from a computational investigation.

    PubMed

    Alamiddine, Zakaria; Selvam, Balaji; Cerón-Carrasco, José P; Mathé-Allainmat, Monique; Lebreton, Jacques; Thany, Steeve H; Laurent, Adèle D; Graton, Jérôme; Le Questel, Jean-Yves

    2015-12-01

    The binding of thiaclopride (THI), a neonicotinoid insecticide, with Aplysia californica acetylcholine binding protein (Ac-AChBP), the surrogate of the extracellular domain of insects nicotinic acetylcholine receptors, has been studied with a QM/QM' hybrid methodology using the ONIOM approach (M06-2X/6-311G(d):PM6). The contributions of Ac-AChBP key residues for THI binding are accurately quantified from a structural and energetic point of view. The importance of water mediated hydrogen-bond (H-bond) interactions involving two water molecules and Tyr55 and Ser189 residues in the vicinity of the THI nitrile group, is specially highlighted. A larger stabilization energy is obtained with the THI-Ac-AChBP complex compared to imidacloprid (IMI), the forerunner of neonicotinoid insecticides. Pairwise interaction energy calculations rationalize this result with, in particular, a significantly more important contribution of the pivotal aromatic residues Trp147 and Tyr188 with THI through CH···π/CH···O and π-π stacking interactions, respectively. These trends are confirmed through a complementary non-covalent interaction (NCI) analysis of selected THI-Ac-AChBP amino acid pairs.

  2. Development of the Statocyst in Aplysia Californica. Part 1; Observations on Statoconial Development

    NASA Technical Reports Server (NTRS)

    Wiederhold, Michael L.; Sharma, Jyotsna S.; Driscoll, Brian P.; Harrison, Jeffrey L.

    1990-01-01

    The gravity receptor organs of gastropod molluscs, such as Aplysia californica, are bilateral paired statocysts, which contain dense statoconia within a fluid-filled cyst. Gravitational forces on the statoconia are sensed through their interaction with ciliated mechanoreceptor cells in the wall of the cyst. Larval Aplysia contain a single statolith within each statocyst; when the animals grow to a critical size, they begin producing multiple statoconia, a process that continues throughout life. The number of statoconia is highly correlated with animal weight but poorly correlated with age, indicating that stone production is related to total metabolism. The single statolith has an amorphous internal structure whereas the multiple statoconia have calcification deposited on concentric layers of membrane or matrix protein. The statolith appears to be produced within the cyst lumen but the multiple statoconia are produced within supporting cells between the receptor cells. Large adult animals have statoconia larger than those in early post-metamorphic animals which have just started producing multiple stones. The maximum statocyst diameter at which the receptor-cell cilia can suspend the statolith in the center of the cyst lumen is 45 micrometers; production of multiple stones begins when the cyst reaches this size. The mechanisms by which statoconia production is initiated and controlled are discussed.

  3. Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

    PubMed Central

    Elliott, J; Blanchard, S G; Wu, W; Miller, J; Strader, C D; Hartig, P; Moore, H P; Racs, J; Raftery, M A

    1980-01-01

    A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 7. PLATE 1 PMID:7387629

  4. Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction

    PubMed Central

    2011-01-01

    Background During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins. Results Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome. Conclusions Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ. PMID:21798097

  5. Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor gamma subunit.

    PubMed Central

    Claudio, T; Ballivet, M; Patrick, J; Heinemann, S

    1983-01-01

    The nucleotide sequence has been determined of a cDNA clone that codes for the 60,000-dalton gamma subunit of Torpedo californica acetylcholine receptor. The length of the cDNA clone is 2,010 base pairs. The 5' and 3' untranslated regions have respective lengths of 31 and 461 base pairs. Data suggest that the putative polyadenylylation consensus sequence A-A-T-A-A-A may not be required for polyadenylylation of the mRNA corresponding to the cDNA clone described in this study. From the DNA sequence data, the amino acid sequence of the gamma subunit was deduced. The subunit is composed of 489 amino acids giving a molecular mass of 56,600 daltons. The deduced amino acid sequence data also indicate the presence of a 17-amino acid extension or signal peptide on this subunit. From these data, structural predictions for the gamma subunit are made such as potential membrane-spanning regions, possible asparagine-linked glycosylation sites, and the assignment of regions of the protein to the extracellular, internal, and cytoplasmic domains of the lipid bilayer. Images PMID:6573658

  6. Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

    SciTech Connect

    Blanton, M.P.; Wang, H.H. )

    1990-02-06

    A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.

  7. Behavioral aging is associated with reduced sensory neuron excitability in Aplysia californica

    PubMed Central

    Kempsell, Andrew T.; Fieber, Lynne A.

    2014-01-01

    Invertebrate models have advantages for understanding the basis of behavioral aging due to their simple nervous systems and short lifespans. The potential usefulness of Aplysia californica in aging research is apparent from its long history of neurobiological research, but it has been underexploited in this model use. Aging of simple reflexes at both single sensory neuron and neural circuit levels was studied to connect behavioral aging to neurophysiological aging. The tail withdrawal reflex (TWR), righting reflex, and biting response were measured throughout sexual maturity in 3 cohorts of hatchery-reared animals of known age. Reflex times increased and reflex amplitudes decreased significantly during aging. Aging in sensory neurons of animals with deficits in measures of the TWR and biting response resulted in significantly reduced excitability in old animals compared to their younger siblings. The threshold for firing increased while the number of action potentials in response to depolarizing current injection decreased during aging in sensory neurons, but not in tail motoneurons. Glutamate receptor-activated responses in sensory neurons also decreased with aging. In old tail motoneurons, the amplitude of evoked EPSPs following tail shock decreased, presumably due to reduced sensory neuron excitability during aging. The results were used to develop stages of aging relevant to both hatchery-reared and wild-caught Aplysia. Aplysia is a viable aging model in which the contributions of differential aging of components of neural circuits may be assessed. PMID:24847260

  8. Development of the Statocyst in Aplysia Californica. Part 1; Observations on Statoconial Development

    NASA Technical Reports Server (NTRS)

    Wiederhold, Michael L.; Sharma, Jyotsna S.; Driscoll, Brian P.; Harrison, Jeffrey L.

    1990-01-01

    The gravity receptor organs of gastropod molluscs, such as Aplysia californica, are bilateral paired statocysts, which contain dense statoconia within a fluid-filled cyst. Gravitational forces on the statoconia are sensed through their interaction with ciliated mechanoreceptor cells in the wall of the cyst. Larval Aplysia contain a single statolith within each statocyst; when the animals grow to a critical size, they begin producing multiple statoconia, a process that continues throughout life. The number of statoconia is highly correlated with animal weight but poorly correlated with age, indicating that stone production is related to total metabolism. The single statolith has an amorphous internal structure whereas the multiple statoconia have calcification deposited on concentric layers of membrane or matrix protein. The statolith appears to be produced within the cyst lumen but the multiple statoconia are produced within supporting cells between the receptor cells. Large adult animals have statoconia larger than those in early post-metamorphic animals which have just started producing multiple stones. The maximum statocyst diameter at which the receptor-cell cilia can suspend the statolith in the center of the cyst lumen is 45 micrometers; production of multiple stones begins when the cyst reaches this size. The mechanisms by which statoconia production is initiated and controlled are discussed.

  9. Transcriptional analysis of a whole-body form of long-term habituation in Aplysia californica

    PubMed Central

    Holmes, Geraldine; Herdegen, Samantha; Schuon, Jonathan; Cyriac, Ashly; Lass, Jamie; Conte, Catherine; Calin-Jageman, Irina E.

    2015-01-01

    Habituation is the simplest form of learning, but we know little about the transcriptional mechanisms that encode long-term habituation memory. A key obstacle is that habituation is relatively stimulus-specific and is thus encoded in small sets of neurons, providing poor signal/noise ratios for transcriptional analysis. To overcome this obstacle, we have developed a protocol for producing whole-body long-term habituation of the siphon-withdrawal reflex (SWR) of Aplysia californica. Specifically, we constructed a computer-controlled brushing apparatus to apply low-intensity tactile stimulation over the entire dorsal surface of Aplysia at regular intervals. We found that 3 d of training (10 rounds of stimulation/day; each round = 15 min brushing at a 10-sec ISI; 15-min rest between rounds) produces habituation with several characteristics favorable for mechanistic investigation. First, habituation is widespread, with SWR durations reduced whether the reflex is evoked by tactile stimulation to the head, tail, or the siphon. Second, long-term habituation is sensitive to the pattern of training, occurring only when brushing sessions are spaced out over 3 d rather than massed into a single session. Using a custom-designed microarray and quantitative PCR, we show that long-term habituation produces long-term up-regulation of an apparent Aplysia homolog of cornichon, a protein important for glutamate receptor trafficking. Our training paradigm provides a promising starting point for characterizing the transcriptional mechanisms of long-term habituation memory. PMID:25512573

  10. No evidence of temporal preferences in caching by Western scrub-jays (Aphelocoma californica).

    PubMed

    Thom, James M; Clayton, Nicola S

    2014-03-01

    Humans and other animals often favour immediate gratification over long-term gain. Primates, including humans, appear more willing to wait for rewards than other animals, such as rats or pigeons. Another group displaying impressive patience are the corvids, which possess large brains and show sophisticated cognitive abilities. Here, we assess intertemporal choice in one corvid species, the Western scrub-jay (Aphelocoma californica). These birds cache food for future consumption and respond flexibly to future needs. Cache-theft and cache-degradation are time-dependent processes in scrub-jay ecology that might necessitate sensitivity to delays between caching and retrieval. We adopt a caching paradigm with delays of up to 49 h. Across two experiments we find no evidence of a preference for earlier recovery. We highlight the possibility that, although scrub-jays can discriminate between the present and the future, they may not understand how far into the future an event will occur. Copyright © 2013. Published by Elsevier B.V.

  11. Morphology, innervation, and peripheral sensory cells of the siphon of aplysia californica.

    PubMed

    Carrigan, Ian D; Croll, Roger P; Wyeth, Russell C

    2015-11-01

    The siphon of Aplysia californica has several functions, including involvement in respiration, excretion, and defensive inking. It also provides sensory input for defensive withdrawals that have been studied extensively to examine mechanisms that underlie learning. To better understand the neuronal bases of these functions, we used immunohistochemistry to catalogue peripheral cell types and innervation of the siphon in stage 12 juveniles (chosen to allow observation of tissues in whole-mounts). We found that the siphon nerve splits into three major branches, leading ultimately to a two-part FMRFamide-immunoreactive plexus and an apparently separate tyrosine hydroxylase-immunoreactive plexus. Putative sensory neurons included four distinct types of tubulin-immunoreactive bipolar cells (one likely also tyrosine hydroxylase immunoreactive) that bore ciliated dendrites penetrating the epithelium. A fifth bipolar neuron type (tubulin- and FMRFamide-immunoreactive) occurred deeper in the tissue, associated with part of the FMRFamide-immunoreactive plexus. Our observations emphasize the structural complexity of the peripheral nervous system of the siphon, and the importance of direct tests of the various components to better understand the functioning of the entire organ, including its role in defensive withdrawal responses. © 2015 Wiley Periodicals, Inc.

  12. The transcriptome of the early life history stages of the California Sea Hare Aplysia californica

    PubMed Central

    Fiedler, T. J.; Hudder, A.; McKay, S. J.; Shivkumar, S.; Capo, T. R.; Schmale, M. C.; Walsh, P.J.

    2010-01-01

    Aplysia californica is a marine opisthobranch mollusc used as a model organism in neurobiology for cellular analyses of learning and behavior because it possesses a comparatively small number of neurons of large size. The mollusca comprise the second largest animal phylum, yet detailed genetic and genomic information is only recently beginning to accrue. Thus developmental and comparative evolutionary biology as well as biomedical research would benefit from additional information on DNA sequences of Aplysia. Therefore, we have constructed a series of unidirectional cDNA libraries from different life stages of Aplysia. These include whole organisms from the egg, veliger, metamorphic, and juvenile stages as well as adult neural tissue for reference. Individual clones were randomly picked, and high-throughput, single pass sequence analysis was performed to generate 7971 sequences. Of these, there were 5507 quality-filtered ESTs that clustered into 1988 unigenes, which are annotated and deposited into GenBank. A significant number (497) of ESTs did not match existing Aplysia ESTs and are thus potentially novel sequences for Aplysia. GO and KEGG analyses of these novel sequences indicated that a large number were involved in protein binding and translation, consistent with the predominant biosynthetic role in development and the presence of stage-specific protein isoforms. PMID:20434970

  13. Synaptic vesicles isolated from the electric organ of Torpedo californica and from the central nervous system of Mus musculus contain small ribonucleic acids (sRNAs).

    PubMed

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

    2017-06-01

    Synaptic vesicles (SVs) are presynaptic organelles that load and release small molecule neurotransmitters at chemical synapses. In addition to classic neurotransmitters, we have demonstrated that SVs isolated from the Peripheral Nervous Systems (PNS) of the electric organ of Torpedo californica, a model cholinergic synapse, and SVs isolated from the Central Nervous System (CNS) of Mus musculus (mouse) contain small ribonucleic acids (sRNAs; ≤ 50 nucleotides) (Scientific Reports, 5:1-14(14918) Li et al. (2015) [1]). Our previous publication provided the five most abundant sequences associated with the T. californica SVs, and the ten most abundant sequences associated with the mouse SVs, representing 59% and 39% of the total sRNA reads sequenced, respectively). We provide here a full repository of the SV sRNAs sequenced from T. californica and the mouse deposited in the NCBI as biosamples. Three data studies are included: SVs isolated from the electric organ of T. californica using standard techniques, SVs isolated from the electric organ of T. californica using standard techniques with an additional affinity purification step, and finally, SVs isolated from the CNS of mouse. The three biosamples are available at https://www.ncbi.nlm.nih.gov/biosample/ SRS1523467, SRS1523466, and SRS1523472 respectively.

  14. Comparison of the chemistry and diversity of endophytes isolated from wild-harvested and greenhouse-cultivated yerba mansa (Anemopsis californica)

    PubMed Central

    Bussey, Robert O.; Kaur, Amninder; Todd, Daniel A.; Egan, Joseph M.; El-Elimat, Tamam; Graf, Tyler N.; Raja, Huzefa A.; Oberlies, Nicholas H.; Cech, Nadja B.

    2015-01-01

    With this study, we explored the identity and chemistry of fungal endophytes from the roots of yerba mansa [Anemopsis californica (Nutt.) Hook. & Arn. (Saururaceae)], a botanical traditionally used to treat infection. We compared the diversity of fungal endophytes isolated from a wild-harvested A. californica population, and those from plants cultivated for one year in a greenhouse environment. The wild-harvested population yielded thirteen fungal strains (eleven unique genotypes). Of the extracts prepared from these fungi, four inhibited growth of Staphylococcus aureus by >25% at 20 µg/mL, and three inhibited growth of Pseudomonas aeruginosa by ≥20% at 200 µg/mL. By comparison, A. californica roots after one year of cultivation in the greenhouse produced only two unique genotypes, neither of which displayed significant antimicrobial activity. The fungus Chaetomium cupreum isolated from wild-harvested A. californica yielded a new antimicrobial spirolactone, chaetocuprum (1). An additional fourteen known compounds were identified using LC-MS dereplication of the various fungal endophytes. This study provides new insights into the identity and chemistry of A. californica fungal endophytes, and demonstrates the importance of considering growing conditions when pursuing natural product drug discovery from endophytic fungi. PMID:25642298

  15. Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica.

    PubMed

    Ikezawa, Nobuhiro; Iwasa, Kinuko; Sato, Fumihiko

    2007-02-01

    (S)-stylopine is an important intermediate in the biosynthesis of benzophenanthridine alkaloids, such as sanguinarine. Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges. Although the methylenedioxy bridge-forming P450 (CYP719) involved in berberine biosynthesis has been cloned from Coptis japonica[Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, Inouye K & Sato F (2003) J Biol Chem278, 38557-38565], no information is available regarding the genes for methylenedioxy bridge-forming enzymes in stylopine biosynthesis. Two cytochrome P450 cDNAs involved in stylopine biosynthesis were isolated using degenerate primers designed for C. japonica CYP719 from cultured Eschscholzia californica cells. Heterologous expression in Saccharomyces cerevisiae showed that both CYP719A2 and CYP719A3 had stylopine synthase activity to catalyze methylenedioxy bridge-formation from cheilanthifoline to stylopine, but not cheilanthifoline synthase activity to convert scoulerine to cheilanthifoline. Functional differences and expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in stylopine biosynthesis. Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)-cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumbamine. An expression analysis in E. californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and S-adenosyl-l-methionine : 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase), whereas the specific expression of CYP719A3 in root was notable. Treatment of E. californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes. The physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis are

  16. Plant-determined variation in cardenolide content and thin-layer chromatography profiles of monarch butterflies,Danaus plexippus reared on milkweed plants in California : 3. Asclepias californica.

    PubMed

    Brower, L P; Seiber, J N; Nelson, C J; Lynch, S P; Hoggard, M P; Cohen, J A

    1984-12-01

    Variation in gross cardenolide concentration of the mature leaves of 85Asclepias californica plants collected in four different areas of California is a positively skewed distribution ranging from 9 to 199 μg of cardenolide per 0.1 g dry weight with a mean of 66 μg/0.1 g. Butterflies reared individually on these plants in their native habitats contained a normal distribution of cardenolide ranging from 59 to 410 μg of cardenolide per 0.1 g dry weight with a mean of 234 μg. Cardenolide uptake by the butterflies was a logarithmic function of plant concentration. Total cardenolide per butterfly ranged from 143 to 823 μg with a mean of 441 μg and also was normally distributed. Populational variation of plant cardenolide concentrations occurs within subspecies, but the northern subspeciesA. c. greenei does not differ significantly from the southernA. c. californica. Generally higher concentrations occur in butterflies from northern populations and in females. No evidence was adduced that cardenolides in the plants adversely affected the butterflies. Low cardenolide concentrations in the leaves and the absence of cardenolides in the latex characterize bothA. californica andA. speciosa, but notA. eriocarpa. Thin-layer chromatography in two solvent systems isolated 24 cardenolide spots in the plants, of which 18 are stored by the butterflies. There was a minor difference in the cardenolide spot patterns due to geographic origin of the plants, but as in our previous studies, none in the sexes of the butterflies. UnlikeA. eriocarpa andA. speciosa, A. californica plants lack cardenolides withRf values greater than digitoxigenin. Overall, the cardenolides of bothA. californica andA. speciosa are more polar than those inA. eriocarpa. A. californica plants contain cardenolides of the calotropagenin series including calotropin, calactin, and uscharidin, and the latter is metabolically transformed by monarch larvae to calactin and calotropin. Cardenolides of this series also

  17. A mechanism of adaptation to hypergravity in the statocyst of Aplysia californica

    NASA Technical Reports Server (NTRS)

    Pedrozo, H. A.; Schwartz, Z.; Luther, M.; Dean, D. D.; Boyan, B. D.; Wiederhold, M. L.

    1996-01-01

    The gravity-sensing organ of Aplysia californica consists of bilaterally paired statocysts containing statoconia, which are granules composed of calcium carbonate crystals in an organic matrix. In early embryonic development, Aplysia contain a single granule called a statolith, and as the animal matures, statoconia production takes place. The objective of this study was to determine the effect of hypergravity on statoconia production and homeostasis and explore a possible physiologic mechanism for regulating this process. Embryonic Aplysia were exposed to normogravity or 3 x g or 5.7 x g and each day samples were analyzed for changes in statocyst, statolith, and body dimensions until they hatched. In addition, early metamorphosed Aplysia (developmental stages 7-10) were exposed to hypergravity (2 x g) for 3 weeks, and statoconia number and statocyst and statoconia volumes were determined. We also determined the effects of hypergravity on statoconia production and homeostasis in statocysts isolated from developmental stage 10 Aplysia. Since prior studies demonstrated that urease was important in the regulation of statocyst pH and statoconia formation, we also evaluated the effect of hypergravity on urease activity. The results show that hypergravity decreased statolith and body diameter in embryonic Aplysia in a magnitude-dependent fashion. In early metamorphosed Aplysia, hypergravity decreased statoconia number and volume. Similarly, there was an inhibition of statoconia production and a decrease in statoconia volume in isolated statocysts exposed to hypergravity in culture. Urease activity in statocysts decreased after exposure to hypergravity and was correlated with the decrease in statoconia production observed. In short, there was a decrease in statoconia production with exposure to hypergravity both in vivo and in vitro and a decrease in urease activity. It is concluded that exposure to hypergravity downregulates urease activity, resulting in a significant

  18. NMR solution structure of attractin, a water-borne protein pheromone from the mollusk Aplysia californica.

    PubMed

    Garimella, Ravindranath; Xu, Yuan; Schein, Catherine H; Rajarathnam, Krishna; Nagle, Gregg T; Painter, Sherry D; Braun, Werner

    2003-08-26

    Water-borne protein pheromones are essential for coordination of reproductive activities in many marine organisms. In this paper, we describe the first structure of a pheromone protein from a marine organism, that of attractin (58 residues) from Aplysia californica. The NMR solution structure was determined from TOCSY, NOESY, and DQF-COSY measurements of recombinant attractin expressed in insect cells. The sequential resonance assignments were done with standard manual procedures. Approximately 90% of the 949 unambiguous NOESY cross-peaks were assigned automatically with simultaneous three-dimensional structure calculation using our NOAH/DIAMOD/FANTOM program suite. The final bundle of energy-refined structures is well-defined, with an average rmsd value to the mean structure of 0.72 +/- 0.12 A for backbone and 1.32 +/- 0.11 A for heavy atoms for amino acids 3-47. Attractin contains two antiparallel helices, made up of residues Ile9-Gln16 and I30-S36. The NMR distance constraints are consistent with the three disulfide bonds determined by mass spectroscopy (C4-C41, C13-C33, and C20-C26), where the first two could be directly determined from NOESY cross-peaks between CH beta protons of the corresponding cysteines. The second helix contains the (L/I)(29)IEECKTS(36) sequence conserved in attractins from five species of Aplysia that could interact with the receptor. The sequence and structure of this region are similar to those of the recognition helix of the Er-11 pheromone of the unicellular ciliate Euplotes raikovi, suggesting a possible common pathway for intercellular communication of these two distinct pheromone families.

  19. Urotensin II in invertebrates: from structure to function in Aplysia californica.

    PubMed

    Romanova, Elena V; Sasaki, Kosei; Alexeeva, Vera; Vilim, Ferdinand S; Jing, Jian; Richmond, Timothy A; Weiss, Klaudiusz R; Sweedler, Jonathan V

    2012-01-01

    Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.

  20. Shading decreases the abundance of the herbivorous California horn snail, Cerithidea californica

    USGS Publications Warehouse

    Lorda, Julio; Lafferty, Kevin D.

    2012-01-01

    Most of the intertidal zone in estuaries of California, USA and Baja California, Mexico is covered with vascular vegetation. Shading by these vascular plants influences abiotic and biotic processes that shape benthic community assemblages. We present data on the effects of shading on the California horn snail, Cerithidea californica. This species is important because it is the most common benthic macrofaunal species in these systems and acts as an obligate intermediate host of several species of rematode parasites that infect several other species. Using observational and experimental studies, we found a negative effect of shade on the distribution and abundance of the California horn snail. We hypothesized that shading reduces the abundance of the epipelic diatoms that the snails feeds on, causing snails to leave haded areas. We observed a negative relationship between vascular plant cover, sub-canopy light levels, and snail density in Mugu Lagoon. Then we experimentally manipulated light regimes, by clipping vegetation and adding shade structures, and found higher snail densities at higher light levels. In Goleta Slough, we isolated the effect of shade from vegetation by documenting a negative relationship between the shade created by two bridges and diatom and snail densities. We also found that snails moved the greatest distances over shaded channel banks compared to unshaded channel banks. Further, we documented the effect of water depth and channel bank orientation on shading in this system. An additional effect of shading is the reduction of temperature, providing an alternative explanation for some of our results. These results broaden our knowledge of how variation in the light environment influences the ecology of estuarine ecosystems.

  1. A mechanism of adaptation to hypergravity in the statocyst of Aplysia californica

    NASA Technical Reports Server (NTRS)

    Pedrozo, H. A.; Schwartz, Z.; Luther, M.; Dean, D. D.; Boyan, B. D.; Wiederhold, M. L.

    1996-01-01

    The gravity-sensing organ of Aplysia californica consists of bilaterally paired statocysts containing statoconia, which are granules composed of calcium carbonate crystals in an organic matrix. In early embryonic development, Aplysia contain a single granule called a statolith, and as the animal matures, statoconia production takes place. The objective of this study was to determine the effect of hypergravity on statoconia production and homeostasis and explore a possible physiologic mechanism for regulating this process. Embryonic Aplysia were exposed to normogravity or 3 x g or 5.7 x g and each day samples were analyzed for changes in statocyst, statolith, and body dimensions until they hatched. In addition, early metamorphosed Aplysia (developmental stages 7-10) were exposed to hypergravity (2 x g) for 3 weeks, and statoconia number and statocyst and statoconia volumes were determined. We also determined the effects of hypergravity on statoconia production and homeostasis in statocysts isolated from developmental stage 10 Aplysia. Since prior studies demonstrated that urease was important in the regulation of statocyst pH and statoconia formation, we also evaluated the effect of hypergravity on urease activity. The results show that hypergravity decreased statolith and body diameter in embryonic Aplysia in a magnitude-dependent fashion. In early metamorphosed Aplysia, hypergravity decreased statoconia number and volume. Similarly, there was an inhibition of statoconia production and a decrease in statoconia volume in isolated statocysts exposed to hypergravity in culture. Urease activity in statocysts decreased after exposure to hypergravity and was correlated with the decrease in statoconia production observed. In short, there was a decrease in statoconia production with exposure to hypergravity both in vivo and in vitro and a decrease in urease activity. It is concluded that exposure to hypergravity downregulates urease activity, resulting in a significant

  2. Genetically based latitudinal clines in Artemisia californica drive parallel clines in arthropod communities.

    PubMed

    Pratt, Jessica D; Datu, Andrew; Tran, Thi; Sheng, Daniel C; Mooney, Kailen A

    2017-01-01

    Intraspecific variation in plant traits has been clearly shown to drive the structure of associated arthropod communities at the spatial scale of individual plant populations. Nevertheless, it is largely unknown whether plant trait variation among populations drives landscape-scale variation in arthropod communities, and how the strength of such plant genetic effects compares to, and interacts with, those of environmental variation. We documented the structure of arthropod communities on Artemisia californica for two consecutive years in a common garden of plants sourced from five populations along a 5° latitudinal gradient and grown under precipitation treatments approximating the four-fold difference between the north and south range margins for this species. Previous study of plant traits from this garden documented clinal genetic variation, suggesting local adaptation to this environmental gradient, as well as effects of precipitation manipulation that were consistent among populations (i.e., no genotype-by-environment interaction). Within the common garden, arthropod density, evenness, and diversity increased clinally with population source latitude, and arthropod community composition (i.e., species relative abundance) showed a north-south divide. The 2.6-fold cline of northward increase in arthropod density in the common garden was mirrored by a 6.4-fold increase in arthropod density on wild plants sampled along the species range. In contrast to the strong influence of plant genotype, the precipitation manipulation only influenced arthropod community composition, and plant genetic effects on arthropods operated independently of precipitation regime (no genotype-by-environment interaction). Accordingly, we conclude that the strongest driver of landscape-level variation in arthropod communities in this foundational plant species is not variation in the abiotic environment itself, but rather variation in plant traits underlain by the evolutionary process of

  3. Localization of a molluscan gonadotropin-releasing hormone in Aplysia californica by in situ hybridization and immunocytochemistry.

    PubMed

    Jung, Lisa H; Kavanaugh, Scott I; Sun, Biao; Tsai, Pei-San

    2014-01-01

    Gonadotropin-releasing hormone (GnRH) plays important roles in vertebrate reproduction. Recently, molecules structurally similar to vertebrate GnRH were discovered in mollusks, including a gastropod, Aplysia californica. As an important step toward understanding the function of A. californica GnRH (ap-GnRH), the present study examined the localization of ap-GnRH peptide and transcript in the central and peripheral tissues. Reverse transcription polymerase chain reaction (RT-PCR) revealed wide expression of ap-GnRH in all ganglia (abdominal, buccal, cerebral, and pedal ganglia) of the central nervous system (CNS) and in multiple peripheral organs. However, in situ hybridization (ISH) revealed that cells positive for ap-GnRH are detectable only in the CNS, with the pedal ganglia containing the highest number of ap-GnRH-positive neurons, followed by the cerebral and abdominal ganglia. Most neurons positive for the transcript were simultaneously positive for the peptide, although some discrepancies were observed in cerebral and abdominal ganglia. Overall, our data suggest the de novo synthesis of ap-GnRH is restricted to the CNS, with the pedal ganglia being the primary source of ap-GnRH. Our results support the notion that ap-GnRH is a bona-fide neuropeptide that may assume diverse central functions, including those unrelated to reproduction.

  4. Sequence analysis of the complete genome of Trichoplusia ni single nucleopolyhedrovirus and the identification of a baculoviral photolyase gene

    SciTech Connect

    Willis, Leslie G.; Siepp, Robyn; Stewart, Taryn M.; Erlandson, Martin A.; Theilmann, David A. . E-mail: TheilmannD@agr.gc.ca

    2005-08-01

    The genome of the Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), a group II NPV which infects the cabbage looper (T. ni), has been completely sequenced and analyzed. The TnSNPV DNA genome consists of 134,394 bp and has an overall G + C content of 39%. Gene analysis predicted 144 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. Comparisons with previously sequenced baculoviruses indicate that 119 TnSNPV ORFs were homologues of previously reported viral gene sequences. Ninety-four TnSNPV ORFs returned an Autographa californica multiple NPV (AcMNPV) homologue while 25 ORFs returned poor or no sequence matches with the current databases. A putative photolyase gene was also identified that had highest amino acid identity to the photolyase genes of Chrysodeixis chalcites NPV (ChchNPV) (47%) and Danio rerio (zebrafish) (40%). In addition unlike all other baculoviruses no obvious homologous repeat (hr) sequences were identified. Comparison of the TnSNPV and AcMNPV genomes provides a unique opportunity to examine two baculoviruses that are highly virulent for a common insect host (T. ni) yet belong to diverse baculovirus taxonomic groups and possess distinct biological features. In vitro fusion assays demonstrated that the TnSNPV F protein induces membrane fusion and syncytia formation and were compared to syncytia formed by AcMNPV GP64.

  5. Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

    PubMed Central

    Lu, Hui; McManus, Jeffrey M.; Chiel, Hillel J.

    2013-01-01

    In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool. This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations. To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction

  6. Extracellularly identifying motor neurons for a muscle motor pool in Aplysia californica.

    PubMed

    Lu, Hui; McManus, Jeffrey M; Chiel, Hillel J

    2013-03-25

    In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool. This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations. To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1

  7. Modulation of potassium conductances by an endogenous neuropeptide in neurones of Aplysia californica.

    PubMed

    Brezina, V; Eckert, R; Erxleben, C

    1987-01-01

    1. Macroscopic and single-channel currents were recorded from voltage-clamped neurones in the abdominal and pleural ganglia of Aplysia californica in order to investigate conductance changes elicited by application of the endogenous peptide FMRFamide (Phe-Met-Arg-Phe-NH2) and related neuropeptides to the cell surface. 2. The Ca-dependent K current, IK(Ca), when elicited at a constant voltage by intracellular injection of Ca2+, was insensitive to FMRFamide or its derivative YGG-FMRFamide (Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2). 3. Under steady voltage clamp, certain cells responded to a brief puff of FMRFamide or YGG-FMRFamide with a transient outward current lasting about 1 min. Unclamped cells responded with a corresponding hyperpolarization. These responses reversed at about -75 mV. Ion substitution indicated that the current is carried by K+. 4. FMRFamide and YGG-FMRFamide were equally effective in activating the outward current, whereas FMRF, met-enkephalin and leu-enkephalin were ineffective. 5. At voltages negative to -30 mV and, in the absence of extracellular Ca2+, also at more positive potentials, the FMRFamide-sensitive current showed no voltage dependence beyond that predicted from constant-field considerations. 6. The response to FMRFamide was relatively insensitive to extracellular tetraethylammonium (TEA, KD approximately 75 mM) and 4-aminopyridine (4-AP, KD approximately 6 mM). It was suppressed in Ba-containing solutions, but was unaffected by injection of the Ca chelating agent EGTA. The response was blocked by serotonin and other agents known to elevate intracellular adenosine 3',5'-phosphate (cyclic AMP) levels, and by direct injection of cyclic AMP into the cell. 7. In its pharmacological properties and lack of voltage dependence, the FMRFamide-activated current resembles the 'S' current, IK(S), a K current suppressed by application of serotonin in Aplysia neurones. 8. The similarity between the FMRFamide-sensitive current and the 'S' current was

  8. Modulation of potassium conductances by an endogenous neuropeptide in neurones of Aplysia californica.

    PubMed Central

    Brezina, V; Eckert, R; Erxleben, C

    1987-01-01

    1. Macroscopic and single-channel currents were recorded from voltage-clamped neurones in the abdominal and pleural ganglia of Aplysia californica in order to investigate conductance changes elicited by application of the endogenous peptide FMRFamide (Phe-Met-Arg-Phe-NH2) and related neuropeptides to the cell surface. 2. The Ca-dependent K current, IK(Ca), when elicited at a constant voltage by intracellular injection of Ca2+, was insensitive to FMRFamide or its derivative YGG-FMRFamide (Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2). 3. Under steady voltage clamp, certain cells responded to a brief puff of FMRFamide or YGG-FMRFamide with a transient outward current lasting about 1 min. Unclamped cells responded with a corresponding hyperpolarization. These responses reversed at about -75 mV. Ion substitution indicated that the current is carried by K+. 4. FMRFamide and YGG-FMRFamide were equally effective in activating the outward current, whereas FMRF, met-enkephalin and leu-enkephalin were ineffective. 5. At voltages negative to -30 mV and, in the absence of extracellular Ca2+, also at more positive potentials, the FMRFamide-sensitive current showed no voltage dependence beyond that predicted from constant-field considerations. 6. The response to FMRFamide was relatively insensitive to extracellular tetraethylammonium (TEA, KD approximately 75 mM) and 4-aminopyridine (4-AP, KD approximately 6 mM). It was suppressed in Ba-containing solutions, but was unaffected by injection of the Ca chelating agent EGTA. The response was blocked by serotonin and other agents known to elevate intracellular adenosine 3',5'-phosphate (cyclic AMP) levels, and by direct injection of cyclic AMP into the cell. 7. In its pharmacological properties and lack of voltage dependence, the FMRFamide-activated current resembles the 'S' current, IK(S), a K current suppressed by application of serotonin in Aplysia neurones. 8. The similarity between the FMRFamide-sensitive current and the 'S' current was

  9. Presynaptic target of Ca2+ action on neuropeptide and acetylcholine release in Aplysia californica.

    PubMed

    Ohnuma, K; Whim, M D; Fetter, R D; Kaczmarek, L K; Zucker, R S

    2001-09-15

    1. When buccal neuron B2 of Aplysia californica is co-cultured with sensory neurons (SNs), slow peptidergic synapses are formed. When B2 is co-cultured with neurons B3 or B6, fast cholinergic synapses are formed. 2. Patch pipettes were used to voltage clamp pre- and postsynaptic neurons and to load the caged Ca2+ chelator o-nitrophenyl EGTA (NPE) and the Ca2+ indicator BTC into presynaptic neurons. The relationships between presynaptic [Ca2+]i and postsynaptic responses were compared between peptidergic and cholinergic synapses formed by cell B2. 3. Using variable intensity flashes, Ca2+ stoichiometries of peptide and acetylcholine (ACh) release were approximately 2 and 3, respectively. The difference did not reach statistical significance. 4. ACh quanta summate linearly postsynaptically. We also found a linear dose-response curve for peptide action, indicating a linear relationship between submaximal peptide concentration and response of the SN. 5. The minimum intracellular calcium concentrations ([Ca2+]i) for triggering peptidergic and cholinergic transmission were estimated to be about 5 and 10 microM, respectively. 6. By comparing normal postsynaptic responses to those evoked by photolysis of NPE, we estimate [Ca2+]i at the release trigger site elicited by a single action potential (AP) to be at least 10 microM for peptidergic synapses and probably higher for cholinergic synapses. 7. Cholinergic release is brief (half-width approximately 200 ms), even in response to a prolonged rise in [Ca2+]i, while some peptidergic release appears to persist for as long as [Ca2+]i remains elevated (for up to 10 s). This may reflect differences in sizes of reserve pools, or in replenishment rates of immediately releasable pools of vesicles. 8. Electron microscopy revealed that most synaptic contacts had at least one morphologically docked dense core vesicle that presumably contained peptide; these were often located within conventional active zones. 9. Both cholinergic and

  10. Organochlorine contaminants and maternal offloading in the lecithotrophic Pacific angel shark (Squatina californica) collected from southern California.

    PubMed

    Lyons, Kady; Lowe, Christopher G

    2015-08-15

    Pacific angel sharks (Squatina californica) are a benthic elasmobranch that occupy intermediate trophic level positions in coastal food webs. Angel sharks' life history characteristics make them susceptible to accumulating high amounts of contaminants. Four angel sharks were opportunistically captured in southern California and their liver and uterine contents were analyzed for PCBs, DDTs and other pesticides. High DDT:PCB ratios were found in the sharks indicating direct or indirect foraging near a local EPA Superfund Site. Organic contaminants were measured in ovulated eggs, indicating that females are able to maternally offload contaminants. Despite the potential mismatch between ovarian and uterine fecundity, we estimated females to offload approximately 13±5% of their total body load, which represents the upper limit of this capability. Although low in sample size, the initial findings from this study suggest that habitat use might play an important role in contaminant accumulation in this species.

  11. Suppression of calcium current by an endogenous neuropeptide in neurones of Aplysia californica.

    PubMed Central

    Brezina, V; Eckert, R; Erxleben, C

    1987-01-01

    1. Actions of the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) and its derivative YGG-FMRFamide (Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2) on Ca2+ current were examined in identified, voltage-clamped neurones in the abdominal ganglion of Aplysia californica. 2. 'Puffed' application of either peptide at concentrations of 1-50 microM was followed by a transient partial suppression of pharmacologically isolated inward Ca2+ current elicited by a depolarizing step. At 20 degrees C, suppression was maximal 10-25 s following the brief puff of peptide, and lasted up to 90 s. Bath application of peptide had a steady suppressing effect, showing little if any desensitization. 3. Alternative sources of inward current suppression were ruled out, indicating that application of FMRFamide or YGG-FMRFamide produces a true decrease in Ca2+ current, rather than enhancement of possible contaminating outward (K+, H+ or Cl-) currents. 4. FMRFamide and YGG-FMRFamide were equally effective in suppressing Ca2+ current (apparent dissociation constant, KD* approximately 10 microM). However, only 30-50% of the total Ca2+ current elicited by voltage steps to above +10 mV appeared to be susceptible to suppression by even saturating concentrations of peptide. This, as well as a reduced effect of the peptides on Ca2+ current which was observed at potentials below +10 mV, may perhaps result from the presence of more than one class of Ca2+ channels, only one of which is sensitive to FMRFamide. 5. FMRFamide eliminated a constant fraction of Ca2+ current at all potentials above +10 mV, and had no direct effect on activation or inactivation of the remaining current. This behaviour is consistent with reduction in the number of functional Ca2+ channels by the peptide. 6. Suppression of Ca2+ current produced a concomitant depression of Ca2+-dependent K+ current, which was shown previously to be insensitive to FMRFamide when activated by direct ionophoretic injection of Ca2+ into the cell. 7. The effect of

  12. Suppression of calcium current by an endogenous neuropeptide in neurones of Aplysia californica.

    PubMed

    Brezina, V; Eckert, R; Erxleben, C

    1987-07-01

    1. Actions of the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) and its derivative YGG-FMRFamide (Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2) on Ca2+ current were examined in identified, voltage-clamped neurones in the abdominal ganglion of Aplysia californica. 2. 'Puffed' application of either peptide at concentrations of 1-50 microM was followed by a transient partial suppression of pharmacologically isolated inward Ca2+ current elicited by a depolarizing step. At 20 degrees C, suppression was maximal 10-25 s following the brief puff of peptide, and lasted up to 90 s. Bath application of peptide had a steady suppressing effect, showing little if any desensitization. 3. Alternative sources of inward current suppression were ruled out, indicating that application of FMRFamide or YGG-FMRFamide produces a true decrease in Ca2+ current, rather than enhancement of possible contaminating outward (K+, H+ or Cl-) currents. 4. FMRFamide and YGG-FMRFamide were equally effective in suppressing Ca2+ current (apparent dissociation constant, KD* approximately 10 microM). However, only 30-50% of the total Ca2+ current elicited by voltage steps to above +10 mV appeared to be susceptible to suppression by even saturating concentrations of peptide. This, as well as a reduced effect of the peptides on Ca2+ current which was observed at potentials below +10 mV, may perhaps result from the presence of more than one class of Ca2+ channels, only one of which is sensitive to FMRFamide. 5. FMRFamide eliminated a constant fraction of Ca2+ current at all potentials above +10 mV, and had no direct effect on activation or inactivation of the remaining current. This behaviour is consistent with reduction in the number of functional Ca2+ channels by the peptide. 6. Suppression of Ca2+ current produced a concomitant depression of Ca2+-dependent K+ current, which was shown previously to be insensitive to FMRFamide when activated by direct ionophoretic injection of Ca2+ into the cell. 7. The effect of

  13. Myogenesis in Aplysia californica (Cooper, 1863) (Mollusca, Gastropoda, Opisthobranchia) with special focus on muscular remodeling during metamorphosis.

    PubMed

    Wollesen, Tim; Wanninger, Andreas; Klussmann-Kolb, Annette

    2008-07-01

    To date only few comparative approaches tried to reconstruct the ontogeny of the musculature in invertebrates. This may be due to the difficulties involved in reconstructing three dimensionally arranged muscle systems by means of classical histological techniques combined with light or transmission electron microscopy. Within the scope of the present study we investigated the myogenesis of premetamorphic, metamorphic, and juvenile developmental stages of the anaspidean opisthobranch Aplysia californica using fluorescence F-actin-labeling in conjunction with modern confocal laser scanning microscopy. We categorized muscles with respect to their differentiation and degeneration and found three true larval muscles that differentiate during the embryonic and veliger phase and degenerate during or slightly after metamorphosis. These are the larval retractor, the accessory larval retractor, and the metapodial retractor muscle. While the pedal retractor muscle, some transversal mantle fibers and major portions of the cephalopedal musculature are continued and elaborated during juvenile and adult life, the buccal musculature and the anterior retractor muscle constitute juvenile/adult muscles which differentiate during or after metamorphosis. The metapodial retractor muscle has never been reported for any other gastropod taxon. Our findings indicate that the late veliger larva of A. californica shares some common traits with veligers of other gastropods, such as a larval retractor muscle. However, the postmetamorphic stages exhibit only few congruencies with other gastropod taxa investigated to date, which is probably due to common larval but different adult life styles within gastropods. Accordingly, this study provides further evidence for morphological plasticity in gastropod myogenesis and stresses the importance of ontogenetic approaches to understand adult conditions and life history patterns.

  14. Comparison of acetylcholine receptor-controlled cation flux in membrane vesicles from Torpedo californica and Electrophorus electricus: chemical kinetic measurements in the millisecond region.

    PubMed

    Hess, G P; Pasquale, E B; Walker, J W; McNamee, M G

    1982-02-01

    In earlier studies with the acetylcholine receptor (AcChoR) of Electrophorus electricus the rate and equilibrium constants for a model that relates the ligand binding to ion translocation were determined, and the dependence of these constants on the concentrations of carbamoylcholine and acetylcholine, over a 200- and 5000-fold range, respectively, could be predicted. AcChoR-controlled cation flux has now been measured in Torpedo californica vesicles by using a pulsed-quench-flow technique with a 2-msec time resolution. Torpedo vesicles on a weight basis may contain several hundred times more receptor sites than do E. electricus vesicles. Techniques have been developed to (i) correct for the kinetic heterogeneity of the vesicle population; (ii) use the inactivation of the receptor by its natural ligand to reduce influx rates at high ligand concentrations to a measurable level (this permitted J(A), the influx rate coefficient before the onset of inactivation, to be measured); and (iii) determine the rate coefficients of two processes that lead to successive inactivations (desensitization) of the receptor and occur in different time regions. An extension of a model proposed for the E. electricus receptor accommodates the ion translocation in T. californica vesicles. The features in common are: (i) A rapid initial flux rate [J(A)(max) for T. californica is 310 sec(-1); for E. electricus it is 7.5 sec(-1)]. These differences in flux rates are consistent with a difference in AcChoR density. (ii) A rapid inactivation process [alpha(max) for T. californica is 2 sec(-1); for E. electricus it is 7 sec(-1)]. (iii) A slow AcChoR-controlled flux that continues after the rapid inactivation [J(I)(max) for T. californica is 1.3 sec(-1); for E. electricus it is 0.015 sec(-1)]. The main difference between the flux in the two types of vesicle is the existence of a second, slower, inactivation process in T. californica with a rate coefficient, beta, of 0.12 sec(-1). The second

  15. Comparison of acetylcholine receptor-controlled cation flux in membrane vesicles from Torpedo californica and Electrophorus electricus: Chemical kinetic measurements in the millisecond region

    PubMed Central

    Hess, George P.; Pasquale, Elena B.; Walker, Jeffrey W.; McNamee, Mark G.

    1982-01-01

    In earlier studies with the acetylcholine receptor (AcChoR) of Electrophorus electricus the rate and equilibrium constants for a model that relates the ligand binding to ion translocation were determined, and the dependence of these constants on the concentrations of carbamoylcholine and acetylcholine, over a 200- and 5000-fold range, respectively, could be predicted. AcChoR-controlled cation flux has now been measured in Torpedo californica vesicles by using a pulsed-quench-flow technique with a 2-msec time resolution. Torpedo vesicles on a weight basis may contain several hundred times more receptor sites than do E. electricus vesicles. Techniques have been developed to (i) correct for the kinetic heterogeneity of the vesicle population; (ii) use the inactivation of the receptor by its natural ligand to reduce influx rates at high ligand concentrations to a measurable level (this permitted JA, the influx rate coefficient before the onset of inactivation, to be measured); and (iii) determine the rate coefficients of two processes that lead to successive inactivations (desensitization) of the receptor and occur in different time regions. An extension of a model proposed for the E. electricus receptor accommodates the ion translocation in T. californica vesicles. The features in common are: (i) A rapid initial flux rate [JA(max) for T. californica is 310 sec-1; for E. electricus it is 7.5 sec-1]. These differences in flux rates are consistent with a difference in AcChoR density. (ii) A rapid inactivation process [α(max) for T. californica is 2 sec-1; for E. electricus it is 7 sec-1]. (iii) A slow AcChoR-controlled flux that continues after the rapid inactivation [JI(max) for T. californica is 1.3 sec-1; for E. electricus it is 0.015 sec-1]. The main difference between the flux in the two types of vesicle is the existence of a second, slower, inactivation process in T. californica with a rate coefficient, β, of 0.12 sec-1. The second process leads to undetectable

  16. Draft de novo transcriptome assembly and proteome characterization of the electric lobe of Tetronarce californica: a molecular tool for the study of cholinergic neurotransmission in the electric organ.

    PubMed

    Stavrianakou, Maria; Perez, Ricardo; Wu, Cheng; Sachs, Matthew S; Aramayo, Rodolfo; Harlow, Mark

    2017-08-14

    The electric organ of Tetronarce californica (an electric ray formerly known as Torpedo californica) is a classic preparation for biochemical studies of cholinergic neurotransmission. To broaden the usefulness of this preparation, we have performed a transcriptome assembly of the presynaptic component of the electric organ (the electric lobe). We combined our assembled transcriptome with a previous transcriptome of the postsynaptic electric organ, to define a MetaProteome containing pre- and post-synaptic components of the electric organ. Sequencing yielded 102 million paired-end 100 bp reads. De novo Trinity assembly was performed at Kmer 25 (default) and Kmers 27, 29, and 31. Trinity, generated around 103,000 transcripts, and 78,000 genes per assembly. Assemblies were evaluated based on the number of bases/transcripts assembled, RSEM-EVAL scores and informational content and completeness. We found that different assemblies scored differently according to the evaluation criteria used, and that while each individual assembly contained unique information, much of the assembly information was shared by all assemblies. To generate the presynaptic transcriptome (electric lobe), while capturing all information, assemblies were first clustered and then combined with postsynaptic transcripts (electric organ) downloaded from NCBI. The completness of the resulting clustered predicted MetaProteome was rigorously evaluated by comparing its information against the predicted proteomes from Homo sapiens, Callorhinchus milli, and the Transporter Classification Database (TCDB). In summary, we obtained a MetaProteome containing 92%, 88.5%, and 66% of the expected set of ultra-conserved sequences (i.e., BUSCOs), expected to be found for Eukaryotes, Metazoa, and Vertebrata, respectively. We cross-annotated the conserved set of proteins shared between the T. californica MetaProteome and the proteomes of H. sapiens and C. milli, using the H. sapiens genome as a reference. This

  17. Occurrence and seasonal variation of 19-norcholest-4-en-3-one and 3 beta-monohydroxy sterols in the Californian gorgonian, Muricea californica.

    PubMed

    Popov, S; Carlson, R M; Djerassi, C

    1983-04-01

    The first natural occurrence of 19-norcholestenone is reported, together with 17 sterols and one other delta 4-3-ketone in the extracts of the Californian gorgonian, Muricea californica (Aurivillius). Six additional demethyl sterols and five additional 4-monomethyl sterols which remain unidentified were also detected. Lipid extracts of M. californica from a winter and summer collection were split by various chromatographic methods into free sterol, steryl ester, and steryl conjugate fractions. Sterol compositions (determined by CG and CG-MS) of each fraction, subsequent to hydrolysis, are tabulated and discussed with respect to plausible origins of observed variations. The possible relationship of the Muricea 19-nor-steroidal ketone to other naturally occurring 19-nor-steroids is discussed.

  18. Studies on the metabolism and toxicological detection of the Eschscholtzia californica alkaloids californine and protopine in urine using gas chromatography-mass spectrometry.

    PubMed

    Paul, Liane D; Maurer, Hans H

    2003-06-05

    Eschscholtzia californica preparations are in use as phytopharmaceuticals and as herbal drugs. Studies are described on the metabolism and the toxicological analysis of the Eschscholtzia californica alkaloids californine and protopine in rat urine using gas chromatography-mass spectrometry. The identified metabolites indicated that californine is extensively metabolized by N-demethylation and/or single or double demethylenation with consecutive catechol-O-methylation of one of the hydroxy groups. Protopine, however, only undergoes extensive demethylenation of the 2,3-methylenedioxy group followed by catechol-O-methylation. All phenolic hydroxy metabolites were found to be partly conjugated. The authors' systematic toxicological analysis procedure using full-scan gas chromatography-mass spectrometry after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of the main metabolites of californine and protopine in rat urine after a dose which should correspond to that of drug users. Therefore, use of Eschscholtzia californica preparations should also be detectable in human urine by the authors' systematic toxicological analysis procedure.

  19. Comparison of the interactions of a specific neurotoxin (alpha-bungarotoxin) with the acetylcholine receptor in Torpedo californica and Electrophorus electricus membrane preparations.

    PubMed

    Leprince, P; Noble, R L; Hess, G P

    1981-09-15

    alpha-Bungarotoxin, a snake neurotoxin, binds irreversibly and specifically to the acetylcholine receptor isolated from the electroplax of Electrophorus electricus and Torpedo species and has been an important tool in the study of the receptor-ligand binding mechanism. Two distinct kinetic processes have been observed in studies with membranes from E. electricus. A minimum mechanism for the toxin reaction involves (i) the reversible binding of two toxin molecules to the receptor prior to the irreversible formation of toxin receptor complexes and (ii) a toxin-induced conformational change of the receptor which leads to an increase in the affinity of the receptor binding sites for toxin [Hess, G. P., Bulger, J. E., Fu, J.-j. L., Hindy, E. F., & Silberstein, R. J. (1975) Biochem. Biophys. Res. Commun. 64, 1018-1027]. Only one process has been detected in Torpedo membranes. Here, we determine whether the receptors in Torpedo californica and E. electricus membranes have different properties or whether the measurements and their interpretation were responsible for the different results. Two methods which are frequently used in binding studies to separate free and bound toxin, a CM-52 cellulose minicolumn assay and DE-81 filter disk assay, have been compared. The results obtained indicate that the interaction of toxin with receptor from T. californica is similar to that observed with receptor from E. electricus. The apparent differences which have been reported in the literature are shown to have arisen from the design of the experiments in which T. californica membranes were used.

  20. Botryosphaeriaceae species associated with dieback and canker disease of bay laurel in northern California with the description of Dothiorella californica sp. nov.

    PubMed

    Lawrence, Daniel P; Peduto Hand, Francesca; Gubler, W Douglas; Trouillas, Florent P

    2017-04-01

    Members of the Botryosphaeriaceae are cosmopolitan fungi that may exist as seemingly innocuous endophytes or as destructive pathogens of numerous woody hosts, including fruit and nut crops, urban ornamental trees and shrubs, and forest trees. Surveys of bay laurel in northern California have revealed symptoms of dieback and branch canker of unknown aetiology. The goals of this study were to identify and clarify the species of Botryosphaeriaceae associated with these symptoms and to confirm their pathogenicity. To understand the role of members of the Botryosphaeriaceae in the dieback and canker disease of bay laurel, 23 isolates were isolated from symptomatic wood. Phylogenetic analyses of ITS, translation elongation factor 1-α, and beta-tubulin revealed three species: Botryosphaeria dothidea, Neofusicoccum nonquaesitum, and the newly described and typified species Dothiorella californica sp. nov. When select isolates were inoculated to 2- to 3-year-old branches of Umbellularia californica in a natural forest, both B. dothidea and N. nonquaesitum were pathogenic with N. nonquaesitum producing the largest lesions at 12- and 18-months post inoculation, respectively, while Do. californica did not cause wood lesions significantly greater than the mock-inoculated controls. This study represents the first attempt to identify and test the pathogenicity of Botryosphaeriaceae species associated with dieback and canker disease of bay laurel in a northern California forest.

  1. Multiscale Structure of the Underwater Adhesive of Phragmatopoma Californica: a Nanostructured Latex with a Steep Microporosity Gradient

    PubMed Central

    Stevens, Mark J.; Steren, Rebekah E.; Hlady, Vladimir; Stewart, Russell J.

    2008-01-01

    Phragmatopoma Californica builds a tubular dwelling by gluing bits of sand and seashell together underwater with a proteinaceous adhesive. In the lab, the animals will build with 0.5 mm glass beads. Two spots of glue with a consistent volume of about 100 pL each are deposited on the glass beads before placement on the end of the tube. The animals wriggled the particles for 20-30 s before letting go, which suggested that the adhesive was sufficiently set within 30 s to support the glass beads. The structure of the adhesive joints was examined at the micro- and nanoscopic length scales using laser scanning confocal and atomic force microscopies. At the microscale, the adhesive was a cellular solid with cell diameters ranging from 0.5 to 6.0 μm, distributed to create a steep porosity gradient that ranged from near zero at the outside edges to about 50% at the center of the adhesive joint. At the nanoscale, the adhesive appeared to be an accretion of trillions of deformable nanospheres, reminiscent of a high-solids-content latex adhesive. The implications of the structure for the functionality of the adhesive is discussed. PMID:17394366

  2. Presynaptic modulating effects of GABA on depression, facilitation, and posttetanic potentiation of a cholinergic synapse in Aplysia californica.

    PubMed

    Tremblay, J P; Plourde, G

    1977-12-01

    The effects of gamma-aminobutyric acid (GABA) have been studied on the synaptic depression, frequency facilitation, and posttetanic potentiation (PTP) of a unitary, monosynaptic, and presumably cholinergic excitatory postsynaptic potential (EPSP). This EPSP, produced by minimal stimulation of the right visceropleural connective, was recorded in cell R 15 of Aplysia californica. Perfusion with GABA (10(-4)-10(-3) M) reduces the size of all EPSPs produced by a train of 100 stimuli at 1/s. It also reduced the synaptic depression and PTP, and increases the frequency facilitation seen during the train. GABA does not significantly effect the membrane resistance (mean 102%) but it slightly depolarizes (mean 6 mV) the postsynaptic cell. GABA does not reduce an acetylcholine iontophoretic potential produced on R15. The effects of GABA are reduction when chloride is replaced by acetate but they remain significant. Picrotoxin and bicuculline fail to antagonize GABA. Addition of sodium azide or dinitrophenol does not reduce the action of GABA and even prolongs it. The effects of GABA are attributed to two sites of action: a postsynaptic one, responsible for the small change in potential and partially responsible for the reduction of EPSP size; and a presynaptic one, responsible for a further reduction of EPSP size and the changes of depression, facilitation, and PTP.

  3. Proteomics reveals selective regulation of proteins in response to memory-related serotonin stimulation in Aplysia californica ganglia.

    PubMed

    Monje, Francisco J; Birner-Gruenberger, Ruth; Darnhofer, Barbara; Divisch, Isabella; Pollak, Daniela D; Lubec, Gert

    2012-02-01

    The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large-scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2-D gel electrophoresis approach. Protein spots were quantified and protein-level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin-1 and deleted in azoospermia-associated protein (DAZAP-1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP-1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK-1 and actin-depolymerizing factor (ADF) in pleural ganglia and DAZAP-1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel-based mass spectrometrical method (FT-MS). Taken together, protein-level changes induced by the learning-related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.

  4. Unique ionotropic receptors for D-aspartate are a target for serotonin-induced synaptic plasticity in Aplysia californica.

    PubMed

    Carlson, Stephen L; Fieber, Lynne A

    2012-01-01

    The non-L-glutamate (L-Glu) receptor component of D-aspartate (D-Asp) currents in Aplysia californica buccal S cluster (BSC) neurons was studied with whole cell voltage clamp to differentiate it from receptors activated by other well-known agonists of the Aplysia nervous system and investigate modulatory mechanisms of D-Asp currents associated with synaptic plasticity. Acetylcholine (ACh) and serotonin (5-HT) activated whole cell excitatory currents with similar current voltage relationships to D-Asp. These currents, however, were pharmacologically distinct from D-Asp. ACh currents were blocked by hexamethonium (C6) and tubocurarine (D-TC), while D-Asp currents were unaffected. 5-HT currents were blocked by granisetron and methysergide (MES), while D-Asp currents were unaffected. Conversely, while (2S,3R)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid(PPDA) blocked D-Asp currents, it had no effect on ACh or 5-HT currents. Comparison of the charge area described by currents induced by ACh or 5-HT separately from, or with, D-Asp suggests activation of distinct receptors by all 3 agonists. Charge area comparisons with L-Glu, however, suggested some overlap between L-Glu and D-Asp receptors. Ten minute exposure to 5-HT induced facilitation of D-Asp-evoked responses in BSC neurons. This effect was mimicked by phorbol ester, suggesting that protein kinase C (PKC) was involved.

  5. Stereoselective L-(3H)quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors

    SciTech Connect

    Murray, T.F.; Mpitsos, G.J.; Siebenaller, J.F.; Barker, D.L.

    1985-12-01

    The muscarinic antagonist L-(/sup 3/H)quinuclidinyl benzilate (L-(/sup 3/H)QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-(/sup 3/H)QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-(/sup 3/H)QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-(/sup 3/H)QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-(/sup 3/H)QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-(/sup 3/H)QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-(/sup 3/H)QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-(/sup 3/H)QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution.

  6. Differential evolutionary rates of neuronal transcriptome in Aplysia kurodai and Aplysia californica as a tool for gene mining.

    PubMed

    Choi, Sun-Lim; Lee, Yong-Seok; Rim, Young-Soo; Kim, Tae-Hyung; Moroz, Leonid L; Kandel, Eric R; Bhak, Jong; Kaang, Bong-Kiun

    2010-07-01

    The marine mollusk Aplysia is a fascinating model organism for studying molecular mechanisms underlying learning and memory. However, evolutionary studies about Aplysia have been limited by the lack of its genomic information. Recently, large-scale expressed sequence tag (EST) databases have been acquired by sequencing cDNA libraries from A. californica and A. kurodai. The closeness between the two species allowed us to investigate rapidly evolving genes by comparing their orthologs. Using this method, we found that a subset of signal transduction genes in neurons showed rates of protein evolution higher than those of housekeeping genes. Moreover, we were also able to find several candidate genes that may be involved in learning and memory or synaptic plasticity among genes showing relatively higher K(a)/K(s) ratios. We also investigated the relationship between evolutionary rates and tissue distribution of Aplysia genes. They propose that the estimation of evolutionary rates cannot be a good marker to assess neuronal expression; however, it still can be an efficient way to narrow down the pool of candidate genes involved in neuronal functions for the further studies.

  7. Multipart copolyelectrolyte adhesive of the sandcastle worm, Phragmatopoma californica (Fewkes): catechol oxidase catalyzed curing through peptidyl-DOPA.

    PubMed

    Wang, Ching Shuen; Stewart, Russell J

    2013-05-13

    Tube-building sabellariid polychaetes have major impacts on the geology and ecology of shorelines worldwide. Sandcastle worms, Phragmatopoma californica (Fewkes), live along the western coast of North America. Individual sabellariid worms build tubular shells by gluing together mineral particles with a multipart polyelectrolytic adhesive. Distinct sets of oppositely charged components are packaged and stored in concentrated granules in separate cell types. Homogeneous granules contain sulfated macromolecules as counter-polyanion to polycationic Pc2 and Pc5 proteins, which become major components of the fully cured glue. Heterogeneous granules contain polyphosphoproteins, Pc3A/B, paired with divalent cations and polycationic Pc1 and Pc4 proteins. Both types of granules contain catechol oxidase that catalyzes oxidative cross-linking of L-DOPA. Co-secretion of catechol oxidase guarantees rapid and spatially homogeneous curing with limited mixing of the preassembled adhesive packets. Catechol oxidase remains active long after the glue is fully cured, perhaps providing an active cue for conspecific larval settlement.

  8. Influence of metal exposure history on the bioaccumulation and subcellular distribution of aqueous cadmium in the insect Hydropsyche californica

    USGS Publications Warehouse

    Cain, D.J.; Buchwalter, D.B.; Luoma, S.N.

    2006-01-01

    The influence of metal exposure history on rates of aqueous Cd accumulation, elimination, and subcellular distribution was examined in the aquatic insect Hydropsyche californica. Specimens were obtained from a reference site and a metal-contaminated site and returned to the laboratory where they were continuously exposed to aqueous Cd (518 ng/L, nominal) for 6 d, followed by 9 d of depuration. Rates of Cd accumulation and elimination were similar in insects from the two sites. Efflux rate constants, ke, ranged from 0.20 to 0.24/d (t1/2 ??? 3 d). Immediately following exposure, the cytosol accounted for 40% of the body burden in insects from both sites; however, 89 ?? 2% of the cytosolic Cd was associated with metallothionein-like proteins (MTLP) in insects from the contaminated site, compared to 60 ?? 0% in insects from the reference site. The concentration of Cd bound to non-MTLPs (representing potentially Cd-sensitive proteins) was significantly greater in the insects from the reference site (134 ?? 7 ng/g) than in those from the contaminated site (42 ?? 2 ng/g). At the end of the depuration period, 90% of the accumulated Cd body burden had been eliminated, and Cd concentrations in MTLPs and non-MTLPs were similar between the sites. Results suggested that differences in exposure history had no influence on the bioaccumulation of Cd, but did affect the concentrations of Cd bound to MTLP during Cd exposure in these insects. ?? 2006 SETAC.

  9. A comparative karyological study of the blue-breasted quail (Coturnix chinensis, Phasianidae) and California quail (Callipepla californica, Odontophoridae).

    PubMed

    Shibusawa, M; Nishida-Umehara, C; Tsudzuki, M; Masabanda, J; Griffin, D K; Matsuda, Y

    2004-01-01

    We conducted comparative chromosome painting and chromosome mapping with chicken DNA probes against the blue-breasted quail (Coturnix chinensis, CCH) and California quail (Callipepla californica, CCA), which are classified into the Old World quail and the New World quail, respectively. Each chicken probe of chromosomes 1-9 and Z painted a pair of chromosomes in the blue-breasted quail. In California quail, chicken chromosome 2 probe painted chromosomes 3 and 6, and chicken chromosome 4 probe painted chromosomes 4 and a pair of microchromosomes. Comparison of the cytogenetic maps of the two quail species with those of chicken and Japanese quail revealed that there are several intrachromosomal rearrangements, pericentric and/or paracentric inversions, in chromosomes 1, 2 and 4 between chicken and the Old World quail. In addition, a pericentric inversion was found in chromosome 8 between chicken and the three quail species. Ordering of the Z-linked DNA clones revealed the presence of multiple rearrangements in the Z chromosomes of the three quail species. Comparing these results with the molecular phylogeny of Galliformes species, it was also cytogenetically supported that the New World quail is classified into a different clade from the lineage containing chicken and the Old World quail.

  10. A conformational change in the peripheral anionic site of Torpedo californica acetylcholinesterase induced by a bis-imidazolium oxime.

    PubMed

    Legler, Patricia M; Soojhawon, Iswarduth; Millard, Charles B

    2015-09-01

    As part of ongoing efforts to design improved nerve agent antidotes, two X-ray crystal structures of Torpedo californica acetylcholinesterase (TcAChE) bound to the bis-pyridinium oxime, Ortho-7, or its experimental bis-imidazolium analogue, 2BIM-7, were determined. Bis-oximes contain two oxime groups connected by a hydrophobic linker. One oxime group of Ortho-7 binds at the entrance to the active-site gorge near Trp279, and the second binds at the bottom near Trp84 and Phe330. In the Ortho-7-TcAChE complex the oxime at the bottom of the gorge was directed towards the nucleophilic Ser200. In contrast, the oxime group of 2BIM-7 was rotated away from Ser200 and the oxime at the entrance induced a significant conformational change in the peripheral anionic site (PAS) residue Trp279. The conformational change alters the surface of the PAS and positions the imidazolium oxime of 2BIM-7 further from Ser200. The relatively weaker binding and poorer reactivation of VX-inhibited, tabun-inhibited or sarin-inhibited human acetylcholinesterase by 2BIM-7 compared with Ortho-7 may in part be owing to the unproductively bound states caught in crystallo. Overall, the reactivation efficiency of 2BIM-7 was comparable to that of 2-pyridine aldoxime methyl chloride (2-PAM), but unlike 2-PAM the bis-imidazolium oxime lacks a fixed charge, which may affect its membrane permeability.

  11. The Vacuolar Proton-Cation Exchanger EcNHX1 Generates pH Signals for the Expression of Secondary Metabolism in Eschscholzia californica1

    PubMed Central

    Roos, Werner

    2016-01-01

    Cell cultures of Eschscholzia californica react to a fungal elicitor by the overproduction of antimicrobial benzophenanthridine alkaloids. The signal cascade toward the expression of biosynthetic enzymes includes (1) the activation of phospholipase A2 at the plasma membrane, resulting in a peak of lysophosphatidylcholine, and (2) a subsequent, transient efflux of vacuolar protons, resulting in a peak of cytosolic H+. This study demonstrates that one of the Na+/H+ antiporters acting at the tonoplast of E. californica cells mediates this proton flux. Four antiporter-encoding genes were isolated and cloned from complementary DNA (EcNHX1–EcNHX4). RNA interference-based, simultaneous silencing of EcNHX1, EcNHX3, and EcNHX4 resulted in stable cell lines with largely diminished capacities of (1) sodium-dependent efflux of vacuolar protons and (2) elicitor-triggered overproduction of alkaloids. Each of the four EcNHX genes of E. californica reconstituted the lack of Na+-dependent H+ efflux in a Δnhx null mutant of Saccharomyces cerevisiae. Only the yeast strain transformed with and expressing the EcNHX1 gene displayed Na+-dependent proton fluxes that were stimulated by lysophosphatidylcholine, thus giving rise to a net efflux of vacuolar H+. This finding was supported by three-dimensional protein homology models that predict a plausible recognition site for lysophosphatidylcholine only in EcNHX1. We conclude that the EcNHX1 antiporter functions in the elicitor-initiated expression of alkaloid biosynthetic genes by recruiting the vacuolar proton pool for the signaling process. PMID:26578709

  12. Immediate and persistent transcriptional correlates of long-term sensitization training at different CNS loci in Aplysia californica.

    PubMed

    Herdegen, Samantha; Conte, Catherine; Kamal, Saman; Calin-Jageman, Robert J; Calin-Jageman, Irina E

    2014-01-01

    Repeated noxious stimulation produces long-term sensitization of defensive withdrawal reflexes in Aplysia californica, a form of long-term memory that requires changes in both transcription and translation. Previous work has identified 10 transcripts which are rapidly up-regulated after long-term sensitization training in the pleural ganglia. Here we use quantitative PCR to begin examining how these transcriptional changes are expressed in different CNS loci related to defensive withdrawal reflexes at 1 and 24 hours after long-term sensitization training. Specifically, we sample from a) the sensory wedge of the pleural ganglia, which exclusively contains the VC nociceptor cell bodies that help mediate input to defensive withdrawal circuits, b) the remaining pleural ganglia, which contain withdrawal interneurons, and c) the pedal ganglia, which contain many motor neurons. Results from the VC cluster show different temporal patterns of regulation: 1) rapid but transient up-regulation of Aplysia homologs of C/EBP, C/EBPγ, and CREB1, 2) delayed but sustained up-regulation of BiP, Tolloid/BMP-1, and sensorin, 3) rapid and sustained up-regulation of Egr, GlyT2, VPS36, and an uncharacterized protein (LOC101862095), and 4) an unexpected lack of regulation of Aplysia homologs of calmodulin (CaM) and reductase-related protein (RRP). Changes in the remaining pleural ganglia mirror those found in the VC cluster at 1 hour but with an attenuated level of regulation. Because these samples had almost no expression of the VC-specific transcript sensorin, our data suggests that sensitization training likely induces transcriptional changes in either defensive withdrawal interneurons or neurons unrelated to defensive withdrawal. In the pedal ganglia, we observed only a rapid but transient increase in Egr expression, indicating that long-term sensitization training is likely to induce transcriptional changes in motor neurons but raising the possibility of different transcriptional

  13. Immediate and Persistent Transcriptional Correlates of Long-Term Sensitization Training at Different CNS Loci in Aplysia californica

    PubMed Central

    Herdegen, Samantha; Conte, Catherine; Kamal, Saman; Calin-Jageman, Robert J.; Calin-Jageman, Irina E.

    2014-01-01

    Repeated noxious stimulation produces long-term sensitization of defensive withdrawal reflexes in Aplysia californica, a form of long-term memory that requires changes in both transcription and translation. Previous work has identified 10 transcripts which are rapidly up-regulated after long-term sensitization training in the pleural ganglia. Here we use quantitative PCR to begin examining how these transcriptional changes are expressed in different CNS loci related to defensive withdrawal reflexes at 1 and 24 hours after long-term sensitization training. Specifically, we sample from a) the sensory wedge of the pleural ganglia, which exclusively contains the VC nociceptor cell bodies that help mediate input to defensive withdrawal circuits, b) the remaining pleural ganglia, which contain withdrawal interneurons, and c) the pedal ganglia, which contain many motor neurons. Results from the VC cluster show different temporal patterns of regulation: 1) rapid but transient up-regulation of Aplysia homologs of C/EBP, C/EBPγ, and CREB1, 2) delayed but sustained up-regulation of BiP, Tolloid/BMP-1, and sensorin, 3) rapid and sustained up-regulation of Egr, GlyT2, VPS36, and an uncharacterized protein (LOC101862095), and 4) an unexpected lack of regulation of Aplysia homologs of calmodulin (CaM) and reductase-related protein (RRP). Changes in the remaining pleural ganglia mirror those found in the VC cluster at 1 hour but with an attenuated level of regulation. Because these samples had almost no expression of the VC-specific transcript sensorin, our data suggests that sensitization training likely induces transcriptional changes in either defensive withdrawal interneurons or neurons unrelated to defensive withdrawal. In the pedal ganglia, we observed only a rapid but transient increase in Egr expression, indicating that long-term sensitization training is likely to induce transcriptional changes in motor neurons but raising the possibility of different transcriptional

  14. Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology.

    PubMed

    Niessen, K V; Muschik, S; Langguth, F; Rappenglück, S; Seeger, T; Thiermann, H; Worek, F

    2016-04-15

    Organophosphorus compounds (OPC), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). Inhibited AChE results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nAChR) in the postsynaptic membrane is provoked. Direct targeting of nAChR to reduce receptor desensitisation might be an alternative therapeutic approach. For drug discovery, functional properties of potent therapeutic candidates need to be investigated in addition to affinity properties. Solid supported membrane (SSM)-based electrophysiology is useful for functional characterisation of ligand-gated ion channels like nAChRs, as charge translocations via capacitive coupling of the supporting membrane can be measured. By varying the agonist (carbamoylcholine) concentration, different functional states of the nAChR were initiated. Using plasma membrane preparations obtained from Torpedo californica electric organ, functional properties of selected nAChR ligands and non-oxime bispyridinium compounds were investigated. Depending on overall-size, the bispyridinium compounds enhanced or inhibited cholinergic signals induced by 100 μM carbamoylcholine. Applying excessive concentrations of the agonist carbamoylcholine provoked desensitisation of the nAChRs, whereas addition of bispyridinium compounds bearing short alkyl linkers exhibited functional recovery of previously desensitised nAChRs. The results suggest that these non-oxime bispyridinium compounds possibly interacted with nAChR subtypes in a manner of a positive allosteric modulator (PAM). The described newly developed functional assay is a valuable tool for the assessment of functional properties of potential compounds such as nAChR modulating ligands, which might be a promising approach in the therapeutically treatment of OPC-poisonings. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Highlighting manganese dynamics in the nervous system of Aplysia californica using MEMRI at ultra-high field.

    PubMed

    Jelescu, Ileana O; Nargeot, Romuald; Le Bihan, Denis; Ciobanu, Luisa

    2013-08-01

    Exploring the pathways of manganese (Mn(2+)) transport in the nervous system becomes of interest as many recent studies use Mn(2+) as a neural tract tracer in mammals. In this study, we performed manganese enhanced MRI (MEMRI) at 17.2 T on the buccal ganglia of Aplysia californica. The main advantage of this model over mammalian systems is that it contains networks of large identified neurons. Using Mn(2+) retrograde transport along selected nerves, we first validated the mapping of motor neurons' axonal projections into peripheral nerves, previously obtained from optical imaging (Morton et al., 1991). This protocol was found not to alter the functional properties of the neuronal network. Second, we compared the Mn(2+) dynamics inside the ganglia in the presence or absence of chemical stimulation. We found that 2h of stimulation with the modulatory transmitter dopamine increased the extent of areas of intermediate signal enhancement caused by manganese accumulation. In the absence of dopamine, an overall decrease of the enhanced areas in favor of non-enhanced areas was found, as a result of natural Mn(2+) washout. This supports the hypothesis that, upon activation, Mn(2+) is released from labeled neurons and captured by other, initially unlabeled, neurons. However, the latter could not be clearly identified due to lack of sensitivity and multiplicity of possible pathways starting from labeled cells. Nonetheless, the Aplysia buccal ganglia remain a well-suited model for attempting to visualize Mn(2+) transport from neuron to neuron upon activation, as well as for studying dopaminergic modulation in a motor network.

  16. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  17. Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica.

    PubMed

    Heinze, Michael; Steighardt, Jörg; Gesell, Andreas; Schwartze, Wieland; Roos, Werner

    2007-12-01

    Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).

  18. Individual synaptic vesicles from the electroplaque of Torpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP

    PubMed Central

    Li, Huinan; Harlow, Mark L.

    2014-01-01

    Abstract The type of neurotransmitter secreted by a neuron is a product of the vesicular transporters present on its synaptic vesicle membranes and the available transmitters in the local cytosolic environment where the synaptic vesicles reside. Synaptic vesicles isolated from electroplaques of the marine ray, Torpedo californica, have served as model vesicles for cholinergic neurotransmission. Many lines of evidence support the idea that in addition to acetylcholine, additional neurotransmitters and/or neuromodulators are also released from cholinergic synapses. We identified the types of vesicular neurotransmitter transporters present at the electroplaque using immunoblot and immunofluoresence techniques with antibodies against the vesicle acetylcholine transporter (VAChT), the vesicular glutamate transporters (VGLUT1, 2, and 3), and the vesicular nucleotide transporter (VNUT). We found that VAChT, VNUT, VGLUT 1 and 2, but not 3 were present by immunoblot, and confirmed that the antibodies were specific to proteins of the axons and terminals of the electroplaque. We used a single‐vesicle imaging technique to determine whether these neurotransmitter transporters were present on the same or different populations of synaptic vesicles. We found that greater than 85% of vesicles that labeled for VAChT colabeled with VGLUT1 or VGLUT2, and approximately 70% colabeled with VNUT. Based upon confidence intervals, at least 52% of cholinergic vesicles isolated are likely to contain all four transporters. The presence of multiple types of neurotransmitter transporters – and potentially neurotransmitters – in individual synaptic vesicles raises fundamental questions about the role of cotransmitter release and neurotransmitter synergy at cholinergic synapses. PMID:24744885

  19. Western scrub-jays (Aphelocoma californica) solve multiple-string problems by the spatial relation of string and reward.

    PubMed

    Hofmann, M M; Cheke, L G; Clayton, N S

    2016-11-01

    String-pulling is a widely used paradigm in animal cognition research to assess what animals understand about the functionality of strings as a means to obtain an out-of-reach reward. This study aimed to systematically investigate what rules Western scrub-jays (Aphelocoma californica) use to solve different patterned string tasks, i.e. tasks in which subjects have to choose between two or more strings of which only one is connected to the reward, or where one is more efficient. Arranging strings in a parallel configuration showed that the jays were generally capable of solving multiple-string tasks and acted in a goal-directed manner. The slanted and crossed configurations revealed a reliance on a "proximity rule", that is, a tendency to choose the string-end closest to the reward. When confronted with strings of different lengths attached to rewards at different distances the birds chose according to the reward distance, preferring the reward closest to them, and were sensitive to the movement of the reward, but did not consistently prefer the shorter and therefore more efficient string. Generally, the scrub-jays were successful in tasks where the reward was closest to the string-ends they needed to pull or when string length and reward distance correlated, but the birds had problems when the wrong string-end was closest to the reward or when the food items were in close proximity to each other. These results show that scrub-jays had a partial understanding of the physical principles underlying string-pulling but relied on simpler strategies such as the proximity rule to solve the tasks.

  20. Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

    PubMed Central

    Paz, Aviv; Roth, Esther; Ashani, Yacov; Xu, Yechun; Shnyrov, Valery L; Sussman, Joel L; Silman, Israel; Weiner, Lev

    2012-01-01

    The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic “anionic” subsite (CAS), the peripheral “anionic” subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both “anionic” subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ∼8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage. PMID:22674800

  1. Clinal adaptation and adaptive plasticity in Artemisia californica: implications for the response of a foundation species to predicted climate change.

    PubMed

    Pratt, Jessica D; Mooney, Kailen A

    2013-08-01

    Local adaptation and plasticity pose significant obstacles to predicting plant responses to future climates. Although local adaptation and plasticity in plant functional traits have been documented for many species, less is known about population-level variation in plasticity and whether such variation is driven by adaptation to environmental variation. We examined clinal variation in traits and performance - and plastic responses to environmental change - for the shrub Artemisia californica along a 700 km gradient characterized (from south to north) by a fourfold increase in precipitation and a 61% decrease in interannual precipitation variation. Plants cloned from five populations along this gradient were grown for 3 years in treatments approximating the precipitation regimes of the north and south range margins. Most traits varying among populations did so clinally; northern populations (vs. southern) had higher water-use efficiencies and lower growth rates, C : N ratios and terpene concentrations. Notably, there was variation in plasticity for plant performance that was strongly correlated with source site interannual precipitation variability. The high-precipitation treatment (vs. low) increased growth and flower production more for plants from southern populations (181% and 279%, respectively) than northern populations (47% and 20%, respectively). Overall, precipitation variability at population source sites predicted 86% and 99% of variation in plasticity in growth and flowering, respectively. These striking, clinal patterns in plant traits and plasticity are indicative of adaptation to both the mean and variability of environmental conditions. Furthermore, our analysis of long-term coastal climate data in turn indicates an increase in interannual precipitation variation consistent with most global change models and, unexpectedly, this increased variation is especially pronounced at historically stable, northern sites. Our findings demonstrate the

  2. Gene identification and evidence for expression of G protein alpha subunits, phospholipase C, and an inositol 1,4,5-trisphosphate receptor in Aplysia californica rhinophore.

    PubMed

    Cummins, Scott F; De Vries, Melissa R; Hill, Kristen S; Boehning, Darren; Nagle, Gregg T

    2007-07-01

    In the marine mollusk Aplysia californica, waterborne protein pheromones that are released during egg laying act in concert to stimulate mate attraction. However, molecular information concerning the cellular receptors and signaling mechanisms that may be involved in waterborne peptide and protein pheromonal communication is lacking. As a first step toward examining whether members of the G protein family and phosphoinositide signaling pathway are present in the primary peripheral chemosensory organs (i.e., rhinophores), we isolated five full-length cDNA clones from an A. californica central nervous system cDNA library. These clones encoded (1) the G protein alpha subunits of the Gq, Gi, and Go families, (2) a protein with homology to phospholipase C (PLC) isoforms, and (3) an inositol 1,4,5-trisphosphate receptor (IP3R). The expression of these genes was examined using laser capture microdissection/reverse transcription-polymerase chain reaction and in situ hybridization. All of them are expressed in the rhinophore sensory epithelium, suggesting that Galphaq, Galphai, Galphao, PLC-like protein, and IP3R may be involved in waterborne protein pheromone detection in Aplysia-possibly via a phosphoinositide signaling mechanism.

  3. Use of chemical modifications and site-directed mutagenesis to probe the functional role of thiol groups on the. gamma. subunit of Torpedo californica acetylcholine receptor

    SciTech Connect

    Pradier, L.; Yee, A.S.; McNamee, M.G. )

    1989-08-08

    Alkylation of Torpedo californica purified nicotinic acetylcholine receptor (AChR) with N-phenylmaleimide (NPM) under nonreducing conditions led to ion flux inhibition without affecting ligand binding properties. The {gamma} subunit was shown to be preferentially labeled by ({sup 3}H)NPM with partial labeling of the {alpha} subunit at higher NPM concentrations. Alkylation occurs at cysteine residues as confirmed by amino acid analysis. Cyanogen bromide peptide mapping of the {gamma} subunit indicates that at least two residues corresponding to Cys-416, -420, or -451 are labeled. Residues 416 and 420 are part of the proposed amphipathic helix, and the functional role of these two cysteines is further investigated by site-directed mutagenesis of T. californica AChR cDNAs and expression of the mutants in Xenopus laevis oocytes following injection of SP6 transcripts. Several features of SP6 transcripts are shown to be important for efficient translation in vivo. Mutations Cys {yields} Ser{gamma}416,420 and Cys {yields} Phe{gamma}416 did not perturb either the receptor functional properties or its expression levels. The double mutant Cys {yields} Phe{gamma}416,420 displayed a 30% decrease of normalized AChR activity. The relatively small effect of large steric mutations in the amphipathic helix argues against its presence in the tightly packed transmembrane domain of the protein.

  4. The effects of low levels of light at night upon the endocrine physiology of western scrub-jays (Aphelocoma californica).

    PubMed

    Schoech, Stephan J; Bowman, Reed; Hahn, Thomas P; Goymann, Wolfgang; Schwabl, Ingrid; Bridge, Eli S

    2013-11-01

    Florida scrub-jays (Aphelocoma coerulescens) in the suburbs breed earlier than jays in native habitat. Amongst the possible factors that influence this advance (e.g., food availability, microclimate, predator regime, etc.), is exposure to artificial lights at night (LAN). LAN could stimulate the reproductive axis of the suburban jays. Alternatively, LAN could inhibit pineal melatonin (MEL), thus removing its inhibitory influence on the reproductive axis. Because Florida scrub-jays are a threatened species, we used western scrub-jays (Aphelocoma californica) to investigate the effects of LAN upon reproductive hormones and melatonin. Jays were held under conditions in which the dark-phase of the light:dark cycle was without illumination and then under low levels of LAN. Under both conditions, birds were exposed first to short-days (9.5L:14.5D) that were gradually increased to long-days (14.5L:9.5D). At various times, blood samples were collected during the light part of the cycle to measure reproductive hormones (luteinizing hormone, LH; testosterone, T; and estradiol, E2 ). Similarly, samples to assess melatonin were collected during the dark. In males, LAN caused a depression in LH levels and levels were ∼4× greater under long- than short-days. In females, there was no effect of LAN or photoperiod upon LH. LAN resulted in depressed T levels in females, although there was no effect on T in males. E2 levels in both sexes were lower under LAN than under an unlighted dark-phase. Paradoxically, MEL was higher in jays under LAN, and under long-days. MEL did not differ by sex. LAN disrupted the extraordinarily strong correlation between T and E2 that existed under unlighted nocturnal conditions. Overall, our findings fail to support the hypothesis that LAN stimulates the reproductive axis. Rather, the data demonstrate that LAN tends to inhibit reproductive hormone secretion, although not in a consistent fashion between the sexes. © 2013 Wiley Periodicals, Inc.

  5. An in vitro preparation for eliciting and recording feeding motor programs with physiological movements in Aplysia californica.

    PubMed

    McManus, Jeffrey M; Lu, Hui; Chiel, Hillel J

    2012-12-05

    Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors(1), allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper(2). However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs(2,3,4), but it is very difficult to directly record from individual neurons(5). Additionally, in vivo, ingestive programs can be further divided into bites and swallows(1,2), a distinction that is difficult to make in most previously described in vitro preparations. The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes(6). The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor

  6. The Sea Slug, Pleurobranchaea californica: A Signpost Species in the Evolution of Complex Nervous Systems and Behavior

    PubMed Central

    Gillette, Rhanor; Brown, Jeffrey W.

    2015-01-01

    How and why did complex brain and behavior evolve? Clues emerge from comparative studies of animals with simpler morphology, nervous system, and behavioral economics. The brains of vertebrates, arthropods, and some annelids have highly derived executive structures and function that control downstream, central pattern generators (CPGs) for locomotion, behavioral choice, and reproduction. For the vertebrates, these structures—cortex, basal ganglia, and hypothalamus—integrate topographically mapped sensory inputs with motivation and memory to transmit complex motor commands to relay stations controlling CPG outputs. Similar computations occur in the central complex and mushroom bodies of the arthropods, and in mammals these interactions structure subjective thought and socially based valuations. The simplest model systems available for comparison are opisthobranch molluscs, which have avoided selective pressure for complex bodies, brain, and behavior through potent chemical defenses. In particular, in the sea-slug Pleurobranchaea californica the functions of vertebrates’ olfactory bulb and pallium are performed in the peripheral nervous system (PNS) of the chemotactile oral veil. Functions of hypothalamus and basal ganglia are combined in Pleurobranchaea’s feeding motor network. The actions of basal ganglia on downstream locomotor regions and spinal CPGs are analogous to Pleurobranchaea’s feeding network actions on CPGs for agonist and antagonist behaviors. The nervous systems of opisthobranch and pulmonate gastropods may conserve or reflect relations of the ancestral urbilaterian. Parallels and contrasts in neuronal circuits for action selection in Pleurobranchaea and vertebrates suggest how a basic set of decision circuitry was built upon in evolving segmentation, articulated skeletons, sociality, and highly invested reproductive strategies. They suggest (1) an origin of olfactory bulb and pallium from head-region PNS; (2) modularization of an ancestral

  7. Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus.

    PubMed

    Valdes, Victor Julian; Sampieri, Alicia; Sepulveda, Jorge; Vaca, Luis

    2003-05-23

    Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA by the RNAi machinery depends upon Watson-Crick base-pairing interactions, RNAi can be exquisitely specific. We took advantage of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific, because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.

  8. Budded baculovirus particle structure revisited.

    PubMed

    Wang, Qiushi; Bosch, Berend-Jan; Vlak, Just M; van Oers, Monique M; Rottier, Peter J; van Lent, Jan W M

    2016-02-01

    Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈ 6-7 nm, which is significantly thicker than a usual biological membrane (<4 nm) as measured by X-ray scanning. Most spikes are densely clustered at the two apical ends of the virion although some envelope proteins are also found more sparsely on the lateral regions. The spikes on the surface of AcMNPV BVs appear distinctly different from those of SeMNPV. Based on our observations we propose a new near-native structural model of baculovirus BVs. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Profile of the alpha-bungarotoxin-binding regions on the extracellular part of the alpha-chain of Torpedo californica acetylcholine receptor.

    PubMed Central

    Mulac-Jericevic, B; Atassi, M Z

    1987-01-01

    The continuous alpha-neurotoxin-binding regions on the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica acetylcholine receptor were localized by reaction of 125I-labelled alpha-bungarotoxin with synthetic overlapping peptides spanning this entire part of the chain. The specificity of the binding was confirmed by inhibition with unlabelled toxin and, for appropriate peptides, with unlabelled anti-(acetylcholine receptor) antibodies. Five toxin-binding regions were localized within residues 1-10, 32-41, 100-115, 122-150 and 182-198. The third, fourth and fifth (and to a lesser extent the first and second) toxin-binding regions overlapped with regions recognized by anti-(acetylcholine receptor) antibodies. The five toxin-binding regions may be distinct sites or, alternatively, different 'faces' in one (or more) sites. PMID:3435488

  10. The nitrogen supply from soils and insects during growth of the pitcher plants Nepenthes mirabilis, Cephalotus follicularis and Darlingtonia californica.

    PubMed

    Schulze, W; Schulze, E D; Pate, J S; Gillison, A N

    1997-11-01

    This study investigated the nitrogen (N) acquisition from soil and insect capture during the growth of three species of pitcher plants, Nepenthes mirabilis, Cephalotus follicularis and Darlingtonia californica. (15)N/(14)N natural abundance ratios (δ(15)N) of plants and pitchers of different age, non-carnivorous reference plants, and insect prey were used to estimate proportional contributions of insects to the N content of leaves and whole plants. Young Nepenthes leaves (phyllodes) carrying closed pitchers comprised major sinks for N and developed mainly from insect N captured elsewhere on the plant. Their δ(15)N values of up to 7.2‰ were higher than the average δ(15)N value of captured insects (mean δ(15)N value = 5.3‰). In leaves carrying old pitchers that are acting as a N source, the δ(15)N decreased to 3.0‰ indicating either an increasing contribution of soil N to those plant parts which in fact captured the insects or N gain from N2 fixation by microorganisms which may exist in old pitchers. The δ(15)N value of N in water collected from old pitchers was 1.2‰ and contained free amino acids. The fraction of insect N in young and old pitchers and their associated leaves decreased from 1.0 to 0.3 mg g(-1). This fraction decreased further with the size of the investigated tiller. Nepenthes contained on average 61.5 ± 7.6% (mean ± SD, range 50-71%) insect N based on the N content of a whole tiller. In the absence of suitable non-carnivorous reference plants for Cephalotus, δ(15)N values were assessed across a developmental sequence from young plants lacking pitchers to large adults with up to 38 pitchers. The data indicated dependence on soil N until 4 pitchers had opened. Beyond that stage, plant size increased with the number of catching pitchers but the fraction of soil N remained high. Large Cephalotus plants were estimated to derive 26 ± 5.9% (mean ± SD of the three largest plants; range: 19-30%) of the N from insects

  11. EST database for early flower development in California poppy (Eschscholzia californica Cham., Papaveraceae) tags over 6,000 genes from a basal eudicot.

    PubMed

    Carlson, John E; Leebens-Mack, James H; Wall, P Kerr; Zahn, Laura M; Mueller, Lukas A; Landherr, Lena L; Hu, Yi; Ilut, Daniel C; Arrington, Jennifer M; Choirean, Stephanie; Becker, Annette; Field, Dawn; Tanksley, Steven D; Ma, Hong; dePamphilis, Claude W

    2006-10-01

    The Floral Genome Project (FGP) selected California poppy (Eschscholzia californica Cham. ssp. Californica) to help identify new florally-expressed genes related to floral diversity in basal eudicots. A large, non-normalized cDNA library was constructed from premeiotic and meiotic floral buds and sequenced to generate a database of 9,079 high quality Expressed Sequence Tags (ESTs). These sequences clustered into 5,713 unigenes, including 1,414 contigs and 4,299 singletons. Homologs of genes regulating many aspects of flower development were identified, including those for organ identity and development, cell and tissue differentiation, cell cycle control, and secondary metabolism. Over 5% of the transcriptome consisted of homologs to known floral gene families. Most are the first representatives of their respective gene families in basal eudicots and their conservation suggests they are important for floral development and/or function. App. 10% of the transcripts encoded transcription factors and other regulatory genes, including nine genes from the seven major lineages of the important MADS-box family of developmental regulators. Homologs of alkaloid pathway genes were also recovered, providing opportunities to explore adaptive evolution in secondary products. Furthermore, comparison of the poppy ESTs with the Arabidopsis genome provided support for putative Arabidopsis genes that previously lacked annotation. Finally, over 1,800 unique sequences had no observable homology in the public databases. The California poppy EST database and library will help bridge our understanding of flower initiation and development among higher eudicot and monocot model plants and provide new opportunities for comparative analysis of gene families across angiosperm species.

  12. Effects of lipid-analog detergent solubilization on the functionality and lipidic cubic phase mobility of the Torpedo californica nicotinic acetylcholine receptor.

    PubMed

    Padilla-Morales, Luis F; Morales-Pérez, Claudio L; De La Cruz-Rivera, Pamela C; Asmar-Rovira, Guillermo; Báez-Pagán, Carlos A; Quesada, Orestes; Lasalde-Dominicci, José A

    2011-10-01

    Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β(2)-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility.

  13. Effects of CO2-induced pH reduction on the exoskeleton structure and biophotonic properties of the shrimp Lysmata californica

    PubMed Central

    Taylor, Jennifer R. A.; Gilleard, Jasmine M.; Allen, Michael C.; Deheyn, Dimitri D.

    2015-01-01

    The anticipated effects of CO2-induced ocean acidification on marine calcifiers are generally negative, and include dissolution of calcified elements and reduced calcification rates. Such negative effects are not typical of crustaceans for which comparatively little ocean acidification research has been conducted. Crustaceans, however, depend on their calcified exoskeleton for many critical functions. Here, we conducted a short-term study on a common caridean shrimp, Lysmata californica, to determine the effect of CO2-driven reduction in seawater pH on exoskeleton growth, structure, and mineralization and animal cryptic coloration. Shrimp exposed to ambient (7.99 ± 0.04) and reduced pH (7.53 ± 0.06) for 21 days showed no differences in exoskeleton growth (percent increase in carapace length), but the calcium weight percent of their cuticle increased significantly in reduced pH conditions, resulting in a greater Ca:Mg ratio. Cuticle thickness did not change, indicating an increase in the mineral to matrix ratio, which may have mechanical consequences for exoskeleton function. Furthermore, there was a 5-fold decrease in animal transparency, but no change in overall shrimp coloration (red). These results suggest that even short-term exposure to CO2-induced pH reduction can significantly affect exoskeleton mineralization and shrimp biophotonics, with potential impacts on crypsis, physical defense, and predator avoidance. PMID:26030212

  14. Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica

    NASA Technical Reports Server (NTRS)

    Pedrozo, H. A.; Schwartz, Z.; Dean, D. D.; Harrison, J. L.; Campbell, J. W.; Wiederhold, M. L.; Boyan, B. D.

    1997-01-01

    To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

  15. Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica

    NASA Technical Reports Server (NTRS)

    Pedrozo, H. A.; Schwartz, Z.; Dean, D. D.; Harrison, J. L.; Campbell, J. W.; Wiederhold, M. L.; Boyan, B. D.

    1997-01-01

    To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

  16. Effects of Lipid-Analog Detergent Solubilization on the Functionality and Lipidic Cubic Phase Mobility of the Torpedo californica Nicotinic Acetylcholine Receptor

    PubMed Central

    Padilla-Morales, Luis F.; Morales-Pérez, Claudio L.; De La Cruz-Rivera, Pamela C.; Asmar-Rovira, Guillermo; Báez-Pagán, Carlos A.

    2011-01-01

    Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β2-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility. PMID:21922299

  17. Analysis of Phenolic Compounds and Antioxidant Abilities of Extracts from Germinating Vitis californica Seeds Submitted to Cold Stress Conditions and Recovery after the Stress

    PubMed Central

    Weidner, Stanisław; Chrzanowski, Sebastian; Karamać, Magdalena; Król, Angelika; Badowiec, Anna; Mostek, Agnieszka; Amarowicz, Ryszard

    2014-01-01

    The material for this study consisted of stratified seeds of Vitis californica submitted to germination under optimum conditions (+25 °C) or under chill stress (+10 °C), also followed by recovery. It has been determined that the germinating seeds contain considerable amounts of tannins, catechins as well as phenolic acids such as gallic, p-coumaric, caffeic and ferulic acids. Gallic acid appeared in the highest amount in the germinating seeds (from 42.40–204.00 µg/g of fresh weight (FW)), followed by caffeic acid (from 6.62–20.13 µg/g FW), p-coumaric acid (from 2.59–5.41 µg/g FW), and ferulic acid (from 0.56–0.92 µg/g FW). The phenolic acids occurred mostly in the ester form. Under chill stress, the germinating seeds were determined to contain an elevated total amount of phenolics, as well as raised levels of condensed tannins, catechins, gallic acid, and gafeic acid. The levels of p-coumoric and ferulic acids were found to have decreased. In extracts isolated from a sample exposed to low temperature, increased antioxidant activity and reduction potential were also demonstrated. Tissue of the germinating seeds which underwent post-stress recovery was found to have less total phenolics. PMID:25222557

  18. Investigation of the subcellular architecture of L7 neurons of Aplysia californica using magnetic resonance microscopy (MRM) at 7.8 microns.

    PubMed

    Lee, Choong H; Flint, Jeremy J; Hansen, Brian; Blackband, Stephen J

    2015-06-10

    Magnetic resonance microscopy (MRM) is a non-invasive diagnostic tool which is well-suited to directly resolve cellular structures in ex vivo and in vitro tissues without use of exogenous contrast agents. Recent advances in its capability to visualize mammalian cellular structure in intact tissues have reinvigorated analytical interest in aquatic cell models whose previous findings warrant up-to-date validation of subcellular components. Even if the sensitivity of MRM is less than other microscopic technologies, its strength lies in that it relies on the same image contrast mechanisms as clinical MRI which make it a unique tool for improving our ability to interpret human diagnostic imaging through high resolution studies of well-controlled biological model systems. Here, we investigate the subcellular MR signal characteristics of isolated cells of Aplysia californica at an in-plane resolution of 7.8 μm. In addition, direct correlation and positive identification of subcellular architecture in the cells is achieved through well-established histology. We hope this methodology will serve as the groundwork for studying pathophysiological changes through perturbation studies and allow for development of disease-specific cellular modeling tools. Such an approach promises to reveal the MR contrast changes underlying cellular mechanisms in various human diseases, for example in ischemic stroke.

  19. Single-cell lipidomics: characterizing and imaging lipids on the surface of individual Aplysia californica neurons with cluster secondary ion mass spectrometry.

    PubMed

    Passarelli, Melissa K; Ewing, Andrew G; Winograd, Nicholas

    2013-02-19

    Neurons isolated from Aplysia californica , an organism with a well-defined neural network, were imaged with secondary ion mass spectrometry, C(60)-SIMS. A major lipid component of the neuronal membrane was identified as 1-hexadecyl-2-octadecenoyl-sn-glycero-3-phosphocholine [PC(16:0e/18:1)] using tandem mass spectrometry (MS/MS). The assignment was made directly off the sample surface using a C(60)-QSTAR instrument, a prototype instrument that combines an ion source with a commercial electrospray ionization/matrix-assisted laser desorption ionization (ESI/MALDI) mass spectrometer. Normal phase liquid chromatography mass spectrometry (NP-LC-MS) was used to confirm the assignment. Cholesterol and vitamin E were also identified with in situ tandem MS analyses that were compared to reference spectra obtained from purified compounds. In order to improve sensitivity on the single-cell level, the tandem MS spectrum of vitamin E reference material was used to extract and compile all the vitamin E related peaks from the cell image. The mass spectrometry images reveal heterogeneous distributions of intact lipid species, PC(16:0e/18:1), vitamin E, and cholesterol on the surface of a single neuron. The ability to detect these molecules and determine their relative distribution on the single-cell level shows that the C(60)-QSTAR is a potential platform for studying important biochemical processes, such as neuron degeneration.

  20. Effects of CO2-induced pH reduction on the exoskeleton structure and biophotonic properties of the shrimp Lysmata californica.

    PubMed

    Taylor, Jennifer R A; Gilleard, Jasmine M; Allen, Michael C; Deheyn, Dimitri D

    2015-06-01

    The anticipated effects of CO2-induced ocean acidification on marine calcifiers are generally negative, and include dissolution of calcified elements and reduced calcification rates. Such negative effects are not typical of crustaceans for which comparatively little ocean acidification research has been conducted. Crustaceans, however, depend on their calcified exoskeleton for many critical functions. Here, we conducted a short-term study on a common caridean shrimp, Lysmata californica, to determine the effect of CO2-driven reduction in seawater pH on exoskeleton growth, structure, and mineralization and animal cryptic coloration. Shrimp exposed to ambient (7.99 ± 0.04) and reduced pH (7.53 ± 0.06) for 21 days showed no differences in exoskeleton growth (percent increase in carapace length), but the calcium weight percent of their cuticle increased significantly in reduced pH conditions, resulting in a greater Ca:Mg ratio. Cuticle thickness did not change, indicating an increase in the mineral to matrix ratio, which may have mechanical consequences for exoskeleton function. Furthermore, there was a 5-fold decrease in animal transparency, but no change in overall shrimp coloration (red). These results suggest that even short-term exposure to CO2-induced pH reduction can significantly affect exoskeleton mineralization and shrimp biophotonics, with potential impacts on crypsis, physical defense, and predator avoidance.

  1. Investigation of the subcellular architecture of L7 neurons of Aplysia californica using magnetic resonance microscopy (MRM) at 7.8 microns

    PubMed Central

    Lee, Choong H.; Flint, Jeremy J.; Hansen, Brian; Blackband, Stephen J.

    2015-01-01

    Magnetic resonance microscopy (MRM) is a non-invasive diagnostic tool which is well-suited to directly resolve cellular structures in ex vivo and in vitro tissues without use of exogenous contrast agents. Recent advances in its capability to visualize mammalian cellular structure in intact tissues have reinvigorated analytical interest in aquatic cell models whose previous findings warrant up-to-date validation of subcellular components. Even if the sensitivity of MRM is less than other microscopic technologies, its strength lies in that it relies on the same image contrast mechanisms as clinical MRI which make it a unique tool for improving our ability to interpret human diagnostic imaging through high resolution studies of well-controlled biological model systems. Here, we investigate the subcellular MR signal characteristics of isolated cells of Aplysia californica at an in-plane resolution of 7.8 μm. In addition, direct correlation and positive identification of subcellular architecture in the cells is achieved through well-established histology. We hope this methodology will serve as the groundwork for studying pathophysiological changes through perturbation studies and allow for development of disease-specific cellular modeling tools. Such an approach promises to reveal the MR contrast changes underlying cellular mechanisms in various human diseases, for example in ischemic stroke. PMID:26059695

  2. Serial reversal learning and the evolution of behavioral flexibility in three species of North American corvids (Gymnorhinus cyanocephalus, Nucifraga columbiana, Aphelocoma californica).

    PubMed

    Bond, Alan B; Kamil, Alan C; Balda, Russell P

    2007-11-01

    In serial reversal learning, subjects learn to respond differentially to 2 stimuli. When the task is fully acquired, reward contingencies are reversed, requiring the subject to relearn the altered associations. This alternation of acquisition and reversal can be repeated many times, and the ability of a species to adapt to this regimen has been considered as an indication of behavioral flexibility. Serial reversal learning of 2-choice discriminations was contrasted in 3 related species of North American corvids: pinyon jays (Gymnorhinus cyanocephalus), which are highly social; Clark's nutcrackers (Nucifraga columbiana), which are relatively solitary but specialized for spatial memory; and western scrub jays (Aphelocoma californica), which are ecological generalists. Pinyon jays displayed significantly lower error rates than did nutcrackers or scrub jays after reversal of reward contingencies for both spatial and color stimuli. The effect was most apparent in the 1st session following each reversal and did not reflect species differences in the rate of initial discrimination learning. All 3 species improved their performance over successive reversals and showed significant transfer between color and spatial tasks, suggesting a generalized learning strategy. The results are consistent with an evolutionary association between behavioral flexibility and social complexity. Copyright 2007 APA.

  3. Is the western scrub-jay (Aphelocoma californica) really an underdog among food-caching corvids when it comes to hippocampal volume and food caching propensity?

    PubMed

    Pravosudov, Vladimir V; de Kort, Selvino R

    2006-01-01

    Food caching has been linked to better performance on spatial memory tasks and enlarged hippocampal volume in both birds and mammals. Within food-caching birds, it has also been predicted that species less reliant on stored food should have inferior spatial memory and a smaller hippocampus compared to species that depend heavily on food caches. Several comparisons suggest that North American corvids have a significantly smaller hippocampus and overall brain volume compared to the Eurasian corvid species and that western scrub-jays (Aphelocoma californica) have a smaller hippocampus compared to the more specialized Clark's nutcracker. Here we present the largest data set of scrub-jay brains and, in contrast to previous reports, show that relative to body mass western scrub-jays have a brain size similar to the largest brain size of Eurasian corvids. The relative hippocampal volume of scrub-jays is also among the largest of all investigated corvids. These findings may not be surprising considering that scrub-jays have been reported to have remarkable cognitive capacities such as episodic-like memory and experience projection. Our data suggest that many previously made assumptions about western scrub-jays as less specialized food hoarders might be an oversimplification and that simple categorization of species into specialized and non-specialized hoarders might not provide useful insights into the evolution of memory and the hippocampus. (c) 2006 S. Karger AG, Basel.

  4. Microclimate Impacts Survival and Prevalence of Phytophthora ramorum in Umbellularia californica, a Key Reservoir Host of Sudden Oak Death in Northern California Forests

    PubMed Central

    DiLeo, Matthew V.; Bostock, Richard M.; Rizzo, David M.

    2014-01-01

    Phytophthora ramorum, an invasive pathogen and the causal agent of Sudden Oak Death, has become established in mixed-evergreen and redwood forests in coastal northern California. While oak and tanoak mortality is the most visible indication of P. ramorum’s presence, epidemics are largely driven by the presence of bay laurel (Umbellularia californica), a reservoir host that supports both prolific sporulation in the winter wet season and survival during the summer dry season. In order to better understand how over-summer survival of the pathogen contributes to variability in the severity of annual epidemics, we monitored the viability of P. ramorum leaf infections over three years along with coincident microclimate. The proportion of symptomatic bay laurel leaves that contained viable infections decreased during the first summer dry season and remained low for the following two years, likely due to the absence of conducive wet season weather during the study period. Over-summer survival of P. ramorum was positively correlated with high percent canopy cover, less negative bay leaf water potential and few days exceeding 30°C but was not significantly different between mixed-evergreen and redwood forest ecosystems. Decreased summer survival of P. ramorum in exposed locations and during unusually hot summers likely contributes to the observed spatiotemporal heterogeneity of P. ramorum epidemics. PMID:25098281

  5. Standardization of the experimental autoimmune myasthenia gravis (EAMG) model by immunization of rats with Torpedo californica acetylcholine receptors — Recommendations for methods and experimental designs

    PubMed Central

    Losen, Mario; Martinez-Martinez, Pilar; Molenaar, Peter C.; Lazaridis, Konstantinos; Tzartos, Socrates; Brenner, Talma; Duan, Rui-Sheng; Luo, Jie; Lindstrom, Jon; Kusner, Linda

    2015-01-01

    Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR) is characterized by a chronic, fatigable weakness of voluntary muscles. The production of autoantibodies involves the dysregulation of T cells which provide the environment for the development of autoreactive B cells. The symptoms are caused by destruction of the postsynaptic membrane and degradation of the AChR by IgG autoantibodies, predominantly of the G1 and G3 subclasses. Active immunization of animals with AChR from mammalian muscles, AChR from Torpedo or Electrophorus electric organs, and recombinant or synthetic AChR fragments generates a chronic model of MG, termed experimental autoimmune myasthenia gravis (EAMG). This model covers cellular mechanisms involved in the immune response against the AChR, e.g. antigen presentation, T cell-help and regulation, B cell selection and differentiation into plasma cells. Our aim is to define standard operation procedures and recommendations for the rat EAMG model using purified AChR from the Torpedo californica electric organ, in order to facilitate more rapid translation of preclinical proof of concept or efficacy studies into clinical trials and, ultimately, clinical practice. PMID:25796590

  6. Analysis of phenolic compounds and antioxidant abilities of extracts from germinating Vitis californica seeds submitted to cold stress conditions and recovery after the stress.

    PubMed

    Weidner, Stanisław; Chrzanowski, Sebastian; Karamać, Magdalena; Król, Angelika; Badowiec, Anna; Mostek, Agnieszka; Amarowicz, Ryszard

    2014-09-12

    The material for this study consisted of stratified seeds of Vitis californica submitted to germination under optimum conditions (+25 °C) or under chill stress (+10 °C), also followed by recovery. It has been determined that the germinating seeds contain considerable amounts of tannins, catechins as well as phenolic acids such as gallic, p-coumaric, caffeic and ferulic acids. Gallic acid appeared in the highest amount in the germinating seeds (from 42.40-204.00 µg/g of fresh weight (FW)), followed by caffeic acid (from 6.62-20.13 µg/g FW), p-coumaric acid (from 2.59-5.41 µg/g FW), and ferulic acid (from 0.56-0.92 µg/g FW). The phenolic acids occurred mostly in the ester form. Under chill stress, the germinating seeds were determined to contain an elevated total amount of phenolics, as well as raised levels of condensed tannins, catechins, gallic acid, and gafeic acid. The levels of p-coumoric and ferulic acids were found to have decreased. In extracts isolated from a sample exposed to low temperature, increased antioxidant activity and reduction potential were also demonstrated. Tissue of the germinating seeds which underwent post-stress recovery was found to have less total phenolics.

  7. Identification of potent bactericidal compounds produced by escapin, an L-amino acid oxidase in the ink of the sea hare Aplysia californica.

    PubMed

    Ko, Ko-Chun; Wang, Binghe; Tai, Phang C; Derby, Charles D

    2008-12-01

    The ink of sea hares (Aplysia californica) contains escapin, an L-amino acid oxidase that metabolizes L-lysine, thereby producing a mixture that kills microbes and deters attacking predators. This secretion contains H2O2,ammonia, and an equilibrium mixture of "escapin intermediate product" (EIP-K) that includes alpha-keto-epsilon-aminocaproic acid and several other molecules. Components of the equilibrium mixture react nonenzymatically with H2O2 to form "escapin end product" (EEP-K), which contains delta-aminovaleric acid and delta-valerolactam. The proportions of the molecules in this equilibrium mixture change with pH, and this is biologically important because the secretion is pH 5 when released but becomes pH 8 when fully diluted in seawater. The goal of the current study was to identify which molecules in this equilibrium mixture are bactericidal. We show that a mixture of H2O2 and EIP-K, but not EEP-K, at low mM concentrations is synergistically responsible for most of the bactericidal activity of the secretion against Escherichia coli, Vibrio harveyi, Staphylococcus aureus,and Pseudomonas aeruginosa. Low pH enhances the bactericidal effect, and this does not result from stress associated with low pH itself. Sequential exposure to low mM concentrations of EIP-K and H2O2, in either order, does not kill E. coli. Reaction products formed when L-arginine is substituted for L-lysine have almost no bactericidal activity. Our results favor the idea that the bactericidal activity is due to unstable intermediates of the reaction of alpha-keto-epsilon-aminocaproic acid with H2O2.

  8. Floral homeotic C function genes repress specific B function genes in the carpel whorl of the basal eudicot California poppy (Eschscholzia californica).

    PubMed

    Yellina, Aravinda L; Orashakova, Svetlana; Lange, Sabrina; Erdmann, Robert; Leebens-Mack, Jim; Becker, Annette

    2010-12-01

    The floral homeotic C function gene AGAMOUS (AG) confers stamen and carpel identity and is involved in the regulation of floral meristem termination in Arabidopsis. Arabidopsis ag mutants show complete homeotic conversions of stamens into petals and carpels into sepals as well as indeterminacy of the floral meristem. Gene function analysis in model core eudicots and the monocots rice and maize suggest a conserved function for AG homologs in angiosperms. At the same time gene phylogenies reveal a complex history of gene duplications and repeated subfunctionalization of paralogs. EScaAG1 and EScaAG2, duplicate AG homologs in the basal eudicot Eschscholzia californica show a high degree of similarity in sequence and expression, although EScaAG2 expression is lower than EScaAG1 expression. Functional studies employing virus-induced gene silencing (VIGS) demonstrate that knock down of EScaAG1 and 2 function leads to homeotic conversion of stamens into petaloid structures and defects in floral meristem termination. However, carpels are transformed into petaloid organs rather than sepaloid structures. We also show that a reduction of EScaAG1 and EScaAG2 expression leads to significantly increased expression of a subset of floral homeotic B genes. This work presents expression and functional analysis of the two basal eudicot AG homologs. The reduction of EScaAG1 and 2 functions results in the change of stamen to petal identity and a transformation of the central whorl organ identity from carpel into petal identity. Petal identity requires the presence of the floral homeotic B function and our results show that the expression of a subset of B function genes extends into the central whorl when the C function is reduced. We propose a model for the evolution of B function regulation by C function suggesting that the mode of B function gene regulation found in Eschscholzia is ancestral and the C-independent regulation as found in Arabidopsis is evolutionarily derived.

  9. Role of protein kinase C in the induction and maintenance of serotonin-dependent enhancement of the glutamate response in isolated siphon motor neurons of Aplysia californica.

    PubMed

    Villareal, Greg; Li, Quan; Cai, Diancai; Fink, Ann E; Lim, Travis; Bougie, Joanna K; Sossin, Wayne S; Glanzman, David L

    2009-04-22

    Serotonin (5-HT) mediates learning-related facilitation of sensorimotor synapses in Aplysia californica. Under some circumstances 5-HT-dependent facilitation requires the activity of protein kinase C (PKC). One critical site of PKC's contribution to 5-HT-dependent synaptic facilitation is the presynaptic sensory neuron. Here, we provide evidence that postsynaptic PKC also contributes to synaptic facilitation. We investigated the contribution of PKC to enhancement of the glutamate-evoked potential (Glu-EP) in isolated siphon motor neurons in cell culture. A 10 min application of either 5-HT or phorbol ester, which activates PKC, produced persistent (> 50 min) enhancement of the Glu-EP. Chelerythrine and bisindolylmaleimide-1 (Bis), two inhibitors of PKC, both blocked the induction of 5-HT-dependent enhancement. An inhibitor of calpain, a calcium-dependent protease, also blocked 5-HT's effect. Interestingly, whereas chelerythrine blocked maintenance of the enhancement, Bis did not. Because Bis has greater selectivity for conventional and novel isoforms of PKC than for atypical isoforms, this result implicates an atypical isoform in the maintenance of 5-HT's effect. Although induction of enhancement of the Glu-EP requires protein synthesis (Villareal et al., 2007), we found that maintenance of the enhancement does not. Maintenance of 5-HT-dependent enhancement appears to be mediated by a PKM-type fragment generated by calpain-dependent proteolysis of atypical PKC. Together, our results suggest that 5-HT treatment triggers two phases of PKC activity within the motor neuron, an early phase that may involve conventional, novel or atypical isoforms of PKC, and a later phase that selectively involves an atypical isoform.

  10. Molecular Cloning, Expression Pattern, and Immunocytochemical Localization of a Gonadotropin-Releasing Hormone-like Molecule in the Gastropod Mollusk, Aplysia californica

    PubMed Central

    Zhang, Lihong; Tello, Javier A.; Zhang, Weimin; Tsai, Pei-San

    2008-01-01

    Successful reproduction in vertebrates depends upon the actions of gonadotropin-releasing hormone (GnRH). Despite the wide presence of GnRH in Phylum Chordata, GnRH has not been isolated in protostomes other than the common octopus. To provide information on the evolution of this critical hormone, we isolated the full-length cDNA of a GnRH-like molecule from the central nervous system of a gastropod mollusk, the sea hare Aplysia californica. The open reading frame of this cDNA encodes a protein of 147 amino acids. The molecular architecture of the deduced protein is highly homologous to that reported for the prepro-octopus GnRH (oct-GnRH) and consists of a putative signal peptide, a GnRH dodecapeptide, a downstream processing site, and a GnRH-associated peptide (GAP). The deduced amino acid sequence of the Aplysia GnRH (ap-GnRH) is QNYHFSNGWYAG and differs from oct-GnRH by only two amino acids. The transcript for ap-GnRH is widely expressed in the central nervous system (CNS), the ovotestis, and the atrial gland, an exocrine gland. Immunocytochemistry (ICC) using an antiserum against oct-GnRH detected immunoreactive neurons in all CNS ganglia examined, and the staining was abolished by the preadsorption of the antiserum with synthetic ap-GnRH. In sum, ap-GnRH sequence is the first gastropod GnRH-like molecule to be elucidated. Further, it represents one of the only two GnRH-like molecules found outside Phylum Chordata. These data refute the possibility that oct-GnRH arose singly in cephalopods by convergent evolution and provide valuable support for an ancient origin of GnRH during metazoan evolution. PMID:18178211

  11. Molecular cloning, expression pattern, and immunocytochemical localization of a gonadotropin-releasing hormone-like molecule in the gastropod mollusk, Aplysia californica.

    PubMed

    Zhang, Lihong; Tello, Javier A; Zhang, Weimin; Tsai, Pei-San

    2008-04-01

    Successful reproduction in vertebrates depends upon the actions of gonadotropin-releasing hormone (GnRH). Despite the wide presence of GnRH in Phylum Chordata, GnRH has not been isolated in protostomes other than the common octopus. To provide information on the evolution of this critical hormone, we isolated the full-length cDNA of a GnRH-like molecule from the central nervous system of a gastropod mollusk, the sea hare Aplysia californica. The open reading frame of this cDNA encodes a protein of 147 amino acids. The molecular architecture of the deduced protein is highly homologous to that reported for the prepro-octopus GnRH (oct-GnRH) and consists of a putative signal peptide, a GnRH dodecapeptide, a downstream processing site, and a GnRH-associated peptide (GAP). The deduced amino acid sequence of the Aplysia GnRH (ap-GnRH) is QNYHFSNGWYAG and differs from oct-GnRH by only two amino acids. The transcript for ap-GnRH is widely expressed in the central nervous system (CNS), the ovotestis, and the atrial gland, an exocrine gland. Immunocytochemistry (ICC) using an antiserum against oct-GnRH detected immunoreactive neurons in all CNS ganglia examined, and the staining was abolished by the preadsorption of the antiserum with synthetic ap-GnRH. In sum, ap-GnRH sequence is the first gastropod GnRH-like molecule to be elucidated. Further, it represents one of the only two GnRH-like molecules found outside Phylum Chordata. These data refute the possibility that oct-GnRH arose singly in cephalopods by convergent evolution and provide valuable support for an ancient origin of GnRH during metazoan evolution.

  12. Tryptophan Substitutions at Lipid-exposed Positions of the Gamma M3 Transmembrane Domain Increase the Macroscopic Ionic Current Response of the Torpedo californica Nicotinic Acetylcholine Receptor

    PubMed Central

    Cruz-Martín, A.; Mercado, J.L.; Rojas, L.V.; McNamee, M.G.; Lasalde-Dominicci, J.A.

    2015-01-01

    Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (γM3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltage-clamped oocytes. For all the substitutions the concentration for half-maximal activation, EC50, is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2- to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC50s were similar to those measured for the αC418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs. PMID:11547353

  13. Expression patterns of STM-like KNOX and Histone H4 genes in shoot development of the dissected-leaved basal eudicot plants Chelidonium majus and Eschscholzia californica (Papaveraceae).

    PubMed

    Groot, Edwin P; Sinha, Neelima; Gleissberg, Stefan

    2005-06-01

    Knotted-like homeobox (KNOX) genes encode important regulators of shoot development in flowering plants. In Arabidopsis, class I KNOX genes are part of a regulatory system that contributes to indeterminacy of shoot development, delimitation of leaf primordia and internode development. In other species, class I KNOX genes have also been recruited in the control of marginal blastozone fractionation during dissected leaf development. Here we report the isolation of class I KNOX genes from two species of the basal eudicot family Papaveraceae, Chelidonium majus and Eschscholzia californica. Sequence comparisons and expression patterns indicate that these genes are orthologs of SHOOTMERISTEMLESS (STM), a class I KNOX gene from Arabidopsis. Both genes are expressed in the center of vegetative and floral shoot apical meristems (SAM), but downregulated at leaf or floral organ initiating sites. While Eschscholzia californica STM (EcSTM) is again upregulated during acropetal pinna formation, in situ hybridization could not detect Chelidonium majus STM (CmSTM) transcripts at any stage of basipetal leaf development, indicating divergent evolution of STM gene function in leaves within Papaveraceae. Immunolocalization of KNOX proteins indicate that other gene family members may control leaf dissection in both species. The contrasting direction of pinna initiation in the two species was also investigated using Histone H4 expression. Leaves at early stages of development did not reveal notable differences in cell division activity of the elongating leaf axis, suggesting that differential meristematic growth may not play a role in determining the observed dissection patterns.

  14. Identification and characterization of the ecdysteroid UDPglucosyltransferase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus

    Treesearch

    Christopher I. Riegel; Carita Lanner-Herrera; James M. Slavicek

    1994-01-01

    We have located, cloned, sequenced and characterized the ecdysteroid UDP-glucosyltransferase gene (egt) gene from the baculovirus Lymantria dispar multinucleocapsid nuclear polyhedrosis virus,(LdMNPV), which is specific for the gypsy moth (L. dispar). The egt gene from the related baculovirus Autographa californica...

  15. Replication and inclusion body characteristics of two Lymantria dispar nuclear polyhedrosis virus plaque variants

    Treesearch

    James M. Slavicek; Carita Lanner-Herrera; Nancy Hayes-Plazolles; Mary Ellen Kelly; Martha Fikes

    1991-01-01

    Propagation of Autographa californica nuclear polyhedrosis virus in cell culture results in the generation of a mutant virus, termed few polyhedra. This plaque variant is characterized by a high budded virus titer, the formation of few polyhedral inclusion bodies (PIBs), and the production of PIBs exhibiting a low potency against its natural host....

  16. Response of gypsy moth larvae to homologous and heterologous nuclear polyhedrosis virus

    Treesearch

    Kathleen S. Shields; Edward M. Dougherty

    1991-01-01

    The gypsy moth, Lymantria dispar, is not particularly susceptible to baculoviruses other than the nuclear polyhedrosis virus originally isolated from the species (LdMNPV). The multiple enveloped nuclear polyhedrosis virus of Autographa californica (AcMNPV), a very virulent baculovirus that replicates in a large number of...

  17. Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar

    Treesearch

    John Kuzio; Margot N. Pearson; Steve H. Harwood; C. Joel Funk; Jay T. Evans; James M. Slavicek; George F. Rohrmann

    1999-01-01

    The genome of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed. It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of ≥150 nucleotides. Homologs were found to 95 of the 155 genes predicted for the Autographa californica...

  18. Baculovirus expressed virus-like particles of Pea eation mosaic virus vary in size and encapsidate baculovirus mRNAs

    USDA-ARS?s Scientific Manuscript database

    Pea enation mosaic virus (PEMV: family Luteoviridae) is transmitted in a persistent, circulative manner by aphids. We inserted cDNAs encoding the structural proteins of PEMV, the coat protein (CP) and coat protein-read through domain (CPRT) into the genome of the baculovirus Autographa californica m...

  19. Floral homeotic C function genes repress specific B function genes in the carpel whorl of the basal eudicot California poppy (Eschscholzia californica)

    PubMed Central

    2010-01-01

    Background The floral homeotic C function gene AGAMOUS (AG) confers stamen and carpel identity and is involved in the regulation of floral meristem termination in Arabidopsis. Arabidopsis ag mutants show complete homeotic conversions of stamens into petals and carpels into sepals as well as indeterminacy of the floral meristem. Gene function analysis in model core eudicots and the monocots rice and maize suggest a conserved function for AG homologs in angiosperms. At the same time gene phylogenies reveal a complex history of gene duplications and repeated subfunctionalization of paralogs. Results EScaAG1 and EScaAG2, duplicate AG homologs in the basal eudicot Eschscholzia californica show a high degree of similarity in sequence and expression, although EScaAG2 expression is lower than EScaAG1 expression. Functional studies employing virus-induced gene silencing (VIGS) demonstrate that knock down of EScaAG1 and 2 function leads to homeotic conversion of stamens into petaloid structures and defects in floral meristem termination. However, carpels are transformed into petaloid organs rather than sepaloid structures. We also show that a reduction of EScaAG1 and EScaAG2 expression leads to significantly increased expression of a subset of floral homeotic B genes. Conclusions This work presents expression and functional analysis of the two basal eudicot AG homologs. The reduction of EScaAG1 and 2 functions results in the change of stamen to petal identity and a transformation of the central whorl organ identity from carpel into petal identity. Petal identity requires the presence of the floral homeotic B function and our results show that the expression of a subset of B function genes extends into the central whorl when the C function is reduced. We propose a model for the evolution of B function regulation by C function suggesting that the mode of B function gene regulation found in Eschscholzia is ancestral and the C-independent regulation as found in Arabidopsis is

  20. Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing L-amino acid oxidase from ink of the sea hare Aplysia californica.

    PubMed

    Yang, Hsiuchin; Johnson, Paul Micah; Ko, Ko-Chun; Kamio, Michiya; Germann, Markus W; Derby, Charles D; Tai, Phang C

    2005-09-01

    A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica was cloned and sequenced, and is the first sea hare antimicrobial protein to be functionally expressed in E. coli. Sequence analysis suggested that this protein is a flavin-containing l-amino acid oxidase (LAAO), with one predicted potential glycosylation site, although the glycosylation could not be experimentally confirmed. This protein, which we call ;escapin', has high sequence similarity to several other gastropod proteins. Escapin was verified by NMR, mass spectroscopy and HPLC to have FAD as its flavin cofactor. Escapin's antimicrobial effects, bacteriostasis and bactericidal, were determined using a combination of two assays: (1) incubation of bacteria on solid media followed by assessment of inhibition by direct observation of zones of inhibition or by turbidity measurements; and (2) incubation of bacteria in liquid media followed by counting viable colonies after growing on agar plates. Native escapin inhibited the growth of Gram-positive and Gram-negative bacteria, including marine bacteria (Vibrio harveyii and Staphylococcus aureus) and pathogenic bacteria (Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa). Escapin also inhibited the growth of yeast and fungi, with different efficacies. Escapin's antimicrobial activity was concentration dependent and did not decrease when stored for more than 5 months at room temperature. Escapin was bacteriostatic and not bactericidal in minimal media (e.g. salt media) with glucose, yeast extract, and a mixture of 20 amino acids each at 50 micromol l(-1), but was bactericidal in media enriched with Tryptone Peptone. Escapin was also strongly bactericidal in media with l-lysine at concentrations as low as 3 mmol l(-1) and slightly bactericidal in 50 mmol l(-1) l-arginine, but not in most other amino acids even at 50 mmol l(-1). Escapin had high oxidase activity (producing hydrogen

  1. Calcium/calmodulin-dependent nitric oxide synthase activity in the CNS of Aplysia californica: biochemical characterization and link to cGMP pathways.

    PubMed

    Bodnárová, Michaela; Martásek, Pavel; Moroz, Leonid L

    2005-04-01

    We characterized enzymatic activity of nitric oxide synthase (NOS) in the central nervous system of Aplysia californica, a popular experimental model in cellular and system neuroscience, and provided biochemical evidence for NO-cGMP signaling in molluscs. Aplysia NOS (ApNOS) activity, determined as citrulline formation, revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence and it was inhibited by 50% with 5mM of W7 hydrochloride (a potent Ca/CaM-dependent phosphodiesterase inhibitor). A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia. Specifically, the ApNOS was inhibited by 65-92% with 500 microM of L-NAME (a competitive NOS inhibitor) whereas d-NAME at the same concentration had no effect. S-Ethylisothiourea hydrobromide (5mM), a selective inhibitor of all NOS isoforms, suppressed ApNOS by 85%, l-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL, 5mM), an iNOS inhibitor, by 78% and L-thiocitrulline (5mM) (an inhibitor of nNOS and iNOS) by greater than 95%. Polyclonal antibodies raised against rat nNOS hybridized with a putative purified ApNOS (160 kDa protein) from partially purified central nervous system homogenates in Western blot studies. Consistent with other studies, the activity of soluble guanylyl cyclase was stimulated as a result of NO interaction with its heme prosthetic group. The basal levels of cGMP were estimated by radioimmunoassay to be 44.47 fmol/microg of protein. Incubation of Aplysia CNS with the NO donors DEA/NONOate (diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate - 1mM) or S-nitroso-N-acetylpenicillamine (1mM) and simultaneous phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (1mM) prior to the assay showed a 26-80 fold increase in basal cGMP levels. Addition of ODQ (1H-[1,2,4]oxadiazolo[4,3-a] quinoxaline-1-one - 1mM), a selective inhibitor of soluble guanylyl cyclase, completely abolished this effect. This confirms that NO may indeed function as a

  2. The seirena B class floral homeotic mutant of California Poppy (Eschscholzia californica) reveals a function of the enigmatic PI motif in the formation of specific multimeric MADS domain protein complexes.

    PubMed

    Lange, Matthias; Orashakova, Svetlana; Lange, Sabrina; Melzer, Rainer; Theißen, Günter; Smyth, David R; Becker, Annette

    2013-02-01

    The products of B class floral homeotic genes specify petal and stamen identity, and loss of B function results in homeotic conversions of petals into sepals and stamens into carpels. Here, we describe the molecular characterization of seirena-1 (sei-1), a mutant from the basal eudicot California poppy (Eschscholzia californica) that shows homeotic changes characteristic of floral homeotic B class mutants. SEI has been previously described as EScaGLO, one of four B class-related MADS box genes in California poppy. The C terminus of SEI, including the highly conserved PI motif, is truncated in sei-1 proteins. Nevertheless, like the wild-type SEI protein, the sei-1 mutant protein is able to bind CArG-boxes and can form homodimers, heterodimers, and several higher order complexes with other MADS domain proteins. However, unlike the wild type, the mutant protein is not able to mediate higher order complexes consisting of specific B, C, and putative E class related proteins likely involved in specifying stamen identity. Within the PI motif, five highly conserved N-terminal amino acids are specifically required for this interaction. Several families lack this short conserved sequence, including the Brassicaceae, and we propose an evolutionary scenario to explain these functional differences.

  3. The seirena B Class Floral Homeotic Mutant of California Poppy (Eschscholzia californica) Reveals a Function of the Enigmatic PI Motif in the Formation of Specific Multimeric MADS Domain Protein Complexes[C][W][OA

    PubMed Central

    Lange, Matthias; Orashakova, Svetlana; Lange, Sabrina; Melzer, Rainer; Theißen, Günter; Smyth, David R.; Becker, Annette

    2013-01-01

    The products of B class floral homeotic genes specify petal and stamen identity, and loss of B function results in homeotic conversions of petals into sepals and stamens into carpels. Here, we describe the molecular characterization of seirena-1 (sei-1), a mutant from the basal eudicot California poppy (Eschscholzia californica) that shows homeotic changes characteristic of floral homeotic B class mutants. SEI has been previously described as EScaGLO, one of four B class–related MADS box genes in California poppy. The C terminus of SEI, including the highly conserved PI motif, is truncated in sei-1 proteins. Nevertheless, like the wild-type SEI protein, the sei-1 mutant protein is able to bind CArG-boxes and can form homodimers, heterodimers, and several higher order complexes with other MADS domain proteins. However, unlike the wild type, the mutant protein is not able to mediate higher order complexes consisting of specific B, C, and putative E class related proteins likely involved in specifying stamen identity. Within the PI motif, five highly conserved N-terminal amino acids are specifically required for this interaction. Several families lack this short conserved sequence, including the Brassicaceae, and we propose an evolutionary scenario to explain these functional differences. PMID:23444328

  4. Application of an integrated LC-UV-MS-NMR platform to the identification of secondary metabolites from cell cultures: benzophenanthridine alkaloids from elicited Eschscholzia californica (california poppy) cell cultures().

    PubMed

    Gathungu, Rose M; Oldham, John T; Bird, Susan S; Lee-Parsons, Carolyn W T; Vouros, Paul; Kautz, Roger

    2012-01-01

    Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for obtaining natural products of medical relevance. Cultures can be optimized for high yields of desired metabolites using rapid profiling assays such as HPLC. We describe an approach to establishing a rapid assay for profiling cell culture expression systems using a novel microscale LC-UV-MS-NMR platform, designed to acquire both MS and NMR each at their optimal sensitivity, by using nanosplitter MS from 4 mm analytical HPLC columns, and offline microdroplet NMR. The approach is demonstrated in the analysis of elicited Eschscholzia californica cell cultures induced with purified yeast extract to produce benzophenanthridine alkaloids. Preliminary HPLC-UV provides an overview of the changes in the production of alkaloids with time after elicitation. At the time point corresponding to the production of the most alkaloids, the integrated LC-MS-microcoil NMR platform is used for structural identification of extracted alkaloids. Eight benzophenanthridine alkaloids were identified at the sub-microgram level. This paper demonstrates the utility of the nanosplitter LC-MS/microdroplet NMR platform when establishing cell culture expression systems.

  5. Chemical kinetic measurements of the effect of trans- and cis-3,3'-Bis[(trimethylammonio)methyl]azobenzene bromide on acetylcholine receptor mediated ion translocation in Electrophorus electricus and Torpedo californica.

    PubMed

    Delcour, A H; Hess, G P

    1986-04-08

    A quench-flow technique was used to study the effect of trans- and cis-3,3'-bis[(trimethylammonio)methyl]azobenzene bromide (trans- and cis-Bis-Q), photoisomerizable ligands, on the acetylcholine receptor in vesicles prepared from the electric organ of Electrophorus electricus and of Torpedo californica. In E. electricus, two rate coefficients of the receptor-mediated translocation of 86Rb+ induced with trans-Bis-Q were measured: JA, the rate coefficient for ion flux, and alpha, the rate coefficient for receptor inactivation (desensitization). Both rate coefficients increase with increasing concentrations of Bis-Q up to 50 microM. At higher concentrations JA decreases in a concentration-dependent manner while alpha remains unchanged. This effect was previously observed with suberyldicholine [Pasquale, E. B., Takeyasu, K., Udgaonkar, J., Cash, D.J., Severski, M.C., & Hess, G. P. (1983) Biochemistry 22, 5967-5973] and with acetylcholine [Takeyasu, K., Udgaonkar, J., & Hess, G. P. (1983) Biochemistry 22, 5973-5978] and was analyzed in terms of a minimum mechanism that accounts for the properties of activation, desensitization, and inhibition of the receptor. Two molecules of trans-Bis-Q must be bound for the channel to open, but at concentrations greater than 50 microM the population of open channels decreases because of the additional binding of one molecule of trans-Bis-Q to a regulatory inhibitory site, independent of the activating sites. cis-Bis-Q does not induce transmembrane ion flux, but it does inhibit the response of the receptor to acetylcholine and induces inactivation (desensitization) in the micromolar range.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen: Identification of N-formylkynurenine tryptophan derivatives within the active-site gorge of its complex with the photosensitizer methylene blue

    PubMed Central

    Triquigneaux, Mathilde M.; Ehrenshaft, Marilyn; Roth, Esther; Silman, Israel; Ashani, Yakov; Mason, Ronald P.; Weiner, Lev; Deterding, Leesa J.

    2013-01-01

    Synopsis The principal role of acetylcholinesterase (AChE) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a catalytic ‘anionic’ subsite (CAS) and a second peripheral ‘anionic’ site (PAS), the gorge mouth, both of which bind acetylcholine via π-cation interactions, primarily with two conserved tryptophans (Trps). It was earlier shown that generation of 1O2 by illumination of methylene blue (MB) causes irreversible inactivation of Torpedo californica AChE (TcAChE), and suggested that photo-oxidation of Trps might be responsible. In the present study, structural modification of the TcAChE Trps induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and mass spectrometry. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp 84 and Trp 279 – present at the CAS and PAS, respectively. Peptides containing these two oxidized Trp residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus, the bound MB elicited selective action of 1O2 on Trp residues facing onto the water-filled active-site gorge. Our findings thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents. PMID:22888904

  7. Efficacy of Three Vaccines in Protecting Western Scrub-Jays (Aphelocoma californica) from Experimental Infection with West Nile Virus: Implications for Vaccination of Island Scrub-Jays (Aphelocoma insularis)

    PubMed Central

    Wheeler, Sarah S.; Langevin, Stanley; Woods, Leslie; Carroll, Brian D.; Vickers, Winston; Morrison, Scott A.; Chang, Gwong-Jen J.; Reisen, William K.

    2011-01-01

    Abstract The devastating effect of West Nile virus (WNV) on the avifauna of North America has led zoo managers and conservationists to attempt to protect vulnerable species through vaccination. The Island Scrub-Jay (Aphelocoma insularis) is one such species, being a corvid with a highly restricted insular range. Herein, we used congeneric Western Scrub-Jays (Aphelocoma californica) to test the efficacy of three WNV vaccines in protecting jays from an experimental challenge with WNV: (1) the Fort Dodge West Nile-Innovator® DNA equine vaccine, (2) an experimental DNA plasmid vaccine, pCBWN, and (3) the Merial Recombitek® equine vaccine. Vaccine efficacy after challenge was compared with naïve and nonvaccinated positive controls and a group of naturally immune jays. Overall, vaccination lowered peak viremia compared with nonvaccinated positive controls, but some WNV-related pathology persisted and the viremia was sufficient to possibly infect susceptible vector mosquitoes. The Fort Dodge West Nile-Innovator DNA equine vaccine and the pCBWN vaccine provided humoral immune priming and limited side effects. Five of the six birds vaccinated with the Merial Recombitek vaccine, including a vaccinated, non-WNV challenged control, developed extensive necrotic lesions in the pectoral muscle at the vaccine inoculation sites, which were attributed to the Merial vaccine. In light of the well-documented devastating effects of high morbidity and mortality associated with WNV infection in corvids, vaccination of Island Scrub-Jays with either the Fort Dodge West Nile-Innovator DNA vaccine or the pCBWN vaccine may increase the numbers of birds that would survive an epizootic should WNV become established on Santa Cruz Island. PMID:21438693

  8. The occlusion-derived virus envelope protein ODV-E56 is required for optimal oral infectivity but is not essential for virus binding and fusion

    USDA-ARS?s Scientific Manuscript database

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine the role of ODV-E56 in oral infectivity, we produced recombinant EGFP-expressing AcMNPV clones (Ac69GFP-e56lacZ and AcIEGFP-e56lac...

  9. Repeat sequences from complex ds DNA viruses can be used as minisatellite probes for DNA fingerprinting.

    PubMed

    Crawford, A M; Buchanan, F C; Fraser, K M; Robinson, A J; Hill, D F

    1991-01-01

    In a search for new fingerprinting probes for use with sheep, repeat sequences derived from five poxviruses, an iridovirus and a baculovirus were screened against DNA from sheep pedigrees. Probes constructed from portions of the parapox viruses, orf virus and papular stomatitis virus and the baculovirus from the alfalfa looper, Autographa californica, nuclear polyhedrosis virus all gave fingerprint patterns. Probes from three other poxviruses and an iridovirus did not give useful banding patterns.

  10. Mechanisms of Action of Escapin, a Bactericidal Agent in the Ink Secretion of the Sea Hare Aplysia californica: Rapid and Long-Lasting DNA Condensation and Involvement of the OxyR-Regulated Oxidative Stress Pathway

    PubMed Central

    Ko, Ko-Chun; Tai, Phang C.

    2012-01-01

    The marine snail Aplysia californica produces escapin, an l-amino acid oxidase, in its defensive ink. Escapin uses l-lysine to produce diverse products called escapin intermediate products of l-lysine (EIP-K), including α-amino-ε-caproic acid, Δ1-piperidine-2-carboxylic acid, and Δ2-piperidine-2-carboxylic acid. EIP-K and H2O2 together, but neither alone, is a powerful bactericide. Here, we report bactericidal mechanisms of escapin products on Escherichia coli. We show that EIP-K and H2O2 together cause rapid and long-lasting DNA condensation: 2-min treatment causes significant DNA condensation and killing, and 10-min treatment causes maximal effect, lasting at least 70 h. We isolated two mutants resistant to EIP-K plus H2O2, both having a single missense mutation in the oxidation regulatory gene, oxyR. A complementation assay showed that the mutated gene, oxyR(A233V), renders resistance to EIP-K plus H2O2, and a gene dosage effect leads to reduction of resistance for strains carrying wild-type oxyR. Temperature stress with EIP-K does not produce the bactericidal effect, suggesting the effect is due to a specific response to oxidative stress. The null mutant for any single DNA-binding protein—Dps, H-NS, Hup, Him, or MukB—was not resistant to EIP-K plus H2O2, suggesting that no single DNA-binding protein is necessary to mediate this bactericidal effect, but allowing for the possibility that EIP-K plus H2O2 could function through a combination of DNA-binding proteins. The bactericidal effect of EIP-K plus H2O2 was eliminated by the ferrous ion chelator 1,10-phenanthroline, and it was reduced by the hydroxyl radical scavenger thiourea, suggesting hydroxyl radicals mediate the effects of EIP-K plus H2O2. PMID:22232273

  11. The chemistry of escapin: identification and quantification of the components in the complex mixture generated by an L-amino acid oxidase in the defensive secretion of the sea snail Aplysia californica.

    PubMed

    Kamio, Michiya; Ko, Ko-Chun; Zheng, Shilong; Wang, Binghe; Collins, Stacy L; Gadda, Giovanni; Tai, Phang C; Derby, Charles D

    2009-01-01

    Escapin is an L-amino acid oxidase in the ink of a marine snail, the sea hare Aplysia californica, which oxidizes L-lysine (1) to produce a mixture of chemicals which is antipredatory and antimicrobial. The goal of our study was to determine the identity and relative abundance of the constituents of this mixture, using molecules generated enzymatically with escapin and also using products of organic syntheses. We examined this mixture under the natural range of pH values for ink-from approximately 5 at full strength to approximately 8 when fully diluted in sea water. The enzymatic reaction likely forms an equilibrium mixture containing the linear form alpha-keto-epsilon-aminocaproic acid (2), the cyclic imine Delta(1)-piperidine-2-carboxylic acid (3), the cyclic enamine Delta(2)-piperidine-2-carboxylic acid (4), possibly the linear enol 6-amino-2-hydroxy-hex-2-enoic acid (7), the alpha-dihydroxy acid 6-amino-2,2-dihydroxy-hexanoic acid (8), and the cyclic aminol 2-hydroxy-piperidine-2-carboxylic acid (9). Using NMR and mass spectroscopy, we show that 3 is the major component of this enzymatic product at any pH, but at more basic conditions, the equilibrium shifts to produce relatively more 4, and at acidic conditions, the equilibrium shifts to produce relatively more 2, 7, and/or 9. Studies of escapin's enzyme kinetics demonstrate that because of the high concentrations of escapin and L-lysine in the ink secretion, millimolar concentrations of 3, H(2)O(2), and ammonia are produced, and also lower concentrations of 2, 4, 7, and 9 as a result. We also show that reactions of this mixture with H(2)O(2) produce delta-aminovaleric acid (5) and delta-valerolactam (6), with 6 being the dominant component under the naturally acidic conditions of ink. Thus, the product of escapin's action on L-lysine contains an equilibrium mixture that is more complex than previously known for any L-amino acid oxidase.

  12. Mechanisms of action of escapin, a bactericidal agent in the ink secretion of the sea hare Aplysia californica: rapid and long-lasting DNA condensation and involvement of the OxyR-regulated oxidative stress pathway.

    PubMed

    Ko, Ko-Chun; Tai, Phang C; Derby, Charles D

    2012-04-01

    The marine snail Aplysia californica produces escapin, an L-amino acid oxidase, in its defensive ink. Escapin uses L-lysine to produce diverse products called escapin intermediate products of L-lysine (EIP-K), including α-amino-ε-caproic acid, Δ¹-piperidine-2-carboxylic acid, and Δ²-piperidine-2-carboxylic acid. EIP-K and H₂O₂ together, but neither alone, is a powerful bactericide. Here, we report bactericidal mechanisms of escapin products on Escherichia coli. We show that EIP-K and H₂O₂ together cause rapid and long-lasting DNA condensation: 2-min treatment causes significant DNA condensation and killing, and 10-min treatment causes maximal effect, lasting at least 70 h. We isolated two mutants resistant to EIP-K plus H₂O₂, both having a single missense mutation in the oxidation regulatory gene, oxyR. A complementation assay showed that the mutated gene, oxyR(A233V), renders resistance to EIP-K plus H₂O₂, and a gene dosage effect leads to reduction of resistance for strains carrying wild-type oxyR. Temperature stress with EIP-K does not produce the bactericidal effect, suggesting the effect is due to a specific response to oxidative stress. The null mutant for any single DNA-binding protein--Dps, H-NS, Hup, Him, or MukB--was not resistant to EIP-K plus H₂O₂, suggesting that no single DNA-binding protein is necessary to mediate this bactericidal effect, but allowing for the possibility that EIP-K plus H₂O₂ could function through a combination of DNA-binding proteins. The bactericidal effect of EIP-K plus H₂O₂ was eliminated by the ferrous ion chelator 1,10-phenanthroline, and it was reduced by the hydroxyl radical scavenger thiourea, suggesting hydroxyl radicals mediate the effects of EIP-K plus H₂O₂.

  13. Overexpression of Bm65 correlates with reduced susceptibility to inactivation by UV light.

    PubMed

    Tang, Qi; Hu, Zhaoyang; Yang, Yanhua; Wu, Huiling; Qiu, Lipeng; Chen, Keping; Li, Guohui

    2015-05-01

    Ultraviolet (UV) light is one of the factors that causes baculovirus inactivation. However, little is known about the response of baculoviruses to UV light. In the present study, Bombyx mori nucleopolyhedrovirus (BmNPV) orf 65 (Bm65), the homolog of Autographa californica nucleopolyhedrovirus orf 79 (Ac79), a predicted endonuclease, was analyzed. Preliminary results indicated that Bm65 mainly accumulated within the nucleus and could improve the survival rate of Escherichia coli (E. coli) and BmNPV BVs after UV radiation, suggesting that Bm65 was involved in the repair of UV-induced DNA damage.

  14. Transposon-mediated mutagenesis of a baculovirus.

    PubMed

    Fraser, M J; Brusca, J S; Smith, G E; Summers, M D

    1985-09-01

    Spontaneous mutants of insect nuclear polyhedrosis viruses of Autographa californica (AcMNPV) and Galleria mellonella (GmMNPV) were analyzed. These mutants produce few polyhedra in infected cells and have insertions of host cell DNA. All the insertions mapped to two adjacent sites in the genome. The junctions between two host insertions and the viral DNA were sequenced. One of the insertions contained a perfect 7-bp inverted terminal repeat, and had caused a direct duplication of 4 bp of viral DNA at the point of insertion. Therefore, this insertion sequence has properties of a transposon of the host cell Trichoplusia ni.

  15. Baculovirus-mediated interferon alleviates dimethylnitrosamine-induced liver cirrhosis symptoms in a murine model.

    PubMed

    Nishibe, Y; Kaneko, H; Suzuki, H; Abe, T; Matsuura, Y; Takaku, H

    2008-07-01

    The wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a range of mammalian cell types in vitro but does not replicate in these cells. The current study investigated the in vivo effect of AcMNPV in the mouse model of liver cirrhosis induced by the mutagen dimethylnitrosamine. Intraperitoneal injection of AcMNPV induced an immune response. The baculovirus was taken up by the liver and spleen where it suppressed liver injury and fibrosis through the induction of interferons. This study presents the first evidence of the feasibility of using baculovirus to treat liver cirrhosis.

  16. Production of human beta interferon in insect cells infected with a Baculovirus expression vector

    SciTech Connect

    Smith, G.E.; Summers, M.D.; Fraser, M.J.

    1983-12-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for human beta interferon. By using specially constructed plasmids, the protein-coding sequences for interferon were linked to the AcNPV promoter for the gene encoding for polyhedrin, the major occlusion protein. The interferon gene was inserted at various locations relative to the AcNPV polyhedrin transcriptional and translational signals, and the interferon-polyhedrin hybrid genes were transferred to infectious AcNPV expression vectors. Biologically active interferon was produced, and greater than 95% was secreted from infected insect cells. A maximum of ca. 5 x 10/sup 6/ U of interferon activity was produced by 10/sup 6/ infected cells. These results demonstrate that AcNPV should be suitable for use as a eucaryotic expression vector for the production of products from cloned genes.

  17. Functional expression of the Aequorea victoria green fluorescent protein in insect cells using the baculovirus expression system.

    PubMed

    Reiländer, H; Haase, W; Maul, G

    1996-02-06

    A DNA fragment encoding the green fluorescent protein (GFP) was isolated via PCR from a jellyfish Aequorea victoria cDNA, cloned and sequenced. Subsequently, a recombinant baculovirus bearing the coding region of the GFP under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin gene promoter was constructed and isolated. High-level expression of GFP could be easily monitored in Spodoptera frugiperda (Sf9) insect cells after infection with recombinant baculovirus, due to the intrinsic fluorescence (lambda(max) = 508 nm) of the recombinant protein after excitation with blue light (lambda(max) = 400 nm). The functional recombinant GFP displayed an apparent molecular mass of approximately 43 kDa and the fluorescence emission spectrum of the recombinant protein was virtually identical to that of the native green fluorescent protein.

  18. Localization of a Baculovirus-Induced Chitinase in the Insect Cell Endoplasmic Reticulum

    PubMed Central

    Thomas, Carole J.; Brown, Helen L.; Hawes, Chris R.; Lee, Bum Yong; Min, Mi-Kyung; King, Linda A.; Possee, Robert D.

    1998-01-01

    Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of p10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected Trichoplusia ni larvae by the same period. PMID:9811762

  19. Actin-based motility drives baculovirus transit to the nucleus and cell surface.

    PubMed

    Ohkawa, Taro; Volkman, Loy E; Welch, Matthew D

    2010-07-26

    Most viruses move intracellularly to and from their sites of replication using microtubule-based mechanisms. In this study, we show that nucleocapsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus undergo intracellular motility driven by actin polymerization. Motility requires the viral P78/83 capsid protein and the host Arp2/3 complex. Surprisingly, the virus directs two sequential and coordinated phases of actin-based motility. Immediately after cell entry, motility enables exploration of the cytoplasm and collision with the nuclear periphery, speeding nuclear entry and the initiation of viral gene expression. Nuclear entry itself requires transit through nuclear pore complexes. Later, after the onset of early gene expression, motility is required for accumulation of a subpopulation of nucleocapsids in the tips of actin-rich surface spikes. Temporal coordination of actin-based nuclear and surface translocation likely enables rapid transmission to neighboring cells during infection in insects and represents a distinctive evolutionary strategy for overcoming host defenses.

  20. Expression from baculovirus and serological reactivity of the nucleocapsid protein of dolphin morbillivirus.

    PubMed

    Grant, Rebecca J; Kelley, Karen L; Maruniak, James E; Garcia-Maruniak, Alejandra; Barrett, Tom; Manire, Charles A; Romero, Carlos H

    2010-07-14

    The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Autographa californica nuclear polyhedrosis virus) vector and shown by SDS-PAGE and Western blot analysis to be about 57 kDa. Transmission electron microscopy revealed fully assembled nucleocapsid-like particles (NLPs) exhibiting the typical helical herringbone morphology. These NLPs were approximately 20-22 nm in diameter and varied in length from 50 to 100 nm. Purified DMV-N protein was used as antigen in an indirect ELISA (iELISA) and shown to react with rabbit and human antisera to measles virus (MV) and dog sera with antibodies to canine distemper virus (CDV). The iELISA was used for the demonstration of morbillivirus antibodies in the serum of cetaceans and manatees, showing potential as a serological tool for the mass screening of morbillivirus antibodies in marine mammals. (c) 2009 Elsevier B.V. All rights reserved.

  1. Aggregation of AcMNPV LEF-10 and Its Impact on Viral Late Gene Expression

    PubMed Central

    Xu, Xiaodong; Zhou, Xinyu; Nan, Hao; Zhao, Yu; Bai, Yu; Ou, Yanmei; Chen, Hongying

    2016-01-01

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor gene lef-10 has been identified to be required for viral late gene expression by transient expression assay. Our previous work has shown that the gene product LEF-10 can form very stable high-molecular-weight complexes, but the structure and function of the protein remain unknown. In this study, we demonstrated that LEF-10 was essential for the replication of AcMNPV, and its truncated fragment containing amino acid residues 1 to 48 were sufficient to support the virus survival. Our data also suggested that the LEF-10 could spontaneously aggregate to form punctate spots in virus infected Sf9 cells at low frequency, and the aggregation of the protein could be induced by LEF-10 over-expression. When the protein aggregated to form punctate spots, soluble LEF-10 proteins were depleted and this could result in the down-regulation of viral late gene expression. PMID:27152613

  2. The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins.

    PubMed

    Hattori, Tomomi; Nakanishi, Kohei; Mori, Takaaki; Tomita, Masahiro; Tsumoto, Kanta

    2016-01-01

    Budded virus (BV) particles of baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) are harvested from the supernatant of liquid culture of Sf9 host cells by ultracentrifugation. Using polyacrylamide gel electrophoresis, Western blot and transmission electron microscopy (TEM) of BV samples fractionated closely by sucrose density gradient centrifugation, we observed that BVs exhibited different qualities depending on whether they had been harvested from the supernatant from a standing (static), shaking (suspension), or standing/shaking (pre-/post-infection) culture of Sf9 cells. The amount of BV protein apparently increased in the order of standing, standing/shaking, and shaking procedure, and the yield of intact particles showed an opposite trend. TEM observation clearly showed that appropriate fractions of the standing and standing/shaking cultures contained more intact BV particles than those from the shaking culture. These results suggest that the qualities of recombinant BV particles may be related to the culture conditions of the host cells.

  3. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.

    PubMed Central

    Kitts, P A; Ayres, M D; Possee, R D

    1990-01-01

    Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location. Images PMID:2216760

  4. Host Range Factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) Is an Essential Viral Factor Required for Productive Infection of NPVs in IPLB-Ld652Y Cells Derived from L. dispar

    PubMed Central

    Ishikawa, Hiroki; Ikeda, Motoko; Felipe Alves, Cristiano A.; Thiem, Suzanne M.; Kobayashi, Michihiro

    2004-01-01

    Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells. PMID:15507661

  5. Identification of a Novel Apoptosis Suppressor Gene from the Baculovirus Lymantria dispar Multicapsid Nucleopolyhedrovirus ▿

    PubMed Central

    Yamada, Hayato; Shibuya, Miyuki; Kobayashi, Michihiro; Ikeda, Motoko

    2011-01-01

    Ld652Y cells from Lymantria dispar readily undergo apoptosis upon infection with a variety of nucleopolyhedroviruses (NPVs), while L. dispar multicapsid NPV (LdMNPV) infection of Ld652Y cells results in the production of a high titer of progeny viruses. Here, we identify a novel LdMNPV apoptosis suppressor gene, apsup, which functions to suppress apoptosis induced in Ld652Y cells by infection with vAcΔp35, a p35-defective recombinant Autographa californica MNPV. apsup also suppresses apoptosis of Ld652Y cells induced by actinomycin D and UV exposure. Apsup is expressed in LdMNPV-infected Ld652Y cells late in infection, and RNA interference-mediated apsup ablation induces apoptosis of LdMNPV-infected Ld652Y cells. PMID:21411519

  6. Baculovirus expression of the small genome segment of Hantaan virus and potential use of the expressed nucleocapsid protein as a diagnostic antigen.

    PubMed

    Schmaljohn, C S; Sugiyama, K; Schmaljohn, A L; Bishop, D H

    1988-04-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for the nucleocapsid protein gene of Hantaan virus. Two different cDNA clones representing the small genome segment of Hantaan virus were inserted into the transfer vector pAcYM1, and recombinants were generated by replacement of a portion of the baculovirus polyhedrin gene with the foreign, Hantaan virus gene. Recombinants containing both the first and second ATG initiation codons of the Hantaan virus gene produced nucleocapsid protein, while those containing only the second codon did not. The expressed nucleocapsid protein was evaluated as a potential diagnostic antigen with a variety of hantavirus-immune sera. The high levels of expression obtained, specific serological reactivity with immune sera and the low level of biological containment required for production of this protein all suggest a significant advantage over authentic viral antigen for diagnosis of hantavirus infection.

  7. Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system.

    PubMed

    Zhang, F; Wolff, M W; Williams, D; Busch, K; Lang, S C; Murhammer, D W; Linhardt, R J

    2001-02-01

    Human secreted alkaline phosphatase (SEAP) was produced in a stably-transformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.

  8. Hyperactivity and tree-top disease induced by the baculovirus AcMNPV in Spodoptera exigua larvae are governed by independent mechanisms

    NASA Astrophysics Data System (ADS)

    van Houte, Stineke; Ros, Vera I. D.; van Oers, Monique M.

    2014-04-01

    Although many parasites are known to manipulate the behavior of their hosts, the mechanisms underlying such manipulations are largely unknown. Baculoviruses manipulate the behavior of caterpillar hosts by inducing hyperactivity and by inducing climbing behavior leading to death at elevated positions (tree-top disease or Wipfelkrankheit). Whether hyperactivity and tree-top disease are independent manipulative strategies of the virus is unclear. Recently, we demonstrated the involvement of the protein tyrosine phosphatase ( ptp) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in the induction of hyperactivity in Spodoptera exigua larvae. Here we show that AcMNPV ptp is not required for tree-top disease, indicating that in S. exigua baculovirus-induced hyperactivity and tree-top disease are independently induced behaviors that are governed by distinct mechanisms.

  9. AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene

    SciTech Connect

    McCarthy, Christina B.; Theilmann, David A.

    2008-05-25

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC{sup ac142REP-ac143KO}). Fluorescence and light microscopy showed that infection by AcBAC{sup ac142REP-ac143KO} is limited to a single cell and titration assays confirmed that AcBAC{sup ac142REP-ac143KO} was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC{sup ac142REP-ac143KO} transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.

  10. AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene.

    PubMed

    McCarthy, Christina B; Theilmann, David A

    2008-05-25

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC(ac142)(REP-ac143KO)). Fluorescence and light microscopy showed that infection by AcBAC(ac142)(REP-ac143KO) is limited to a single cell and titration assays confirmed that AcBAC(ac142)(REP-ac143KO) was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC(ac142)(REP-ac143KO) transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.

  11. Characterization of glycolipids synthesized in an identified neuron of Aplysia californica.

    PubMed

    Sherbany, A A; Ambron, R T; Schwartz, J H

    1984-07-01

    Because radioactive precursors can be injected directly into the cell body or axon of R2, a giant, identified neuron of the Aplysia abdominal ganglion, it was possible to show that glycolipid is synthesized in the cell body, inserted into membranes along with glycoprotein, and then exported into the axon within organelles that are moved by fast axonal transport. After intrasomatic injection of N-[3H]-acetyl-D-galactosamine, five major 3H-glycolipids were identified using thin layer polysilicic acid glass fiber chromatography. At least two of the lipids are negatively charged. Analysis of 32P-labeled lipid from the abdominal ganglion revealed the presence of 2-aminoethylphosphonate, indicating that these polar substances are sphingophosphonoglycolipids. The major 3H-glycolipids synthesized in R2 are similar to a family of phospholipids isolated from the skin of A. kurodai, previously characterized by Araki et al. (Araki, S., Y. Komai, and M. Satake (1980) Biochem J. 87: 503-510). Since sialic acid is absent in Aplysia as in other invertebrates, these polar glycolipids may function like gangliosides in vertebrates. The polar 3H-glycolipids are synthesized and incorporated into intracytoplasmic membranes solely in the cell body. Direct injection of the labeled sugar into the axon revealed no local synthesis or exchange of glycolipid. Moreover, there was no indication for transfer from glial cells into axoplasm. Although the incorporation of N-[3H]-acetyl-D-galactosamine into glycolipid is not affected by anisomycin, an effective inhibitor of protein synthesis, the export into the axon of membranes containing the newly synthesized lipid is completely blocked by the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Functional and structural correlates of cell size in paracerebral neurons of Pleurobranchaea californica.

    PubMed

    Kovac, M P; Davis, W J; Matera, E; Gillette, R

    1982-05-01

    1. The paracerebral neurons (PCNs) in the brain of the mollusk Pleurobranchaea are a population of 12-16 interneurons that send axons to the buccal ganglion and control cyclic feeding behavior (9). In the present study we show that the PCNs differ in size and that a number of functional and structural properties of the PCNs are closely correlated with cell size. 2. PCN soma diameter varies from about 30 to 120 micrometers. The diameters segregate into two distinct but overlapping populations, which correspond to independently assigned functional classifications of "tonic" and "phasic" PCNs. The mean soma diameters of two populations were 63 and 84 micrometers, respectively. 3. Two morphological features vary systematically with PCN soma size. First, soma diameter, axonal conduction velocity, and extracellular spike amplitude were positively correlated; therefore, PCN axon diameter presumably increases with soma diameter. Second, intrasomatic injection of lucifer yellow revealed that the small, tonic PCNs are multipolar, while the large, phasic PCNs are generally monopolar neurons. 4. Small PCNs discharge tonically in response to sustained current injection and have a weak effect on cyclic motor output recorded from nerves that innervate feeding muscles. In contrast, the large PCNs discharge phasically in bursts of action potentials that are coordinated with the cyclic motor output and have a comparatively strong effect on the rhythm. The motor effects of simultaneous tonic and phasic PCN stimulation are additive. 5. Tonic and phasic PCNs innervate different but partially overlapping populations of feeding motor neurons. Phasic PCNs typically inhibit motor neurons exiting buccal root 3, while tonic PCNs either have no effect or are weakly excitatory. 6. Tonic and phasic PCNs exhibit different intrinsic properties. In comparison with phasic PCNs, tonic PCNs have higher input resistances, higher spontaneous discharge rates at rest potential, lower firing thresholds to intrasomatically injected current, lower absolute voltage thresholds, greater pacemaker sensitivity, and greater total capacitance. 7. Tonic and phasic PCNs exhibit different input properties. Tonic PCNs are recruited before phasic ones during cycylic buccal motor output induced by stomatogastric nerve stimulation. Phasic PCNs receive powerful, cycylic inhibition that is not shared by tonic PCNs. In addition, extracellular stimulation of the large oral veil nerve of the brain excites tonic PCNs but causes a biphasic postsynaptic potential (PSP) in phasic PCNs that has a net inhibitory effect. Some excitatory synaptic input to phasic and tonic PCNs is unshared, while some is shared. 8. It is concluded that these command interneurons obey the size principle discovered earlier in motor neurons (4, 13-16). Cell size per se is not the causal variable, however; instead the underlying causes of the differences between small and large PCNs include different input and output organizations as well as different intrinsic functional and morphological properties.

  13. Connecting Model Species to Nature: Predator-Induced Long-Term Sensitization in "Aplysia Californica"

    ERIC Educational Resources Information Center

    Mason, Maria J.; Watkins, Amanda J.; Wakabayashi, Jordann; Buechler, Jennifer; Pepino, Christine; Brown, Michelle; Wright, William G.

    2014-01-01

    Previous research on sensitization in "Aplysia" was based entirely on unnatural noxious stimuli, usually electric shock, until our laboratory found that a natural noxious stimulus, a single sublethal lobster attack, causes short-term sensitization. We here extend that finding by demonstrating that multiple lobster attacks induce…

  14. Transcriptional Analysis of a Whole-Body Form of Long-Term Habituation in "Aplysia Californica"

    ERIC Educational Resources Information Center

    Holmes, Geraldine; Herdegen, Samantha; Schuon, Jonathan; Cyriac, Ashly; Lass, Jamie; Conte, Catherine; Calin-Jageman, Irina E.; Calin-Jageman, Robert J.

    2015-01-01

    Habituation is the simplest form of learning, but we know little about the transcriptional mechanisms that encode long-term habituation memory. A key obstacle is that habituation is relatively stimulus-specific and is thus encoded in small sets of neurons, providing poor signal/noise ratios for transcriptional analysis. To overcome this obstacle,…

  15. Functional magnetic resonance microscopy at single-cell resolution in Aplysia californica

    PubMed Central

    Radecki, Guillaume; Nargeot, Romuald; Jelescu, Ileana Ozana; Le Bihan, Denis; Ciobanu, Luisa

    2014-01-01

    In this work, we show the feasibility of performing functional MRI studies with single-cell resolution. At ultrahigh magnetic field, manganese-enhanced magnetic resonance microscopy allows the identification of most motor neurons in the buccal network of Aplysia at low, nontoxic Mn2+ concentrations. We establish that Mn2+ accumulates intracellularly on injection into the living Aplysia and that its concentration increases when the animals are presented with a sensory stimulus. We also show that we can distinguish between neuronal activities elicited by different types of stimuli. This method opens up a new avenue into probing the functional organization and plasticity of neuronal networks involved in goal-directed behaviors with single-cell resolution. PMID:24872449

  16. The Influence of Increased Nitrogen Tensions on Properties of Identified Neurons in ’Aplysia Californica’.

    DTIC Science & Technology

    Aplysia are unaffected by increases in nitrogen tensions to 10 atmospheres absolute. Since no significantly large or consistent alteration occurred in...are stable and well controlled in these neurons to this degree of pressure. The fact that Aplysia is considered an intertidal animal that has not

  17. Connecting model species to nature: predator-induced long-term sensitization in Aplysia californica.

    PubMed

    Mason, Maria J; Watkins, Amanda J; Wakabayashi, Jordann; Buechler, Jennifer; Pepino, Christine; Brown, Michelle; Wright, William G

    2014-08-01

    Previous research on sensitization in Aplysia was based entirely on unnatural noxious stimuli, usually electric shock, until our laboratory found that a natural noxious stimulus, a single sublethal lobster attack, causes short-term sensitization. We here extend that finding by demonstrating that multiple lobster attacks induce long-term sensitization (≥24 h) as well as similar, although not identical, neuronal correlates as observed after electric shock. Together these findings establish long- and short-term sensitization caused by sublethal predator attack as a natural equivalent to sensitization caused by artificial stimuli.

  18. Functional magnetic resonance microscopy at single-cell resolution in Aplysia californica.

    PubMed

    Radecki, Guillaume; Nargeot, Romuald; Jelescu, Ileana Ozana; Le Bihan, Denis; Ciobanu, Luisa

    2014-06-10

    In this work, we show the feasibility of performing functional MRI studies with single-cell resolution. At ultrahigh magnetic field, manganese-enhanced magnetic resonance microscopy allows the identification of most motor neurons in the buccal network of Aplysia at low, nontoxic Mn(2+) concentrations. We establish that Mn(2+) accumulates intracellularly on injection into the living Aplysia and that its concentration increases when the animals are presented with a sensory stimulus. We also show that we can distinguish between neuronal activities elicited by different types of stimuli. This method opens up a new avenue into probing the functional organization and plasticity of neuronal networks involved in goal-directed behaviors with single-cell resolution.

  19. Circadian Rhythm of Neuron R15 of Aplysia californica: In Vivo Photoentrainment.

    PubMed

    Audesirk, G; Strumwasser, F

    1975-06-01

    (1) The neuron R15 in the parietovisceral ganglion of Aplysia has a circadian rhythm of spiking activity when recorded in the isolated ganglion. The rhythm is entrained in vivo by light-dark cycles. (2) The phase of the R15 rhythm is a function not only of the entraining light schedule, but also of the time of dissection. Changes in the dissection time during the light portion of the light-dark cycle yield little change in the subsequent R15 peak time. Dissections during the dark portion produce peak times that vary with dissection time with a slope that is approximately one. (3) The circadian rhythm of R15 can be phase-shifted in vivo by changes in the phase of the entraining light-dark cycle in one to two weeks. R15 neurons of blinded Aplysia, however, show little or no phase shift in this time. (4) It is concluded that the eyes are important as receptors for the photoentrainment of the R15 rhythm in vivo, but that neural connections from the eyes to R15 are not required.

  20. Circadian Rhythm of Neuron R15 of Aplysia californica: In Vivo Photoentrainment

    PubMed Central

    Audesirk, Gerald; Strumwasser, Felix

    1975-01-01

    (1) The neuron R15 in the parietovisceral ganglion of Aplysia has a circadian rhythm of spiking activity when recorded in the isolated ganglion. The rhythm is entrained in vivo by light-dark cycles. (2) The phase of the R15 rhythm is a function not only of the entraining light schedule, but also of the time of dissection. Changes in the dissection time during the light portion of the light-dark cycle yield little change in the subsequent R15 peak time. Dissections during the dark portion produce peak times that vary with dissection time with a slope that is approximately one. (3) The circadian rhythm of R15 can be phase-shifted in vivo by changes in the phase of the entraining light-dark cycle in one to two weeks. R15 neurons of blinded Aplysia, however, show little or no phase shift in this time. (4) It is concluded that the eyes are important as receptors for the photoentrainment of the R15 rhythm in vivo, but that neural connections from the eyes to R15 are not required. PMID:16592252

  1. Consequences and mechanisms of spike broadening of R20 cells in Aplysia californica.

    PubMed

    Ma, M; Koester, J

    1995-10-01

    We studied frequency-dependent spike broadening in the two electrically coupled R20 neurons in the abdominal ganglion of Aplysia. The peptidergic R20 cells excite the R25/L25 interneurons (which trigger respiratory pumping) and inhibit the RB cells. When fired at 1-10 Hz, the duration of the falling phase of the action potential in R20 neurons increases 2-10 fold during a spike train. Spike broadening recorded from the somata of the R20 cells affected synaptic transmission to nearby follower cells. Chemically mediated synaptic output was reduced by approximately 50% when recorded trains of nonbroadened action potentials were used as command signals for a voltage-clamped R20 cell. Electrotonic EPSPs between the R20 cells, which normally facilitated by two- to fourfold during a high frequency spike train, showed no facilitation when spike broadening was prevented under voltage-clamp control. To examine the mechanism of frequency-dependent spike broadening, we applied two-electrode voltage-clamp and pharmacological techniques to the somata of R20 cells. Several voltage-gated ionic currents were isolated, including INa, a multicomponent ICa, and three K+ currents--a high threshold, fast transient A-type K+ current (IAdepol), a delayed rectifier K+ current (IK-V), and a Ca(2+)-sensitive K+ current (IK-Ca), made up of two components. The influences of different currents on spike broadening were determined by using the recorded train of gradually broadening action potentials as the command for the voltage clamp. We found the following. (1) IAdepol is the major outward current that contributes to repolarization of nonbroadened spikes. It undergoes pronounced cumulative inactivation that is a critical determinant of spike broadening. (2) Activity-dependent changes in IK-V, IK-Ca, and ICa have complex effects on the kinetics and extent of broadening. (3) The time integral of ICa during individual action potentials increases approximately threefold during spike broadening.

  2. Connecting Model Species to Nature: Predator-Induced Long-Term Sensitization in "Aplysia Californica"

    ERIC Educational Resources Information Center

    Mason, Maria J.; Watkins, Amanda J.; Wakabayashi, Jordann; Buechler, Jennifer; Pepino, Christine; Brown, Michelle; Wright, William G.

    2014-01-01

    Previous research on sensitization in "Aplysia" was based entirely on unnatural noxious stimuli, usually electric shock, until our laboratory found that a natural noxious stimulus, a single sublethal lobster attack, causes short-term sensitization. We here extend that finding by demonstrating that multiple lobster attacks induce…

  3. Transcriptional Analysis of a Whole-Body Form of Long-Term Habituation in "Aplysia Californica"

    ERIC Educational Resources Information Center

    Holmes, Geraldine; Herdegen, Samantha; Schuon, Jonathan; Cyriac, Ashly; Lass, Jamie; Conte, Catherine; Calin-Jageman, Irina E.; Calin-Jageman, Robert J.

    2015-01-01

    Habituation is the simplest form of learning, but we know little about the transcriptional mechanisms that encode long-term habituation memory. A key obstacle is that habituation is relatively stimulus-specific and is thus encoded in small sets of neurons, providing poor signal/noise ratios for transcriptional analysis. To overcome this obstacle,…

  4. Habituation in the Tail Withdrawal Reflex Circuit is Impaired During Aging in Aplysia californica.

    PubMed

    Kempsell, Andrew T; Fieber, Lynne A

    2016-01-01

    The relevance of putative contributors to age-related memory loss are poorly understood. The tail withdrawal circuit of the sea hare, a straightforward neural model, was used to investigate the aging characteristics of rudimentary learning. The simplicity of this neuronal circuit permits attribution of declines in the function of specific neurons to aging declines. Memory was impaired in advanced age animals compared to their performance at the peak of sexual maturity, with habituation training failing to attenuate the tail withdrawal response or to reduce tail motoneuron excitability, as occurred in peak maturity siblings. Baseline motoneuron excitability of aged animals was significantly lower, perhaps contributing to a smaller scope for attenuation. Conduction velocity in afferent fibers to tail sensory neurons (SN) decreased during aging. The findings suggest that age-related changes in tail sensory and motor neurons result in deterioration of a simple form of learning in Aplysia.

  5. Biocompatibility of adhesive complex coacervates modeled after the sandcastle glue of Phragmatopoma californica for craniofacial reconstruction.

    PubMed

    Winslow, Brent D; Shao, Hui; Stewart, Russell J; Tresco, Patrick A

    2010-12-01

    Craniofacial reconstruction would benefit from a degradable adhesive capable of holding bone fragments in three-dimensional alignment and gradually being replaced by new bone without loss of alignment or volume changes. Modeled after a natural adhesive secreted by the sandcastle worm, we studied the biocompatibility of adhesive complex coacervates in vitro and in vivo with two different rat calvarial models. We found that the adhesive was non-cytotoxic and supported the attachment, spreading, and migration of a commonly used osteoblastic cell line over the course of several days. In animal studies we found that the adhesive was capable of maintaining three-dimensional bone alignment in freely moving rats over a 12 week indwelling period. Histological evidence indicated that the adhesive was gradually resorbed and replaced by new bone that became lamellar across the defect without loss of alignment, changes in volume, or changes in the adjacent uninjured bone. The presence of inflammatory cells was consistent with what has been reported with other craniofacial fixation methods including metal plates, screws, tacks, calcium phosphate cements and cyanoacrylate adhesives. Collectively, the results suggest that the new bioadhesive formulation is degradable, osteoconductive and appears suitable for use in the reconstruction of craniofacial fractures. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Biocompatibility of adhesive complex coacervates modeled after the Sandcastle glue of P. californica for craniofacial reconstruction

    PubMed Central

    Winslow, Brent D.; Shao, Hui; Stewart, Russell J.; Tresco, Patrick A.

    2011-01-01

    Craniofacial reconstruction would benefit from a degradable adhesive capable of holding bone fragments in three-dimensional alignment and gradually being replaced by new bone without loss of alignment or volume changes. Modeled after a natural adhesive secreted by the sandcastle worm, we studied the biocompatibility of adhesive complex coacervates in vitro and in vivo with two different rat calvarial models. We found that the adhesive was non-cytotoxic and supported the attachment, spreading, and migration of a commonly used osteoblastic cell line over the course of several days. In animal studies we found that the adhesive was capable of maintaining three-dimensional bone alignment in freely moving rats over a 12 week indwelling period. Histological evidence indicated that the adhesive was gradually resorbed and replaced by new bone that became lamellar across the defect without loss of alignment, changes in volume, or changes in the adjacent uninjured bone. The presence of inflammatory cells was consistent with what has been reported with other craniofacial fixation methods including metal plates, screws, tacks, calcium phosphate cements and cyanoacrylate adhesives. Collectively, the results suggest that the new bioadhesive formulation is degradable, osteoconductive and appears suitable for use in the reconstruction of craniofacial fractures. PMID:20950851

  7. Characterization of glycolipids synthesized in an identified neuron of Aplysia californica

    SciTech Connect

    Sherbany, A.A.; Ambron, R.T.; Schwartz, J.H.

    1984-07-01

    Because radioactive precursors can be injected directly into the cell body or axon of R2, a giant, identified neuron of the Aplysia abdominal ganglion, it was possible to show that glycolipid is synthesized in the cell body, inserted into membranes along with glycoprotein, and then exported into the axon within organelles that are moved by fast axonal transport. After intrasomatic injection of N-(/sup 3/H)-acetyl-D-galactosamine, five major /sup 3/H-glycolipids were identified using thin layer polysilicic acid glass fiber chromatography. At least two of the lipids are negatively charged. Analysis of /sup 32/P-labeled lipid from the abdominal ganglion revealed the presence of 2-aminoethylphosphonate, indicating that these polar substances are sphingophosphonoglycolipids. The major /sup 3/H-glycolipids synthesized in R2 are similar to a family of phospholipids isolated from the skin of A. kurodai. Since sialic acid is absent in Aplysia as in other invertebrates, these polar glycolipids may function like gangliosides in vertebrates. The polar /sup 3/H-glycolipids are synthesized and incorporated into intracytoplasmic membranes solely in the cell body. Direct injection of the labeled sugar into the axon revealed no local synthesis or exchange of glycolipid. Moreover, there was no indication for transfer from glial cells into axoplasm. Although the incorporation of N-(/sup 3/H)-acetyl-D-galactosamine into glycolipid is not affected by anisomycin, an effective inhibitor of protein synthesis, the export into the axon of membranes containing the newly synthesized lipid is completely blocked by the drug.

  8. Efficient perturbation analysis of elastic network models - Application to acetylcholinesterase of T. californica

    NASA Astrophysics Data System (ADS)

    Hamacher, K.

    2010-09-01

    Elastic network models in their different flavors have become useful models for the dynamics and functions of biomolecular systems such as proteins and their complexes. Perturbation to the interactions occur due to randomized and fixated changes (in molecular evolution) or designed modifications of the protein structures (in bioengineering). These perturbations are modifications in the topology and the strength of the interactions modeled by the elastic network models. We discuss how a naive approach to compute properties for a large number of perturbed structures and interactions by repeated diagonalization can be replaced with an identity found in linear algebra. We argue about the computational complexity and discuss the advantages of the protocol. We apply the proposed algorithm to the acetylcholinesterase, a well-known enzyme in neurobiology, and show how one can gain insight into the "breathing dynamics" of a structural funnel necessary for the function of the protein. The computational speed-up was a 60-fold increase in this example.

  9. Parallel processing in an identified neural circuit: the Aplysia californica gill-withdrawal response model system.

    PubMed

    Leonard, Janet L; Edstrom, John P

    2004-02-01

    The response of the gill of Aplysia calfornica Cooper to weak to moderate tactile stimulation of the siphon, the gill-withdrawal response or GWR, has been an important model system for work aimed at understanding the relationship between neural plasticity and simple forms of non-associative and associative learning. Interest in the GWR has been based largely on the hypothesis that the response could be explained adequately by parallel monosynaptic reflex arcs between six parietovisceral ganglion (PVG) gill motor neurons (GMNs) and a cluster of sensory neurons termed the LE cluster. This hypothesis, the Kupfermann-Kandel model, made clear, falsifiable predictions that have stimulated experimental work for many years. Here, we review tests of three predictions of the Kupfermann-Kandel model: (1) that the GWR is a simple, reflexive behaviour graded with stimulus intensity; (2) that central nervous system (CNS) pathways are necessary and sufficient for the GWR; and (3) that activity in six identified GMNs is sufficient to account for the GWR. The available data suggest that (1) a variety of action patterns occur in the context of the GWR; (2) the PVG is not necessary and the diffuse peripheral nervous system (PNS) is sufficient to mediate these action patterns; and (3) the role of any individual GMN in the behaviour varies. Both the control of gill-withdrawal responses, and plasticity in these responses, are broadly distributed across both PNS and CNS pathways. The Kupfermann-Kandel model is inconsistent with the available data and therefore stands rejected. There is, no known causal connection or correlation between the observed plasticity at the identified synapses in this system and behavioural changes during non-associative and associative learning paradigms. Critical examination of these well-studied central pathways suggests that they represent a 'wetware' neural network, architecturally similar to the neural network models of the widely used 'Perceptron' and/or 'Back-propagation' type. Such models may offer a more biologically realistic representation of nervous system organisation than has been thought. In this model, the six parallel GMNs of the CNS correspond to a hidden layer within one module of the gill-control system. That is, the gill-control system appears to be organised as a distributed system with several parallel modules, some of which are neural networks in their own right. A new model is presented here which predicts that the six GMNs serve as components of a 'push-pull' gain control system, along with known but largely unidentified inhibitory motor neurons from the PVG. This 'push-pull' gain control system sets the responsiveness of the peripheral gill motor system. Neither causal nor correlational links between specific forms of neural plasticity and behavioural plasticity have been demonstrated in the GWR model system. However, the GWR model system does provide an opportunity to observe and describe directly the physiological and biochemical mechanisms of distributed representation and parallel processing in a largely identifiable 'wetware' neural network.

  10. Characterization of a unique OpMNPV-specific early gene not required for viral infection in tissue culture.

    PubMed

    Shippam, C; Wu, X; Stewart, S; Theilmann, D A

    1997-01-20

    opep-2 is an Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) early gene in the ie1-ie2 gene region for which there is no homolog in either the archetype virus, Autographa californica MNPV, or Bombyx mori NPV. opep-2 is transcribed immediately upon infection as three mRNAs which initiate from a early gene motif (TATA-N27-CAGT). The expression of multiple transcripts at very early times postinfection has only been previously described for the baculovirus early gene ie1, which produces spliced mRNAs. However, distinct from ie1, the multiple mRNAs of opep-2 are due to multiple termination sites and not splicing. Western blot analysis of steady-state levels of OPEP-2 showed that in OpMNPV-infected Ld652Y cells maximum levels are obtained at 8-12 hr postinfection (p.i.) prior to DNA replication. By 48 hr p.i. OPEP-2 is shut off and is undetectable. To aid in elucidating the function of this OpMNPV-specific gene an opep-2 deletion mutant was generated and was compared to wild-type virus to determine if its absence affects viral growth in Ld652Y tissue culture cells.

  11. Mucosal delivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liver abscess in hamster.

    PubMed

    Meneses-Ruiz, D M; Laclette, J P; Aguilar-Díaz, H; Hernández-Ruiz, J; Luz-Madrigal, A; Sampieri, A; Vaca, L; Carrero, J C

    2011-01-01

    Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

  12. Mucosal Delivery of ACNPV Baculovirus Driving Expression of the Gal-Lectin LC3 Fragment Confers Protection against Amoebic Liver Abscess in Hamster

    PubMed Central

    Meneses-Ruiz, DM; Laclette, JP; Aguilar-Díaz, H; Hernández-Ruiz, J; Luz-Madrigal, A; Sampieri, A; Vaca, L; Carrero, JC

    2011-01-01

    Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine. PMID:22110386

  13. Comparative studies of lepidopteran baculovirus-specific protein FP25K: development of a novel Bombyx mori nucleopolyhedrovirus-based vector with a modified fp25K gene.

    PubMed

    Nakanishi, Tadashi; Goto, Chie; Kobayashi, Michihiro; Kang, Wonkyung; Suzuki, Takehiro; Dohmae, Naoshi; Matsumoto, Shogo; Shimada, Toru; Katsuma, Susumu

    2010-05-01

    Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.

  14. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

    SciTech Connect

    Germann, U.A.; Willingham, M.C.; Pastan, I.; Gottesman, M.M. )

    1990-03-06

    The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. The authors expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by ({sup 3}H)azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents compete with the ({sup 3}H)azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.

  15. A novel recombinant baculovirus overexpressing a Bacillus thuringiensis Cry1Ab toxin enhances insecticidal activity

    PubMed Central

    2014-01-01

    Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of killing. In this study a truncated form of cry1Ab gene derived from Bacillus thuringinsis (Bt) subsp. aegypti isolate Bt7 was engineered into the genome of the baculovirus Autographa californica multiple nuclearpolyhedrosis wild type virus, in place of the polyhedrin gene by using homologous recombination in Spodoptera frugiperda (Sf) cells between a transfer vector carrying the Bt gene and the wild type virus linearized DNA. Recombinant wild type virus containing the cry1Ab gene was detected as blue occlusion-negative plaques in monolayers of Sf cells grown in the presence of X-Gal. In Sf cells infected with plaque-purified recombinant virus, the cry1Ab gene was expressed to yield a protein of approximately 82-kDa, as determined by immunoblot analysis. The toxicity of the recombinant virus expressing the insecticidal crystal protein (ICP) was compared to that of the wild-type virus. Infected-cell extract was toxic to cotton leaf worm Spodoptera littoralis second instar larvae and the estimated LC50 was 1.7 μg/ml for the recombinant virus compared with that of wild-type virus which was 10 μg/ml. PMID:24735532

  16. Deletion of the AcMNPV core gene ac109 results in budded virions that are non-infectious

    SciTech Connect

    Fang Minggang; Nie, Yingchao; Theilmann, David A.

    2009-06-20

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 is a core gene and its function in the virus life cycle is unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac109 deletion virus (vAc{sup 109KO}). Fluorescence and light microscopy showed that transfection of vAc{sup 109KO} results in a single-cell infection phenotype. Viral DNA replication is unaffected and the development of occlusion bodies in vAc{sup 109KO}-transfected cells evidenced progression to the very late phases of viral infection. Western blot and confocal immunofluorescence analysis showed that AC109 is expressed in the cytoplasm and nucleus throughout infection. In addition, AC109 is a structural protein as it was detected in both budded virus (BV) and occlusion derived virus in both the envelope and nucleocapsid fractions. Titration assays by qPCR and TCID{sub 50} showed that vAc{sup 109KO} produced BV but the virions are non-infectious. The vAc{sup 109KO} BV were indistinguishable from the BV of repaired and wild type control viruses as determined by negative staining and electron microscopy.

  17. A highly conserved baculovirus gene p48 (ac103) is essential for BV production and ODV envelopment

    SciTech Connect

    Yuan Meijin; Wu Wenbi; Liu Chao; Wang Yanjie; Hu Zhaoyang; Yang Kai Pang Yi

    2008-09-15

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) is a highly conserved baculovirus gene of unknown function. In the present study, we generated a knockout of the p48 gene in an AcMNPV bacmid and investigated the role of P48 in baculovirus life cycle. The p48-null Bacmid vAc{sup P48-KO-PH-GFP} was unable to propagate in cell culture, while a 'repair' Bacmid vAc{sup P48-REP-PH-GFP} was able to replicate in a manner similar to a wild-type Bacmid vAc{sup PH-GFP}. Titration assays and Western blotting confirmed that vAc{sup P48-KO-PH-GFP} was unable to produce budded viruses (BVs). qPCR analysis showed that p48 deletion did not affect viral DNA replication. Electron microscopy indicated that P48 was required for nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and their subsequent occlusion. Confocal analysis showed that P48 prominently condensed in the centre of the nucleus. Our results demonstrate that P48 plays an essential role in BV production and ODV envelopment in the AcMNPV life cycle.

  18. Actin-based motility drives baculovirus transit to the nucleus and cell surface

    PubMed Central

    Ohkawa, Taro; Volkman, Loy E.

    2010-01-01

    Most viruses move intracellularly to and from their sites of replication using microtubule-based mechanisms. In this study, we show that nucleocapsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus undergo intracellular motility driven by actin polymerization. Motility requires the viral P78/83 capsid protein and the host Arp2/3 complex. Surprisingly, the virus directs two sequential and coordinated phases of actin-based motility. Immediately after cell entry, motility enables exploration of the cytoplasm and collision with the nuclear periphery, speeding nuclear entry and the initiation of viral gene expression. Nuclear entry itself requires transit through nuclear pore complexes. Later, after the onset of early gene expression, motility is required for accumulation of a subpopulation of nucleocapsids in the tips of actin-rich surface spikes. Temporal coordination of actin-based nuclear and surface translocation likely enables rapid transmission to neighboring cells during infection in insects and represents a distinctive evolutionary strategy for overcoming host defenses. PMID:20660627

  19. Genome sequence of Perigonia lusca single nucleopolyhedrovirus: insights into the evolution of a nucleotide metabolism enzyme in the family Baculoviridae

    PubMed Central

    Ardisson-Araújo, Daniel M. P.; Lima, Rayane Nunes; Melo, Fernando L.; Clem, Rollie J.; Huang, Ning; Báo, Sônia Nair; Sosa-Gómez, Daniel R.; Ribeiro, Bergmann M.

    2016-01-01

    The genome of a novel group II alphabaculovirus, Perigonia lusca single nucleopolyhedrovirus (PeluSNPV), was sequenced and shown to contain 132,831 bp with 145 putative ORFs (open reading frames) of at least 50 amino acids. An interesting feature of this novel genome was the presence of a putative nucleotide metabolism enzyme-encoding gene (pelu112). The pelu112 gene was predicted to encode a fusion of thymidylate kinase (tmk) and dUTP diphosphatase (dut). Phylogenetic analysis indicated that baculoviruses have independently acquired tmk and dut several times during their evolution. Two homologs of the tmk-dut fusion gene were separately introduced into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, which lacks tmk and dut. The recombinant baculoviruses produced viral DNA, virus progeny, and some viral proteins earlier during in vitro infection and the yields of viral occlusion bodies were increased 2.5-fold when compared to the parental virus. Interestingly, both enzymes appear to retain their active sites, based on separate modeling using previously solved crystal structures. We suggest that the retention of these tmk-dut fusion genes by certain baculoviruses could be related to accelerating virus replication and to protecting the virus genome from deleterious mutation. PMID:27273152

  20. Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein

    SciTech Connect

    Au, Victoria; Yu Mei; Carstens, Eric B.

    2009-03-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143.

  1. Complex dynamics of defective interfering baculoviruses during serial passage in insect cells.

    PubMed

    Zwart, Mark P; Pijlman, Gorben P; Sardanyés, Josep; Duarte, Jorge; Januário, Cristina; Elena, Santiago F

    2013-03-01

    Defective interfering (DI) viruses are thought to cause oscillations in virus levels, known as the 'Von Magnus effect'. Interference by DI viruses has been proposed to underlie these dynamics, although experimental tests of this idea have not been forthcoming. For the baculoviruses, insect viruses commonly used for the expression of heterologous proteins in insect cells, the molecular mechanisms underlying DI generation have been investigated. However, the dynamics of baculovirus populations harboring DIs have not been studied in detail. In order to address this issue, we used quantitative real-time PCR to determine the levels of helper and DI viruses during 50 serial passages of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in Sf21 cells. Unexpectedly, the helper and DI viruses changed levels largely in phase, and oscillations were highly irregular, suggesting the presence of chaos. We therefore developed a simple mathematical model of baculovirus-DI dynamics. This theoretical model reproduced patterns qualitatively similar to the experimental data. Although we cannot exclude that experimental variation (noise) plays an important role in generating the observed patterns, the presence of chaos in the model dynamics was confirmed with the computation of the maximal Lyapunov exponent, and a Ruelle-Takens-Newhouse route to chaos was identified at decreasing production of DI viruses, using mutation as a control parameter. Our results contribute to a better understanding of the dynamics of DI baculoviruses, and suggest that changes in virus levels over passages may exhibit chaos.

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

    PubMed

    Elgaied, Lamiaa; Salem, Reda; Elmenofy, Wael

    2017-08-01

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

  3. Baculovirus-mediated expression of human apolipoprotein E in Manduca sexta larvae generates particles that bind to the low density lipoprotein receptor.

    PubMed Central

    Gretch, D G; Sturley, S L; Friesen, P D; Beckage, N E; Attie, A D

    1991-01-01

    Human apolipoprotein E (apoE) is a ligand for the low density lipoprotein (LDL) receptor and mediates the catabolism of several classes of lipoprotein particles. Binding of apoE to the LDL receptor requires association of apoE with lipid in a vesicle or a lipoprotein particle. Because of this requirement, purified apoE or apoE derived directly from bacterial expression systems does not bind to the LDL receptor. To overcome this problem and to facilitate analysis of apoE structure, recombinant baculoviruses containing the human apoE cDNA fused to the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus were constructed. The recombinant viruses were used to infect larvae of the tobacco hornworm Manduca sexta in vivo. High levels of lipoprotein particles containing human apoE were present in the hemolymph of infected larvae. In contrast to apoE produced by recombinant baculovirus-infected insect cells in vitro, these particles were excellent ligands for the LDL receptor. Images PMID:1924311

  4. The gene encoding the capsid protein P82 of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus: sequencing, transcription and characterization by immunoblot analysis.

    PubMed

    Li, X; Pang, A; Lauzon, H A; Sohi, S S; Arif, B M

    1997-10-01

    A gene encoding a capsid-associated viral structural protein has been identified and sequenced in the genome of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV). The gene has a 1872 nucleotide open reading frame (ORF) encoding 624 amino acids with a predicted molecular mass of 71.4 kDa. Transcription, which appeared to be initiated from a conserved GTAAG motif of baculovirus late genes, was detected at 12 h, reached a maximum at 48 h and declined at 72 h post-infection (p.i.). Part of the ORF was cloned in frame into a prokaryotic expression vector, pMAL-c2, and the fusion protein was used to generate antibodies in rabbits. It was shown, with the aid of the polyclonal antiserum, that this viral protein was detectable at 24 h p.i. in infected cells. The protein appeared as an 82 kDa band in occlusion-derived virus and as an 82 kDa band and a 72 kDa band in budded virus. Amino acid sequence comparisons revealed that this ORF had high homology with the ORF p87 (77% similarity) of Orgyia pseudotsugata (Op) MNPV and the ORF p80 (60% similarity) of Autographa californica (Ac) MNPV. Immunoblots confirmed that the CfMNPV protein had antigenic similarities to the P87 protein of OpMNPV, but not to the P80 of AcMNPV.

  5. Identification, localization, transcription, and sequence analysis of the Choristoneura fumiferana nuclear polyhedrosis virus DNA polymerase gene.

    PubMed

    Liu, J J; Carstens, E B

    1995-06-01

    The location of the Choristoneura fumiferana baculovirus DNA polymerase gene was determined by hybridization analysis using a probe prepared from the previously identified polymerase gene from the Autographa californica multiple nuclear polyhedrosis virus. DNA sequence analysis revealed that the Choristoneura fumiferana baculovirus DNA polymerase gene consists of 2970 base pairs encoding 990 amino acids (114.2 kDa). Transcriptional analysis demonstrated that overlapping transcripts of 3.2 and 4.6 kb, first detected at 6 hr postinfection, potentially coded for the DNA polymerase gene. The major transcription starts sites, identified at 6 hr postinfection, mapped to baculovirus consensus early start sites CGTGCTCA and CAGT. The relatively low level and late initiation of the DNA polymerase gene coupled with our previous data on the temporal control of DNA replication and late gene synthesis (Liu and Carstens, 1993) suggests that the low virulence of the spruce budworm baculovirus may be related to the regulation of its gene expression at the transcriptional level.

  6. Choristoneura fumiferana granulovirus: sequence analysis and 5' characterization of ORF891.

    PubMed

    Bah, A; Lucarotti, C J; Arella, M; Guertin, C

    1999-01-01

    A gene located immediately upstream of the granulin gene of Choristoneura fumiferana (ChfuGV) granulovirus was identified, sequenced and named ORF891. The determined, putative open reading frame (ORF) of 891 bp encodes an estimated 34.6 kDa protein. The 5' end transcript of the gene was mapped and analysed. A putative promoter region organization of ChfuGV ORF891 contains a consensus late baculovirus promoter element, TAAG, and two putative early TATA boxes similar to the promoters of ORF909 of Cryptophlebia leucotreta granulovirus (ClGV). Sequence comparisons of ChfuGV ORF891 with ClGV ORF909 and Cydia pomonella granulovirus (CpGV) ORF124R showed respective homologies of 60.9 and 63.9% for nucleotides and 46.3% and 49.3% for amino acids. Homology of ChfuGV ORF891 with ME53 ORF of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was 68.2% for nucleotides but a total lack of homology for amino acid sequences. Two zinc finger motifs are also associated with ChfuGV ORF891.

  7. Choristoneura fumiferana multiple nucleopolyhedrovirus LEF-3-P143 complex can complement DNA replication and budded virus in an AcMNPV LEF-3-P143 double knockout bacmid.

    PubMed

    Yu, Mei; Carstens, Eric B

    2012-02-01

    Transient replication assays using Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) genes suggested that the interactions between P143, the viral helicase and LEF-3, a ssDNA-binding protein, may represent virus species specificity determinants. P143 and LEF-3 are essential for DNA replication in these assays and together with IE-1, the major immediate-early transcription factor, may be part of the viral replisome. In the current report, a lef-3/p143 double-knockout AcMNPV bacmid was constructed that was defective for viral DNA replication and late gene expression. When the homologous lef-3/p143 CfMNPV genes were introduced into this double-knockout bacmid, DNA replication was restored but the level of replication was lower, budded virus production was delayed, and the yields were reduced from those in an AcMNPV-rescue bacmid. These results suggest that to maximize virus replication, baculovirus replisome assembly and function requires protein-protein interactions between P143 and LEF-3, and other viral proteins.

  8. Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV.

    PubMed Central

    French, T J; Roy, P

    1990-01-01

    The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7. Images PMID:2157041

  9. Myocyte enhancer factor 2 (MEF2) is a key modulator of the expression of the prothoracicotropic hormone gene in the silkworm, Bombyx mori.

    PubMed

    Shiomi, Kunihiro; Fujiwara, Yoshihiro; Atsumi, Tsutomu; Kajiura, Zenta; Nakagaki, Masao; Tanaka, Yoshiaki; Mizoguchi, Akira; Yaginuma, Toshinobu; Yamashita, Okitsugu

    2005-08-01

    Prothoracicotropic hormone (PTTH) plays a central role in controlling molting, metamorphosis, and diapause termination in insects by stimulating the prothoracic glands to synthesize and release the molting hormone, ecdysone. Using Autographa californica nucleopolyhedrovirus (AcNPV)-mediated transient gene transfer into the central nervous sytem (CNS) of the silkworm, Bombyx mori, we identified two cis-regulatory elements that participate in the decision and the enhancement of PTTH gene expression in PTTH-producing neurosecretory cells (PTPCs). The cis-element mediating the enhancement of PTTH gene expression binds the transcription factor Bombyx myocyte enhancer factor 2 (BmMEF2). The BmMEF2 gene was expressed in various tissues including the CNS. In brain, the BmMEF2 gene was expressed at elevated levels in two types of lateral neurosecretory cells, namely PTPCs and corazonin-like immunoreactive lateral neurosecretory cells. Overexpression of BmMEF2 cDNA caused an increase in the transcription of PTTH. Therefore, BmMEF2 appears to be particularly important in the brain where it is responsible for the differentiation of lateral neurosecretory cells, including the enhancement of PTTH gene expression. This is the first report to identify a target gene of MEF2 in the invertebrate nervous system.

  10. MultiBac turns sweet

    PubMed Central

    Palmberger, Dieter; Klausberger, Miriam; Berger, Imre; Grabherr, Reingard

    2013-01-01

    The baculovirus/insect cell system has proven to be a powerful tool for the expression of eukaryotic proteins. Therapeutics, especially in the field of vaccinology, are often composed of several different protein subunits. Conventional baculoviral expression schemes largely lack efficient strategies for simultaneous multi-gene expression. The MultiBac technology which is based on an engineered genome of Autographa californica nuclear polyhedrosis virus in combination with specially designed transfer vectors is an elegant way for flexible generation of multi-subunit proteins in insect cells. Yet, the glycosylation pattern of insect cell-derived products is not favorable for many applications. Therefore, a modified version of MultiBac, SweetBac, was generated allowing for a flexible glycosylation of target proteins in insect cells. Beyond the SweetBac technology MultiBac can further be designed for bridging the gap between cell engineering and transient modulation of host genes for improved and product tailored expression of recombinant proteins. PMID:23018636

  11. Identification of AcMNPV EXON0 (ac141) domains required for efficient production of budded virus, dimerization and association with BV/ODV-C42 and FP25.

    PubMed

    Fang, Minggang; Nie, Yingchao; Dai, Xiaojiang; Theilmann, David A

    2008-05-25

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late gene exon0 (ac141) is required for the efficient production of budded virus (BV). EXON0 interacts with nucleopcapsid protein BV/ODV-C42 and FP25 and enables egress of nucleocapsids from the nucleus to the cytoplasm. This study examines the functional domains of EXON0 that play a role in BV production. Six putative domains of the 261 amino acid EXON0 were deleted and examined for functionality by determining their ability to rescue an AcMNPV exon0 knockout bacmid. Domain mapping results showed that all the six domains were required but deletion of the N-terminal acidic region and the leucine zipper domains had the greatest impact on BV production. Yeast 2-hybrid and co-immunoprecipitation demonstrated that EXON0 formed dimers. Point mutation analysis demonstrated that the leucine zipper was required for dimer formation and interaction with BV/ODV-C42 and FP25. The charged domain was also required for BV/ODV-C42 interaction.

  12. In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector

    SciTech Connect

    Wang Shengpeng; Guo Tingqing; Guo Xiuyang; Huang Junting; Lu Changde . E-mail: cdlu@sibs.ac.cn

    2006-03-24

    In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.

  13. Recombinant baculovirus as a highly potent vector for gene therapy of human colorectal carcinoma: molecular cloning, expression, and in vitro characterization.

    PubMed

    Paul, Arghya; Jardin, Barbara A; Kulamarva, Arun; Malhotra, Meenakshi; Elias, Cynthia B; Prakash, Satya

    2010-06-01

    Present therapeutic strategies for most cancers are restricted mainly to the primary tumors and are also not very effective in controlling metastatic states. Alternatively, gene therapy can be a potential option for treating such cancers. Currently mammalian viral-based cancer gene therapy is the most popular approach, but the efficacy has been shown to be quite low in clinical trials. In this study, for the first time, the insect cell-specific baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been evaluated as a vector for gene delivery to colorectal cancer cells. Experiments involving factorial design were employed to study the individual and combined effects of different parameters such as multiplicity of infection (MOI), viral incubation time and epigenetic factors on transduction efficiency. The results demonstrate that baculovirus gene delivery system holds immense potential for development of a new generation of highly effective virotherapy for colorectal, as well as other major carcinomas (breast, pancreas, and brain), and offers significant benefits to traditional animal virus-based vectors with respect to safety concerns.

  14. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

    SciTech Connect

    Long, C.M.; Rohrmann, G.F.; Merrill, G.F.

    2009-06-05

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

  15. Characterization of a Trichoplusia ni hexamerin-derived promoter in the AcMNPV baculovirus vector.

    PubMed

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Sánchez-Ramos, Ismael; Escribano, José M

    2013-06-10

    The promoter sequences of the encoding genes for the three most abundant hexamerins of the Lepidoptera Trichoplusia ni were isolated and cloned into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-derived baculovirus expression vector. From the sequences analyzed, the DNA region driving the expression of the Basic juvenile hormone-suppressible protein 2 (BJHSP-2), denominated pB2, presented the highest promoter strength in the context of the baculovirus vector in Sf21 insect cells. This promoter activity occurred earlier in baculovirus-infected cells than that achieved by a conventional polyhedrin promoter (polh), but surprisingly stopped at 48h post-infection. A mapping of pB2 essential promoter elements determined that a region of about 400bp, denominated pB29, retained and even increased the transcriptional activity with respect to the parental full-length sequence. Finally, several chimeric combinations of the insect-derived pB2 with the virus-derived conventional polh or p10 promoters were constructed and incorporated into an AcMNPV baculovirus vector. The pB2-p10 combination showed increased recombinant protein expression at early times post-infection and similar expression levels at very late times post-infection in Sf21 cells with respect to conventional late promoters. To the best of our knowledge, pB2 is the first promoter isolated from the Lepidoptera T. ni, the natural host of AcMNPV, to be assayed in a baculovirus expression vector.

  16. Baculovirus envelope protein ODV-E66 is a novel chondroitinase with distinct substrate specificity.

    PubMed

    Sugiura, Nobuo; Setoyama, Yuka; Chiba, Mie; Kimata, Koji; Watanabe, Hideto

    2011-08-19

    Chondroitin sulfate is a linear polysaccharide of alternating D-glucuronic acid and N-acetyl-D-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4-9) and temperature (30-60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism.

  17. Live imaging of baculovirus infection of midgut epithelium cells: a functional assay of per os infectivity factors.

    PubMed

    Mu, Jingfang; van Lent, Jan W M; Smagghe, Guy; Wang, Yun; Chen, Xinwen; Vlak, Just M; van Oers, Monique M

    2014-11-01

    The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host-range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with EGFP fused to the nucleocapsid to monitor virus entry into primary midgut epithelium cells ex vivo using confocal fluorescence microscopy. The EGFP-labelled virus showed similar BV virulence and ODV infectivity as WT virus. The ability to bind and enter host cells was then visualized for WT AcMNPV and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, whilst PIF1 and PIF2 play important roles in the entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.

  18. Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

    PubMed Central

    Chateigner, Aurélien; Bézier, Annie; Labrousse, Carole; Jiolle, Davy; Barbe, Valérie; Herniou, Elisabeth A.

    2015-01-01

    Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%). K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs). Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential. PMID:26198241

  19. A novel baculovirus-derived promoter with high activity in the baculovirus expression system

    PubMed Central

    Martínez-Solís, María; Gómez-Sebastián, Silvia; Escribano, José M.; Jakubowska, Agata K.

    2016-01-01

    The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS. PMID:27375973

  20. A highly conserved baculovirus gene p48 (ac103) is essential for BV production and ODV envelopment.

    PubMed

    Yuan, Meijin; Wu, Wenbi; Liu, Chao; Wang, Yanjie; Hu, Zhaoyang; Yang, Kai; Pang, Yi

    2008-09-15

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) is a highly conserved baculovirus gene of unknown function. In the present study, we generated a knockout of the p48 gene in an AcMNPV bacmid and investigated the role of P48 in baculovirus life cycle. The p48-null Bacmid vAc(P48-KO-PH-GFP) was unable to propagate in cell culture, while a 'repair' Bacmid vAc(P48-REP-PH-GFP) was able to replicate in a manner similar to a wild-type Bacmid vAc(PH-GFP). Titration assays and Western blotting confirmed that vAc(P48-KO-PH-GFP) was unable to produce budded viruses (BVs). qPCR analysis showed that p48 deletion did not affect viral DNA replication. Electron microscopy indicated that P48 was required for nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and their subsequent occlusion. Confocal analysis showed that P48 prominently condensed in the centre of the nucleus. Our results demonstrate that P48 plays an essential role in BV production and ODV envelopment in the AcMNPV life cycle.

  1. Open reading frame 94 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus encodes a novel conserved occlusion-derived virion protein, ODV-EC43.

    PubMed

    Fang, Minggang; Wang, Hanzhong; Wang, Hualin; Yuan, Li; Chen, Xinwen; Vlak, Just M; Hu, Zhihong

    2003-11-01

    Open reading frame 94 (Ha94) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) is 1086 bp long and a homologue of Autographa californica multiple NPV ORF109. The gene is conserved among all baculoviruses whose genomes have been completely sequenced so far and is thus considered a baculovirus core gene. Ha94 transcripts were detected from 24 to 96 h post-infection (p.i.) of HzAM1 cells with HaSNPV. Polyclonal antiserum raised to a GST-HA94 fusion protein recognized a 43 kDa protein, HA94, in infected cell lysates from 36 to 96 h p.i., suggesting that Ha94 is a late gene. Western blot analysis of proteins present in budded virus and occlusion-derived virus (ODV) showed that Ha94 encodes a structural component of ODV. When ODVs were fractionated further into nucleocapsid and envelope components, Western blot analysis indicated that the encoded protein was associated with both the nucleocapsid and the envelope. In summary, data available indicated that Ha94 encodes a novel ODV-specific protein of HaSNPV, designated ODV-EC43.

  2. Molecular biology of the baculovirus occlusion-derived virus envelope.

    PubMed

    Braunagel, Sharon C; Summers, Max D

    2007-10-01

    Study of the biology of the occlusion-derived virus (ODV) of the baculovirus Autographa californica nucleopolyhedrovirus provides opportunities to reveal new discoveries into the mechanism of several cellular pathways. The synchronous pulse of multiple ODV envelope proteins that integrate into the endoplasmic reticulum (ER) and traffic to the nuclear membranes on their way to the ODV envelope provide a unique tool to study the mechanisms of integral membrane protein trafficking from the ER to the outer and inner nuclear membrane. Studies of the formation of virus-induced, intranuclear membrane microvesicles provide insight on mechanisms that alter fluidity and regulate budding of the inner nuclear membrane. Since ODV is specially adapted for primary infection of the insect gut, studies of the structure and function of ODV envelope proteins reveals insights on the mechanism of viral invasion of the gut and this knowledge is fundamental for the development of new strategies for insect control. This review focuses on recent advances in understanding the source of the ODV envelope and the molecular events that sort and traffic integral membrane proteins from the ER to the ODV envelope. The composition of ODV is reviewed, however it is worth noting that the function of many ODV proteins are currently unknown.

  3. In situ cleavage of baculovirus occlusion-derived virus receptor binding protein P74 in the peroral infectivity complex.

    PubMed

    Peng, Ke; van Lent, Jan W M; Vlak, Just M; Hu, Zhihong; van Oers, Monique M

    2011-10-01

    Proteolytic processing of viral membrane proteins is common among enveloped viruses and facilitates virus entry. The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) protein P74 is part of a complex of essential peroral infectivity factors (PIFs). Here we report that P74 is efficiently cleaved into two fragments of about equal size by an occlusion body (OB) endogenous alkaline protease during ODV release when AcMNPV OBs are derived from larvae. The cleavage is specific for P74, since the other known peroral infectivity factors in the same complex (PIF1, PIF2, and PIF3) were not cleaved under the same conditions. P74 cleavage was not observed in OBs produced in three different insect cell lines, suggesting a larval host origin of the responsible protease. P74 in OBs produced in larvae of two different host species was cleaved into fragments with the same apparent molecular mass, indicating that the virus incorporates a similar alkaline protease from different hosts. Coimmunoprecipitation analysis revealed that the two P74 subunit fragments remain associated with the recently discovered PIF complex. We propose that under in vivo ODV infection conditions, P74 undergoes two sequential cleavage events, the first one being performed by an ODV-associated host alkaline protease and the second carried out by trypsin in the host midgut.

  4. Trafficking of ODV-E66 is mediated via a sorting motif and other viral proteins: facilitated trafficking to the inner nuclear membrane.

    PubMed

    Braunagel, Sharon C; Williamson, Shawn T; Saksena, Suraj; Zhong, Zhenping; Russell, William K; Russell, David H; Summers, Max D

    2004-06-01

    The N-terminal 33 aa of the envelope protein ODV-E66 are sufficient to traffic fusion proteins to intranuclear membranes and the ODV envelope during infection with Autographa californica nucleopolyhedrovirus. This sequence has two distinct features: (i) an extremely hydrophobic sequence of 18 aa and (ii) positively charged amino acids close to the C-terminal end of the hydrophobic sequence. In the absence of infection, this sequence is sufficient to promote protein accumulation at the inner nuclear membrane. Covalent cross-linking results show that the lysines of the motif are proximal to FP25K and/or BV/ODV-E26 during transit from the endoplasmic reticulum to the nuclear envelope. We propose that the 33 aa comprise a signature for sorting proteins to the inner nuclear membrane (sorting motif) and that, unlike other resident proteins of the inner nuclear membrane, ODV-E66 and sortingmotif fusions do not randomly diffuse from their site of insertion at the endoplasmic reticulum to the nuclear envelope and viral-induced intranuclear membranes. Rather, during infection, trafficking is mediated by protein-protein interactions.

  5. AcMNPV AC16 (DA26, BV/ODV-E26) regulates the levels of IE0 and IE1 and binds to both proteins via a domain located within the acidic transcriptional activation domain.

    PubMed

    Nie, Yingchao; Fang, Minggang; Theilmann, David A

    2009-03-15

    IE0 and IE1 are the primary viral regulatory proteins of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) involved in the transactivation of early genes, stimulation of late gene expression, and viral DNA replication. The protein interactions required for IE0 or IE1 to achieve these varied roles are not well defined, so to identify proteins that interact with IE0 and IE1, tandem affinity purification (TAP) and LC-MS/MS was used. Analysis of purified proteins identified AC16 (DA26, BV/ODV-E26) from TAP tagged IE0 virus infected Sf9 cells. Co-immunoprecipitation confirmed that AC16 interacts with both IE0 and IE1 and yeast 2-hybrid analysis mapped the domain required for interaction with AC16. Mutation of the AC16 binding domain enhanced BV production by viruses expressing only IE0 but had no effect if only IE1 is expressed. An ac16 deletion virus was constructed and was shown not to affect the temporal expression of IE0 and IE1; however the relative level of IE0 to IE1 was significantly increased.

  6. AcMNPV-mediated expression of BmK IT promotes the apoptosis of Sf9 cells and replication of AcMNPV.

    PubMed

    Fu, Yue-Jun; Zhao, Jie; Liang, Ai-Hua; Hu, Feng-Yun

    2015-06-25

    Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neurotoxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating or channel currents. To promote the insecticidal effects of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the gene encoding an excitatory insect toxin, BmK IT, was inserted into the genome of AcMNPV to construct a recombinant baculovirus, AcMNPV-BmK IT. Spodopter frugiperda 9 (Sf9) cells were infected with AcMNPV and AcMNPV-BmK IT respectively for 24 h. Results from the MTT assay, TUNEL assay, analysis of the expression level of apoptosis-related proteins (c-Myc, cleaved-Caspase3, Bcl-2 and Bax) of Sf9 cells, the transcription level of key genes (38K, C42, P78, F) of AcMNPV, and viral propagation assay demonstrated that AcMNPV-mediated expression of BmK IT promoted the apoptosis of Sf9 cells and replication of AcMNPV. The results laid a foundation for further structural and functional analysis of BmK IT.

  7. Effects of recombinant baculovirus AcMNPV-BmK IT on the formation of early cables and nuclear polymerization of actin in Sf9 cells.

    PubMed

    Fu, Yuejun; Lin, Taotao; Liang, Aihua; Hu, Fengyun

    2016-05-01

    Autographa californica nuclearpoly hedrosis virus (AcMNPV) is one of the most important baculoviridae. However, the application of AcMNPV as a biocontrol agent has been limited. Previously, we engineered Buthus martensii Karsch insect toxin (BmK IT) gene into the genome of AcMNPV. The bioassay data indicated that the recombinant baculovirus AcMNPV-BmK IT significantly enhanced the anti-insect efficacy of the virus. The actin cytoskeleton is the major component beneath the surface of eukaryotic cells. In this report, the effects of AcMNPV-BmK IT on the formation of early cables of actin and nuclear filamentous-actin (F-actin) were studied. The results indicated that these baculovirus induced rearrangement of the actin cytoskeleton of host cells during infection and actin might participate in the transportation of baculovirus from cytoplasm to the nuclei. AcMNPV-BmK IT delayed the formation of early cables of actin and nuclear F-actin and accelerated the clearance of actin in the nuclei.

  8. Virus-free transient protein production in Sf9 cells.

    PubMed

    Shen, Xiao; Hacker, David L; Baldi, Lucia; Wurm, Florian M

    2014-02-10

    A method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed with the gene of interest being expressed from a plasmid carrying the homologous region 5 enhancer (hr5) and the immediate early 1 (ie1) promoter from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Under the optimal conditions described in the study, cells were transfected at a density of 30×10⁶ cells/mL with 0.9 μg DNA and 1.35 μg of linear 25 kD polyethylenimine (PEI) per million cells. Following transfection, the culture was diluted to 4×10⁶ cells/mL for the protein production phase. The volumetric yield of tumor necrosis factor receptor (ectodomain) fused to an Fc domain (TNFR-Fc) was about 100 μg/mL for cultures at volumes up to 300 mL. As expected, the molecular weight of the dimeric TNFR-Fc produced from Sf9 cells was about 6 kDa less than that produced from a recombinant Chinese hamster ovary (CHO) cell line due to differences in glycosylation between the two hosts. Transient transfection provides an alternative to the baculovirus expression vector system (BEVS) for the rapid production of recombinant proteins from Sf9 cells.

  9. Functional analysis of Spodoptera frugiperda nucleopolyhedrovirus late expression factors in Sf9 cells.

    PubMed

    Berretta, Marcelo F; López, M Gabriela; Taboga, Oscar; Sciocco-Cap, Alicia; Romanowski, Víctor

    2013-02-01

    We used transient expression assays to assess the function of the baculovirus Spodoptera frugiperda M nucleopolyhedrovirus (SfMNPV) homologs of Autographa californica MNPV (AcMNPV) factors involved in late gene expression (lefs), in the Sf9 insect cell-line, which is permissive for both viruses. It is well-established that nineteen AcMNPV lefs support optimal levels of activity from a late promoter-reporter gene cassette in this assay. A subgroup of SfMNPV lefs predicted to function in transcription-specific events substituted the corresponding AcMNPV lefs very efficiently. When all SfMNPV lefs were assayed, including replication lefs, activity was low, but addition of two AcMNPV lefs not encoded in SfMNPV genome, resulted in augmented reporter activity. SfMNPV IE-1 was able to activate an early promoter cis-linked to an hr-derived element from SfMNPV but not from AcMNPV. However, the level of early promoter activation with SfMNPV IE-1 was lower compared to AcMNPV IE-1.

  10. Synthesis and Functional Characterization of MAF-1A Peptide Derived From the Larvae of Housefly, Musca domestica (Diptera: Muscidae).

    PubMed

    Zhou, Jing; Kong, Lina; Fang, Nainai; Mao, Bin; Ai, Hui

    2016-11-01

    The Musca domestica antifungal peptide-1A (MAF-1A peptide) from housefly larvae was synthesized by solid-phase synthesis technique, and antiviral, antioxidant, and antifungal properties were evaluated in this study. Present results indicated that it could significantly inhibit the infection of influenza virus H1N1, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), and Bombyx mori nuclear polyhydrosis virus (BmNPV), which displayed excellent virucidal activities. Antioxidant results demonstrated that the MAF-1A peptide had effective scavenging activity for hydroxyl and superoxide radicals, which were similar to that of ascorbic acid. Besides, antifungal results showed that it can also significantly inhibit the growth of four fungi, and the half inhibitory concentrations (IC50) values were ∼59.3, 84.2, 144.9, and 48.5 μg/ml, respectively, highlighting an important role of MAF-1A peptide in the defense of M domestica against pathogenic microorganisms. These results revealed that the MAF-1A peptide from housefly larvae has great potential as a natural ingredient for the exploitation of antiviral and antifungal therapeutic agents, avoiding abuse of chemical agents and environmental pollution. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. A novel baculovirus-derived promoter with high activity in the baculovirus expression system.

    PubMed

    Martínez-Solís, María; Gómez-Sebastián, Silvia; Escribano, José M; Jakubowska, Agata K; Herrero, Salvador

    2016-01-01

    The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

  12. Temporal characterization of protein production levels from baculovirus vectors coding for GFP and RFP genes under non-conventional promoter control.

    PubMed

    George, Steve; Jauhar, Altamash M; Mackenzie, Jennifer; Kieβlich, Sascha; Aucoin, Marc G

    2015-09-01

    The ease of use and versatility of the Baculovirus Expression Vector System (BEVS) has made it one of the most widely used systems for recombinant protein production However, co-expression systems currently in use mainly make use of the very strong very late p10 and polyhedron (polh) promoters to drive expression of foreign genes, which does not provide much scope for tailoring expression ratios within the cell. This work demonstrates the use of different Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) promoters to control the timing and expression of two easily traceable fluorescent proteins, the enhanced green fluorescent protein (eGFP), and a red fluorescent protein (DsRed2) in a BEVS co-expression system. Our results show that gene expression levels can easily be controlled using this strategy, and also that modulating the expression level of one protein can influence the level of expression of the other protein within the system, thus confirming the concept of genes "competing" for limited cellular resources. Plots of "expression ratios" of the two model genes over time were obtained, and may be used in future work to tightly control timing and levels of foreign gene expression in an insect cell co-expression system.

  13. Bacterial, but not baculoviral infections stimulate Hemolin expression in noctuid moths.

    PubMed

    Terenius, Olle; Popham, Holly J R; Shelby, Kent S

    2009-11-01

    Lepidopteran larvae are regularly infected by baculoviruses during feeding on infected plants. The differences in sensitivity to these infections can be substantial, even among closely related species. For example, the noctuids Cotton bollworm (Helicoverpa zea) and Tobacco budworm (Heliothis virescens), have a 1000-fold difference in sensitivity to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection. Recent data were interpreted to indicate that the lepidopteran immunoglobulin protein, Hemolin, is synthesized upon viral injection and therefore to participate in anti-viral responses. To investigate whether Hemolin synthesis is affected by a natural viral infection, specific transcription in fat bodies and hemocytes of H. zea and H. virescens larvae was monitored following per os infection with the baculovirus HzSNPV (H. zea single nucleopolyhedrovirus). Both moths showed the same expression pattern as seen in uninfected animals and coincided with ecdysone responses, previously known to induce Hemolin expression. In contrast, injection of lyophilized Micrococcus luteus resulted in increased Hemolin expression supporting a role for Hemolin as an immuno-responsive protein in these species. The combined data are consistent with the suggestion that while Hemolin seems to participate in the response to virus infection in the superfamily Bombycoidea, this is not true in the Noctuoidea.

  14. Characterization of a baculovirus gene encoding a small conotoxinlike polypeptide.

    PubMed Central

    Eldridge, R; Li, Y; Miller, L K

    1992-01-01

    We identified a gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) that encodes a small cysteine-rich polypeptide which has size and sequence similarity to omega-conotoxins, a class of calcium ion (Ca2+) channel inhibitors, found in the venom of cone snails. Transcriptional analysis indicated that the 159-bp open reading frame, which we named ctl, and a downstream 984-bp open reading frame are transcribed as a single 1.3-kb bicistronic late RNA. The mature ctl gene product was identified as a small secreted protein by high-pressure liquid chromatography fractionation of extracellular fluid. Viruses with a site-specific deletion in ctl appeared normal with regard to the kinetics and virulence of infection, both in vitro and in vivo. Although we studied the behavior of wild-type and mutant virus-infected insects in some detail, a biological role for ctl in AcMNPV infection remains to be established. Images PMID:1404603

  15. How baculovirus polyhedra fit square pegs into round holes to robustly package viruses.

    PubMed

    Ji, Xiaoyun; Sutton, Geoff; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Stuart, David I

    2010-01-20

    Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.

  16. Virus separation using membranes.

    PubMed

    Grein, Tanja A; Michalsky, Ronald; Czermak, Peter

    2014-01-01

    Industrial manufacturing of cell culture-derived viruses or virus-like particles for gene therapy or vaccine production are complex multistep processes. In addition to the bioreactor, such processes require a multitude of downstream unit operations for product separation, concentration, or purification. Similarly, before a biopharmaceutical product can enter the market, removal or inactivation of potential viral contamination has to be demonstrated. Given the complexity of biological solutions and the high standards on composition and purity of biopharmaceuticals, downstream processing is the bottleneck in many biotechnological production trains. Membrane-based filtration can be an economically attractive and efficient technology for virus separation. Viral clearance, for instance, of up to seven orders of magnitude has been reported for state of the art polymeric membranes under best conditions.This chapter summarizes the fundamentals of virus ultrafiltration, diafiltration, or purification with adsorptive membranes. In lieu of an impractical universally applicable protocol for virus filtration, application of these principles is demonstrated with two examples. The chapter provides detailed methods for production, concentration, purification, and removal of a rod-shaped baculovirus (Autographa californica M nucleopolyhedrovirus, about 40 × 300 nm in size, a potential vector for gene therapy, and an industrially important protein expression system) or a spherical parvovirus (minute virus of mice, 22-26 nm in size, a model virus for virus clearance validation studies).

  17. Baculovirus VP80 protein and the F-actin cytoskeleton interact and connect the viral replication factory with the nuclear periphery.

    PubMed

    Marek, Martin; Merten, Otto-Wilhelm; Galibert, Lionel; Vlak, Just M; van Oers, Monique M

    2011-06-01

    Recently, we showed that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) VP80 protein is essential for the formation of both virion types, budded virus (BV) and occlusion-derived virus (ODV). Deletion of the vp80 gene did not affect assembly of nucleocapsids. However, these nucleocapsids were not able to migrate from the virogenic stroma to the nuclear periphery. In the current paper, we constructed a baculovirus recombinant with enhanced-green fluorescent protein (EGFP)-tagged VP80, allowing visualization of the VP80 distribution pattern during infection. In baculovirus-infected cells, the EGFP-VP80 protein is entirely localized in nuclei, adjacent to the virus-triggered F-actin scaffold that forms a highly organized three-dimensional network connecting the virogenic stroma physically with the nuclear envelope. Interaction between VP80 and host actin was confirmed by coimmunoprecipitation. We further showed that VP80 is associated with the nucleocapsid fraction of both BVs and ODVs, typically at one end of the nucleocapsids. In addition, the presence of sequence motifs with homology to invertebrate paramyosin proteins strongly supports a role for VP80 in the polar transport of nucleocapsids to the periphery of the nucleus on their way to the plasma membrane to form BVs and for assembly in the nuclear periphery to form ODVs for embedding in viral occlusion bodies.

  18. Characterization of an Extremely Basic Protein Derived from Granulosis Virus Nucleocapsids †

    PubMed Central

    Tweeten, Kathleen A.; Bulla, Lee A.; Consigli, Richard A.

    1980-01-01

    Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex. Images PMID:16789190

  19. Baculovirus studies in new, indigenous lepidopteran cell lines.

    PubMed

    Pant, U; Sudeep, A B; Athawale, S S; Vipat, V C

    2002-01-01

    Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines.

  20. Maternal effects, flight versus fecundity trade-offs, and offspring immune defence in the Speckled Wood butterfly, Pararge aegeria

    PubMed Central

    2010-01-01

    Background Maternal condition can generate resource-related maternal effects through differential egg provisioning, and can greatly affect offspring performance. In the present study, the speckled wood butterfly Pararge aegeria (L.) was used to investigate whether (after controlling for egg size) maternal age, and increased flight during the oviposition period, resulted in changes in egg provisioning and whether this contributed to variation in offspring performance, i) early in development (egg stage and early post-hatching development), and ii) later in larval development after being exposed to the model viral pathogen system; the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). Results Age-related changes in maternal egg provisioning were observed to influence egg stage development only. Flight-induced changes in maternal egg provisioning had direct consequences for offspring growth and survival across each life stage from egg to adulthood; offspring from forced flight mothers had lower larval masses and longer development times. Offspring with lower larval masses also had reduced survival after exposure to the viral pathogen. Conclusion The present study demonstrates that a change in maternal provisioning as a result of increased flight during the oviposition period has the potential to exert non-genetic cross-generational fitness effects in P. aegeria. This could have important consequences for population dynamics, particularly in fragmented anthropogenic landscapes. PMID:21067561

  1. Cloning of complete genomes of large dsDNA viruses by in vitro transposition of an F factor containing transposon.

    PubMed

    Wang, Yongjie; Stojiljković, Nina; Jehle, Johannes A

    2010-07-01

    An improved bacmid technology for cloning complete genomes of large dsDNA viruses with circular genomes has been developed and tested. The system, termed EZ::BAC, is based on Escherichia coli F factor replicon, a chloramphenicol resistant marker gene with the mosaic ends recognized specifically by the transposase of the Tn5. In vitro transposition was carried out for the baculovirus shuttle vector pMON14272 (136kb) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome (134kb) as target DNAs. Transposon EZ::BAC was inserted randomly into the target DNAs, leading to 9bp duplication of the flanking end at the insertion site. One of the obtained AcMNPV::BACs replicated in Sf21 cells after transfection. The random in vitro generation of viral bacmids using EZ::BAC facilitates the host-independent propagation of intact and functional viral genomes in E. coli cells and does not require sequence information of the target DNA as is necessary for the generation of bacmids in conventional systems. Copyright 2009 Elsevier B.V. All rights reserved.

  2. Prorenin processing enzyme (PPE) produced by Baculovirus-infected Sf-9 insect cells: PPE is the cysteine protease encoded in the acMNPV gene.

    PubMed

    Gotoh, Takeshi; Awa, Hirono; Kikuchi, Ken-Ichi; Nirasawa, Satoru; Takahashi, Saori

    2010-01-01

    In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610-2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells.

  3. The Chitinase A from the baculovirus AcMNPV enhances resistance to both fungi and herbivorous pests in tobacco.

    PubMed

    Corrado, Giandomenico; Arciello, Stefania; Fanti, Paolo; Fiandra, Luisa; Garonna, Antonio; Digilio, Maria Cristina; Lorito, Matteo; Giordana, Barbara; Pennacchio, Francesco; Rao, Rosa

    2008-08-01

    Biotechnology has allowed the development of novel strategies to obtain plants that are more resistant to pests, fungal pathogens and other agents of biotic stress. The obvious advantages of having genotypes with multiple beneficial traits have recently fostered the development of gene pyramiding strategies, but less attention has been given to the study of genes that can increase resistance to different types of harmful organisms. Here we report that a recombinant Chitinase A protein of the Autographa californica nuclear polyhedrosis virus (AcMNPV) has both antifungal and insecticide properties in vitro. Transgenic tobacco plants expressing an active ChiA protein showed reduced damages caused by fungal pathogens and lepidopteran larvae, while did not have an effect on aphid populations. To our knowledge, this is the first report on the characterisation and expression in plants of a single gene that increases resistance against herbivorous pests and fungal pathogens and not affecting non-target insects. The implications and the potential of the ChiA gene for plant molecular breeding and biotechnology are discussed.

  4. Expression of the Lassa virus nucleocapsid protein in insect cells infected with a recombinant baculovirus: application to diagnostic assays for Lassa virus infection.

    PubMed

    Barber, G N; Clegg, J C; Lloyd, G

    1990-01-01

    The coding region of the gene for the nucleocapsid protein of Lassa virus has been inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) using the transfer vector pAcYM1, so that expression of the foreign DNA is under the control of the promoter of the AcNPV polyhedrin gene. Infection of cultured Spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of a protein that was indistinguishable from the authentic Lassa virus protein by SDS gel electrophoresis and immunoblotting with a variety of specific immune sera and monoclonal antibodies (MAbs). The kinetics of appearance of the protein were comparable to those of polyhedrin production in wild-type AcNPV-infected cells. The recombinant material was antigenic when used in ELISA for Lassa virus-specific antibodies, reacting well with MAbs specific for the nucleocapsid protein and with sera from experimentally infected guinea-pigs. The recombinant ELISA was able to clearly distinguish sera from human cases of Lassa fever against a panel of known negative sera of African origin. Recombinant-infected insect cells were an effective substitute for mammalian cells infected with Lassa virus itself in the immunofluorescence assay for Lassa virus-specific antibodies. This system offers attractive alternatives to the use of Lassa virus-infected materials as reagents in diagnostic procedures.

  5. Mixed infections and the competitive fitness of faster-acting genetically modified viruses

    PubMed Central

    Zwart, Mark P; Van Der Werf, Wopke; Van Oers, Monique M; Hemerik, Lia; Van Lent, Jan M V; De Visser, J Arjan G M; Vlak, Just M; Cory, Jenny S

    2009-01-01

    Faster-acting recombinant baculoviruses have shown potential for improved suppression of insect pests, but their ecological impact on target and nontarget hosts and naturally occurring pathogens needs to be assessed. Previous studies have focused on the fitness of recombinants at the between-hosts level. However, the population structure of the transmission stages will also be decided by within-host selection. Here we have experimentally quantified the within-host competitive fitness of a fast-acting recombinant Autographa californica multicapsid nucleopolyhedrovirus missing the endogenous egt gene (vEGTDEL), by means of direct competition in single- and serial-passage experiments with its parental virus. Quantitative real-time PCR was employed to determine the ratio of these two viruses in passaged mixtures. We found that vEGTDEL had reduced within-host fitness: per passage the ratio of wild type to vEGTDEL was on average enhanced by a factor of 1.53 (single passage) and 1.68 (serial passage). There is also frequency-dependence: the higher the frequency of vEGTDEL, the stronger the selection against it is. Additionally, the virus ratio is a predictor of time to host death and virus yield. Our results show that egt is important to within-host fitness and allow for a more complete assessment of the ecological impact of recombinant baculovirus release. PMID:25567862

  6. Conserved structural motifs at the C-terminus of baculovirus protein IE0 are important for its functions in transactivation and supporting hr5-mediated DNA replication.

    PubMed

    Luria, Neta; Lu, Liqun; Chejanovsky, Nor

    2012-05-01

    IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  7. Identification of a Conserved Non-Protein-Coding Genomic Element that Plays an Essential Role in Alphabaculovirus Pathogenesis

    PubMed Central

    Kikhno, Irina

    2014-01-01

    Highly homologous sequences 154–157 bp in length grouped under the name of “conserved non-protein-coding element” (CNE) were revealed in all of the sequenced genomes of baculoviruses belonging to the genus Alphabaculovirus. A CNE alignment led to the detection of a set of highly conserved nucleotide clusters that occupy strictly conserved positions in the CNE sequence. The significant length of the CNE and conservation of both its length and cluster architecture were identified as a combination of characteristics that make this CNE different from known viral non-coding functional sequences. The essential role of the CNE in the Alphabaculovirus life cycle was demonstrated through the use of a CNE-knockout Autographa californica multiple nucleopolyhedrovirus (AcMNPV) bacmid. It was shown that the essential function of the CNE was not mediated by the presumed expression activities of the protein- and non-protein-coding genes that overlap the AcMNPV CNE. On the basis of the presented data, the AcMNPV CNE was categorized as a complex-structured, polyfunctional genomic element involved in an essential DNA transaction that is associated with an undefined function of the baculovirus genome. PMID:24740153

  8. An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells.

    PubMed

    Aslanidi, George; Lamb, Kenneth; Zolotukhin, Sergei

    2009-03-31

    Production of clinical-grade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases. We describe a system based on a stably transformed insect cell lines harboring helper genes required for vector production. Integrated genes remain silent until the cell is infected with a single baculovirus expression vector (BEV). The induction of expression results from a combination of the amplification of integrated resident genes (up to 1,200 copies per cell) and the enhancement of the expression mediated by the immediate-early trans-regulator 1 (IE-1) encoded by BEV. The integration cassette incorporates an IE-1 binding target sequence from wild-type Autographa californica multiple nuclear polyhedrosis virus, a homologous region 2 (hr2). A feed-forward loop is initiated by one of the induced proteins, Rep78, boosting the amplification of the integrated genes. The system was tested for the coordinated expression of 7 proteins required to package recombinant adeno-associated virus (rAAV)2 and rAAV1. The described arrangement provided high levels of Rep and Cap proteins, thus improving rAAV yield by 10-fold as compared with the previously described baculovirus/rAAV production system.

  9. Uses of flow cytometry in virology.

    PubMed Central

    McSharry, J J

    1994-01-01

    This article reviews some of the published applications of flow cytometry for in vitro and in vivo detection and enumeration of virus-infected cells. Sample preparation, fixation, and permeabilization techniques for a number of virus-cell systems are evaluated. The use of flow cytometry for multiparameter analysis of virus-cell interactions for simian virus 40, herpes simplex viruses, human cytomegalovirus, and human immunodeficiency virus and its use for determining the effect of antiviral compounds on these virus-infected cells are reviewed. This is followed by a brief description of the use of flow cytometry for the analysis of several virus-infected cell systems, including blue tongue virus, hepatitis C virus, avian reticuloendotheliosis virus, African swine fever virus, woodchuck hepatitis virus, bovine viral diarrhea virus, feline leukemia virus, Epstein-Barr virus, Autographa californica nuclear polyhedrosis virus, and Friend murine leukemia virus. Finally, the use of flow cytometry for the rapid diagnosis of human cytomegalovirus and human immunodeficiency virus in peripheral blood cells of acutely infected patients and the use of this technology to monitor patients on antiviral therapy are reviewed. Future prospects for the rapid diagnosis of in vivo viral and bacterial infections by flow cytometry are discussed. Images PMID:7530594

  10. Baculovirus Capsid Display Potentiates OVA Cytotoxic and Innate Immune Responses

    PubMed Central

    Molinari, Paula; Crespo, María I.; Gravisaco, María J.

    2011-01-01

    Baculoviruses (BV) are DNA viruses that are pathogenic for insects. Although BV infect a range of mammalian cell types, they do not replicate in these cells. Indeed, the potential effects of these insect viruses on the immune responses of mammals are only just beginning to be studied. We show in this paper that a recombinant Autographa californica multiple nuclear polyhedrosis virus carrying a fragment of ovalbumin (OVA) on the VP39 capsid protein (BV-OVA) has the capacity to act as an adjuvant and vector of antigens in mice, thereby promoting specific CD4 and cytotoxic T cell responses against OVA. BV also induced in vivo maturation of dendritic cells and the production of inflammatory cytokines, thus promoting innate and adaptive immune responses. The OVA-specific response induced by BV-OVA was strong enough to reject a challenge with OVA-expressing melanoma cells (MO5 cells) and effectively prolonged survival of MO5 bearing mice. All these findings, together with the absence of pre-existing immunity to BV in humans and the lack of viral gene expression in mammalian cells, make BV a candidate for vaccination. PMID:21918683

  11. Evaluation of Cre Recombinase Delivery in Mammalian Cells using Baculovirus Infection

    PubMed Central

    Erbs, Eric; Pradhan, Amynah A.; Matifas, Audrey; Kieffer, Brigitte L.; Massotte, Dominique

    2013-01-01

    In vivo conditional knock-out of a protein is a method of choice to decipher its biological function. It can be achieved by encoding the cre-recombinase on a recombinant virus to exert spatio-temporal control of its expression and enzymatic activity and, subsequently, of the target gene deletion. Recombinant baculoviruses have been successfully used to express a wide range of proteins in insect cells. More recently, their potential to infect mammalian cells has been addressed but, so far, their ability to yield a conditional knock-out as a result of efficient in vivo cre-recombinase gene delivery has not been examined. Cre-recombinase fused to the green fluorescent protein was cloned under the control of the CAG promoter in a recombinant Autographa californica baculovirus expressing the vesicular stomatitis virus envelope G protein for increased mammalian cell infection. Gene delivery was evaluated in vitro in mammalian cells, neuroblastoma and mouse primary neuronal cultures as well as in vivo in the mouse brain. Infection with adeno-associated viruses encoding the cre-recombinase fused to the green fluorescent protein was performed as a positive control. Our results indicate that baculovirus infection leads to functional cre-recombinase expression in non-neuronal and neuroblastoma cell lines but not in mouse primary neuronal cultures or brain. PMID:23732834

  12. A novel expression system based on host-range expansion of baculovirus.

    PubMed

    Zhu, Y; Qi, Y; Liu, D; Joshua, M N; Wang, Y

    1998-12-01

    A host range expanded recombinant Autographa californica multiple-nucleocapsid nucleopolyhedrosis virus AcMNPV/r2 was obtained by cotransfection of the bacmid DNA from Escherichia coli DH10Bac along with a plasmid pBmH-M containing HindIII M fragment of Bombyx mori nuclear polyhedrosis virus (BmNPV) genomic DNA. A recombinant transposon vector carrying a mutant green fluorescent protein gene (GFP) and a polyhedrin gene was constructed. Transposition was carried out in both E. coli DH10Bac and E. coli DH10BmH, which contains AcMNPV/r2 and a helper plasmid. Recombinant DNAs were transfected into Sf-9 cells to generate recombinant virus AcMNPV/r3 and AcMNPV/r4 respectively. Viral stock of AcMNPV/r4 was then infected into Bombyx mori cells (BmN) and Bombyx mori larvae (silkworm). Analysis shows that GFP was highly expressed in Bombyx mori larvae. This expression system, is practicable therefore for mass production of foreign gene products.

  13. Expression and post-translational processing of a broad-spectrum organophosphorus-neurotoxin-degrading enzyme in insect tissue culture

    SciTech Connect

    Dave, K.I.; Phillips, L.; Luckow, V.A.; Wild, J.R.

    1994-12-31

    A recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), has been utilized to express the opd (organophosphate-degrading) gene from Pseudomonas diminuta in insect tissue-culture cells (Sf9) of the fall armyworm (Spodoptera frugiperda). The broad-spectrum organophosphate hydrolase (EC 3.1.8.1) encoded by this gene is a member of a general class of enzymes (organophosphate (OP) anhyorolases) that include parathion hydrolases, di-isopropyl-fluorophosphatases (DFpases), somanases, and OP phosphotrlesterases. This particular enzyme possesses the ability to hydrolyse paraoxon (P-O bond), DFP, sarin (P-F bond), VX (P-S bond) and tabun (P-CN bond), as well as a number of other extensively used organophosphorus pesticides. The enzyme produced in infected Sf9 cells is post-translationally processed and resembles the mature form of the enzyme expressed in various bacterial cells as identified by immunoprecipitation on Western blots. N-terminal sequence analysis of enzyme expressed in insect cells revealed Gly-29 as the terminal residue, whereas expression in Escherichia coli removes this residue, exposing Ser-30 at the N-terminus. Conditions for optimal expression of the enzyme in this system are described. Furthermore, hydrolytic efficiency of some OPs with purified enzyme from this system is discussed in relation to the in situ activity of Pseudomonas diminuta MG cells.

  14. The Development of Caching and Object Permanence in Western Scrub-Jays (Aphelocoma californica): Which Emerges First?

    PubMed Central

    Salwiczek, Lucie H.; Schlinger, Barney; Emery, Nathan J.; Clayton, Nicola S.

    2010-01-01

    Recent studies on the food-caching behavior of corvids have revealed complex physical and social skills, yet little is known about the ontogeny of food caching in relation to the development of cognitive capacities. Piagetian object permanence is the understanding that objects continue to exist even when they are no longer visible. Here, the authors focus on Piagetian Stages 3 and 4, because they are hallmarks in the cognitive development of both young children and animals. Our aim is to determine in a food-caching corvid, the Western scrub-jay, whether (1) Piagetian Stage 4 competence and tentative caching (i.e., hiding an item invisibly and retrieving it without delay), emerge concomitantly or consecutively; (2) whether experiencing the reappearance of hidden objects enhances the timing of the appearance of object permanence; and (3) discuss how the development of object permanence is related to behavioral development and sensorimotor intelligence. Our findings suggest that object permanence Stage 4 emerges before tentative caching, and independent of environmental influences, but that once the birds have developed simple object-permanence, then social learning might advance the interval after which tentative caching commences. PMID:19685971

  15. Botrytis californica, a new cryptic species in the B. cinerea species complex causing gray mold in blueberries and table grapes

    USDA-ARS?s Scientific Manuscript database

    Botrytis cinerea consists of two cryptic species, referred to as Group I and Group II based on Bc-hch gene RFLP haplotyping, and Group I has been described as a new cryptic species B. pseudocinerea. During a survey for Botrytis spp. causing gray mold in blueberries and table grapes in the Central Va...

  16. Transforming Growth Factor ß Recruits Persistent MAPK Signaling to Regulate Long-Term Memory Consolidation in "Aplysia Californica"

    ERIC Educational Resources Information Center

    Shobe, Justin; Philips, Gary T.; Carew, Thomas J.

    2016-01-01

    In this study, we explore the mechanistic relationship between growth factor signaling and kinase activity that supports the protein synthesis-dependent phase of long-term memory (LTM) consolidation for sensitization of "Aplysia." Specifically, we examine LTM for tail shock-induced sensitization of the tail-elicited siphon withdrawal…

  17. Transforming Growth Factor ß Recruits Persistent MAPK Signaling to Regulate Long-Term Memory Consolidation in "Aplysia Californica"

    ERIC Educational Resources Information Center

    Shobe, Justin; Philips, Gary T.; Carew, Thomas J.

    2016-01-01

    In this study, we explore the mechanistic relationship between growth factor signaling and kinase activity that supports the protein synthesis-dependent phase of long-term memory (LTM) consolidation for sensitization of "Aplysia." Specifically, we examine LTM for tail shock-induced sensitization of the tail-elicited siphon withdrawal…

  18. Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments

    PubMed Central

    Asmar-Rovira, Guillermo A.; Asseo-García, Aloysha M.; Quesada, Orestes; Hanson, Michael A.; Nogueras, Carlos; Lasalde-Dominicci, José A.; Stevens, Raymond C.

    2009-01-01

    The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, the molecular mechanisms by which detergents influence membrane protein stability and function remain poorly understood, and elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examined nine detergents upon nAChR solubilization and purification, to assess receptor lipid composition using GC (Gas Chromatography)-FID (Flame Ionization) and/or GC-MSD (Mass Selective Detectors), stability and aggregation state using A-SEC (Analytical Size-Exclusion Chromatography) and EM (Electron Microscopy), and planar lipid bilayers to measure ion channel function. Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analog detergents [CHAPS (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate), FC-12 (n-Dodecylphosphocholine) and sodium cholate (3α,7α,12α-Trihydroxy-5β-cholan-24-oic acid)] maintaining stability and supporting ion channel function, while non-lipid analog detergents [Cymal-6 (6-Cyclohexyl-1-hexyl-β-d-maltoside), DDM (n-Dodecyl-β-d-maltopyranoside), LDAO (Lauryldimethylamine-N-oxide) and OG (n-Octyl-β-d-glucopyranoside)] showed decreased stability and significant reduction or loss of ion channel function. Anapoe-C12E9 (Polyoxyethylene-(9)-dodecyl ether) and BigCHAP (N,N′-bis-(3-d-Gluconamidopropyl) cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function when compared to lipid-analog detergents. Overall, these results show that the nAChR can be stable and functional in lipid-analog detergents or in detergents that retain moderate amounts of residual native lipids, while the opposite is true about non-lipid analog detergents. These results highlight the importance of careful biophysical characterization of membrane proteins for future functional or structural studies in the detergent-solubilized state. PMID:18581036

  19. DIETARY NITROGEN AVAILABILITY IN MACROALGAE AFFECTS GROWTH OF THE SEA HARE APLYSIA CALIFORNICA (OPISTHOBRANCHIA:ANASPIDEA). (R830414)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  20. Transforming growth factor β recruits persistent MAPK signaling to regulate long-term memory consolidation in Aplysia californica.

    PubMed

    Shobe, Justin; Philips, Gary T; Carew, Thomas J

    2016-05-01

    In this study, we explore the mechanistic relationship between growth factor signaling and kinase activity that supports the protein synthesis-dependent phase of long-term memory (LTM) consolidation for sensitization ofAplysia Specifically, we examine LTM for tail shock-induced sensitization of the tail-elicited siphon withdrawal (T-SW) reflex, a form of memory that requires both (i) extracellular signal-regulated kinase (ERK1/2; MAPK) activity within identified sensory neurons (SNs) that mediate the T-SW and (ii) the activation of transforming growth factor β (TGFβ) signaling. We now report that repeated tail shocks that induce intermediate-term (ITM) and LTM for sensitization, also induce a sustained post-training phase of MAPK activity in SNs (lasting at least 1 h). We identified two mechanistically distinct phases of post-training MAPK: (i) an immediate phase that does not require ongoing protein synthesis or TGFβ signaling, and (ii) a sustained phase that requires both protein synthesis and extracellular TGFβ signaling. We find that LTM consolidation requires sustained MAPK, and is disrupted by inhibitors of protein synthesis and TGFβ signaling during the consolidation window. These results provide strong evidence that TGFβ signaling sustains MAPK activity as an essential mechanistic step for LTM consolidation.

  1. Distinct Growth Factor Families Are Recruited in Unique Spatiotemporal Domains during Long-Term Memory Formation in Aplysia californica.

    PubMed

    Kopec, Ashley M; Philips, Gary T; Carew, Thomas J

    2015-06-03

    Several growth factors (GFs) have been implicated in long-term memory (LTM), but no single GF can support all of the plastic changes that occur during memory formation. Because GFs engage highly convergent signaling cascades that often mediate similar functional outcomes, the relative contribution of any particular GF to LTM is difficult to ascertain. To explore this question, we determined the unique contribution of distinct GF families (signaling via TrkB and TGF-βr-II) to LTM formation in Aplysia. We demonstrate that TrkB and TGF-βr-II signaling are differentially recruited during two-trial training in both time (by trial 1 or 2, respectively) and space (in distinct subcellular compartments). These GFs independently regulate MAPK activation and synergistically regulate gene expression. We also show that trial 1 TrkB and trial 2 TGF-βr-II signaling are required for LTM formation. These data support the view that GFs engaged in LTM formation are interactive components of a complex molecular network.

  2. DIETARY NITROGEN AVAILABILITY IN MACROALGAE AFFECTS GROWTH OF THE SEA HARE APLYSIA CALIFORNICA (OPISTHOBRANCHIA:ANASPIDEA). (R830414)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  3. Kinetics of calcium-dependent inactivation of calcium current in voltage-clamped neurones of Aplysia californica.

    PubMed Central

    Chad, J; Eckert, R; Ewald, D

    1984-01-01

    Ca currents flowing during voltage-clamp depolarizations were examined in axotomized Aplysia neurones under conditions that virtually eliminated other currents. Moderate to large currents exhibited a two-component time course of relaxation that can be approximated reasonably well by the sum of two exponentials. The rapid phase (tau 1 approximately equal to 70 ms at 0 mV) plus the slower phase (tau 2 approximately equal to 300 ms at 0 mV) ride upon a steady, non-inactivating current, I infinity. Conditions that diminish the peak current amplitude, such as reduced stimulus depolarization, inactivation remaining from a prior depolarization, or partial blockade of the Ca conductance by Cd, slowed both phases of inactivation, and all selectively eliminated the tau 1 phase, such that weak currents exhibited only the slower phase of decline. Injection of EGTA slowed both phases of inactivation, decreased the extent of the tau 1 phase, and increased the intensity of I infinity and of the current during the tau 2 phase. For a given voltage, the rate of inactivation increased as the peak current strength was increased, and decreased as the peak current strength was decreased. For a given peak current the rate of inactivation decreased as depolarization was increased. The relation of inactivation to prior Ca2+ entry was essentially linear for small currents, but decreased in slope with time during strong currents. The relation also became shallower with increasing depolarization, suggesting an apparent decrease in the efficacy of Ca in causing inactivation at more positive potentials. The basic kinetics of Ca current inactivation along with experimentally induced changes in those kinetics were simulated with a binding-site model in which inactivation develops during current flow as a function of the entry and accumulation of free Ca2+. This demonstrated that a single Ca-mediated process can account for the two-component time course of inactivation, and that the nearly bi-exponential shape need not arise from two separate processes. The two-component time course emerges as a consequence of a postulated hyperbolic reaction between diminishing probability of channels remaining open and the accumulation of intracellular free Ca2+. The occurrence of a single- or a two-component time course of inactivation thus appears to depend on the levels of internal free Ca2+ traversed during current flow. PMID:6323696

  4. Screening of candidate proteins interacting with IE-2 of Bombyx mori nucleopolyhedrovirus.

    PubMed

    Wu, Yejun; Wu, Yu; Wu, Yan; Tang, Hui; Wu, Huiling; Zhang, Guozheng; Wang, Wenbing

    2013-10-01

    IE-2 of Bombyx mori nucleopolyhedrovirus (BmNPV) has been shown to play important roles in baculovirus infection, which are involved in gene expression and viral replication. However, the mechanism remains unknown. In this paper, by TargetP software, four genes, i.e.-2, odv-e26, odv-e56 and BmNPV-gp101 (Ac-orf116) of BmNPV and Autographa californica multiple NPV (AcMNPV) were predicted to be located in mitochondria. By BLAST tool using BmNPV IE-2 protein sequence, 14 NPVs were found to have IE-2 homologues in GenBank, and most of them were predicted to be located in mitochondria, except for that of Antheraea pernyi NPV (AnpeNPV) and Anticarsia gemmatalis NPV (AngeNPV). To observe the subcellular localization of BmNPV IE-2, a recombinant virus overexpressed the IE-2 and eGFP fusion protein was constructed. In infected BmN cells, the fluorescence specifically enriched in the cellular mitochondria. This evidence was accordant with the prediction. Further, Pull-down assay was used to select protein candidates interacting with IE-2 in B. mori cells infected with BmNPV. Of several isolated protein components, sixteen candidates were identified by MALDI-TOF mass-spectrometry, eight baculoviral proteins (ALK-EXO, F protein, IAP-1, LEF-3, LEF-9, ODV-NC42, TLP, and VP39), and eight proteins from B. mori (Actin, ADP/ATP translocase, ATP synthase subunit beta, Beta-tubulin, DNA topoisomerase 2, Histone H4, Soluble guanylyl cyclae alpha-1 subunit, Transketolase). From the functional point of view, most of these proteins were generally divided into two groups, mitochondrial interaction proteins and viral DNA replication proteins. These results implied that the IE-2 had multiple functions involved in regulating viral gene expression, viral replication and also as a component of mitochondrial factors to regulate the cellular energy supply and apoptosis.

  5. Effects of temperature on the susceptibility of insect cells to infection by baculoviruses.

    PubMed

    Lynn, D E

    2001-01-01

    Three insect cell lines were tested for susceptibility to baculovirus infection by use of a typical endpoint assay procedure. Cell lines from Spodoptera frugiperda (IPLB-Sf21AE), Lymantria dispar (IPLB-LdEIta), and Heliothis virescens (IPLB-HvE6s) in 96-well tissue culture plates were each infected with dilutions of extra cellular virus suspensions of the Autographa californica nucleopolyhedrovirus (AcMNPV). In addition, the L. dispar and H. virescens cells were also infected with L. dispar nucleopolyhedrovirus, and Helicoverpa zea nucleopolyhedrovirus, respectively. Each cell/virus combination was incubated at three temperatures: 22, 27 and 32 degrees C and wells were scored for positive infection (presence of occlusion bodies in cell nuclei) at 2 to 4 day intervals for up to 4 weeks. The resulting data were analyzed by the Spearman-Kärber method, providing virus titers for each combination of virus, cell line, and temperature. The results were categorized by accuracy (assuming the highest titer achieved was the most accurate) and by rapidity of maximum titer. AcMNPV reached the highest titer in each line at 22 degrees C although equivalent titers were reached with both AcMNPV and HzSNPV in the HvE6a line at all three temperatures. This line actually reported about 100-fold less AcMNPV than the other two lines with the same virus sample. Alternatively, the Sf21AE and LdEIta lines reached 10-fold higher titers at the lowest temperature as compared with the higher temperatures, although also at a slower rate.

  6. Verifying the stability of selected genes for normalization in Q PCR experiments of Spodoptera frugiperda cells during AcMNPV infection.

    PubMed

    Salem, Tamer Z; Allam, Walaa R; Thiem, Suzanne M

    2014-01-01

    It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection. Autographa californica nucleopolyhedrovirus (AcMNPV) is known to globally shut off host gene transcription in Sf21 cells and to modify their cytoskeletons. In this study, seven host genes were selected for validation as references for gene expression experiments using qRT-PCR. Two of them, ecdysoneless (ECD) and myosin showed stable RNA levels in our previous microarray study at 6, 12, and 24 hpi for both genes and 48 hpi for ECD. The others, actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S ribosome (28S), are commonly employed as reference genes for qRT-PCR. Ribosomal protein L35 (L35) gene was selected to test if ribosomal protein genes show stable RNA transcript levels similar to 28S and 18S rRNA and to validate the microarray data. In addition to 28S, previously known to have stable transcript levels, qRT-PCR showed that ECD transcript levels remained constant throughout the time course of AcMNPV infection. Transcripts of cytoskeleton genes such as actin, tubulin, and myosin declined dramatically as the infection progressed. GAPDH and L35 transcripts also declined over time. These results indicate that ECD is a reliable reference gene for qRT-PCR experiments during AcMNPV infection of Spodoptera frugiperda cells. Although 28S could be used as a reference gene for these experiments, it is less useful than ECD because of its abundance, which might make it difficult to establish an accurate baseline value for data analysis.

  7. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35

    PubMed Central

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-01-01

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its ‘reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro. PMID:22170098

  8. The 91-205 amino acid region of AcMNPV ORF34 (Ac34), which comprises a potential C3H zinc finger, is required for its nuclear localization and optimal virus multiplication.

    PubMed

    Qiu, Jianxiang; Tang, Zhimin; Yuan, Meijin; Wu, Wenbi; Yang, Kai

    2017-01-15

    During baculovirus infection, most viral proteins must be imported to the nucleus to support virus multiplication. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf34 (ac34) is an alphabaculovirus unique gene that is required for optimal virus production. Ac34 distributes in both the cytoplasm and the nuclei of virus-infected Sf9 cells, but contains no conventional nuclear localization signal (NLS). In this study, we investigated the nuclear targeting domains in Ac34. Transient expression assays showed that Ac34 localized in both the cytoplasm and the nuclei of Sf9 cells, indicating that no viral protein is required for Ac34 nuclear localization. Subcellular localization analysis of Ac34 truncations and internal deletions fused with green fluorescent protein in plasmid-transfected Sf9 cells identified that the 91-205 amino acid (aa) region is required for Ac34 nuclear localization. Mutations in a potential C3H zinc finger (aa 116-131) in Ac34 resulted in exclusive cytoplasmic distribution of GFP:Ac34, suggesting that the zinc finger is required for Ac34 nuclear localization. To assess the functional importance of Ac34 in the nucleus during virus replication, recombinant AcMNPV bacmids containing a series of Ac34 truncations, internal deletions, or site mutations fused with HA tags were constructed. Subcellular localization analysis showed that Ac34 with internal deletions in aa 91-205 or site mutations in the potential zinc finger was predominantly distributed in the cytoplasm. Viral plaque assays and virus growth curves indicated that disruption of Ac34 nuclear localization significantly impaired virus replication. Taken together, our findings demonstrated that the nuclear localization of Ac34 requires the 91-205 aa region and its nuclear localization is essential for optimal virus replication.

  9. Studies of Choristoneura fumiferana nuclear polyhedrosis virus gene expression in insect cells.

    PubMed

    Qiu, W; Liu, J J; Carstens, E B

    1996-03-15

    To investigate the mechanisms regulating baculovirus virulence and host range we have begun to study Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) and its gene expression in permissive and nonpermissive cells. We have identified and mapped three genes on the CfMNPV genome. The polyhedrin gene is located from 0.0 to 2.0 m.u. and two other genes, dnapol and p143, both of which are essential for baculovirus DNA replication, are located from 35.3 to 40.9 m.u. and 55.5 to 63.4 m.u., respectively. To gain insight into the expression of CfMNPV genes in permissive C. fumiferana and nonpermissive Spodoptera frugiperda cells, we constructed three expression plasmids in which the promoter region of the dnapol, the p143, and polyhedrin genes were placed in front of a chloramphenicol acetyltransferase reporter gene. All three CfMNPV promoters were active in nonpermissive cells in the presence of Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA, but no activity was detected in permissive cells either in the presence of CfMNPV DNA or AcMNPV DNA. This lack of promoter activity was not due to failure of viral or plasmid DNA to enter the cell nucleus. It was possible that the reporter plasmids were inefficient templates for transcriptional transactivation so we developed a CfMNPV transfer vector and generated a recombinant virus in which the polyhedrin promoter driving CAT gene cassette was integrated into the CfMNPV genome. In this case, the CfMNPV polyhedrin promoter was highly active in the permissive cells.

  10. A molt-associated chitinase cDNA from the spruce budworm, Choristoneura fumiferana.

    PubMed

    Zheng, Y; Zheng, S; Cheng, X; Ladd, T; Lingohr, E J; Krell, P J; Arif, B M; Retnakaran, A; Feng, Q

    2002-12-01

    Chitinase (CfChitinase) cDNA from the spruce budworm, Choristoneura fumiferana, was cloned using reverse transcription PCR and cDNA library screening. The CfChitinase cDNA was determined to be 2856 nucleotides long with the longest open reading frame made up of 1671 nucleotides that encoded a protein that was 557 amino acid long with a predicted molecular mass of 62 kDa. The deduced amino acid sequence showed 76-79% identity with other lepidopteran chitinases. Northern blots revealed that transcripts of CfChitinase appeared prior to each molt and peaked on the day of ecdysis from the second instar to the pupal stage but disappeared immediately after the molt. No transcripts could be detected in the early first instar prior to the spinning of the hibernaculum or in the diapausing second instars or during the intermolt periods of the other instars. Western blot analysis revealed that the protein appeared 12 h prior to ecdysis and disappeared 12 h after ecdysis from the sixth instar to pupal stage. The 20-hydroxyecdysone analog, tebufenozide (RH5992), induced expression of CfChitinase in the early stage of the sixth instar and caused a precocious and incomplete molt into an extra larval stage. During the sixth instar to the pupal molt, transcripts could be detected only in the epidermis and fat bodies, but not in the midgut. Western blots showed that the protein was present in the epidermis and midgut, but not in the fat bodies. The recombinant protein expressed in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) showed high levels of chitinolytic activity with an optimal pH range 6-9. Glycosylation appeared to be necessary for the chitinolytic activity and secretion of the recombinant protein.

  11. Identification and molecular characterization of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus genomic region encoding the regulatory genes pkip, p47, lef-12, and gta.

    PubMed

    Lapointe, R; Back, D W; Ding, Q; Carstens, E B

    2000-05-25

    Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV) is a baculovirus pathogenic to spruce budworm, the most damaging insect pest in Canadian forestry. CfMNPV is less virulent to its host insect and its replication cycle is slower than the baculovirus type species Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) but the basis of these characteristics is not known. We have now identified, localized, and determined the sequence of the region of CfMNPV carrying potentially important regulatory genes including p47, lef-12, gta, and pkip. DNA database searches revealed that this region of CfMNPV is most closely related to the homologous OpMNPV genes. Transcription analysis demonstrated that CfMNPV P47 is encoded by a 1.6-kb transcript, LEF-12 is encoded by a 2.6-kb transcript, and GTA is encoded by a 2.1-kb transcript. Transcripts for these genes were detectable at 6 h postinfection but all of them showed a burst in expression levels between 12 and 24 h postinfection corresponding to the time of initiation of CfMNPV DNA replication. A polyclonal antibody, raised against CfMNPV P47, detected a nuclear 43-kDa polypeptide from 12 to 72 h postinfection, demonstrating that the CfMNPV p47 gene product is first expressed at a time corresponding to the burst of transcriptional activity between the early and the late phases. Both AcMNPV and CfMNPV P47 translocate to the nucleus of infected cells.

  12. Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

    PubMed

    Lyupina, Yulia V; Zatsepina, Olga G; Serebryakova, Marina V; Erokhov, Pavel A; Abaturova, Svetlana B; Kravchuk, Oksana I; Orlova, Olga V; Beljelarskaya, Svetlana N; Lavrov, Andrey I; Sokolova, Olga S; Mikhailov, Victor S

    2016-06-01

    Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection.

  13. Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication.

    PubMed

    Mitchell, Jonathan K; Friesen, Paul D

    2012-12-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals.

  14. Functional characterization of Bombyx mori nucleopolyhedrovirus late gene transcription and genome replication factors in the non-permissive insect cell line SF-21

    SciTech Connect

    Berretta, Marcelo F.; Deshpande, Mandar; Crouch, Erin A.; Passarelli, A. Lorena . E-mail: lpassar@ksu.edu

    2006-04-25

    We compared the abilities of late gene transcription and DNA replication machineries of the baculoviruses Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) in SF-21 cells, an insect-derived cell line permissive for AcMNPV infection. It has been well established that 19 AcMNPV late expression factors (lefs) stimulate substantial levels of late gene promoter activity in SF-21 cells. Thus, we constructed a set of clones containing the BmNPV homologs of the AcMNPV lefs under control of the constitutive Drosophila heat shock 70 protein promoter and tested their ability to activate an AcMNPV late promoter-reporter gene cassette in SF-21 cells. We tested the potential of individual or predicted functional groups of BmNPV lefs to successfully replace the corresponding AcMNPV gene(s) in transient late gene expression assays. We found that most, but not all, BmNPV lefs were able to either fully or partially substitute for the corresponding AcMNPV homolog in the context of the remaining AcMNPV lefs with the exception of BmNPV p143, ie-2, and p35. BmNPV p143 was unable to support late gene expression or be imported into the nucleus of cells in the presence of the AcMNPV or the BmNPV LEF-3, a P143 nuclear shuttling factor. Our results suggest that host-specific factors may affect the function of homologous proteins.

  15. Expression of the lef5 gene from Spodoptera exigua multiple nucleopolyhedrovirus contributes to the baculovirus stability in cell culture.

    PubMed

    Martínez-Solís, María; Jakubowska, Agata K; Herrero, Salvador

    2017-09-09

    Baculoviruses are a broad group of viruses infecting insects, predominately of the order Lepidoptera. They are used worldwide as biological insecticides and as expression vectors to produce recombinant proteins. Baculoviruses replicate in their host, although several cell lines have been developed for in vitro replication. Nevertheless, replication of baculoviruses in cell culture involves the generation of defective viruses with a decrease in productivity and virulence. Transcriptional studies of the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infective process revealed differences in the expression patterns when the virus replicated under in vitro (Se301 cells) or in vivo (S. exigua larvae) conditions. The late expression factor 5 (lef5) gene was found to be highly overexpressed when the virus replicates in larvae. To test the possible role of lef5 expression in viral stability, recombinant AcMNPV expressing the lef5 gene from SeMNPV (Se-lef5) was generated and its stability was monitored during successive infection passages in Sf21 cells by evaluating the loss of several essential and non-essential genes. The gfp transgene was more stable in those viruses expressing the Se-LEF5 protein and the GFP-defective viruses were accumulated at a lower level when compared to its control viruses, confirming the positive influence of lef5 in viral stability during the multiplication process. This work describes for the first time a viral factor involved in transgene stability when baculoviruses replicate in cell culture, opening new ways to facilitate the in vitro production of recombinant proteins using baculovirus.

  16. Phylogenetic analysis and possible function of bro-like genes, a multigene family widespread among large double-stranded DNA viruses of invertebrates and bacteria.

    PubMed

    Bideshi, Dennis K; Renault, Sylvaine; Stasiak, Karine; Federici, Brian A; Bigot, Yves

    2003-09-01

    Baculovirus repeated open reading frame (bro) genes and their relatives constitute a multigene family, typically with multiple copies per genome, known to occur among certain insect dsDNA viruses and bacteriophages. Little is known about the evolutionary history and function of the proteins encoded by these genes. Here we have shown that bro and bro-like (bro-l) genes occur among viruses of two additional invertebrate viral families, Ascoviridae and Iridoviridae, and in prokaryotic class II transposons. Analysis of over 100 sequences showed that the N-terminal region, consisting of two subdomains, is the most conserved region and contains a DNA-binding motif that has been characterized previously. Phylogenetic analysis indicated that these proteins are distributed among eight groups, Groups 1-7 consisting of invertebrate virus proteins and Group 8 of proteins in bacteriophages and bacterial transposons. No bro genes were identified in databases of invertebrate or vertebrate genomes, vertebrate viruses and transposons, nor in prokaryotic genomes, except in prophages or transposons of the latter. The phylogenetic relationship between bro genes suggests that they have resulted from recombination of viral genomes that allowed the duplication and loss of genes, but also the acquisition of genes by horizontal transfer over evolutionary time. In addition, the maintenance and diversity of bro-l genes in different types of invertebrate dsDNA viruses, but not in vertebrate viruses, suggests that these proteins play an important role in invertebrate virus biology. Experiments with the unique orf2 bro gene of Autographa californica multicapsid nucleopolyhedrovirus showed that it is not required for replication, but may enhance replication during the occlusion phase of reproduction.

  17. The "11K" gene family members sf68, sf95 and sf138 modulate transmissibility and insecticidal properties of Spodoptera frugiperda multiple nucleopolyhedrovirus.

    PubMed

    Beperet, Inés; Simón, Oihane; Williams, Trevor; López-Ferber, Miguel; Caballero, Primitivo

    2015-05-01

    The "11K" gene family is notable for having homologs in both baculoviruses and entomopoxviruses and is classified as either type 145 or type 150, according to their similarity with the ac145 or ac150 genes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). One homolog of ac145 (sf138) and two homologs of ac150 (sf68 and sf95) are present in Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Recombinant bacmids lacking sf68, sf95 or sf138 (Sf68null, Sf95null and Sf138null, respectively) and the respective repair bacmids were generated from a bacmid comprising the complete virus genome. Occlusion bodies (OBs) of the Sf138null virus were ∼15-fold less orally infective to insects, which was attributed to a 100-fold reduction in ODV infectious titer. Inoculation of insects with Sf138null OBs in mixtures with an optical brightener failed to restore the pathogenicity of Sf138null OBs to that of the parental virus, indicating that the effects of sf138 deletion on OB pathogenicity were unlikely to involve an interaction with the gut peritrophic matrix. In contrast, deletion of sf68 and sf95 resulted in a slower speed-of-kill by 9h, and a concurrent increase in the yield of OBs. Phylogenetic analysis indicated that sf68 and sf95 were not generated after a duplication event of an ancestral gene homologous to the ac150 gene. We conclude that type 145 genes modulate the primary infection process of the virus, whereas type 150 genes appear to have a role in spreading systemic infection within the insect. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Characterization of novel components of the baculovirus per os infectivity factor complex.

    PubMed

    Peng, Ke; van Lent, Jan W M; Boeren, Sjef; Fang, Minggang; Theilmann, David A; Erlandson, Martin A; Vlak, Just M; van Oers, Monique M

    2012-05-01

    Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.

  19. Protein-Protein Interactions of the Baculovirus Per Os Infectivity Factors in the PIF Complex.

    PubMed

    Zheng, Qin; Shen, Yunwang; Kon, Xiangshuo; Zhang, Jianjia; Feng, Min; Wu, Xiaofeng

    2017-01-28

    After ingestion of occlusion bodies, the occlusion-derived viruses (ODVs) of baculoviruses establish the first round of infection within the larval host midgut cells. Several ODV envelope proteins, called per os infectivity factors (PIFs), have been shown to be essential for oral infection. Eight PIFs have been identified to date, including P74, PIFs1-6, and Ac110. At least six PIFs: P74, PIFs1-4, PIF6, together with three other ODV-specific proteins: Ac5, P95 (Ac83), and Ac108, have been reported to form a complex on the ODV surface. In this study, in order to understand the interactions of these PIFs, the direct protein-protein interactions of the nine components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex were investigated using yeast two-hybrid (Y2H) combined with bimolecular fluorescence complementation (BiFC) assay. Six direct interactions comprising PIF1-PIF2, PIF1-PIF3, PIF1-PIF4, PIF1-P95, PIF2-PIF3, and PIF3-PIF4, were identified in Y2H analysis, and these results were further verified by BiFC. For P74, PIF6, Ac5 and Ac108, no direct interaction was identified. P95 (Ac83) was identified to interact with PIF1 and further Y2H analysis of the truncations and deletion mutants showed that the predicted P95 chitin-binding domain and PIF1 100-200aa were responsible for P95 interaction with PIF1. Furthermore, a summary of the protein-protein interactions of PIFs reported so far, comprising 10 reciprocal interactions and 2 self-interactions, is presented, which will facilitate our understanding of the characteristic of PIF complex.

  20. Baculovirus FP25K Localization: Role of the Coiled-Coil Domain.

    PubMed

    Garretson, Tyler A; McCoy, Jason C; Cheng, Xiao-Wen

    2016-11-01

    Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking

  1. Chondroitinase from baculovirus Bombyx mori nucleopolyhedrovirus and chondroitin sulfate from silkworm Bombyx mori.

    PubMed

    Sugiura, Nobuo; Ikeda, Motoko; Shioiri, Tatsumasa; Yoshimura, Mayumi; Kobayashi, Michihiro; Watanabe, Hideto

    2013-12-01

    Chondroitin sulfate (CS) is a linear polysaccharide composed of repeating disaccharide units of glucuronic acid (GlcUA) and N-acetyl-d-galactosamine (GalNAc) with sulfate groups at various positions. Baculovirus is an insect-pathogenic virus that infects Lepidoptera larvae. Recently, we found that the occlusion-derived virus envelope protein 66 (ODV-E66) from Autographa californica nucleopolyhedrovirus (AcMNPV) exhibits chondroitin (CH)-digesting activity with distinct substrate specificity. Here, we demonstrate that the ODV-E66 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) exhibits 92% homology to the amino acid sequence and 83% of the CH lyase activity of ODV-E66 from AcMNPV. ODV-E66 cleaves glycosyl bonds at nonreducing sides of disaccharide units consisting of nonsulfated and 6-O-sulfated GalNAc residues. We then investigated CS in the silkworm, Bombyx mori, which is the host of BmNPV. CS was present in insect tissues such as the midgut, peritrophic membrane, silk gland and skin. The polysaccharide consisted of a nonsulfated disaccharide unit, mono-sulfated disaccharide at Position 4 of the GalNAc residue and mono-sulfated disaccharide at Position 6 of the GalNAc residue. With regard to immunohistochemical analysis, the staining patterns of the silkworm tissues were different among anti-CS antibodies. Chondroitn sulfate that is digestible by ODV-E66 exists sufficiently in the peritrophic membrane protecting the midgut epithelium from ingested pathogens. Our results suggest that ODV-E66 facilitates the primary infection of the virus by digestion of CS in the peritrophic membrane.

  2. Baculovirus per os infectivity factors form a complex on the surface of occlusion-derived virus.

    PubMed

    Peng, Ke; van Oers, Monique M; Hu, Zhihong; van Lent, Jan W M; Vlak, Just M

    2010-09-01

    Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% beta-mercaptoethanol with heating at 50 degrees C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infection.

  3. Identification of protein-protein interactions of the occlusion-derived virus-associated proteins of Helicoverpa armigera nucleopolyhedrovirus.

    PubMed

    Peng, Ke; Wu, Minzhi; Deng, Fei; Song, Jingjiao; Dong, Chunsheng; Wang, Hualin; Hu, Zhihong

    2010-03-01

    The purpose of this study was to identify protein-protein interactions among the components of the occlusion-derived virus (ODV) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV), a group II alphabaculovirus in the family Baculoviridae. To achieve this, 39 selected genes of potential ODV structural proteins were cloned and expressed in the Gal4 yeast two-hybrid (Y2H) system. The direct-cross Y2H assays identified 22 interactions comprising 13 binary interactions [HA9-ODV-EC43, ODV-E56-38K, ODV-E56-PIF3, LEF3-helicase, LEF3-alkaline nuclease (AN), GP41-38K, GP41-HA90, 38K-PIF3, 38K-PIF2, VP80-HA100, ODV-E66-PIF3, ODV-E66-PIF2 and PIF3-PIF2] and nine self-associations (IE1, HA44, LEF3, HA66, GP41, CG30, 38K, PIF3 and P24). Five of these interactions - LEF3-helicase and LEF3-AN, and the self-associations of IE1, LEF3 and 38K - have been reported previously in Autographa californica multiple nucleopolyhedrovirus. As HA44 and HA100 were two newly identified ODV proteins of group II viruses, their interactions were further confirmed. The self-association of HA44 was verified with a His pull-down assay and the interaction of VP80-HA100 was confirmed by a co-immunoprecipitation assay. A summary of the protein-protein interactions of baculoviruses reported so far, comprising 68 interactions with 45 viral proteins and five host proteins, is presented, which will facilitate our understanding of the molecular mechanisms of baculovirus infection.

  4. The Host Specificities of Baculovirus per os Infectivity Factors

    PubMed Central

    Song, Jingjiao; Wang, Xi; Hou, Dianhai; Huang, Huachao; Liu, Xijia; Deng, Fei; Wang, Hualin; Arif, Basil M.; Hu, Zhihong; Wang, Manli

    2016-01-01

    Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host’s midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses. PMID:27454435

  5. The effect of cell line, phylogenetics and medium on baculovirus budded virus yield and quality.

    PubMed

    Matindoost, Leila; Hu, Hao; Chan, Leslie C L; Nielsen, Lars K; Reid, Steven

    2014-01-01

    The performance of bioprocesses involving baculoviruses largely depends on an efficient infection of cells by concentrated budded virus (BV) inoculums. Baculovirus expression vector systems have been established using Autographa californica nucleopolyhedrovirus (AcMNPV), a group I NPV that displays rapid virus kinetics, whereas bioprocesses using group II baculovirus-based biopesticides such as Helicoverpa armigera nucleopolyhedrovirus (HearNPV) have the limitation of low levels of BV, as these viruses often display poor BV production kinetics. In this study, the effect of key parameters involved in the quality of progeny virions, including cell line, virus phylogenetics and medium, on viral DNA replication, virus trafficking to the extracellular environment, and the yield of recombinant protein or polyhedra were investigated in synchronous infections of HearNPV and AcMNPV. HearNPV showed higher vDNA replication in its optimum medium, SF900III, when compared to AcMNPV, but both viruses had similar specific extracellular virion content. However, the ratio of AcMNPV extracellular virions to the total number of progeny virions produced was higher, and their quality was tenfold higher than that of HearNPV extracellular virions. The results of infection of two different cell lines, High Five and Sf9, with AcMNPV, along with HearNPV infection of HzAM1 cells in three different media, suggest that the host cells and the nutritional state of the medium as well as the phylogenetics of the virus affect the BV yields produced by different baculovirus/cell line/medium combinations.

  6. Generating a host range-expanded recombinant baculovirus

    PubMed Central

    Wu, Chunfeng; Deng, Zihao; Long, Zhao; Cai, Yi; Ying, Zhongfu; Yin, Hanqi; Yuan, Meijin; Clem, Rollie J.; Yang, Kai; Pang, Yi

    2016-01-01

    As baculoviruses usually have a narrow insecticidal spectrum, knowing the mechanisms by which they control the host-range is prerequisite for improvement of their applications as pesticides. In this study, from supernatant of culture cells transfected with DNAs of an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant lacking the antiapoptotic gene p35 (vAc∆P35) and a cosmid representing a fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), a viral strain was plaque-purified and named vAcRev. vAcRev had a broader host range than either vAc∆P35 or SeMNPV parental virus, being able to infect not only the permissive hosts of its parental viruses but also a nonpermissive host (Spodoptera litura). Genome sequencing indicated that vAcRev comprises a mixture of two viruses with different circular dsDNA genomes. One virus contains a genome similar to vAc∆P35, while in the other viral genome, a 24.4 kbp-fragment containing 10 essential genesis replaced with a 4 kbp-fragment containing three SeMNPV genes including a truncated Se-iap3 gene. RNA interference and ectopic expression assays found that Se-iap3 is responsible for the host range expansion of vAcRev, suggesting that Se-iap3 inhibits the progression of apoptosis initiated by viral infection and promotes viral propagation in hosts both permissive and non-permissive for AcMNPV and SeMNPV. PMID:27321273

  7. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

    PubMed

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-12-15

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro.

  8. A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

    PubMed Central

    2013-01-01

    Background Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. Results In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. Conclusions The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are

  9. Novel Apoptosis Suppressor Apsup from the Baculovirus Lymantria dispar Multiple Nucleopolyhedrovirus Precludes Apoptosis by Preventing Proteolytic Processing of Initiator Caspase Dronc

    PubMed Central

    Yamada, Hayato; Kitaguchi, Koji; Hamajima, Rina; Kobayashi, Michihiro

    2013-01-01

    We previously identified a novel baculovirus-encoded apoptosis suppressor, Apsup, from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Apsup inhibits the apoptosis of L. dispar Ld652Y cells triggered by infection with p35-defective Autographa californica MNPV (vAcΔp35) and exposure to actinomycin D or UV light. Here, we examined the functional role of Apsup in apoptosis regulation in insect cells. Apsup prevented apoptosis and the proteolytic processing of L. dispar initiator caspase Dronc (Ld-Dronc) in Ld652Y cells triggered by overexpression of Ld-Dronc, LdMNPV inhibitor-of-apoptosis 3 (IAP3), or Hyphantria cunea MNPV IAP1. In vAcΔp35-infected apoptotic Ld652Y cells, Apsup restricted apoptosis induction and prevented processing of endogenous Ld-Dronc. Conversely, upon RNA interference (RNAi)-mediated silencing of apsup, LdMNPV-infected Ld652Y cells, which typically support high-titer virus replication, underwent apoptosis, accompanied by the processing of endogenous Ld-Dronc. Furthermore, endogenous Ld-Dronc coimmunoprecipitated with transiently expressed Apsup, indicating that Apsup physically interacts with Ld-Dronc. Apsup prevented the apoptosis of Sf9 cells triggered by vAcΔp35 infection but did not inhibit apoptosis or activation of caspase-3-like protease in vAcΔp35-infected Drosophila melanogaster S2 cells. Apsup also inhibited the proteolytic processing of L. dispar effector caspase Ld-caspase-1 in the transient expression assay but did not physically interact with Ld-caspase-1. These results demonstrate that Apsup inhibits apoptosis in Ld652Y cells by preventing the proteolytic processing of Ld-Dronc. Together with our previous findings showing that Apsup prevents the processing of both overexpressed Ld-Dronc and Bombyx mori Dronc, these results also demonstrate that Apsup functions as an effective apoptotic suppressor in various lepidopteran, but not dipteran, insect cells. PMID:24067961

  10. Loading of iron into recombinant rat liver ferritin heteropolymers by ceruloplasmin.

    PubMed

    Juan, S H; Guo, J H; Aust, S D

    1997-05-15

    We have reported previously that the heavy chain of ferritin is required for iron incorporation by ceruloplasmin (J.-H. Guo, M. Abedi, and S. D. Aust (1996) Arch. Biochem. Biophys. 335(1)). The purpose of this study was to determine how many heavy chains were required for ceruloplasmin to interact with ferritin such that iron loading occurred. The cDNA sequences encoding the heavy and light chains of rat liver ferritin were cloned into the baculovirus transfer vector pA-cUW51 under the control of polyhedrin and p10 promoters, respectively, which was then incorporated by homologous recombination into the infections Autographa californica nuclear polyhedrosis virus genome. Both ferritin chains were expressed and assembled into two heteropolymers following the infection of insect cells by recombinant virus, which were separated by DEAE-Sepharose chromatography. The percentage of heavy (H) and light (L) chains making up the two heteropolymers, determined by gel scanning following the resolution of chains on SDS-PAGE, were equivalent to 1 H and 23 L chains and 2 H and 22 L chains. The maximal extent of iron loading was observed using 1 mol of rat ceruloplasmin per mole of H chain in the two heteropolymers. The extent of iron incorporation decreased with additional ceruloplasmin. Iron incorporation into rat liver ferritin, found to contain 10 H chains, increased as the molar ratio of ceruloplasmin to ferritin increased to 4:1 and remained the same up to 8:1. Iron loading into horse spleen ferritin, found to have one H chain, appeared similar to that for recombinant ferritin, having only one H chain. Therefore, we propose that the optimal molar ratio of ceruloplasmin to ferritin depends upon the numbers of H chain making up the ferritin molecule for the maximal incorporation of iron into ferritin. These results also suggest that the iron loading channel is contained within a single H chain subunit.

  11. Baculovirus-induced tree-top disease: how extended is the role of egt as a gene for the extended phenotype?

    PubMed

    Ros, Vera I D; van Houte, Stineke; Hemerik, Lia; van Oers, Monique M

    2015-01-01

    Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP-glucosyl transferase (egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree-top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree-top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree-top disease. Here, we studied tree-top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree-top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting-dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree-top disease in lepidopteran larvae. The conclusion that egt is a 'gene for an extended phenotype' is therefore not generally applicable for all baculovirus-host interactions. We hypothesize that in some baculovirus-host systems (including LdMNPV in L. dispar), an effect of egt on tree-top disease can be observed through indirect effects of egt on moulting-related climbing behaviour.

  12. Display of a Maize cDNA library on baculovirus infected insect cells

    PubMed Central

    Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

    2008-01-01

    Background Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. Results We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 × 105 independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. Conclusion The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence. PMID:18700036

  13. Recombinant protein production by the baculovirus-insect cell system in Basal media without serum supplementation.

    PubMed

    Nishikawa, Norikatsu; Yamaji, Hideki; Fukuda, Hideki

    2003-11-01

    The production of beta-galactosidase by Sf9 cells infected with recombinant Autographa californica nucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the beta-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted beta-galactosidase production, their removal by medium replacement on post-infection day 1 gave a beta-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing beta-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.

  14. Active Depletion of Host Cell Inhibitor-of-Apoptosis Proteins Triggers Apoptosis upon Baculovirus DNA Replication▿

    PubMed Central

    Vandergaast, Rianna; Schultz, Kimberly L. W.; Cerio, Rebecca J.; Friesen, Paul D.

    2011-01-01

    Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda, respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects. PMID:21653668

  15. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    SciTech Connect

    Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E. )

    1990-10-01

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4.

  16. Disruption of the baculovirus core gene ac78 results in decreased production of multiple nucleocapsid-enveloped occlusion-derived virions and the failure of primary infection in vivo.

    PubMed

    Li, Sai-Nan; Wang, Jin-Yu; Yuan, Mei-Jin; Yang, Kai

    2014-10-13

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac78 gene is one of the baculovirus core genes. Recent studies showed that ac78 is essential for budded virion (BV) production and the embedding of occlusion-derived virion (ODV) into occlusion body during the AcMNPV life cycle. Here, we report that an ac78-knockout AcMNPV (vAc78KO) constructed in this study had different phenotypes than those described in the previous studies. A few infectious BVs were detected using titer assays, immunoblot analyses and plaque assays, indicating that ac78 is not essential for BV formation. Electron microscopy confirmed that the ac78 deletion did not affect nucleocapsid assembly and ODV formation. However, the numbers of multiple nucleocapsid-enveloped ODVs and ODV-embedded occlusion bodies were significantly decreased. Subsequently, the highly conserved amino acid residues 2-25 and 64-88 of Ac78, which are homologous to an oxidoreductase and cytochrome c oxidase, respectively, were demonstrated to play a crucial role in the morphogenesis of multiple nucleocapsid-enveloped ODV. Immunoblot analysis found that Ac78 was an ODV envelope-associated protein. Consistently, amino acid residues 56-93 of Ac78 were identified as an inner nuclear membrane sorting motif, which may direct the localization of Ac78 to the ODV envelope. In vivo infectivity assays showed that the occlusion bodies of vAc78KO were unable to establish primary infection in the midgut of Trichoplusia ni larvae. Taken together, our results suggest that ac78 plays an important role in BV production and proper multiple nucleocapsid-enveloped ODV formation, as well as AcMNPV primary infection in vivo.

  17. Characterization of Novel Components of the Baculovirus Per Os Infectivity Factor Complex

    PubMed Central

    Peng, Ke; van Lent, Jan W. M.; Boeren, Sjef; Fang, Minggang; Theilmann, David A.; Erlandson, Martin A.; Vlak, Just M.

    2012-01-01

    Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed. PMID:22379094

  18. Baculovirus Per Os Infectivity Factors Form a Complex on the Surface of Occlusion-Derived Virus ▿

    PubMed Central

    Peng, Ke; van Oers, Monique M.; Hu, Zhihong; van Lent, Jan W. M.; Vlak, Just M.

    2010-01-01

    Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% β-mercaptoethanol with heating at 50°C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infection. PMID:20610731

  19. Effect of culture conditions on the degree of sialylation of a recombinant glycoprotein expressed in insect cells.

    PubMed

    Joosten, Christoph E; Shuler, Michael L

    2003-01-01

    Secreted human placental alkaline phosphatase (SEAP) was produced in a nonengineered Trichoplusia ni insect cell line, Tn-4s, using a recombinant Autographa californica baculovirus expression vector. The effect of culture conditions on SEAP specific yield and glycosylation was studied. When cultured in the high aspect ratio vessel (HARV) or in tissue culture flasks (T-flasks), baculovirus-infected Tn-4s cells produced high levels of SEAP (13 and 23 U/10(6) cells, respectively; 4 days postinfection), but in those conditions SEAP possessed only high mannose, paucimannosidic, and hybrid structures. In spinner flasks, lower SEAP yields were obtained (<4 U/10(6) cells, 3 days postinfection), but in such cultures, sialylation of SEAP could be achieved. Several spinner-flask culture conditions were tested and resulted in different SEAP specific yields and levels of sialylation. The highest level of sialylation (9%) was obtained in the culture with the lowest agitation rate and lowest yield (1.2 U/10(6) cells), suggesting a limiting capacity of the Tn-4s cells to process glycoproteins to sialylation. High specific yield, low passage number Tn5B1-4 cells did not produce SEAP with complex glycosylation when cultured in a low agitation rate spinner-flask. On the basis of these results, we propose that the Golgi apparatus has a limited capacity for processing proteins to complex glycosylation and sialylation and that this capacity is easily overwhelmed by high levels of foreign protein productivity. Selected media additives such as Pluronic F-68, dextran sulfate (MW 12 500) and a lipids premix did not allow improvement of the specific yield of sialylated SEAP when supplemented to spinner-flask cultures.

  20. Transport of Wild-Type and Recombinant Nucleopolyhedroviruses by Scavenging and Predatory Arthropods.

    PubMed

    Lee; Fuxa

    2000-05-01

    Wild-type and recombinant nucleopolyhedroviruses (NPVs) were compared in their capability to be transported over limited distances by the predator Podisus maculiventris (Say) and scavengers Sarcophaga bullata (Parker) and Acheta domesticus (Linnaeus) in Trichoplusia ni (Hübner) larvae infesting collards in a greenhouse microcosm. Viruses tested were variants of Autographa californica (Speyer) NPV (AcNPV): wild-type virus (AcNPV.WT), AcNPV expressing a scorpion toxin (AcNPV.AaIT), and AcNPV expressing juvenile hormone esterase (AcJHE.SG). Podisus maculiventris transported AcNPV.WT and S. bullata transported AcNPV.WT and AcNPV.AaIT. Prevalence and transport of AcNPV.WT were greater than those of AcNPV.AaIT and AcJHE.SG, regardless of whether the nontarget organism carriers were present or absent. Podisus maculiventris and S. bullata transported recombinant and wild-type NPVs at a rate of up to 62.5 cm/day, and A. domesticus transported wild-type NPV at 125 cm/day. The infected host insects, T. ni, undoubtedly contributed to viral transport in the current research. In every experiment, both the wild-type and recombinant virus spread to some degree in the plots without predators or scavengers. The relative amounts of NPVs that accumulated in soil, as indicated by bioassay mortality percentages, generally exhibited spatial patterns similar to those of T. ni mortality due to NPV on the collards plants. Thus, the predator and scavengers in the current research demonstrated some capacity to transport wild-type as well as recombinant viruses at significant rates in a greenhouse microcosm.

  1. A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

    PubMed Central

    Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

    2013-01-01

    Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID

  2. Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria

    PubMed Central

    Brand, Ingo L.; Civciristov, Srgjan; Taylor, Nicole L.; Talbo, Gert H.; Pantaki-Eimany, Delara; Levina, Vita; Clem, Rollie J.; Perugini, Matthew A.; Kvansakul, Marc; Hawkins, Christine J.

    2012-01-01

    Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a “reactive site loop” within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity. PMID:22720082

  3. Activity-Dependent Inhibitory Gating in Molecular Signaling Cascades Induces a Novel Form of Intermediate-Term Synaptic Facilitation in "Aplysia Californica"

    ERIC Educational Resources Information Center

    Fischbach, Soren; Kopec, Ashley M.; Carew, Thomas J.

    2014-01-01

    Mechanistically distinct forms of long-lasting plasticity and memory can be induced by a variety of different training patterns. Although several studies have identified distinct molecular pathways that are engaged during these different training patterns, relatively little work has explored potential interactions between pathways when they are…

  4. Phytophthora ramorum and Phytophthora gonapodyides differently colonize and contribute to decay of California bay laurel (Umbellularia californica) leaf litter in stream ecosystems

    Treesearch

    Kamyar Aram; David M. Rizzo

    2017-01-01

    The prevalence of Phytophthora species in surface waters has earned increasing attention in the past decades, in great part as a result of “stream monitoring” programs for detection and monitoring of Phytophthora ramorum and other invasive species. The potential for Phytophthora ...

  5. Activity-Dependent Inhibitory Gating in Molecular Signaling Cascades Induces a Novel Form of Intermediate-Term Synaptic Facilitation in "Aplysia Californica"

    ERIC Educational Resources Information Center

    Fischbach, Soren; Kopec, Ashley M.; Carew, Thomas J.

    2014-01-01

    Mechanistically distinct forms of long-lasting plasticity and memory can be induced by a variety of different training patterns. Although several studies have identified distinct molecular pathways that are engaged during these different training patterns, relatively little work has explored potential interactions between pathways when they are…

  6. Baculovirus display of functional antibody Fab fragments.

    PubMed

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  7. Rapid and efficient filtration-based procedure for separation and safe analysis of CBRN mixed samples.

    PubMed

    Bentahir, Mostafa; Laduron, Frederic; Irenge, Leonid; Ambroise, Jérôme; Gala, Jean-Luc

    2014-01-01

    Separating CBRN mixed samples that contain both chemical and biological warfare agents (CB mixed sample) in liquid and solid matrices remains a very challenging issue. Parameters were set up to assess the performance of a simple filtration-based method first optimized on separate C- and B-agents, and then assessed on a model of CB mixed sample. In this model, MS2 bacteriophage, Autographa californica nuclear polyhedrosis baculovirus (AcNPV), Bacillus atrophaeus and Bacillus subtilis spores were used as biological agent simulants whereas ethyl methylphosphonic acid (EMPA) and pinacolyl methylphophonic acid (PMPA) were used as VX and soman (GD) nerve agent surrogates, respectively. Nanoseparation centrifugal devices with various pore size cut-off (30 kD up to 0.45 µm) and three RNA extraction methods (Invisorb, EZ1 and Nuclisens) were compared. RNA (MS2) and DNA (AcNPV) quantification was carried out by means of specific and sensitive quantitative real-time PCRs (qPCR). Liquid chromatography coupled to time-of-flight mass spectrometry (LC/TOFMS) methods was used for quantifying EMPA and PMPA. Culture methods and qPCR demonstrated that membranes with a 30 kD cut-off retain more than 99.99% of biological agents (MS2, AcNPV, Bacillus Atrophaeus and Bacillus subtilis spores) tested separately. A rapid and reliable separation of CB mixed sample models (MS2/PEG-400 and MS2/EMPA/PMPA) contained in simple liquid or complex matrices such as sand and soil was also successfully achieved on a 30 kD filter with more than 99.99% retention of MS2 on the filter membrane, and up to 99% of PEG-400, EMPA and PMPA recovery in the filtrate. The whole separation process turnaround-time (TAT) was less than 10 minutes. The filtration method appears to be rapid, versatile and extremely efficient. The separation method developed in this work constitutes therefore a useful model for further evaluating and comparing additional separation alternative procedures for a safe handling and

  8. High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells.

    PubMed

    Sisk, W P; Bradley, J D; Leipold, R J; Stoltzfus, A M; Ponce de Leon, M; Hilf, M; Peng, C; Cohen, G H; Eisenberg, R J

    1994-02-01

    Two forms of herpes simplex virus glycoprotein gD were recombined into Autographa californica nuclear polyhedrosis virus (baculovirus) and expressed in infected Spodoptera frugiperda (Sf9) cells. Each protein was truncated at residue 306 of mature gD. One form, gD-1(306t), contains the coding sequence of Patton strain herpes simplex virus type 1 gD; the other, gD-1(QAAt), contains three mutations which eliminate all signals for addition of N-linked oligosaccharides. Prior to recombination, each gene was cloned into the baculovirus transfer vector pVT-Bac, which permits insertion of the gene minus its natural signal peptide in frame with the signal peptide of honeybee melittin. As in the case with many other baculovirus transfer vectors, pVT-Bac also contains the promoter for the baculovirus polyhedrin gene and flanking sequences to permit recombination into the polyhedrin site of baculovirus. Each gD gene was engineered to contain codons for five additional histidine residues following histidine at residue 306, to facilitate purification of the secreted protein on nickel-containing resins. Both forms of gD-1 were abundantly expressed and secreted from infected Sf9 cells, reaching a maximum at 96 h postinfection for gD-1(306t) and 72 h postinfection for gD-1(QAAt). Secretion of the latter protein was less efficient than gD-1(306t), possibly because of the absence of N-linked oligosaccharides from gD-1(QAAt). Purification of the two proteins by a combination of immunoaffinity chromatography, nickel-agarose chromatography, and gel filtration yielded products that were > 99% pure, with excellent recovery. We are able to obtain 20 mg of purified gD-1(306t) and 1 to 5 mg of purified gD-1(QAAt) per liter of infected insect cells grown in suspension. Both proteins reacted with monoclonal antibodies to discontinuous epitopes, indicating that they retain native structure. Use of this system for gD expression makes crystallization trials feasible.

  9. Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies.

    PubMed

    Yang, Shili; Zhao, Lijuan; Ma, Ruipeng; Fang, Wei; Hu, Jia; Lei, Chengfeng; Sun, Xiulian

    2017-07-15

    The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity.IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this

  10. Superinfection Exclusion in Alphabaculovirus Infections Is Concomitant with Actin Reorganization

    PubMed Central

    Beperet, Inés; Irons, Sarah L.; Simón, Oihane; King, Linda A.; Williams, Trevor; Possee, Robert D.; Caballero, Primitivo

    2014-01-01

    ABSTRACT Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses—AcMNPV and SfMNPV—but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses. IMPORTANCE Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an

  11. Baculovirus-challenge and poor nutrition inflict within-generation fitness costs without triggering transgenerational immune priming.

    PubMed

    Shikano, Ikkei; Hua, Kevin Ngoc; Cory, Jenny S

    2016-05-01

    Invertebrate hosts that survive pathogen challenge can produce offspring that are more resistant to the same pathogen via immune priming, thereby improving the fitness of their offspring in the same pathogen environment. Most evidence for immune priming comes from exposure to bacteria and there are limited data on other groups of pathogens. Poor parental nutrition has also been shown to result in the transgenerational transfer of pathogen resistance and increased immunocompetence. Here, we combine exposure to an insect DNA virus with a change in the parental diet to examine both parental costs and transgenerational immune priming. We challenged the cabbage looper, Trichoplusia ni, with a low dose of the baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and altered dietary protein to carbohydrate ratio (p:c ratio) after virus exposure. Insects fed a low protein diet had lower haemolymph protein concentrations, and exhibited costs of smaller pupae and slower development, while survivors of virus challenge developed more slowly, irrespective of p:c ratio, and those that were virus-challenged and fed on a low protein diet showed a reduction in haemocyte density. In addition, AcMNPV-challenged parents laid fewer eggs earlier in egg laying although egg size was the same as for unchallenged parents. There was no evidence for increased resistance to AcMNPV (immune priming) or changes in haemocyte number (as proxy for constitutive cellular immunity) in the offspring either as a result of parental AcMNPV-challenge or low dietary p:c ratio. Therefore, although pathogen-challenge and nutritional changes can affect host development and reproduction, this does not necessarily translate into transgenerational immune priming. Our findings contrast with an earlier study on another type of baculovirus, a granulovirus, where immune priming was suggested. This indicates that transgenerational immune priming is not universal in invertebrates and is likely to

  12. Rapid and Efficient Filtration-Based Procedure for Separation and Safe Analysis of CBRN Mixed Samples

    PubMed Central

    Bentahir, Mostafa; Laduron, Frederic; Irenge, Leonid; Ambroise, Jérôme; Gala, Jean-Luc

    2014-01-01

    Separating CBRN mixed samples that contain both chemical and biological warfare agents (CB mixed sample) in liquid and solid matrices remains a very challenging issue. Parameters were set up to assess the performance of a simple filtration-based method first optimized on separate C- and B-agents, and then assessed on a model of CB mixed sample. In this model, MS2 bacteriophage, Autographa californica nuclear polyhedrosis baculovirus (AcNPV), Bacillus atrophaeus and Bacillus subtilis spores were used as biological agent simulants whereas ethyl methylphosphonic acid (EMPA) and pinacolyl methylphophonic acid (PMPA) were used as VX and soman (GD) nerve agent surrogates, respectively. Nanoseparation centrifugal devices with various pore size cut-off (30 kD up to 0.45 µm) and three RNA extraction methods (Invisorb, EZ1 and Nuclisens) were compared. RNA (MS2) and DNA (AcNPV) quantification was carried out by means of specific and sensitive quantitative real-time PCRs (qPCR). Liquid chromatography coupled to time-of-flight mass spectrometry (LC/TOFMS) methods was used for quantifying EMPA and PMPA. Culture methods and qPCR demonstrated that membranes with a 30 kD cut-off retain more than 99.99% of biological agents (MS2, AcNPV, Bacillus Atrophaeus and Bacillus subtilis spores) tested separately. A rapid and reliable separation of CB mixed sample models (MS2/PEG-400 and MS2/EMPA/PMPA) contained in simple liquid or complex matrices such as sand and soil was also successfully achieved on a 30 kD filter with more than 99.99% retention of MS2 on the filter membrane, and up to 99% of PEG-400, EMPA and PMPA recovery in the filtrate. The whole separation process turnaround-time (TAT) was less than 10 minutes. The filtration method appears to be rapid, versatile and extremely efficient. The separation method developed in this work constitutes therefore a useful model for further evaluating and comparing additional separation alternative procedures for a safe handling and

  13. Superinfection exclusion in alphabaculovirus infections is concomitant with actin reorganization.

    PubMed

    Beperet, Inés; Irons, Sarah L; Simón, Oihane; King, Linda A; Williams, Trevor; Possee, Robert D; López-Ferber, Miguel; Caballero, Primitivo

    2014-03-01

    Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses--AcMNPV and SfMNPV--but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses. Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period

  14. The Baculovirus Antiapoptotic p35 Protein Functions as an Inhibitor of the Host RNA Interference Antiviral Response

    PubMed Central

    Mehrabadi, Mohammad; Hussain, Mazhar; Matindoost, Leila

    2015-01-01

    ABSTRACT RNA interference (RNAi) is considered an ancient antiviral defense in diverse organisms, including insects. Virus infections generate double-strand RNAs (dsRNAs) that trigger the RNAi machinery to process dsRNAs into virus-derived short interfering RNAs (vsiRNAs), which target virus genomes, mRNAs, or replication intermediates. Viruses, in turn, have evolved viral suppressors of RNAi (VSRs) to counter host antiviral RNAi. Following recent discoveries that insects mount an RNAi response against DNA viruses, in this study, we found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection similarly induces an RNAi response in Spodoptera frugiperda cells by generating a large number of vsiRNAs postinfection. Interestingly, we found that AcMNPV expresses a potent VSR to counter RNAi. The viral p35 gene, which is well known as an inhibitor of apoptosis, was found to be responsible for the suppression of RNAi in diverse insect and mammalian cells. The VSR activity of p35 was further confirmed by a p35-null AcMNPV that did not suppress the response. In addition, our results showed that the VSR activity is not due to inhibition of dsRNA cleavage by Dicer-2 but acts downstream in the RNAi pathway. Furthermore, we found that the VSR activity is not linked to the antiapoptotic activity of the protein. Overall, our results provide evidence for the existence of VSR activity in a double-stranded DNA virus and identify the responsible gene, which is involved in the inhibition of RNAi as well as apoptosis. IMPORTANCE Our findings demonstrate the occurrence of an insect RNAi response against a baculovirus (AcMNPV) that is highly utilized in microbial control, biological and biomedical research, and protein expression. Moreover, our investigations led to the identification of a viral suppressor of RNAi activity and the gene responsible for the activity. Notably, this gene is also a potent inhibitor of apoptosis. The outcomes signify the dual role of a

  15. The Baculovirus Antiapoptotic p35 Protein Functions as an Inhibitor of the Host RNA Interference Antiviral Response.

    PubMed

    Mehrabadi, Mohammad; Hussain, Mazhar; Matindoost, Leila; Asgari, Sassan

    2015-08-01

    RNA interference (RNAi) is considered an ancient antiviral defense in diverse organisms, including insects. Virus infections generate double-strand RNAs (dsRNAs) that trigger the RNAi machinery to process dsRNAs into virus-derived short interfering RNAs (vsiRNAs), which target virus genomes, mRNAs, or replication intermediates. Viruses, in turn, have evolved viral suppressors of RNAi (VSRs) to counter host antiviral RNAi. Following recent discoveries that insects mount an RNAi response against DNA viruses, in this study, we found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection similarly induces an RNAi response in Spodoptera frugiperda cells by generating a large number of vsiRNAs postinfection. Interestingly, we found that AcMNPV expresses a potent VSR to counter RNAi. The viral p35 gene, which is well known as an inhibitor of apoptosis, was found to be responsible for the suppression of RNAi in diverse insect and mammalian cells. The VSR activity of p35 was further confirmed by a p35-null AcMNPV that did not suppress the response. In addition, our results showed that the VSR activity is not due to inhibition of dsRNA cleavage by Dicer-2 but acts downstream in the RNAi pathway. Furthermore, we found that the VSR activity is not linked to the antiapoptotic activity of the protein. Overall, our results provide evidence for the existence of VSR activity in a double-stranded DNA virus and identify the responsible gene, which is involved in the inhibition of RNAi as well as apoptosis. Our findings demonstrate the occurrence of an insect RNAi response against a baculovirus (AcMNPV) that is highly utilized in microbial control, biological and biomedical research, and protein expression. Moreover, our investigations led to the identification of a viral suppressor of RNAi activity and the gene responsible for the activity. Notably, this gene is also a potent inhibitor of apoptosis. The outcomes signify the dual role of a virus-encoded protein

  16. Serum-Free Culture of the Suspension Cell Line QB-Tn9-4s of the Cabbage Looper, Trichoplusia ni, is Highly Productive for Virus Replication and Recombinant Protein Expression

    PubMed Central

    Zheng, Gui-Ling; Zhou, Hong-Xu; Li, Chang-You

    2014-01-01

    Serum-free cultures of insect cells play an important role in the fields of protein engineering, medicine, and biology. In this paper, the suspension cell line QB-Tn9-4s of Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) was successfully adapted to serum-free Sf-900 III medium and passaged for 52 generations. The adapted QB-Tn9-4s cells grew faster. Their population doubling time shortened from 27.4 hr in serum-containing medium to 24.1 hr, and their maximal density increased by 1.83-fold, reaching 3.50 × 106 cells/mL in serum-free culture in T-flasks. The cells readily adapted to spinner culture, with maximum cell density of 4.40 × 106 cells/mL in a spinner flask. Although the infection rate of Autographa californica multiple nucleopolyhedrovirus and production of occlusion bodies (OBs) of the adapted QB-Tn9-4s cells were 91.0% and 85.4 OBs/cell, respectively, similar to those of QB-Tn9-4s cells cultured in serum-containing medium and control BTI-Tn5B1-4 cells, their budded virus titer was 4.97 × 107 TCID50/mL, significantly higher than those of the latter two. In addition, the expression levels of β-galactosidase at six days post-infection and secreted alkaline phosphatase at seven days postinfection in the adapted QB-Tn9-4s cells reached 2.98 ± 0.15×104 IU/mL and 3.34 ± 0.13 IU/mL, respectively, significantly higher than those of QB-Tn9-4s and control BTI-Tn5B1-4 cultured in serum-containing media. The above findings establish a foundation for industrial production of virus and recombinant proteins in QB-Tn9-4s serum-free culture. PMID:25373171

  17. Identification of a High-Efficiency Baculovirus DNA Replication Origin That Functions in Insect and Mammalian Cells

    PubMed Central

    Wu, Yueh-Lung; Wu, Carol-P; Huang, Yu-Hui; Huang, Sheng-Ping; Lo, Huei-Ru; Chang, Hao-Shuo; Lin, Pi-Hsiu; Wu, Ming-Cheng; Chang, Chia-Jung

    2014-01-01

    ABSTRACT The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells. IMPORTANCE Baculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143

  18. 77 FR 51042 - Endangered Species Recovery Permit Applications

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-23

    ...-mallow) Monardella viminea (=M. linoides subsp. v.) (willowy monardella) Orcuttia californica (California... filifolia (Santa Cruz Island rockcress) Sidalcea pedata (pedate checker-mallow) Taraxacum californicum...

  19. 50 CFR 17.41 - Special rules-birds.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 2 2012-10-01 2012-10-01 false Special rules-birds. 17.41 Section 17.41 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR (CONTINUED... rules—birds. (a) (b) Coastal California gnatcatcher (Polioptila californica californica). (1) Except as...

  20. 50 CFR 17.41 - Special rules-birds.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 2 2013-10-01 2013-10-01 false Special rules-birds. 17.41 Section 17.41 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR (CONTINUED... rules—birds. (a) (b) Coastal California gnatcatcher (Polioptila californica californica). (1) Except as...

  1. Biosafety of Recombinant and Wild Type Nucleopolyhedroviruses as Bioinsecticides

    PubMed Central

    Ashour, Mohamed-Bassem; Ragheb, Didair A.; El-Sheikh, El-Sayed A.; Gomaa, El-Adarosy A.; Kamita, Shizuo G.; Hammock, Bruce D.

    2007-01-01

    The entomopathogenic Autographa californica (Speyer) nucleopolyhedrovirus (AcMNPV) has been genetically modified to increase its speed of kill. The potential adverse effects of a recombinant AcMNPV (AcAaIT) as well as wild type AcMNPV and wild type Spodoptera littoralis NPV (SlNPV) were studied. Cotton plants were treated with these viruses at concentrations that were adjusted to resemble the recommended field application rate (4 × 1012 PIBs/feddan, feddan = 4,200 m2) and 3rd instar larvae of S. littoralis were allowed to feed on the contaminated plants. SDS-PAGE, ELISA, and DNA analyses were used to confirm that larvae that fed on these plants were virus-infected. Polyhedra that were purified from the infected larvae were subjected to structural protein analysis. A 32 KDa protein was found in polyhedra that were isolated from all of the viruses. Subtle differences were found in the size and abundance of ODV proteins. Antisera against polyhedral proteins isolated from AcAaIT polyhedra were raised in rabbits. The terminal bleeds from rabbits were screened against four coating antigens (i.e., polyhedral proteins from AcAaIT, AcAaIT from field-infected larvae (AcAaIT-field), AcMNPV, and SlNPV) using a two-dimensional titration method with the coated antigen format. Competitive inhibition experiments were conducted in parallel to optimize antibody and coating antigen concentrations for ELISA. The IC50 values for each combination ranged from 1.42 to 163 μg/ml. AcAaIT-derived polyhedrin gave the lowest IC50 value, followed by those of SlNPV, AcAaIT-field, and AcMNPV. The optimized ELISA system showed low cross reactivity for AcMNPV (0.87%), AcAaIT-field (1.2%), and SlNPV (4.0%). Genomic DNAs isolated from AcAaIT that were passaged in larvae of S. littoralis that were reared in the laboratory or field did not show any detectable differences. Albino rats (male and female) that were treated with AcAaIT, AcMNPV or SlNPV (either orally or by intraperitoneal injection at

  2. Biosafety of recombinant and wild type nucleopolyhedroviruses as bioinsecticides.

    PubMed

    Ashour, Mohamed-Bassem; Ragheb, Didair A; El-Sheikh, El-Sayed A; Gomaa, El-Adarosy A; Kamita, Shizuo G; Hammock, Bruce D

    2007-06-01

    The entomopathogenic Autographa californica (Speyer) nucleopolyhedrovirus (AcMNPV) has been genetically modified to increase its speed of kill. The potential adverse effects of a recombinant AcMNPV (AcAaIT) as well as wild type AcMNPV and wild type Spodoptera littoralis NPV (SlNPV) were studied. Cotton plants were treated with these viruses at concentrations that were adjusted to resemble the recommended field application rate (4 x 10(12) PIBs/feddan, feddan = 4,200 m2) and 3rd instar larvae of S. littoralis were allowed to feed on the contaminated plants. SDS-PAGE, ELISA, and DNA analyses were used to confirm that larvae that fed on these plants were virus-infected. Polyhedra that were purified from the infected larvae were subjected to structural protein analysis. A 32 KDa protein was found in polyhedra that were isolated from all of the viruses. Subtle differences were found in the size and abundance of ODV proteins. Antisera against polyhedral proteins isolated from AcAaIT polyhedra were raised in rabbits. The terminal bleeds from rabbits were screened against four coating antigens (i.e., polyhedral proteins from AcAaIT, AcAaIT from field-infected larvae (AcAaIT-field), AcMNPV, and SlNPV) using a two-dimensional titration method with the coated antigen format. Competitive inhibition experiments were conducted in parallel to optimize antibody and coating antigen concentrations for ELISA. The IC50 values for each combination ranged from 1.42 to 163 microg/ml. AcAaIT-derived polyhedrin gave the lowest IC50 value, followed by those of SlNPV, AcAaIT-field, and AcMNPV. The optimized ELISA system showed low cross reactivity for AcMNPV (0.87%), AcAaIT-field (1.2%), and SlNPV (4.0%). Genomic DNAs isolated from AcAaIT that were passaged in larvae of S. littoralis that were reared in the laboratory or field did not show any detectable differences. Albino rats (male and female) that were treated with AcAaIT, AcMNPV or SlNPV (either orally or by intraperitoneal injection at

  3. Women's Health Among the Chumash

    PubMed Central

    Adams, James D.; Garcia, Cecilia

    2006-01-01

    Plants were, and still are, widely used for a number of conditions affecting women in California. This article discusses traditional remedies of the Chumash for dysmenorrhea, premenstrual syndrome, feminine hygiene, heavy menstruation, urinary tract infections, parturition, lactation, infant care, menopause, sexually transmitted diseases, fertility, contraception and abortions. Many plants are presented including Artemisia douglasiana, Paeonia californica, Trichostema lanatum, Salvia apiana, Ephedra viridis, Leymus condensatus, Vitis californica, Eschscholzia californica, Rosa californica, Scirpus acutus, Anemopsis californica and Phoradendron macrophyllum. By providing the specific uses of plants for specific diseases and discussing chemistry, efficacy and safety concerns for each plant, we hope that this article gives direction to women seeking to use plants in their health care. PMID:16550233

  4. Display of VP1 on the Surface of Baculovirus and Its Immunogenicity against Heterologous Human Enterovirus 71 Strains in Mice

    PubMed Central

    Kiener, Tanja K.; Chow, Vincent T. K.; Kwang, Jimmy

    2011-01-01

    Background Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and has caused high mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no effective vaccine and antiviral agents available against EV71 infections. VP1 is one of the major immunogenic capsid protein of EV71 and plays a crucial role in viral infection. Antibodies against VP1 are important for virus neutralization. Methodology/Principal Finding In the present study, infectious EV71 viruses were generated from their synthetic complementary DNA using the human RNA polymerase I reverse genetics system. Secondly, the major immunogenic capsid protein (VP1) of EV71-Fuyang (subgenogroup C4) was displayed on the surface of recombinant baculovirus Bac-Pie1-gp64-VP1 as gp64 fusion protein under a novel White Spot Syndrome Virus (WSSV) immediate early ie1 promoter. Baculovirus expressed VP1 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that VP1 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired VP1 from the insect cell membrane via the budding process. After two immunizations in mice, Bac-Pie1-gp64-VP1 elicited neutralization antibody titer of 1∶64 against EV71 (subgenogroup C4) in an in vitro neutralization assay. Furthermore, the antisera showed high cross-neutralization activities against all 11 subgenogroup EV71 strains. Conclusion Our results illustrated that Bac-Pie1-gp64-VP1 retained native epitopes of VP1 and acted as an effective EV71 vaccine candidate which would enable rapid production without any biosafety concerns. PMID:21747954

  5. Environmental Impact Statement/Environmental Impact Report for the Disposal and Reuse of Mare Island Naval Shipyard Vallejo, California. Volume 2.

    DTIC Science & Technology

    1998-04-01

    family Salix lasiolepis arroyo willow Salix gooddingii black willow Scrophulariaceae - Figwort family *Bellardia trixago bellardia Castilleja exerta...canadensis blue toadflax Mimulus aurantiacus sticky monkey flower Scrophularia californica coast figwort Tripbysaria pusilla dwarf orthocarpus

  6. Sacramento River Flood Control Project, California, Mid-Valley Area, Phase III. Design Memorandum, Volume 2 of 2

    DTIC Science & Technology

    1995-08-01

    corridor, tree canopy consists primarily of valley oak, sycamore, cottonwood, and willow. California grape and mistletoe are sometimes present. A well...sycamore (Platanus racemosa), cottonwood (Populus fremontii), and willow (Salix spp.). California grape (Vitus californica) and mistletoe (Phoradendron

  7. Distributions and habitat models of epiphytic physconia in north-central California.

    Treesearch

    Sarah. Jovan

    2003-01-01

    I examined the distributions of eight Physconia species in northern and central California: Physconia americana, P. californica, P. enteroxantha, P. fallax, P. isidiigera, P. isidiomuscigena, P. leucoleiptes, and P...

  8. Predicted fire behavior and societal benefits in three eastern Sierra Nevada vegetation types

    Treesearch

    C.A. Dicus; K. Delfino; D.R. Weise

    2009-01-01

    We investigated potential fire behavior and various societal benefits (air pollution removal, carbon sequestration, and carbon storage) provided by woodlands of pinyon pine (Pinus monophylla) and juniper (Juniperus californica), shrublands of Great Basin sagebrush (Artemisia tridentata) and rabbitbrush (Ericameria nauseosa...

  9. Attraction of pest moths (Lepidoptera: Noctuidae, Crambidae) to floral lures on the island of Hawaii

    USDA-ARS?s Scientific Manuscript database

    Traps baited with floral chemicals on the island of Hawaii captured several pest moth species. Chrysodeixis eriosoma (Doubleday)(green garden looper), Autographa biloba (Doubleday)(bi-lobed looper), and Mythimna unipuncta (Haworth)(true armyworm), all Noctuidae, as well as Hymenia recurvalis (L.)(be...

  10. Extracts of Phenolic Compounds from Seeds of Three Wild Grapevines—Comparison of Their Antioxidant Activities and the Content of Phenolic Compounds

    PubMed Central

    Weidner, Stanisław; Powałka, Anna; Karamać, Magdalena; Amarowicz, Ryszard

    2012-01-01

    Phenolic compounds were extracted from three wild grapevine species: Vitis californica, V. riparia and V. amurensis seeds using 80% methanol or 80% acetone. The total content of phenolic compounds was determined utilizing the Folin-Ciocalteu’s phenol reagent while the content of tannins was assayed with the vanillin and BSA precipitation methods. Additionally, the DPPH free radical scavenging activity and the reduction power of the extracts were measured. The RP-HPLC method was applied to identify the phenolic compounds in the extracts, such as phenolic acids and catechins. The seeds contained large amounts of tannins, catechins and gallic acid and observable quantities of p-coumaric acid. The total content of phenolic compounds and tannins was similar in the extracts from V. californica and V. riparia seeds. However, the total content of total phenolic compounds and tannins in the extracts from V. californica and V. riperia seeds were about two-fold higher than that in the extracts from V. amurensis seeds. Extracts from seeds of the American species (V. californica and V. riparia) contained similarly high concentrations of tannins, whereas extracts from seeds of V. amurensis had approximately half that amount of these compounds. The content of catechin and epicatechin was similar in all extracts. The highest DPPH• anti-radical scavenging activity was observed in the acetonic and methanolic extracts of V. californica and V. riparia seeds— while the acetonic extract from the V. californica seeds was the strongest reducing agent. PMID:22489161

  11. Structural and functional analysis of Aplysia attractins, a family of water-borne protein pheromones with interspecific attractiveness

    PubMed Central

    Painter, Sherry D.; Cummins, Scott F.; Nichols, Amy E.; Akalal, David-B. G.; Schein, Catherine H.; Braun, Werner; Smith, John S.; Susswein, Abraham J.; Levy, Miriam; de Boer, Pamela A. C. M.; ter Maat, Andries; Miller, Mark W.; Scanlan, Cory; Milberg, Richard M.; Sweedler, Jonathan V.; Nagle, Gregg T.

    2004-01-01

    Mate attraction in Aplysia involves a long-distance water-borne signal (the protein pheromone attractin), which is released during egg laying. Aplysia californica attractin attracts species that produce closely related attractins, such as Aplysia brasiliana, whose geographic distribution does not overlap that of A. californica. This finding suggests that other mollusks release attractin-related pheromones to form and maintain breeding aggregations. We describe four additional members of the attractin family: A. brasiliana, Aplysia fasciata, Aplysia depilans (which aggregates with A. fasciata aggregations), and Aplysia vaccaria (which aggregates with A. californica aggregations). On the basis of their sequence similarity with A. californica attractin, the attractin proteins fall into two groups: A. californica, A. brasiliana, and A. fasciata (91–95% identity), and A. depilans and A. vaccaria (41–43% identity). The sequence similarity within the attractin family, the conserved six cysteines, and the compact fold of the NMR solution structure of A. californica attractin suggest a common fold for this pheromone family containing two antiparallel helices. The second helix contains the IEECKTS sequence conserved in Aplysia attractins. Mutating surface-exposed charged residues within this heptapeptide sequence abolishes attractin activity, suggesting that the second helix is an essential part of the receptor-binding interface. PMID:15118100

  12. Abrogation of contaminating RNA activity in HIV-1 Gag VLPs.

    PubMed

    Valley-Omar, Ziyaad; Meyers, Ann E; Shephard, Enid G; Williamson, Anna-Lise; Rybicki, Edward P

    2011-10-06

    HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/μg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat

  13. The localization of a heterologous displayed antigen in the baculovirus-budded virion determines the type and strength of induced adaptive immune response.

    PubMed

    Tavarone, Eugenia; Molina, Guido Nicolás; Amalfi, Sabrina; Peralta, Andrea; Molinari, Paula; Taboga, Oscar

    2017-02-17

    In the search of strategies of presentation of heterologous antigens to elicit humoral or cellular immune responses that modulate and properly potentiate each type of response, researchers have been studying baculovirus (BV) as vaccine vectors with promising results. For some years, several research groups explored different antigen presentation approaches using the BV AcNPV by expressing polypeptides on the surface of budded virions or by de novo synthesis of heterologous antigens by transduction of mammalian cells. In the case of expression on the surface of budded virions, for example, researchers have expressed polypeptides in peplomers as GP64 glycoprotein fusions or distributed throughout the entire surface by fusions to portion