Science.gov

Sample records for autographa californica gp64

  1. Betabaculovirus F proteins showed different efficiencies when rescuing the infectivity of gp64-null Autographa californica nucleopolyhedrovirus.

    PubMed

    Yin, Feifei; Wang, Manli; Tan, Ying; Deng, Fei; Vlak, Just M; Hu, Zhihong; Wang, Hualin

    2013-02-01

    The Agrotis segetum granulovirus (AgseGV) F protein was previously identified as the first betabaculovirus F protein with functional homology to Autographa californica nucleopolyhedrovirus (AcMNPV) GP64. In the current study, F proteins from Xestia c-nigrum granulovirus (XecnGV), Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), Choristoneura occidentalis granulovirus (ChocGV) and Plutella xylostella GV (PlxyGV) were studied for their ability to rescue the infectivity of gp64-null AcMNPV. Our results showed that most studied betabaculovirus F proteins could replace the function of AcMNPV GP64, however, their efficiencies to rescue the infectivity of gp64-null AcMNPV were substantially different. PlxyF, although fusogenic, was the only protein that failed to substitute the function of AcMNPV GP64. Further studies using Sf9(0p1D) cell line showed that PlxyF appeared to be properly incorporated into AcMNPV virions and underwent correct post-translational cleavage and N-linked glycosylation. However, the gp64-null AcMNPV containing PlxyF could not be propagated in either Sf9 or P. xylostella cells. PMID:23245471

  2. Functional Analysis of the Autographa californica Multiple Nucleopolyhedrovirus GP64 Terminal Fusion Loops and Interactions with Membranes

    PubMed Central

    Dong, Sicong

    2012-01-01

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) glycoprotein GP64 is the major envelope protein of the budded virus (BV). GP64 is a class III fusion protein that mediates BV attachment to the cell surface and low-pH-triggered membrane fusion between the BV envelope and the endosome membrane during entry. Class III fusion proteins contain terminal looped structures that are believed to interact with membranes. To examine the functions of 3 loops found at the apex of the GP64 postfusion structure, we generated 2-alanine substitutions that scanned the two so-called fusion loops (loop 1 and loop 2) plus an adjacent loop structure (loop 3) that is closely attached to loop 2 and is also found at the apex of the GP64 postfusion structure. We identified essential residues from Y75 to T86 (loop 1) and N149 to H156 (loop 2) that are required for fusion activity, but no essential residues in loop 3. Further analysis revealed that critical fusion loop residues fall within two groups that are associated with either membrane merger (hemifusion) or fusion pore expansion. We next examined the interactions of soluble GP64 proteins and BV with membranes composed of various phospholipids. BV interacted directly with small unilamellar vesicles (SUVs) comprised of phospholipids phosphatidylcholine and phosphatidic acid (PC/PA) or phosphatidylcholine and phosphatidylserine (PC/PS) under neutral and acidic pH. We also examined the interactions of soluble GP64 constructs containing substitutions of the most hydrophobic residues within each of the two fusion loops. We found that a 2-residue substitution in either single loop (loop 1 [positions 81 and 82] or loop 2 [positions 153 and 154]) was not sufficient to substantially reduce the GP64-liposome interaction, but the same substitutions in both fusion loops severely reduced the GP64-liposome association at neutral pH. These results suggest that critical hydrophobic residues in both fusion loops may be involved in the

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

    PubMed

    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. PMID:26655244

  4. 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.

  5. Gene transduction in mammalian cells using Bombyx mori nucleopolyhedrovirus assisted by glycoprotein 64 of Autographa californica multiple nucleopolyhedrovirus

    PubMed Central

    Kato, Tatsuya; Sugioka, Saki; Itagaki, Kohei; Park, Enoch Y.

    2016-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV), an alphabaculovirus, has been widely utilized for protein expression in not only insect cells but also mammalian cells. AcMNPV is closely related to Bombyx mori nucleopolyhedrovirus (BmNPV), and nucleotide sequences of AcMNPV genes have high similarity with those of BmNPV. However, the transduction of BmNPV into mammalian cells has not been reported. In this study, we constructed a recombinant BmNPV (BmNPVΔbgp/AcGP64/EGFP) whose surface 64 kDa glycoprotein (BmGP64) was substituted with that from AcMNPV (AcGP64). BmNPVΔbgp/AcGP64/EGFP also carried an EGFP gene under the control of the CMV promoter. BmNPVΔbgp/AcGP64/EGFP successfully transduced HEK293T cells. In comparison, a control construct (BmNPVΔbgp/BmGP64/EGFP) which possessed BmGP64 instead of AcGP64 did not express EGFP in HEK293T cells. The transduction efficiency of BmNPVΔbgp/AcGP64/EGFP was lower than that of an AcMNPV based-BacMam GFP transduction control. This result indicates that AcGP64 facilitates BmNPV transduction into HEK293T cells. BmNPV can be prepared easily on a large scale because BmNPV can infect silkworm larvae without any special equipment, even though specific diet is needed for silkworm rearing. BmNPV gene transduction into mammalian cells can potentially be applied easily for gene delivery into mammalian cells. PMID:27562533

  6. 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.

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

    PubMed

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi

    2006-05-01

    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. PMID:16545777

  8. PHYSICAL MAPS OF 'AUTOGRAPHA CALIFORNICA' AND 'RACHIPLUSIA OU' NUCLEAR POLYHEDROSIS VIRUS RECOMBINANTS

    EPA Science Inventory

    TN-368 cells were infected simultaneously with the closely related Autographa californica (AcMNPV) and Rachiplusia ou (RoMNPV) nuclear polyhedrosis viruses. Progeny viral isolates were plaque purified, and their DNAs were analyzed with restriction endonucleases. Of 100 randomly c...

  9. AUTOGRAPHA CALIFORNICA NUCLEAR POLYHEDROSIS VIRUS EFFICIENTLY ENTERS BUT DOES NOT REPLICATE IN POIKILOTHERMIC VERTEBRATE CELLS

    EPA Science Inventory

    The host range of the insect virus Autographa californica nuclear polyhedrosis virus (AcMNPV) was examined. AcMNPV could not initiate a productive infection in frog, turtle, trout, or moth cell lines. After exposure to AcMNPV, neither viral DNA nor RNA synthesis could be detected...

  10. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  12. 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.

  13. Analysis of an Autographa californica Nucleopolyhedrovirus lef-11 Knockout: LEF-11 Is Essential for Viral DNA Replication

    PubMed Central

    Lin, Guangyun; Blissard, Gary W.

    2002-01-01

    The Autographa californica nucleopolyhedrovirus (AcMNPV) lef-11 gene was previously identified by transient late expression assays as a gene important for viral late gene expression. The lef-11 gene was not previously identified as necessary for DNA replication in transient origin-dependent plasmid DNA replication assays. To examine the role of lef-11 in the context of the infection cycle, we generated a deletion of the lef-11 gene by recombination in an AcMNPV genome propagated as a BACmid in Escherichia coli. The resulting AcMNPV lef-11-null BACmid (vAclef11KO) was unable to propagate in cell culture, although a “repair” AcMNPV BACmid (vAclef11KO-REP), which was generated by transposition of the lef-11 gene into the polyhedrin locus of the vAclef11KO BACmid, was able to replicate in a manner similar to wild-type or control AcMNPV viruses. Thus, the lef-11 gene is essential for viral replication in Sf9 cells. The vAclef11KO BACmid was examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in late transcription. The lef-11-null BACmid and control BACmids were transfected into Sf9 cells, and viral DNA replication was monitored. The viral DNA genome of the lef-11-null BACmid (vAclef11KO) was not amplified, whereas replication and amplification of the genomes of the repair BACmid (vAclef11KO-REP), wild-type AcMNPV, and a nonpropagating gp64-null control BACmid (vAcGUSgp64KO) were readily detected. Northern blot analysis of transcripts from selected early, late, and very late genes showed that late and very late transcription was absent in cells transfected with the lef-11-null BACmid. Thus, in contrast to prior studies using transient replication and late expression assays, studies of a lef-11-null BACmid indicate that LEF-11 is required for viral DNA replication during the infection cycle. PMID:11861844

  14. 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.

  15. Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

    PubMed Central

    Smith, Gale E.; Fraser, M. J.; Summers, Max D.

    1983-01-01

    We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus. Images PMID:16789242

  16. Autographa californica multiple nucleopolyhedrovirus gene ac81 is required for nucleocapsid envelopment.

    PubMed

    Dong, Fang; Wang, Jinwen; Deng, Riqiang; Wang, Xunzhang

    2016-08-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly pathogenic Baculoviridae that targets insects, whose core gene, ac81, has an unknown function. To determine the role of ac81 in the life cycle of AcMNPV, an ac81-knockout (Ac-81KO-GP) was constructed through homologous recombination in Escherichia coli. We determined that no budded virions were produced in Ac-81KO-GP-transfected Sf9 cells, while there was no effect on viral DNA replication. Electron microscopy (EM) analysis revealed that occlusion-derived virions (ODVs) envelopment and the subsequent embedding of virions into occlusion bodies (OBs) were aborted due to ac81 deletion. Interestingly, confocal microscopy and immunofluorescence analysis revealed that Ac81 was predominantly localized to the ring zone of nuclei during the late phase of infection. In addition, Ac81 was localized to the mature and premature ODVs in virus-infected cells within the ring zone as revealed by immuno-electron microscopy (IEM) analysis. Furthermore, we determined that Ac81 contained a functional hydrophobic transmembrane (TM) domain, whose deletion resulted in a phenotype similar to that of Ac-81KO-GP. These results suggest that Ac81 might be a TM protein that played an important role in nucleocapsid envelopment. PMID:27212683

  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. The Autographa californica multiple nucleopolyhedrovirus ac110 gene encodes a new per os infectivity factor.

    PubMed

    Jiantao Liu; Zhu, Leyuan; Zhang, Shan; Deng, Zihao; Huang, Zhihong; Yuan, Meijin; Wu, Wenbi; Yang, Kai

    2016-08-01

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac110 gene is especially conserved in lepidopteran-specific baculoviruses and is uncharacterized. To investigate the role of ac110 in the baculovirus life cycle, an ac110-knockout (vAc110KO) and a repair (vAc110:HA) viruses were constructed in this study. Budded virion production and occlusion body formation were unaffected in vAc110KO-transfected or infected Sf9 cells. Intrahemocoelic injection of budded virions of vAc110KO killed Trichoplusia ni larvae as efficiently as the repair or the wild-type viruses. However, per os inoculation of occlusion bodies of vAc110KO failed to establish infection in T. ni larvae, while the repair virus was as efficient as the wild-type virus. Treatment with calcofluor white, a reagent that damages the peritrophic membrane, did not rescue the oral infectivity of vAc110KO. These results suggested that Ac110 is a new per os infectivity factor that may play a role after occlusion-derived virions pass through the peritrophic membrane during oral infection. PMID:27212681

  19. 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.

  20. 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.

  1. 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.

  2. Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin

    PubMed Central

    Moreno-Habel, Daniela A.; Biglang-awa, Ivan M.; Dulce, Angelica; DeeLuu, Dee; Garcia, Peter; Weers, Paul M. M.; Haas-Stapleton, Eric J.

    2012-01-01

    Antimicrobial peptides are generated in insects exposed to pathogens for combating infection. Gloverin is a small cationic antibacterial protein whose expression is induced in the hemocytes and fat body cells of Trichoplusia ni larvae exposed to bacteria. The purpose of this study was to determine the role of gloverin during baculovirus infection. We found that gloverin expression is induced in T. ni systemically infected with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Two gloverin genes were cloned using RNA isolated from the hemocytes of T. ni larvae that were systemically infected AcMNPV budded virus (BV) and C-terminal 6x-His and V5 epitope tags were incorporated to facilitate gloverin isolation, detection and functional studies. The supernatants of Sf9 cells stably transfected with the two gloverin expression plasmids and affinity purified gloverin proteins reduced the quantity of infectious AcMNPV BV as measured in vitro by plaque assay with untransfected Sf9 cells. Nanomolar concentrations of affinity column purified gloverin protein caused calcein to be rapidly released from unilamellar vesicles comprised of phosphatidylglycerol, but not from vesicles made up of phosphatidylcholine, suggesting that gloverin interaction with membranes is rapid and affected by membrane charge. Both the BV inactivation and calcein release activities of gloverin increased with higher concentrations of gloverin. These results demonstrate that gloverin is an antiviral protein that interacts with vesicle membranes to cause the contents to be released. PMID:22401766

  3. 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.

  4. RESTRICTION MAPS OF FIVE AUTOGRAPHA CALIFORNICA MNPV VARIANTS, TRICHOPLUSIA NI MNPV, AND GALLERIA MELLONELLA MNPV DNAS WITH ENDONUCLEASES SMAI, KPNI, BAMHI, SACI, XHOI, AND ECORI

    EPA Science Inventory

    The restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) E2 DNA were mapped for the endonucleases SmaI, KpnK, BamHI, SacI, XhoI, and EcoRI. The restriction maps of four other AcMNPV variants, Trichoplusia ni (TnMNPV), and Galleria mellonella (GmMNPV) ge...

  5. SELECTION KINETICS DURING SERIAL CELL CULTURE PASSAGE OF MIXTURES OF WILD TYPE AUTOGRAPHA CALIFORNICA NUCLEAR POLYHEDROSIS VIRUS AND ITS RECOMBINANT AC360-B-GAL

    EPA Science Inventory

    Detailed analysis of the selection process in serial co-infections of cell cultures by wild type Autographa californica nuclear polyhedrosis virus, AcNPV/E2, and Ac36O-B-gal, a "genetically engineered" strain, shows that the unaltered strain was clearly dominant even when it bega...

  6. IN VITRO COMPETITION OF NATURAL 'AUTOGRAPHA CALIFORNICA' NUCLEAR POLYHEDROSIS VIRUS AND A RECOMBINANT EXPRESSING A POLYHEDRIN-BETA GALACTOSIDASE FUSION PROTEIN

    EPA Science Inventory

    The paper describes results of experiments conducted to investigate the kinetics of in vitro competition between natural progenitor Autographa californica (Acv E-2) and the recombinant Ac360-Bgal virus strains. The following conclusions can be drawn from the analysis: Selection p...

  7. Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase C Terminus Is Required for Nuclear Localization and Viral DNA Replication

    PubMed Central

    Feng, Guozhong

    2014-01-01

    ABSTRACT The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. IMPORTANCE The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are

  8. 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

  9. 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

    2016-01-01

    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. PMID:27164127

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

    PubMed Central

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

    2016-01-01

    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. PMID:27164127

  11. 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.

  12. 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

  13. Identification of three late expression factor genes within the 33.8- to 43.4-map-unit region of Autographa californica nuclear polyhedrosis virus.

    PubMed Central

    Lu, A; Miller, L K

    1994-01-01

    A transient transactivation assay system was used in combination with an overlapping Autographa californica nuclear polyhedrosis virus clone library to identify genes involved in late and very late baculovirus gene expression. We have identified three genes within the 33.8- to 43.4-map-unit region of the A. californica nuclear polyhedrosis virus genome which contribute to expression from promoters of the vp39 major capsid protein and polyhedrin genes. One of these three genes corresponds to the previously identified DNA polymerase gene, while the other two genes encode previously unidentified polypeptides of 59,418 and 8,706 Da. None of these genes were required for expression from the early etl promoter. Images PMID:8084003

  14. The Baculovirus Core Gene ac83 Is Required for Nucleocapsid Assembly and Per Os Infectivity of Autographa californica Nucleopolyhedrovirus

    PubMed Central

    Zhu, Shimao; Wang, Wei; Wang, Yan; Yuan, Meijin

    2013-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection. PMID:23864639

  15. Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

    PubMed

    Yu, Qian; Xiong, Youhua; Liu, Jianliang; Wang, Qin; Qiu, Yuanxin; Wen, Dongling

    2016-06-01

    Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields. PMID:26922645

  16. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. 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

  19. Nucleotide sequence and transcriptional analysis of the HindIII P region of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus.

    PubMed

    Carstens, E B; Lu, A

    1990-12-01

    DNA sequence analysis of the HindIII P region of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus confirmed the specific amplification of 1.4 kb of viral DNA from this region of the genome. The sequenced region included an open reading frame, translated in a counterclockwise direction, which would potentially encode a 74K protein. The amplified DNA was contained within this open reading frame, resulting in in-frame amplifications of a domain within the protein. Transcription studies revealed the presence of a ladder of viral RNA species corresponding to a 2.5 kb transcript carrying tandem repeats of about 1.4 kb. This indicated that the duplicated DNA was transcribed in the same orientation as the p10 gene. We predict that transcripts synthesized from the opposite DNA strand also consist of a ladder of related mRNAs which would be translated to produce a family of p74 proteins with multiple internal domains. PMID:2273394

  20. 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

  1. Effects of Substituting Granulin or a Granulin-Polyhedrin Chimera for Polyhedrin on Virion Occlusion and Polyhedral Morphology in Autographa californica Multinucleocapsid Nuclear Polyhedrosis Virus

    PubMed Central

    Eason, Jane E.; Hice, Robert H.; Johnson, Jeffrey J.; Federici, Brian A.

    1998-01-01

    Substitution of granulin from the Trichoplusia ni granulosis virus (TnGV) for polyhedrin of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) yielded a few very large (2 to 5 μm) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type AcMNPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses. PMID:9621097

  2. 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

  3. 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.

  4. Autographa californica multiple nucleopolyhedrovirus Ac92 (ORF92, P33) is required for budded virus production and multiply enveloped occlusion-derived virus formation.

    PubMed

    Wu, Wenbi; Passarelli, A Lorena

    2010-12-01

    The Autographa californica multiple nucleopolyhedrovirus orf92 (p33), ac92, is one of 31 genes carried in all sequenced baculovirus genomes, thus suggesting an essential function. Ac92 has homology to the family of flavin adenine dinucleotide-linked sulfhydryl oxidases and is related to the ERV/ALR family of sulfhydryl oxidases. The role of ac92 during virus replication is unknown. Ac92 was associated with the envelope of both budded and occlusion-derived virus (ODV). To investigate the role of Ac92 during virus replication, an ac92-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration and plaque assays showed no virus spread in ac92-knockout bacmid DNA-transfected insect cells. Deletion of ac92 did not affect viral DNA replication. However, ac92-knockout bacmid DNA-transfected cells lacked multiply enveloped occlusion-derived nucleocapsids; instead, singly enveloped nucleocapsids were detected. To gain insight into the requirement for sulfhydryl oxidation during virus replication, a virus was constructed in which the Ac92 C(155)XXC(158) amino acids, important for sulfhydryl oxidase activity, were mutated to A(155)XXA(158). The mutant virus exhibited a phenotype similar to that of the knockout virus, suggesting that the C-X-X-C motif was essential for sulfhydryl oxidase activity and responsible for the altered ODV phenotype. PMID:20861245

  5. 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.

  6. 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. PMID:15831946

  7. Analysis of an Autographa californica Multicapsid Nucleopolyhedrovirus lef-6-Null Virus: LEF-6 Is Not Essential for Viral Replication but Appears To Accelerate Late Gene Transcription

    PubMed Central

    Lin, Guangyun; Blissard, Gary W.

    2002-01-01

    The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-6 gene was previously shown to be necessary for optimal transcription from an AcMNPV late promoter in transient late expression assays. In the present study, we examined the expression and cellular localization of lef-6 during the AcMNPV infection cycle and generated a lef-6-null virus for studies of the role of lef-6 in the infection cycle. Transcription of lef-6 was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor. To examine lef-6 in the context of the AcMNPV infection cycle, we deleted the lef-6 gene from an AcMNPV genome propagated as an infectious BACmid in Escherichia coli. Unexpectedly, the resulting AcMNPV lef-6-null BACmid (vAclef6KO) was able to propagate in cell culture, although virus yields were substantially reduced. Thus, the lef-6 gene is not essential for viral replication in Sf9 cells. Two “repair” AcMNPV BACmids (vAclef6KO-REP-P and vAclef6KO-REP-ie1P) were generated by transposition of the lef-6 gene into the polyhedrin locus of the vAclef6KO BACmid. Virus yields from the two repair viruses were similar to those from wild-type AcMNPV or a control (BACmid-derived) virus. The lef-6-null BACmid (vAclef6KO) was further examined to determine whether the deletion of lef-6 affected DNA replication or late gene transcription in the context of an infection. The lef-6 deletion did not appear to affect viral DNA replication. Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the lef-6-null BACmid. This phenotype was rescued in viruses containing the lef-6 gene reinserted into the polyhedrin locus. Thus, the lef-6 gene was not essential for either viral DNA replication or late gene transcription, but the absence of

  8. 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

  9. 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.

  10. Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen.

    PubMed Central

    Martin, D W; Weber, P C

    1997-01-01

    Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes. PMID:8985377

  11. 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

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

    PubMed

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

    2016-02-01

    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

  13. 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

  14. Effects of Deletion and Overexpression of the Autographa californica Nuclear Polyhedrosis Virus FP25K Gene on Synthesis of Two Occlusion-Derived Virus Envelope Proteins and Their Transport into Virus-Induced Intranuclear Membranes

    PubMed Central

    Rosas-Acosta, Germán; Braunagel, Sharon C.; Summers, Max D.

    2001-01-01

    Partial deletions within Autographa californica open reading frame 61 (FP25K) alter the expression and accumulation profile of several viral proteins and the transport of occlusion-derived virus (ODV)-E66 to intranuclear membranes during infection (S. C. Braunagel et al., J. Virol. 73:8559–8570, 1999). Here we show the effects of a full deletion and overexpression of FP25K on the transport and expression of two ODV envelope proteins, ODV-E66 (E66) and ODV-E25 (E25). Deletion and overexpression of FP25K substantially altered the levels of expression of E66 during infection. Compared with cells infected with wild-type (wt) virus, the levels of E66 were reduced fivefold in cells infected with a viral mutant lacking FP25K (ΔFP25K) and were slightly increased in cells infected with a viral mutant overexpressing FP25K (FP25Kpolh). In contrast, no significant changes were observed in the levels of E25 among wt-, ΔFP25K-, and FP25Kpolh-infected cells. The changes observed in the levels of E66 among the different viral mutants were not accompanied by changes in either the time of synthesis, membrane association, protein turnover, or steady-state transcript abundance. Deletion of FP25K also substantially altered the transport and localization of E66 during infection. In cells infected with the ΔFP25K mutant virus, E66 accumulated in localized regions at the nuclear periphery and the outer nuclear membrane and did not traffic to intranuclear membranes. In contrast, in cells infected with the FP25Kpolh mutant virus E66 trafficked to intranuclear membranes. For comparison, E25 was normally transported to intranuclear membranes in both ΔFP25K- and FP25Kpolh-infected cells. Altogether these studies suggest that FP25K affects the synthesis of E66 at a posttranscriptional level, probably by altering the translation of E66; additionally, the block in transport of E66 at the nuclear envelope in ΔFP25K-infected cells suggests that the pathway of E66 trafficking to the inner

  15. 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. PMID:10542319

  16. Control of baculovirus gp64-induced syncytium formation by membrane lipid composition.

    PubMed Central

    Chernomordik, L; Leikina, E; Cho, M S; Zimmerberg, J

    1995-01-01

    We have investigated the effects of membrane lipid composition on biological membrane fusion triggered by low pH and mediated by the baculovirus envelope glycoprotein gp64. Lysolipids, either added exogenously or produced in situ by phospholipase A2 treatment of cell membranes, reversibly inhibited syncytium formation. Lysolipids also decreased the baculovirus infection rate. In contrast, oleic and arachidonic acids and monoolein promoted cell-cell fusion. Membrane lipid composition affected pH-independent processes which followed the low-pH-induced change in fusion protein conformation. Inhibition and promotion of membrane fusion by a number of lipids could not be explained by mere binding or incorporation into membranes, but rather was correlated with the effective molecular shape of exogenous lipids. Our data are consistent with the hypothesis that membrane fusion proceeds through highly bent membrane intermediates (stalks) having a net negative curvature. Consequently, inverted cone-shaped lysolipids inhibit and cone-shaped cis-unsaturated fatty acids promote stalk formation and, ultimately, membrane fusion. PMID:7707532

  17. Antisense RNA inactivation of gene expression of a cell-cell adhesion protein (gp64) in the cellular slime mold Polysphondylium pallidum.

    PubMed

    Funamoto, S; Ochiai, H

    1996-05-01

    The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cell-cell adhesion. To explore the functional role of gp64, we made an antisense RNA expression construct designed to prevent the gene expression of gp64; the construct was introduced into P. pallidum cells and the transformants were characterised. The antisense RNA-expressing clone L3mc2 which had just been harvested at the growth phase tended to re-form in aggregates smaller in size than did the parental cells in either the presence or absence of 10 mM EDTA. In contrast, 6.5-hour starved L3mc2 cells remained considerably dissociated from each other after 5 minutes gyrating, although aggregation gradually increased by 50% during a further 55 minutes gyrating in the presence of 10 mM EDTA. Correspondingly, L3mc2 lacked specifically the cell-cell adhesion protein, gp64. We therefore conclude that the gp64 protein is involved in forming the EDTA-resistant cell-cell contact. In spite of the absence of gp64, L3mc2 exhibited normal developmental processes, a fact which demonstrates that another cell-cell adhesion system exists in the development of Polysphondylium. This is the first report in which an antisense RNA technique was successfully applied to Polysphondylium. PMID:8743948

  18. Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody

    PubMed Central

    2013-01-01

    Background Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. Results A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Conclusions Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer

  19. 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.

  20. The transcriptome of the baculovirus Autographa californica multiple nucleopolyhedrovirus in Trichoplusia ni cells.

    PubMed

    Chen, Yun-Ru; Zhong, Silin; Fei, Zhangjun; Hashimoto, Yoshifumi; Xiang, Jenny Z; Zhang, Shiying; Blissard, Gary W

    2013-06-01

    Baculoviruses are important insect pathogens that have been developed as protein expression vectors in insect cells and as transduction vectors for mammalian cells. They have large double-stranded DNA genomes containing approximately 156 tightly spaced genes, and they present significant challenges for transcriptome analysis. In this study, we report the first comprehensive analysis of AcMNPV transcription over the course of infection in Trichoplusia ni cells, by a combination of strand-specific RNA sequencing (RNA-Seq) and deep sequencing of 5' capped transcription start sites and 3' polyadenylation sites. We identified four clusters of genes associated with distinctive patterns of mRNA accumulation through the AcMNPV infection cycle. A total of 218 transcription start sites (TSS) and 120 polyadenylation sites (PAS) were mapped. Only 29 TSS were associated with a canonical TATA box, and 14 initiated within or near the previously identified CAGT initiator motif. The majority of viral transcripts (126) initiated within the baculovirus late promoter motif (TAAG), and late transcripts initiated precisely at the second position of the motif. Analysis of 3' ends showed that 92 (77%) of the 3' PAS were located within 30 nucleotides (nt) downstream of a consensus termination signal (AAUAAA or AUUAAA). A conserved U-rich region was found approximately 2 to 10 nt downstream of the PAS for 58 transcripts. Twelve splicing events and an unexpectedly large number of antisense RNAs were identified, revealing new details of possible regulatory mechanisms controlling AcMNPV gene expression. Combined, these data provide an emerging global picture of the organization and regulation of AcMNPV transcription through the infection cycle. PMID:23536684

  1. 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

    2013-02-01

    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. PMID:23123012

  2. 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.

  3. 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

  4. In vitro anticancer activity of Anemopsis californica

    PubMed Central

    KAMINSKI, CATHERINE N.; FERREY, SETH L.; LOWREY, TIMOTHY; GUERRA, LEO; VAN SLAMBROUCK, SEVERINE; STEELANT, WIM F.A.

    2010-01-01

    Three different extract conditions (aqueous, EtOH and EtOAc) of four different parts (bracts, leaves, roots and stems) of the plant Anemopsis californica (A. californica) were evaluated for their effect on the growth and migration of human colon cancer cells, HCT-8, and the breast cancer cell lines Hs 578T and MCF-7/AZ. Our aim was to identify potential anticancer activity in crude A. californica extracts, given that this plant is used by Native Americans to treat a variety of diseases, including cancer. Our results demonstrated that for each of the cell lines tested, the majority of ethyl acetate extracts of all the plant parts are more toxic than the aqueous and ethanol extracts. Furthermore, significant growth inhibitory activity against the three cell lines was found for the ethyl acetate extract of the roots, while the aqueous extract of the roots influenced the migratory capacity of the three cell lines. This study provides evidence for the anticancer properties of A. californica when extracted in water and ethyl acetate, and supports the importance for further purification of the crude extracts and isolation of potential new anticancer compounds through bio-guided fractionation. PMID:21941602

  5. 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

  6. Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer

    PubMed Central

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

    2005-01-01

    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 106 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 107 to 109 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (∼109 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 ∼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. PMID:16188984

  7. 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

  8. Composition and Antimicrobial Activity of Anemopsis californica leaf oil.

    PubMed

    Medina, Andrea L; Lucero, Mary E; Holguin, F Omar; Estell, Rick E; Posakony, Jeff J; Simon, Julian; O'Connell, Mary A

    2005-11-01

    Isolation and characterization of leaf volatiles in Anemopsis californica (Nutt.) Hook. and Arn. (A. californica) was performed using steam distillation, solid-phase microextraction, and supercritical fluid extraction. Thirty-eight compounds were detected and identified by gas chromatography; elemicin was the major component of the leaf volatiles. While the composition of the leaf volatiles varied with method of extraction, alpha-pinene, sabinene, beta-phellandrene, 1,8-cineole, piperitone, methyl eugenol, (E)-caryophyllene, and elemicin were usually present in readily detectable amounts. Greenhouse-reared clones of a wild population of A. californica had an identical leaf volatile composition with the parent plants. Steam-distilled oil had antimicrobial properties against 3 (Staphylococcus aureus, Streptococcus pneumoniae, and Geotrichim candidum) of 11 microbial species tested. Some of this bioactivity could be accounted for by the alpha-pinene in the oil. PMID:16248573

  9. 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. PMID:26122312

  10. Headspace Volatiles of Scutellaria californica A. Gray Flowers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Volatile constituents of California skullcap (Scutellaria californica A. Gray) flowers were isolated by solid-phase microextraction (SPME) and analyzed by GC and GC/MS. A total of 52 constituents were identified (constituting 90.79% of the total area), 12 of which were tentatively identified. Cary...

  11. Topographical studies of the nicotinic acetylcholine receptor. [Torpedo californica

    SciTech Connect

    Middlemas, D.S.

    1987-01-01

    All four subunits of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with the photoactivated hydrophobic probe, (/sup 3/H)adamantanediazirine, which selectively labels regions of integral membrane proteins in contact with the hydrocarbon core of the lipid bilayer. All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with (/sup 3/H)cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. Since the photogenerated carbene is situated near the lipid-water interface, this probe has potential as a topographic tool for mapping membrane protein structure. The labeling studies with both (/sup 3/H)adamantanediazirine and (/sup 3/H)cholesteryl diazoacetate support the concept that the acetylcholine receptor is a pseudosymmetric complex of homologous subunits, all of which interact with and span the membrane. The synthesis of the fluorine-containing agonists for the Torpedo californica nicotinic acetylcholine receptor, fluoroacetylcholine bromide and p-fluorophenyltrimethylammonium iodide, are described. It is demonstrated that both are agonists using a cation flux assay with acetylcholine receptor enriched membrane vesicles. The affinity cleavage reagent, p-thiocyanophenyltrimethylammonium iodide, specifically cleaves a peptide bond of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica. It is demonstrated that this reagent is an agonist using a cation flux assay. The cleavage is blocked by stoichiometric quantities of ..cap alpha..-bungarotoxin.

  12. Antimycobacterial Furofuran Lignans from the Roots of Anemopsis californica

    PubMed Central

    Bussey, Robert O; Sy-Cordero, Arlene A; Figueroa, Mario; Carter, Fredrick S.; Falkinham, Joseph O.; Oberlies, Nicholas H.; Cech, Nadja B.

    2015-01-01

    Topical preparations of Anemopsis californica have been used by Native American tribes in the southwestern United States and northern Mexico to treat inflammation and infections. We report results of bioassay-guided isolation conducted on a sample of A. californica roots. The furofuran lignans sesamin (1) and asarinin (2) were isolated and shown to have MIC values ranging from 23 to 395 µM against five different species of environmental nontuberculous mycobacteria. These findings are significant given that these bacteria can cause skin, pulmonary, and lymphatic infections. Crude A. californica extracts were analyzed by liquid chromatography - mass spectrometry (LC-MS), and it was determined that sesamin and asarinin were extracted at relatively high levels from roots (1.7–3.1g/kg and 1.1–1.7 g/kg, respectively), but lower levels from leaves (0.13 g/kg for both compounds). Our findings suggest that the majority of activity of crude A. californica root extracts against nontuberculous mycobacteria can be attributed to the presence of sesamin and asarinin. This paper is the first to report isolation of these compounds from a member of the Saururaceae family, and the first to describe their activity against nontuberculous mycobacteria. PMID:24687738

  13. Assessment of a Basement Membrane-Degrading Protease on Dissemination and Secondary Infection of Autographa californica Multiple Nucleopolyhedrovirus in Heliothis virescens L.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, that digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus that expresses ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significant...

  14. The Ac124 protein is not essential for the propagation of Autographa californica multiple nucleopolyhedrovirus, but it is a viral pathogenicity factor.

    PubMed

    Liang, Changyong; Lan, Dandan; Zhao, Shuling; Liu, Lulu; Xue, Yanan; Zhang, Yongli; Wang, Yun; Chen, Xinwen

    2015-01-01

    orf124 (ac124) of AcMNPV is one of the highly conserved unique genes in group I lepidopteran nucleopolyhedroviruses. So far, its function remains unknown. In this study, infection with a virus expressing an ac124-gfp fusion showed that Ac124 localized to the cytoplasm throughout the infection. In addition, an ac124 knockout virus was generated to determine the role of ac124 in the baculovirus life cycle. Our results showed that an ac124 knockout AcMNPV could produce infectious budded viruses (BVs) and occlusion bodies (OBs) like those produced by the wild virus and ac124 repair virus. These three viruses had similar growth kinetics during the infection phase. There was no significant difference in nucleocapsids, occlusion-derived viruses and OBs visualized by electron microscopy. The ac124 deletion mutant did not reduce AcMNPV infectivity for S. exigua in an LD50 bioassay. However, it took 20 h longer for the ac124 deletion mutant to kill S. exigua than wild-type virus in the LT50 bioassay. Altogether, these results demonstrate that ac124 is not required for viral replication, but it accelerates the killing of infected larvae. PMID:25380680

  15. Spectral diversity of fluorescent proteins from the anthozoan Corynactis californica.

    PubMed

    Schnitzler, Christine E; Keenan, Robert J; McCord, Robert; Matysik, Artur; Christianson, Lynne M; Haddock, Steven H D

    2008-01-01

    Color morphs of the temperate, nonsymbiotic corallimorpharian Corynactis californica show variation in pigment pattern and coloring. We collected seven distinct color morphs of C. californica from subtidal locations in Monterey Bay, California, and found that tissue- and color-morph-specific expression of at least six different genes is responsible for this variation. Each morph contains at least three to four distinct genetic loci that code for these colors, and one morph contains at least five loci. These genes encode a subfamily of new GFP-like proteins, which fluoresce across the visible spectrum from green to red, while sharing between 75% to 89% pairwise amino-acid identity. Biophysical characterization reveals interesting spectral properties, including a bright yellow protein, an orange protein, and a red protein exhibiting a "fluorescent timer" phenotype. Phylogenetic analysis indicates that the FP genes from this species evolved together but that diversification of anthozoan fluorescent proteins has taken place outside of phylogenetic constraints, especially within the Corallimorpharia. The discovery of more examples of fluorescent proteins in a non-bioluminescent, nonsymbiotic anthozoan highlights possibilities of adaptive ecological significance unrelated to light regulation for algal symbionts. The patterns and colors of fluorescent proteins in C. californica and similar species may hold meaning for organisms that possess the visual pigments to distinguish them. PMID:18330643

  16. Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors.

    PubMed

    Fedurco, Milan; Gregorová, Jana; Šebrlová, Kristýna; Kantorová, Jana; Peš, Ondřej; Baur, Roland; Sigel, Erwin; Táborská, Eva

    2015-01-01

    The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α 1 β 2 γ 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S)-reticuline behaved as positive allosteric modulator at the α 3, α 5, and α 6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α 3 β 2 γ 2 and α 5 β 2 γ 2 GABAA receptors. Interestingly, α 1, α 3, and α 5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine-suspected (S)-reticuline metabolites in the rodent brain. PMID:26509084

  17. Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors

    PubMed Central

    Fedurco, Milan; Gregorová, Jana; Šebrlová, Kristýna; Kantorová, Jana; Peš, Ondřej; Baur, Roland; Sigel, Erwin; Táborská, Eva

    2015-01-01

    The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α1β2γ2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S)-reticuline behaved as positive allosteric modulator at the α3, α5, and α6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α3β2γ2 and α5β2γ2 GABAA receptors. Interestingly, α1, α3, and α5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine—suspected (S)-reticuline metabolites in the rodent brain. PMID:26509084

  18. 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.

  19. Comparative analysis of early ontogeny in Bursatella leachii and Aplysia californica

    PubMed Central

    Vue, Zer; Capo, Thomas R.; Bardales, Ana T.

    2014-01-01

    Opisthobranch molluscs exhibit fascinating body plans associated with the evolution of shell loss in multiple lineages. Sea hares in particular are interesting because Aplysia californica is a well-studied model organism that offers a large suite of genetic tools. Bursatella leachii is a related tropical sea hare that lacks a shell as an adult and therefore lends itself to comparative analysis with A. californica. We have established an enhanced culturing procedure for B. leachii in husbandry that enabled the study of shell formation and loss in this lineage with respect to A. californica life staging. PMID:25538871

  20. INITIAL SIZE AND DYNAMICS OF VIRAL FUSION PORES ARE A FUNCTION OF THE FUSION PROTEIN MEDIATING MEMBRANE FUSION

    PubMed Central

    Plonsky, I.; Kingsley, D. H.; Rashtian, A.; Blank, P.S.; Zimmerberg, J.

    2013-01-01

    To investigate the role of the fusogenic protein in the initial size and dynamics of the pore that widens to finalize membrane fusion, two different fusion proteins expressed in the same cell line were investigated: the major glycoprotein of baculovirus Autographa californica (GP64) and the hemaggluttinin of influenza X31 (HA). The host Sf9 cells expressing these viral proteins, irrespective of protein species, fused to human red blood cells (RBC) upon acidification of the medium. High time resolution electrophysiological study of fusion pore conductance revealed fundamental differences in a) the initial pore conductance (pores created by HA were smaller than those created by GP64), b) the ability of pores to flicker (only HA-mediated pores flickered), and c) the time required for pore formation (HA-mediated pores took much longer to form following acidification). Thus 1) HA and GP64 have divergent electrophysiological phenotypes even when they fuse identical membranes, and 2) fusion proteins play a crucial role in determining initial fusion pore characteristics. The structure of the initial fusion pore detected by electrical conductance measurements is sensitive to the nature of the fusion protein. PMID:18208404

  1. Baculoviral display of the green fluorescent protein and rubella virus envelope proteins.

    PubMed

    Mottershead, D; van der Linden, I; von Bonsdorff, C H; Keinänen, K; Oker-Blom, C

    1997-09-29

    The ability to display heterologous proteins and peptides on the surface of different types of bacteriophage has proven extremely useful in protein structure/function studies. To display such proteins in a eucaryotic environment, we have produced a vector allowing for fusion of proteins to the amino-terminus of the Autographa californica nuclear polyhedrosis virus (AcNPV) major envelope glycoprotein, gp64. Such fusion proteins incorporate into the baculoviral virion and display the FLAG epitope tag. We have further produced recombinant baculoviruses displaying the green fluorescent protein (GFP) and the rubella virus envelope proteins, E1 and E2. The incorporation of the GFPgp64, E1gp64, and E2gp64 fusion proteins into the baculovirus particle was demonstrated by western blot analysis of purified budded virus. This is the first report of the display of the GFP protein or the individual rubella virus spike proteins on the surface of an enveloped virus. Such a eucaryotic viral display system may be useful for the display of proteins dependent on glycosylation for activity and for targeting of recombinant baculoviruses to novel host cell types as a gene transfer vehicle. PMID:9325155

  2. 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.

  3. 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)

  4. 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. PMID:24919845

  5. 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.

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

    PubMed

    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

    2015-08-01

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. 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.

  10. 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

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

    PubMed

    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

  12. Two partially unfolded states of Torpedo californica acetylcholinesterase.

    PubMed

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

    1996-09-01

    Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physiochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. Composition of Pteryxia terebinthina var. californica (Coult. and Rose) mathias essential oils

    USGS Publications Warehouse

    Beauchamp, P.E.; Dev, V.; Munevar-Mendoza, E.; Moore, P.E.

    2000-01-01

    ??-Pinene (35.0%, 53.8%) was the major component of both the aerial parts and the root oils of Pteryxia terebinthina var. californica, respectively. ??-Phellandrene (12.2%) was the other most abundant component of the oil from aeial parts while ??-3-carene (14.2%) was the second abundant component of the root oil. ?? 2000 Allured Publishing Corp.

  16. Chemotypic Variation of Essential Oils in the Medicinal Plant, Anemopsis californica

    PubMed Central

    Medina-Holguín, Andrea L.; Holguín, F. Omar; Micheletto, Sandra; Goehle, Sondra; Simon, Julian A.; O’Connell, Mary A.

    2008-01-01

    Anemopsis californica (Saururaceae) commonly called yerba mansa, is an important medicinal plant in many deserts in the southwestern region of North America. Populations of A. californica, collected throughout New Mexico, were examined for chemical variability in roots and rhizomes for select monocyclic (cymene, limonene, piperitone and thymol) and bicyclic (α-pinene, 1,8-cineole and myrtenol) monoterpenoid and phenylpropanoid (methyleugenol, isoeugenol and elemicin) derived essential oil components. Three distinct chemotypes were detected using a hierarchical clustering analysis on the concentration of 10 different analytes in three individuals from each of 17 populations. One chemotype was characterized by high elemicin concentrations, a second chemotype by high methyleugenol concentrations and the third by high piperitone and thymol concentrations. Steam distilled oil was used to screen for anticancer bioactivity. A. californica root oils demonstrated anti-proliferative activity against AN3CA and HeLa cells in vitro but no activity against lung, breast, prostate or colon cancer cells. The IC50 values for the root oil were 0.056% and 0.052% (v/v) for the AN3CA and HeLa cells respectively. PMID:18177907

  17. Chemotypic variation of essential oils in the medicinal plant, Anemopsis californica.

    PubMed

    Medina-Holguín, Andrea L; Holguín, F Omar; Micheletto, Sandra; Goehle, Sondra; Simon, Julian A; O'Connell, Mary A

    2008-02-01

    Anemopsis californica (Saururaceae) commonly called yerba mansa, is an important medicinal plant in many deserts in the southwestern region of North America. Populations of A. californica, collected throughout New Mexico, were examined for chemical variability in roots and rhizomes for select monocyclic (cymene, limonene, piperitone and thymol) and bicyclic (alpha-pinene, 1,8-cineole and myrtenol) monoterpenoid and phenylpropanoid (methyleugenol, isoeugenol and elemicin) derived essential oil components. Three distinct chemotypes were detected using a hierarchical clustering analysis on the concentration of 10 different analytes in three individuals from each of 17 populations. One chemotype was characterized by high elemicin concentrations, a second chemotype by high methyleugenol concentrations and the third by high piperitone and thymol concentrations. Steam distilled oil was used to screen for anticancer bioactivity. A. californica root oils demonstrated anti-proliferative activity against AN3CA and HeLa cells in vitro but no activity against lung, breast, prostate or colon cancer cells. The IC(50) values for the root oil were 0.056% and 0.052% (v/v) for the AN3CA and HeLa cells, respectively. PMID:18177907

  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. PMID:6759370

  19. 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. PMID:27054913

  20. 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

  1. Shotgun proteomic analysis of yeast-elicited California poppy (Eschscholzia californica) suspension cultures producing enhanced levels of benzophenanthridine alkaloids.

    PubMed

    Oldham, John T; Hincapie, Marina; Rejtar, Tomas; Wall, P Kerr; Carlson, John E; Lee-Parsons, Carolyn W T

    2010-09-01

    The California poppy, Eschscholzia californica, produces benzophenanthridine alkaloids (BPAs), an important class of biologically active compounds. Cell cultures of E. californica were investigated as an alternative and scalable method for producing these valuable compounds; treatment with yeast extract increased production from low levels to 23 mg/g dry weight (DW) of BPAs. A shotgun proteomic analysis of E. californica cell cultures was undertaken to explore changes in metabolism associated with enhanced BPA production. We implemented differential centrifugation and then shotgun proteomics based on nanoliquid chromatography/mass spectrometry (nano-LC-MS/MS) for peptide separation and analysis. A unigene database available for E. californica was translated and utilized for protein identification. Approximately 646 proteins (3% false discovery rate at the protein level) were identified. Differentially abundant proteins observed with elicitation included enzymes involved in (S)-adenosyl methionine (SAM) biosynthesis and BPA biosynthesis. These results demonstrate (1) the identification of proteins from a medicinal plant using shotgun proteomics combined with a well-annotated, translated unigene database and (2) the potential utility of proteomics for exploring changes in metabolism associated with enhanced secondary metabolite production. PMID:20690678

  2. 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. PMID:24266426

  3. 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. PMID:26644027

  4. 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.

  5. 1H NMR Relaxation Investigation of Inhibitors Interacting with Torpedo californica Acetylcholinesterase

    NASA Astrophysics Data System (ADS)

    Delfini, Maurizio; Gianferri, Raffaella; Dubbini, Veronica; Manetti, Cesare; Gaggelli, Elena; Valensin, Gianni

    2000-05-01

    Two naphthyridines interacting with Torpedo californica acetylcholinesterase (AChE) were investigated. 1H NMR spectra were recorded and nonselective, selective, and double-selective spin-lattice relaxation rates were measured. The enhancement of selective relaxation rates could be titrated by different ligand concentrations at constant AChE (yielding 0.22 and 1.53 mM for the dissociation constants) and was providing evidence of a diverse mode of interaction. The double-selective relaxation rates were used to evaluate the motional correlation times of bound ligands at 34.9 and 36.5 ns at 300 K. Selective relaxation rates of bound inhibitors could be interpreted also in terms of dipole-dipole interactions with protons in the enzyme active site.

  6. Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

    PubMed

    Hartig, P R; Raftery, M A

    1979-04-01

    Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes. PMID:427105

  7. Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

    PubMed Central

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

    2013-01-01

    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. PMID:23892672

  8. 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. PMID:25970633

  9. 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

  10. 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. PMID:26740541

  11. 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

  12. 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. PMID:27445997

  13. 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

  14. Localization of GABA-like immunoreactivity in the central nervous system of Aplysia californica.

    PubMed

    Díaz-Ríos, M; Suess, E; Miller, M W

    1999-10-18

    Gamma-aminobutyric acid (GABA) is present in the central nervous system of Aplysia californica (Gastropoda, Opisthobranchia) where its role as a neurotransmitter is supported by pharmacological, biochemical, and anatomical investigations. In this study, the distribution of GABA-immunoreactive (GABAi) neurons and fiber systems in Aplysia was examined by using wholemount immunohistochemistry and nerve backfill methods. GABAi neurons were located in the buccal, cerebral, and pedal ganglia. Major commissural fiber systems were present in each of these ganglia, whereas more limited fiber systems were observed in the ganglionic connectives. Some of the interganglionic fibers were found to originate from two unpaired GABAi neurons, one in the buccal ganglion and one in the right pedal ganglion, each of which exhibited bilateral projections. No GABAi fibers were found in the nerves that innervate peripheral sensory, motor, or visceral organs. Although GABAi cells were not observed in the pleural or abdominal ganglia, these ganglia did receive limited projections of GABAi fibers originating from neurons in the pedal ganglia. The distribution of GABAi neurons suggests that this transmitter system may be primarily involved in coordinating certain bilateral central pattern generator (CPG) systems related to feeding and locomotion. In addition, the presence of specific interganglionic GABAi projections also suggests a role in the regulation or coordination of circuits that produce components of complex behaviors. PMID:10524338

  15. Serotonin immunoreactivity in the central nervous system of the marine molluscs Pleurobranchaea californica and Tritonia diomedea.

    PubMed

    Sudlow, L C; Jing, J; Moroz, L L; Gillette, R

    1998-06-15

    The central nervous systems of the marine molluscs Pleurobranchaea californica (Opisthobranchia: Notaspidea) and Tritonia diomedea (Opisthobranchia: Nudibranchia) were examined for serotonin-immunoreactive (5-HT-IR) neurons and processes. Bilaterally paired clusters of 5-HT-IR neuron somata were distributed similarly in ganglia of the two species. In the cerebropleural ganglion complex, these were the metacerebral giant neurons (both species), a dorsal anterior cluster (Pleurobranchaea only), a dorsal medial cluster including identified neurons of the escape swimming network (both species), and a dorsal lateral cluster in the cerebropleural ganglion (Pleurobranchaea only). A ventral anterior cluster (both species) adjoined the metacerebral giant somata at the anterior ganglion edge. Pedal ganglia had the greatest number of 5-HT-IR somata, the majority located near the roots of the pedal commissure in both species. Most 5-HT-IR neurons were on the dorsal surface of the pedal ganglia in Pleurobranchaea and were ventral in Tritonia. Neither the buccal ganglion of both species nor the visceral ganglion of Pleurobranchaea had 5-HT-IR somata. Afew asymmetrical 5-HT-IR somata were found in cerebropleural and pedal ganglia in both species, always on the left side. The clustering of 5-HT-IR neurons, their diverse axon pathways, and the known physiologic properties of their identified members are consistent with a loosely organized arousal system of serotonergic neurons whose components can be generally or differentially active in expression of diverse behaviors. PMID:9619500

  16. 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.

  17. 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.

  18. Quantitative reflection imaging of fixed Aplysia californica pedal ganglion neurons on nanostructured plasmonic crystals.

    PubMed

    Le, An-Phong; Kang, Somi; Thompson, Lucas B; Rubakhin, Stanislav S; Sweedler, Jonathan V; Rogers, John A; Nuzzo, Ralph G

    2013-10-24

    Studies of the interactions between cells and surrounding environment including cell culture surfaces and their responses to distinct chemical and physical cues are essential to understanding the regulation of cell growth, migration, and differentiation. In this work, we demonstrate the capability of a label-free optical imaging technique-surface plasmon resonance (SPR)-to quantitatively investigate the relative thickness of complex biomolecular structures using a nanoimprinted plasmonic crystal and laboratory microscope. Polyelectrolyte films of different thicknesses deposited by layer-by-layer assembly served as the model system to calibrate the reflection contrast response originating from SPRs. The calibrated SPR system allows quantitative analysis of the thicknesses of the interface formed between the cell culture substrate and cellular membrane regions of fixed Aplysia californica pedal ganglion neurons. Bandpass filters were used to isolate spectral regions of reflected light with distinctive image contrast changes. Combining of the data from images acquired using different bandpass filters leads to increase image contrast and sensitivity to topological differences in interface thicknesses. This SPR-based imaging technique is restricted in measurable thickness range (∼100-200 nm) due to the limited plasmonic sensing volume, but we complement this technique with an interferometric analysis method. Described here simple reflection imaging techniques show promise as quantitative methods for analyzing surface thicknesses at nanometer scale over large areas in real-time and in physicochemical diverse environments. PMID:23647567

  19. 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. PMID:25921857

  20. Lipoxygenase activity and sanguinarine production in cell suspension cultures of California poppy (Eschscholtzia californica CHAM.).

    PubMed

    Kollárová, R; Oblozinský, M; Kováciková, V; Holková, I; Balazová, A; Pekárová, M; Hoffman, P; Bezáková, L

    2014-08-01

    In this study we investigated the influence of biotic elicitor (phytopathogenic fungus Botrytis cinerea) and abiotic elicitors (methyljasmonate [MJ] and salicylic acid [SA]) on lipoxygenase (LOX) activity and sanguinarine production in cell suspension cultures of California poppy (Eschscholtzia californica CHAM.). We have observed different time effects of elicitors (10, 24, 48 and 72 h) on LOX activity and production of sanguinarine in in vitro cultures. All elicitors used in the experiments evidently increased the LOX activity and sanguinarine production in contrast to control samples. The highest LOX activities were determined in samples elicitated by MJ after 48 h and 72 h and the lowest LOX activities (in contrast to control samples) were detected after biotic elicitation by Botrytis cinerea. These activities showed about 50% lower level against the activities after MJ elicitation. The maximal amount of sanguinarine was observed after 48 h in MJ treated cultures (429.91 mg/g DCW) in comparision with control samples. Although all elicitors affect the sanguinarine production, effect of SA and biotic elicitor on sanguinarine accumulation in in vitrocultures was not so significant than after MJ elicitation. PMID:25158577

  1. Photoaffinity labeling and quaternary structure of the acetylcholine receptor from Torpedo californica.

    PubMed Central

    Hucho, F; Layer, P; Kiefer, H R; Bandini, G

    1976-01-01

    Membrane fragments from electric tissue of Torpedo californica containing nicotinic acetylcholine receptor are composed of four different polypeptide chains with molecular weights of 40,000 (alpha), 48,000 (beta), 62,000 (gamma), and 66,000 (delta). The alpha and beta chains are still present in all and gamma and delta in some of the receptor preparations after Triton X-100 extraction and purification by affinity chromatography. All components of the receptor react covalently with the photoaffinity label 4-azido-2-nitrobenzyltrimethylammonium fluoroborate, the delta chain incorporating less of the reagent as compared to the alpha and beta chains. Agonists and antagonists containing a quaternary ammonium group protect all chains against the label; the principal neurotoxin from Naja naja siamensis protects the alpha chain only. We conclude that the alpha chain binds the neurotoxin from Naja naja, the alpha and beta chains are involved in the binding of ligands with quaternary ammonium groups, and the function of the gamma and delta chains remains to be determined. Images PMID:1066671

  2. Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones.

    PubMed

    Millard, Charles B; Shnyrov, Valery L; Newstead, Simon; Shin, Irina; Roth, Esther; Silman, Israel; Weiner, Lev

    2003-10-01

    Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is "native-like," and inactivation can be reversed by exposure to reduced glutathione. The allicin-modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t(1/2) approximately 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine-N-oxide, glycerol, and sucrose, and the divalent cations, Ca(2+), Mg(2+), and Mn(2+) can prevent this transition of the native-like state for >24 h at room temperature. Trimethylamine-N-oxide and Mg(2+) can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39 degrees C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native-like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry. PMID:14500892

  3. Reproductive parameters of the Pacific angel shark Squatina californica (Selachii: Squatinidae).

    PubMed

    Romero-Caicedo, A F; Galván-Magaña, F; Hernández-Herrera, A; Carrera-Fernández, M

    2016-04-01

    Reproductive characteristics of the Pacific angel shark, Squatina californica, were evaluated from 420 specimens obtained from the artisanal fishery in La Paz Bay, Gulf of California, Mexico. Females (99 cm, 6000 g) were larger than males (95 cm, 5000 g) in terms of both total length (LT ) and body mass (MT ). The overall sex ratio was significantly different from the expected 1:1, suggesting sexual segregation of mature individuals in La Paz Bay. Males had developed reproductive organs and calcified claspers from 72 cm LT ; the median size at maturity (LT50 ) was 75·6 cm. In females, only the left ovary was functional and mature ovarian follicles were present from 77 cm LT ; the estimated LT50 was 77·7 cm. For the 10 gravid females sampled, uterine fecundity was between two and 10 embryos. Mature, non-gravid females with small and large ovarian follicles appeared simultaneously with gravid females with follicles that did not exceed 1·9 cm diameter. PMID:26931737

  4. 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.

  5. Application of molecular techniques to identification of three plusiine species, Autographa nigrisigna, Macdunnoughia confusa, and Thysanoplusia intermixta (Lepidoptera: Noctuidae), found in integrated pest management lettuce fields in Japan.

    PubMed

    Hashiyama, Aoi; Nomura, Masashi; Kurihara, Jun; Toyoshima, Goro

    2011-08-01

    Three plusiine species, Autographa nigrisigna, Macdunnoughia confusa, and Thysanoplusia intermixta (Lepidoptera: Noctuidae), are commonly found together in lettuce, Lactuca sativa L., fields in Japan. Given the marked morphological similarities between these species and the difficulty associated with discriminating between them using only visual cues, we used multiplex polymerase chain reaction (PCR) assay to distinguish between the three target species. Multiplex PCR uses four primers to simultaneously amplify a specific region of the mitochondrial DNA and produce species-specific banding patterns. The stringency of the method was tested using specimens of different sex, location, and developmental stage, and consistent results were obtained for all samples. Indeed, our method has the potential to clarify the species structure of plusiine species in lettuce fields. PMID:21882693

  6. Identification of the Lymantria dispar Nucleopolyhedrovirus Envelope Fusion Protein Provides Evidence for a Phylogenetic Division of the Baculoviridae†

    PubMed Central

    Pearson, Margot N.; Groten, Christoph; Rohrmann, George F.

    2000-01-01

    The complete genome sequences of a number of diverse members of the Baculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV (LdMNPV), revealed a single open reading frame (ld130) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length ld130 gene and of an ld130-enhanced green fluorescent protein gene (egfp) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with LdMNPV or transfected with ld130-egfp. In addition, cells transfected with either ld130 or ld130-egfp or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution of gp64 appears to be confined to a relatively closely related group of NPVs, homologs of ld130 are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein. PMID:10846096

  7. 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

  8. 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

  9. Serotonin increases intracellular Ca2+ transients in voltage-clamped sensory neurons of Aplysia californica.

    PubMed Central

    Boyle, M B; Klein, M; Smith, S J; Kandel, E R

    1984-01-01

    Noxious stimulation of the tail of Aplysia californica produces behavioral sensitization; it enhances several related defensive reflexes. This reflex enhancement involves heterosynaptic facilitation of transmitter release from sensory neurons of the reflex. The facilitation is stimulated by serotonin (5-HT) and involves suppression of a 5-HT-sensitive K+ current (the S current). Suppression of the S current broadens the action potential of the sensory neurons and is thought to enhance transmitter release by prolonging entry of Ca2+ in the presynaptic terminals. We now report a component of enhanced Ca2+ accumulation that is independent of changes in spike shape. We have measured intracellular free Ca2+ transients during long depolarizing steps in voltage-clamped sensory neuron cell bodies injected with the Ca2+-sensitive dye arsenazo III. The free Ca2+ transients elicited by a range of depolarizing voltage-clamp steps increase in amplitude by 75% following application of 5-HT. Since it is observed under voltage-clamp conditions, this increase in the free Ca2+ transients is not merely secondary to the changes in K+ current but must reflect an additional mechanism, an intrinsic change in the handling of Ca2+ by the cell. We have not yet determined whether this change in Ca2+ handling reflects an increase in Ca2+ influx, a reduction in intracellular Ca2+ uptake, or a release of Ca2+ from intracellular stores. Regardless of the underlying mechanism, however, it seems possible that the enhancement of Ca2+ accumulation and the reduction in K+ current act synergistically in producing short-term presynaptic facilitation. Alternatively, this additional modulation of Ca2+ by 5-HT might contribute to processes such as classical conditioning or long-term sensitization that may depend on Ca2+. PMID:6594707

  10. CjbHLH1 homologs regulate sanguinarine biosynthesis in Eschscholzia californica cells.

    PubMed

    Yamada, Yasuyuki; Motomura, Yukiya; Sato, Fumihiko

    2015-05-01

    Isoquinoline alkaloids (IQAs), terpenoid indole alkaloid and nicotine are some of the most studied alkaloids. Recently, several groups have reported that the biosynthesis of these alkaloids is regulated by basic helix-loop-helix (bHLH) transcription factors. Whereas the biosyntheses of nicotine and terpenoid indole alkaloid in Nicotiana plants and Catharanthus roseus are directly or indirectly regulated by Arabidopsis thaliana MYC2 homologs, a non-MYC2-type bHLH transcription factor, CjbHLH1, comprehensively regulates berberine biosynthesis in Coptis japonica. Interestingly, CjbHLH1 homologous genes were found in many IQA-producing plant species, which suggests that non-MYC2-type CjbHLH homologs are specifically associated with IQA biosynthesis. To test whether CjbHLH1 homologs are involved in the biosynthesis of IQA in a plant other than C. japonica, we isolated two genes homologous to CjbHLH1, i.e. EcbHLH1-1 and EcbHLH1-2, from Eschscholzia californica (California poppy). Stable transformants in which the expression levels of EcbHLH1 genes were constitutively suppressed by RNA interference (RNAi) showed a reduced expression of some IQA biosynthetic enzyme genes. A metabolite analysis confirmed that the suppression of EcbHLH1, particularly EcbHLH1-2, caused a decrease in sanguinarine accumulation in transgenic cultured cells. These results indicate that non-MYC2-type EcbHLH1s regulate IQA biosynthesis in California poppy like CjbHLH1 in C. japonica. PMID:25713177

  11. Isolation and characterization of haemoporin, an abundant haemolymph protein from Aplysia californica.

    PubMed Central

    Jaenicke, Elmar; Walsh, Patrick J; Decker, Heinz

    2003-01-01

    In the present study, we show the isolation and characterization of the protein haemoporin, which constitutes the second most abundant protein fraction in the haemolymph of the marine gastropod Aplysia californica. Although Aplysia is commonly used to investigate the molecular basis of learning, not much is known about the proteins in its haemolymph, which is in contact with the neurons owing to the open circulatory system of molluscs. In the native state, haemoporin is a macromolecular complex forming a cylinder with a central solvent-filled pore. The native complex most probably is a homopentamer made up from 70 kDa subunits with a molecular mass of 360 kDa and a sedimentation coefficient of 11.7 S. Prediction of the secondary structure by CD spectroscopy revealed that haemoporin contains 36% alpha-helices and 19% beta-strands. An absorption band in the 300-400 nm region indicates that haemoporin probably contains a bound substance. Haemoporin also contains a below average amount of tryptophan as evident from absorption and fluorescence spectra. The specific absorption coefficient at 280 nm (a (280 nm, 1 mg/ml)) varies between 0.42 and 0.59 l x g(-1) x cm(-1) depending on the method. The function of the protein is not yet known, but there are structural parallels between haemoporin and a pore protein reported previously in the haemolymph of another marine gastropod Megathura crenulata. The alanine-rich N-terminal sequence (AAVPEAAAEATAEAAPVSEF) is unique among protein sequences and indicates an alpha-helical structure. Whereas one side of the helix is hydrophobic and faces the interior of the protein, the other side contains a glutamic cluster, which may form the channel of the pore in the quaternary structure. Thus both proteins might belong to a new class of haemolymph proteins present in the haemolymph of marine gastropods. PMID:12889987

  12. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    SciTech Connect

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F. . E-mail: rohrmanng@orst.edu

    2007-03-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

  13. 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.

  14. 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

  15. 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. PMID:25986655

  16. RADIOIMMUNOASSAY ANALYSIS OF BACULOVIRUS GRANULINS AND POLYHEDRINS

    EPA Science Inventory

    Granulin and polyhedrin proteins were purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis from the baculoviruses Autographa californica, Rachiplusia ou, Heliothis zea, Heliothis armigera, Trichoplusia ni, and Spodotera frugiperda. Antisera were...

  17. 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. PMID:18157859

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

    PubMed

    Dangl, Gerald S; Mendum, Mary Lou; Yang, Judy; Walker, M Andrew; Preece, John E

    2015-12-01

    Hybridization of introduced domesticates and closely related natives is well documented in annual crops. The widespread introduction of the domesticated grapevine, Vitis vinifera, into California where it overlaps with two native congenerics, with which it is interfertile, provides opportunity to investigate hybridization between woody perennials. Although geographically widespread, the introduction over the past two centuries has been limited to a few elite clonal cultivars, providing a unique opportunity to study the effects of hybridization on the native species. The amount of hybridization with V. vinifera and the genetic diversity of wild-growing Vitis californica and Vitis girdiana were examined using nineteen microsatellite markers. STRUCTURE analysis was used to define hybrid and introgressed individuals and to analyze genetic structure of the native species. FAMOZ software was used to identify which V. vinifera cultivars served as parents of F 1 hybrids. The three species were clearly distinguished by STRUCTURE analysis. Thirty percent of 119 V. californica vines were hybrids. The domesticated parent was identified for 16 F 1 hybrid vines; the original California cultivar, 'Mission', was the parent of eight. Backcrosses were also found, showing introgression into subsequent generations. Similar results were obtained for a small sample of V. girdiana. Removing hybrids greatly reduced the genetic variation of the presumed pure species, among which there was essentially no genetic structure. Limited genetic variability indicates the California natives may be threatened by genetic erosion. The discovery of F 1 hybrids of 'Mission', a cultivar not grown in the areas for ~100 years, suggests long generation times for wild vines that, often, grow into expansive liana and propagate by layering, all factors that limit recruitment in populations already disjunct by habitat lose. Hermaphroditic flowers and fruit that is more attractive to birds may favor the

  19. Larval growth, development, and survival of laboratory-reared Aplysia californica: Effects of diet and veliger density*

    PubMed Central

    Capo, Thomas R.; Bardales, Ana T.; Gillette, Phillip R.; Lara, Monica R.; Schmale, Michael C.; Serafy, Joseph E.

    2009-01-01

    Over the last three decades, the California sea hare, Aplysia californica, has played an increasingly important role as a model organism in the neurosciences. Since 1995, the National Resource for Aplysia has supported a growing research community by providing a consistent supply of laboratory-reared individuals of known age, reproductive status, and environmental history. The purpose of the present study was to resolve the key biological factors necessary for successful culture of large numbers of high quality larval Aplysia. Data from a sequence of five experiments demonstrated that algal diet, food concentration, and veliger density significantly affected growth, attainment of metamorphic competency, and survival of Aplysia larvae. The highest growth and survival were achieved with a mixed algal diet of 1:1 Isochrysis sp (TISO) and Chaetoceros muelleri (CHGRA) at a total concentration of 250 x 103 cells/mL and a larval density of 0.5 – 1.0 per mL. Rapid growth was always correlated with faster attainment of developmental milestones and increased survival, indicating that the more rapidly growing larvae were healthier. Trials conducted with our improved protocol resulted in larval growth rates of >14 μm/d, which yielded metamorphically competent animals within 21 days with survival rates in excess of 90%. These data indicate the important effects of biotic factors on the critical larval growth period in the laboratory and show the advantages of developing optimized protocols for culture of such marine invertebrates. PMID:19000779

  20. Stereoselective L-[3H]quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors.

    PubMed

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

    1985-12-01

    The muscarinic antagonist L-[3H]quinuclidinyl benzilate (L-[3H]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-[3H]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-[3H]QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-[3H]QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-[3H]QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-[3H]QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-[3H]QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-[3H]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. PMID:4078624

  1. Taste-mediated behavioral and electrophysiological responses by the predatory fish Ariopsis felis to deterrent pigments from Aplysia californica ink.

    PubMed

    Nusnbaum, Matthew; Aggio, Juan F; Derby, Charles D

    2012-04-01

    Chemical defenses are used by many organisms to avoid predation, and these defenses may function by stimulating predators' chemosensory systems. Our study examined detection mechanisms for components of defensive ink of sea hares, Aplysia californica, by predatory sea catfish, Ariopsis felis. Behavioral analyses show aplysioviolin and phycoerythrobilin are detected intra-orally and by barbels and are deterrent at concentrations as low as 0.1% full strength. We performed electrophysiological recordings from the facial-trigeminal nerve complex innervating the maxillary barbel and tested aplysioviolin, phycoerythrobilin, amino acids, and bile salts in cross-adaptation experiments. Amino acids and bile salts are known stimulatory compounds for teleost taste systems. Our results show aplysioviolin and phycoerythrobilin are equally stimulatory and completely cross-adapt to each other's responses. Adaptation to aplysioviolin or phycoerythrobilin reduced but did not eliminate responses to amino acids or bile salts. Adaptation to amino acids or bile salts incompletely reduced responses to aplysioviolin or phycoerythrobilin. The fact that cross-adaptations with aplysioviolin and phycoerythrobilin were not completely reciprocal indicates there are amino acid and bile salt sensitive fibers insensitive to aplysioviolin and phycoerythrobilin. These results indicate two gustatory pathways for aplysioviolin and phycoerythrobilin: one independent of amino acids and bile salts and another shared with some amino acids. PMID:22200975

  2. 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.

  3. 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. PMID:26327369

  4. 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.

  5. 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. PMID:23530959

  6. [Effect of abiotic elicitation on the sanguinarine production and polyphenol oxidase activity in the suspension culture of Eschscholtzia californica CHAM].

    PubMed

    Bilka, František; Balážová, Andrea; Bilková, Andrea; Holková, Ivana

    2013-08-01

    Elicitation of plant in vitro cultures represents a biotechnological tool to improve the production of secondary metabolites. In this study, the effect of AgNO3 and CdCl2 on the sanguinarine production by the suspension culture of Eschscholtzia californica CHAM. was investigated. Elicitors were added to the cultures at the 14th day of subcultivation and their effect on the sanguinarine production was evaluated after a 48 h exposure. AgNO3 at the concentration of 0.075 mmol.l-1 and CdCl2 at the concentration of 4 mmol.l-1 induced a ca. 5.2- and 5.6-multiple increase in sanguinarine synthesis, respectively. This amount represents probably the maximal production, because a further increase in the elicitors concentrations did not increase sanguinarine production. Both abiotic elicitors induced a polyphenol oxidase specific activity increase. Polyphenol oxidase is probably involved in the biosynthesis of sanguinarine at the level of dopamine formation. Dopamine is a precursor of (S)-norcoclaurine, the first intermediate with the benzylisoquinoline structure. PMID:24047145

  7. New insights on the molecular recognition of imidacloprid with Aplysia californica AChBP: a computational study.

    PubMed

    Cerón-Carrasco, José P; Jacquemin, Denis; Graton, Jérôme; Thany, Steeve; Le Questel, Jean-Yves

    2013-04-18

    The binding of imidacloprid (IMI), the forerunner of neonicotinoid insecticides, with the acetylcholine binding protein (AChBP) from Aplysia californica, the established model for the extracellular domain of insects nicotinic acetylcholine receptors, has been studied with a two-layer ONIOM partition approach (M06-2X/6-311G(d):PM6). Our calculations allow delineating the contributions of the key residues of AChBP for IMI binding. In particular, the importance of Trp147 and Cys190-191, through weak CH···π interactions and both van der Waals and hydrogen-bond (H-bond) interactions, respectively, are highlighted. Furthermore, H-bonds between hydroxyl groups of both Ser189 and Tyr55 and the IMI nitro group are pointed out. The participation of Ile118, whose main chain NH and carbonyl group are hydrogen-bonded with the IMI pyridinic nitrogen through a water molecule, is characterized. Our simulations also indicate the presence of a significant contribution of this residue through van der Waals interactions. The various trends obtained by the calculations of the pairwise interaction energies are confirmed through a complementary noncovalent interaction (NCI) analysis of selected IMI-AChBP amino acid pairs. Indeed, the contribution of a halogen-bond interaction between IMI and AChBP, recently proposed in the literature, is corroborated by our NCI analysis. PMID:23521537

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

    PubMed

    Weigl, Sophie; Brandt, Wolfgang; Langhammer, Renate; Roos, Werner

    2016-02-01

    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

  9. 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

  10. 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 100μM 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. PMID:26851639

  11. 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.

  12. 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

  13. Cloning of the non-neuronal intermediate filament protein of the gastropod Aplysia californica; identification of an amino acid residue essential for the IFA epitope.

    PubMed

    Riemer, D; Dodemont, H; Weber, K

    1991-12-01

    We describe the isolation and characterization of a full-length cDNA corresponding to the larger non-neuronal (nn) intermediate filament (IF) protein of the gastropod Aplysia californica. Comparison of the sequences of the nn-IF proteins from Aplysia californica and Helix aspersa shows a strong evolutionary drift. At a 72% sequence identity level, the IF proteins of Opisthobranchia and Pulmonata show a larger distance than vimentins from Xenopus and mammals. The sequence comparison of the two snail proteins provides an important step in understanding the epitope of the monoclonal antibody IFA mapped by previous studies to the consensus sequence at the carboxy-terminal end of the rod domain of IF proteins. We identify for the first time in a naturally occurring IF protein a single amino acid exchange which leads to the loss of the epitope. The consensus sequence YRKLLEGEE present in IFA-positive proteins such as the Helix IF protein is changed in the IFA-negative Aplysia protein only by the conservative substitution of the arginine (R) by a lysine (K). Thus, the IFA epitope is not a necessity of IF structure, and its presence or absence on different IF proteins reflects only small changes in an otherwise conserved consensus sequence. Consequently, lack of IFA reactivity does not exclude the presence of IF. This result predicts that IF are much more universally expressed in lower eukaryotes than currently expected from immunological results with the monoclonal antibody IFA. PMID:1724961

  14. How to produce a chemical defense: structural elucidation and anatomical distribution of aplysioviolin and phycoerythrobilin in the sea hare Aplysia californica.

    PubMed

    Kamio, Michiya; Nguyen, Linh; Yaldiz, Seymanur; Derby, Charles D

    2010-05-01

    We previously used bioassay-guided fractionation to identify phycoerythrobilin (1) and its monomethyl ester, aplysioviolin (2), as components in the ink secretion of a marine gastropod, the sea hare Aplysia californica, that act as chemical deterrents against predatory blue crabs. This was the first report of 1 as a natural product. Compound 2 was previously reported as a natural product from three species of Aplysia (A. fasciata, A. dactylomela, and A. parvula), but the reported structure and composition of stereoisomers of 2 are different among these species. Sea hares are thought to produce 2 from phycoerythrin, a photosynthetic pigment in their red-algal diet composed of a phycobiliprotein covalently linked to the chromophore 1, by cleavage of the covalent bond and methylation of 1, but neither the sequence nor the anatomical location of the cleavage and methylation is known. In this study, we clarify the structure of 1 and 2 in ink secretion of A. californica, and describe the distribution of 1 and 2 in the tissues of sea hares. We conclude that cleavage of the covalent bond in phycoerythrin occurs first, forming 1 in the digestive gland, followed by methylation of 1 to yield 2 in the ink gland. PMID:20491075

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

    PubMed

    Gillette, Rhanor; Brown, Jeffrey W

    2015-12-01

    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 feeding

  16. 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. PMID:26743500

  17. 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

  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

    2015-01-01

    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

  19. 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

  20. Complete DNA sequence of the mitochondrial genome of the sea-slug, Aplysia californica: conservation of the gene order in Euthyneura.

    PubMed

    Knudsen, Bjarne; Kohn, Andrea B; Nahir, Ben; McFadden, Catherine S; Moroz, Leonid L

    2006-02-01

    We have sequenced and characterized the complete mitochondrial genome of the sea slug, Aplysia californica, an important model organism in experimental biology and a representative of Anaspidea (Opisthobranchia, Gastropoda). The mitochondrial genome of Aplysia is in the small end of the observed sizes of animal mitochondrial genomes (14,117 bp, NCBI Accession No. NC_005827). The Aplysia genome, like most other mitochondrial genomes, encodes genes for 2 ribosomal subunit RNAs (small and large rRNAs), 22 tRNAs, and 13 protein subunits (cytochrome c oxidase subunits 1-3, cytochrome b apoenzyme, ATP synthase subunits 6 and 8, and NADH dehydrogenase subunits 1-6 and 4L). The gene order is virtually identical between opisthobranchs and pulmonates, with the majority of differences arising from tRNA translocations. In contrast, the gene order from representatives of basal gastropods and other molluscan classes is significantly different from opisthobranchs and pulmonates. The Aplysia genome was compared to all other published molluscan mitochondrial genomes and phylogenetic analyses were carried out using a concatenated protein alignment. Phylogenetic analyses using maximum likelihood based analyses of the well aligned regions of the protein sequences support both monophyly of Euthyneura (a group including both the pulmonates and opisthobranchs) and Opisthobranchia (as a more derived group). The Aplysia mitochondrial genome sequenced here will serve as an important platform in both comparative and neurobiological studies using this model organism. PMID:16230032

  1. 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.

  2. 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

    2015-01-01

    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

  3. 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

  4. 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

  5. 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

    2014-01-01

    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

  6. Microclimate impacts survival and prevalence of Phytophthora ramorum in Umbellularia californica, a key reservoir host of sudden oak death in Northern California forests.

    PubMed

    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

  7. 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

  8. Application of high resolution NMR, ESR, and gamma-ray scintillation spectroscopy to the study of ligand binding in proteins. [Torpedo californica

    SciTech Connect

    Lancione, G.V.

    1982-01-01

    Electron spin resonance spectroscopy has been employed to study the nature of the ligand binding site of alpha-1-antitrypsin. Spectra of spin-labeled alpha-1-antitrypsin were recorded at pH's ranging from 2.4 to 12.5. This data demonstrates the tight binding of the spin-label to the protease, and the sensitivity of the bound spin-label to informational changes in the protease inhibitor. A molecular dipstick approach has also been applied to this system and has yielded information on the geometry of the cleft accommodating the spin-label. /sup 160/Terbium(III) exchange experiments have been performed on the acetylcholine receptor protein isolated from Torpedo californica, employing a specially designed flow dialysis apparatus constructed in the laboratory. The apparatus is designed to allow continuous monitoring of /sup 160/Tb(III) gamma-ray emission from the protein compartment of the flow dialysis cell. Nicotinic ligand-induced displacement of /sup 160/Tb(III) from the nicotinic binding site of the receptor was monitored as a funtion of (1) the concentration of nicotinic ligand in the washout buffer, and (2) the nature of the nicotinic ligand in the buffer. Measured /sup 160/Tb(III) exchange half-lives indicate (1) a direct relationship between /sup 160/Tb(III) displacement and nicotinic ligand concentration in the wash-out buffer, and (2) an enhanced /sup 160/Tb(III) displacement for nicotinic agents possessing quaternary ammonium functions.

  9. TRANSFECTION WITH BACULOVIRUS DNA

    EPA Science Inventory

    Purified DNA from the nuclear polyhedrosis viruses of Autographa californica (AcM NPV) and Rachiplusia ou (RoM NPV) were found to be infectious in TN-368 cells employing the calcium phosphate precipitation technique (F.L. Graham and A.J. van der Eb, Virology, 52, 456-467, 1973). ...

  10. Genomic sequence analysis of a nucleopolyhedrovirus isolated from the diamondback moth, Plutella xylostella.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The CL3 plaque isolate of Plutella xylostella multiple nucleopolyhedrovirus (PlxyMNPV-CL3) is a variant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) but exhibits a much higher degree of virulence against the diamondback moth, Plutella xylostella. To identify genetic differences ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Genomic sequence analysis of a nucleopolyhedrovirus isolated from the diamondback moth, Plutella xylostella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The CL3 plaque isolate of Plutella xylostella multiple nucleopolyhedrovirus (PlxyMNPV-CL3) exhibits a high degree of genetic similarity with the Autographa californica MNPV but is significantly more virulent against the diamondback moth, P. xylostella, than AcMNPV. To identify genetic differences b...

  13. 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

  14. 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

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

    2011-08-01

    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

  15. Highly Efficient Virus-induced Gene Silencing (VIGS) in California Poppy (Eschscholzia californica): An Evaluation of VIGS as a Strategy to Obtain Functional Data from Non-model Plants

    PubMed Central

    Wege, Stefanie; Scholz, Andrea; Gleissberg, Stefan; Becker, Annette

    2007-01-01

    Background and Aims Eschscholzia californica (California poppy) is an emerging model plant for ‘evo–devo’ studies from the basal eudicot clade of Papaveraceae. California poppy has a relatively small genome, a short life cycle and, most importantly, it is amenable for transformation. However, since this transformation protocol is time consuming, virus-induced gene silencing (VIGS) was evaluated as a fast method to obtain functional data for California poppy genes. Methods Commercially available California poppy plants were infiltrated with Agrobacterium tumefaciens carrying the tobacco rattle virus plasmids pTRV1 and pTRV2. pTRV2 contained part of the eschscholzia Phytoene Desaturase (EcPDS) gene whose loss of function results in photobleaching of the green parts of the plant and in a lack of floral coloration. The degree and duration of these symptoms was evaluated for vegetative rosettes and plants in flower. Key Results It is shown that VIGS is able to effectively down-regulate the EcPDS gene in eschscholzia. Various degrees of silencing were observed starting <2 weeks after infiltration with Agrobacterium tumefaciens in 92 % of the plants. Tissue with silencing symptoms also showed complete or strong reduction of EcPDS transcripts. Strong silencing resulted in almost completely white petals, fruits, shoots and leaves. Plants with a strong degree of silencing will eventually die off; however, others are able to produce EcPDS gene product even after a strong initial silencing and will recover. Silencing was found to be not always systemic, but was often restricted to certain organs or parts of organs. Conclusions VIGS is an effective, fast and transient method to down-regulate gene expression in eschscholzia. It serves well to detect prominent phenotypes which may become obvious even if some target gene transcript remains in the plant tissue. However, subtle phenotypes will be more difficult to detect, as extremely strong silencing effects occur in <10 % of

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. 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. PMID:22232273

  18. Viruses that ride on the coat-tails of actin nucleation.

    PubMed

    Newsome, Timothy P; Marzook, N Bishara

    2015-10-01

    Actin nucleation drives a diversity of critical cellular processes and the motility of a select group of viral pathogens. Vaccinia virus and baculovirus, Autographa californica multiple nucleopolyhedrovirus, recruit and activate the cellular actin nucleator, the Arp2/3 complex, at the surface of virus particles thereby instigating highly localized actin nucleation. The extension of these filaments provides a mechanical force that bestows the ability to navigate the intracellular environment and promote their infectious cycles. This review outlines the viral and cellular proteins that initiate and regulate the signalling networks leading to viral modification of the actin cytoskeleton and summarizes recent insights into the role of actin-based virus transport. PMID:26459972

  19. Regulation of expression of a baculovirus ecdysteroid UDPglucosyltransferase gene.

    PubMed Central

    O'Reilly, D R; Miller, L K

    1990-01-01

    The Autographa californica nuclear polyhedrosis virus egt gene encodes an ecdysteroid UDPglucosyltransferase which catalyzes the transfer of glucose from UDPglucose to ecdysteroid insect molting hormones. Expression of this gene allows the virus to block molting and pupation of infected insect larvae. In this study, we present the nucleotide sequence of the A. californica nuclear polyhedrosis virus egt gene and characterize egt gene expression at the transcriptional and translational levels. egt was transcribed as two 5'-coterminal mRNAs early in infection. Transferase activity was detected in infected cells and in the extracellular fluid by 3 h after infection. The majority of the activity accumulated in the extracellular fluid. We show that the egt gene product is a 60-kilodalton protein which is secreted from the infected cell. The egt gene is located in a region of the A. californica nuclear polyhedrosis virus genome which exhibited hypervariability in serially passaged virus stocks. Nucleotide sequence analysis revealed that the most common deletion occurring in these serially passaged virus isolates is located in the egt gene. Images PMID:2106039

  20. 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

  1. The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

    PubMed

    de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

    2015-11-01

    In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects. PMID:26323262

  2. Biologically active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein A.

    PubMed Central

    Andersons, D; Engström, A; Josephson, S; Hansson, L; Steiner, H

    1991-01-01

    A synthetic antibody-binding part derived from protein A from Staphylococcus aureus was used as a fusion partner in a eukaryotic expression system employing Autographa californica nuclear polyhedrosis as a vector. This, in conjunction with an efficient signal sequence, facilitated the purification of the antibacterial peptide cecropin A from the medium of Spodoptera frugiperda cells infected with a recombinant virus. In order to increase further the concentrations of fusion protein, Trichoplusia ni larvae were used as host. Cecropin A could be obtained after cleavage of the fusion protein with CNBr. Biological activity as well as the correct structure including the C-terminal amide group was shown using electrophoresis with detection of antibacterial proteins and mass spectroscopy. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1720614

  3. 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.

  4. 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.

  5. Secretion of green fluorescent protein from recombinant baculovirus-infected insect cells.

    PubMed

    Laukkanen, M L; Oker-Blom, C; Keinänen, K

    1996-09-24

    Trichoplusia ni (High Five) and Spodoptera frugiperda (Sf21) cells were engineered for expression of epitope (Flag)-tagged signal peptide-green fluorescent protein (GFP) fusions to examine the suitability of GFP as a secretory marker. The recombinant baculovirus-infected cells became fluorescent, and the High Five cells but not Sf21 cells secreted GFP in the culture medium as detected by the presence in the culture supernatant of a Flag-immunoreactive 30-kDa species and the characteristic 510-nm GFP fluorescence peak. Signal peptides derived from ecdysteroid UDP-glucosyltransferase of Autographa californica nuclear polyhedrosis virus and from rat brain glutamate receptor were both able to promote secretion of GFP. GFP may thus be used as a research tool in the study of the secretory process in insect cells both in cell biology and in biotechnological applications. PMID:8831686

  6. Phosphatase activity of Bombyx mori nucleopolyhedrovirus PTP is dispensable for enhanced locomotory activity in B. mori larvae.

    PubMed

    Katsuma, Susumu

    2015-11-01

    Baculovirus-induced enhanced locomotory activity (ELA) is not induced in caterpillars infected with a mutant Bombyx mori nucleopolyhedrovirus (BmNPV) or Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lacking a functional protein tyrosine phosphatase gene (ptp). Previous studies suggest that the PTP proteins from BmNPV and AcMNPV act in different ways to induce ELA, i.e., BmNPV PTP is utilized as a virion structural component, whereas AcMNPV PTP requires its phosphatase activity. Here, I generated and characterized two new BmNPV mutants expressing enzymatically inactive PTP proteins and confirmed that the phosphatase activity of PTP is not required for ELA induction in BmNPV-infected B. mori larvae. PMID:26550695

  7. 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. PMID:26498840

  8. Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system.

    PubMed

    Motohashi, Tomoko; Shimojima, Tsukasa; Fukagawa, Tatsuo; Maenaka, Katsumi; Park, Enoch Y

    2005-01-21

    Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. PMID:15596136

  9. Establishment of tetracycline-inducible gene expression systems in the silkworm, Bombyx mori.

    PubMed

    Karasaki, Noriko; Mon, Hiroaki; Takahashi, Masateru; Lee, Jae Man; Koga, Katsumi; Kawaguchi, Yutaka; Kusakabe, Takahiro

    2009-04-01

    Tetracycline-inducible gene expression (Tet-on) system has become one of the first choices for the control of transgenes expression in mammal and drosophila. However, the Tet-on systems that have been established in mammalian system or tuned into drosophila do not function in the silkworm, Bombyx mori. To construct a functional Tet-on system in B. mori, we modified rtTA by introducing a transcription activation domain of immediate-early gene 1 of Autographa californica nuclear polyhedrosis virus and nuclear localization signal of SV40 large T-antigen. The modified rtTA can activate the transcription from 9 x tetO promoter in the silkworm cells up to 250-fold in the presence of doxycycline. PMID:19066730

  10. 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. PMID:20005643

  11. The heptad repeats region is essential for AcMNPV P10 filament formation and not the proline-rich or the C-terminus basic regions

    SciTech Connect

    Dong Chunsheng; Deng Fei; Li Dan; Wang Hualin; Hu Zhihong

    2007-09-01

    Baculovirus P10 protein is a small conserved protein and is expressed as bundles of filaments in the host cell during the late phase of virus infection. So far the published results on the domain responsible for filament structural formation have been contradictory. Electron microscopy revealed that the C-terminus basic region was involved in filament structural formation in the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) [van Oers, M.M., Flipsen, J.T., Reusken, C.B., Sliwinsky, E.L., Vlak, J.M., 1993. Functional domains of the p10 protein of Autographa californica nuclear polyhedorsis virus. J. Gen. Virol. 74, 563-574.]. While in the Helicoverpa armigera nucleopolyhedrovirus (HearNPV), the heptad repeats region but not the C-terminus domain was proven to be responsible for filament formation [Dong, C., Li, D., Long, G., Deng, F., Wang, H., Hu, Z., 2005. Identification of functional domains required for HearNPV P10 filament formation. Virology 338, 112-120.]. In this manuscript, fluorescence confocal microscopy was applied to study AcMNPV P10 filament formation. A set of plasmids containing different P10 structural domains fused with a fluorescent protein were constructed and transfected into Sf-9 cells. The data indicated that the heptad repeats region, but not the proline-rich region or the C-terminus basic region, is essential for AcMNPV P10 filament formation. Co-transfection of P10s tagged with different fluorescent revealed that P10s with defective heptad repeats region could not interact with intact heptad repeats region or even full-length P10s to form filament structure. Within the heptad repeats region, deletion of the three amino acids spacing of AcMNPV P10 appeared to have no significant impact on the formation of filament structures, but the content of the heptad repeats region appeared to play a role in the morphology of the filaments.

  12. Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: comparative neutralization comparative infectivity, and in vitro growth studies.

    PubMed Central

    Volkman, L E; Summers, M D; Hsieh, C H

    1976-01-01

    Nuclear polyhedrosis virus infections of lepidopteran cells often result in the production of both occluded and nonoccluded virus. The characterization of these two different forms has been the subject of several papers. We have divided the nonoccluded virus (NOV) category further into plasma membrane-budded non-occluded virus (PMB-NOV), intracellular NOV, and hemolymph-derived NOV, and have done additional studies investigating the differences between these nonoccluded forms and the alkali-liberated forms from occlusions of the nuclear polyhedrosis viruses of Autographa californica and Rachiplusa ou. The methods used to discern differences and similarities among the forms were serological, biochemical, and visual, all related to their biological acitivity. Neutralization studies revealed that alkali-liberated virus and PMB-NOV had both similar and different antigens. Antisera raised against alkali-liberated virus from occlusions neutralized the alkali-liberated form of the virus, but did not neutralize the intracellular or extracellular nonoccluded forms. Antisera raised against the TN-368-13 PMB-NOV, however, neutralized the alkali-liberated forms as well as all forms of the NOV. Adsorption of this antisera with alkali-liberated virus did not diminish the neutralization titer against the nonoccluded forms, thus confirming the antigenic differences between the alkali-liberated and nonoccluded forms of the virus. Physical-infectious particle ratio calculations indicated that the PMB-NOV of Autographa californica are about 1,900-fold more infectious than the single-nucleocapsid-per-envelope alkali-liberated particles and about 1,700-fold more infectious than the multiple-nucleocapsid-per-envelope particles, as assayed in vitro. In addition, a study of viral growth kinetics monitored concurrently with the appearance of polyhedra showed that PMB-NOV production is shut down with the onset of polyhedron formation. PMID:787558

  13. Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene.

    PubMed Central

    Guarino, L A; Summers, M D

    1986-01-01

    The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1. Images PMID:3944847

  14. Reexamination of the gill withdrawal reflex of Aplysia californica Cooper (Gastropoda; Opisthobranchia).

    PubMed

    Leonard, J L; Edstrom, J; Lukowiak, K

    1989-06-01

    The gill withdrawal reflex (GWR), an important model system for neural mechanisms of learning, varies in form and amplitude within as well as between preparations and is therefore a heterogeneous collection of action patterns, not a reflex. At least 4 action patterns occur in response to mechanical stimulation of the siphon. It is often impossible to categorize a particular movement unambiguously. All may occur spontaneously. Gill movements may be described as combinations of 10 actions; 4 involving vein movements are described here. All actions and action patterns can occur in preparations lacking the central nervous system. Some vein movements may generate considerable force without markedly altering gill area. It is suggested that this explains why some early studies failed to identify the important role of the peripheral nervous system in the GWR. Studies based on the assumption that the GWR involves a single type of movement controlled by cells of the parietovisceral ganglion require reevaluation. PMID:2544202

  15. Connecting model species to nature: predator-induced long-term sensitization in Aplysia californica

    PubMed Central

    Mason, Maria J.; Watkins, Amanda J.; Wakabayashi, Jordann; Buechler, Jennifer; Pepino, Christine; Brown, Michelle

    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 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. PMID:25028394

  16. Peptide cotransmitter release from motorneuron B16 in aplysia californica: costorage, corelease, and functional implications.

    PubMed

    Vilim, F S; Cropper, E C; Price, D A; Kupfermann, I; Weiss, K R

    2000-03-01

    Many neurons contain multiple peptide cotransmitters in addition to their classical transmitters. We are using the accessory radula closer neuromuscular system of Aplysia, which participates in feeding in these animals, to define the possible consequences of multiple modulators converging on single targets. How these modulators are released onto their targets is of critical importance in understanding the outcomes of their modulatory actions and their physiological role. Here we provide direct evidence that the partially antagonistic families of modulatory peptides, the myomodulins and buccalins, synthesized by motorneuron B16 are costored and coreleased in fixed ratios. We show that this release is calcium-dependent and independent of muscle contraction. Furthermore, we show that peptide release is initiated at the low end of the physiological range of motorneuron firing frequency and that it increases with increasing motorneuron firing frequency. The coordination of peptide release with the normal operating range of a neuron may be a general phenomenon and suggests that the release of peptide cotransmitters may exhibit similar types of regulation and plasticity as have been observed for classical transmitters. Stimulation paradigms that increase muscle contraction amplitude or frequency also increase peptide release from motor neuron B16. The net effect of the modulatory peptide cotransmitters released from motorneuron B16 would be to increase relaxation rate and therefore allow more frequent and/or larger contractions to occur without increased resistance to antagonist muscles. The end result of this modulation could be to maximize the efficiency of feeding. PMID:10684904

  17. 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.

  18. 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. PMID:26903863

  19. 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. PMID:25028394

  20. 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…

  1. 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,…

  2. The Use of California Sagebrush (Artemisia californica) Liniment to Control Pain

    PubMed Central

    Adams, James D.

    2012-01-01

    The incidence of arthritis is increasing every year, as does the need for pain medication. The current work reviews an American Indian liniment that is traditionally used for pain therapy. The chemistry, therapeutic use and safety of the liniment are reviewed. The liniment contains monoterpenoids, sesquiterpenes, flavonoids, alkaloids and other compounds. PMID:24281255

  3. Habituation in the Tail Withdrawal Reflex Circuit is Impaired During Aging in Aplysia californica

    PubMed Central

    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. PMID:26903863

  4. Channel properties of the purified acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayer membranes.

    PubMed Central

    Montal, M; Labarca, P; Fredkin, D R; Suarez-Isla, B A

    1984-01-01

    The electrophysiological properties of the cation channel of the purified nicotinic acetylcholine receptor (AChR) reconstituted in planar lipid bilayers were characterized. Single-channel currents were activated by acetylcholine, carbamylcholine and suberyldicholine. The single channel conductance (28 pS in 0.3 M NaCl) was ohmic and independent of the agonist. Single channel currents increased with Na+ concentration to a maximum conductance of 95 pS and showed a half-saturation point of 395 mM. The apparent ion selectivity sequence, derived from single-channel current recordings, is: NH+4 greater than Cs+ greater than Rb+ greater than or equal to Na+ Cl-, F-, SO2-(4). The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of at least two distinct open states. The time constants depend on the choice of agonist, being consistently longer for suberyldicholine than for carbamylcholine. Similar channel properties were recorded in bilayers formed from monolayers at the tip of patch pipets . Single-channel currents occur in paroxysms of channel activity followed by quiescent periods. This pattern is more pronounced as the agonist concentration increases, and is reflected in histograms of channel-opening frequencies. Computer simulations with a three-state model, consisting of two closed (unliganded and liganded) and one open state, do not resemble the recorded pattern of channel activity, especially at high agonist concentration. Inclusion of a desensitized liganded state reproduces the qualitative features of channel recordings. The occurrence of paroxysms of channel activity thus seems to result from the transit of AChR through its active conformation, from which it can open several times before desensitizing. PMID:6324900

  5. 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.

  6. 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

  7. Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

    PubMed

    Kompier, R; Kislev, N; Segal, I; Kadouri, A

    1991-10-01

    Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells. PMID:1367637

  8. A simple plasmid-based transient gene expression method using High Five cells.

    PubMed

    Shen, Xiao; Pitol, Ana K; Bachmann, Virginie; Hacker, David L; Baldi, Lucia; Wurm, Florian M

    2015-12-20

    The High Five (H5) cell line, derived from the lepidopteran Trichoplusia ni, is one of the major insect cell hosts for the production of recombinant proteins using the baculovirus expression vector system (BEVS). Here, we describe a simple polyethylenimine (PEI)-based transient gene expression (TGE) process for the rapid production of recombinant proteins from suspension-adapted H5 cells. The method was optimized using two model proteins, enhanced green fluorescent protein (EGFP) and human tumor necrosis factor receptor-Fc fusion protein (TNFR-Fc). After screening several promoter and enhancer combinations for high levels of TNFR:Fc production, an expression vector containing the Autographa californica multicapsid nucleopolyhedrovirus immediate early 1 (ie1) promoter and homologous region 5 (hr5) enhancer was selected. Cells were transfected at a density of 2×10(6) cells/mL by direct addition of DNA and PEI. Under optimized conditions, a 90% transfection efficiency (percentage of EGFP-positive cells) was obtained. In addition, we observed volumetric TNFR-Fc yields over 150μg/mL within 4 days of transfection. The method was found to be reproducible and scalable to 300mL. This plasmid-based transient transfection process is a simple and efficient alternative to the BEVS for recombinant protein production in H5 cells. PMID:26476358

  9. Infection, transfection, and co-transfection of baculoviruses by microprojectile bombardment of larvae.

    PubMed

    Obregón-Barboza, Verónica; Del Rincón-Castro, Ma Cristina; Cabrera-Ponce, José L; Ibarra, Jorge E

    2007-03-01

    The use of baculoviruses as expression vectors for heterologous proteins has been practically limited to the use of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this work, infection, transfection and co-transfection events with the baculoviruses AcMNPV and Trichoplusia ni granulovirus (TnGV) were accomplished by bombardment of T. ni first-instar larvae with microprojectiles coated with virions, viral DNA, and viral DNA and a transfer vector, respectively. A series of shooting conditions were tested until positive results were obtained. The use of 1.6 microm gold particles at 900 psi shooting pressure, 400 Torr vacuum, 7 cm distance to target, on sets of 20 first-instar larvae held in a 16 mm diameter container, proved to be the best shooting conditions. Typical infection symptoms were shown by larvae when shot with viruses or viral DNA from AcMNPV or TnGV. Co-transfected recombinant AcMNPV and TnGV were identified by the formation of occlusion bodies and GFP, respectively, in bombarded larvae. This technique opens a wide range of possibilities, not only to use an extensive number of baculoviruses as expression vectors for heterologous proteins, but also be used to infect, transfect or co-transfect a wide variety of viruses into animal cells. PMID:17184851

  10. Eri silkworm (Samia ricini), a non-mulberry host system for AcMNPV mediated expression of recombinant proteins.

    PubMed

    Hosamani, Madhusudan; Basagoudanavar, Suresh H; Sreenivasa, B P; Inumaru, Shigeki; Ballal, Chandish R; Venkataramanan, Ramamurthy

    2015-12-20

    The baculovirus expression system (BVES) based on Autographa californica nucleopolyhedrovirus (AcMNPV) is widely used for the expression of eukaryotic proteins. Several insect cells/larvae that are permissive to AcMNPV have been routinely used as hosts to express heterologous proteins. Domesticated Eri silkworm (Samia ricini), reared in many parts of India, Japan and China, is a non-mulberry silkworm. The present study shows that the Eri silkworm larvae are susceptible to intra-haemocoelical inoculation of AcMNPV. The virus replicates in the larva, as indicated by an increased viral loads in the haemolymph upon injection of a recombinant AcMNPV carrying green fluorescent protein gene. The virus showed localized replication in different tissues including the fat body, haemocytes, tracheal matrix and in the Malphigian tubules. The larval system was successfully used to express heterologous protein, by infecting with a recombinant AcMNPV carrying the 3ABC coding sequence of foot-and-mouth disease virus (FMDV). The study shows that the Eri silkworm larva can be a potential alternative bioreactor, for scaling up of the recombinant proteins employing the baculovirus system. PMID:26467714

  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. PMID:22110386

  12. Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector.

    PubMed

    Urakawa, T; Ferguson, M; Minor, P D; Cooper, J; Sullivan, M; Almond, J W; Bishop, D H

    1989-06-01

    A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected insect cells made poliovirus proteins that included the structural proteins VP0, VP1 and VP3. Poliovirus particles were recovered from extracts of the infected cells and demonstrated to be free from detectable levels of RNA and to be non-infectious in tissue culture. After particle purification by CsCl gradient centrifugation and immunization of outbred mice, antibodies to the structural proteins, including neutralizing antibodies, were obtained. Other recombinant baculoviruses, containing the majority of the capsid coding region of P3/Leon/37 (e.g. AcCAP21, nucleotide residues 742 to 3318), made an unprocessed precursor to the poliovirus structural proteins. These data suggested that processing of the poliovirus gene product by the AcLeon construct was catalysed by the poliovirus-encoded proteases. The results demonstrated that antigenic and immunogenic poliovirus proteins and empty particles can be made in insect cells by recombinant baculoviruses. PMID:2543785

  13. Specificity of Baculovirus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation▿ †

    PubMed Central

    Wang, Manli; Tuladhar, Era; Shen, Shu; Wang, Hualin; van Oers, Monique M.; Vlak, Just M.; Westenberg, Marcel

    2010-01-01

    The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9-null mutant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alphabaculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that AcMNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9-null AcMNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly. PMID:20519380

  14. The Lymantria dispar nucleopolyhedrovirus contains the capsid-associated p24 protein gene.

    PubMed

    Slavicek, James M; Hayes-Plazolles, Nancy

    2003-01-01

    During the course of investigations on a wild-type strain of Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV), a region of the viral genome was analyzed and found to contain 697 bp that is lacking in the sequenced strain (5-6) of LdMNPV (Kuzio et al., Virology 253, 17-34, 1999). The sequenced strain of LdMNPV contains a mutation in the 25 K few polyhedra (FP) gene, and exhibits the phenotype of a FP mutant. The additional sequence was located at approximately 81.4 map units within the viral genome, and was found in 10 different wild-type LdMNPV genotypic variants analyzed. Since the additional sequence wasfound in all wild-type virus strains analyzed, this sequence should be included in the representative LdMNPV genome. Sequence analysis of the genomic region containing the additional sequences revealed the presence of a homologue of the Autographa californica MNPV capsid-associated p24 gene (ORF 129). This gene, absent in LdMNPV isolate 5-6, is also present in the Orgyia pseudotsugata MNPV, Bombyx mori NPV, Spodoptera exigua MNPV, S. litura MNPV, Mamestra configurata MNPV, Helicoverpa armigera SNPV, H. zea SNPV, Buzura suppressaria SNPV, Xestia c-nigrum granulovirus, Plutella xylostella GV, and Cydia pomonella GV. PMID:12680688

  15. Control of programmed cell death by the baculovirus genes p35 and iap.

    PubMed Central

    Clem, R J; Miller, L K

    1994-01-01

    The SF-21 insect cell line undergoes rapid and widespread apoptosis when treated with actinomycin D or when infected with a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus lacking a p35 gene or a functionally active iap (inhibitor of apoptosis) gene. Here we provide evidence that the basis for the induction of apoptosis by these two different stimuli is the cessation of RNA synthesis. We also show that expression of either p35 or two different functional iap homologs blocks apoptosis independently of other viral genes, indicating that these gene products act directly on the cellular apoptotic pathway. The iap genes encode a C3HC4 (or RING) finger motif found in a number of transcriptional regulatory proteins, as well as two additional Cys/His motifs (baculovirus iap repeats). We show that specific amino acids within both the C3HC4 finger and the N-terminal baculovirus iap repeat are critical for anti-apoptosis function. Overexpression of either mammalian bcl-2 or adenovirus E1B-19K, genes which block apoptosis when overexpressed in a number of mammalian cells, does not block actinomycin D-induced apoptosis in SF-21 cells. Images PMID:8035800

  16. Characterization of an Egyptian Spodoptera littoralis nucleopolyhedrovirus and a possible use of a highly conserved region from polyhedrin gene for nucleopolyhedrovirus detection

    PubMed Central

    Seufi, AlaaEddeen M

    2008-01-01

    An Egyptian isolate of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was tested for its potential as biocontrol agent in comparison to Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Comparative assays of SpliNPV and AcMNPV against 2nd instar larvae of Spodoptera littoralis revealed 4-fold greater susceptibility of S. littoralis to AcMNPV than to SpliNPV based on LC50 values for the two viruses. The LT50s determined for SpliNPV and AcMNPV using LC50 of the virus against 2nd instar larvae were 4.2 and 5.8 days, respectively. A DNA segment of 405 bp containing highly conserved region from polyhedrin gene of SpliNPV (Polh-cr) was successfully amplified by PCR. Subsequently, this DNA segment was cloned and sequenced. Nucleotide sequence and its deduced amino acid sequence were compared to all available sequences in GenBank. Sequence alignment results revealed that Polh-cr showed significant similarities with 91 different baculovirus isolates. The percentage of homology ranged from 78% for Plusia orichalcea NPV to 99% for SpliNPV. This highly conserved region provides a candidate that could be used in easy, fast and economic prospective systems for virus detection as well as in biological control strategies. PMID:18215282

  17. Comparative Studies of Lepidopteran Baculovirus-Specific Protein FP25K: Development of a Novel Bombyx mori Nucleopolyhedrovirus-Based Vector with a Modified fp25K Gene▿

    PubMed Central

    Nakanishi, Tadashi; Goto, Chie; Kobayashi, Michihiro; Kang, WonKyung; Suzuki, Takehiro; Dohmae, Naoshi; Matsumoto, Shogo; Shimada, Toru; Katsuma, Susumu

    2010-01-01

    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. PMID:20219904

  18. A baculovirus anti-apoptosis gene homolog of the Trichoplusia ni granulovirus.

    PubMed

    Bideshi, D K; Anwar, A T; Federici, B A

    1999-01-01

    An inhibitor of apoptosis (iap) gene homolog (Tn-iap) of the Trichoplusia ni granulovirus (TnGV) was cloned, sequenced and mapped on the genome of TnGV. Tn-iap encoded a protein (Tn-IAP) of 301 amino acids with a predicted molecular mass of 35 kDa. The Tn-IAP contained the two sequence motifs, BIRs and RING finger, characteristic of IAP proteins, and shared identities of 21-27% and similarities of 28-53% with IAP proteins of Cydia pomonella GV (Cp-IAP), Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus (MNPV) (Op-IAP1, 3), Autographa californica MNPV (Ac-IAP1), Bombyx mori NPV (Bm-IAP1), Lymantria dispar MNPV (Ld-IAP3) and Buzura suppressaria single nucleocapsid NPV (Bs-IAP1). However, Tn-IAP shared no significant homology with baculovirus IAP2 proteins. Using an antisense Tn-iap probe, two major transcripts of approximately 800 nt and 1600 nt were detected by Northern blot analysis of RNA extracted from the fat body of T. ni larvae infected with the TnGV. Unlike Cp-IAP and Op-IAP3, however, Tn-IAP did not rescue virion occlusion in SF21 cells infected with a p35-deficient AcMNPV mutant. Tn-IAP's synthesis in vivo but failure to rescue p35-deficient AcMNPV in SF21 cells suggests it is a functional IAP that is only effective in certain cell types. PMID:10541013

  19. Genome sequence of Perigonia lusca single nucleopolyhedrovirus: insights into the evolution of a nucleotide metabolism enzyme in the family Baculoviridae.

    PubMed

    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. A baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper.

    PubMed Central

    Thiem, S M; Miller, L K

    1989-01-01

    An Autographa californica nuclear polyhedrosis virus gene encoding a 30-kilodalton polypeptide with two different sequence motifs characteristic of DNA-binding proteins was identified immediately downstream of the major capsid protein gene (vp39). The gene, CG30, was characterized by sequencing, transcriptional mapping, in vitro translation of hybrid-selected RNA, and comparison of the derived polypeptide sequence with published data bases. The initial ATG of the 792-base-pair CG30 open reading frame is two nucleotides downstream of the vp39 terminal TAA codon. Early transcripts of CG30 initiate within the vp39 coding sequence. At late times, bicistronic transcripts initiate from the vp39 promoter, continue through CG30, and terminate at the same site as the early transcripts. In vitro translation of hybrid-selected early CG30 RNA yields a polypeptide of 30 kilodaltons. The predicted CG30 polypeptide sequence has characteristics of a eucaryotic transcriptional activator and is novel in having two potential DNA-binding domains. A stretch of acidic residues bridges a zinc finger at the amino terminus and a leucine zipper with a flanking basic region at the carboxyl terminus. Images PMID:2507791

  1. Identification and functional analysis of LsMNPV anti-apoptosis genes.

    PubMed

    Kim, Yu-Sin; Xiao, Hua-Zhong; Du, En-Qi; Cai, Guo-Shuai; Lu, Song-Ya; Qi, Yi-Peng

    2007-07-31

    Three anti-apoptosis genes, Ls-iap2, iap3 and p49 were found in Leucania separata multiple nuclear polyhedrovirus. Amino acid sequence homology of Ls-IAP2 and Ls-IAP3 with Op-IAP2 and Op-IAP3 from Orgyia pseddotsugata MNPV were 20% and 42%, while that of Ls-P49 is 28% with Sl-P49 from Spodoptera littorolis MNPV. Ls-IAP2 contains one baculoviral IAP repeat (BIR) domain followed by a RING domain, while Ls-IAP3 contains two BIRs and a RING. Ls-P49 contains a reactive site loop, predicted cleavage site (KKLD(74) downward arrow G) that is different from Sl-P49 (TVID(94) downward arrow G). Expressed Ls-iap3 or Ls-p49 under presence of actinomycin D in SF9 cells, DNA ladder assay revealed that Ls- IAP3 or Ls-P49 could block the apoptosis of SF9 cells induced by actinomycin D. Replication of p35 deficient-mutant Autographa californica MNPV in SF9 cells was also rescued when Ls-iap3 or Ls-p49 was expressed transiently. No anti-apoptotic activity was observed for Ls-IAP2. The results showed that both of Ls-IAP3 and Ls-P49 were functional apoptotic suppressors in SF9 cells. PMID:17669274

  2. A Role for the Anti-Viral Host Defense Mechanism in the Phylogenetic Divergence in Baculovirus Evolution.

    PubMed

    Nagamine, Toshihiro; Sako, Yasushi

    2016-01-01

    Although phylogenic analysis often suggests co-evolutionary relationships between viruses and host organisms, few examples have been reported at the microevolutionary level. Here, we show a possible example in which a species-specific anti-viral response may drive phylogenic divergence in insect virus evolution. Two baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV), have a high degree of DNA sequence similarity, but exhibit non-overlapping host specificity. In our study of their host-range determination, we found that BmNPV replication in B. mori cells was prevented by AcMNPV-P143 (AcP143), but not BmNPV-P143 (BmP143) or a hybrid P143 protein from a host-range expanded phenotype. This suggests that AcMNPV resistance in B. mori cells depends on AcP143 recognition and that BmNPV uses BmP143 to escapes this recognition. Based on these data, we propose an insect-baculovirus co-evolution scenario in which an ancestor of silkworms exploited an AcMNPV-resistant mechanism; AcMNPV counteracted this resistance via P143 mutations, resulting in the birth of BmNPV. PMID:27244571

  3. 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.

  4. Purification and characterization of a viral chitinase active against plant pathogens and herbivores from transgenic tobacco.

    PubMed

    Di Maro, Antimo; Terracciano, Irma; Sticco, Lucia; Fiandra, Luisa; Ruocco, Michelina; Corrado, Giandomenico; Parente, Augusto; Rao, Rosa

    2010-05-01

    The Autographa californica nucleopolyhedrovirus chitinase A (AcMNPV ChiA) is a chitinolytic enzyme with fungicidal and insecticidal properties. Its expression in transgenic plants enhances resistance against pests and fungal pathogens. We exploited tobacco for the production of a biologically active recombinant AcMNPV ChiA (rChiA), as such species is an alternative to traditional biological systems for large-scale enzyme production. The protein was purified from leaves using ammonium sulfate precipitation followed by anion exchange and gel-filtration chromatography. Transgenic plants produced an estimated 14 mg kg(-1) fresh leaf weight, which represents 0.2% of total soluble proteins. The yield of the purification was about 14% (2 mg kg(-1) fresh leaf weight). The comparison between the biochemical and kinetic properties of the rChiA with those of a commercial Serratia marcescens chitinase A indicated that the rChiA was thermostable and more resistant at basic pH, two positive features for agricultural and industrial applications. Finally, we showed that the purified rChiA enhanced the permeability of the peritrophic membrane of larvae of two Lepidoptera (Bombyx mori and Heliothis virescens) and inhibited spore germination and growth of the phytopatogenic fungus Alternaria alternata. The data indicated that tobacco represents a suitable platform for the production of rChiA, an enzyme with interesting features for future applications as "eco-friendly" control agent in agriculture. PMID:20302895

  5. Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori.

    PubMed

    Huang, Q; Deveraux, Q L; Maeda, S; Stennicke, H R; Hammock, B D; Reed, J C

    2001-01-15

    We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP, suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase in the mitochondria/cytochrome-c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream initiator caspase in the conserved mitochondria/cytochrome-c pathway for apoptosis. PMID:11341966

  6. α-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

    PubMed Central

    Grula, Marjori A.; Buller, Patricia L.; Weaver, Robert F.

    1981-01-01

    [3H]RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of Heliothis zea larvae infected with H. zea nuclear polyhedrosis virus and from cultured Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [3H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from H. zea larval fat bodies and S. frugiperda cells was only inhibited 20 to 25% by concentrations of α-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in S. frugiperda cells grown in the presence of an α-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to α-amanitin during infection, and there was no evidence that a virus-coded, α-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II. PMID:16789208

  7. Differential requirements of two insect cell lines for growth in serum-free medium.

    PubMed

    Vaughn, J L; Fan, F

    1997-06-01

    The development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 x 10(6) TCID50 extracellular virus and 4.4 x 10(6) polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects. PMID:9201517

  8. Antiviral, immunomodulatory, and free radical scavenging activities of a protein-enriched fraction from the larvae of the housefly, Musca domestica.

    PubMed

    Ai, Hui; Wang, Furong; Zhang, Na; Zhang, Lingyao; Lei, Chaoliang

    2013-01-01

    In our previous study, protein-enriched fraction (PEF) that was isolated from the larvae of the housefly, Musca domestica L. (Diptera: Muscidae), showed excellent hepatoprotective activity as well as the potential for clinical application in therapy for liver diseases. In this study, antiviral, immunomodulatory, and free radical scavenging activities of PEF were evaluated. The antiviral results demonstrated that PEF inhibited the infection of avian influenza virus H9N2 and had a virucidal effect against the multicapsid nucleopolyhedrovirus of the alfalfa looper, Autographa californica Speyer (Lepidoptera: Noctuidae) in vitro. The mortality of silkworm larve in a PEF treatment group decreased significantly compared with a negative control. PEF showed excellent scavenging activity for 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radicals, which were similar to those of ascorbic acid. The imunomodulatory results suggested that PEF could effectively improve immune function in experimental mice. Our results indicated that PEF could possibly be used for the prophylaxis and treatment of diseases caused by avian influenza virus infection. In addition, PEF with virucidal activity against insect viruses might provide useful for the development of antimicrobial breeding technology for economically important insects. As a natural product from insects, PEF could be a potential source for the discovery of potent antioxidant and immunomodulatory agents. PMID:24735244

  9. 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.

  10. The utility of camptothecin as a synergist of Bacillus thuringiensis var. kurstaki and nucleopolyhedroviruses against Trichoplusia ni and Spodoptera exigua.

    PubMed

    Sun, Shifeng; Cheng, Zhongshan; Fan, Jing; Cheng, Xinghu; Pang, Yi

    2012-08-01

    We studied the effect of combining microbial pesticides with camptothecin (CPT) on the mortality of two lepidopteran insects: Trichoplusia ni (Hübner) and Spodoptera exigua (Hübner). CPT is an alkaloid that is often used as an anticancer agent. Here, CPT was evaluated as a microbial pesticide synergist of Bacillus thuringiensis (Bt) and insect baculovirus. The toxicity of CPT and its synergistic effects on two microbial pesticides were studied using the diet overlay method. Bioassay results showed that CPT significantly enhances the toxicity of Bt variety kurstaki to S. exigua and T ni. In addition, CPT strongly enhanced the infectivity of Autographa californica (Speyer) multinucleocapsid nucleopolyhedrovirus (AcMNPV) and S. exigua nucleopolyhedrovirus (SeMNPV). Using light microscopy, we found that CPT disrupts the peritrophic membrane of T. ni larvae and severely affects the structure of the midgut, resulting in an abnormal gut lumen morphology. We speculate that CPT increases toxicity by affecting the permeability of the peritrophic membrane. PMID:22928294

  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. PMID:27375973

  12. 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

  13. Gene organization and transcription of TED, a lepidopteran retrotransposon integrated within the baculovirus genome.

    PubMed Central

    Friesen, P D; Nissen, M S

    1990-01-01

    A single copy of the retrotransposon TED, from the moth Trichoplusia ni (a lepidopteran noctuid), was identified within the DNA genome of the baculovirus Autographa californica nuclear polyhedrosis virus. Determination of the complete nucleotide sequence (7,510 base pairs) of the integrated copy indicated that TED belongs to the family of retrotransposons that includes Drosophila melanogaster elements 17.6 and gypsy and thus represents the first nondipteran member of this invertebrate group to be identified. The internal portion of TED, flanked by long terminal repeats (LTRs), is composed of three long open reading frames comparable in size and location to the gag, pol, and env genes of the vertebrate retroviruses. Sequence similarity with the dipteran elements was the highest within individual domains of TED open reading frame 2 (pol region) that are also conserved among the retroviruses and encode protease, reverse transcriptase, and integrase functions, respectively. Mapping the 5' and 3' termini of TED RNAs indicated that the LTRs have a retroviral U3-R-U5 structural organization that is capable of directing the synthesis of transcripts that represent potential substrates for reverse transcription and intermediates in transposition. Abundant RNAs were also initiated from a site within the 5' LTR that matches the consensus motif for the promoter of late, hyperexpressed baculovirus genes. The presence of this viruslike promoter within TED and its subsequent activation only after integration within the viral genome suggest a possible symbiotic relationship with the baculovirus that could extend transposon host range. Images PMID:1692964

  14. Characterization of the viral fibroblast growth factor homolog of Helicoverpa armigera single nucleopolyhedrovirus.

    PubMed

    Yin, Feifei; Du, Ruikun; Kuang, Wenhua; Yang, Guang; Wang, Hualin; Deng, Fei; Hu, Zhihong; Wang, Manli

    2016-06-01

    Fibroblast growth factor (FGF) is found throughout multicellular organisms; however, fgf homologs (vfgf) have only been identified among viruses in lepidopteran baculoviruses. The function of vFGFs from Group I alphabaculoviruses, including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV), involves accelerated killing of infected larvae by both viruses. The vFGF of Group II alphabaculovirus is structurally different from that of Group I alphabaculovirus, with a larger C-terminal region and additional N-linked glycosylation sites. In this study, we characterized the Group II alphabaculovirus vFGF of Helicoverpa armigera single nucleopolyhedrovirus (HearNPV). The transcription and expression of vfgf was detected at 3 h and 16 h post-infection in HearNPV-infected cells. To further study vFGF function, we constructed vfgf-knockout and -repaired HearNPV bacmids and investigated their affect in both cultured cells and insects. Deletion of vfgf had no effect on budded-virus production or viral DNA replication in cultured HzAM1 cells. However, bioassays showed that HearNPV vfgf deletion significantly increased the median lethal dose and delayed the median lethal time by ∼12 h in the host insect when the virus was delivered orally. These results suggested that vFGF is an important virulent factor for HearNPV infection and propagation in vivo. PMID:27142667

  15. Immunomodulatory effect of baculovirus in chickens: How it modifies the immune response against infectious bursal disease virus.

    PubMed

    Chimeno Zoth, Silvina; Carballeda, Juan Manuel; Gravisaco, María José; Lucero, María Soledad; Richetta, Matías; Gómez, Evangelina; Berinstein, Analía

    2016-07-01

    Several reports have shown that baculoviruses (BVs) have strong adjuvant properties on the mammalian immune system. Recent studies of our group demonstrated the ability of BV to stimulate the innate immunity in chickens. In this investigation, we aimed to assess the potential antiviral effect of BV given both, before and after infectious bursal disease virus (IBDV). In the first case, specific pathogen free chickens were intravenously inoculated with 5 × 10(7) pfu of Autographa californica nuclear polyhedrosis virus and 3 h later were orally administered 2.5 × 10(5) egg infectious doses 50 of IBDV. In the second case, chickens received IBDV 3 h before BV inoculation. Five days later, chickens were bled and euthanized. RNA from the bursa was analyzed for cytokine production. Also, bursae were used for virus recovery, and processed for lymphocyte isolation. The results showed that the administration of BV 3 h after the inoculation with IBDV produced important changes in the effect that IBDV causes in the bursa. BV reduced the infiltration of T lymphocytes, decreased the expression pattern of IL-6 and IFN-γ and inhibited IBDV replication. The results herein presented demonstrate that this Lepidopteran virus shows antiviral activity in chickens under experimental conditions. Investigations under field conditions have to be done to probe this strategy as a valuable sanitary tool for the treatment and prevention of chicken diseases. PMID:27063861

  16. Insecticidal effects of Buthus occitanus tunetanus BotIT6 toxin expressed in Escherichia coli and baculovirus/insect cells.

    PubMed

    Bel Haj Rhouma, Rahima; Cérutti-Duonor, Martine; Benkhadir, Khadija; Goudey-Perrière, Françoise; El Ayeb, Mohamed; Lopez-Ferber, Miguel; Karoui, Habib

    2005-12-01

    BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV. PMID:16216259

  17. Baculovirus phosphoprotein pp31 is associated with virogenic stroma.

    PubMed Central

    Guarino, L A; Dong, W; Xu, B; Broussard, D R; Davis, R W; Jarvis, D L

    1992-01-01

    The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle. Images PMID:1433508

  18. 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.

  19. 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. PMID:25006078

  20. New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus.

    PubMed

    Regev, Avital; Rivkin, Hadassah; Gurevitz, Michael; Chejanovsky, Nor

    2006-12-22

    Baculoviruses are orally infectious to insects and considered to be natural insecticides. To enhance their speed-of-kill these viruses were engineered to express arthropod neurotoxins under the control of various strong promoters. Although this strategy proved to be efficient, it raised recently concerns about safety. We analyzed the speed-of-kill and safety of Autographa californica multiple nucleopolyhedrovirus expressing the insecticidal scorpion neurotoxin AaIT and found that the mortality of Helicoverpa armigera larvae was enhanced significantly when the expression was controlled by the baculovirus delayed-early promoter 39K rather than the very late promoter p10. This improvement was also reflected in better protection of cotton leaves on which these insects were fed. Using lacZ as a sensitive reporter we also found that expression driven by the 39K promoter was detected in insect but not in mammalian cells. These results imply that by selection of an appropriate viral promoter, engineered baculoviruses may comply with the high standard biosafety requirements from a genetically modified organism (GMO). Our results provide further support for the potential use of engineered baculoviruses in insect pest control in a safely manner. PMID:17141223

  1. 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

  2. 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

  3. Antiviral, Immunomodulatory, and Free Radical Scavenging Activities of a Protein-Enriched Fraction from the Larvae of the Housefly, Musca domestica

    PubMed Central

    Ai, Hui; Wang, Furong; Zhang, Na; Zhang, Lingyao; Lei, Chaoliang

    2013-01-01

    In our previous study, protein-enriched fraction (PEF) that was isolated from the larvae of the housefly, Musca domestica L. (Diptera: Muscidae), showed excellent hepatoprotective activity as well as the potential for clinical application in therapy for liver diseases. In this study, antiviral, immunomodulatory, and free radical scavenging activities of PEF were evaluated. The antiviral results demonstrated that PEF inhibited the infection of avian influenza virus H9N2 and had a virucidal effect against the multicapsid nucleopolyhedrovirus of the alfalfa looper, Autographa californica Speyer (Lepidoptera: Noctuidae) in vitro. The mortality of silkworm larve in a PEF treatment group decreased significantly compared with a negative control. PEF showed excellent scavenging activity for 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radicals, which were similar to those of ascorbic acid. The imunomodulatory results suggested that PEF could effectively improve immune function in experimental mice. Our results indicated that PEF could possibly be used for the prophylaxis and treatment of diseases caused by avian influenza virus infection. In addition, PEF with virucidal activity against insect viruses might provide useful for the development of antimicrobial breeding technology for economically important insects. As a natural product from insects, PEF could be a potential source for the discovery of potent antioxidant and immunomodulatory agents. PMID:24735244

  4. 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

  5. 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). PMID:24297430

  6. 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.

  7. A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

    PubMed

    Au, Shelly; Wu, Wei; Zhou, Lixin; Theilmann, David A; Panté, Nelly

    2016-08-01

    The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle. PMID:27284005

  8. 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.

  9. Recombinant Outer Capsid Glycoprotein (VP7) of Rotavirus Expressed in Insect Cells Induces Neutralizing Antibodies in Rabbits

    PubMed Central

    Khodabandehloo, M; Shahrabadi, M Shamsi; Keyvani, H; Bambai, B; Sadigh, ZA

    2012-01-01

    Background: Rotaviruses cause diarrhea in infants and young children worldwide. Rotavirus outer capsid protein, VP7 is major neutralizing antigen that is important component of subunit vaccine to prevent rotavirus infection. Many efforts have been done to produce recombinant VP7 that maintain native characteristics. We used baculovirus expression system to produce rotavirus VP7 protein and to study its immunogenicity. Methods: Simian rotavirus SA11 full-length VP7 ORF was cloned into a cloning plasmid and then the cloned gene was inserted into the linear DNA of baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) downstream of the polyhedrin promoter by in vitro recombination reactions. The expressed VP7 in the insect cells was recognized by rabbit hyperimmune serum raised against SA11 rotavirus by Immunofluorescence and western blotting assays. Rabbits were immunized subcutaneously by cell extracts expressing VP7 protein. Results: Reactivity with anti-rotavirus antibody suggested that expressed VP7 protein had native antigenic determinants. Injection of recombinant VP7 in rabbits elicited the production of serum antibodies, which were able to recognize VP7 protein from SA11 rotavirus by Western blotting test and neutralized SA11 rotavirus in cell culture. Conclusion: Recombinant outer capsid glycoprotein (VP7) of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits and may be a candidate of rotavirus vaccine. PMID:23113180

  10. Baculovirus Envelope Protein ODV-E66 Is a Novel Chondroitinase with Distinct Substrate Specificity*

    PubMed Central

    Sugiura, Nobuo; Setoyama, Yuka; Chiba, Mie; Kimata, Koji; Watanabe, Hideto

    2011-01-01

    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. PMID:21715327

  11. 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. PMID:25850946

  12. A Role for the Anti-Viral Host Defense Mechanism in the Phylogenetic Divergence in Baculovirus Evolution

    PubMed Central

    Nagamine, Toshihiro; Sako, Yasushi

    2016-01-01

    Although phylogenic analysis often suggests co-evolutionary relationships between viruses and host organisms, few examples have been reported at the microevolutionary level. Here, we show a possible example in which a species-specific anti-viral response may drive phylogenic divergence in insect virus evolution. Two baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV), have a high degree of DNA sequence similarity, but exhibit non-overlapping host specificity. In our study of their host-range determination, we found that BmNPV replication in B. mori cells was prevented by AcMNPV-P143 (AcP143), but not BmNPV-P143 (BmP143) or a hybrid P143 protein from a host-range expanded phenotype. This suggests that AcMNPV resistance in B. mori cells depends on AcP143 recognition and that BmNPV uses BmP143 to escapes this recognition. Based on these data, we propose an insect-baculovirus co-evolution scenario in which an ancestor of silkworms exploited an AcMNPV-resistant mechanism; AcMNPV counteracted this resistance via P143 mutations, resulting in the birth of BmNPV. PMID:27244571

  13. Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies.

    PubMed Central

    Meulenberg, J J; Bende, R J; Pol, J M; Wensvoort, G; Moormann, R J

    1995-01-01

    The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV. PMID:8574824

  14. 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

  15. 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.

  16. 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

  17. 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. PMID:27084925

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  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 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…

  1. Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells.

    PubMed

    Tran, Trinh T B; Dietmair, Stefanie; Chan, Leslie C L; Huynh, Hoai T; Nielsen, Lars K; Reid, Steven

    2012-03-01

    Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus-insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl+P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell-baculovirus systems for bioprocess optimization. PMID:22166686

  2. Transcriptional enhancer activity of hr5 requires dual-palindrome half sites that mediate binding of a dimeric form of the baculovirus transregulator IE1.

    PubMed

    Rodems, S M; Friesen, P D

    1995-09-01

    The hr5 enhancer element stimulates early viral transcription and may function as an origin of DNA replication for Autographa californica nuclear polyhedrosis virus (AcMNPV). The smallest functional unit of hr5 is a 28-bp repeat consisting of an imperfect palindrome (28-mer). To identify essential sequences and examine the molecular basis of hr5 activity, the effects of site-directed mutations on transcriptional enhancement by the 28-mer and binding of the AcMNPV transregulator IE1 were investigated. In transfection assays and infections with AcMNPV recombinants, activation of a basal viral promoter required sequences within both halves of the 28-mer. Basal promoter activation also required a critical spacing between these half sites. Mobility shift assays indicated that hr5 probes containing a single 28-mer were bound by in vitro-synthesized IE1. Competition assays using DNA fragments that contained mutated 28-mers demonstrated that both half sites were required for optimal binding of IE1. Similar assays using mutated 28-mer DNAs and nuclear extracts indicated that the relative affinity with which AcMNPV infection-specific proteins bound to the 28-mer was similar to that of in vitro-synthesized IE1. By using a combination of DNA binding and antibody supershift assays, it was demonstrated that IE1 binds to the 28-mer as a dimer. Collectively, these findings support a model in which symmetrical IE1 binding and simultaneous interaction with each half site are required for IE1-mediated transcriptional enhancement by hr5. Thus, sequence-specific binding may be one of the mechanisms by which IE1 directly or indirectly transregulates baculovirus gene expression. PMID:7636981

  3. 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. PMID:26945516

  4. Identification and characterization of vlf-1, a baculovirus gene involved in very late gene expression.

    PubMed Central

    McLachlin, J R; Miller, L K

    1994-01-01

    We have identified a gene required for strong expression of the polyhedrin gene by characterizing a mutant, tsB837, of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) which is temperature sensitive (ts) for occluded virus production at the nonpermissive temperature. Marker rescue experiments utilizing an overlapping set of AcMNPV genomic clones revealed that the gene responsible for the ts mutant phenotype mapped to a region between 46 and 48 map units. Fragments (2.2 kb) from both wild-type AcMNPV and tsB837 genomes spanning the mutated region were sequenced, and a single nucleotide difference was observed. This mutation is predicted to substitute a single amino acid within a 44.4-kDa polypeptide. Analysis of protein synthesis in wild-type- and mutant-infected cells at the nonpermissive temperature indicated that polyhedrin synthesis was dramatically reduced in the mutant. Northern (RNA) blot analysis revealed that the mutant had markedly reduced levels of polyhedrin transcripts. Transcripts of another very late gene, p10, were also reduced but to a lesser degree. The transcription of two late genes (603 ORF and vp39) was neither reduced nor temporally delayed. Thus, the gene encoding this very late expression factor, designated vlf-1, regulates the levels of very late gene transcripts, and the tsB837 mutation affects the levels of polyhedrin gene transcripts more strongly than those of p10 transcripts. The product of the newly identified gene has a surprising but significant relationship to a family of integrases and resolvases. Images PMID:7966564

  5. 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. PMID:25443568

  6. Silencing of the Baculovirus Op-iap3 Gene by RNA Interference Reveals that It Is Required for Prevention of Apoptosis during Orgyia pseudotsugata M Nucleopolyhedrovirus Infection of Ld652Y Cells†

    PubMed Central

    Means, John C.; Muro, Israel; Clem, Rollie J.

    2003-01-01

    The Op-iap3 gene from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) inhibits apoptosis induced by a mutant of Autographa californica MNPV (AcMNPV) that lacks the antiapoptotic gene p35, as well as apoptosis induced by a wide range of other stimuli in both mammalian and insect cells. However, the role of Op-iap3 during OpMNPV infection has not been previously examined. To determine the function of the Op-IAP3 protein during OpMNPV infection, we used RNA interference (RNAi) to silence Op-iap3 expression during OpMNPV infection of Ld652Y cells. Infected cells treated with Op-iap3 double-stranded RNA (dsRNA) did not accumulate detectable Op-iap3 mRNA, confirming that the Op-iap3 gene was effectively silenced. Op-IAP3 protein was found to be a component of the budded virion; however, in OpMNPV-infected cells treated with Op-iap3 dsRNA, the Op-IAP3 protein that was introduced by the inoculum virus decreased to almost undetectable levels by 12 h after dsRNA addition. Apoptosis was observed in infected cells treated with Op-iap3 dsRNA beginning at 12 h, and by 48 h, almost all of the cells had undergone apoptosis. These results show for the first time that Op-IAP3 is necessary to prevent apoptosis during OpMNPV infection. In addition, our results demonstrate that the RNAi technique can be an effective tool for studying baculovirus gene function. PMID:12663755

  7. Analysis of Genes Expression of Spodoptera exigua Larvae upon AcMNPV Infection

    PubMed Central

    Wang, Yong; Zhen, Zou; Tao, Xue Ying; Lee, Joo Hyun; Liu, Qin; Kim, Jae Su; Shin, Sang Woon; Je, Yeon Ho

    2012-01-01

    Background The impact of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection on host gene expression in Spodoptera exigua 4th instar larvae was investigated through the use of 454 sequencing-based RNA-seq of cDNA libraries developed from insects challenged with active AcMNPV or heat-inactivated AcMNPV. Methodology/Principal Findings By comparing the two cDNA libraries, we show that 201 host genes are significantly up-regulated and 234 genes are significantly down-regulated by active AcMNPV infection. Down-regulated host genes included genes encoding antimicrobial peptides, namely three gloverin isoforms and an attacin, indicating that the viral infection actively repressed the expression of a portion of the host immune gene repertoire. Another interesting group of down-regulated host genes included genes encoding two juvenile hormone binding proteins and a hexamerin, all of which are involved in juvenile hormone regulation. The expression of these genes was enhanced by the topical application of Juvenile Hormone III (JHIII) in the insects challenged with heat-inactivated AcMNPV. However, infection with the active virus strongly suppresses the expression of these three genes, regardless of the absence or presence of JHIII. Conclusions/Significance Using RNA-seq, we have identified groups of immune-regulated and juvenile hormone-regulated genes that are suppressed by infection with active AcMNPV. This information and further studies on the regulation of host gene expression by AcMNPV will provide the tools needed to enhance the utility of the virus as an effective protein expression system and as an insecticide. PMID:22860129

  8. Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions.

    PubMed

    Theilmann, D A; Chantler, J K; Stweart, S; Flipsen, H T; Vlak, J M; Crook, N E

    1996-04-01

    A highly conserved baculovirus late gene called odvp-6e was shown to be a structural protein that is specific for occlusion-derived virus (ODV) envelopes. The complete sequence of this gene is presented for both Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) and Cydia pomonella granulosis virus (CpGV). The predicted sizes of the OpMNPV and CpGV ODVP-6E are 40, 241, and 38,655 respectively. The OpMNPV odvp-6e gene was transcriptionally mapped and was shown to initiate from a consensus late gene motif, TTAAG, and is expressed from 18-120 hr postinfection. Polyclonal antiserum was generated against a bacterial fusion protein and used to analyze the cellular steady-state levels of ODVP-6E and to determine if this protein was a component of either budded virus (BV) or ODV. Western blots showed that ODVP-6E is a component of the ODV but not BV. This was confirmed by immunoelectron microscopy of ODV from Autographa californica NPV (AcMNPV) which localized ODVP-6E to the ODV envelope. The sequences of the odvp-6e gene from the baculoviruses Choristoneura fumiferana NPV (CfMNPV), AcMNPV, and Helicoverpa zea NPV (HzSNPV) were obtained from GenBank. Comparisons of the predicted amino acid sequences of OpMNPV, CpGV, AcMNPV, CfMNPV, and HzSNPV show that there are two possible membrane-spanning domains and a cysteine-rich domain that are conserved in all of the proteins. PMID:8615018

  9. The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication.

    PubMed Central

    Lu, A; Miller, L K

    1995-01-01

    A set of 18 plasmid subclones of the Autographa californica nuclear polyhedrosis virus genome supports expression from a late viral promoter in transient expression assays (J. W. Todd, A. L. Passarelli, and L. K. Miller, J. Virol. 69:968-974, 1995). Using this set of plasmids, we have assigned a role for each of the 18 genes required for optimal late gene expression with respect to its involvement at the levels of transcription, translation, and/or DNA replication. RNase protection analyses demonstrated that all of the known late expression factor genes (lefs) affected the steady-state level of reporter gene RNA. Thus, none of the lefs appeared to be specifically involved in translation. A subset of the lefs supported plasmid replication; ie-1, lef-1, lef-2, lef-3, p143, and p35 were essential for plasmid replication, while ie-n, lef-7, and dnapol had stimulatory effects. The predicted sequence of lef-7 suggests that it is a homolog of herpesvirus single-stranded DNA-binding protein (UL29). The role of p35 in plasmid replication appears to be suppression of apoptosis, because p35 could be functionally replaced in the replication assay by either Cp-iap or Op-iap, two heterologous baculovirus genes which suppress apoptosis by a mechanism which appears to differ from that of p35. Thus, one or more of the replication-related lefs or the process of plasmid replication appears to induce cellular apoptosis. Our results indicate that the remaining lefs, lefs 4 through 11, p47, and 39K (pp31), function either at the level of transcription or at that of mRNA stabilization. PMID:7815565

  10. Influence of infection route on the infectivity of baculovirus mutants lacking the apoptosis-inhibiting gene p35 and the adjacent gene p94.

    PubMed Central

    Clem, R J; Robson, M; Miller, L K

    1994-01-01

    The infectivity of Autographa californica nuclear polyhedrosis virus mutants lacking the apoptosis-inhibiting gene p35 is decreased 1,000-fold or more in larvae of the insect Spodoptera frugiperda if the budded form of the virus is administered by hemocoelic injection; this decrease is correlated with the antiviral effects of apoptosis (R. J. Clem and L. K. Miller, J. Virol. 67:3730-3738, 1993). We have extended this correlation by showing that the infectivity of p35 mutant budded virus is restored to wild-type levels by expression of an unrelated baculovirus apoptosis-inhibiting gene, Cp-iap. We have also examined the oral infectivity of the occluded form of mutants lacking p35, the neighboring p94 gene, or both genes by feeding insects occluded virus. The oral infectivity of the p35 mutant was significantly reduced in S. frugiperda larvae, but this reduction (25-fold) was less than that observed for the hemocoelic route of infection (1,000-fold). The disruption of p94 alone had no apparent effect on infectivity by either route. Unexpectedly, however, the disruption of both p35 and p94 restored oral infectivity to nearly wild-type levels but did not exert this compensatory effect on infectivity by hemocoelic injection. Thus, the infectivity of the double p35/p94 mutant is affected in a route-specific manner in S. frugiperda larvae, suggesting a tissue-specific response to p35 and/or p94. Infectivity in a different host, Trichoplusia ni, was unaffected by all the mutants tested, consistent with previous studies indicating a lack of sensitivity to apoptosis in this species. However, T. ni and S. frugiperda larvae infected with p35 mutants failed to exhibit the symptom of morphological disintegration ("melting") typical of a wild-type infection, suggesting that p35 is required for the infection of some tissues in both species. PMID:8084009

  11. Identification and characterization of the Cydia pomonella granulovirus cathepsin and chitinase genes.

    PubMed

    Kang, W; Tristem, M; Maeda, S; Crook, N E; O'Reilly, D R

    1998-09-01

    A 3.2 kb BamHI-EcoRI fragment of the Cydia pomonella granulovirus (CpGV) genome was subcloned and characterized. Sequence analysis revealed two complete and one partial open reading frames (ORFs). ORF7L is predicted to encode a 66.7 kDa protein (594 amino acid residues) that is 57% identical (amino acid sequence) to the chiA gene (ORF126) of Autographa californica nucleopolyhedrovirus (AcMNPV), encoding a chitinase. ORF8R is 333 amino acids in length and shows high similarity (between 64% and 67%) with baculovirus cathepsins. The partial ORF, ORF5L, is related to AcMNPV ORF145 of unknown function. Phylogenetic trees were constructed for both chitinase and cathepsin sequences from baculoviruses and other species. In both cases, the baculovirus sequences were monophyletic but with a deep division between the GVs and NPVs, suggesting both genes were present in an ancestral virus prior to the separation of the two genera. However, these studies did not provide definitive evidence for the origin of either protein in baculoviruses. To investigate CpGV cathepsin function, a rescue experiment was performed using a Bombyx mori NPV (BmNPV) mutant (BmCysPD) which lacks a functional cathepsin (cath) gene. Larvae infected with BmCysPD-Cp.cat, a BmCysPD derivative carrying CpGV cath, showed similar symptoms to wild-type BmNPV infected insects, confirming that CpGV cath encodes a functional cathepsin. Primer extension analysis of mRNA from BmCysPD-Cp.cat infected cells showed that CpGV cath transcription was initiated from a consensus late transcription motif (ATAAG) within the CpGV sequences, indicating that a CpGV late promoter motif was recognized in this NPV system. PMID:9747739

  12. Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

    PubMed Central

    Du, X; Thiem, S M

    1997-01-01

    Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection. PMID:9311875

  13. An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs.

    PubMed Central

    Birnbaum, M J; Clem, R J; Miller, L K

    1994-01-01

    Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA. Images PMID:8139034

  14. An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs.

    PubMed

    Birnbaum, M J; Clem, R J; Miller, L K

    1994-04-01

    Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA. PMID:8139034

  15. Isolation of an Apoptosis Suppressor Gene of the Spodoptera littoralis Nucleopolyhedrovirus†

    PubMed Central

    Du, Quansheng; Lehavi, Dana; Faktor, Ouriel; Qi, Yipeng; Chejanovsky, Nor

    1999-01-01

    Spodoptera frugiperda SF9 cells infected with mutants of the Autographa californica nucleopolyhedrovirus (AcMNPV) which lack a functional p35 gene undergo apoptosis, aborting the viral infection. The Spodoptera littoralis nucleopolyhedrovirus (SlNPV) was able to suppress apoptosis triggered by vΔP35K/pol+, an AcMNPV p35 null mutant. To identify the putative apoptotic suppressor gene of SlNPV, overlapping cosmid clones representing the entire SlNPV genome were individually cotransfected along with genomic DNA of vΔP35K/pol+. Using this complementation assay, we isolated a SlNPV DNA fragment that was able to rescue the vΔP35K/pol+ infection in SF9 cells. By further subcloning and rescue, we identified a novel SlNPV gene, Slp49. The Slp49 sequence predicted a 49-kDa polypeptide with about 48.8% identity to the AcMNPV apoptotic suppressor P35. SLP49 displays a potential recognition site, TVTDG, for cleavage by death caspases. Recombinant AcMNPVs deficient in p35 bearing the Slp49 gene did not induce apoptosis and showed successful productive infections in SF9 cells, indicating that Slp49 is a functional homologue of p35. A 1.5-kbp Slp49-specific transcript was identified in SF9 cells infected with SlNPV or with vAc496, a vΔP35K/pol+-recombinant bearing Slp49. The discovery of Slp49 contributes to the identification of important functional motifs conserved in p35-like apoptotic suppressors and to the future isolation of p35-like genes from other baculoviruses. PMID:9882332

  16. 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

  17. 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

    2011-01-01

    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

  18. Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.) (Lepidoptera: Noctuidae).

    PubMed

    Cheng, Xiao-Wen; Carner, Gerald R; Lange, Martin; Jehle, Johannes A; Arif, Basil M

    2005-02-01

    A multicapsid nucleopolyhedrovirus (ThorMNPV) that was co-isolated with a single nucleocapid ThorSNPV from mixed infected larvae of Thysanoplusia orichalcea L. (Lepidoptea: Noctuidae) is characterized. Scanning electron microscopy of ThorMNPV showed a dodecahedral-shaped occlusion body (OB). The occluded virions contained one to as many as eight nucleocapsids/virion. Virion band profiles in gradient centrifugation were consistent in at least 10 rounds of centrifugation from different virion sample preparations. The ThorMNPV had high virulence to third instar Trichoplusia ni and Pseudoplusia includens with LD50 values of 17 and 242OBs per larva, respectively. However, ThorMNPV did not cause mortality in Spodoptera exigua, Spodoptera frugiperda, Spodoptera eridania, Anticarsia gemmatalis, and Helicoverpa zea. ThorMNPV replicates in cells of various tissues such as the fat body and tracheal epithelium cells. T. ni High 5 cells were permissive to ThorMNPV in terms of infection and viral DNA transfection, but SF-21 was less permissive and the infection process was slower. Production of OBs by ThorMNPV in the nuclei of SF-21 was not well pronounced. The genome size of ThorMNPV was estimated to be 136 kb. The polyhedrin gene open reading frame (ORF) was cloned and completely sequenced. The promoter sequence is identical to that of Autographa californica MNPV. Phylogenetic analyses using partial sequences of the polh, lef-8, and lef-9 revealed that ThorMNPV is a member of the Group I NPVs and is related but distinct from the AcMNPV/Rachiplusia ou NPV/Bombyx mori NPV cluster. PMID:15766929

  19. Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

    PubMed Central

    Mitchell, Jonathan K.

    2012-01-01

    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. PMID:23035220

  20. 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

  1. 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. PMID:20139610

  2. 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

  3. Use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles.

    PubMed

    An, K; Gillock, E T; Sweat, J A; Reeves, W M; Consigli, R A

    1999-04-01

    The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells. PMID:10211971

  4. 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.

  5. Generating a host range-expanded recombinant baculovirus.

    PubMed

    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

  6. 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

  7. 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

  8. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

    PubMed Central

    Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

  9. Heterogeneous Host Susceptibility Enhances Prevalence of Mixed-Genotype Micro-Parasite Infections

    PubMed Central

    Vlak, Just M.; Zwart, Mark P.

    2011-01-01

    Dose response in micro-parasite infections is usually shallower than predicted by the independent action model, which assumes that each infectious unit has a probability of infection that is independent of the presence of other infectious units. Moreover, the prevalence of mixed-genotype infections was greater than predicted by this model. No probabilistic infection model has been proposed to account for the higher prevalence of mixed-genotype infections. We use model selection within a set of four alternative models to explain high prevalence of mixed-genotype infections in combination with a shallow dose response. These models contrast dependent versus independent action of micro-parasite infectious units, and homogeneous versus heterogeneous host susceptibility. We specifically consider a situation in which genome differences between genotypes are minimal, and highly unlikely to result in genotype-genotype interactions. Data on dose response and mixed-genotype infection prevalence were collected by challenging fifth instar Spodoptera exigua larvae with two genotypes of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), differing only in a 100 bp PCR marker sequence. We show that an independent action model that includes heterogeneity in host susceptibility can explain both the shallow dose response and the high prevalence of mixed-genotype infections. Theoretical results indicate that variation in host susceptibility is inextricably linked to increased prevalence of mixed-genotype infections. We have shown, to our knowledge for the first time, how heterogeneity in host susceptibility affects mixed-genotype infection prevalence. No evidence was found that virions operate dependently. While it has been recognized that heterogeneity in host susceptibility must be included in models of micro-parasite transmission and epidemiology to account for dose response, here we show that heterogeneity in susceptibility is also a fundamental principle explaining

  10. 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. PMID:10882435

  11. Baculovirus DNA Replication-Specific Expression Factors Trigger Apoptosis and Shutoff of Host Protein Synthesis during Infection▿

    PubMed Central

    Schultz, Kimberly L. W.; Friesen, Paul D.

    2009-01-01

    Apoptosis is an important antivirus defense. To define the poorly understood pathways by which invertebrates respond to viruses by inducing apoptosis, we have identified replication events that trigger apoptosis in baculovirus-infected cells. We used RNA silencing to ablate factors required for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Transfection with double-stranded RNA (dsRNA) complementary to the AcMNPV late expression factors (lefs) that are designated as replicative lefs (lef-1, lef-2, lef-3, lef-11, p143, dnapol, and ie-1/ie-0) blocked virus DNA synthesis and late gene expression in permissive Spodoptera frugiperda cells. dsRNAs specific to designated nonreplicative lefs (lef-8, lef-9, p47, and pp31) blocked late gene expression without affecting virus DNA replication. Thus, both classes of lefs functioned during infection as defined. Silencing the replicative lefs prevented AcMNPV-induced apoptosis of Spodoptera cells, whereas silencing the nonreplicative lefs did not. Thus, the activity of replicative lefs or virus DNA replication is sufficient to trigger apoptosis. Confirming this conclusion, AcMNPV-induced apoptosis was suppressed by silencing the replicative lefs in cells from a divergent species, Drosophila melanogaster. Silencing replicative but not nonreplicative lefs also abrogated AcMNPV-induced shutdown of host protein synthesis, suggesting that virus DNA replication triggers inhibition of host biosynthetic processes and that apoptosis and translational arrest are linked. Our findings suggest that baculovirus DNA replication triggers a host cell response similar to the DNA damage response in vertebrates, which causes translational arrest and apoptosis. Pathways for detecting virus invasion and triggering apoptosis may therefore be conserved between insects and mammals. PMID:19706708

  12. Improvement in the UV resistance of baculoviruses by displaying nano-zinc oxide-binding peptides on the surfaces of their occlusion bodies.

    PubMed

    Li, Jin; Zhou, Yin; Lei, Chengfeng; Fang, Wei; Sun, Xiulian

    2015-08-01

    The sensitivity of baculoviruses to UV radiation severely limits their large-scale application as biological insecticides. The polyhedron envelope of a baculovirus, which is composed of carbohydrate and polyhedron envelope protein (PEP), is a significant structure for the stability and persistence of occlusion bodies (OBs) under environmental conditions. The results of this study revealed that the rough pitted surface phenotype of a pep-null Autographa californica multiple nucleopolyhedrovirus (AcMNPV) could not be rescued by any of its homologues, such as Helicoverpa armigera nucleopolyhedrovirus pep or Cydia pomonella granulovirus putative peps. In contrast, the N-terminal and middle flexible region (NM region, 1-167 aa) of AcMNPV PEP were able to form an intact OB envelope. Furthermore, this region was capable of carrying eGFP to the surfaces of the OBs. To improve the UV resistance of AcMNPV OBs, two peptides capable of specifically binding to nano-ZnO were separately fused to the NM region of PEP. Under laboratory conditions, infectivity of the recombinant viruses binding to nano-ZnO particles was about ninefold higher than that without the nano-ZnO particles after UV-B irradiation. Pot experiments revealed that the half-life of the recombinant baculovirus binding nano-ZnO particles was 3.3 ± 0.15 days, which was significantly longer than that of the control virus (0.49 ± 0.06 days). These results therefore represent a new approach for the protection the baculoviral insecticides against UV irradiation in the field. PMID:25895092

  13. The N Terminus of the Vaccinia Virus Protein F1L Is an Intrinsically Unstructured Region That Is Not Involved in Apoptosis Regulation.

    PubMed

    Caria, Sofia; Marshall, Bevan; Burton, Robyn-Lee; Campbell, Stephanie; Pantaki-Eimany, Delara; Hawkins, Christine J; Barry, Michele; Kvansakul, Marc

    2016-07-01

    Subversion of host cell apoptotic responses is a prominent feature of viral immune evasion strategies to prevent premature clearance of infected cells. Numerous poxviruses encode structural and functional homologs of the Bcl-2 family of proteins, and vaccinia virus harbors antiapoptotic F1L that potently inhibits the mitochondrial apoptotic checkpoint. Recently F1L has been assigned a caspase-9 inhibitory function attributed to an N-terminal α helical region of F1L spanning residues 1-15 (1) preceding the domain-swapped Bcl-2-like domains. Using a reconstituted caspase inhibition assay in yeast we found that unlike AcP35, a well characterized caspase-9 inhibitor from the insect virus Autographa californica multiple nucleopolyhedrovirus, F1L does not prevent caspase-9-mediated yeast cell death. Furthermore, we found that deletion of the F1L N-terminal region does not impede F1L antiapoptotic activity in the context of a viral infection. Solution analysis of the F1L N-terminal regions using small angle x-ray scattering indicates that the region of F1L spanning residues 1-50 located N-terminally from the Bcl-2 fold is an intrinsically unstructured region. We conclude that the N terminus of F1L is not involved in apoptosis inhibition and may act as a regulatory element in other signaling pathways in a manner reminiscent of other unstructured regulatory elements commonly found in mammalian prosurvival Bcl-2 members including Bcl-xL and Mcl-1. PMID:27151220

  14. New anamorphic yeast species: Candida infanticola sp. nov., Candida polysorbophila sp. nov., Candida transvaalensis sp. nov., and Trigonopsis californica sp. nov.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three new species of Candida and a new species of Trigonopsis are described based on their recognition from phylogenetic analysis of gene sequences from large subunit ribosomal RNA, ITS1/ITS2 rRNA, mitochondrial small subunit rRNA and cytochrome oxidase II. Candida infanticola sp. nov. (type strain...

  15. Overexpression of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in Benzylisoquinoline alkaloid biosynthesis in cultured Eschscholzia californica.

    PubMed

    Inui, Takayuki; Tamura, Ken-Ichi; Fujii, Nanae; Morishige, Takashi; Sato, Fumihiko

    2007-02-01

    Benzylisoquinoline alkaloids are one of the most important secondary metabolite groups, and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the production of these alkaloids, we investigated the effect of the overexpression of putative rate-limiting step enzymes in benzylisoquinoline alkaloid biosynthesis. We introduced two O-methyltransferase [Coptis japonica norcoclaurine 6-O-methyltransferase (6OMT) and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT)] expression vectors into cultured California poppy cells to avoid the gene silencing effect of endogenous genes. We established 20 independent lines for 6OMT transformants and 15 independent lines for 4'OMT transformants. HPLC/liquid chromatography-mass spectrometry (LC-MS) analysis revealed that the overexpression of C. japonica 6OMT was associated with an average alkaloid content 7.5 times greater than that in the wild type, whereas the overexpression of C. japonica 4'OMT had only a marginal effect. Further characterization of 6OMT in California poppy cells indicated that a 6OMT-specific gene is missing and 4OMT catalyzes the 6OMT reaction with low activity in California poppy, which supports the notion that the 6OMT reaction is important for alkaloid biosynthesis in this plant species. We discuss the importance of 6OMT in benzylisoquinoline alkaloid biosynthesis and the potential for using a rate-limiting step gene to improve alkaloid production. PMID:17189286

  16. 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…

  17. SURVIVAL OF 'DAPHNIA', CRAYFISH, AND STONEFLIES IN AIR-SUPERSATURATED WATER

    EPA Science Inventory

    Daphnia magna, the crayfish Pacifastacus leniusculus, and nymphs of the stoneflies, Acroneuria californica, A. pacifica, and Pteronarcys californica were tested in the laboratory to determine their survival in different concentrations of air-supersaturated water. The mean 96-h LC...

  18. Effect of the peak cell density of recombinant AcMNPV-infected Hi5 cells on baculovirus yields.

    PubMed

    Huynh, Hoai T; Tran, Trinh T B; Chan, Leslie C L; Nielsen, Lars K; Reid, Steven

    2015-02-01

    The phenomenon of the cell density effect is not readily explained by an obvious nutrient limitation, and a recent study has suggested that for recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV)-infected Sf9 cells, a drop in messenger RNA (mRNA) levels may be sufficient to explain the cell density effect for this system. The current study aims to investigate the response in cell-specific yields (viral DNA (vDNA), LacZ mRNA and β-galactosidase (β-Gal) protein) with increasing infection cell density (ICD) for rAcMNPV-infected Hi5 cells, where the rAcMNPV expresses the β-Gal gene under control of the polyhedral promoter. Hi5 cells in suspension culture of Express Five® medium were synchronously infected with a rAcMNPV at multiple ICDs between 0.5 and 6 × 10(6) cells/mL and a multiplicity of infection of 10 plaque-forming units (PFU)/cell either in the original or fresh medium conditions. There were negative correlations between the three key virus infection indicators (vDNA, mRNA and β-Gal) and the peak cell density (PCD). However, unlike infected Sf9 cells, the yield decline started at the lowest PCD investigated (0.6 × 10(6) cells/mL). Generally, the yield decline with increasing PCD was most pronounced for β-Gal followed by mRNA and was more moderate for vDNA. The decline was significantly reduced but not totally arrested when fresh medium replacement was used. The results suggest that the reduction in recombinant protein-specific yields at high PCDs is associated with limitations during the up-stream processes of replication and transcription rather than entirely caused by limitations during translation. In addition, low production rates at late infection stages of moderate to high ICDs are a probable cause of the cell density effect. PMID:25472440

  19. Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

    SciTech Connect

    Vanarsdall, Adam L.; Mikhailov, Victor S.; Rohrmann, George F. . E-mail: rohrmanng@orst.edu

    2007-08-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout

  20. A new cell line derived from embryonic tissues of Holotrichia parallela (Coleoptera:Scarabaeidae).

    PubMed

    Li, Miao-Miao; Zheng, Gui-Ling; Su, Rui; Wan, Fang-Hao; Li, Chang-You

    2016-06-01

    Holotrichia parallela is an important agricultural underground insect pest and also an edible and medicinal insect. Establishing a new cell line of H. parallela will provide a rapid and convenient tool for the studies on its physiology, pathology, and gene functions. In this study, by using the embryonic tissue of H. parallela as the material, we established a new cell line named Hp-E-1. The microscopic observation of its morphological characteristics revealed that its cellular morphology was mainly in the spherical morphology with a mean cellular diameter of 17.71 ± 2.34 μm, accounting for 67% of the total cells. The spindle-shaped cells accounted for 33% of the total cells with a mean size of 23.51 ± 4.37 × 13.98 ± 2.05 μm. The chromosomal number varied from 7 to 40, with about 50% of the cells having a diploid chromosome number of 2n = 20. Random amplified polymorphic DNA (RAPD) analysis indicated that the profiles of PCR-amplified fragments of this cell line were basically similar to those of the embryonic tissues of H. parallela but were obviously different from those of cell line BTI-Tn5B1-4 of Trichoplusia ni and cell line Sf-9 of Spodoptera frugiperda. The DNA fragment encoding mitochondrial cytochrome C oxidase subunit I (COI) gene of this cell line shared 99.7% homology with that of the embryonic tissue of H. parallela, confirming that this cell line is indeed derived from H. parallela. The results of growth curve measurement indicated that the population doubling time of this cell line was 136.7 h. Cell line Hp-E-1 could not be infected by three viruses Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Bombyx mori nucleopolyhedrovirus (BmNPV), and Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV). PMID:27083164

  1. 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

  2. Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

    PubMed Central

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

    2009-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using pulsed-field gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 hours post-infection, DBP co-localized with replicated DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp

  3. Comparative insecticidal properties of two nucleopolyhedrovirus vectors encoding a similar toxin gene chimer.

    PubMed

    Treacy, M F; Rensner, P E; All, J N

    2000-08-01

    Laboratory, greenhouse and field studies were conducted to characterize the insecticidal properties of genetically altered forms of Autographa californica (Speyer) nucleopolyhedrovirus (AcNPV) and Helicoverpa zea (Boddie) NPV (HzNPV) against selected heliothine species. The altered viruses each contained a chimeric 0.8-kb fragment encoding the insect-specific, sodium channel neurotoxin from the Algerian scorpion Androctonus australis Hector (AaIT, hence recombinant viruses designated Ac-AaIT and Hz-AaIT). Based on LD50 values, results from diet-overlay bioassays showed Ac-AaIT and Hz-AaIT to be equally virulent against larval tobacco budworm, Heliothis virescens (F.), but Hz-AaIT averaged 1,335-fold greater bioactivity than Ac-AaIT against larval cotton bollworm, Helicoverpa zea (Boddie). Hz-AaIT killed larvae of both heliothine species at rates significantly faster than those imparted by HzNPV (viral LT50 values averaged 2.5 and 5.6 d, respectively). In greenhouse studies, foliar sprays of Ac-AaIT and Hz-AaIT were equally effective in controlling H. virescens on cotton; however, Hz-AaIT provided control of H. zea on cotton at a level superior to that of Ac-AaIT. For example, after three weekly sessions of foliar application and H. zea artificial infestation, cotton treated with Ac-AaIT or Hz-AaIT at 10 x 10(11) occulsion bodies (OB)/ha averaged 2.5 and 16.2 nondamaged flower buds per plant, respectively. Another greenhouse study conducted against heliothine species on cotton showed that the quicker killing speed exhibited by Hz-AaIT led to improved plant protection versus HzNPV. Finally, results from three field trials demonstrated that Hz-AaIT at 5-12 x 10(11) OB/ha provided control of the heliothine complex in cotton at levels slightly better than Bacillus thuringiensis, equal to the macrolide, spinosad, and only slightly less than that of selected pyrethroid and carbamate insecticides. Overall, results from these studies indicate that, because of host range

  4. 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

  5. 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...

  6. 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

  7. Women's health among the Chumash.

    PubMed

    Adams, James D; Garcia, Cecilia

    2006-03-01

    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

  8. 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

  9. 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

  10. Volatile Growth Inhibitors Produced by Aromatic Shrubs.

    PubMed

    Muller, C H; Muller, W H; Haines, B L

    1964-01-31

    Root growth of Cucumis and Avena seedlings is inhibited by volatile materials produced by leaves of Salvia leucophylla, S. apiana, and Artemisia californica. The toxic substance may be deposited when dew condenses on affected seedlings in the field. PMID:17833745

  11. CO-OCCLUSION AND PERSISTENCE OF A BACULOVIRUS MUTANT LACKING THE POLYHEDRIN GENE

    EPA Science Inventory

    A co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. oinfection of Spodoptera frugiperda (IPLB-SF-21) tissue culture cells with Aucographa californica nuclear polyhedrosis...

  12. Inactivation studies of acetylcholinesterase with phenylmethylsulfonyl fluoride.

    PubMed

    Kraut, D; Goff, H; Pai, R K; Hosea, N A; Silman, I; Sussman, J L; Taylor, P; Voet, J G

    2000-06-01

    Acetylcholinesterase (AChE), a serine hydrolase, is potentially susceptible to inactivation by phenylmethylsulfonyl fluoride (PMSF) and benzenesulfonyl fluoride (BSF). Although BSF inhibits both mouse and Torpedo californica AChE, PMSF does not react measurably with the T. californica enzyme. To understand the residue changes responsible for the change in reactivity, we studied the inactivation of wild-type T. californica and mouse AChE and mutants of both by BSF and PMSF both in the presence and absence of substrate. The enzymes investigated were wild-type mouse AChE, wild-type T. californica AChE, wild-type mouse butyrylcholinesterase, mouse Y330F, Y330A, F288L, and F290I, and the double mutant T. californica F288L/F290V (all mutants given T. californica numbering). Inactivation rate constants for T. californica AChE confirmed previous reports that this enzyme is not inactivated by PMSF. Wild-type mouse AChE and mouse mutants Y330F and Y330A all had similar inactivation rate constants with PMSF, implying that the difference between mouse and T. californica AChE at position 330 is not responsible for their differing PMSF sensitivities. In addition, butyrylcholinesterase and mouse AChE mutants F288L and F290I had increased rate constants ( approximately 14 fold) over those of wild-type mouse AChE, indicating that these residues may be responsible for the increased sensitivity to inactivation by PMSF of butyrylcholinesterase. The double mutant T. californica AChE F288L/F290V had a rate constant nearly identical with the rate constant for the F288L and F290I mouse mutant AChEs, representing an increase of approximately 4000-fold over the T. californica wild-type enzyme. It remains unclear why these two positions have more importance for T. californica AChE than for mouse AChE. PMID:10825396

  13. Attraction of pest moths (Lepidoptera: Noctuidae, Crambidae) to floral lures on the island of Hawaii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

    SciTech Connect

    Chen, Hong-Zhang; Wu, Carol P.; Chao, Yu-Chan; Liu, Catherine Yen-Yen

    2011-02-11

    Research highlights: {yields} Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. {yields} Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. {yields} CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. {yields} Virus entry and gene expression can be separate events in different cell types. -- Abstract: The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

  15. Adaptive Immune Responses Elicited by Baculovirus and Impacts on Subsequent Transgene Expression In Vivo

    PubMed Central

    Luo, Wen-Yi; Lin, Shih-Yeh; Lo, Kai-Wei; Lu, Chia-Hsin; Hung, Chang-Lin; Chen, Chi-Yuan; Chang, Chien-Chung

    2013-01-01

    Baculovirus (BV) is a promising gene therapy vector and typically requires readministration because BV mediates transient expression. However, how the prime-boost regimen triggers BV-specific adaptive responses and their impacts on BV readministration, transgene expression, and therapeutic/vaccine efficacy remain unknown. Here we unraveled that BV injection into BALB/c mice induced the production of BV-specific antibodies, including IgG1 and IgG2a, which could neutralize BV by antagonizing the envelope protein gp64 and impede BV-mediated transgene expression. Moreover, humans did not possess preexisting anti-BV antibodies. BV injection also elicited BV-specific Th1 and Th2 responses as well as CD4+ and CD8+ T cell responses. gp64 was a primary immunogen to activate the antibody and CD8+ T cell response, with its peptide at positions 457 to 465 (peptide 457-465) being the major histocompatibility complex (MHC) class I epitope to stimulate CD8+ T cell and cytotoxic responses. Nonetheless, a hybrid Sleeping Beauty-based BV enabled long-term expression for >1 year by a single injection, indicating that the T cell responses did not completely eradicate BV-transduced cells and implicating the potential of this hybrid BV vector for gene therapy. These data unveil that BV injection triggers adaptive immunity and benefit rational design of BV administration schemes for gene therapy and vaccination. PMID:23408634

  16. Complement regulatory proteins are incorporated into lentiviral vectors and protect particles against complement inactivation.

    PubMed

    Schauber-Plewa, C; Simmons, A; Tuerk, M J; Pacheco, C D; Veres, G

    2005-02-01

    Lentiviral vectors pseudotyped with G glycoprotein from vesicular stomatitis virus (VSV-G) and baculovirus gp64 are inactivated by human complement. The extent of vector inactivation in serum from individual donors was examined and results showed wide donor-dependent variation in complement sensitivity for VSV-G-pseudotyped lentivectors. Amphotropic envelope (Ampho)-pseudotyped vectors were generally resistant to serum from all donors, while gp64-pseudotyped vectors were inactivated but showed less donor-to-donor variation than VSV-G. In animal sera, the vectors were mostly resistant to inactivation by rodent complement, whereas canine complement caused a moderate reduction in titer. In a novel advance for the lentiviral vector system, human complement-resistant-pseudotyped lentivector particles were produced through incorporation of complement regulatory proteins (CRPs). Decay accelerating factor (DAF)/CD55 provided the most effective protection using this method, while membrane cofactor protein (MCP)/CD46 showed donor-dependent protection and CD59 provided little or no protection against complement inactivation. Unlike previous approaches using CRPs to produce complement-resistant viral vectors, CRP-containing lentivectors particles were generated for this study without engineering the CRP molecules. Thus, through overexpression of native DAF/CD55 in the viral producer cell, an easy method was developed for generation of lentiviral vectors that are almost completely resistant to inactivation by human complement. Production of complement-resistant lentiviral particles is a critical step toward use of these vectors for in vivo gene therapy applications. PMID:15550926

  17. Lentiviral vector gene transfer to porcine airways.

    PubMed

    Sinn, Patrick L; Cooney, Ashley L; Oakland, Mayumi; Dylla, Douglas E; Wallen, Tanner J; Pezzulo, Alejandro A; Chang, Eugene H; McCray, Paul B

    2012-01-01

    In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).Molecular Therapy - Nucleic Acids (2012) 1, e56; doi:10.1038/mtna.2012.47; published online 27 November 2012. PMID:23187455

  18. Complete Genomic Sequence of Bacteriophage Felix O1†

    PubMed Central

    Whichard, Jean M.; Weigt, Lee A.; Borris, Douglas J.; Li, Ling Ling; Zhang, Qing; Kapur, Vivek; Pierson, F. William; Lingohr, Erika J.; She, Yi-Min; Kropinski, Andrew M.; Sriranganathan, Nammalwar

    2010-01-01

    Bacteriophage O1 is a Myoviridae A1 group member used historically for identifying Salmonella. Sequencing revealed a single, linear, 86,155-base-pair genome with 39% average G+C content, 131 open reading frames, and 22 tRNAs. Closest protein homologs occur in Erwinia amylovora phage φEa21-4 and Escherichia coli phage wV8. Proteomic analysis indentified structural proteins: Gp23, Gp36 (major tail protein), Gp49, Gp53, Gp54, Gp55, Gp57, Gp58 (major capsid protein), Gp59, Gp63, Gp64, Gp67, Gp68, Gp69, Gp73, Gp74 and Gp77 (tail fiber). Based on phage-host codon differences, 7 tRNAs could affect translation rate during infection. Introns, holin-lysin cassettes, bacterial toxin homologs and host RNA polymerase-modifying genes were absent. PMID:21994654

  19. Identification of subunits of acetylcholine receptor that interact with a cholesterol photoaffinity probe

    SciTech Connect

    Middlemas, D.S.; Raftery, M.A.

    1987-03-10

    All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with (/sup 3/H)cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. This investigation also demonstrates that this probe is a useful reagent for studying the interaction of cholesterol with membrane proteins.

  20. Sporulation on plant roots by Phytophthora ramorum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytophthora ramorum has been shown to infect the roots of many of the pathogen’s foliar hosts. Methods of detecting inoculum in runoff and of quantifying root colonization were tested using Viburnum tinus, Camellia oleifera, Quercus prinus, Umbellularia californica, and Epilobium ciliatum. Plants...

  1. Cercosporoid leaf pathogens from whorled milkweed and spineless safflower in California.

    PubMed

    Koike, Steven T; Baameur, Aziz; Groenewald, Johannes Z; Crous, Pedro W

    2011-06-01

    Two cercosporoid species are respectively described from Mexican whorled milkweed (Asclepias fascicularis), and spineless safflower (Carthamus tinctorius) from California. Passalora californica represents a new pathogen on Asclepias fascicularis, while Ramularia cynarae is confirmed on Carthamus tinctorius and Cynara cardunculus (Asteraceae), and an epitype designated. Pathogenicity is also established for both pathogens based on Koch's postulate. PMID:22679582

  2. The Tail-Elicited Tail Withdrawal Reflex of "Aplysia" Is Mediated Centrally at Tail Sensory-Motor Synapses and Exhibits Sensitization across Multiple Temporal Domains

    ERIC Educational Resources Information Center

    Philips, Gary T.; Sherff, Carolyn M.; Menges, Steven A.; Carew, Thomas J.

    2011-01-01

    The defensive withdrawal reflexes of "Aplysia californica" have provided powerful behavioral systems for studying the cellular and molecular basis of memory formation. Among these reflexes the (T-TWR) has been especially useful. In vitro studies examining the monosynaptic circuit for the T-TWR, the tail sensory-motor (SN-MN) synapses, have…

  3. YIELD POTENTIAL OF SELECTED MEDICINAL HERBS AT THREE PLANT SPACINGS IN NEW MEXICO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Field studies were conducted to determine the production potential of echinacea (Echinacea purpurea), valerian (Valeriana officinalis), mullein (Verbascum thapsus) and yerba mansa (Anemopsis californica) medicinal herbs at two sites in New Mexico. Las Cruces, N.M. is at an elevation of 1,186 m and h...

  4. TEMPORAL VARIATION IN THE DIETS OF CALIFORNIA QUAIL IN WESTERN OREGON

    EPA Science Inventory

    This study was designed to describe dietary changes by comparison of information on long-term diet and plant food availability of California Quail (Callipepla californica) in western Oregon. he authors examined crops from 222 California Quail collected in 197678 and 1985-87. iets...

  5. FEEDING RATES OF THE MUD SHRIMP UPOGEBIA PUGETTENSIS AND IMPLICATIONS FOR ESTUARINE PHYTOPLANKTON ABUNDANCE

    EPA Science Inventory

    The burrowing shrimp Upogebia pugettensis is an abundant inhabitant of Pacific Northwest bays and estuaries where it lives commensally with the clam Cryptomya californica. Suspension-feeding activities of the shrimp and its commensal clam, as well as particle settlement within t...

  6. PARTICLE REMOVAL RATES BY THE MUD SHRIMP UPOGEBIA PUGETTENSIS, ITS BURROW, AND A COMMENSAL CLAM: EFFECTS ON ESTUARINE PHYTOPLANKTON ABUNDANCE

    EPA Science Inventory

    The burrowing shrimp Upogebia pugettensis is an abundant intertidal inhabitant of Pacific Northwest bays and estuaries where it lives commensally with the bivalve Cryptomya californica. Suspension-feeding activities by the shrimp and by its commensal clam, as well as particle se...

  7. The Roots of Defense: Plant Resistance and Tolerance to Belowground Herbivory

    PubMed Central

    Watts, Sean M.; Dodson, Craig D.; Reichman, O. J.

    2011-01-01

    Background There is conclusive evidence that there are fitness costs of plant defense and that herbivores can drive selection for defense. However, most work has focused on above-ground interactions, even though belowground herbivory may have greater impacts on individual plants than above-ground herbivory. Given the role of belowground plant structures in resource acquisition and storage, research on belowground herbivores has much to contribute to theories on the evolution of plant defense. Pocket gophers (Geomyidae) provide an excellent opportunity to study root herbivory. These subterranean rodents spend their entire lives belowground and specialize on consuming belowground plant parts. Methodology and Principal Findings We compared the root defenses of native forbs from mainland populations (with a history of gopher herbivory) to island populations (free from gophers for up to 500,000 years). Defense includes both resistance against herbivores and tolerance of herbivore damage. We used three approaches to compare these traits in island and mainland populations of two native California forbs: 1) Eschscholzia californica populations were assayed to compare alkaloid deterrents, 2) captive gophers were used to test the palatability of E. californica roots and 3) simulated root herbivory assessed tolerance to root damage in Deinandra fasciculata and E. californica. Mainland forms of E. californica contained 2.5 times greater concentration of alkaloids and were less palatable to gophers than island forms. Mainland forms of D. fasciculata and, to a lesser extent, E. californica were also more tolerant of root damage than island conspecifics. Interestingly, undamaged island individuals of D. fasciculata produced significantly more fruit than either damaged or undamaged mainland individuals. Conclusions and Significance These results suggest that mainland plants are effective at deterring and tolerating pocket gopher herbivory. Results also suggest that both forms of

  8. Properties and substrate specificities of the phenylalanyl-transfer-ribonucleic acid synthetases of Aesculus species

    PubMed Central

    Anderson, J. W.; Fowden, L.

    1970-01-01

    1. Phenylalanyl-tRNA synthetases have been partially purified from cotyledons of seeds of Aesculus californica, which contains 2-amino-4-methylhex-4-enoic acid, and from four other species of Aesculus that do not contain this amino acid. The A. californica preparation was free from other aminoacyl-tRNA synthetases, and the contaminating synthetase activity in preparations from A. hippocastanum was decreased to acceptable limits by conducting assays of pyrophosphate exchange activity in 0.5m-potassium chloride. 2. The phenylalanyl-tRNA synthetase from each species activated 2-amino-4-methylhex-4-enoic acid with Km 30–40 times that for phenylalanine. The maximum velocity for 2-amino-4-methylhex-4-enoic acid was only 30% of that for phenylalanine with the A. californica enzyme, but the maximum velocities for the two substrates were identical for the other four species. 3. 2-Amino-4-methylhex-4-enoic acid was not found in the protein of A. californica, so discrimination against this amino acid probably occurs in the step of transfer to tRNA, though subcellular localization, or subsequent steps of protein synthesis could be involved. 4. Crotylglycine, methallylglycine, ethallylglycine, 2-aminohex-4,5-dienoic acid, 2-amino-5-methylhex-4-enoic acid, 2-amino-4-methylhex-4-enoic acid, β-(thien-2-yl)alanine, β-(pyrazol-1-yl)alanine, phenylserine and m-fluorophenylalanine were substrates for pyrophosphate exchange catalysed by the phenylalanyl-tRNA synthetases of A. californica or A. hippocastanum. Allylglycine, phenylglycine and 2-amino-4-phenylbutyric acid were inactive. PMID:5493504

  9. First record of a louse fly, Stilbometopa impressa (Bigot), and new host for Microlynchia pusilla (Speiser) (Hippoboscidae) from the Cape Region, Baja California Sur, México.

    PubMed

    Llinas, J; Jiménez, M L

    1996-04-01

    Nine of thirty California quail (Callipepla californica achrustera) captured in autumn of 1992, 17 km west of La Paz, Baja California Sur, México, were parasitized by louse flies. We identified eight Microlynchia pusilla and three Stilbometopa impressa from 30 quails in the ratio of 2.75:1. These are the first records of S. impressa for Cape Region and the first time either fly has been reported from the California quail in Baja California Sur. PMID:8722274

  10. Floral visitation by the Argentine ant reduces pollinator visitation and seed set in the coast barrel cactus, Ferocactus viridescens.

    PubMed

    LeVan, Katherine E; Hung, Keng-Lou James; McCann, Kyle R; Ludka, John T; Holway, David A

    2014-01-01

    Mounting evidence indicates that trade-offs between plant defense and reproduction arise not only from resource allocation but also from interactions among mutualists. Indirect costs of plant defense by ants, for example, can outweigh benefits if ants deter pollinators. Plants can dissuade ants from occupying flowers, but such arrangements may break down when novel ant partners infiltrate mutualisms. Here, we examine how floral visitation by ants affects pollination services when the invasive Argentine ant (Linepithema humile) replaces a native ant species in a food-for-protection mutualism with the coast barrel cactus (Ferocactus viridescens), which, like certain other barrel cacti, produces extrafloral nectar. We compared the effects of floral visitation by the Argentine ant with those of the most prevalent native ant species (Crematogaster californica). Compared to C. californica, the Argentine ant was present in higher numbers in flowers. Cactus bees (Diadasia spp.), the key pollinators in this system, spent less time in flowers when cacti were occupied by the Argentine ant compared to when cacti were occupied by C. californica. Presumably as a consequence of decreased duration of floral visits by Diadasia, cacti occupied by L. humile set fewer seeds per fruit and produced fewer seeds overall compared to cacti occupied by C. californica. These data illustrate the importance of mutualist identity in cases where plants balance multiple mutualisms. Moreover, as habitats become increasingly infiltrated by introduced species, the loss of native mutualists and their replacement by non-native species may alter the shape of trade-offs between plant defense and reproduction. PMID:23892582

  11. Photosynthetic responses of field-grown Pinus radiata trees to artificial and aphid-induced defoliation.

    PubMed

    Eyles, Alieta; Smith, David; Pinkard, Elizabeth A; Smith, Ian; Corkrey, Ross; Elms, Stephen; Beadle, Chris; Mohammed, Caroline

    2011-06-01

    The phloem-feeding aphid Essigella californica represents a potential threat to the productivity of Pinus radiata plantations in south-eastern Australia. Five- and nine-year-old field trials were used to characterize the effects of artificial and natural aphid-induced (E. californica) defoliation, respectively, on shoot photosynthesis and growth. Photosynthetic capacity (A(max)) was significantly greater following a 25% (D25) (13.8 µmol m(-2) s(-1)) and a 50% (D50) (15.9 µmol m(-2) s(-1)) single-event upper-crown artificial defoliation, 3 weeks after defoliation than in undefoliated control trees (12.9 µmol m(-2) s(-1)). This response was consistently observed for up to 11 weeks after the defoliation event; by Week 16, there was no difference in A(max) between control and defoliated trees. In the D50 treatment, this increased A(max) was not sufficient to fully compensate for the foliage loss as evidenced by the reduced diameter increment (by 15%) in defoliated trees 36 weeks after defoliation. In contrast, diameter increment of trees in the D25 treatment was unaffected by defoliation. The A(max) of trees experiencing upper-crown defoliation by natural and repeated E. californica infestations varied, depending on host genotype. Despite clear differences in defoliation levels between resistant and susceptible genotypes (17 vs. 35% of tree crown defoliated, respectively), growth of susceptible genotypes was not significantly different from that of resistant genotypes. The observed increases in A(max) in the lower crown of the canopy following attack suggested that susceptible genotypes were able to partly compensate for the loss of foliage by compensatory photosynthesis. The capacity of P. radiata to regulate photosynthesis in response to natural aphid-induced defoliation provides evidence that the impact of E. californica attack on stem growth will be less than expected, at least for up to 35% defoliation. PMID:21697147

  12. Non-virulence of a recombinant shrimp nidovirus is associated with its non structural gene sequence and not a large structural gene deletion

    SciTech Connect

    Gangnonngiw, Warachin; Anantasomboon, Gun; Sang-oum, Wiwat; Sriurairatana, Siriporn; Sritunyalucksana, Kallaya; Flegel, Timothy W.

    2009-03-01

    RT-PCR using a commercial kit for yellow head virus (YHV) detection in growth-retarded shrimp yielded an unusual 777 bp amplicon instead of expected amplicons of 277 bp for YHV type-1 (YHV-1) or 406 bp for YHV type-2 (YHV-2). Cloning and sequencing (GenBank (EU170438)) revealed approximately 80% identity to non-structural (NS) ORF1b sequences of both YHV-1 (GenBank (AA083987)) and YHV-2 (GenBank (AF227196)), indicating an atypical YHV type (A-YHV) phylogenetically equidistant from both types. An RT-PCR test specifically designed for A-YHV revealed that it was uncommon and that its occurrence in shrimp culture ponds did not correlate with growth retardation or mortality. By immunohistochemistry with YHV-specific monoclonal antibodies, the A-YHV gave positive reactions for envelope protein gp64 and capsid protein p20, but not for envelope protein gp116, even though gp116 and gp64 originate from a polyprotein of ORF3. Lack of gp116 immunoreactivity correlated with a large ORF3 deletion (GenBank (EU123854)) in the region of the protein targeted by an MAb against gp116. Transmission electron microscopy of A-YHV-infected shrimp revealed only unenveloped pre-virions. During manuscript revision, information received revealed that typing of YHV isolates based on sequences of ORF1b and ORF3 had yielded several geographical types, including one virulent type (YHV-1b) with an ORF3 deletion sequence that matched the sequence of A-YHV. Using these sequences and an additional A-YHV sequence ( (EU853170)) from the ORF1b typing region, A-YHV potentially represents a recombinant between type 1b and type 5. SDS-PAGE and Western blot analysis revealed that type 1b produced a gp116 deletion protein that did not bind with the MAb or polyclonal Ab to normal gp116. Overall, the information suggested that lack of A-YHV virulence was associated with the NS gene sequence linked to ORF1b rather than the deletion in ORF3.

  13. Intracellular self-assembly based multi-labeling of key viral components: Envelope, capsid and nucleic acids.

    PubMed

    Wen, Li; Lin, Yi; Zhang, Zhi-Ling; Lu, Wen; Lv, Cheng; Chen, Zhi-Liang; Wang, Han-Zhong; Pang, Dai-Wen

    2016-08-01

    Envelope, capsid and nucleic acids are key viral components that are all involved in crucial events during virus infection. Thus simultaneous labeling of these key components is an indispensable prerequisite for monitoring comprehensive virus infection process and dissecting virus infection mechanism. Baculovirus was genetically tagged with biotin on its envelope protein GP64 and enhanced green fluorescent protein (EGFP) on its capsid protein VP39. Spodoptera frugiperda 9 (Sf9) cells were infected by the recombinant baculovirus and subsequently fed with streptavidin-conjugated quantum dots (SA-QDs) and cell-permeable nucleic acids dye SYTO 82. Just by genetic engineering and virus propagation, multi-labeling of envelope, capsid and nucleic acids was spontaneously accomplished during virus inherent self-assembly process, significantly simplifying the labeling process while maintaining virus infectivity. Intracellular dissociation and transportation of all the key viral components, which was barely reported previously, was real-time monitored based on the multi-labeling approach, offering opportunities for deeply understanding virus infection and developing anti-virus treatment. PMID:27209260

  14. Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection.

    PubMed

    Xu, Xin-Gang; Tong, De-Wen; Wang, Zhi Sheng; Zhang, Qi; Li, Zhao-Cai; Zhang, Kuan; Li, Wei; Liu, Hung-Jen

    2011-06-01

    Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections. PMID:21793437

  15. Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.

    PubMed

    Xu, Xin-Gang; Wang, Zhi-Sheng; Zhang, Qi; Li, Zhao-Cai; Ding, Li; Li, Wei; Wu, Hung-Yi; Chang, Ching-Dong; Lee, Long-Huw; Tong, De-Wen; Liu, Hung-Jen

    2012-02-01

    The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections. PMID:22172969

  16. Retinal transduction profiles by high-capacity viral vectors

    PubMed Central

    Puppo, Agostina; Cesi, Giulia; Marrocco, Elena; Piccolo, Pasquale; Jacca, Sarah; Shayakhmetov, Dmitry M.; Parks, Robin J.; Davidson, Beverly L.; Colloca, Stefano; Brunetti-Pierri, Nicola; Ng, Philip; Donofrio, Gaetano; Auricchio, Alberto

    2014-01-01

    Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, the limited cargo capacity of AAV prevents their use for therapy of those inherited retinopathies (IRs) due to mutations in large (>5kb) genes. Viral vectors derived from Adenovirus (Ad), Lentivirus (LV) and Herpesvirus (HV) can package large DNA sequences but do not target efficiently retinal photoreceptors (PRs) where the majority of genes responsible for IRs are expressed. Here, we have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes, and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium (RPE). We found that LV-GP64 tends to transduce more PRs than the canonical LV-VSVG albeit this was restricted to a narrow region. We observed more extensive PR transduction with HdAd1, 2 and 5/F35++ than with LV, although none of them outperformed the canonical HdAd5 or matched the extension of PR transduction achieved with AAV2/8. PMID:24989814

  17. Differential Susceptibilities to BmNPV Infection of Two Cell Lines Derived from the Same Silkworm Ovarian Tissues

    PubMed Central

    Zhang, Chun-Dong; He, Qian; Dong, Zhan-Qi; Cao, Ming-Ya; Dong, Xiao-Long; Pan, Cai-Xia; Lu, Cheng; Pan, Min-Hui

    2014-01-01

    We previously established and characterized two insect cell lines (BmN-SWU1 and BmN-SWU2) from Bombyx mori ovaries. Here, we examined their differential susceptibilities to Bombyx mori nucleopolyhedrovirus (BmNPV) despite having originated from the same tissue source. BmN-SWU1 cells were susceptible and supported high titers of BmNPV replication, while BmN-SWU2 cells were resistant to BmNPV infection. Subcellular localization analysis demonstrated that very few BmNPV particles could be imported into BmN-SWU2 cells. However, initiation of BmNPV DNA replication but not amplification was detected in BmN-SWU2 cells after transfection with vA4prm-VP39-EGFP bacmid DNA. BmNPV transcription assays showed that late and very late but not early viral genes apparently were blocked in BmNSWU2 cells by unknown mechanisms. Further syncytium formation assays demonstrated that the BmNPV envelope fusion protein GP64 could not mediate BmN-SWU2 host cell-cell membrane fusion. Taken together, these results indicate that these two cell lines represent optimal tools for investigating host-virus interactions and insect antiviral mechanisms. PMID:25221982

  18. Characterization of rbcL group IA introns from two colonial volvocalean species (Chlorophyceae).

    PubMed

    Nozaki, H; Ohta, N; Yamada, T; Takano, H

    1998-05-01

    Group I introns were reported for the first time in the large subunit of Rubisco (rbcL) genes, using two colonial green algae, Pleodorina californica and Gonium multicoccum (Volvocales). The rbcL gene of P. californica contained an intron (PIC intron) of 1320 bp harboring an open reading frame (ORF). The G. multicoccum rbcL gene had two ORF-lacking introns of 549 (GM1 intron) and 295 (GM2 intron) base pairs. Based on the conserved nucleotide sequences of the secondary structure, the PIC and GM1 introns were assigned to group IA2 whereas the GM2 intron belonged to group IA1. Southern hybridization analyses of nuclear and chloroplast DNAs indicated that such intron-containing rbcL genes are located in the chloroplast genome. Sequencing RNAs from the two algae revealed that these introns are spliced out during mRNA maturation. In addition, the PIC and GM1 introns were inserted in the same position of the rbcL exons, and phylogenetic analysis of group IA introns indicated a close phylogenetic relationship between the PIC and GM1 introns within the lineage of bacteriophage group IA2 introns. However, P. californica and G. multicoccum occupy distinct clades in the phylogenetic trees of the colonial Volvocales, and the majority of other colonial volvocalean species do not have such introns in the rbcL genes. Therefore, these introns might have been recently inserted in the rbcL genes independently by horizontal transmission by viruses or bacteriophage. PMID:9620266

  19. A test system to evaluate the susceptibility of Oregon, USA, native stream invertebrataes to triclopyr and carbaryl.

    PubMed

    Peterson, J L; Jepson, P C; Jenkins, J J

    2001-10-01

    The susceptibility of six indigenous macroinvertebrate species representative of U.S. Pacific Northwest streams (Ameletus sp., Brachycentrus americanus, Calineuria californica, Cinygma sp., Lepidostoma unicolor, Psychoglypha sp. early and late instar) to formulated triclopyr ester (herbicide) and carbaryl (insecticide) was determined using laboratory bioassays. Acute toxicity was expressed as the lethal concentration to 50% (LC50) and 1% (LC1) of the test population based on a 96-h exposure duration. Carbaryl was found to be 1,000 times more toxic than triclopyr for all the organisms tested. The LCI values (7.5, 28.8, 9.0, 3.0, 9.5, 14.8, 33.8 microg/L, respectively, for carbaryl and 1.8, 3.9, 4.0, 4.2, 29.0, 16.1 mg/L, respectively, for triclopyr) were used in the calculation of hazardous concentration to 5% of the stream macroinvertebrate community (HC5) based on the lower 95% confidence limit (HC5/95). The hazardous concentration (HC5/95) for triclopyr was 0.11 mg/L and for carbaryl ranged from 0.43 to 0.66 microg/L, respectively. Triclopyr and carbaryl symptomology were analyzed for two organisms, C. californica and Cinygma sp. Carbaryl symptomology included knockdown and moribund states with severity and time of appearance being a function of dose. In triclopyr poisoning, death occurred suddenly with little or no symptomology. Time to 50% mortality (LT50) values were consistently higher for C. californica than for Cinygma sp. exposed to both chemicals at similar concentrations. PMID:11596752

  20. Two new desert Eschscholzia (Papaveraceae) from southwestern North America

    PubMed Central

    Still, Shannon M.

    2014-01-01

    Abstract Two new species of Eschscholzia are described. Both are found in the deserts of California and one extends outside the state boundary into Arizona. Eschscholzia androuxii Still, sp. nov. is found mainly in and around Joshua Tree National Park in Riverside and San Bernardino counties. Eschscholzia papastillii Still, sp. nov. is found from the northern Mojave south through Joshua Tree National Park to central Imperial County. Both are annuals found in coarse, sandy soil and have yellow flowers typical of desert Eschscholzia. Eschscholzia papastillii has an expanded receptacular rim similar to that of Eschscholzia californica. Eschscholzia androuxii has anthocyanin bands around the stamen filaments. PMID:24843288

  1. Infrared neural stimulation (INS) inhibits electrically evoked neural responses in the deaf white cat

    NASA Astrophysics Data System (ADS)

    Richter, Claus-Peter; Rajguru, Suhrud M.; Robinson, Alan; Young, Hunter K.

    2014-03-01

    Infrared neural stimulation (INS) has been used in the past to evoke neural activity from hearing and partially deaf animals. All the responses were excitatory. In Aplysia californica, Duke and coworkers demonstrated that INS also inhibits neural responses [1], which similar observations were made in the vestibular system [2, 3]. In deaf white cats that have cochleae with largely reduced spiral ganglion neuron counts and a significant degeneration of the organ of Corti, no cochlear compound action potentials could be observed during INS alone. However, the combined electrical and optical stimulation demonstrated inhibitory responses during irradiation with infrared light.

  2. Helix peptide immunoreactivity pattern in the nervous system of juvenile aplysia.

    PubMed

    Ierusalimsky, V N; Boguslavsky, D V; Belyavsky, A V; Balaban, P M

    2003-12-12

    Distribution of neurons immunopositive to antibody against the small peptides encoded by the Helix Command-Specific 2 (HCS2) gene in the central nervous system of juvenile Aplysia californica was investigated. The HCS2 gene is specifically expressed in the withdrawal behavior neurons of the terrestrial snail Helix lucorum. In Aplysia, 20-25 immunopositive neuronal somata were observed on dorsal surface of each pleural ganglion (including a giant pleural neuron). The HCS2-encoded peptide immunopositive fibers were observed in neuropiles of all ganglia and in many nerves. Functional significance of Aplysia immunopositive cells is discussed. PMID:14667582

  3. The 'headache tree' via umbellulone and TRPA1 activates the trigeminovascular system.

    PubMed

    Nassini, Romina; Materazzi, Serena; Vriens, Joris; Prenen, Jean; Benemei, Silvia; De Siena, Gaetano; la Marca, Giancarlo; Andrè, Eunice; Preti, Delia; Avonto, Cristina; Sadofsky, Laura; Di Marzo, Vincenzo; De Petrocellis, Luciano; Dussor, Greg; Porreca, Frank; Taglialatela-Scafati, Orazio; Appendino, Giovanni; Nilius, Bernd; Geppetti, Pierangelo

    2012-02-01

    The California bay laurel or Umbellularia californica (Hook. & Arn.) Nutt., is known as the 'headache tree' because the inhalation of its vapours can cause severe headache crises. However, the underlying mechanism of the headache precipitating properties of Umbellularia californica is unknown. The monoterpene ketone umbellulone, the major volatile constituent of the leaves of Umbellularia californica, has irritating properties, and is a reactive molecule that rapidly binds thiols. Thus, we hypothesized that umbellulone stimulates the transient receptor potential ankyrin 1 channel in a subset of peptidergic, nocioceptive neurons, activating the trigeminovascular system via this mechanism. Umbellulone, from µM to sub-mM concentrations, selectively stimulated transient receptor potential ankyrin 1-expressing HEK293 cells and rat trigeminal ganglion neurons, but not untransfected cells or neurons in the presence of the selective transient receptor potential ankyrin 1 antagonist, HC-030031. Umbellulone evoked a calcium-dependent release of calcitonin gene-related peptide from rodent trigeminal nerve terminals in the dura mater. In wild-type mice, umbellulone elicited excitation of trigeminal neurons and released calcitonin gene-related peptide from sensory nerve terminals. These two responses were absent in transient receptor potential ankyrin 1 deficient mice. Umbellulone caused nocioceptive behaviour after stimulation of trigeminal nerve terminals in wild-type, but not transient receptor potential ankyrin 1 deficient mice. Intranasal application or intravenous injection of umbellulone increased rat meningeal blood flow in a dose-dependent manner; a response selectively inhibited by systemic administration of transient receptor potential ankyrin 1 or calcitonin gene-related peptide receptor antagonists. These data indicate that umbellulone activates, through a transient receptor potential ankyrin 1-dependent mechanism, the trigeminovascular system, thereby causing

  4. Two new desert Eschscholzia (Papaveraceae) from southwestern North America.

    PubMed

    Still, Shannon M

    2014-01-01

    Two new species of Eschscholzia are described. Both are found in the deserts of California and one extends outside the state boundary into Arizona. Eschscholzia androuxii Still, sp. nov. is found mainly in and around Joshua Tree National Park in Riverside and San Bernardino counties. Eschscholzia papastillii Still, sp. nov. is found from the northern Mojave south through Joshua Tree National Park to central Imperial County. Both are annuals found in coarse, sandy soil and have yellow flowers typical of desert Eschscholzia. Eschscholzia papastillii has an expanded receptacular rim similar to that of Eschscholzia californica. Eschscholzia androuxii has anthocyanin bands around the stamen filaments. PMID:24843288

  5. Eurasian jays (Garrulus glandarius) overcome their current desires to anticipate two distinct future needs and plan for them appropriately

    PubMed Central

    Cheke, Lucy G.; Clayton, Nicola S.

    2012-01-01

    Western scrub-jays (Aphelocoma californica) have been shown to overcome present satiety to cache food they will desire in the future. Here, we show that another corvid, the Eurasian jay (Garrulus glandarius), can distinguish between two distinct future desires and plan for each appropriately, despite experiencing a conflicting current motivation. We argue that these data address the criticisms of previous work, and suggest a way in which associative learning processes and future-oriented cognition may combine to allow prospective behaviour. PMID:22048890

  6. Culture and maintenance of selected invertebrates in the laboratory and classroom.

    PubMed

    Smith, Stephen A; Scimeca, Joseph M; Mainous, Mary E

    2011-01-01

    Invertebrate species have been used for many years in the laboratory and teaching environment. We discuss some of the most commonly maintained invertebrates--the nematode (Caenorhabditis elegans), the California sea hare (Aplysia californica), the fruit fly (Drosophila melanogaster), terrestrial hermit crabs, the horseshoe crab (Limulus polyphemus), and cephalopods--and briefly describe general techniques for culturing them in captivity. The aim of this article is to give potential users an idea of the materials, methods, and effort required to maintain each type of organism in a laboratory or classroom setting. PMID:21709308

  7. Haemocyanins in spiders, III. Chemical and physical properties of the proteins in Dugesiella and Cupiennius blood.

    PubMed

    Markl, J; Schmid, R; Czichos-Tiedt, S; Linzen, B

    1976-12-01

    The haemolymph of the tarantulas, Dugesiella (Eurypelma) californica and Dugesiella (Eurypelma) helluo contains high molecular weight haemocyanin (80-82% of total blood proteins) and a second protein not related to haemocyanin (18-20%). In the Lycosid spider, Cupiennius salei, haemocyanin (75% of total blood protein) occurs in two states of association. The haemocyanins were isolated by ultracentrifugation, gel filtration, isoelectric focusing, or preparative gel electrophoresis. Their sedimentation constants are 36.7 S (both tarantulas), 23.4 S and 15.9 S (Cupiennius). After alkaline dissociation, polypeptides sedimenting at 5.8 S (D. californica) and 4.7 S (Cupiennius) were obtained. The molecular weight of the intact functional subunit is (by sedimentation equilibrium) 70 300 (D. californica) and 69 900 (Cupiennius). Copper analysis results in closely similar values. By sodium dodecylsulphate gel electrophoresis, molecular weights of 71 000 (D. californica), 72 000 (Cupiennius) and 74 000 (D. helluo) were obtained. Denaturation with various agents did not lead to smaller polypeptides. The amino acid composition of the haemocyanins was determined (Table 1). The amino end group is blocked. The haemocyanins contain 1.2-1.5% of neutral carbohydrates and 0.3-0.5% of glucosamine (possibly acetylated). The neutral carbohydrates were identified with glucose, mannose, fucose, and arabinose, glucose being the dominant species. Neuraminic acid was not detected. The haemocyanins of the three species cannot be distinguished by their carbohydrate moieties, while there is a significant difference in amino acid composition between tarantula and Cupiennius haemocyanins. The second, non-respiratory protein isolated from spider blood sediments with 16.1 S (Dugesiella) or 15.9 S (Cupiennius). Its isoelectric point is at pH 5.5 It is stable in weakly alkaline solutions but can be denatured to yield polypeptide chains with molecular weights of 95 000 and 110 000. The amino acid

  8. Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors.

    PubMed

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Shenkarev, Zakhar O; Kasheverov, Igor E; Chugunov, Anton O; Kulbatskii, Dmitrii S; Myshkin, Mikhail Yu; Utkin, Yuri N; Efremov, Roman G; Tsetlin, Victor I; Arseniev, Alexander S; Kirpichnikov, Mikhail P; Dolgikh, Dmitry A

    2016-09-01

    'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs. PMID:27343701

  9. Host Phenology and Leaf Effects on Susceptibility of California Bay Laurel to Phytophthora ramorum.

    PubMed

    Johnston, Steven F; Cohen, Michael F; Torok, Tamas; Meentemeyer, Ross K; Rank, Nathan E

    2016-01-01

    Spread of the plant pathogen Phytophthora ramorum, causal agent of the forest disease sudden oak death, is driven by a few competent hosts that support spore production from foliar lesions. The relationship between traits of a principal foliar host, California bay laurel (Umbellularia californica), and susceptibility to P. ramorum infection were investigated with multiple P. ramorum isolates and leaves collected from multiple trees in leaf-droplet assays. We examined whether susceptibility varies with season, leaf age, or inoculum position. Bay laurel susceptibility was highest during spring and summer and lowest in winter. Older leaves (>1 year) were more susceptible than younger ones (8 to 11 months). Susceptibility was greater at leaf tips and edges than the middle of the leaf. Leaf surfaces wiped with 70% ethanol were more susceptible to P. ramorum infection than untreated leaf surfaces. Our results indicate that seasonal changes in susceptibility of U. californica significantly influence P. ramorum infection levels. Thus, in addition to environmental variables such as temperature and moisture, variability in host plant susceptibility contributes to disease establishment of P. ramorum. PMID:26439707

  10. Serotonin-like immunoreactivity in the central and peripheral nervous systems of the interstitial acochlidean Asperspina sp. (Opisthobranchia).

    PubMed

    Hochberg, Rick

    2007-08-01

    Species of Acochlidea are common members of the marine interstitial environment and defined in part by their minuscule size and highly divergent morphology relative to other benthic opisthobranchs. Despite these differences, acochlideans such as species of Asperspina display many plesiomorphic characteristics, including an unfused condition of their neural ganglia. To gain insight into the distribution of specific neural subsets within acochlidean ganglia, a species of Asperspina was studied by using anti-serotonin immunohistochemistry and epifluorescence and confocal laser scanning microscopy. Results reveal similarities between Asperspina and larger opisthobranchs in the general distribution of serotonergic perikarya in the central nervous system. Specifically, the arrangement of perikarya into regional clusters within the cerebral and pedal ganglia and the absence of immunoreactive perikarya in the pleural ganglia are similar to the model species of Aplysia californica, Pleurobranchaea californica, and Tritonia diomedea. Moreover, serotonergic innervation of the rhinophores in all opisthobranchs, including Asperspina sp., originates from the cerebral ganglion instead of directly from the rhinophoral ganglion. Serotonergic innervation of the body wall, including the epithelium, muscles, and pedal sole, appears to arise exclusively from pedal and accessory ganglia. These observations indicate a general conservation of serotonin-like immunoreactivity in the central and peripheral nervous systems of acochlidean and other benthic opisthobranchs. PMID:17679719

  11. Chemical composition of inks of diverse marine molluscs suggests convergent chemical defenses.

    PubMed

    Derby, Charles D; Kicklighter, Cynthia E; Johnson, P M; Zhang, Xu

    2007-05-01

    Some marine molluscs, notably sea hares, cuttlefish, squid, and octopus, release ink when attacked by predators. The sea hare Aplysia californica releases secretions from the ink gland and opaline gland that protect individuals from injury or death from predatory spiny lobsters through a combination of mechanisms that include chemical deterrence, sensory disruption, and phagomimicry. The latter two mechanisms are facilitated by millimolar concentrations of free amino acids (FAA) in sea hare ink and opaline, which stimulate the chemosensory systems of predators, ultimately leading to escape by sea hares. We hypothesize that other inking molluscs use sensory disruption and/or phagomimicry as a chemical defense. To investigate this, we examined concentrations of 21 FAA and ammonium in the defensive secretions of nine species of inking molluscs: three sea hares (Aplysia californica, Aplysia dactylomela, Aplysia juliana) and six cephalopods (cuttlefish: Sepia officinalis; squid: Loligo pealei, Lolliguncula brevis, Dosidicus gigas; octopus: Octopus vulgaris, Octopus bimaculoides). We found millimolar levels of total FAA and ammonium in these secretions, and the FAA in highest concentration were taurine, aspartic acid, glutamic acid, alanine, and lysine. Crustaceans and fish, which are major predators of these molluscs, have specific receptor systems for these FAA. Our chemical analysis supports the hypothesis that inking molluscs have the potential to use sensory disruption and/or phagomimicry as a chemical defense. PMID:17393278

  12. Social Organization in Parasitic Flatworms--Four Additional Echinostomoid Trematodes Have a Soldier Caste and One Does Not.

    PubMed

    Garcia-Vedrenne, Ana E; Quintana, Anastasia C E; DeRogatis, Andrea M; Martyn, Kayla; Kuris, Armand M; Hechinger, Ryan F

    2016-02-01

    Complex societies where individuals exhibit division of labor with physical polymorphism, behavioral specialization, and caste formation have evolved several times throughout the animal kingdom. Recently, such complex sociality has been recognized in digenean trematodes; evidence is limited to 6 marine species. Hence, the extent to which a soldier caste is present throughout the Trematoda is sparsely documented, and there are no studies detailing the structure of a species lacking such a social structure. Here we examine colony structure for an additional 5 echinostomoid species, 4 of which infect the marine snail Cerithidea californica and 1 (Echinostoma liei) that infects the freshwater snail Biomphalaria glabrata . For all species, we present redia morphology (pharynx and body size) and the distribution of individuals of different castes throughout the snail body. When morphological evidence indicated the presence of a soldier caste, we assessed behavior by measuring attack rates of the different morphs toward heterospecific trematodes. Our findings indicate that each of the 4 species from C. californica have a permanent soldier caste while E. liei does not. The observed intra- and inter-specific variation of caste structure for those species with soldiers, and the documentation of colony structure for a species explicitly lacking permanent soldiers, emphasizes the diverse nature of trematode sociality and the promise of the group to permit comparative investigations of the evolution and ecology of sociality. PMID:26560890

  13. Revalidation of the spider genus Citharoceps Chamberlin, 1924 (Araneae, Segestriidae)

    PubMed Central

    Giroti, André Marsola; Brescovit, Antonio Domingos

    2015-01-01

    Abstract Citharoceps Chamberlin was diagnosed by the presence of a very distinctive stridulatory apparatus composed of two patches of ridges on the sides of the cephalic region, and a stridulatory thorn on the prolateral region of the femur I. Currently, this genus is a junior synonym of Ariadna Audouin, with the assumption that the stridulatory apparatus could constitute an exclusive feature of its unique known species, Citharoceps fidicina Chamberlin, currently senior synonym of Citharoceps californica Chamberlin & Ivie. In the present study, Citharoceps is revalidated and redescribed based on the occurrence of the stridulatory apparatus in Citharoceps fidicina and Segestria cruzana Chamberlin & Ivie, and also on the presence of distinguishable characters, such as the length of the labium-sternum junction, ventral median spine on male metatarsi I, and strong sclerotized interpulmonary fold in females, forming a conspicuous median flap. Segestria cruzana is transfered to Citharoceps, with Citharoceps californica removed from the synonym of Citharoceps fidicina, and proposed as a junior synonym of Citharoceps cruzana, due to the similarity between the additional material examined and the original description. Males of Citharoceps fidicina and Citharoceps cruzana are described for the first time. PMID:25901118

  14. Review of Canadian species of the genera Gnathusa Fenyes, Mniusa Mulsant & Rey and Ocyusa Kraatz (Coleoptera, Staphylinidae, Aleocharinae)

    PubMed Central

    Klimaszewski, Jan; Webster, Reginald P.; Langor, David W.; Bourdon, Caroline; Hammond, H.E. James; Pohl, Greg R.; Godin, Benoit

    2014-01-01

    Abstract Four species of Gnathusa Fenyes (G. alfacaribou Klimaszewski & Langor, G. caribou Lohse, G. eva Fenyes, and G. tenuicornis Fenyes) occur in the Nearctic and in Canada. Three species of Ocyusa Kraatz (O. asperula Casey, O. californica Bernhauer, O. canadensis Lohse), and three species of Mniusa Mulsant and Ray (M. minutissima (Klimaszewski & Langor), M. yukonensis (Klimaszewski & Godin), and M. odelli Klimaszewski & Webster, sp. n.), are known from the Nearctic and all but O. californica occur in Canada. The recently described Gnathusa minutissima Klimaszewski and Langor and Ocyusa yukonensis Klimaszewski and Godin, are transferred here to the genus Mniusa Mulsant & Rey. New provincial and state records are reported for: G. eva (Alberta), G. tenuicornis (Alberta, Oregon, and New Brunswick), O. canadensis (New Brunswick and Newfoundland), M. minutissima (New Brunswick), and M. yukonensis (Nova Scotia, New Brunswick, Quebec, and British Columbia). The female of M. yukonensis was discovered and is illustrated for the first time. The genus Mniusa is reported for the first time from Canada and represents the first confirmed generic record for North America. Keys for identification of all Canadian species, images of body and genital structures, maps showing distribution mainly in Canada, and new bionomics data are provided. PMID:24899860

  15. BmREEPa Is a Novel Gene that Facilitates BmNPV Entry into Silkworm Cells

    PubMed Central

    Wang, Wei; Pan, Cai-xia; Wu, Yun-fei; Du, Guo-yu; Chen, Peng; Lu, Cheng; Pan, Min-hui

    2015-01-01

    We previously established two silkworm cell lines, BmN-SWU1 and BmN-SWU2, from Bombyx mori ovaries. BmN-SWU1 cells are susceptible while BmN-SWU2 cells are highly resistant to BmNPV infection. Interestingly, we found that the entry of BmNPV into BmN-SWU2 cells was largely inhibited. To explore the mechanism of this inhibition, in this study we used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative protein expression profiling and identified 629 differentially expressed proteins between the two cell lines. Among them, we identified a new membrane protein termed BmREEPa. The gene encoding BmREEPa transcribes two splice variants; a 573 bp long BmREEPa-L encoding a protein with 190 amino acids and a 501 bp long BmREEPa-S encoding a protein with 166 amino acids. BmREEPa contains a conserved TB2/DP, HVA22 domain and three transmembrane domains. It is localized in the plasma membrane with a cytoplasmic C-terminus and an extracellular N-terminus. We found that limiting the expression of BmREEPa in BmN-SWU1 cells inhibited BmNPV entry, whereas over-expression of BmREEPa in BmN-SWU2 cells promoted BmNPV entry. Our results also indicated that BmREEPa can interact with GP64, which is the key envelope fusion protein for BmNPV entry. Taken together, the findings of our study revealed that BmREEPa is required for BmNPV to gain entry into silkworm cells, and may provide insights for the identification of BmNPV receptors. PMID:26656276

  16. BmREEPa Is a Novel Gene that Facilitates BmNPV Entry into Silkworm Cells.

    PubMed

    Dong, Xiao-long; Liu, Tai-hang; Wang, Wei; Pan, Cai-xia; Wu, Yun-fei; Du, Guo-yu; Chen, Peng; Lu, Cheng; Pan, Min-hui

    2015-01-01

    We previously established two silkworm cell lines, BmN-SWU1 and BmN-SWU2, from Bombyx mori ovaries. BmN-SWU1 cells are susceptible while BmN-SWU2 cells are highly resistant to BmNPV infection. Interestingly, we found that the entry of BmNPV into BmN-SWU2 cells was largely inhibited. To explore the mechanism of this inhibition, in this study we used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative protein expression profiling and identified 629 differentially expressed proteins between the two cell lines. Among them, we identified a new membrane protein termed BmREEPa. The gene encoding BmREEPa transcribes two splice variants; a 573 bp long BmREEPa-L encoding a protein with 190 amino acids and a 501 bp long BmREEPa-S encoding a protein with 166 amino acids. BmREEPa contains a conserved TB2/DP, HVA22 domain and three transmembrane domains. It is localized in the plasma membrane with a cytoplasmic C-terminus and an extracellular N-terminus. We found that limiting the expression of BmREEPa in BmN-SWU1 cells inhibited BmNPV entry, whereas over-expression of BmREEPa in BmN-SWU2 cells promoted BmNPV entry. Our results also indicated that BmREEPa can interact with GP64, which is the key envelope fusion protein for BmNPV entry. Taken together, the findings of our study revealed that BmREEPa is required for BmNPV to gain entry into silkworm cells, and may provide insights for the identification of BmNPV receptors. PMID:26656276

  17. Plant and Root Growth Responses to Heterogeneous Supplies of Soil Water in Two Coastal Shrubs of California.

    NASA Astrophysics Data System (ADS)

    Cole, S.; Mahall, B. E.

    2007-05-01

    Much effort has been focused on identifying plant and root growth responses to heterogeneous supplies of soil nutrients. However, in many circumstances, soil water may limit plant growth and it too can have a patchy distribution. In our research we asked: 1) What is the ecological significance of soil moisture heterogeneity to plant growth in a California coastal dune habitat? 2) How does growth of whole plants and roots respond to soil moisture heterogeneity? and 3) Can roots of these species sense and grow towards moisture-rich areas (hydrotropism) in a natural medium? To address these questions: we conducted comparative field studies of water relations and growth of Artemisia californica and Eriogonum parvifolium; we performed a growth rate study of roots and plants in experimental pots with either patchy or homogeneous distributions of soil water; and we analyzed individual root growth in sand-filled observation chambers in response to moisture-rich patches and resultant soil water gradients. In the field, correlations between daily photosynthetic rates, active leaf display and predawn xylem pressure potentials (ΨPD) indicated that access to water limited growth in A. californica and E. parvifolium. These species, common in habit and habitat, differed in their ability to access water with E. parvifolium having overall higher ΨPD than A. californica (repeated measures ANOVA, P < 0.01). Our growth rate study revealed that patchy supplies of water did not reduce the relative growth rate or average size of E. parvifolium (two-tailed t-tests, P > 0.25). It appears that modified partitioning of growth both at the whole plant and root system level permitted E. parvifolium to maintain growth in patchy soil water conditions. We found that E. parvifolium increased allocation to roots and proliferated in moisture-rich patches in the patchy soil water treatment. Root length density and the proportion of root mass present in the patch was 20- to >100-fold greater in and

  18. An Expedient Synthesis, Acetylcholinesterase Inhibitory Activity, and Molecular Modeling Study of Highly Functionalized Hexahydro-1,6-naphthyridines

    PubMed Central

    Almansour, Abdulrahman I.; Suresh Kumar, Raju; Arumugam, Natarajan; Basiri, Alireza; Kia, Yalda; Ashraf Ali, Mohamed

    2015-01-01

    A series of hexahydro-1,6-naphthyridines were synthesized in good yields by the reaction of 3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones with cyanoacetamide in the presence of sodium ethoxide under simple mixing at ambient temperature for 6–10 minutes and were assayed for their acetylcholinesterase (AChE) inhibitory activity using colorimetric Ellman's method. Compound 4e with methoxy substituent at ortho-position of the phenyl rings displayed the maximum inhibitory activity with IC50 value of 2.12 μM. Molecular modeling simulation of 4e was performed using three-dimensional structure of Torpedo californica AChE (TcAChE) enzyme to disclose binding interaction and orientation of this molecule into the active site gorge of the receptor. PMID:25710037

  19. Effects of pelletized anticoagulant rodenticides on California quail

    USGS Publications Warehouse

    Blus, L.J.; Henny, C.J.; Grove, R.A.

    1985-01-01

    A moribund, emaciated California quail (Callipepla californica) that was found in an orchard in the state of Washington had an impacted crop and gizzard. Pellets containing the anticoagulant chlorophacinone (Rozol, RO) were in the crop; the gizzard contents consisted of a pink mass of paraffin that was selectively accumulated from the paraffinized pellets. The plasma prothrombin time of 28 sec was near that determined for control quail. The signs of RO intoxication seen in the moribund wild quail were duplicated in captive quail given ad libitum diets of either RO or another paraffinized chlorophacinone pellet (Mr. Rat Guard II, MRG). This left little doubt that paraffin impaction of the gizzard was the primary problem. All captive quail fed RO or MRG pellets showed no increases in prothrombin times compared to control values, died in an emaciated condition, and had gizzards impacted with paraffin.

  20. Characterization of alpha-conotoxin interactions with the nicotinic acetylcholine receptor and monoclonal antibodies.

    PubMed Central

    Ashcom, J D; Stiles, B G

    1997-01-01

    The venoms of predatory marine cone snails, Conus species, contain numerous peptides and proteins with remarkably diverse pharmacological properties. One group of peptides are the alpha-conotoxins, which consist of 13-19 amino acids constrained by two disulphide bonds. A biologically active fluorescein derivative of Conus geographus alpha-conotoxin GI (FGI) was used in novel solution-phase-binding assays with purified Torpedo californica nicotinic acetylcholine receptor (nAchR) and monoclonal antibodies developed against the toxin. The binding of FGI to nAchR or antibody had apparent dissociation constants of 10-100 nM. Structure-function studies with alpha-conotoxin GI analogues composed of a single disulphide loop revealed that different conformational restraints are necessary for effective toxin interactions with nAchR or antibodies. PMID:9359860

  1. Soils and vegetation of Santa Barbara Island, Channel Islands National Park, California, USA

    NASA Astrophysics Data System (ADS)

    Halvorson, William L.; Fenn, Dennis B.; Allardice, William R.

    1988-01-01

    The multifaceted development of an erosion surface on Santa Barbara Island, Channel Islands National Park, California, has led to this study of the relationship between soils and vegetation. A dry Mediterranean climate and past attempts at farming and introductions of alien species have led to vegetative degradation accompanied by both gully and surface erosion. Soil and vegetation analyses show this erosion to be in a location of transition. The soils are Typic Chromoxererts (Vertisol Order) with high clay, salinity, and sodium contents. The vegetation is ecotonal in nature, grading from a principally alien annual grassland with Avena fatua and Atriplex semibaccata to a shrub community dominated by the native Suaeda californica. Management toward revegetation and stabilization of this island ecosystem will be difficult with high clay, saline-sodic soils and disturbed vegetation.

  2. The influence of Aster x salignus Willd. Invasion on the diversity of soil yeast communities

    NASA Astrophysics Data System (ADS)

    Glushakova, A. M.; Kachalkin, A. V.; Chernov, I. Yu.

    2016-07-01

    The annual dynamics of yeast communities were studied in the soddy-podzolic soil under the thickets of Aster x salignus Willd., one of the widespread invasive plant species in central Russia. Yeast groups in the soils under continuous aster thickets were found to differ greatly from the yeast communities in the soils under the adjacent indigenous meadow vegetation. In both biotopes the same species ( Candida vartiovaarae, Candida sake, and Cryptococcus terreus) are dominants. However, in the soils under indigenous grasses, eurybiontic yeasts Rhodotorula mucilaginosa, which almost never occur in the soil under aster, are widespread. In the soil under aster, the shares of other typical epiphytic and pedobiontic yeast fungi (ascomycetic species Wickerhamomyces aniomalus, Barnettozyma californica and basidiomycetic species Cystofilobasidium macerans, Guehomyces pullulans) significantly increase. Thus, the invasion of Aster x salignus has a clear effect on soil yeast complexes reducing their taxonomic and ecological diversity.

  3. Biophysical discussions: ionic channels in membranes held at Airlie, Virginia on 2-5 October 1983

    SciTech Connect

    Not Available

    1983-10-05

    Partial contents include: Light-activated channels in limulus ventral photoreceptors; Paramagnetic hydrophobic ions as probes for electrically active conformational transitions in Ion channels; Acetylcholine receptor. Dynamic properties; Acetylcholine-activated channel current-voltage relations in symmetrical Na(+) solutions; A molecular model for an acetylcholine binding site. Ion channel and the bilayer helices of the acetylcholine receptor assigned using single group rotation theory and electrostatic interactions; Effects of halothane on the acetylcholine receptor channel in cultured xenopus myocytes; Deuterium oxide effects frog endplate channels; Activation and inactivation kinetics or torpedo Californica acetylcholine receptor in reconstituted membranes; Acetylcholine-induced K(+) current in amphibian atrial cells; Functional reconstitution of rat striatal dopamine agonist receptors into artificial lipid bimolecular membranes; Blocking kinetics at excitatory acetylcholine responses on Aplysia Neurons; The secondary structure of Acetycholine receptor reconstituted in a single lipid component as determined by raman spectroscopy; Molecular and cellular mapping of the voltage-dependent Na(+) channel.

  4. Alternating current and infrared produce an onset-free reversible nerve block

    PubMed Central

    Lothet, Emilie H.; Kilgore, Kevin L.; Bhadra, Niloy; Bhadra, Narendra; Vrabec, Tina; Wang, Yves T.; Jansen, E. Duco; Jenkins, Michael W.; Chiel, Hillel J.

    2014-01-01

    Abstract. Nerve block can eliminate spasms and chronic pain. Kilohertz frequency alternating current (KHFAC) produces a safe and reversible nerve block. However, KHFAC-induced nerve block is associated with an undesirable onset response. Optical inhibition using infrared (IR) laser light can produce nerve block without an onset response, but heats nerves. Combining KHFAC with IR inhibition [alternating current and infrared (ACIR)] produces a rapidly reversible nerve block without an onset response. ACIR can be used to rapidly and reversibly provide onset-free nerve block in the unmyelinated nerves of the marine mollusk Aplysia californica and may have significant advantages over either modality alone. ACIR may be of great clinical utility in the future. PMID:26157966

  5. Caprella suprapiscis sp. nov. (Crustacea: Amphipoda: Caprellidae) from the Pacific coast of Mexico.

    PubMed

    Galván-Villa, Cristian M; Ayón-Parente, Manuel

    2015-01-01

    A new species of caprellid, Caprella suprapiscis sp. nov., is described based on several specimens collected from Bahía Chamela, Jalisco, Mexico. All specimens were found in association with the scorpionfish Scorpaena mystes. Caprellids are set on the dorsal surface of fishes. The species is distinguished by head with a short dorsal projection, eyes distinctive, body slender and smooth, peduncular articles of antenna 1 not setose, antenna 2 with swimming setae, gnathopod 2 with three ventral projections in males. The species is close to C. californica, C. mercedesae, and C. scaura for a sharp spine on the forehead but can be distinguished by gnathopod 2 finely setose, and basis of gnathopod 2 shorter. PMID:26248940

  6. Uranium series ages of corals from the upper Pleistocene Mulege terrace, Baja California Sur, Mexico

    SciTech Connect

    Ashby, J.R.; Ku, T.L.; Minch, J.A.

    1987-02-01

    Specimens of Porites californica contained in the sediments of upper Pleistocene, +12-m marine terrace deposits developed on the east coast of the Baja California (Mexico) peninsula at Mulege have yielded /sup 239/Th//sup 234/U dates of 124 +/- 5 and 144 +/- 7 ka (+/- 1 sigma). These dates can be assigned to the well-documented late Pleistocene oxygen-isotope stage 5e high sea stand. Differences between the eustatic and present elevations of this terrace indicate average uplift rates since terrace formation of approximately 4 to 5 cm/1000 yr, indicating a relative stability and lack of major vertical deformation since the late Pleistocene. This terrace in the Mulege area can now be correlated with other marine terraces throughout the Baja California peninsula and southern California.

  7. The relative toxicities of several pesticides to naiads of three species of stoneflies

    USGS Publications Warehouse

    Sanders, Herman O.; Cope, Oliver B.

    1968-01-01

    Static bioassays were conducted to determine the relative acute toxicities of some insecticides, herbicides, fungicides, a defoliant, and a molluscicide to the naiads of three species of stonef!y, Pteronarcys califomica, Pteronarcella badia, and Claassenia sabulosa. Toxic effects were measured by determination of median lethal concn (Lcoo) for 24-, 48-, and 96-hr exposures, at 15.5C. Endrin and dieldrin were the most and DDT the least toxic of the chlorinated hydrocarbon insecticides tested. Parathion was the most toxic organophosphate insecticide to P. califomica naiads, but dursban was the most toxic to P. badia and C. sabulosa naiads. Trichlorofon ( Dipterex) was the least toxic to all three species. P. badia, the species of smallest size, was the species most susceptible to most pesticides, followed in descending order of sensitivity by C. sabulosa and P. califomica. Smaller specimens of P. californica naiads were consistently more susceptible to some insecticides than larger specimens of the same species.

  8. A new species of Helobdella (Hirudinida: Glossiphoniidae) from Oregon

    USGS Publications Warehouse

    Moser, William E.; Fend, Steven V.; Richardson, Dennis J.; Hammond, Charlette I.; Lazo-Wasem, Eric A.; Govedich, Fredric R.; Gullo, Bettina S.

    2013-01-01

    Helobdella bowermani n. sp. is described from specimens collected in fine sediment of open water benthos of Upper Klamath Lake, Klamath County, Oregon. The new species has pale yellow/buff coloration with scattered chromatophore blotches throughout the dorsal surface, lateral extensions or papillae only on the a2 annulus, dorsal medial row of papillae with small papilla on a1 and larger papillae on a2 and a3, and a small oval scute (rarely triangular). Helobdella bowermani n. sp. is morphologically similar to Helobdella atli and Helobdella simplex. Molecular comparison of CO-I sequence data from H. bowermani n. sp. revealed differences of 10.6%–10.8% with Helobdella californica, differences of 12.2%–13.7% with H. atli, and differences of 12.7%–13.2% with H. simplex.

  9. Small estuarine fishes feed on large trematode cercariae: Lab and field investigations

    USGS Publications Warehouse

    Kaplan, A.T.; Rebhal, S.; Lafferty, K.D.; Kuris, A.M.

    2009-01-01

    In aquatic ecosystems, dense populations of snails can shed millions of digenean trematode cercariae every day. These short-lived, free-living larvae are rich in energy and present a potential resource for consumers. We investigated whether estuarine fishes eat cercariae shed by trematodes of the estuarine snail Cerithidea californica. In aquaria we presented cercariae from 10 native trematode species to 6 species of native estuarine fishes. Many of these fishes readily engorged on cercariae. To determine if fishes ate cercariae in the field, we collected the most common fish species, Fundulus parvipinnis (California killifish), from shallow water on rising tides when snails shed cercariae. Of 61 killifish, 3 had recognizable cercariae in their gut. Because cercariae are common in this estuary, they could be frequent sources of energy for small fishes. In turn, predation on cercariae by fishes (and other predators) could also reduce the transmission success of trematodes. ?? 2009 American Society of Parasitologists.

  10. Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry

    PubMed Central

    Nemes, Peter; Rubakhin, Stanislav S.; Aerts, Jordan T.; Sweedler, Jonathan V.

    2013-01-01

    Single-cell mass spectrometry (MS) empowers metabolomic investigations by decreasing analytical dimensions to the size of individual cells and subcellular structures. We describe a protocol for investigating and quantifying metabolites in individual isolated neurons using single-cell capillary electrophoresis hyphenated to electrospray ionization time-of-flight MS. The protocol requires ~2 h for sample preparation, neuron isolation, and metabolite extraction, and 1 h for metabolic measurement. The approach was used to detect more than 300 distinct compounds in the mass range of typical metabolites in various individual neurons (25–500-µm in diameter) isolated from the sea slug (Aplysia californica) central and rat (Rattus norvegicus) peripheral nervous systems. A subset of identified compounds was sufficient to reveal metabolic differences among freshly isolated neurons of different types and changes in the metabolite profiles of cultured neurons. The protocol can be applied to the characterization of the metabolome in a variety of smaller cells and/or subcellular domains. PMID:23538882

  11. Cloning and characterization of an abalone (Haliotis discus hannai) actin gene

    NASA Astrophysics Data System (ADS)

    Ma, Hongming; Xu, Wei; Mai, Kangsen; Liufu, Zhiguo; Chen, Hong

    2004-10-01

    An actin encoding gene was cloned by using RT-PCR, 3‧ RACE and 5‧ RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3‧ untranslated region of 307 base pairs and 79 base pairs of 5‧ untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin (β-actin) than to human muscle actin.

  12. Hybrid Viability and Fertility in Co-occuring Plant Species

    NASA Astrophysics Data System (ADS)

    Hernandez, E.; Garcia, C.; Yost, J.

    2012-12-01

    Similar species of plants can co-exist due to reproductive barriers that keep them from hybridizing. In the case of Lasthenia gracilis and L. californica, certain reproductive barriers allow them to co-exist at Jasper Ridge without hybridization. The two species are locally adapted to different regions of the same hillside, and have slight differences in flowering time but hybrids can be created at low rate in the green house. We tested the viability and fertility of green house produced hybrids to quantify post-zygotic reproductive isolation at Jasper Ridge. We planted 10 hybrid seeds and 10 control seeds from 11 different families. We measured the percent germination, survival to flowering and pollen fertility of the seeds. We expect lower germination, lower survival to flowering, and lower pollen viability of hybrid seeds as compared to control seeds.

  13. Development of a high specific activity radioligand, /sup 125/I-LSD, and its application to the study of serotonin receptors

    SciTech Connect

    Kadan, M.J.

    1987-01-01

    /sup 125/I-Labeled receptor ligands can be synthesized with specific activities exceeding 2000 Ci/mmol, making them nearly 70-fold more sensitive in receptor site assays than (mono) tritiated ligands. We have synthesized and characterized /sup 125/I-lysergic acid diethylamide (/sup 125/I-LSD), the first radioiodinated ligand for serotonin receptor studies. The introduction of /sup 125/I at the 2 position of LSD increased both the affinity and selectivity of this compound for serotonin 5-HT/sub 2/ receptors in rat cortex. The high specific activity of /sup 125/I-LSD and its high ratio of specific to nonspecific binding make this ligand especially useful for autoradiographic studies of serotonin receptor distribution. We have found that /sup 125/I-LSD binds with high affinity to a class of serotonin receptors in the CNS of the marine mollusk Aplysia californica.

  14. Complex coacervates as a foundation for synthetic underwater adhesives

    PubMed Central

    Stewart, Russell J.; Wang, Ching Shuen; Shao, Hui

    2011-01-01

    Complex coacervation was proposed to play a role in the formation of the underwater bioadhesive of the Sandcastle worm (Phragmatopoma californica) based on the polyacidic and polybasic nature of the glue proteins and the balance of opposite charges at physiological pH. Morphological studies of the secretory system suggested the natural process does not involve complex coacervation as commonly defined. The distinction may not be important because electrostatic interactions likely play an important role in formation of the sandcastle glue. Complex coacervation has also been invoked in the formation of adhesive underwater silk fibers of caddisfly larvae and the adhesive plaques of mussels. A process similar to complex coacervation, that is, condensation and dehydration of biopolyelectrolytes through electrostatic associations, seems plausible for the caddisfly silk. This much is clear, the sandcastle glue complex coacervation model provided a valuable blueprint for the synthesis of a biomimetic, waterborne, underwater adhesive with demonstrated potential for repair of wet tissue. PMID:21081223

  15. Visitor center at the Antelope Valley California Poppy Reserve, Lancaster, California

    SciTech Connect

    Colyer, R.D.; Freeman, S.P.

    1981-01-01

    The Antelope Valley California Poppy Reserve contains the largest remaining stand of the California Poppy (Eschschozia Californica), the state flower of California. To welcome the thousands of people viewing the desert wildflowers each spring, the State of California decided to build a visitor/interpretive center. This building deals primarily with the question of fit; a building's fit aesthetically with its site and the fit of a building's design response to the climate of the site. In this case, both aspects of this question led the client and architects to seek an earth sheltered solution using materials at least metaphorically indigenous to the region. On both a technical and formal level, this building seeks to fit the unique climate and historical heritage of its site.

  16. Statoconia formation in molluscan statocysts

    NASA Technical Reports Server (NTRS)

    Wiederhold, M. L.; Sheridan, C. E.; Smith, N. K.

    1986-01-01

    The gravity sensors of all molluscs phylogenetically below the cephalopods are spherical organs called statocysts. The wall of the sphere contains mechanosensory cells whose sensory cilia project into the lumen of the cyst. The lumen is filled with fluid and dense "stones", the statoconia or statoliths, which sink under the influence of gravity to load, and stimulate, those receptor cells which are at the bottom. The statoconia of Aplysia californica are shown to be calcified about a lamellar arrangement of membranes. Similar lamellar membrane arrangements are seen within the receptor cells, and their possible role in the formation of the statoconia is discussed. SEM of unfixed statoconia reveals plate-like crystallization on their surface. Elemental analysis shows a relatively high Sr content, which is of interest, since others have recently reported that Sr is required in the culture medium of several laboratory reared molluscs in order for the statoconia to develop.

  17. Elevated atmospheric CO{sub 2} and soil nutrients alter competitive performance of California annual grassland species

    SciTech Connect

    Reynolds, H.L.; Chapin, F.S. III; Field, C.B.

    1995-06-01

    Atmospheric CO{sub 2} and soil nutrients altered interspecific competitive performance of three grassland annuals, all exhibiting the C{sub 3} metabolic pathway. Plantago erecta, an herbaceous dicot dominant in low-fertility serpentine grassland, was the superior interspecific competitor at low soil nutrients. Bromus hordeaceus, an introduced grass dominant in higher fertility sandstone grassland, was the superior interspecific competitor at high soil nutrients. Interspecific competitive ability of Plantago was slightly enhanced under elevated CO{sub 2}, but only at high soil nutrients, whereas interspecific competitive ability of Bromus was stimulated under elevated CO{sub 2} at both low and high soil nutrients. Interspecific competitive ability of Lasthenia californica, another herbaceous dicot common in serpentine grassland, was low in all treatments, and tended to decrease with elevated CO{sub 2} at low soil nutrients. Our results suggest that elevated CO{sub 2} may shift plant species abundance of serpentine grassland in favor of Bromus hordeaceus.

  18. Mechanism, dynamics, and biological existence of multistability in a large class of bursting neurons

    PubMed Central

    Newman, Jonathan P.; Butera, Robert J.

    2010-01-01

    Multistability, the coexistence of multiple attractors in a dynamical system, is explored in bursting nerve cells. A modeling study is performed to show that a large class of bursting systems, as defined by a shared topology when represented as dynamical systems, is inherently suited to support multistability. We derive the bifurcation structure and parametric trends leading to multistability in these systems. Evidence for the existence of multirhythmic behavior in neurons of the aquatic mollusc Aplysia californica that is consistent with our proposed mechanism is presented. Although these experimental results are preliminary, they indicate that single neurons may be capable of dynamically storing information for longer time scales than typically attributed to nonsynaptic mechanisms. PMID:20590314

  19. Uranium-series age of the Eel Point terrace, San Clemente Island, California

    SciTech Connect

    Muhs, D.R.; Szabo, B.J.

    1982-01-01

    Uranium-series analysis of the coral Allopora californica Verrill from the 2nd, 32-m Eel Point terrace on San Clemente Island, California, has yielded an age of 127,000 +/- 7,000 yr. The Eel Point terrace is thus correlative with numerous terrace localities on the southern California mainland, with coral reefs on Barbados and New Guinea dated about 120,000 yr, and with substage 5e of the marine oxygen-isotope record. A tectonic uplift rate of about 0.20 m/1,000 yr has been calculated assuming a sea level slightly higher than the present one at the time of terrace formation. Extrapolation of this uplift rate allows age estimates to be made for other terraces on the island.

  20. Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species

    PubMed Central

    Anderson, J. W.; Fowden, L.

    1970-01-01

    1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [32P]pyrophosphate-ATP exchange technique. 3. β-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and γ-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for β-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. α-Cyclopropylglycine and α-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase. PMID:5493505

  1. Reaction sequence and molecular mass of a Cl(-)-translocating P-type ATPase.

    PubMed Central

    Gerencser, G A; Zelezna, B

    1993-01-01

    The basolateral membranes of Aplysia californica foregut absorptive cells contain both Cl(-)-stimulated ATPase and ATP-dependent Cl- transport activities, and each was inhibited by orthovanadate. Both of these orthovanadate-sensitive activities were reconstituted into proteoliposomes. The reaction sequence kinetics were determined by [gamma-32P]ATP-induced phosphorylation of the reconstituted Cl- pump. Rapid phosphorylation and dephosphorylation kinetics of acyl phosphate bonding were confirmed by destabilization of the phosphoprotein by either hydroxylamine or high pH. Mg2+ caused phosphorylation of the enzyme; Cl- caused dephosphorylation. Orthovanadate almost completely inhibited the Mg(2+)-driven phosphorylation reaction. The molecular mass of the catalytic unit (subunit) of the enzyme appeared to be 110 kDa, which is in agreement with molecular masses of all other catalytic units (subunits) of P-type ATPases. Images Fig. 4 Fig. 7 Fig. 8 PMID:8367450

  2. Trematodes in snails near raccoon latrines suggest a final host role for this mammal in California Salt Marshes

    USGS Publications Warehouse

    Lafferty, K.D.; Dunham, E.J.

    2005-01-01

    Of the 18 trematode species that use the horn snail, Cerithidea californica, as a first intermediate host, 6 have the potential to use raccoons as a final host. The presence of raccoon latrines in Carpinteria Salt Marsh, California, allowed us to investigate associations between raccoons and trematodes in snails. Two trematode species, Probolocoryphe uca and Stictodora hancocki, occurred at higher prevalences in snails near raccoon latrines than in snails away from latrines, suggesting that raccoons may serve as final hosts for these species. Fecal remains indicated that raccoons fed on shore crabs, the second intermediate host for P. uca, and fish, the second intermediate host for S. hancocki. The increase in raccoon populations in the suburban areas surrounding west coast salt marshes could increase their importance as final hosts for trematodes in this system. ?? American Society of Parasitologists 2005.

  3. Clustered voltage-gated Na+ channels in Aplysia axons.

    PubMed

    Johnston, W L; Dyer, J R; Castellucci, V F; Dunn, R J

    1996-03-01

    Clustering of voltage-gated Na+ channels is critical for the fast saltatory conduction of action potentials in vertebrate myelinated axons. However, the mechanisms responsible for the generation and maintenance of Na+ channel clustering are not well understood. In this study we have raised an antibody against the cloned SCAP-1 voltage-gated Na+ channel of the marine invertebrate Aplysia californica and used it to examine Na+ channel localization in Aplysia ganglia and in cultured Aplysia sensory neurons. Our results show that there is a large cytoplasmic pool of Na+ channels in the soma of Aplysia neurons. Furthermore, we show that Na+ channels in Aplysia axons are not homogeneously distributed but, rather, are present in distinct clusters. Theoretical considerations indicate that Na+ channel clustering may enhance action potential conduction. We propose that clustered Na+ channels may be a fundamental property of many axons, and perhaps of many membranes that conduct Na(+)-dependent action potentials. PMID:8774441

  4. Cholinergic ligand interactions with acetylcholine receptor proteins and solvent interactions with N,N-dialkylnicotinamides

    SciTech Connect

    Bean, J.W.

    1987-01-01

    A dual-chambered flow dialysis nuclear counting apparatus was used to monitor cholinergic ligand induced displacement of {sup 155}Eu{sup 3+} from acetylcholine receptor proteins. Acetylcholine, nicotine and carbamylcholine induced similar rates of displacement of {sup 155}Eu{sup 3+} probes of calcium binding sites in receptor proteins from wild type Drosophila melanogaster and Torpedo californica. The receptor isolated from a nicotine resistant strain of Drosophila melanogaster displayed an altered dependency of cholinergic ligand induced cation displacement with respect to the other two receptor proteins. Both Drosophila strains' solubilized receptor proteins migrated as three bands of molecular weights 68,000, 66,000, and 60,000 on denaturing polyacrylamide gels. Carbon-13 NMR techniques were employed to examine the effects of solvent environment on rotational energy barriers in a series of molecules related to the analeptic, nikethamide: N,N-dimethylnicotinamide, 1-nicotinoyl piperidine, and N,N-dipropylnicotinamide.

  5. Characterization of the peptidylglycine α-amidating monooxygenase (PAM) from the venom ducts of neogastropods, Conus bullatus and Conus geographus

    PubMed Central

    Ul-Hasan, Sabah; Burgess, Daniel M.; Gajewiak, Joanna; Li, Qing; Hu, Hao; Yandell, Mark; Olivera, Baldomero M.; Bandyopadhyay, Pradip K.

    2014-01-01

    Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus. PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells. PMID:23994590

  6. Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

    PubMed

    Wall, K A; Klis, M; Kornet, J; Coyle, D; Amé, J C; Jacobson, M K; Slama, J T

    1998-11-01

    Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells. PMID:9794804

  7. Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

    PubMed Central

    Wall, K A; Klis, M; Kornet, J; Coyle, D; Amé, J C; Jacobson, M K; Slama, J T

    1998-01-01

    Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells. PMID:9794804

  8. Using larval trematodes that parasitize snails to evaluate a saltmarsh restoration project

    USGS Publications Warehouse

    Huspeni, T.C.; Lafferty, K.D.

    2004-01-01

    We conducted a Before-After-Control-Impact (BACI) study using larval digeneans infecting the California horn snail, Cerithidea californica, to evaluate the success of an ecological restoration project at Carpinteria Salt Marsh in California, USA. Digenean trematodes are parasites with complex life cycles requiring birds and other vertebrates as final hosts. We tested two hypotheses for prevalence and species richness of larval trematodes in C. californica: (1) prior to the restoration, sites to be restored would have lower trematode prevalence and species richness relative to unimpacted control sites, and (2) that these differences would diminish after restoration. The sites to be restored were initially degraded for trematode species. They had a mean trematode prevalence (12%) and species richness (4.5 species) that were lower than control sites (28% trematode prevalence and 7 species). Despite the differences in prevalence, the proportional representation of each trematode species in the total community was similar between sites to be restored and control sites. Over the six years following restoration, trematode prevalence nearly quadrupled at restored sites (43%) while the prevalence at control sites (26%) remained unchanged. In addition, species richness at restored sites doubled (9 species), while species richness at the control sites (7.8 species) did not change. Immediately after restoration, the relative abundance of trematode species using fishes as second intermediate hosts declined while those using molluscs as second intermediate hosts increased. Trematode communities at restored and control sites gradually returned to being similar. We interpret the increase in trematode prevalence and species richness at restored sites to be a direct consequence of changes in bird use of the restored habitat. This study demonstrates a new comparative technique for assessing wetlands, and while it does not supplant biotic surveys, it informs such taxonomic lists. Most

  9. Flowering responses of insect-pollinated plants to elevated CO{sub 2} levels

    SciTech Connect

    Cushman, J.H.; Koch, G.W.; Chiariello, N.R. ||

    1995-06-01

    Elevated atmospheric CO{sub 2} concentrations have been predicted or shown to substantially influence plants, communities and ecosystems in a variety of ways. Here, we examined the effects of elevated CO{sub 2} levels on the timing and magnitude of flowering for two insect-pollinated annual plant species in a serpentine grassland. We focused on Lasthenia californica and Linanthus parviflorus and addressed three questions: (1) Do elevated CO{sub 2} levels influence flowering phenologies and is this species specific? (2) Do elevated CO{sub 2} levels affect flower production and is this due to altered numbers of individuals, flowers per plant, or both? and (3) Are effects on flowering due to elevated CO{sub 2} levels per se or changes in environmental conditions associated with methods used to manipulate CO{sub 2} levels? To address these questions, we used the ecosystem experiment at Stanford University`s Jasper Ridge Biological Preserve (San Mateo Co., CA). This system consists of 20 open-topped chambers - half receiving ambient CO{sub 2} (360 ppm) and half receiving elevated CO{sub 2} (720 ppm) - and 10 untreated plots serving as chamber controls. Results from the 1994 season demonstrated that there were species-specific responses to elevated CO{sub 2} levels and the field chambers. For Lasthenia californica, elevated CO{sub 2} per se did not affect relative abundance, inflorescence production, or phenology, but chambers did significantly increase inflorescence production and extend the duration of flowering. For Linanthus parviflorus, elevated CO{sub 2} levels significantly increased relative abundance and flower production, and extended the flowering period slightly, while the chambers significantly decreased flower production early in the season and increased it later in the season.

  10. Single-Cell Metabolomics: Changes in the Metabolome of Freshly Isolated and Cultured Neurons

    PubMed Central

    2012-01-01

    Metabolites are involved in a diverse range of intracellular processes, including a cell’s response to a changing extracellular environment. Using single-cell capillary electrophoresis coupled to electrospray ionization mass spectrometry, we investigated how placing individual identified neurons in culture affects their metabolic profile. First, glycerol-based cell stabilization was evaluated using metacerebral neurons from Aplysia californica; the measurement error was reduced from ∼24% relative standard deviation to ∼6% for glycerol-stabilized cells compared to those isolated without glycerol stabilization. In order to determine the changes induced by culturing, 14 freshly isolated and 11 overnight-cultured neurons of two metabolically distinct cell types from A. californica, the B1 and B2 buccal neurons, were characterized. Of the more than 300 distinctive cell-related signals detected, 35 compounds were selected for their known biological roles and compared among each measured cell. Unsupervised multivariate and statistical analysis revealed robust metabolic differences between these two identified neuron types. We then compared the changes induced by overnight culturing; metabolite concentrations were distinct for 26 compounds in the cultured B1 cells. In contrast, culturing had less influence on the metabolic profile of the B2 neurons, with only five compounds changing significantly. As a result of these culturing-induced changes, the metabolic composition of the B1 neurons became indistinguishable from the cultured B2 cells. This observation suggests that the two cell types differentially regulate their in vivo or in vitro metabolomes in response to a changing environment. PMID:23077722

  11. Ecology of larval trematodes in three marine gastropods.

    PubMed

    Curtis, Lawrence A

    2002-01-01

    To comprehend natural host-parasite systems, ecological knowledge of both hosts and parasites is critical. Here I present a view of marine systems based on the snail Ilyanassa obsoleta and its trematodes. This system is reviewed and two others, those of the snails Cerithidea californica and Littorina littorea, are then summarized and compared. Trematodes can profoundly affect the physiology, behaviour and spatial distribution of hosts. Studying these systems is challenging because trematodes are often embedded in host populations in unappreciated ways. Trematode prevalence is variable, but can be high in populations of all three hosts. Conditions under which single- and multiple-species infections can accumulate are considered. Adaptive relations between species are likely the most important and potentials for adaptation of parasites to hosts, hosts to parasites, and parasites to other parasites are also considered. Even if colonization rate is low, a snail population can develop high trematode prevalence, if infections persist long and the host is long-lived and abundant. Trematodes must be adapted to use their snail hosts. However, both I. obsoleta and L. littorea possess highly dispersed planktonic larvae and trematode prevalence is variable among snail populations. Host adaptation to specific infections, or even to trematodes in general, is unlikely because routine exposure to trematodes is improbable. Crawl-away juveniles of C. californica make adaptation to trematodes in that system a possibility. Trematode species in all three systems are not likely adapted to each other. Multiple-species infections are rare and definitive hosts scatter parasite eggs among snail populations with variable prevalences. Routine co-occurrence of trematodes in snails is thus unlikely. Adaptations of these larval trematodes to inhabit the snail host must, then, be the basis for what happens when they do co-occur. PMID:12396215

  12. Phylogeny of the pollinating yucca moths, with revision of Mexican species (Tegeticula and Parategeticula; Lepidoptera, Prodoxidae)

    SciTech Connect

    Pellmyr, Olof; Balcazar-Lara, Manuel; Segraves, Kari A.; Althoff, David M.; Littlefield, Rik J.

    2008-02-01

    ABSTRACT The yucca moths (Tegeticula and Parategeticula; Lepidoptera, Prodoxidae) are well-known for their obligate relationship as exclusive pollinators of yuccas. Revisionary work in recent years has revealed far higher species diversity than historically recognized, increasing the number of described species from four to 21. Based on field surveys in Mexico and examination of collections, we describe five additional species: T. californica Pellmyr sp. nov., T. tehuacana Pellmyr & Balcázar-Lara sp. nov., T. tambasi Pellmyr & Balcázar-Lara sp. nov., T. baja Pellmyr & Balcázar-Lara sp. nov., and P. californica Pellmyr & Balcázar-Lara sp. nov. Tegeticula treculeanella Pellmyr is identified as a junior synonym of T. mexicana Bastida. A diagnostic key to the adults of all species of the T. yuccasella complex is provided. A phylogeny based on a 2104-bp segment of mitochondrial DNA (mtDNA) in the cytochrome oxidase I and II region supported monophyly of the two pollinator genera, and strongly supported monophyly of the 17 recognized species of the T. yuccasella complex. Most relationships are well-supported, but some relationships within a recent and rapidly diversified group of 11 taxa are less robust, and in one case conflicts with a whole-genome data set (AFLP). The current mtDNA-based analyses, together with previously published AFLP data, provide a robust phylogenetic foundation for future studies of life history evolution and host interactions in one of the classical models of coevolution and obligate mutualism. ADDITIONAL KEY WORDS: mutualism, pollination, molecular phylogenetics, mitochondrial DNA

  13. Identification, characterization, and expression levels of putative adhesive proteins from the tube-dwelling polychaete Sabellaria alveolata.

    PubMed

    Becker, Pierre T; Lambert, Aurélie; Lejeune, Annabelle; Lanterbecq, Déborah; Flammang, Patrick

    2012-10-01

    The shelter of the tube-dwelling polychaete Sabellaria alveolata is composed of mineral particles assembled with spots of a proteinaceous cement. The adhesive proteins constituting the cement were identified on the basis of their sequence similarity with proteins of a phylogenetically related species, Phragmatopoma californica. Two positively charged proteins, Sa-1 and Sa-2, share common features: they both have a mass of 22 kDa; are rich in glycine, tyrosine and basic residues; and show repeated peptide motifs. The consensus repeat of Sa-1 is KGAYGAKGLGYGNKAGYGAYG (occurring 6-8 times), while Sa-2 displays the consensus heptapeptide VHKAAWG (5 times) and undecapeptide VHKAAGYGGYG (8 times). Two variants of a serine-rich protein, Sa-3A (22 kDa) and Sa-3B (21 kDa), were also identified. Their serine residues account for 75 mol% and are probably phosphorylated, meaning that Sa-3 is very acidic and negatively charged. Moreover, tyrosine residues of all adhesive proteins are presumably modified into DOPA. Although protein sequences are not well-conserved between S. alveolata and P. californica, their main characteristics (including amino acid composition, post-translational modifications, repeated patterns, isoelectric point, and mass) are shared by both species. This suggests that these features are more important for their function than the primary structure of the proteins. The mRNA abundance for each protein was estimated by quantitative real-time PCR, revealing relative expression levels of about 5, 11, 1.5, and 1 for Sa-1, -2, -3A, and -3B, respectively. These levels could be indicative of charge neutralization phenomena or could reflect their function (interface vs. bulk) in the cement. PMID:23111133

  14. Electrophysiology of chloride transport in Aplysia (mollusk) intestine.

    PubMed

    Gerencser, G A

    1983-02-01

    This investigation was principally undertaken to examine the mechanism of Cl- transport across the Aplysia californica intestinal epithelium. Previous results have shown: 1) the transmural potential difference (psi m leads to s) and the mucosal membrane potential difference (psi m) to be negative relative to the mucosal solution, 2) mucosal D-glucose hyperpolarized psi m leads to s and depolarized psi m, 3) mucosal D-glucose significantly increased intracellular Cl- activity (aiCl), however the electrochemical potential (-mu i) for intracellular Cl- was significantly less in both cases, than the -muCl in the mucosal solution, 4) replacing Cl- in the bathing medium with SO-4(2) significantly reduced both psi m and psi m leads to s, and 5) the energy within the electrochemical potential difference for Na+ (delta -mu Na) directed from mucosa to cytosol was energetically adequate so that intracellular Cl- accumulation could occur. New results showed: 1) psi m and psi m leads to s to significantly hyperpolarize when Na+ was replaced with Tris+ in the bathing medium, 2) aiCl decreased from 13.9 +/- 0.5 to 9.1 +/- 0.3 mM when Na+ was replaced with Tris+ in the bathing medium. The intracellular -muCl, both in the presence and absence of Na+, was significantly less than -muCl in the mucosal medium. These results suggest that Na+ and Cl- transport across the mucosal membrane are not mechanistically coupled and that an active extrusion mechanism for Cl- exists in the lateral-serosal membrane of the surface epithelial cells of A. californica intestine. PMID:6824101

  15. Effects of five southern California macroalgal diets on consumption, growth, and gonad weight, in the purple sea urchin Strongylocentrotus purpuratus

    PubMed Central

    Byrnes, Jarrett E.K.; Reed, Daniel C.

    2015-01-01

    Consumer growth and reproductive capacity are direct functions of diet. Strongylocentrotid sea urchins, the dominant herbivores in California kelp forests, strongly prefer giant kelp (Macrocystis pyrifera), but are highly catholic in their ability to consume other species. The biomass of Macrocystis fluctuates greatly in space and time, and the extent to which urchins can use alternate species of algae or a mixed diet of multiple algal species to maintain fitness when giant kelp is unavailable is unknown. We experimentally examined the effects of single and mixed species diets on consumption, growth and gonad weight in the purple sea urchin Strongylocentrotus purpuratus. Urchins were fed single species diets consisting of one of four common species of macroalgae (the kelps Macrocystis pyrifera and Pterygophora californica, and the red algae Chondracanthus corymbiferus and Rhodymenia californica (hereafter referred to by genus)) or a mixed diet containing all four species ad libitum over a 13-week period in a controlled laboratory setting. Urchins fed Chondracanthus, Macrocystis and a mixed diet showed the highest growth (in terms of test diameter, wet weight and jaw length) and gonad weight, while urchins fed Pterygophora and Rhodymenia showed the lowest. Urchins consumed their preferred food, Macrocystis, at the highest rate when offered a mixture, but consumed Chondracanthus or Macrocystis at similar rates when the two algae were offered alone. The differences in urchin feeding behavior and growth observed between these diet types suggest the relative availability of the algae tested here could affect urchin populations and their interactions with the algal assemblage. The fact that the performance of urchins fed Chondracanthus was similar or higher than those fed the preferred Macrocystis suggests that the availability of the former could could sustain growth and reproduction of purple sea urchins during times of low Macrocystis abundance as is common following

  16. PLusiinae (Excl. Abrostolini) (Lepidoptera: Noctuidae) of Ethiopia. A faunistical survey with biogeographical comments.

    PubMed

    Kravchenko, Vasiliy D; Ronkay, Laszlo; Behounek, Gottfried; Müller, Günter C

    2015-01-01

    The extensive survey in different regions of Ethiopia between 1987-1990 and 2005-2011 resulted in the recognition of 39 species of Plusiinae. The majority of the species belong to two large genera, Ctenoplusia (15 species) and Thysanoplusia (16 species). A new synonymy is established, Plusiotricha gorilla (Holland, 1894) is proved to represent the female sex of Plusiotricha livida Holland, 1894 (syn. nov.). The present paper does not include the records of the species of the tribe Abrostolini. Eighteen species are recorded for the first time from Ethiopia. Twenty species of the identified taxa are known only from tropical and subtropical Africa, while the areas of ten species extend from Africa to the Arabian Peninsula or even further to the north. Eight species are widespread not only in Africa but also in the Palearctic and Oriental regions. One species-Autographa gamma, a well-known Palearctic pest of different vegetables-is found in the Afrotropical region only in Ethiopia, at medium and high mountain elevations but not in the tropical lowlands. PMID:26623895

  17. Female dietary bias towards large migratory moths in the European free-tailed bat (Tadarida teniotis).

    PubMed

    Mata, Vanessa A; Amorim, Francisco; Corley, Martin F V; McCracken, Gary F; Rebelo, Hugo; Beja, Pedro

    2016-03-01

    In bats, sexual segregation has been described in relation to differential use of roosting and foraging habitats. It is possible that variation may also exist between genders in the use of different prey types. However, until recently this idea was difficult to test owing to poorly resolved taxonomy of dietary studies. Here, we use high-throughput sequencing to describe gender-related variation in diet composition of the European free-tailed bat (Tadarida teniotis), while controlling for effects of age and season. We analysed guano pellets collected from 143 individuals mist-netted from April to October 2012 and 2013, in northeast Portugal. Moths (Lepidoptera; mainly Noctuidae and Geometridae) were by far the most frequently recorded prey, occurring in nearly all samples and accounting for 96 out of 115 prey taxa. There were significant dietary differences between males and females, irrespective of age and season. Compared to males, females tended to consume larger moths and more moths of migratory behaviour (e.g.Autographa gamma). Our study provides the first example of gender-related dietary variation in bats, illustrating the value of novel molecular tools for revealing intraspecific variation in food resource use in bats and other insectivores. PMID:27009885

  18. Cyclic Avian Mass Mortality in the Northeastern United States Is Associated with a Novel Orthomyxovirus

    PubMed Central

    Ballard, Jennifer R.; Tesh, Robert B.; Brown, Justin D.; Ruder, Mark G.; Keel, M. Kevin; Munk, Brandon A.; Mickley, Randall M.; Gibbs, Samantha E. J.; Travassos da Rosa, Amelia P. A.; Ellis, Julie C.; Ip, Hon S.; Shearn-Bochsler, Valerie I.; Rogers, Matthew B.; Ghedin, Elodie; Holmes, Edward C.; Parrish, Colin R.; Dwyer, Chris

    2014-01-01

    ABSTRACT Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, MA, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here, we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein that was demonstrated to have fusion activity; this also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined. IMPORTANCE The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and

  19. Two year field study to evaluate the efficacy of Mamestra brassicae nucleopolyhedrovirus combined with proteins derived from Xestia c-nigrum granulovirus.

    PubMed

    Goto, Chie; Mukawa, Shigeyuki; Mitsunaga, Takayuki

    2015-03-01

    Japan has only three registered baculovirus biopesticides despite its long history of studies on insect viruses. High production cost is one of the main hindrances for practical use of baculoviruses. Enhancement of insecticidal effect is one possible way to overcome this problem, so there have been many attempts to develop additives for baculoviruses. We found that alkaline soluble proteins of capsules (GVPs) of Xestia c-nigrum granulovirus can increase infectivity of some viruses including Mamestra brassicae nucleopolyhedrovirus (MabrNPV), and previously reported that MabrNPV mixed with GVPs was highly infectious to three important noctuid pests of vegetables in the following order, Helicoverpa armigera, M. brassicae, and Autographa nigrisigna. In this study, small-plot experiments were performed to assess concentrations of MabrNPV and GVPs at three cabbage fields and a broccoli field for the control of M. brassicae. In the first experiment, addition of GVPs (10 µg/mL) to MabrNPV at 106 OBs/mL resulted in a significant increase in NPV infection (from 53% to 66%). In the second experiment, the enhancing effect of GVP on NPV infection was confirmed at 10-times lower concentrations of MabrNPV. In the third and fourth experiments, a 50% reduction in GVPs (from 10 µg/mL to 5 µg/mL) did not result in a lowering of infectivity of the formulations containing MabrNPV at 105 OBs/mL. These results indicate that GVPs are promising additives for virus insecticides. PMID:25760139

  20. Comparison of field-collected ascovirus isolates by DNA hybridization, host range, and histopathology.

    PubMed

    Hamm, J J; Styer, E L; Federici, B A

    1998-09-01

    Six field-collected ascovirus isolates obtained from five noctuid species in the continental United States were compared with respect to the general relatedness of their DNA, host range, and histopathology. Two isolates were from Spodoptera frugiperda, and the other four were from Autographa precationis, Heliothis virescens, Helicoverpa zea, and Trichoplusia ni. DNA-DNA hybridization studies showed that the six isolates belonged to three distinct viral species, with the isolates from S. frugiperda composing one species, those from A. precationis and H. virescens a second species, and those from H. zea and T. ni a third species. The host range and histopathology of each isolate was studied in eight noctuid species, S. frugiperda, Spodoptera ornithogalli, Spodoptera exigua, Spodoptera eridania, H. virescens, H. zea, A. precationis, and Feltia subterranea. Though some variation existed between the different isolates of each viral species, distinct patterns were apparent for each. The viral species from S. frugiperda had a host range that was limited primarily to Spodoptera species and both isolates of this virus only replicated and caused significant pathology in the fat body, whereas the viral species from A. precationis and H. virescens had a much broader host range that included most of the species tested, but also had a tissue tropism primarily restricted to the fat body. The viral species from T. ni and H. zea readily infected all the hosts tested, where the principal site of replication and significant pathology was the epidermis. In many test hosts, however, this viral species also replicated and caused significant pathology in the tracheal epithelium and to a lesser extent in the fat body. Aside from contributing to knowledge of ascovirus biology, these studies indicate that DNA hybridization profiles combined with studies of host range and tissue tropism can be used as characters for defining ascovirus species. PMID:9709014

  1. Adaptive strategies in nocturnally migrating insects and songbirds: contrasting responses to wind.

    PubMed

    Chapman, Jason W; Nilsson, Cecilia; Lim, Ka S; Bäckman, Johan; Reynolds, Don R; Alerstam, Thomas

    2016-01-01

    Animals that use flight as their mode of transportation must cope with the fact that their migration and orientation performance is strongly affected by the flow of the medium they are moving in, that is by the winds. Different strategies can be used to mitigate the negative effects and benefit from the positive effects of a moving flow. The strategies an animal can use will be constrained by the relationship between the speed of the flow and the speed of the animal's own propulsion in relation to the surrounding air. Here we analyse entomological and ornithological radar data from north-western Europe to investigate how two different nocturnal migrant taxa, the noctuid moth Autographa gamma and songbirds, deal with wind by analysing variation in resulting flight directions in relation to the wind-dependent angle between the animal's heading and track direction. Our results, from fixed locations along the migratory journey, reveal different global strategies used by moths and songbirds during their migratory journeys. As expected, nocturnally migrating moths experienced a greater degree of wind drift than nocturnally migrating songbirds, but both groups were more affected by wind in autumn than in spring. The songbirds' strategies involve elements of both drift and compensation, providing some benefits from wind in combination with destination and time control. In contrast, moths expose themselves to a significantly higher degree of drift in order to obtain strong wind assistance, surpassing the songbirds in mean ground speed, at the cost of a comparatively lower spatiotemporal migratory precision. Moths and songbirds show contrasting but adaptive responses to migrating through a moving flow, which are fine-tuned to the respective flight capabilities of each group in relation to the wind currents they travel within. PMID:26147535

  2. Two Year Field Study to Evaluate the Efficacy of Mamestra brassicae Nucleopolyhedrovirus Combined with Proteins Derived from Xestia c-nigrum Granulovirus

    PubMed Central

    Goto, Chie; Mukawa, Shigeyuki; Mitsunaga, Takayuki

    2015-01-01

    Japan has only three registered baculovirus biopesticides despite its long history of studies on insect viruses. High production cost is one of the main hindrances for practical use of baculoviruses. Enhancement of insecticidal effect is one possible way to overcome this problem, so there have been many attempts to develop additives for baculoviruses. We found that alkaline soluble proteins of capsules (GVPs) of Xestia c-nigrum granulovirus can increase infectivity of some viruses including Mamestra brassicae nucleopolyhedrovirus (MabrNPV), and previously reported that MabrNPV mixed with GVPs was highly infectious to three important noctuid pests of vegetables in the following order, Helicoverpa armigera, M. brassicae, and Autographa nigrisigna. In this study, small-plot experiments were performed to assess concentrations of MabrNPV and GVPs at three cabbage fields and a broccoli field for the control of M. brassicae. In the first experiment, addition of GVPs (10 µg/mL) to MabrNPV at 106 OBs/mL resulted in a significant increase in NPV infection (from 53% to 66%). In the second experiment, the enhancing effect of GVP on NPV infection was confirmed at 10-times lower concentrations of MabrNPV. In the third and fourth experiments, a 50% reduction in GVPs (from 10 µg/mL to 5 µg/mL) did not result in a lowering of infectivity of the formulations containing MabrNPV at 105 OBs/mL. These results indicate that GVPs are promising additives for virus insecticides. PMID:25760139

  3. Multiple Mutations on the Second Acetylcholinesterase Gene Associated With Dimethoate Resistance in the Melon Aphid, Aphis gossypii (Hemiptera: Aphididae).

    PubMed

    Lokeshwari, D; Krishna Kumar, N K; Manjunatha, H

    2016-04-01

    The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is an important cosmopolitan and extremely polyphagous species capable of causing direct and indirect damage to various crops. Insecticide resistance in melon aphids is of particular concern. To determine the basis of resistance, organophosphate (OP)-resistant strains of A. gossypii were obtained by continuous selection with dimethoate in the laboratory, and resistance mechanisms were investigated along with susceptible strains. Three resistant strains LKR-1, LKR-2, and LKR-3 exhibiting 270-, 243-, and 210-fold resistance obtained after 30 generations of selection with dimethoate, respectively, were utilized in this study. The role of acetylcholinesterase (AChE), a target enzyme for OPs and carbamates (CMs), was investigated. AChE enzyme assay revealed that there was no significant change in the activities of AChE in resistant and susceptible strains. However, AChE inhibitory assay showed that 50% of the enzyme activity in resistant strains was inhibited at significantly higher concentration of dimethoate (131.87, 158.65, and 99.29 µmolL(−1)) as compared with susceptible strains (1.75 and 2.01 µmolL(−1)), indicating AChE insensitivity owing to altered AChE. Molecular diagnostic tool polymerase chain reaction-restriction fragment length polymorphism revealed the existence of two consistent non-synonymous point mutations, single-nucleotide polymorphism, viz., A302S (equivalent to A201 in Torpedo californica Ayres) and S431F (equivalent to F331 in T. californica), in the AChE gene Ace2 of resistant strains. Further, cloning and sequencing of a partial fragment of Ace2 (897 bp) gene from susceptible and resistant strains revealed an additional novel mutation G221A in resistant strains, LKR-1 and LKR-2. Susceptible Ace2 genes shared 99.6 and 98.9% identity at the nucleic acid and amino acid levels with resistant ones, respectively. Functional analysis of these point mutations was assessed by in

  4. Speciation in Western Scrub-Jays, Haldane’s rule, and genetic clines in secondary contact

    PubMed Central

    2014-01-01

    Background Haldane’s Rule, the tendency for the heterogametic sex to show reduced fertility in hybrid crosses, can obscure the signal of gene flow in mtDNA between species where females are heterogametic. Therefore, it is important when studying speciation and species limits in female-heterogametic species like birds to assess the signature of gene flow in the nuclear genome as well. We studied introgression of microsatellites and mtDNA across a secondary contact zone between coastal and interior lineages of Western Scrub-Jays (Aphelocoma californica) to test for a signature of Haldane’s Rule: a narrower cline of introgression in mtDNA compared to nuclear markers. Results Our initial phylogeographic analysis revealed that there is only one major area of contact between coastal and interior lineages and identified five genetic clusters with strong spatial structuring: Pacific Slope, Interior US, Edwards Plateau (Texas), Northern Mexico, and Southern Mexico. Consistent with predictions from Haldane’s Rule, mtDNA showed a narrower cline than nuclear markers across a transect through the hybrid zone. This result is not being driven by female-biased dispersal because neutral diffusion analysis, which included estimates of sex-specific dispersal rates, also showed less diffusion of mtDNA. Lineage-specific plumage traits were associated with nuclear genetic profiles for individuals in the hybrid zone, indicating that these differences are under genetic control. Conclusions This study adds to a growing list of studies that support predictions of Haldane’s Rule using cline analysis of multiple loci of differing inheritance modes, although alternate hypotheses like selection on different mtDNA types cannot be ruled out. That Haldane’s Rule appears to be operating in this system suggests a measure of reproductive isolation between the Pacific Slope and interior lineages. Based on a variety of evidence from the phenotype, ecology, and genetics, we recommend elevating

  5. Baculovirus display of single chain antibody (scFv) using a novel signal peptide

    PubMed Central

    2010-01-01

    baculoviral progeny displaying scFvE2/p17. The function required for BV envelope incorporation was carried by the N-terminal octadecapeptide of scFvE2/p17, which acted as a signal peptide for BV display. Fusion of this peptide to the N-terminus of scFv molecules of interest could be applied as a general method for BV-display of scFv in a GP64- and VSV-G-independent manner. PMID:21092083

  6. The biological soil crusts of the San Nicolas Island: Enigmatic algae from a geographically isolated ecosystem

    USGS Publications Warehouse

    Flechtner, V.R.; Johansen, J.R.; Belnap, J.

    2008-01-01

    Composite soil samples from 7 sites on San Nicolas Island were evaluated quantitatively and qualitatively for the presence of cyanobacteria and eukaryotic microalgae. Combined data demonstrated a rich algal flora with 19 cyanobacterial and 19 eukaryotic microalgal genera being identified, for a total of 56 species. Nine new species were identified and described among the cyanobacteria and the eukaryotic microalgae that were isolated: Leibleinia edaphica, Aphanothece maritima, Chroococcidiopsis edaphica, Cyanosarcina atroveneta, Hassallia californica, Hassallia pseudoramosissima, Microchaete terrestre, Palmellopsis californiens, and Pseudotetracystis compactis. Distinct distributional patterns of algal taxa existed among sites on the island and among soil algal floras of western North America. Some algal taxa appeared to be widely distributed across many desert regions, including Microcoleus vaginatus, Nostoc punctiforme, Nostoc paludosum, and Tolypothrix distorta, Chlorella vulgaris, Diplosphaera cf. chodatii, Myrmecia astigmatica, Myrmecia biatorellae, Hantzschia amphioxys, and Luticola mutica. Some taxa share a distinctly southern distribution with soil algae from southern Arizona, southern California, and Baja California (e.g., Scenedesmus deserticola and Eustigmatos magnus). The data presented herein support the view that the cyanobacterial and microalgal floras of soil crusts possess significant biodiversity, much of it previously undescribed.

  7. Live cell imaging of neuronal growth cone motility and guidance in vitro

    PubMed Central

    Suter, Daniel M.

    2013-01-01

    Summary The neuronal growth cone, a highly motile structure at the tip of neuronal processes, is an excellent model system for studying directional cell movements. While biochemical and genetic approaches unveiled molecular interactions between ligand, receptor, signaling and cytoskeleton-associated proteins controlling axonal growth and guidance, in vitro live cell imaging has emerged as a crucial approach for dissecting cellular mechanisms of growth cone motility and guidance. Important insights into these mechanisms have been gained from studies using the large growth cones elaborated by Aplysia californica neurons, an outstanding model system for live cell imaging for a number of reasons. Identified neurons can be isolated and imaged at room temperature. Aplysia growth cones are 5–10 times larger than growth cones from other species, making them suitable for quantitative high-resolution imaging of cytoskeletal protein dynamics and biophysical approaches. Lastly, protein, RNA, fluorescent probes and small molecules can be microinjected into the neuronal cell body for localization and functional studies. The following chapter describes culturing of Aplysia bag cell neurons, live cell imaging of neuronal growth cones using differential interference contrast and fluorescent speckle microscopy as well as the restrained bead interaction assay to induce adhesion-mediated growth cone guidance in vitro. PMID:21748670

  8. Putative lateral inhibition in sensory processing for directional turns

    PubMed Central

    Yafremava, Liudmila S.

    2011-01-01

    Computing targeted responses is a general problem in goal-directed behaviors. We sought the sensory template for directional turning in the predatory sea slug Pleurobranchaea californica, which calculates precise turn angles by averaging multiple stimulus sites on its chemotactile oral veil (Yafremava LS, Anthony CW, Lane L, Campbell JK, Gillette R. J Exp Biol 210: 561–569, 2007). Spiking responses to appetitive chemotactile stimulation were recorded in the two bilateral pairs of oral veil nerves, the large oral veil nerve (LOVN) and the tentacle nerve (TN). The integrative abilities of the peripheral nervous system were significant. Nerve spiking responses to punctate, one-site stimulation of the oral veil followed sigmoid relations as stimuli moved between lateral tentacle and the midline. Receptive fields of LOVN and TN were unilateral, overlapping, and oppositely weighted for responsiveness across the length of oral veil. Simultaneous two-site stimulation caused responses of amplitudes markedly smaller than the sum of corresponding one-site responses. Plots of two-site nerve responses against the summed approximate distances from midline of each site were markedly linear. Thus the sensory paths in the peripheral nervous system show reciprocal occlusion similar to lateral inhibition. This outcome suggests a novel neural function for lateral inhibitory mechanisms, distinct from simple contrast enhancement, in computation of both sensory maps and targeted motor actions. PMID:21490281

  9. Effects of low levels of herbicides on prairie species of the Willamette Valley, Oregon.

    PubMed

    Olszyk, David; Blakeley-Smith, Matthew; Pfleeger, Thomas; Lee, E Henry; Plocher, Milton

    2013-11-01

    The relative sensitivity of 17 noncrop plant species from Oregon's Willamette Valley was determined in response to glyphosate, tribenuron methyl (tribenuron), and fluazifop-p-butyl (fluazifop) herbicides. For glyphosate, Elymus trachycaulus, Festuca arundinacea, Madia elegans, Potentilla gracilis, and Ranunculus occidentalis were the most sensitive species, based on a concentration calculated to reduce shoot dry weight by 25% (IC25 values) of 0.02 to 0.04 × a field application rate of 1112 g active ingredient (a.i.) per hectare. Clarkia amoena and Lupinus albicaulis were the most tolerant to glyphosate, with IC25 values near the field application rate. Clarkia amoena, Prunella vulgaris, and R. occidentalis were the most sensitive to tribenuron, with IC25 values of 0.001 to 0.004 × a field application rate of 8.7 g a.i. ha(-1) for shoot dry weight. Five grass species were tolerant to tribenuron with no significant IC25 values. For fluazifop, 2 native grasses, E. trachycaulus and Danthonia californica, were the most sensitive species, with IC25 values of 0.007 and 0.010 × a field application rate of 210 g a.i. ha(-1) , respectively, for shoot dry weight, while a native grass, Festuca roemeri, and nearly all forbs showed little or no response. These results also indicated that the 3 introduced species used in the present study may be controlled with 1 of the tested herbicides: glyphosate (F. arundinacea), tribenuron (Leucanthemum vulgare), and fluazifop (Cynosurus echinatus). PMID:23881750

  10. Intraguild predation by shore crabs affects mortality, behavior, growth, and densities of California horn snails

    USGS Publications Warehouse

    Lorda, J.; Hechinger, R.F.; Cooper, S. D.; Kuris, A. M.; Lafferty, Kevin D.

    2016-01-01

    The California horn snail, Cerithideopsis californica, and the shore crabs, Pachygrapsus crassipesand Hemigrapsus oregonensis, compete for epibenthic microalgae, but the crabs also eat snails. Such intraguild predation is common in nature, despite models predicting instability. Using a series of manipulations and field surveys, we examined intraguild predation from several angles, including the effects of stage-dependent predation along with direct consumptive and nonconsumptive predator effects on intraguild prey. In the laboratory, we found that crabs fed on macroalgae, snail eggs, and snails, and the size of consumed snails increased with predator crab size. In field experiments, snails grew less in the presence of crabs partially because snails behaved differently and were buried in the sediment (nonconsumptive effects). Consistent with these results, crab and snail abundances were negatively correlated in three field surveys conducted at three different spatial scales in estuaries of California, Baja California, and Baja California Sur: (1) among 61 sites spanning multiple habitat types in three estuaries, (2) among the habitats of 13 estuaries, and (3) among 34 tidal creek sites in one estuary. These results indicate that shore crabs are intraguild predators on California horn snails that affect snail populations via predation and by influencing snail behavior and performance.

  11. Yeast Biodiversity in Vineyard Environments Is Increased by Human Intervention.

    PubMed

    Drumonde-Neves, João; Franco-Duarte, Ricardo; Lima, Teresa; Schuller, Dorit; Pais, Célia

    2016-01-01

    One hundred and five grape samples were collected during two consecutive years from 33 locations on seven oceanic islands of the Azores Archipelago. Grape samples were obtained from vineyards that were either abandoned or under regular cultivation involving common viticultural interventions, to evaluate the impact of regular human intervention on grape yeast biota diversity in vineyards. A total of 3150 yeast isolates were obtained and 23 yeast species were identified. The predominant species were Hanseniaspora uvarum, Pichia terricola, Starmerella bacillaris and Issatchenkia hanoiensis. The species Barnettozyma californica, Candida azymoides and Pichia cecembensis were reported in grapes or wine-associated environments for the first time. A higher biodiversity was found in active vineyards where regular human intervention takes place (Shannon index: 1.89 and 1.53 in the first and second years, respectively) when compared to the abandoned ones (Shannon index: 0.76 and 0.31). This finding goes against the assumptions that human intervention can destroy biodiversity and lead to homogeneity in the environment. Biodiversity indices were considerably lower in the year with the heaviest rainfall. This study is the first to report on the grape yeast communities from several abandoned vineyards that have undergone no human intervention. PMID:27500638

  12. Exploring natural products for new taste sensations.

    PubMed

    Starkenmann, Christian; Cayeux, Isabelle; Birkbeck, Anthony A

    2011-01-01

    This paper discusses the discovery of uncommon taste or trigeminal active compounds and their associated sensory analysis using human tasting panels with the aim of enhancing the overall taste experience whilst reducing where possible the sugar and salt content of foods. The first example outlines the discovery of the sensory quality attributes of (R)-2-(carboxymethylamino)propanoic acid, named (R)-strombine, as assessed by a panel of 47 subjects to confirm its contribution to the typical taste of scallop muscle. The second example discusses the pungency and trigeminal effect of polygodial, which is compared with piperine and capsaicin, as well as the elucidation of a new structure eliciting a trigeminal effect, (+/-)-trans-2,3,3a,7a-tetrahydro-1 H-indene-4-carbaldehyde, discovered in Amomum tsao-ko. Finally, the time intensity trigeminal effect of (-)-menthol is compared with (1R,2RS,4RS)-1-isopropyl-4-methylbicyclo[3.1.0]hexan-2-ol, named dihydroumbellulol, a new cooling compound obtained by hemi-synthesis from umbellulone extracted from Umbellularia californica Nutt. PMID:21797169

  13. A neuron-in-capillary platform for facile collection and mass spectrometric characterization of a secreted neuropeptide

    PubMed Central

    Lee, Chang Young; Fan, Yi; Rubakhin, Stanislav S.; Yoon, Sook; Sweedler, Jonathan V.

    2016-01-01

    The integration of microfluidic devices—which efficiently handle small liquid volumes—with separations/mass spectrometry (MS) is an effective approach for profiling the neurochemistry occurring in selected neurons. Interfacing the microfluidic cell culture to the mass spectrometer is challenging because of geometric and scaling issues. Here we demonstrate the hyphenation of a neuron-in-capillary platform to a solid phase extraction device and off-line MS. A primary neuronal culture of Aplysia californica neurons was established directly inside a cylindrical polyimide capillary. The approach also uses a particle-embedded monolith to condition neuropeptide releasates collected from several Aplysia neurons cultured in the capillary, with the subsequent characterization of released peptides via MS. This system presents a number of advances compared to more traditional microfluidic devices fabricated with polydimethylsiloxane. These include low cost, easy access to cell culture, rigidity, ease of transport, and minimal fluid handling. The cylindrical geometry of the platform allows convenient interface with a wide range of analytical tools that utilize capillary columns. PMID:27245782

  14. Parallel evolution of serotonergic neuromodulation underlies independent evolution of rhythmic motor behavior.

    PubMed

    Lillvis, Joshua L; Katz, Paul S

    2013-02-01

    Neuromodulation can dynamically alter neuronal and synaptic properties, thereby changing the behavioral output of a neural circuit. It is therefore conceivable that natural selection might act upon neuromodulation as a mechanism for sculpting the behavioral repertoire of a species. Here we report that the presence of neuromodulation is correlated with the production of a behavior that most likely evolved independently in two species: Tritonia diomedea and Pleurobranchaea californica (Mollusca, Gastropoda, Opisthobranchia, Nudipleura). Individuals of both species exhibit escape swimming behaviors consisting of repeated dorsal-ventral whole-body flexions. The central pattern generator (CPG) circuits underlying these behaviors contain homologous identified neurons: DSI and C2 in Tritonia and As and A1 in Pleurobranchaea. Homologs of these neurons also can be found in Hermissenda crassicornis where they are named CPT and C2, respectively. However, members of this species do not exhibit an analogous swimming behavior. In Tritonia and Pleurobranchaea, but not in Hermissenda, the serotonergic DSI homologs modulated the strength of synapses made by C2 homologs. Furthermore, the serotonin receptor antagonist methysergide blocked this neuromodulation and the swimming behavior. Additionally, in Pleurobranchaea, the robustness of swimming correlated with the extent of the synaptic modulation. Finally, injection of serotonin induced the swimming behavior in Tritonia and Pleurobranchaea, but not in Hermissenda. This suggests that the analogous swimming behaviors of Tritonia and Pleurobranchaea share a common dependence on serotonergic neuromodulation. Thus, neuromodulation may provide a mechanism that enables species to acquire analogous behaviors independently using homologous neural circuit components. PMID:23392697

  15. Associative memory in three aplysiids: correlation with heterosynaptic modulation.

    PubMed

    Hoover, Brian A; Nguyen, Hoang; Thompson, Laura; Wright, William G

    2006-01-01

    Much recent research on mechanisms of learning and memory focuses on the role of heterosynaptic neuromodulatory signaling. Such neuromodulation appears to stabilize Hebbian synaptic changes underlying associative learning, thereby extending memory. Previous comparisons of three related sea-hares (Mollusca, Opisthobranchia) uncovered interspecific variation in neuromodulatory signaling: strong in Aplysia californica, immeasureable in Dolabrifera dolabrifera, and intermediate in Phyllaplysia taylori. The present study addressed whether this interspecific variation in neuromodulation is correlated with memory of associative (classical conditioning) learning. We differentially conditioned the tail-mantle withdrawal reflex of each of the three species: Mild touch to one side of the tail was paired with a noxious electrical stimulus to the neck. Mild touch to the other side served as an internal control. Post-training reflex amplitudes were tested 15-30 min after training and compared with pre-test amplitudes. All three species showed conditioning: training increased the paired reflex more than the unpaired reflex. However, the temporal pattern of conditioning varied between species. Aplysia showed modest conditioning that grew across the post-test period. Dolabrifera showed distinctly short-lived conditioning, present only on the first post-test. The time course of memory in Phyllaplysia was intermediate, although not statistically distinguishable from the other two species. Taken together, these experiments suggest that evolutionary changes in nonassociative heterosynaptic modulation may contribute to evolutionary changes in the stability of the memory of classical conditioning. PMID:17142308

  16. Evidence for homologous peptidergic neurons in the buccal ganglia of diverse nudibranch mollusks.

    PubMed

    Watson, W H; Willows, A O

    1992-03-01

    The buccal ganglia of seven nudibranches (Aeolidia papillosa, Armina californica, Dirona albolineata, D. picta, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea) were examined to explore possible homologies between large cells that reacted with antibodies directed against small cardioactive peptide B (SCPB). The buccal ganglion of each species possessed a pair of large, dorsal-lateral, whitish neurons that contained an SCPB-like peptide. We refer to these neurons as the SLB (SCPB-immunoreactive Large Buccal) cells. In all species examined, the SLB cells project out the gastroesophageal nerves and appear to innervate the esophagus. In each species, an apparent rhythmic feeding motor program (FMP) was observed by intracellular recording from both SLB neurons and other neurons in isolated preparations of the buccal ganglia. SLB cells often fire at a high frequency, and usually burst in a specific phase relation to the FMP activity. Stimulation of SLB cells enhances expression of the feeding motor program, either by potentiating existing activity or eliciting the FMP in quiescent preparations. Finally, perfusion of isolated buccal ganglia with SCPB excites the SLB cells and activates FMPs. Thus, both the immunohistochemical and electrophysiological data suggest that the SLB cells within three suborders of the opisthobranchia (Dendronotacea, Arminacea, and Aeolidacea) are homologous. A comparison of our data with previously published studies indicates that SLB cell homologs may exist in other gastropods as well. PMID:1527526

  17. Molluscan mobile elements similar to the vertebrate recombination-activating genes

    PubMed Central

    Panchin, Yuri; Moroz, Leonid L.

    2009-01-01

    Animal genomes contain ~20,000 genes. Additionally millions of genes for antigen receptors are generated in cells of the immune system from the sets of separate gene segments by a mechanism known as the V(D)J somatic recombination. The components of the V(D)J recombination system, Recombination-Activating Gene proteins (RAG1 and RAG2) and recombination signal sequence (RSS), are thought to have “entered” the vertebrate genome as a hypothetical “RAG transposon”. Recently discovered mobile elements have terminal inverted repeats (TIRs) similar to RSS and may encode proteins with a different degree of similarity to RAG1. We describe a novel N-RAG-TP transposon identified from the sea slug Aplysia californica that encodes a protein similar to the N-terminal part of RAG1 in vertebrates. This refines the “RAG transposon” hypothesis and allows us to propose a scenario for V(D)J recombination machinery evolution from a relic transposon related to the existing mobile elements N-RAG-TP, Chapaev and Transib. PMID:18313399

  18. House Finch (Haemorhous mexicanus) Conjunctivitis, and Mycoplasma spp. Isolated from North American Wild Birds, 1994-2015.

    PubMed

    Ley, David H; Hawley, Dana M; Geary, Steven J; Dhondt, André A

    2016-07-01

    Sampling wild birds for mycoplasma culture has been key to the study of House Finch (Haemorhous mexicanus) conjunctivitis, yielding isolates of Mycoplasma gallisepticum spanning the temporal and geographic ranges of disease from emergence to endemicity. Faced with the challenges and costs of sample collection over time and from remote locations for submission to our laboratory for mycoplasma culture, protocols evolved to achieve a practical optimum. Herein we report making M. gallisepticum isolates from House Finches almost every year since the disease emerged in 1994, and we now have 227 isolates from 17 states. Our wild bird host range for M. gallisepticum isolates includes Blue Jay ( Cyanocitta cristata ), American Goldfinch (Spinus tristis), Lesser Goldfinch (Spinus psaltria), Purple Finch (Haemorhous purpureus), Evening Grosbeak ( Coccothraustes vespertinus ), and herein first reports for Western Scrub-jay ( Aphelocoma californica ), and American Crow ( Corvus brachyrhynchos ). By collecting and identifying isolates from birds with clinical signs similar to those of House Finch conjunctivitis, we also expanded the known host range of Mycoplasma sturni and obtained isolates from additional wild bird species. Accumulating evidence shows that a diverse range of wild bird species may carry or have been exposed to M. gallisepticum in the US, as in Europe and Asia. Therefore, the emergence of a pathogenic M. gallisepticum strain in House Finches may actually be the exception that has allowed us to identify the broader epidemiologic picture. PMID:27285414

  19. Yeast Biodiversity in Vineyard Environments Is Increased by Human Intervention

    PubMed Central

    Drumonde-Neves, João; Lima, Teresa; Schuller, Dorit; Pais, Célia

    2016-01-01

    One hundred and five grape samples were collected during two consecutive years from 33 locations on seven oceanic islands of the Azores Archipelago. Grape samples were obtained from vineyards that were either abandoned or under regular cultivation involving common viticultural interventions, to evaluate the impact of regular human intervention on grape yeast biota diversity in vineyards. A total of 3150 yeast isolates were obtained and 23 yeast species were identified. The predominant species were Hanseniaspora uvarum, Pichia terricola, Starmerella bacillaris and Issatchenkia hanoiensis. The species Barnettozyma californica, Candida azymoides and Pichia cecembensis were reported in grapes or wine-associated environments for the first time. A higher biodiversity was found in active vineyards where regular human intervention takes place (Shannon index: 1.89 and 1.53 in the first and second years, respectively) when compared to the abandoned ones (Shannon index: 0.76 and 0.31). This finding goes against the assumptions that human intervention can destroy biodiversity and lead to homogeneity in the environment. Biodiversity indices were considerably lower in the year with the heaviest rainfall. This study is the first to report on the grape yeast communities from several abandoned vineyards that have undergone no human intervention. PMID:27500638

  20. Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

    PubMed Central

    Papadouli, I; Potamianos, S; Hadjidakis, I; Bairaktari, E; Tsikaris, V; Sakarellos, C; Cung, M T; Marraud, M; Tzartos, S J

    1990-01-01

    The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible. PMID:1695844

  1. Oral administration of an immunodominant T-cell epitope downregulates Th1/Th2 cytokines and prevents experimental myasthenia gravis

    PubMed Central

    Baggi, Fulvio; Andreetta, Francesca; Caspani, Elisabetta; Milani, Monica; Longhi, Renato; Mantegazza, Renato; Cornelio, Ferdinando; Antozzi, Carlo

    1999-01-01

    The mucosal administration of the native antigen or peptide fragments corresponding to immunodominant regions is effective in preventing or treating several T cell–dependent models of autoimmune disease. No data are yet available on oral tolerance with immunodominant T-cell peptides in experimental autoimmune myasthenia gravis (EAMG), an animal model of B cell–dependent disease. We report that oral administration of the T-cell epitope α146-162 of the Torpedo californica acetylcholine receptor (TAChR) α-subunit suppressed T-cell responses to AChR and ameliorated the disease in C57Bl/6 (B6) mice. Protection from EAMG was associated with reduced serum Ab’s to mouse AChR and reduced AChR loss in muscle. The effect of Tα146-162 feeding was specific; treatment with a control peptide did not affect EAMG manifestations. The protective effect induced by peptide Tα146-162 was mediated by reduced production of IFN-γ, IL-2, and IL-10 by TAChR-reactive cells, suggesting T-cell anergy. TGF-β–secreting Th3 cells did not seem to be involved in tolerance induction. We therefore demonstrate that feeding a single immunodominant epitope can prevent an Ab-mediated experimental model of autoimmune disease. PMID:10545527

  2. Minimum number of lipids are required to support the functional properties of the nicotinic acetylcholine receptor

    SciTech Connect

    Jones, O.T.; Eubanks, J.H.; Earnest, J.P.; McNamee, M.G.

    1988-05-17

    The detergent sodium cholate was used to both solubilize and partially delipidate the nicotinic acetylcholine receptor from Torpedo californica. Using both native membranes and reconstituted membranes, it is shown that the detergent to lipid molar ratio is the most important parameter in determining the effect of the detergent on the functional properties of the receptor. Receptor-lipid complexes were quantitatively separated from detergent and excess lipids by centrifugation through detergent-free sucrose gradients. The lipid to protein molar ratio of the complexes could be precisely controlled by adjusting the cholate and lipid concentrations of the starting membranes. Analyses of both ion influx activity and ligand binding revealed that a minimum of 45 lipids per receptor was required for stabilization of the receptor in a fully functional state. Progressive irreversible inactivation occurred as the lipid to protein mole ratio was decreased below 45, and complete inactivation occurred below a ratio of 20. The results are consistent with a functional requirement for a single shell of lipids around the perimeter of the receptor.

  3. The Insect Chemoreceptor Superfamily Is Ancient in Animals.

    PubMed

    Robertson, Hugh M

    2015-11-01

    The insect chemoreceptor superfamily consists of 2 gene families, the highly diverse gustatory receptors (GRs) found in all arthropods with sequenced genomes and the odorant receptors that evolved from a GR lineage and have been found only in insects to date. Here, I describe relatives of the insect chemoreceptor superfamily, specifically the basal GR family, in diverse other animals, showing that the superfamily dates back at least to early animal evolution. GR-Like (GRL) genes are present in the genomes of the placozoan Trichoplax adhaerens, an anemone Nematostella vectensis, a coral Acropora digitifera, a polychaete Capitella teleta, a leech Helobdella robusta, the nematode Caenorhabditis elegans (and many other nematodes), 3 molluscs (a limpet Lottia gigantea, an oyster Crassostrea gigas, and the sea hare Aplysia californica), the sea urchin Strongylocentrotus purpuratus, and the sea acorn Saccoglossus kowalevskii. While some of these animals contain multiple divergent GRL lineages, GRLs have been lost entirely from other animal lineages such as vertebrates. GRLs are absent from the ctenophore Mnemiopsis leidyi, the demosponge Amphimedon queenslandica, and 2 available chaonoflagellate genomes, so it remains unclear whether this superfamily originated before or during animal evolution. PMID:26354932

  4. Lesions of Copper Toxicosis in Captive Marine Invertebrates With Comparisons to Normal Histology.

    PubMed

    LaDouceur, E E B; Wynne, J; Garner, M M; Nyaoke, A; Keel, M K

    2016-05-01

    Despite increasing concern for coral reef ecosystem health within the last decade, there is scant literature concerning the histopathology of diseases affecting the major constituents of coral reef ecosystems, particularly marine invertebrates. This study describes histologic findings in 6 species of marine invertebrates (California sea hare [Aplysia californica], purple sea urchin [Strongylocentrotus purpuratus], sunburst anemone [Anthopleura sola], knobby star [Pisaster giganteus], bat star [Asterina miniata], and brittle star [Ophiopteris papillosa]) with spontaneous copper toxicosis, 4 purple sea urchins with experimentally induced copper toxicosis, and 1 unexposed control of each species listed. The primary lesions in the California sea hare with copper toxicosis were branchial and nephridial necrosis. Affected echinoderms shared several histologic lesions, including epidermal necrosis and ulceration and increased numbers of coelomocytes within the water-vascular system. The sunburst anemone with copper toxicosis had necrosis of both epidermis and gastrodermis, as well as expulsion of zooxanthellae from the gastrodermis. In addition to the lesions attributed to copper toxicosis, our results describe normal microscopic features of these animals that may be useful for histopathologic assessment of marine invertebrates. PMID:26459519

  5. Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

    PubMed Central

    Machold, J; Utkin, Y; Kirsch, D; Kaufmann, R; Tsetlin, V; Hucho, F

    1995-01-01

    A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis. Images Fig. 2 PMID:7543679

  6. Selective control of small versus large diameter axons using infrared laser light (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Lothet, Emilie H.; Shaw, Kendrick M.; Horn, Charles C.; Lu, Hui; Wang, Yves T.; Jansen, E. Duco; Chiel, Hillel J.; Jenkins, Michael W.

    2016-03-01

    Sensory information is conveyed to the central nervous system via small diameter unmyelinated fibers. In general, smaller diameter axons have slower conduction velocities. Selective control of such fibers could create new clinical treatments for chronic pain, nausea in response to chemo-therapeutic agents, or hypertension. Electrical stimulation can control axonal activity, but induced axonal current is proportional to cross-sectional area, so that large diameter fibers are affected first. Physiologically, however, synaptic inputs generally affect small diameter fibers before large diameter fibers (the size principle). A more physiological modality that first affected small diameter fibers could have fewer side effects (e.g., not recruiting motor axons). A novel mathematical analysis of the cable equation demonstrates that the minimum length along the axon for inducing block scales with the square root of axon diameter. This implies that the minimum length along an axon for inhibition will scale as the square root of axon diameter, so that lower radiant exposures of infrared light will selectively affect small diameter, slower conducting fibers before those of large diameter. This prediction was tested in identified neurons from the marine mollusk Aplysia californica. Radiant exposure to block a neuron with a slower conduction velocity (B43) was consistently lower than that needed to block a faster conduction velocity neuron (B3). Furthermore, in the vagus nerve of the musk shrew, lower radiant exposure blocked slow conducting fibers before blocking faster conducting fibers. Infrared light can selectively control smaller diameter fibers, suggesting many novel clinical treatments.

  7. Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor.

    PubMed Central

    Poulter, L; Earnest, J P; Stroud, R M; Burlingame, A L

    1989-01-01

    Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the alpha subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the gamma subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up approximately equal to 22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide. Images PMID:2771948

  8. Lysophosphatidic Acid Acyltransferase from Coconut Endosperm Mediates the Insertion of Laurate at the sn-2 Position of Triacylglycerols in Lauric Rapeseed Oil and Can Increase Total Laurate Levels

    PubMed Central

    Knutzon, Deborah S.; Hayes, Thomas R.; Wyrick, Annette; Xiong, Hui; Maelor Davies, H.; Voelker, Toni A.

    1999-01-01

    Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 and sn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999–1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at the sn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels. PMID:10398708

  9. Cost-benefit analysis potential in feeding behavior of a predatory snail by integration of hunger, taste, and pain

    NASA Astrophysics Data System (ADS)

    Gillette, Rhanor; Huang, Rong-Chi; Hatcher, Nathan; Moroz, Leonid L.

    2000-03-01

    Hunger/satiation state interacts with appetitive and noxious stimuli to determine feeding and avoidance responses. In the predatory marine snail Pleurobranchaea californica, food chemostimuli induced proboscis extension and biting at concentration thresholds that varied directly with satiation state. However, food stimuli also tended to elicit avoidance behavior (withdrawal and avoidance turns) at concentration thresholds that were relatively low and fixed. When the feeding threshold for active feeding (proboscis extension with biting) was exceeded, ongoing avoidance and locomotion were interrupted and suppressed. Noxious chemostimuli usually stimulated avoidance, but, in animals with lower feeding thresholds for food stimuli, they often elicited feeding behavior. Thus, sensory pathways mediating appetitive and noxious stimuli may have dual access to neural networks of feeding and avoidance behavior, but their final effects are regulated by satiation state. These observations suggest that a simple cost-benefit computation regulates behavioral switching in the animal's foraging behavior, where food stimuli above or below the incentive level for feeding tend to induce feeding or avoidance, respectively. This decision mechanism can weigh the animal's need for nutrients against the potential risk from other predators and the cost of relative energy outlay in an attack on prey. Stimulation of orienting and attack by low-level noxious stimuli in the hungriest animals may reflect risk-taking that can enhance prey capture success. A simple, hedonically structured neural network model captures this computation.

  10. Secondary Ion Mass Spectrometry Imaging of Molecular Distributions in Cultured Neurons and their Processes: Comparative Analysis of Sample Preparation

    PubMed Central

    Tucker, Kevin R.; Li, Zhen; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2012-01-01

    Neurons often exhibit a complex chemical distribution and topography; therefore, sample preparation protocols that preserve structures ranging from relatively large cell somata to small neurites and growth cones are an important factor in secondary ion mass spectrometry (SIMS) imaging studies. Here, SIMS was used to investigate the subcellular localization of lipids and lipophilic species in neurons from Aplysia californica. Using individual neurons cultured on silicon wafers, we compared and optimized several SIMS sampling approaches. After an initial step to remove the high salt culturing media, formaldehyde, paraformaldehyde and glycerol, and various combinations thereof, were tested for their ability to achieve cell stabilization during and after the removal of extracellular media. These treatments improved the preservation of cellular morphology as visualized with SIMS imaging. For analytes >250 Da, coating the cell surface with a 3.2 nm-thick gold layer increased the ion intensity; multiple analytes previously not observed or observed at low abundance were detected, including intact cholesterol and vitamin E molecular ions. However, once a sample was coated, many of the lower molecular mass (<200 Da) analyte signals were suppressed. The optimum approach depended on the analyte being studied, and these approaches included rinsing with water and cell stabilization with glycerol and 4% paraformaldehyde. The sample preparation methods described here enhance SIMS imaging of processes of individual cultured neurons over a broad mass range with enhanced image contrast. PMID:22930440

  11. A feather precipitation hydrogen isoscape for New Zealand

    NASA Astrophysics Data System (ADS)

    Rogers, K. M.; Wassenaar, L. I.; Soto, D. X.; Bartle, J. A.

    2012-04-01

    Forensic isotopic assays of feathers from historical Maori cloaks are a potential tool to link historical artefacts back to their native locales (Iwi) in New Zealand. In order to test this approach, we sampled feathers from extant museum archived birds of known origin for their feather hydrogen isotopes (δyHf) to assign their regional origin and location over time. We obtained feathers from two non-migratory bird species widely distributed around New Zealand, tui (Prosthemadera novaeseelandiae) and quail (Callipepla californica). Feathers were sampled from archived birds collected between 1880-2002 held in 3 New Zealand museum collections. We determined regression coefficients of δ2H on location, latitude, δ2Hprecipitation, and age. The data showed that ground dwelling quail had higher regression coefficients with respect to latitude (r2=0.46) than the nectar feeding tui (r2=0.39). On the whole, both resident birds showed promise as regional geographical indicators of their habitat (r2=0.58). Year of collection had no meaningful effect on isotopic composition. We conclude that isotopic assays may therefore be used to aid in regional assignments relevant to the interpretation of historical artefacts.

  12. Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

    PubMed Central

    Akhmedov, Komol; Kadakkuzha, Beena M.; Puthanveettil, Sathyanarayanan V.

    2014-01-01

    A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia. PMID:24457225

  13. Electron microscopy of complexes of isolated acetylcholine receptor, biotinyl-toxin, and avidin

    SciTech Connect

    Holtzman, E.; Wise, D.; Wall, J.; Karlin, A.

    1982-01-01

    The principal curarimimetic toxin of Naja naja siamensis derivatized with biothinyl groups binds specifically both to acetylcholine receptor, isolated from Torpedo californica electric tissue, and to avidin. Isolated complexes of receptor monomer or dimer, biotinyl-toxin, and avidin were negatively stained and examined in the scanning transmission electron microscope. We measured the angle made by the radius to each avidin bound at the periphery of a monomeric unit in dimer to the axis connecting the centers of the monomers, starting at the crosslink between the monomers. We infer from the distribution of these angles that one toxin binding site is located in the range of 45/sup 0/ to 85/sup 0/ and another at about 100/sup 0/ further from the crosslink between the monomers. Because it is known that there are two toxin binding sites per monomer, associated with the two ..cap alpha.. chains, the bound avidins presumably point to portions of the ..cap alpha.. chains, indicating their positions relative to that portion of the delta chain located at the crosslink between monomers in dimer.

  14. Selective extracellular stimulation of individual neurons in ganglia

    NASA Astrophysics Data System (ADS)

    Lu, Hui; Chestek, Cynthia A.; Shaw, Kendrick M.; Chiel, Hillel J.

    2008-09-01

    Selective control of individual neurons could clarify neural functions and aid disease treatments. To target specific neurons, it may be useful to focus on ganglionic neuron clusters, which are found in the peripheral nervous system in vertebrates. Because neuron cell bodies are found primarily near the surface of invertebrate ganglia, and often found near the surface of vertebrate ganglia, we developed a technique for controlling individual neurons extracellularly using the buccal ganglia of the marine mollusc Aplysia californica as a model system. We experimentally demonstrated that anodic currents can selectively activate an individual neuron and cathodic currents can selectively inhibit an individual neuron using this technique. To define spatial specificity, we studied the minimum currents required for stimulation, and to define temporal specificity, we controlled firing frequencies up to 45 Hz. To understand the mechanisms of spatial and temporal specificity, we created models using the NEURON software package. To broadly predict the spatial specificity of arbitrary neurons in any ganglion sharing similar geometry, we created a steady-state analytical model. A NEURON model based on cat spinal motor neurons showed responses to extracellular stimulation qualitatively similar to those of the Aplysia NEURON model, suggesting that this technique could be widely applicable to vertebrate and human peripheral ganglia having similar geometry.

  15. From the Cover: Specific chemical and structural damage to proteins produced by synchrotron radiation

    NASA Astrophysics Data System (ADS)

    Weik, Martin; Ravelli, Raimond B. G.; Kryger, Gitay; McSweeney, Sean; Raves, Maria L.; Harel, Michal; Gros, Piet; Silman, Israel; Kroon, Jan; Sussman, Joel L.

    2000-01-01

    Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

  16. Habitat Evaluation Procedures (HEP) Report; Forrest Conservation Area, Technical Report 2003-2004.

    SciTech Connect

    Smith, Brent

    2005-01-01

    The Habitat Evaluation Procedure (HEP) study was performed to determine baseline habitat units on the 4,232-acre Forrest Conservation Area managed by the Confederated Tribes of Warm Springs Reservation of Oregon (Tribe) in Grant County, Oregon. The habitat evaluation is required as part of the Memorandum of Agreement between the Confederated Tribes of the Warm Springs and Bonneville Power Administration. Representatives from the Washington Department of Fish and Wildlife and the Tribes conducted the field surveys for the HEP. The survey collected data for habitat variables contained in habitat suitability index (HIS) models for wildlife species; the key species were black-capped chickadee (Poecile atricapilla), mallard (Anas platyrhynchos), mink (Mustela vison), western meadowlark (Sturnella neglecta), mule deer (Odocoileus hemionus), California Quail (Callipepla californica), and yellow warbler (Dendroica petechia). Cover types surveyed were grassland, meadow grassland, conifer forest, riparian tree shrub, shrub steppe, juniper forest, and juniper steppe. Other cover types mapped, but not used in the models were open water, roads, gravel pits, corrals, and residential.

  17. Novel Tacrine-Benzofuran Hybrids as Potent Multitarget-Directed Ligands for the Treatment of Alzheimer's Disease: Design, Synthesis, Biological Evaluation, and X-ray Crystallography.

    PubMed

    Zha, Xiaoming; Lamba, Doriano; Zhang, Lili; Lou, Yinghan; Xu, Changxu; Kang, Di; Chen, Li; Xu, Yungen; Zhang, Luyong; De Simone, Angela; Samez, Sarah; Pesaresi, Alessandro; Stojan, Jure; Lopez, Manuela G; Egea, Javier; Andrisano, Vincenza; Bartolini, Manuela

    2016-01-14

    Twenty-six new tacrine-benzofuran hybrids were designed, synthesized, and evaluated in vitro on key molecular targets for Alzheimer's disease. Most hybrids exhibited good inhibitory activities on cholinesterases and β-amyloid self-aggregation. Selected compounds displayed significant inhibition of human β-secretase-1 (hBACE-1). Among the 26 hybrids, 2e showed the most interesting profile as a subnanomolar selective inhibitor of human acetylcholinesterase (hAChE) (IC50 = 0.86 nM) and a good inhibitor of both β-amyloid aggregation (hAChE- and self-induced, 61.3% and 58.4%, respectively) and hBACE-1 activity (IC50 = 1.35 μM). Kinetic studies showed that 2e acted as a slow, tight-binding, mixed-type inhibitor, while X-ray crystallographic studies highlighted the ability of 2e to induce large-scale structural changes in the active-site gorge of Torpedo californica AChE (TcAChE), with significant implications for structure-based drug design. In vivo studies confirmed that 2e significantly ameliorates performances of scopolamine-treated ICR mice. Finally, 2e administration did not exhibit significant hepatotoxicity. PMID:26632651

  18. Mortality and community changes drive sudden oak death impacts on litterfall and soil nitrogen cycling.

    PubMed

    Cobb, Richard C; Eviner, Valerie T; Rizzo, David M

    2013-10-01

    Few studies have quantified pathogen impacts to ecosystem processes, despite the fact that pathogens cause or contribute to regional-scale tree mortality. We measured litterfall mass, litterfall chemistry, and soil nitrogen (N) cycling associated with multiple hosts along a gradient of mortality caused by Phytophthora ramorum, the cause of sudden oak death. In redwood forests, the epidemiological and ecological characteristics of the major overstory species determine disease patterns and the magnitude and nature of ecosystem change. Bay laurel (Umbellularia californica) has high litterfall N (0.992%), greater soil extractable NO3 -N, and transmits infection without suffering mortality. Tanoak (Notholithocarpus densiflorus) has moderate litterfall N (0.723%) and transmits infection while suffering extensive mortality that leads to higher extractable soil NO3 -N. Redwood (Sequoia sempervirens) has relatively low litterfall N (0.519%), does not suffer mortality or transmit the pathogen, but dominates forest biomass. The strongest impact of pathogen-caused mortality was the potential shift in species composition, which will alter litterfall chemistry, patterns and dynamics of litterfall mass, and increase soil NO3 -N availability. Patterns of P. ramorum spread and consequent mortality are closely associated with bay laurel abundances, suggesting this species will drive both disease emergence and subsequent ecosystem function. PMID:23790136

  19. Fish Synucleins: An Update

    PubMed Central

    Toni, Mattia; Cioni, Carla

    2015-01-01

    Synucleins (syns) are a family of proteins involved in several human neurodegenerative diseases and tumors. Since the first syn discovery in the brain of the electric ray Torpedo californica, members of the same family have been identified in all vertebrates and comparative studies have indicated that syn proteins are evolutionary conserved. No counterparts of syns were found in invertebrates suggesting that they are vertebrate-specific proteins. Molecular studies showed that the number of syn members varies among vertebrates. Three genes encode for α-, β- and γ-syn in mammals and birds. However, a variable number of syn genes and encoded proteins is expressed or predicted in fish depending on the species. Among biologically verified sequences, four syn genes were identified in fugu, encoding for α, β and two γ (γ1 and γ2) isoforms, whereas only three genes are expressed in zebrafish, which lacks α-syn gene. The list of “non verified” sequences is much longer and is often found in sequence databases. In this review we provide an overview of published papers and known syn sequences in agnathans and fish that are likely to impact future studies in this field. Indeed, fish models may play a key role in elucidating some of the molecular mechanisms involved in physiological and pathological functions of syn proteins. PMID:26528989

  20. Functional expression of soluble forms of human CD38 in Escherichia coli and Pichia pastoris.

    PubMed

    Fryxell, K B; O'Donoghue, K; Graeff, R M; Lee, H C; Branton, W D

    1995-06-01

    Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes calcium from intracellular stores in many cells. The synthesis of cADPR from NAD+ and its subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclase and a cADPR hydrolase, respectively. The ADP-ribosyl cyclase cloned from the ovotestis of the marine invertebrate Aplysia californica has amino acid sequence homology to the human lymphocyte surface antigen CD38. CD38 has been shown to catalyze both the formation and the hydrolysis of cADPR. In this study, we produced soluble, enzymatically active CD38 using recombinant expression techniques in bacteria and yeast. We engineered a gene coding for a soluble form of CD38 by excision of the region of the gene coding for the N-terminal amino acids representing the putative membrane spanning sequence and short putative intracellular sequence. For expression in bacteria (Escherichia coli), this construct was cloned into the pFlag-1 plasmid which allows induced, periplasmic expression and relatively simple purification of the soluble CD38. For expression in yeast (Pichia pastoris) the CD38 sequence was further modified to eliminate four putative N-linked glycosylation sites and the resulting construct was expressed as a secreted protein. Both systems produce soluble enzymes of approximately 30 kDa and both recombinant enzymes display similar cyclase and hydrolase activities. PMID:7663169

  1. "Self" and "non-self" in the control of phytoalexin biosynthesis: plant phospholipases A2 with alkaloid-specific molecular fingerprints.

    PubMed

    Heinze, Michael; Brandt, Wolfgang; Marillonnet, Sylvestre; Roos, Werner

    2015-02-01

    The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the "self" plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate "self-made" from "foreign" alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins. PMID:25670767

  2. GIS-based niche modeling for mapping species' habitats

    USGS Publications Warehouse

    Rotenberry, J.T.; Preston, K.L.; Knick, S.

    2006-01-01

    Ecological a??niche modelinga?? using presence-only locality data and large-scale environmental variables provides a powerful tool for identifying and mapping suitable habitat for species over large spatial extents. We describe a niche modeling approach that identifies a minimum (rather than an optimum) set of basic habitat requirements for a species, based on the assumption that constant environmental relationships in a species' distribution (i.e., variables that maintain a consistent value where the species occurs) are most likely to be associated with limiting factors. Environmental variables that take on a wide range of values where a species occurs are less informative because they do not limit a species' distribution, at least over the range of variation sampled. This approach is operationalized by partitioning Mahalanobis D2 (standardized difference between values of a set of environmental variables for any point and mean values for those same variables calculated from all points at which a species was detected) into independent components. The smallest of these components represents the linear combination of variables with minimum variance; increasingly larger components represent larger variances and are increasingly less limiting. We illustrate this approach using the California Gnatcatcher (Polioptila californica Brewster) and provide SAS code to implement it.

  3. Ultrastructure of secretion in the atrial gland of a mollusc (Aplysia).

    PubMed

    Beard, M; Millecchia, L; Masuoka, C; Arch, S

    1982-01-01

    Extracts of the atrial gland of the sea hare Aplysia californica (Mollusca) induce egg laying when injected into mature individuals. Since egg laying is controlled endogenously by a peptide secreted by neuroendocrine cells in the central nervous system, the relationship between the atrial gland and these central neurons has become an issue of interest. With the particular objective of examining secretory structures we undertook an ultrastructural study of the atrial gland and adjacent tissues. This study revealed that the atrial gland epithelium in composed of two major cell types: 'goblet-like' exocrine cells containing large electron-dense granules, and ciliated 'capping cells'. A non-secretory, and possibly post-secretory, cell containing electron-lucent granules was noted. A region of the large hermaphroditic duct contiguous ot the atrial gland, known as the red hemiduct, also displayed capping cells and secretory cells with large granules. The content of these granules is organized into crista-like condensations. The cell also contains iron-rich pigment inclusions. PMID:7112536

  4. Simulated weightlessness in fish and neurophysiological studies on memory storage

    NASA Technical Reports Server (NTRS)

    Vonbaumgarten, R. J.

    1973-01-01

    Simulated weightlessness was used to study the different types of gravity responses in blind fish. It was found that a shift in the direction of low magnitude acceleration in weightlessness causes a rapid 180 deg turn in the blind fish, while a shift in the direction of the applied acceleration in the earth's gravitational field is not significant because of a higher acceleration magnitude threshold than during the zero g condition. This increased responsiveness seems to be explained by a combination of directional sensitivity with a Weber-Fechner relationship of increased receptor sensitivity at diminished levels of background stimulation. Neurophysical studies of the statocyst nerve of the gastropod Mollusc Pleurobranchaea Californica were undertaken in order to understand how complex otolith systems operate. Information storage was investigated on relatively simple neuronal networks in the mollusc Aplysia. Intracellular electrical stimulation of isolated neurons show that a manipulation of autoditonous rhymicity is possible. It was also found that glycolysis and oxidative phosphorylation are involved in inherent rhymicity of Aplysis neurons.

  5. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed

    McCrea, P D; Popot, J L; Engelman, D M

    1987-12-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  6. Widespread genome duplications throughout the history of flowering plants

    PubMed Central

    Cui, Liying; Wall, P. Kerr; Leebens-Mack, James H.; Lindsay, Bruce G.; Soltis, Douglas E.; Doyle, Jeff J.; Soltis, Pamela S.; Carlson, John E.; Arumuganathan, Kathiravetpilla; Barakat, Abdelali; Albert, Victor A.; Ma, Hong; dePamphilis, Claude W.

    2006-01-01

    Genomic comparisons provide evidence for ancient genome-wide duplications in a diverse array of animals and plants. We developed a birth–death model to identify evidence for genome duplication in EST data, and applied a mixture model to estimate the age distribution of paralogous pairs identified in EST sets for species representing the basal-most extant flowering plant lineages. We found evidence for episodes of ancient genome-wide duplications in the basal angiosperm lineages including Nuphar advena (yellow water lily: Nymphaeaceae) and the magnoliids Persea americana (avocado: Lauraceae), Liriodendron tulipifera (tulip poplar: Magnoliaceae), and Saruma henryi (Aristolochiaceae). In addition, we detected independent genome duplications in the basal eudicot Eschscholzia californica (California poppy: Papaveraceae) and the basal monocot Acorus americanus (Acoraceae), both of which were distinct from duplications documented for ancestral grass (Poaceae) and core eudicot lineages. Among gymnosperms, we found equivocal evidence for ancient polyploidy in Welwitschia mirabilis (Gnetales) and no evidence for polyploidy in pine, although gymnosperms generally have much larger genomes than the angiosperms investigated. Cross-species sequence divergence estimates suggest that synonymous substitution rates in the basal angiosperms are less than half those previously reported for core eudicots and members of Poaceae. These lower substitution rates permit inference of older duplication events. We hypothesize that evidence of an ancient duplication observed in the Nuphar data may represent a genome duplication in the common ancestor of all or most extant angiosperms, except Amborella. PMID:16702410

  7. Identification and characterization of mutations in housefly (Musca domestica) acetylcholinesterase involved in insecticide resistance.

    PubMed

    Walsh, S B; Dolden, T A; Moores, G D; Kristensen, M; Lewis, T; Devonshire, A L; Williamson, M S

    2001-10-01

    Acetylcholinesterase (AChE) insensitive to organophosphate and carbamate insecticides has been identified as a major resistance mechanism in numerous arthropod species. However, the associated genetic changes have been reported in the AChE genes from only three insect species; their role in conferring insecticide insensitivity has been confirmed, using functional expression, only for those in Drosophila melanogaster. The housefly, Musca domestica, was one of the first insects shown to have this mechanism; here we report the occurrence of five mutations (Val-180-->Leu, Gly-262-->Ala, Gly-262-->Val, Phe-327-->Tyr and Gly-365-->Ala) in the AChE gene of this species that, either singly or in combination, confer different spectra of insecticide resistance. The baculovirus expression of wild-type and mutated housefly AChE proteins has confirmed that the mutations each confer relatively modest levels of insecticide insensitivity except the novel Gly-262-->Val mutation, which results in much stronger resistance (up to 100-fold) to certain compounds. In all cases the effects of mutation combinations are additive. The mutations introduce amino acid substitutions that are larger than the corresponding wild-type residues and are located within the active site of the enzyme, close to the catalytic triad. The likely influence of these substitutions on the accessibility of the different types of inhibitor and the orientation of key catalytic residues are discussed in the light of the three-dimensional structures of the AChE protein from Torpedo californica and D. melanogaster. PMID:11563981

  8. p63 gene structure in the phylum mollusca.

    PubMed

    Baričević, Ana; Štifanić, Mauro; Hamer, Bojan; Batel, Renato

    2015-08-01

    Roles of p53 family ancestor (p63) in the organisms' response to stressful environmental conditions (mainly pollution) have been studied among molluscs, especially in the genus Mytilus, within the last 15 years. Nevertheless, information about gene structure of this regulatory gene in molluscs is scarce. Here we report the first complete genomic structure of the p53 family orthologue in the mollusc Mediterranean mussel Mytilus galloprovincialis and confirm its similarity to vertebrate p63 gene. Our searches within the available molluscan genomes (Aplysia californica, Lottia gigantea, Crassostrea gigas and Biomphalaria glabrata), found only one p53 family member present in a single copy per haploid genome. Comparative analysis of those orthologues, additionally confirmed the conserved p63 gene structure. Conserved p63 gene structure can be a helpful tool to complement or/and revise gene annotations of any future p63 genomic sequence records in molluscs, but also in other animal phyla. Knowledge of the correct gene structure will enable better prediction of possible protein isoforms and their functions. Our analyses also pointed out possible mis-annotations of the p63 gene in sequenced molluscan genomes and stressed the value of manual inspection (based on alignments of cDNA and protein onto the genome sequence) for a reliable and complete gene annotation. PMID:25936268

  9. Specific Stimulated Uptake of Acetylcholine by Torpedo Electric Organ Synaptic Vesicles

    NASA Astrophysics Data System (ADS)

    Parsons, Stanley M.; Koenigsberger, Robert

    1980-10-01

    The specificity of acetylcholine uptake by synaptic vesicles isolated from the electric organ of Torpedo californica was studied. In the absence of cofactors, [3H]acetylcholine was taken up identically to [14C]choline in the same solution (passive uptake), and the equilibrium concentration achieved inside the vesicles was equal to the concentration outside. In the presence of MgATP, [3H]acetylcholine and [14C]choline in the same solution were taken up identically, except only about half as much of each was taken up (suppressed uptake). [3H]Acetylcholine uptake was stimulated by MgATP and HCO3 about 4-fold relative to suppressed uptake, for a net concentrative uptake of about 2:1 (stimulated uptake). Uptake of [14C]choline in the same solution remained at the suppressed level. [3H]Acetylcholine taken up under stimulated conditions migrated with vesicles containing [14C]mannitol on analytical glycerol density gradients during centrifugation. Vesicles were treated with nine protein modification reagents under mild conditions. Two reagents had no effect on, dithiothreitol potentiated, and six reagents strongly inhibited subsequent stimulated uptake of [3H]acetylcholine. The results indicate that uptake of acetylcholine is conditionally specific for the transported substrate, is carried out by the synaptic vesicles rather than a contaminant of the preparation, and requires a functional protein system containing a critical sulfhydryl group.

  10. Opioid peptides in the nervous system of Aplysia: a combined biochemical, immunocytochemical, and electrophysiological study.

    PubMed

    Carpenter, D O; Kemenes, G; Elekes, K; Leung, M; Stefano, G; Rózsa, K S; Salánki, J

    1995-04-01

    1. We have used biochemical, immunocytochemical, and electrophysiological techniques to evaluate the role of opioid peptides in the central nervous system of the marine mollusc, Aplysia californica. 2. Binding studies using 3H-D-Ala2, met-enkephalinamide (3H-DAMA) showed a single class of high-affinity binding sites with a Kd of 1.3 nM and a binding density of 45 pmol/g. 3. HPLC extracts of ganglia revealed multiple peaks with immunoreactivity for either leu (LEU-IR)- or met-enkephalin (MET-IR), but the amounts were not uniformly distributed in all ganglia. 4. LEU-IR and MET-IR neurons were demonstrated immunocytochemically in all ganglia, but MET-IR neurons were more frequent and were concentrated in pedal and pleural ganglia. While absorption control studies abolished MET-IR, LEU-IR was only partially abolished in the neuropil. 5. In electrophysiological studies, both depolarizing and hyperpolarizing responses were found to D-Ala2-leu-enkephalin (DALEU) and D-Ala2-met enkephalin (DAMET) on some and different neurons. 6. HPLC fractions from regions with retention times corresponding to authentic leu- or met-enkephalin showed physiologic responses similar to those of DALEU and DAMET, respectively. 7. These studies suggest that a variety of endogeneous opioid peptides play physiologically important roles in the nervous system of Aplysia, including but not necessarily limited to leu- and met-enkephalin. PMID:8590454

  11. Perspectives for the structure-based design of acetylcholinesterase reactivators.

    PubMed

    Ochoa, Rodrigo; Rodriguez, Carlos A; Zuluaga, Andres F

    2016-07-01

    Rational design of active molecules through structure-based methods has been gaining adepts during the last decades due to the wider availability of protein structures, most of them conjugated with relevant ligands. Acetylcholinesterase (AChE) is a molecular target with a considerable amount of data related to its sequence and 3-dimensional structure. In addition, there are structural insights about the mechanism of action of the natural substrate and drugs used in Alzheimer's disease, organophosphorus compounds, among others. We looked for AChE structural data useful for in silico design of potential interacting molecules. In particular, we focused on information regarding the design of ligands aimed to reactivate AChE catalytic activity. The structures of 178 AChE were annotated and categorized on different subsets according to the nature of the ligand, source organisms and experimental details. We compared sequence homology among the active site from Torpedo californica, Mus musculus and Homo sapiens with the latter two species having the closest relationship (88.9% identity). In addition, the mechanism of organophosphorus binding and the design of effective reactivators are reviewed. A curated data collection obtained with information from several sources was included for researchers working on the field. Finally, a molecular dynamics simulation with human AChE indicated that the catalytic pocket volume stabilizes around 600 Å(3), providing additional clues for drug design. PMID:27450771

  12. Comparing Wild American Grapes with Vitis vinifera: A Metabolomics Study of Grape Composition.

    PubMed

    Narduzzi, Luca; Stanstrup, Jan; Mattivi, Fulvio

    2015-08-01

    We analyzed via untargeted UHPLC-ESI-Q-TOF-MS the metabolome of the berry tissues (skin, pulp, seeds) of some American Vitis species (Vitis cinerea, Vitis californica, Vitis arizonica), together with four interspecific hybrids, and seven Vitis vinifera cultivars, aiming to find differences in the metabolomes of the American Vitis sp. versus Vitis vinifera. Apart from the known differences, that is, more complex content of anthocyanins and stilbenoids in the American grapes, we observed higher procyanidin accumulation (tens to hundreds of times) in the vinifera skin and seeds in comparison to American berries, and we confirmed this result via phloroglucinolysis. In the American grapes considered, we did not detect the accumulation of pleasing aroma precursors (terpenoids, glycosides), whereas they are common in vinifera grapes. We also found accumulation of hydrolyzable tannins and their precursors in the skin of the wild American grapes, which has never been reported earlier in any of the species under investigation. Such information is needed to improve the design of new breeding programs, lowering the risk of retaining undesirable characteristics in the chemical phenotype of the offspring. PMID:26158394

  13. Grapevine red blotch-associated virus is Present in Free-Living Vitis spp. Proximal to Cultivated Grapevines.

    PubMed

    Perry, Keith L; McLane, Heather; Hyder, Muhammad Z; Dangl, Gerald S; Thompson, Jeremy R; Fuchs, Marc F

    2016-06-01

    Red blotch is an emerging disease of grapevine associated with grapevine red blotch-associated virus (GRBaV). The virus spreads with infected planting stocks but no vector of epidemiological significance has been conclusively identified. A vineyard block of red-blotch-affected Vitis vinifera 'Cabernet franc' clone 214 was observed in California, with a clustering of infected, symptomatic vines focused along one edge of the field proximal to a riparian habitat with free-living Vitis spp. No genetic heterogeneity was observed in a 587-nucleotide region of the GRBaV genome in a population of 44 Cabernet franc clone 214 isolates. By contrast, genetic differences were observed in isolates from other cultivars and clones growing in adjacent blocks. GRBaV was confirmed infecting four free-living vines, two of which were shown to be V. californica × V. vinifera hybrids. The genomes of three free-living GRBaV vine isolates and seven from V. vinifera cultivars were compared; free-living vine isolates were shown to be more similar to each other and a 'Merlot' isolate than to the other cultivated vine isolates. The finding that GRBaV is present in free-living Vitis spp. indicates the virus can be spread by natural (nonhuman-mediated) means, and we hypothesize that in-field spread of GRBaV is occurring. PMID:26960112

  14. α-Bungarotoxin Binding to Acetylcholine Receptor Membranes Studied by Low Angle X-Ray Diffraction

    PubMed Central

    Young, Howard S.; Herbette, Leo G.; Skita, Victor

    2003-01-01

    The nicotinic acetylcholine receptor (nAChR) carries two binding sites for snake venom neurotoxins. α-Bungarotoxin from the Southeast Asian banded krait, Bungarus multicinctus, is a long neurotoxin which competitively blocks the nAChR at the acetylcholine binding sites in a relatively irreversible manner. Low angle x-ray diffraction was used to generate electron density profile structures at 14-Å resolution for Torpedo californica nAChR membranes in the absence and presence of α-bungarotoxin. Analysis of the lamellar diffraction data indicated a 452-Å lattice spacing between stacked nAChR membrane pairs. In the presence of α-bungarotoxin, the quality of the diffraction data and the lamellar lattice spacing were unchanged. In the plane of the membrane, the nAChRs packed together with a nearest neighbor distance of 80 Å, and this distance increased to 85 Å in the presence of toxin. Electron density profile structures were calculated in the absence and presence of α-bungarotoxin, revealing a location for the toxin binding sites. In native, fully-hydrated nAChR membranes, α-bungarotoxin binds to the nAChR outer vestibule and contacts the surface of the membrane bilayer. PMID:12885641

  15. Drug binding to the acetylcholine receptor: Nitroxide analogs of phencyclidine and a local anesthetic

    SciTech Connect

    Palma, A.L.

    1988-01-01

    The interaction of noncompetitive inhibitors (NCIs) with Torpedo californica native nicotinic acetylcholine receptor (nAChR) membranes was examined primarily by the technique of electron paramagnetic resonance (EPR) spectroscopy. The goal of this work being to define some of the physical characteristics for the site(s) of association between an NCI and the nAChR membrane. A nitroxide labeled analog of a quaternary amine local anesthetic, 2-(N,N-dimethyl-N-4-(2,2,6,6-tetramethylpiperidinoxyl)amino)-ethyl 4-hexyloxybenzoate iodide (C6SLMeI), displays a strongly immobilized EPR component when added to nAChR membranes in the presence of carbamylcholine (carb). To further this work, a nitroxide labeled analog of phencyclidine (PCP), a potent NCI, was synthesized. 4-phenyl-4-(1-piperidinyl)-2,2,6,6-tetramethylpiperidinoxyl (PPT) exhibited one-third the potency of PCP in inhibiting nAChR mediated ion flux, and from competition binding studies with ({sup 3}H)PCP displayed a K{sub D} of 0.21 {mu}M towards a carb desensitized nAChR and a K{sub 0.5} of 18 {mu}M for a resting {alpha}-bungarotoxin treated nAChR.

  16. A neuron-in-capillary platform for facile collection and mass spectrometric characterization of a secreted neuropeptide

    NASA Astrophysics Data System (ADS)

    Lee, Chang Young; Fan, Yi; Rubakhin, Stanislav S.; Yoon, Sook; Sweedler, Jonathan V.

    2016-06-01

    The integration of microfluidic devices—which efficiently handle small liquid volumes—with separations/mass spectrometry (MS) is an effective approach for profiling the neurochemistry occurring in selected neurons. Interfacing the microfluidic cell culture to the mass spectrometer is challenging because of geometric and scaling issues. Here we demonstrate the hyphenation of a neuron-in-capillary platform to a solid phase extraction device and off-line MS. A primary neuronal culture of Aplysia californica neurons was established directly inside a cylindrical polyimide capillary. The approach also uses a particle-embedded monolith to condition neuropeptide releasates collected from several Aplysia neurons cultured in the capillary, with the subsequent characterization of released peptides via MS. This system presents a number of advances compared to more traditional microfluidic devices fabricated with polydimethylsiloxane. These include low cost, easy access to cell culture, rigidity, ease of transport, and minimal fluid handling. The cylindrical geometry of the platform allows convenient interface with a wide range of analytical tools that utilize capillary columns.

  17. Analysis of endogenous nucleotides by single cell capillary electrophoresis-mass spectrometry

    PubMed Central

    Liu, Jing-Xin; Aerts, Jordan T.; Rubakhin, Stanislav S.; Zhang, Xin-Xiang; Sweedler, Jonathan V.

    2015-01-01

    Analytical technologies that enable investigations at the single cell level facilitate a range of studies; here a lab-fabricated capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) platform was used to analyze anionic metabolites from individual Aplysia californica neurons. The system employs a customized coaxial sheath-flow nanospray interface connected to a separation capillary, with the sheath liquid and separation buffer optimized to ensure a stable spray. The method provided good repeatability of separation and reliable detection sensitivity for 16 mono-, di- and triphosphate nucleosides. For a range of anionic analytes, including cyclic adenosine monophosphate (cAMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), the detection limits were in the low nanomolar range (<22 nM). A large Aplysia R2 neuron was used to demonstrate the ability of CE-ESI-MS to quantitatively characterize anionic metabolites within individual cells, with 15 nucleotides and derivatives detected. Following the method validation process, we probed smaller, 60-μm diameter Aplysia sensory neurons where sample stacking was used as a simple on-line analyte preconcentration approach. The calculated energy balance ([ATP] + 0.5 × [ADP])/([AMP] + [ADP] + [ATP]) of these cells was comparable with the value obtained from bulk samples. PMID:25212237

  18. Secondary ion mass spectrometry imaging of molecular distributions in cultured neurons and their processes: comparative analysis of sample preparation.

    PubMed

    Tucker, Kevin R; Li, Zhen; Rubakhin, Stanislav S; Sweedler, Jonathan V

    2012-11-01

    Neurons often exhibit a complex chemical distribution and topography; therefore, sample preparation protocols that preserve structures ranging from relatively large cell somata to small neurites and growth cones are important factors in secondary ion mass spectrometry (SIMS) imaging studies. Here, SIMS was used to investigate the subcellular localization of lipids and lipophilic species in neurons from Aplysia californica. Using individual neurons cultured on silicon wafers, we compared and optimized several SIMS sampling approaches. After an initial step to remove the high salt culturing media, formaldehyde, paraformaldehyde, and glycerol, and various combinations thereof, were tested for their ability to achieve cell stabilization during and after the removal of extracellular media. These treatments improved the preservation of cellular morphology as visualized with SIMS imaging. For analytes >250 Da, coating the cell surface with a 3.2 nm-thick gold layer increased the ion intensity; multiple analytes previously not observed or observed at low abundance were detected, including intact cholesterol and vitamin E molecular ions. However, once a sample was coated, many of the lower molecular mass (<200 Da) analyte signals were suppressed. The optimum approach depended on the analyte being studied; the approaches evaluated included rinsing with water and cell stabilization with glycerol and 4 % paraformaldehyde. The sample preparation methods described here enhance SIMS imaging of processes of individual cultured neurons over a broad mass range with enhanced image contrast. PMID:22930440

  19. Differences in dissolved cadmium and zinc uptake among stream insects: Mechanistic explanations

    USGS Publications Warehouse

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

    2005-01-01

    This study examined the extent to which dissolved Cd and Zn uptake rates vary in several aquatic insect taxa commonly used as indicators of ecological health. We further attempted to explain the mechanisms underlying observed differences. By comparing dissolved Cd and Zn uptake rates in several aquatic insect species, we demonstrated that species vary widely in these processes. Dissolved uptake rates were not related to gross morphological features such as body size or gill size-features that influence water permeability and therefore have ionoregulatory importance. However, finer morphological features, specifically, the relative numbers of ionoregulatory cells (chloride cells), appeared to be related to dissolved metal uptake rates. This observation was supported by Michaelis-Menten type kinetics experiments, which showed that dissolved Cd uptake rates were driven by the numbers of Cd transporters and not by the affinities of those transporters to Cd. Calcium concentrations in exposure media similarly affected Cd and Zn uptake rates in the caddisfly Hydropsyche californica. Dissolved Cd and Zn uptake rates strongly co-varied among species, suggesting that these metals are transported by similar mechanisms.

  20. Parasites reduce food web robustness because they are sensitive to secondary extinction as illustrated by an invasive estuarine snail.

    PubMed

    Lafferty, Kevin D; Kuris, Armand M

    2009-06-27

    A robust food web is one in which few secondary extinctions occur after removing species. We investigated how parasites affected the robustness of the Carpinteria Salt Marsh food web by conducting random species removals and a hypothetical, but plausible, species invasion. Parasites were much more likely than free-living species to suffer secondary extinctions following the removal of a free-living species from the food web. For this reason, the food web was less robust with the inclusion of parasites. Removal of the horn snail, Cerithidea californica, resulted in a disproportionate number of secondary parasite extinctions. The exotic Japanese mud snail, Batillaria attramentaria, is the ecological analogue of the native California horn snail and can completely replace it following invasion. Owing to the similarities between the two snail species, the invasion had no effect on predator-prey interactions. However, because the native snail is host for 17 host-specific parasites, and the invader is host to only one, comparison of a food web that includes parasites showed significant effects of invasion on the native community. The hypothetical invasion also significantly reduced the connectance of the web because the loss of 17 native trematode species eliminated many links. PMID:19451117

  1. Determining nest predators of the Least Bell's Vireo through point counts, tracking stations, and video photography

    USGS Publications Warehouse

    Peterson, B.L.; Kus, B.E.; Deutschman, D.H.

    2004-01-01

    We compared three methods to determine nest predators of the Least Bell's Vireo (Vireo bellii pusillus) in San Diego County, California, during spring and summer 2000. Point counts and tracking stations were used to identify potential predators and video photography to document actual nest predators. Parental behavior at depredated nests was compared to that at successful nests to determine whether activity (frequency of trips to and from the nest) and singing vs. non-singing on the nest affected nest predation. Yellow-breasted Chats (Icteria virens) were the most abundant potential avian predator, followed by Western Scrub-Jays (Aphelocoma californica). Coyotes (Canis latrans) were abundant, with smaller mammalian predators occurring in low abundance. Cameras documented a 48% predation rate with scrub-jays as the major nest predators (67%), but Virginia opossums (Didelphis virginiana, 17%), gopher snakes (Pituophis melanoleucus, 8%) and Argentine ants (Linepithema humile, 8%) were also confirmed predators. Identification of potential predators from tracking stations and point counts demonstrated only moderate correspondence with actual nest predators. Parental behavior at the nest prior to depredation was not related to nest outcome.

  2. Loss of Genetic Diversity and Increased Subdivision in an Endemic Alpine Stonefly Threatened by Climate Change

    PubMed Central

    Jordan, Steve; Giersch, J. Joseph; Muhlfeld, Clint C.; Hotaling, Scott; Fanning, Liz; Tappenbeck, Tyler H.; Luikart, Gordon

    2016-01-01

    Much remains unknown about the genetic status and population connectivity of high-elevation and high-latitude freshwater invertebrates, which often persist near snow and ice masses that are disappearing due to climate change. Here we report on the conservation genetics of the meltwater stonefly Lednia tumana (Ricker) of Montana, USA, a cold-water obligate species. We sequenced 1530 bp of mtDNA from 116 L. tumana individuals representing “historic” (>10 yr old) and 2010 populations. The dominant haplotype was common in both time periods, while the second-most-common haplotype was found only in historic samples, having been lost in the interim. The 2010 populations also showed reduced gene and nucleotide diversity and increased genetic isolation. We found lower genetic diversity in L. tumana compared to two other North American stonefly species, Amphinemura linda (Ricker) and Pteronarcys californica Newport. Our results imply small effective sizes, increased fragmentation, limited gene flow, and loss of genetic variation among contemporary L. tumana populations, which can lead to reduced adaptive capacity and increased extinction risk. This study reinforces concerns that ongoing glacier loss threatens the persistence of L. tumana, and provides baseline data and analysis of how future environmental change could impact populations of similar organisms. PMID:27348125

  3. Molecular Dynamics of Mouse Acetylcholinesterase Complexed with Huperzine A

    SciTech Connect

    Tara, Sylvia; Helms, Volkhard H.; Straatsma, TP; Mccammon, J Andrew A.

    1999-03-16

    Two molecular dynamics simulations were performed for a modeled complex of mouse acetylcholinesterase liganded with huperzine A (HupA). Analysis of these simulations shows that HupA shifts in the active site toward Tyr 337 and Phe 338, and that several residues in the active site area reach out to make hydrogen bonds with the inhibitor. Rapid fluctuations of the gorge width are observed, ranging from widths that allow substrate access to the active site, to pinched structures that do not allow access of molecules as small as water. Additional openings or channels to the active site are found. One opening is formed in the side wall of the active site gorge by residues Val 73, Asp 74, Thr 83, Glu 84, and Asn 87. Another opening is formed at the base of the gorge by residues Trp 86, Val 132, Glu 202, Gly 448, and Ile 451. Both of these openings have been observed separately in the Torpedo californica form of the enzyme. These channels could allow transport of waters and ions to and from the bulk solution.

  4. Comparison of rumen microbial inhibition resulting from various essential oils isolated from relatively unpalatable plant species.

    PubMed

    Oh, H K; Jones, M B; Longhurst, W M

    1968-01-01

    Essential oils were isolated from eight plant species which were relatively unpalatable to sheep and deer. The inhibitory potency of these essential oils upon sheep and deer rumen microorganisms was compared, in terms of total gas and volatile fatty acid (VFA) production, by use of an anaerobic manometric technique. Inhibitory effects of oils from the eight plant species may be placed in four groups: (i) essential oils from vinegar weed (Trichostema lanceoletum) and California bay (Umbellularia californica) inhibited rumen microbial activity most; (ii) lesser inhibition was exhibited by rosemary (Rosmarinus officinalis) and California mugwort (Artemisia douglasiana) oils, followed by (iii) blue-gum eucalyptus (Eucalyptus globulus) and sagebrush (Artemisia tridentata) oils; and (iv) oils from Douglas fir (Psuedotsuga menziesii) and Jerusalem oak (chenopodium botrys) resulted in the least inhibition, when 0.3 ml of each oil was used. A highly significant correlation coefficient (r = 0.98(**)) between total gas and VFA production indicated the validity of either method to measure the activity of rumen microorganisms. Our results are discussed in relation to the hypothesis that the selectivity and voluntary consumption of ruminants are related to the characteristic odor and antibacterial action of essential oils isolated from relatively unpalatable plant species. PMID:5636470

  5. Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore

    SciTech Connect

    Talley, Todd T.; Harel, Michal; Hibbs, Ryan E.; Radi, Zoran; Tomizawa, Motohiro; Casida, John E.; Taylor, Palmer

    2008-07-28

    Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 {angstrom} in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids.

  6. California Rare Endemics and Climate Change

    NASA Astrophysics Data System (ADS)

    Espinoza, M.

    2010-12-01

    California is known for its wide variety of endemic flora, from its annuals such as the Eschscholzia californica (California poppy) to the perennials like the Arctostaphylos pallida (Alameda manzanita), which happens to be a rare species. Each species plays an important role in the biodiversity of California, yet there are species that are threatened, not only by human interaction and urbanization, but by climate change. Species that we seldom see are now on the verge of becoming eradicated; rare endemics similar to Arctostaphylos pallida are now facing a new challenge that may severely impair their survival. The climate has changed significantly over the twentieth century and it has affected the distribution of rare endemics in California, both geographically as well as within their climatic and edaphic niches. Lilaeopsis masonii is just one rare endemic, however it serves as a representative of the other 23 species that were studied. Using Maxent, a climate-modeling program, it was viable to construct two climate envelopes of the masonii species: the early century envelope (1930-1959) and the later century envelope (1990-2009). When these two climate envelopes were compared, it became clear that the later century climate envelope had contracted radically, reshaping the climate niche of all rare endemics in California due to an increase in temperature. It is possible to conclude that the future of rare endemics hangs in the balance, where one degree higher in temperature is enough to topple the scale.

  7. Secondary Ion Mass Spectrometry Imaging of Molecular Distributions in Cultured Neurons and Their Processes: Comparative Analysis of Sample Preparation

    NASA Astrophysics Data System (ADS)

    Tucker, Kevin R.; Li, Zhen; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2012-11-01

    Neurons often exhibit a complex chemical distribution and topography; therefore, sample preparation protocols that preserve structures ranging from relatively large cell somata to small neurites and growth cones are important factors in secondary ion mass spectrometry (SIMS) imaging studies. Here, SIMS was used to investigate the subcellular localization of lipids and lipophilic species in neurons from Aplysia californica. Using individual neurons cultured on silicon wafers, we compared and optimized several SIMS sampling approaches. After an initial step to remove the high salt culturing media, formaldehyde, paraformaldehyde, and glycerol, and various combinations thereof, were tested for their ability to achieve cell stabilization during and after the removal of extracellular media. These treatments improved the preservation of cellular morphology as visualized with SIMS imaging. For analytes >250 Da, coating the cell surface with a 3.2 nm-thick gold layer increased the ion intensity; multiple analytes previously not observed or observed at low abundance were detected, including intact cholesterol and vitamin E molecular ions. However, once a sample was coated, many of the lower molecular mass (<200 Da) analyte signals were suppressed. The optimum approach depended on the analyte being studied; the approaches evaluated included rinsing with water and cell stabilization with glycerol and 4 % paraformaldehyde. The sample preparation methods described here enhance SIMS imaging of processes of individual cultured neurons over a broad mass range with enhanced image contrast.

  8. Loss of Genetic Diversity and Increased Subdivision in an Endemic Alpine Stonefly Threatened by Climate Change.

    PubMed

    Jordan, Steve; Giersch, J Joseph; Muhlfeld, Clint C; Hotaling, Scott; Fanning, Liz; Tappenbeck, Tyler H; Luikart, Gordon

    2016-01-01

    Much remains unknown about the genetic status and population connectivity of high-elevation and high-latitude freshwater invertebrates, which often persist near snow and ice masses that are disappearing due to climate change. Here we report on the conservation genetics of the meltwater stonefly Lednia tumana (Ricker) of Montana, USA, a cold-water obligate species. We sequenced 1530 bp of mtDNA from 116 L. tumana individuals representing "historic" (>10 yr old) and 2010 populations. The dominant haplotype was common in both time periods, while the second-most-common haplotype was found only in historic samples, having been lost in the interim. The 2010 populations also showed reduced gene and nucleotide diversity and increased genetic isolation. We found lower genetic diversity in L. tumana compared to two other North American stonefly species, Amphinemura linda (Ricker) and Pteronarcys californica Newport. Our results imply small effective sizes, increased fragmentation, limited gene flow, and loss of genetic variation among contemporary L. tumana populations, which can lead to reduced adaptive capacity and increased extinction risk. This study reinforces concerns that ongoing glacier loss threatens the persistence of L. tumana, and provides baseline data and analysis of how future environmental change could impact populations of similar organisms. PMID:27348125

  9. Helminths of the ocelot from southern Texas.

    PubMed

    Pence, Danny B; Tewes, Michael E; Laack, Linda L

    2003-07-01

    In the USA, the ocelot (Leopardus pardalis) is a highly endangered felid found only in a few remaining vestiges of native thornshrub brushland in the Lower Rio Grande Valley (LRGV) of extreme southern Texas. From 1987-1998, carcasses of 15 adult ocelots that died of vehicular accidents or natural causes were examined for helminths. All cats had 1-8 (mean = 3) helminth species. All were infected with 1-101 (mean +/- SE = 32 +/- 7) Toxascaris leonina. Other helminths from these ocelots were Alaria marcianae, Brachylaima sp., Mesocestoides lineatus, Taenia rileyi, Oncicola canis, Dirofilaria immitis, Physaloptera rara, Ancylostoma tubaeformae, Cylicospirura chevreuxi, Vogeloides felis, and Metathelazia californica. Additionally, two cats had scarring of the aorta with lesions typical of those caused by Spriocerca lupi, although larval nematodes were not seen. A clinal variation in size of nearly three orders of magnitude was noted in the diplostomatid trematodes in the small intestine of one adult male ocelot. Despite the differences in size, all specimens appeared morphologically identical and were regarded as A. marcianae. Helminth prevalences and abundances, including those of potentially pathogenic species like D. immitis, were low. Although a single heartworm infection may have contributed to the death of one ocelot, helminth infections in general seemed to be of no great consequence to this endangered ocelot population. The helminth fauna of ocelots in the LRGV is reflective of that from wild felids in general; all have been reported previously from the bobcat (Lynx rufus) and mountain lion (Puma concolor) elsewhere in Texas. PMID:14567231

  10. Hemisynthesis of dihydroumbellulols from umbellulone: new cooling compounds.

    PubMed

    Starkenmann, Christian; Cayeux, Isabelle; Brauchli, Robert; Mayenzet, Fabienne

    2011-01-26

    Although menthol is a common ingredient used in food products, other molecules also evoke coolness through stimulation of the somatosensory system. To discover new molecules having cooling properties, we virtually screened the chemical structures of terpenes and sesquiterpenes to find structures that are similar to (-)-menthol. We realized that dihydroumbellulols could be good candidates. Although their occurrence was reported in Hyptis pectinata Poit, we were unable to obtain these molecules from the plant or to prove their natural occurrence. Therefore, we extracted (-)-(R)-umbellulone from Umbellularia californica Nutt. The (-)-(R)-umbellulone was reduced to prepare (1R,2R/S)-1-isopropyl-4-methylbicyclo[3.1.0]hex-3-en-2-ol, (1R,4R/S)-1-isopropyl-4-methylbicyclo[3.1.0]hexan-2-one, and (1R,2RS,4RS)-1-isopropyl-4-methylbicyclo[3.1.0]hexan-2-ols, named dihydroumbellulols. Sensory analysis suggested that (1R,2R,4S)-dihydroumbellulol has a pleasant, trigeminal cooling effect, about 2-3 times less cooling than (-)-menthol, with a weak odor slightly reminiscent of eucalyptol. In addition, a previously unreported compound was discovered, (-)-(1R)-1-isopropyl-4-methylenebicyclo[3.1.0]hexan-2-one. PMID:21190364

  11. Acetylcholinesterase: From 3D Structure to Function

    PubMed Central

    Dvir, Hay; Silman, Israel; Harel, Michal; Rosenberry, Terrone L.; Sussman, Joel L.

    2010-01-01

    By rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase terminates neurotransmission at cholinergic synapses. Acetylcholinesterase is a very fast enzyme, functioning at a rate approaching that of a diffusion-controlled reaction. The powerful toxicity of organophosphate poisons is attributed primarily to their potent inhibition of acetylcholinesterase. Acetylcholinesterase inhibitors are utilized in the treatment of various neurological disorders, and are the principal drugs approved thus far by the FDA for management of Alzheimer’s disease. Many organophosphates and carbamates serve as potent insecticides, by selectively inhibiting insect acetylcholinesterase. The determination of the crystal structure of Torpedo californica acetylcholinesterase permitted visualization, for the first time, at atomic resolution, of a binding pocket for acetylcholine. It also allowed identification of the active site of acetylcholinesterase, which, unexpectedly, is located at the bottom of a deep gorge lined largely by aromatic residues. The crystal structure of recombinant human acetylcholinesterase in its apo-state is similar in its overall features to that of the Torpedo enzyme; however, the unique crystal packing reveals a novel peptide sequence which blocks access to the active-site gorge. PMID:20138030

  12. A neuron-in-capillary platform for facile collection and mass spectrometric characterization of a secreted neuropeptide.

    PubMed

    Lee, Chang Young; Fan, Yi; Rubakhin, Stanislav S; Yoon, Sook; Sweedler, Jonathan V

    2016-01-01

    The integration of microfluidic devices-which efficiently handle small liquid volumes-with separations/mass spectrometry (MS) is an effective approach for profiling the neurochemistry occurring in selected neurons. Interfacing the microfluidic cell culture to the mass spectrometer is challenging because of geometric and scaling issues. Here we demonstrate the hyphenation of a neuron-in-capillary platform to a solid phase extraction device and off-line MS. A primary neuronal culture of Aplysia californica neurons was established directly inside a cylindrical polyimide capillary. The approach also uses a particle-embedded monolith to condition neuropeptide releasates collected from several Aplysia neurons cultured in the capillary, with the subsequent characterization of released peptides via MS. This system presents a number of advances compared to more traditional microfluidic devices fabricated with polydimethylsiloxane. These include low cost, easy access to cell culture, rigidity, ease of transport, and minimal fluid handling. The cylindrical geometry of the platform allows convenient interface with a wide range of analytical tools that utilize capillary columns. PMID:27245782

  13. Identity and phylogenetic placement of Spirogyra species (Zygnematophyceae, Charophyta) from California streams and elsewhere(1).

    PubMed

    Stancheva, Rosalina; Hall, John D; McCourt, Richard M; Sheath, Robert G

    2013-06-01

    Diversity of the filamentous green algae in the genus Spirogyra (Zygnematophyceae) was investigated from more than 1,200 stream samples from California. We identified 12 species of Spirogyra not previously known for California (CA), including two species new to science, Spirogyra californica sp. nov. and Spirogyra juliana sp. nov. Environmental preferences of the Californian species are discussed in the light of their restricted distribution to stream habitats with contrasting nutrient levels. We also investigated the systematic relationships of Spirogyra species from several continents using the chloroplast-encoded genes ribulose-1,5-bisphosphate carboxylase/hydrogenase large subunit (rbcL) and the beta subunit of the ATP synthase (atpB). Californian species were positioned in most major clades of Spirogyra. The phylogeny of Spirogyra and its taxonomic implications are discussed, such as the benefits of combining structural and molecular data for more accurate and consistent species identification. Considerable infraspecific genetic variation of globally distributed Spirogyra species was observed across continental scales. This finding suggests that structurally similar species from distant regions may be genetically dissimilar and that Spirogyra may contain a large number of cryptic species. Correlating the morphological and genetic variation within the genus will be a major challenge for future researchers. PMID:27007047

  14. Correlation of 125I-LSD autoradiographic labeling with serotonin voltage clamp responses in Aplysia neurons

    SciTech Connect

    Evans, M.L.; Kadan, M.J.; Hartig, P.R.; Carpenter, D.O. )

    1991-05-01

    Autoradiographic receptor binding studies using 125I-LSD (2-(125I)lysergic acid diethyamide) revealed intense labelling on the soma of a symmetrically located pair of cells in the abdominal ganglion of Aplysia californica. This binding was blocked by micromolar concentrations of serotonin and lower concentrations of the serotonergic antagonists, cyproheptadine and mianserin. Electrophysiological investigation of responses to serotonin of neurons in the left upper quadrant, where one of the labeled neurons is located, revealed a range of serotonin responses. Cells L3 and L6 have a K+ conductance increase in response to serotonin that is not blocked by cyproheptadine or mianserin. Cells L2 and L4 have a biphasic response to serotonin: a Na+ conductance increase, which can be blocked by cyproheptadine and mianserin, followed by a voltage dependent Ca2+ conductance which is blocked by Co2+ but not the serotonergic antagonists. Cell L1, and its symmetrical pair, R1, have in addition to the Na+ and Ca2+ responses observed in L2 and L4, a Cl- conductance increase blocked by LSD, cyproheptadine and mianserin. LSD had little effect on the other responses. The authors conclude that the symmetrically located cells L1 and R1 have a Cl- channel linked to a cyproheptadine- and mianserin-sensitive serotonin receptor that is selectively labelled by 125I-LSD. This receptor has many properties in common with the mammalian serotonin 1C receptor.

  15. Hyperpolarization-activated, cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

    PubMed Central

    Yang, Qizong; Kuzyk, Pavlo; Antonov, Igor; Bostwick, Caleb J.; Kohn, Andrea B.; Moroz, Leonid L.; Hawkins, Robert D.

    2015-01-01

    Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K+ and Na+ ions, and is selectively blocked by Cs+ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway. PMID:26668355

  16. The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Delta1-tetrahydrocannabinolic acid synthase from Cannabis sativa L.

    PubMed

    Sirikantaramas, Supaart; Morimoto, Satoshi; Shoyama, Yoshinari; Ishikawa, Yu; Wada, Yoshiko; Shoyama, Yukihiro; Taura, Futoshi

    2004-09-17

    Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis. PMID:15190053

  17. Determining the Structure of a Cytoplasmic Polyadenylation Element Binding Protein via AMBER9

    NASA Astrophysics Data System (ADS)

    Saunders, Alison

    2010-03-01

    The neurons of Aplysia californica contain cytoplasmic polyadenylation element binding protein (CPEB). CPEB shows prion-like properties when expressed in yeast cells. Because prions have misfolded and normally folded forms, prions can code in neurons like binary codes in computers, with ``present'' and ``not present'' signals available. CPEB thus provides a candidate protein for the molecular basis of memory. I attempt to determine CPEB's structure, by first threading the known protein sequence around a β-helical structure. Threading is preformed by hand, and by a program written to minimize the energy cost of building the structure. I then analyze the stability of the thread using the molecular dynamics program AMBER9. I also analyze a protein of only glutamine (PolyQ) in a β-helical structure to substantiate my use of a β-helix with the glutamine-rich CPEB. I found PolyQ to be stable in a left-handed β-helical structure with eighteen residues per turn. A candidate structure for CPEB was located with the same β-helical structure.

  18. “Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints

    PubMed Central

    Heinze, Michael; Brandt, Wolfgang; Marillonnet, Sylvestre; Roos, Werner

    2015-01-01

    The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins. PMID:25670767

  19. Flood induced phenotypic plasticity in amphibious genus Elatine (Elatinaceae)

    PubMed Central

    Sramkó, Gábor; Horváth, Orsolya; Popiela, Agnieszka; Mesterházy, Attila; Lukács, Balázs András

    2015-01-01

    Vegetative characters are widely used in the taxonomy of the amphibious genus Elatine L. However, these usually show great variation not just between species but between their aquatic and terrestrial forms. In the present study we examine the variation of seed and vegetative characters in nine Elatine species (E. brachysperma, E. californica, E. gussonei, E. hexandra, E. hungarica, E. hydropiper, E. macropoda, E. orthosperma and E. triandra) to reveal the extension of plasticity induced by the amphibious environment, and to test character reliability for species identification. Cultivated plant clones were kept under controlled conditions exposed to either aquatic or terrestrial environmental conditions. Six vegetative characters (length of stem, length of internodium, length of lamina, width of lamina, length of petioles, length of pedicel) and four seed characters (curvature, number of pits / lateral row, 1st and 2nd dimension) were measured on 50 fruiting stems of the aquatic and on 50 stems of the terrestrial form of the same clone. MDA, NPMANOVA Random Forest classification and cluster analysis were used to unravel the morphological differences between aquatic and terrestrial forms. The results of MDA cross-validated and Random Forest classification clearly indicated that only seed traits are stable within species (i.e., different forms of the same species keep similar morphology). Consequently, only seed morphology is valuable for taxonomic purposes since vegetative traits are highly influenced by environmental factors. PMID:26713235

  20. Characterization of a Crabs Claw Gene in basal eudicot species Epimedium sagittatum (Berberidaceae).

    PubMed

    Sun, Wei; Huang, Wenjun; Li, Zhineng; Lv, Haiyan; Huang, Hongwen; Wang, Ying

    2013-01-01

    The Crabs Claw (CRC) YABBY gene is required for regulating carpel development in angiosperms and has played an important role in nectary evolution during core eudicot speciation. The function or expression of CRC-like genes has been explored in two basal eudicots, Eschscholzia californica and Aquilegia formosa. To further investigate the function of CRC orthologous genes related to evolution of carpel and nectary development in basal eudicots, a CRC ortholog, EsCRC, was isolated and characterized from Epimedium sagittatum (Sieb. and Zucc.) Maxim. A phylogenetic analysis of EsCRC and previously identified CRC-like genes placed EsCRC within the basal eudicot lineage. Gene expression results suggest that EsCRC is involved in the development of sepals and carpels, but not nectaries. Phenotypic complementation of the Arabidopsis mutant crc-1 was achieved by constitutive expression of EsCRC. In addition, over-expression of EsCRC in Arabidopsis and tobacco gave rise to abaxially curled leaves. Transgenic results together with the gene expression analysis suggest that EsCRC may maintain a conserved function in carpel development and also play a novel role related to sepal formation. Absence of EsCRC and ElCRC expression in nectaries further indicates that nectary development in non-core eudicots is unrelated to expression of CRC-like genes. PMID:23299438

  1. Different channel properties of Torpedo acetylcholine receptor monomers and dimers reconstituted in planar membranes.

    PubMed Central

    Schindler, H; Spillecke, F; Neumann, E

    1984-01-01

    It is demonstrated that the monomeric and dimeric structures of the nicotinic acetylcholine receptor of Torpedo californica electric tissue, reconstituted in planar lipid bilayers, are functionally different. The native dimer D of Mr 500,000 (heavy-form) exhibits a "single" channel conductance about twice as large as that of the monomer M of Mr 250,000 (light form). Under conditions where monomers aggregate, the conductance changes from the level of the monomer M to that of dimers M2. The dimer conductances (D and M2) seem to result from synchronous opening and closing of the two channels in the dimer, giving the impression of "single channel" activity. This channel cooperativity is apparently mediated by noncovalent interactions between the two monomers, since it requires no disulfide linkage between monomers. Both the monomers M and the dimers D and M2 show at least one substate of lower conductivity. The relative population of the two conductance levels depends on the ion type (Na+ and K+), indicating ion-specific channel states. Since the channel conductance of isolated dimers resembles those obtained from unextracted microsacs, the dimer with two synchronized channels appears to be the in vivo predominant gating unit. In the linear association of dimers, observed in the native membrane, channel synchronization may extend to more than two channels as suggested by oligomeric channel cooperativity in associations of monomers and dimers. PMID:6091143

  2. A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane.

    PubMed Central

    Buckley, K M; Schweitzer, E S; Miljanich, G P; Clift-O'Grady, L; Kushner, P D; Reichardt, L F; Kelly, R B

    1983-01-01

    The plasma membrane of electric organ nerve terminals has two domains that can be distinguished by monoclonal antibodies. A library of 111 mouse monoclonal antibodies raised to nerve terminals from Torpedo californica contains 4 antibodies that bind specifically to the outside of intact synaptosomes. The distribution of the binding sites of these monoclonal antibodies on the outside of intact nerve terminals was examined by immunofluorescence and immunoelectron microscopy. The binding sites of 3 (tor23, 25, and 132) are distributed uniformly over nerve trunks and fine terminal branches. The binding site of the fourth (tor70) is restricted to synaptic junctional regions. This antibody, but not the other 3, recognizes a major component of synaptic vesicles, a proteoglycan associated with the inner surface of the vesicle membrane. The difference in the pattern of binding of these monoclonal antibodies suggests that the region of the plasma membrane containing active zones is antigenically distinguishable from other nerve terminal plasma membrane. We suggest that the antigen recognized by tor70 is externalized by exocytosis of synaptic vesicles while other plasma antigens take a different route to the surface. The unexpected observation that the vesicle antigen remains on the surface after exocytosis and is prevented from diffusion from the synaptic junctional region would be consistent with an interaction between the vesicle proteoglycan and elements of the synaptic cleft. Images PMID:6359167

  3. Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

    SciTech Connect

    Perez-Ramirez, B.; Martinez-Carrion, M. )

    1989-06-13

    A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with ({sup 3}H)pyridoxine 5-phosphate (({sup 3}H)PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of ({sup 3}H)PNP at 37{degree}C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the {alpha}-subunit, internal surface labeling by PLP was greatest for the highest molecular weight ({delta}) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles.

  4. Vegetation history along the eastern, desert escarpment of the Sierra San Pedro Mártir, Baja California, Mexico

    USGS Publications Warehouse

    Holmgren, Camille A.; Betancourt, Julio L.; Rylander, Kate A.

    2011-01-01

    Plant macrofossils from 38 packrat middens spanning the last ~ 33,000 cal yr BP record vegetation between ~ 650 and 900 m elevation along the eastern escarpment of the Sierra San Pedro Mártir, northern Baja California. The middens span most of the Holocene, with a gap between ~ 4600 and 1800 cal yr BP, but coverage in the Pleistocene is uneven with a larger hiatus between 23,100 and 14,400 cal yr BP. The midden flora is relatively stable from the Pleistocene to Holocene. Exceptions include Pinus californiarum, Juniperus californica and other chaparral elements that were most abundant > 23,100 cal yr BP and declined after 14,400 cal yr BP. Despite being near the chaparral/woodland-desertscrub ecotone during glacial times, the midden assemblages reflect none of the climatic reversals evident in the glacial or marine record, and this is corroborated by a nearby semi-continuous pollen stratigraphy from lake sediments. Regular appearance of C4 grasses and summer-flowering annuals since 13,600 cal yr BP indicates occurrence of summer rainfall equivalent to modern (JAS average of ~ 80–90 mm). This casts doubt on the claim, based on temperature proxies from marine sediments in the Guaymas Basin, that monsoonal development in the northern Gulf and Arizona was delayed until after 6200 cal yr BP.

  5. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA)

    PubMed Central

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

    2015-01-01

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5′ ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission. PMID:26446566

  6. Decreased Response to Acetylcholine during Aging of Aplysia Neuron R15

    PubMed Central

    Kadakkuzha, Beena M.; Carter, Christopher J.; Magoski, Neil S.; Capo, Thomas R.; Puthanveettil, Sathyanarayanan V.

    2013-01-01

    How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron. PMID:24386417

  7. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed Central

    McCrea, P D; Popot, J L; Engelman, D M

    1987-01-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  8. Homologues of serotonergic central pattern generator neurons in related nudibranch molluscs with divergent behaviors.

    PubMed

    Newcomb, James M; Katz, Paul S

    2007-04-01

    Homologues of a neuron that contributes to a species-specific behavior were identified and characterized in species lacking that behavior. The nudibranch Tritonia diomedea swims by flexing its body dorsally and ventrally. The dorsal swim interneurons (DSIs) are components of the central pattern generator (CPG) underlying this rhythmic motor pattern and also activate crawling. Homologues of the DSIs were identified in six nudibranchs that do not exhibit dorsal-ventral swimming: Tochuina tetraquetra, Melibe leonina, Dendronotus iris, D. frondosus, Armina californica, and Triopha catalinae. Homology was based upon shared features that distinguish the DSIs from all other neurons: (1) serotonin immunoreactivity, (2) location in the Cerebral serotonergic posterior (CeSP) cluster, and (3) axon projection to the contralateral pedal ganglion. The DSI homologues, named CeSP-A neurons, share additional features with the DSIs: irregular basal firing, synchronous inputs, electrical coupling, and reciprocal inhibition. Unlike the DSIs, the CeSP-A neurons were not rhythmically active in response to nerve stimulation. The CeSP-A neurons in Tochuina and Triopha also excited homologues of the Tritonia Pd5 neuron, a crawling efferent. Thus, the CeSP-A neurons and the DSIs may be part of a conserved network related to crawling that may have been co-opted into a rhythmic swim CPG in Tritonia. PMID:17180703

  9. Sequences and phylogeny analysis of rbcL gene in marine chlorophyta

    NASA Astrophysics Data System (ADS)

    Shen, Songdong; Li, Yanyan; Wu, Xunjian; Ding, Lanping

    2010-06-01

    The rbcL gene of Ulva pertusa, Enteromorpha prolifera and Monostroma grevillei was amplified, sequenced and analyzed. By comparing the rbcL sequences with seven other Ulvales species retrieved from GenBank, the sequence divergences and the phyletic evolution were analyzed and the phylogenetic tree was constructed. From the phylogenetic tree, it can be found that U. pertusa, E. prolifera and U. californica group in one branch, while E. compressa, U. rigida and U. fenestrata cluster in another clade. Obviously, unlike the Enteomorpha species, the Ulva species do not gather in one branch. Therefore Ulva and Enteomorpha might be affiliates of one genus. E. compressa and E. intestinalis gathered together, which coincided with the morphological characters. However, the thallus of U. pertusa is thick and with many holes, which is different from E. prolifera in morphology. They cluster together in the phylogenetic tree with a genetic distance of 0.005. The results indicate that Ulva and Enteromorpha are not distinguished strictly.

  10. Receptor-mediated presynaptic facilitation of quantal release of acetylcholine induced by pralidoxime in Aplysia.

    PubMed

    Fossier, P; Baux, G; Poulain, B; Tauc, L

    1990-09-01

    1. Possible interactions of contrathion (pralidoxime sulfomethylate), a reactivator of phosphorylated acetylcholinesterase (AChE), with the regulation of cholinergic transmission were investigated on an identified synapse in the buccal ganglion of Aplysia californica. 2. Transmitter release was evoked either by a presynaptic action potential or, under voltage clamp, by a long depolarization of the presynaptic cell. At concentrations higher than 10(-5) M, bath-applied contrathion decreased the amplitude of miniature postsynaptic currents and increased their decay time. At the same time, the quantal release of ACh was transiently facilitated. The facilitatory effect of contrathion was prevented by tubocurarine but not by atropine. Because in this preparation, these drugs block, respectively, the presynaptic nicotinic-like and muscarinic-like receptors involved in positive and negative feedback of ACh release, we proposed that contrathion activates presynaptic nicotinic-like receptors. 3. Differential desensitization of the presynaptic receptors is proposed to explain the transience of the facilitatory action of contrathion on ACh release. 4. The complexity of the synaptic action of contrathion raises the possibility that its therapeutic effects in AChE poisonings are not limited to AChE reactivation. PMID:2253262

  11. Enhancement of alkaloid production in opium and California poppy by transactivation using heterologous regulatory factors.

    PubMed

    Apuya, Nestor R; Park, Joon-Hyun; Zhang, Liping; Ahyow, Maurice; Davidow, Patricia; Van Fleet, Jennifer; Rarang, Joel C; Hippley, Matthew; Johnson, Thomas W; Yoo, Hye-Dong; Trieu, Anthony; Krueger, Shannon; Wu, Chuan-yin; Lu, Yu-ping; Flavell, Richard B; Bobzin, Steven C

    2008-02-01

    Genes encoding regulatory factors isolated from Arabidopsis, soybean and corn have been screened to identify those that modulate the expression of genes encoding for enzymes involved in the biosynthesis of morphinan alkaloids in opium poppy (Papaver somniferum) and benzophenanthridine alkaloids in California poppy (Eschscholzia californica). In opium poppy, the over-expression of selected regulatory factors increased the levels of PsCOR (codeinone reductase), Ps4'OMT (S-adenosyl-l-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase) and Ps6OMT [(R,S)-norcoclaurine 6-O-methyltransferase] transcripts by 10- to more than 100-fold. These transcriptional activations translated into an enhancement of alkaloid production in opium poppy of up to at least 10-fold. In California poppy, the transactivation effect of regulatory factor WRKY1 resulted in an increase of up to 60-fold in the level of EcCYP80B1 [(S)-N-methylcoclaurine 3'-hydroxylase] and EcBBE (berberine bridge enzyme) transcripts. As a result, the accumulations of selected alkaloid intermediates were enhanced up to 30-fold. The transactivation effects of other regulatory factors led to the accumulation of the same intermediates. These regulatory factors also led to the production of new alkaloids in California poppy callus culture. PMID:17961129

  12. Holocene vegetation and climate in the Puerto Blanco Mountains, southwestern Arizona

    NASA Astrophysics Data System (ADS)

    Van Devender, Thomas R.

    1987-01-01

    Plant macrofossils from 21 pack rat ( Neotoma sp.) middens at 535-605 m from the Puerto Blanco Mountains, southwestern Arizona, provide and excellent history of vegetation and climate for the last 14, 120 yr B.P. in the Sonoran Desert. A late Wisconsin juniper-Joshua tree woodland gave way to a transitional early Holocene desertscrub with sparse Juniperus californica (California juniper) by 10,540 yr B.P. Important Sonoran Desert plants including Carnegiea gigantea (saguaro) and Encelia farinosa (brittle bush) were dominants. Riparian trees such as Acacia greggii (catclaw acacia), Prosopis velutina (velvet mesquite), and Cerdicium floridum (blue palo verde) grew on dry, south-facing slopes in a middle Holocene Sonoran desertscrub in a warm, wet summer climate with frequent winter freezes. Modern subtropical Sonoran desertscrub formed about 4000 yr B.P. as summer rainfall and winter freezes declined. Cercidium microphyllum (foothills palo verde), Sapium biloculare (Mexican jumping bean), Olneya tesota (ironwood) and Stenocereus thurberi (organ pipe cactus) became dominant as riparian trees retreated to wash habitats. The inferences of a latest Wisconsin/early Holocene summer monsoonal maximum by J. E. Kutzbach (1983), Modeling of Holocene climates. In "Late-Quaternary Environments of the United States," Vol. 2, "The Holocene" (H. E. Wright, Ed.), pp. 271-277. Univ. of Minnesota Press, Minneapolis) are not supported for the Southwest. Apparently the persistence of late Wisconsin circulation patterns offset any increases in insolation.

  13. Analysis of isoquinoline alkaloids in medicinal plants by capillary electrophoresis-mass spectrometry.

    PubMed

    Sturm, S; Stuppner, H

    1998-11-01

    The technique of capillary electrophoresis - mass spectrometry (CE-MS) was applied for determination of isoquinoline alkaloids in crude methanolic extracts of medicinal plants. For the CE separations ammonium formate buffer solutions (70 or 100 mM, pH 3.0 or 4.0) containing 10% methanol or 20-60% acetonitrile as additives were used. The applied voltage was 25 kV, the thermostating temperature was kept constant at 25 degrees C. Coupling with the mass spectrometer was performed via an atmospherical pressure ionization (API) interface and the electrospray ionization technique (ESI). As sheath liquid 5 mM formic acid in acetonitrile at a flow rate of 3 microL/min was used. The spray voltage was 4.5 kV and the temperature of the heated capillary was chosen to be 200 degrees C. Detection in the positive ionization mode resulted in mass spectra showing either the molecular ions [M]+ or the protonated molecular ions [M+H]+. The presented method allows detection and identification of isoquinoline alkaloids in crude methanolic extracts of medicinal plants as Eschscholzia californica CHAM. (Papaveraceae), Hydrastis canadensis L. (Ranunculaceae), Berberis vulgaris L. (Berberidaceae), Jateorhiza palmata (LAM.) MIERS (Menispermaceae) and Chelidonium majus L. (Papaveraceae). PMID:9870408

  14. Taspine: Bioactivity-Guided Isolation and Molecular Ligand–Target Insight of a Potent Acetylcholinesterase Inhibitor from Magnolia x soulangiana

    PubMed Central

    Rollinger, Judith M.; Schuster, Daniela; Baier, Elisabeth; Ellmerer, Ernst P.; Langer, Thierry; Stuppner, Hermann

    2012-01-01

    A bioactivity-guided approach was taken to identify the acetylcholinesterase (AChE, EC 3.1.1.7) inhibitory agent in a Magnolia x soulangiana extract using a microplate enzyme assay with Ellman’s reagent. This permitted the isolation of the alkaloids taspine (1) and (−)-asimilobine (2), which were detected for the first time in this species. Compound 1 showed a significantly higher effect on AChE than the positive control galanthamine and selectively inhibited the enzyme in a long-lasting and concentration-dependent fashion with an IC50 value of 0.33 ± 0.07 μM. Extensive molecular docking studies were performed with human and Torpedo californica-AChE employing Gold software to rationalize the binding interaction. The results suggested ligand 1 to bind in an alternative binding orientation when compared to galanthamine. While this is located in close vicinity to the catalytic amino acid triad, the 1–AChE complex was found to be stabilized by (i) sandwich-like π-stacking interactions between the planar aromatic ligand (1) and the Trp84 and Phe330 of the enzyme, (ii) an esteratic site anchoring with the amino side chain, and (iii) a hydrogen-bonding network. PMID:16989531

  15. Karyotypic evolution in the Galliformes: an examination of the process of karyotypic evolution by comparison of the molecular cytogenetic findings with the molecular phylogeny.

    PubMed

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

    2004-01-01

    To define the process of karyotypic evolution in the Galliformes on a molecular basis, we conducted genome-wide comparative chromosome painting for eight species, i.e. silver pheasant (Lophura nycthemera), Lady Amherst's pheasant (Chrysolophus amherstiae), ring-necked pheasant (Phasianus colchicus), turkey (Meleagris gallopavo), Western capercaillie (Tetrao urogallus), Chinese bamboo-partridge (Bambusicola thoracica) and common peafowl (Pavo cristatus) of the Phasianidae, and plain chachalaca (Ortalis vetula) of the Cracidae, with chicken DNA probes of chromosomes 1-9 and Z. Including our previous data from five other species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and blue-breasted quail (Coturnix chinensis) of the Phasianidae, guinea fowl (Numida meleagris) of the Numididae and California quail (Callipepla californica) of the Odontophoridae, we represented the evolutionary changes of karyotypes in the 13 species of the Galliformes. In addition, we compared the cytogenetic data with the molecular phylogeny of the 13 species constructed with the nucleotide sequences of the mitochondrial cytochrome b gene, and discussed the process of karyotypic evolution in the Galliformes. Comparative chromosome painting confirmed the previous data on chromosome rearrangements obtained by G-banding analysis, and identified several novel chromosome rearrangements. The process of the evolutionary changes of macrochromosomes in the 13 species was in good accordance with the molecular phylogeny, and the ancestral karyotype of the Galliformes is represented. PMID:15218250

  16. Philinidae, Laonidae and Philinorbidae (Gastropoda: Cephalaspidea: Philinoidea) from the northeastern Pacific Ocean and the Beaufort Sea (Arctic Ocean).

    PubMed

    Valdés, Ángel; Cadien, Donald B; Gosliner, Terrence M

    2016-01-01

    Based on morphological data a total of nine native species of Philinidae are recognized from the northeastern Pacific including the Bering Sea and the adjacent Arctic Ocean (Beaufort Sea). Four of them have been previously described: Philine ornatissima Yokoyama, 1927, Philine bakeri Dall, 1919, Philine polystrigma (Dall, 1908), and Philine hemphilli Dall, 1919. Five of them are new and described herein: Philine mcleani sp. nov., Philine baxteri sp. nov., Philine malaquiasi sp. nov., Philine wareni sp. nov., and Philine harrisae sp. nov. These species display a substantial degree of variation in internal and external morphological traits (i.e., presence/absence of gizzard plates, different radular structure and tooth morphology, various reproductive anatomical features) and it is likely that they belong to different clades (genera). However, in the absence of a comprehensive phylogeny for Philine, they are here provisionally regarded as Philine sensu lato. In addition to the nine native species, two introduced species: Philine orientalis A. Adams, 1854 and Philine auriformis Suter, 1909 are here illustrated and compared to the native species to facilitate identification. Finally, two species previously considered members of Philinidae are examined anatomically and confirmed as members of Laonidae, Laona californica (Willett, 1944) and Philinorbidae, Philinorbis albus (Mattox, 1958), based on morphological data. PMID:27515632

  17. Sources of inoculum for Phytophthora ramorum in a redwood forest.

    PubMed

    Davidson, J M; Patterson, H A; Rizzo, D M

    2008-08-01

    ABSTRACT Sources of inoculum were investigated for dominant hosts of Phytophthora ramorum in a redwood forest. Infected trunks, twigs, and/or leaves of bay laurel (Umbellularia californica), tanoak (Lithocarpus densiflorus), and redwood (Sequoia sempervirens) were tested in the laboratory for sporangia production. Sporangia occurred on all plant tissues with the highest percentage on bay laurel leaves and tanoak twigs. To further compare these two species, field measurements of inoculum production and infection were conducted during the rainy seasons of 2003-04 and 2004-05. Inoculum levels in throughfall rainwater and from individual infections were significantly higher for bay laurel as opposed to tanoak for both seasons. Both measurements of inoculum production from bay laurel were significantly greater during 2004-05 when rainfall extended longer into the spring, while inoculum quantities for tanoak were not significantly different between the 2 years. Tanoak twigs were more likely to be infected than bay laurel leaves in 2003-04, and equally likely to be infected in 2004-05. These results indicate that the majority of P. ramorum inoculum in redwood forest is produced from infections on bay laurel leaves. Years with extended rains pose an elevated risk for tanoak because inoculum levels are higher and infectious periods continue into late spring. PMID:18943203

  18. Direct measurement of agonist binding to genetically engineered peptides of the acetylcholine receptor by selective T sub 1 NMR relaxation

    SciTech Connect

    Fraenkel, Y.; Navon, G. ); Aronheim, A.; Gershoni, J.M. )

    1990-03-13

    Interactions of four ligands of the nicotinic acetylcholine receptor with genetically engineered peptides have been studied by NMR. A recombinant cholinergic binding site was prepared as a fusion protein between a truncated form of the bacterial protein trpE and a peptide corresponding to the sequence {alpha}184-200 from the Torpedo californica receptor. This construct binds {alpha}-bungarotoxin while the trpE protein alone does not, and thus serves as a negative control. In this study agonist binding to {alpha}184-200 is demonstrated by monitoring the T{sub 1} relaxation of the ligand's protons in the presence and absence of the recombinant binding site. This binding is specific as it can be competed with {alpha}-bungarotoxin. Quantitative analyses of such competitions yielded the concentration of binding sites, which corresponded to 3.3% and 16.5% of the total protein, for partially purified and affinity-purified {alpha}184-200 constructs, respectively. The K{sub D} values for the binding of acetylcholine, nicotine, d-tubocurarine, and gallamine to the affinity-purified construct were 1.4, 1.4, 0.20, and 0.21 mM, respectively, while K{sub D}'s with the nontoxin binding protein were all above 10 mM. Thus, this is a direct demonstration that the toxin binding domain {alpha}184-200 may comprise a major component of the cholinergic agonist site.

  19. Expression and characterization of biologically active human hepatocyte growth factor (HGF) by insect cells infected with HGF-recombinant baculovirus.

    PubMed

    Yee, C J; DeFrances, M C; Bell, A; Bowen, W; Petersen, B; Michalopoulos, G K; Zarnegar, R

    1993-08-10

    A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by

  20. Comparative Analysis and Distribution of Omega-3 lcPUFA Biosynthesis Genes in Marine Molluscs

    PubMed Central

    Surm, Joachim M.; Prentis, Peter J.; Pavasovic, Ana

    2015-01-01

    Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the