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At least 739 records · Page 41Linked to original sources
PubMed2003

A possible overwintering mechanism for bluetongue virus in the absence of the insect vector.

Bluetongue virus (BTV) and several other Orbivirus species are transmitted between mammalian hosts via bites from adults of certain species of Culicoides midges. However, BTV can survive for 9-12 months (typically during the winter), in the absence of adult vectors, with no detectable cases of viraemia, disease or seroconversion in the host. The survival of the virus from one 'vector season' to the next is called 'overwintering' but the mechanism involved is not fully understood. It is demonstrated that BTV can persistently infect ovine gammadelta T-cells in vitro, a process that may also occur during infection and viraemia in mammalian hosts, thus providing a mechanism for virus persistence. Interaction of persistently BTV-infected gammadelta T-cells with antibody to the gammadelta T-cell-specific surface molecule WC-1 resulted in conversion to a lytic infection and increased virus release. Skin fibroblasts induce a similar conversion, indicating that they express a counter ligand for WC-1. Feeding of Culicoides midges induces skin inflammation, which is accompanied by recruitment of large numbers of activated gammadelta T-cells. The interaction of persistently infected gammadelta T-cells with skin fibroblasts would result in increased virus production at 'biting sites', favouring transmission to the insect vector. This suggested mechanism might also involve up-regulation of the WC-1 ligand at inflamed sites. It has been shown previously that cleavage of virus surface proteins by protease enzymes (which may also be associated with inflammation) generates infectious subvirus particles that have enhanced infectivity (100 times) for the insect vector.

Animals↗
PubMed1999

Stabilization of chromatin structure by PRC1, a Polycomb complex.

The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. We have purified a complex, termed PRC1 (Polycomb repressive complex 1), that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocked the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF did not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 was active on nucleosomal arrays formed with tailless histones.

Animals↗
PubMed2001

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells.

The bioluminescent Ca(2+)-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca(2)(+)-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC(50) value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca(2)(+)-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I.

Aequorin↗
PubMed1998

Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures.

Rabies virus nucleoprotein (N) was produced in insect cells using the baculovirus expression system described by Préhaud et al. (Virology 178, 486-497, 1990). The protein was either purified on a CsCl gradient, resulting in a mixture of nucleocapsid-like structures and beaded rings, as observed by electron microscopy, or on a glycerol gradient that resulted in a preparation of the rings only. The rings and nucleocapsid-like structures had the same morphological characteristics as viral nucleocapsids. N in these structures is an 84 A long and thin molecule that is spaced at around 34 A along the length of the nucleocapsid, identical in shape and spacing as the nucleoprotein in nucleocapsids of rabies virus and very similar to those of vesicular stomatitis virus. The recombinant nucleocapsids contained RNA with a stoichiometry similar to that found in viral nucleocapsids. The RNA bound in the beaded rings was a subset of the insect cellular RNA. One of the RNA species was partially sequenced and, although a positive identification could not be made, could correspond to a tRNA. With respect to sensitivity to trypsin and RNase digestion, the recombinant and viral nucleocapsids behaved similar. Trypsin cleaved a 17 kDa fragment from the carboxy terminus of N with only a very small effect on the morphology of the nucleocapsids. RNase A completely digested the resident RNA in both viral and recombinant nucleocapsids into fragments of 4-5 nt long, again with no effect on the morphology of the nucleocapsids. Thus, when the RNA is cleaved, the structure must be maintained by protein-protein contacts. Experiments to remove the resident RNA from viral and recombinant rabies virus nucleocapsids failed, whereas the same methods used to eliminate the RNA from vesicular stomatitis virus nucleocapsids was successful.

Animals↗
PubMed2006

Expression, purification, and characterization of mouse CYP2d22.

Metabolism of the prototype human CYP2D6 substrates debrisoquine and bufuralol proceeds at a much slower rate in mice; therefore, the mouse has been proposed as an animal model for the human CYP2D6 genetic deficiency. To interpret the molecular mechanism of this deficiency, a cDNA belonging to the CYP2D gene subfamily (Cyp2d22) has been cloned and sequenced from a mouse mammary tumor-derived cell line. In the current study, Cyp2d22 enzyme was overexpressed and purified from insect cells using a baculovirus-mediated system. The activity of this purified enzyme was directly compared with purified human CYP2D6 toward codeine, dextromethorphan, and methadone as substrates. Purified Cyp2d22 was found to catalyze the O-demethylation of dextromethorphan with significantly higher K(m) values (250 microM) than that (4.2 microM) exhibited by purified human CYP2D6. The K(m) for dextromethorphan N-demethylation by Cyp2d22 was found to be 418 microM, much lower than that observed with human CYP2D6 and near the K(m) for dextromethorphan N-demethylation catalyzed by CYP3A4. CYP2D6 catalyzed codeine O-demethylation, whereas Cyp2d22 and CYP3A4 mediated codeine N-demethylation. Furthermore, methadone, a known CYP3A4 substrate and CYP2D6 inhibitor, was N-demethylated by Cyp2d22 with a K(m) of 517 microM and V(max) of 4.9 pmol/pmol/min. Quinidine and ketoconazole, potent inhibitors to CYP2D6 and CYP3A4, respectively, did not show strong inhibition toward Cyp2d22-mediated dextromethorphan O- or N-demethylation. These results suggest that mouse Cyp2d22 has its own substrate specificity beyond CYP2D6-like-deficient activity.

Amino Acid Sequence↗
PubMed1990

Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector.

The phosphorylation pattern of simian virus 40 (SV40) large tumor (T) antigen purified from insect cells infected with a recombinant baculovirus was compared with that reported previously for T antigen from SV40-infected monkey cells. The specific activity of metabolic phosphate labeling of baculovirus T antigen was reduced, and the phosphopeptide map of the baculovirus protein, while qualitatively similar to that of lytic T, revealed several quantitative differences. The most striking difference was the prominence in the baculovirus map of peptides containing phosphothreonine 124. These peptides are known to arise from other phosphopeptides upon dephosphorylation of neighboring serines, suggesting that baculovirus T may be underphosphorylated at these serines and perhaps other sites. Functional assays used to further investigate the phosphorylation state of the baculovirus protein included SV40 DNA binding after enzymatic dephosphorylation with alkaline phosphatase and after phosphorylation by a murine homolog of cdc2 protein kinase. The results imply that baculovirus T antigen is underphosphorylated, in particular at those serine residues whose phosphorylation is responsible for down regulation of DNA-binding activity at site II in the core origin of DNA replication. In contrast, no evidence for a functionally significant underphosphorylation at threonine 124 could be found.

Animals↗
PubMed2006

A pheromone-binding protein mediates the bombykol-induced activation of a pheromone receptor in vitro.

The enormous capacity of the male silkmoth Bombyx mori in recognizing and discriminating bombykol and bombykal is based on distinct sensory neurons in the antennal sensilla hairs. The hydrophobic pheromonal compounds are supposed to be ferried by soluble pheromone-binding proteins (PBPs) through the sensillum lymph toward the receptors in the dendritic membrane. We have generated stable cell lines expressing the candidate pheromone receptors of B. mori, BmOR-1 or BmOR-3, and assessed their responses to hydrophobic pheromone compounds dissolved by means of dimethyl sulfoxide. BmOR-1-expressing cells were activated by bombykol but also responded to bombykal, whereas cells expressing BmOR-3 responded to bombykal only. In experiments employing the B. mori PBP, no organic solvent was necessary to mediate an activation of BmOR-1 by bombykol, indicating that the PBP solubilizes the hydrophobic compound. Furthermore, the employed PBP selectively mediated a response to bombykol but not to bombykal, supporting a ligand specificity of PBPs. This study provides evidence that both distinct pheromone receptors and PBPs play an important role in insect pheromone recognition.

Alkadienes↗
PubMed2001

Structure and activity of the insect cytokine growth-blocking peptide. Essential regions for mitogenic and hemocyte-stimulating activities are separate.

Growth-blocking peptide (GBP) is a 25-amino acid insect cytokine found in Lepidopteran insects that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells (plasmatocytes). The tertiary structure of GBP consists of a structured core that contains a disulfide bridge and a short antiparallel beta-sheet (Tyr(11)-Arg(13) and Cys(19)-Pro(21)) and flexible N and C termini (Glu(1)-Gly(6) and Phe(23)-Gln(25)). In this study, deletion and point mutation analogs of GBP were synthesized to investigate the relationship between the structure of GBP and its mitogenic and plasmatocyte spreading activity. The results indicated that deletion of the N-terminal residue, Glu(1), eliminated all plasmatocyte spreading activity but did not reduce mitogenic activity. In contrast, deletion of Phe(23) along with the remainder of the C terminus destroyed all mitogenic activity but only slightly reduced plasmatocyte spreading activity. Therefore, the minimal structure of GBP containing mitogenic activity is 2-23 GBP, whereas that with plasmatocyte spreading activity is 1-22 GBP. NMR analysis indicated that these N- and C-terminal deletion mutants retained a similar core structure to wild-type GBP. Replacement of Asp(16) with either a Glu, Leu, or Asn residue similarly did not alter the core structure of GBP. However, these mutants had no mitogenic activity, although they retained about 50% of their plasmatocyte spreading activity. We conclude that specific residues in the unstructured and structured domains of GBP differentially affect the biological activities of GBP, which suggests the possibility that multifunctional properties of this peptide may be mediated by different forms of a GBP receptor.

Amino Acid Sequence↗
PubMed1998

Plasmodium gallinaceum: effect of insect cells on ookinete development in vitro.

In vitro culture conditions affecting the transformation efficiency from zygote to ookinete for Plasmodium gallinaceum were examined, as a step toward improving the overall efficiency of in vitro culture systems for sporogonic stages. Gametocytes from infected chickens were allowed to fertilize in vitro and the resulting zygotes were purified and cultured. The time course for ookinete development in vitro was similar to that seen in Aedes aegypti mosquitoes. Supplementing a basal M-199 culture medium with heat-inactivated chicken serum and glucose did not affect transformation efficiency, but resulted in a four-fold increase in infectivity to mosquitoes when fed back to Ae. aegypti. Transformation from zygote to ookinete increased 5- to 10-fold when zygotes were cocultured with one of six different mosquito cell lines or a Drosophila cell line. Under optimal conditions, transformation efficiencies of up to 75% were observed. The presence of insect cells also increased the longevity of ookinetes in culture up to 42 h, while in acellular cultures ookinetes degenerated after about 24 h. The stimulatory effect was apparently not due to a factor secreted into the medium by the cells.

Aedes↗
PubMed1988

RH 5849, a nonsteroidal ecdysone agonist: effects on a Drosophila cell line.

The steroid molting hormone 20-hydroxyecdysone is the physiological inducer of molting and metamorphosis in insects. In ecdysone-sensitive Drosophila Kc cells, the insecticide RH 5849 (1,2-dibenzoyl-1-tert-butylhydrazine) mimics the action of 20-hydroxyecdysone by causing the formation of processes, an inhibition of cell proliferation, and induction of acetylcholinesterase. RH 5849 also competes with [3H]ponasterone A for high-affinity ecdysone receptor sites from Kc cell extracts. Resistant cell populations selected by growth in the continued presence of either RH 5849 or 20-hydroxyecdysone are insensitive to both compounds and exhibit a decreased titer of measurable ecdysone receptors. Although it is less potent than 20-hydroxyecdysone in both whole-cell and cell-free receptor assays, RH 5849 is the first nonsteroidal ecdysone agonist.

Acetylcholinesterase↗
PubMed1983

Morphogenesis of nuclear polyhedrosis virus from Autographa californica in a cell line from Mamestra brassicae (cabbage moth). Further aspects on baculovirus assembly.

Cultures of cell line IZD-MB 0503 from Mamestra brassicae were inoculated with nonoccluded nuclear polyhedrosis virus of Autographa californica (AcNOV). At 1 h postinoculation (p.i.) nucleocapsids were found in the cytoplasm near nuclear pores and within the nucleoplasm. Formation of virogenic stroma was observed at 7 h p.i. The first short empty capsids were seen at 10 h postinoculation (p.i.), followed by partially and completely filled nucleocapsids (11-12 h p.i.) with most capsids filled at 12 h and later. This suggests that nucleocapsid components, such as capsid and DNA-core, assemble in successive stages rather than simultaneously. Viral progeny, being released from the cells by budding, were observed at 13 h p.i.

Animals↗
PubMed1981

Processing of cricket paralysis virus induced polypeptides in Drosophila cells: production of high molecular weight polypeptides by treatment with iodoacetamide.

Infection of Drosophila cells with Cricket paralysis virus in the presence of Actinomycin D results in virtual complete inhibition of host cell protein synthesis by four hours post-infection. Using 35S-methionine or 14C-amino acids to pulse infected cells three major classes of viral induced proteins can be detected, (A) high molecular weight precursor proteins, (B) viral structural proteins and (C) low molecular weight cleavage products. The large number of high molecular weight proteins found in the infected cells suggests that a multiple cleavage cascade mechanism is partially utilized to produce virus structural proteins. In infected cells, even with short pulses, the largest viral induced protein obtained has a molecular weight of 144,000. However with pretreatment of the infected cells with iodoacetamide before pulsing, two further proteins are obtained with molecular weights of 205,000 and 190,000. Other changes occur in viral protein precursors in the presence of iodoacetamide.

Animals↗
PubMed2006

Mosquito La protein binds to the 3' untranslated region of the positive and negative polarity dengue virus RNAs and relocates to the cytoplasm of infected cells.

The untranslated regions (UTRs) of the positive and negative strand RNAs of several viruses are major binding sites for cellular and viral proteins. Human La autoantigen is one of the cellular proteins that interacts with various positive strand RNA viral genomes including that of dengue virus (DEN) within the 5'- and 3'-UTRs of positive (+) and the 3'-UTR of negative strand (-) RNA, and with the nonstructural proteins NS3 and NS5, that form DEN replicase complex. Since DEN replicates in human and mosquito cells, some functional interactions have to be conserved in both hosts. In the present report, we demonstrate that mosquito La protein interacts with the 3'-UTRs of (+) and (-) polarity viral RNAs. The localization of La protein, examined by confocal microscopy, indicates that La protein is redistributed in DEN-infected cells. Furthermore, the presence of La protein in an in vitro replication system inhibited RNA synthesis in a dose-dependent manner, suggesting that La protein plays an important role in dengue virus replicative cycle.

3' Untranslated Regions↗
PubMed2000

Anopheles gambiae pilot gene discovery project: identification of mosquito innate immunity genes from expressed sequence tags generated from immune-competent cell lines.

Together with AIDS and tuberculosis, malaria is at the top of the list of devastating infectious diseases. However, molecular genetic studies of its major vector, Anopheles gambiae, are still quite limited. We have conducted a pilot gene discovery project to accelerate progress in the molecular analysis of vector biology, with emphasis on the mosquito's antimalarial immune defense. A total of 5,925 expressed sequence tags were determined from normalized cDNA libraries derived from immune-responsive hemocyte-like cell lines. The 3,242 expressed sequence tag-containing cDNA clones were grouped into 2,380 clone clusters, potentially representing unique genes. Of these, 1,118 showed similarities to known genes from other organisms, but only 27 were identical to previously known mosquito genes. We identified 38 candidate genes, based on sequence similarity, that may be implicated in immune reactions including antimalarial defense; 19 of these were shown experimentally to be inducible by bacterial challenge, lending support to their proposed involvement in mosquito immunity.

Animals↗
PubMed1999

Fluorescence in situ hybridization landmarks for chromosomes of culicoides variipennis (Diptera: Ceratopogonidae).

Because the three chromosomes of Culicoides variipennis (Coquillett) are morphologically indistinguishable, physical landmarks were needed so that the chromosomes can be identified uniquely and oriented unambiguously before initiating the construction of a physical map based on FISH (fluorescence in situ hybridization). When repetitive sequence clones p1887 and K8.1a8 were labeled with digoxigenin and probe K8a.2G2 was labeled with biotin-11-dUTP and digoxigenin, the 3 chromosomes could be differentiated unambiguously when visualized with specific band-pass filters. This provided the basis for C. variipennis FISH landmark probes that enabled the identification and orientation of all 3 pairs of chromosomes. Using this multicolor FISH labeling strategy, probes that provide unique landmarks for the C. variipennis FISH physical map have been found and may be used in all FISH reactions where an unknown probe is placed to metaphase chromosomes of C. variipennis. A physical map of the C. variipennis genome will provide the foundation for map-based positional cloning of the gene(s) that control vector competence for the bluetongue viruses.

Animals↗
PubMed1984

Plaque assay for black beetle virus.

A rapidly growing strain of virus was used to develop a reliable plaque assay for Black beetle virus on monolayers of cultured Drosophila cells. Cell density of the monolayer was critical for successful plaque formation. The dose-response curve for plaque formation was linear, supporting earlier proposals that both RNA segments of the split genome reside in the same particle. The method greatly facilitates isolation of reassortant and variant strains of virus.

Animals↗