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At least 613 records · Page 34Linked to original sources

Insecticidal activity of the pyrimidine nucleoside analogue (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU).

The insecticidal activity of the antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was assessed in in vivo assays against the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera, Noctuidae). BVDU, mixed into an artificial diet, caused a variety of effects, depending on the concentration used. Compared with controls, food intake was lower, larval growth was retarded and larval development was prolonged. The treated larvae formed smaller pupae and the hatching moths often showed morphogenetic defects. A higher mortality could be found in larval and pupal stages and was generally caused by moult disruption. A choice assay showed that BVDU has very slight feeding-deterrent properties, which only partly explain the toxic effects. The agent most probably acts through its cytostatic activity that has been described previously using cell lines of different insect species.

Animals↗

A recombinant H1 histone-based system for efficient delivery of nucleic acids.

We describe here a unique transfer system based on a truncated form of the human linker histone H1F4 for the delivery of nucleic acids to a variety of cells. The efficiency of truncated histone H1.4F was assessed using both primary mammalian and immortalised insect and mammalian cell lines. Our results indicated that recombinant histone H1.4F was able to deliver DNA, dsRNA and siRNA in all cells tested. Quantitative analysis based on reporter gene expression or silencing of target genes revealed that the transfection efficiency of histone H1.4F was comparable to, or better than, liposome-based systems. Notably, the efficiency of histone H1.4F was associated with very low toxicity for transfected cells. The human H1.4F recombinant protein is easily purified in large-scale from bacterial lysates using inexpensive simplified processing. This versatile transfection system represents an important advance in the field of gene delivery and an improvement over earlier nucleic acid delivery methods.

Animals↗

Evaluation of different biological test systems to assess the toxicity of metabolites from fungal biocontrol agents.

The development of fungal biocontrol agents (BCAs) as alternatives to chemical pesticides is of increasing public interest. Tools to assess the toxicity of the secondary metabolites that these BCAs produce are often not available or existing methods have not yet been evaluated for these compounds. This study compares five different test systems, which include a representative bacterium, protozoan, arthropod and insect and human cell lines, as regards their sensitivity. It also compares the cost in time and resources for conducting the tests. Pure metabolites and crude extracts from two fungal BCAs as well as two chemical pesticides (hoestar and chlorpyrifos) and the mycotoxin patulin were employed as test compounds. All tests systems proved to be suitable for toxicity studies of metabolites from fungal BCAs and showed different grades of sensitivity to the different substances. The possibility of employing an array of test systems to determine ecotoxicological properties is discussed.

Animals↗

Secretion of active human lecithin-cholesterol acyltransferase by insect cells infected with a recombinant baculovirus.

Difficulties in purifying the plasma enzyme lecithin-cholesterol acyltransferase (LCAT) have hampered detailed studies of its (patho)physiological role in lipoprotein metabolism and of structure-function relationships. Potentially, baculovirus-driven expression systems offer a powerful means to produce significant amounts of LCAT. Accordingly, full-length LCAT cDNA was cloned into pVL 1392, a high-level expression derivative of Autographa californica nuclear polyhedrosis virus (AcNPV), and the resultant plasmid was co-transfected into Trichoplusia ni insect cells (High 5 line) with a linearized viral DNA using lipofectin. Such viral DNA had a lethal mutation and grew only when recombined with a pVL1392-type rescue plasmid; cells infected with recombinant Autographa californica LCAT virus changed from a fibroblast-like morphology to rounded, but lacked the polyhedrin occlusion bodies characteristic of wild-type AcNPV infections. Enzymically active recombinant LCAT (rLCAT), sensitive to sulphydryl reagents, was secreted in the late phase of infection (36-48 h) but was absent with wild-type infections. The secreted protein had an apparent molecular mass of 53 kDa by SDS/PAGE, lower than that of native plasma LCAT; it was susceptible to endoglycosidase H digestion and was bound by concanavalin A, suggesting that precursor high-mannose N-glycan chains had not undergone full maturation to complex types. Pretreatment of the cells with tunicamycin to inhibit the first step of N-glycosylation led to intracellular accumulation of immature rLCAT (approximately 46-48 kDa) and a marked reduction in enzyme secreted. We conclude that the baculovirus gene-expression system will permit production of biologically active normal and mutant forms of LCAT protein.

Animals↗

Regulation of Raf-1 kinase by TNF via its second messenger ceramide and cross-talk with mitogenic signalling.

Raf-1 kinase is a central regulator of mitogenic signal pathways, whereas its general role in signal transduction of tumour necrosis factor (TNF) is less well defined. We have investigated mechanisms of Raf-1 regulation by TNF and its messenger ceramide in cell-free assays, insect and mammalian cell lines. In vitro, ceramide specifically bound to the purified catalytic domain and enhanced association with activated Ras proteins, but did not affect the kinase activity of Raf-1. Cell-permeable ceramides induced a marked increase of Ras-Raf-1 complexes in cells co-expressing Raf-1 and activated Ras. Likewise, a fast elevation of the endogeneous ceramide level, induced by TNF treatment of human Kym-1 rhabdomyosarcoma cells, was followed by stimulation of Ras-Raf-1 association without significant Raf-1 kinase activation. Failure of TNF or ceramide to induce Raf-1 kinase was observed in several TNF-responsive cell lines. Both TNF and exogeneous C6-ceramide interfered with the mitogenic activation of Raf-1 and ERK by epidermal growth factor and down-regulated v-Src-induced Raf-1 kinase activity. TNF also induced the translocation of Raf-1 from the cytosolic to the particulate fraction, indicating that this negative regulatory cross-talk occurs at the cell membrane. Interference with mitogenic signals at the level of Raf-1 could be an important initial step in TNF's cytostatic action.

3T3 Cells↗

Interactions of bovine viral diarrhoea virus glycoprotein E(rns) with cell surface glycosaminoglycans.

Recombinant E(rns) glycoprotein of bovine viral diarrhoea virus (BVDV) has been tagged with a marker epitope or linked to an immunoglobulin Fc tail and expressed in insect and mammalian cell lines. The product was shown to be functional, both having ribonuclease activity and binding to a variety of cells that were permissive and non-permissive for replication of BVDV. Addition of soluble E(rns) to the medium blocked replication of BVDV in permissive cells. Binding of epitope-tagged E(rns) to permissive calf testes (CTe) cells was abolished and virus infection was reduced when cells were treated with heparinases I or III. E(rns) failed to bind to mutant Chinese hamster ovary (CHO) cells that lacked glycosaminoglycans (pgsA-745 cells) or heparan sulphate (pgsD-677 cells) but bound to normal CHO cells. E(rns) also bound to heparin immobilized on agarose and could be eluted by heparin and by a high concentration of salt. Flow cytometric analysis of E(rns) binding to CTe cell cultures showed that glycosaminoglycans such as heparin, fucoidan and dermatan sulphate all inhibit binding but dextran sulphate, keratan sulphate, chondroitin sulphate and mannan fail to inhibit binding. The low molecular mass polysulphonated inhibitor suramin also inhibited binding to CTe cells but poly-L-lysine did not. Furthermore, suramin, the suramin analogue CPD14, fucoidan and pentosan polysulphate inhibited the infectivity of virus. It is proposed that binding of E(rns) to cells is through an interaction with glycosaminoglycans and that BVDV may bind to cells initially through this interaction.

Animals↗

An Sp1 response element in the Kaposi's sarcoma-associated herpesvirus open reading frame 50 promoter mediates lytic cycle induction by butyrate.

Kaposi's sarcoma-associated herpesvirus (KSHV) can be driven into the lytic cycle in vitro by phorbol esters and sodium butyrate. This report begins to analyze the process by which butyrate activates the promoter of KSHV open reading frame 50 (ORF50), the key viral regulator of the KSHV latency to lytic cycle switch. A short fragment of the promoter, 134 nucleotides upstream of the translational start of ORF50, retained basal uninduced activity and conferred maximal responsiveness to sodium butyrate. The butyrate response element was mapped to a consensus Sp1-binding site. By means of electrophoretic mobility shift assays, both Sp1 and Sp3 were shown to form complexes in vitro with the ORF50 promoter at the Sp1 site. Butyrate induced the formation of a group of novel complexes, including several Sp3-containing complexes, one Sp1-containing complex, and several other complexes that were not identified with antibodies to Sp1 or Sp3. Formation of all butyrate-induced DNA-protein complexes was mediated by the consensus Sp1 site. In insect and mammalian cell lines, Sp1 significantly activated the ORF50 promoter linked to luciferase. Chromatin immunoprecipitation experiments in a PEL cell line showed that butyrate induced Sp1, CBP, and p300 binding to the ORF50 promoter in vivo in an on-off manner. The results suggest that induction of the KSHV lytic cycle by butyrate is mediated through interactions at the Sp1/Sp3 site located 103 to 112 nucleotides upstream of the translational initiation of ORF50 presumably by enhancing the binding of Sp1 to this site.

Base Sequence↗

A baculovirus-expressed fusion protein containing the antibody-binding domain of protein A and insect luciferase.

A fusion construct encoding two antibody-binding sites of protein A from Staphylococcus aureus and click beetle, Pyrophorus plagiophthalamus, luciferase (LucGR) was designed and expressed using the baculovirus system. The construct was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in the insect Spodoperta frugiperda cell line during viral infection. The properties of the resultant chimeric protein product, protA-LucGR, were studied both in vivo and in vitro by using i) luminometry, ii) immunoblot analysis, iii) immunoprecipitation, iv) metabolic labeling procedures and v) luminescent immunoassays. Together, the results clearly demonstrate that the light-emitting properties of the fused luciferase construct remain intact. Further, the antibody-binding domain of protein A retains its activity as it binds to both rabbit and goat as well as human immunoglobulins. Due to the dual biological function of this fusion protein, it should provide a potential reagent within the field of molecular biology and diagnostics.

Animals↗

Eukaryotic expression systems: a comparison.

Eukaryotic expression systems are frequently employed for the production of recombinant proteins as therapeutics as well as research tools. Most commonly used expression systems are based on stably transfected adherent CHO cells or nonadherent lymphoid cell lines. An efficient alternative is the infection of insect cells by recombinant baculoviruses. Transient expression in mammalian cells, e.g., COS cells, is often used for the production of smaller quantities of proteins. The choice of a suitable expression system depends largely on the biochemical and biological properties of the protein of interest, as well as on the nature of the planned experiments and the amount of recombinant protein required. We summarize here the expression of the cytokine human Leukemia Inhibitory Factor (hu-LIF) in five of the most commonly used systems, namely in CHO, Sp2/0, MEL, COS, and insect cells, in conjunction with an outline of the principles and characteristics of each of these expression systems. In result, the stably transfected cell lines, CHO, Sp2/0, and MEL cells, gave rise to production of fully glycosylated hu-LIF at variable product titers; incompletely glycosylated, albeit biological action hu-LIF could be rapidly produced by transient expression in COS cells or by baculovirus-mediated infection of insect cells.

Animals↗

Development and partial characterization of heliothine cell lines from embryonic and differentiated tissues.

The goal of this study was to generate cell lines from a variety of insect tissues that could be useful for developing in vitro assays with tissue-specific properties. In this article, we describe the establishment of new cell cultures from differentiated (primarily neural) and undifferentiated tissues (primarily embryonic) and their initial characterization. Cell lines were established from the following tissues of the budworm, Heliothis virescens, and the bollworm, Helicoverpa zea: larval ventral nerve cords (4 lines), larval midguts (1 line), adult ovaries (1 line), and embryonic tissues (11 lines). Cell lines were primarily characterized by morphological examination and polymerase chain reaction (PCR) (both deoxyribonucleic acid amplification fingerprinting and inter-simple sequence repeats PCR).

Animals↗

Recombinant human IgA expressed in insect cells.

IgA serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities in serum. To map the sites of interaction of immune effector molecules with the IgA constant region (C alpha), we have expressed soluble, chimeric human IgA in insect cells using recombinant baculoviruses. This antibody is correctly assembled into heavy chain/light chain heterodimers, N-glycosylated, and secreted by the insect cells; further, when coexpressed with a human J chain, the antibodies can assemble into dimers. The recombinant protein is authentic by a number of criteria, including antigen-binding, recognition by monoclonal antibodies, complement fixation via the alternative pathway, and specific binding to the monocyte IgA Fc receptor. We have also constructed viruses which encode structurally altered IgA heavy chains. Using one of these variant viruses, we have shown that glycosylation of the second domain of C alpha is required for interaction with the monocyte IgA Fc receptor. This system should prove useful in further characterization of the structure-function relationships in human C alpha.

Animals↗

Baculovirus studies in new, indigenous lepidopteran cell lines.

Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines.

Animals↗

Application of on-line OUR measurements to detect actions points to improve baculovirus-insect cell cultures in bioreactors.

The continuous monitoring of a process based on the culture of Sf9 insect cells and infection by a baculovirus as a vector to obtain recombinant VP2 protein is studied. On-line OUR determination is based on the direct oxygen measurement in the cell culture vessel and the application of the dynamic method. This approximation allows a proper description of cell growth, with precise identification of the balanced cell growth end and the most important action times in the process, as virus infection time and final cell harvesting. A detailed study of the OUR profiles allows on-line monitoring of the effects of infection and expression protein process, a tool enabling the automatisation of the protein production process in a baculovirus-insect cell system. These parameters have been defined as time of action (TOAs), and include the most relevant actions to take in these type of processes: time of infection (TOI), time of feeding (TOF) and time of harvesting (TOH).

Animals↗

A comparison of techniques for detecting Invertebrate iridescent virus 6.

The aim of this study was to compare the sensitivity and precision of various methods for the detection and quantification of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae) isolated from a the stem-boring moth Chilo suppressalis, and to apply these techniques to the detection of covert infections in the wax moth, Galleria mellonella. The relationship between the virus concentration and absorbance at 260 nm was linear over the range of 1.6 x 10(9)-5.6 x 10(10) particles/ml. TCID(50) assays using 12 different cell lines indicated that two Drosophila lines, DL2 and DR1, had the highest susceptibility whereas cell lines from Aedes albopictus and Plutella xylostella were four orders of magnitude less sensitive. TCID(50) values for IIV-6 in Spodoptera frugiperda Sf9 cells gave the particle-infectivity ratios of 15-64 virus particles/IU. An insect bioassay involved injecting doses of 1-100 IIV-6 particles into the third instar G. mellonella larvae. The prevalence of patent infection was 20-26% at a dose of 1 particle per larva rising to 86-92% at 10 particles and 100% at doses of 50 or 100 particles. Of the insects that survived to adulthood, between 5.8 and 75% caused patent infections when injected into G. mellonella larvae, indicating that they were covertly infected. A PCR technique resulted in 95% detection at 1000 virus particles per insect. Of the insects that proved positive for covert infection by insect bioassay, 41% also proved positive by PCR analysis. It is concluded that the G. mellonella bioassay is highly reliable for detection of doses of 10 particles or more and for determining the relative activity of IIV-6 preparations at doses as low as 1 particle per insect. PCR had a slightly lower sensitivity followed by the insect cell culture assay.

Aedes↗

Lysosomes as the target of yessotoxin in invertebrate and vertebrate cell lines.

The toxic effects of the algal polyether phycotoxin yessotoxin (YTX) are studied in the insect fat body IPLB-LdFB and the mouse fibroblast NIH3T3 cell lines. Our experiments confirm the cytotoxic action exerted by the toxin in both insect and mammalian cells, but morphological observations, TUNEL experiments and electrophoretic evalution of DNA integrity failed to evidence a clear pro-apoptotic role for YTX. In both IPLB-LdFB and NIH3T3 cell lines, neutral red and acridine orange stainings, together with evaluation of acid phosphatase activity demonstrate that YTX first damages lysosomal vesicles. This is then followed by a progressive depolymerization of actin microfilaments, as shown by phalloidin fluorescent immunostaining. Overall, our data identify in early lysosomal damage and the subsequent cytoskeletal disruption two common steps related to YTX toxicity towards metazoan cells.

3T3 Cells↗

Studies on overwintering of bluetongue viruses in insects.

Bluetongue viruses (BTVs) are economically important arboviruses that affect sheep and cattle. The overwintering mechanism of BTVs in temperate climates has eluded researchers for many years. Many arboviruses overwinter in their invertebrate vectors. To test the hypothesis that BTVs overwinter in their vertically infected insect vectors, Culicoides sonorensis larvae were collected from long-term study sites in northern Colorado, USA, and assayed for the presence of BTV RNA by nested RT-PCR. Sequences from BTV RNA segment 7 were detected in 30 % (17/56) of pools composed of larvae and pupae collected in 1998 and in 10 % (31/319) of pools composed of adults reared from larvae collected in 1996. BTV was not isolated from the insects. Additionally, Culicoides cell-culture lines derived from material collected at one of the sites, or derived from insect samples collected during a BTV outbreak, contained BTV RNA segment 7. In contrast, segment 2 RNA was detected at half the rate of segment 7 RNA in the field-collected larvae and was only detected in the Culicoides cell lines with one of two primer sets. These data suggest that BTVs could overwinter in the insect vector and that there is reduced expression of the outer capsid genes during persistent infection.

Animals↗

Species-specific effects of the hcf-1 gene on baculovirus virulence.

The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) is required for the efficient replication and/or stability of reporter plasmids carrying an AcMNPV-derived origin of DNA replication in a cell-specific manner; hcf-1 is required for reporter plasmid replication or stability in TN-368 cells, a cell line derived from the cabbage looper Trichoplusia ni, but not in IPLB-SF-21 (SF-21) cells, a cell line derived from the fall armyworm Spodoptera frugiperda (A. Lu and L. K. Miller, J. Virol. 69:6265-6272, 1995). To further define the function of hcf-1, recombinant viruses with null mutations in hcf-1 were constructed in SF-21 cells and the phenotype of the mutants was determined in selected cell lines as well as in insect larvae. In S.frugiperda larvae and SF-21 cells, the phenotype of hcf-1 mutants was indistinguishable from that of wild-type AcMNPV. In T. ni larvae as well as T. ni-derived cell lines, hcf-1 mutants exhibited a mutant phenotype. In TN-368 cells, the replication of hcf-1 mutants was extremely impaired; the phenotype included a defect in viral DNA replication, late gene transcription, and virus production as well as a complete cessation of host and viral protein synthesis. In another cell line derived from T. ni, the BTI-TN5B1-4 cell line, the hcf-1 mutants exhibited a less severe phenotype. In T. ni larvae, the infectivity of the budded form of hcf-1 mutants was decreased significantly (50-fold), although no difference in the oral infectivity of the occluded form was observed. T. ni larvae infected with hcf-1 mutants by either oral or hemocoelic routes, however, died 20 to 30% more slowly than those infected with wild-type AcMNPV. These data indicate that there is a host-specific requirement for hcf-1 and that it exerts cell line-specific effects and possibly tissue-specific effects on the rate at which the virus replicates, thereby affecting the virulence of the virus in a species-specific manner.

Animals↗