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Isoforms of cyclic AMP response element binding proteins in Drosophila S2 cells.

Activation or inhibition of the cyclic AMP (cAMP)-protein kinase A (PKA) pathway can ultimately regulate the transcription of a variety of genes. In vertebrates, the best characterized nuclear targets of PKA are the 'cAMP response element' (CRE) binding proteins (CREB). Differences in the transcriptional response to this pathway between cells and tissues can be based on the presence of distinct CREB isoforms. In this context, we have now investigated the presence of different dCREB transcripts in a stable, embryonic insect cell line, i.e., Drosophila Schneider 2 (S2) cells. In addition, we have studied the possible effect of cellular cAMP- and Ca2+ increases on the expression of a luciferase reporter in cells transfected with a CRE-containing reporter gene construct. In combination with recent data from the literature, our results indicate that the regulation of CRE-dependent gene expression shows some important differences between insects and vertebrates.

Amino Acid Sequence↗

Functional and comparative analysis of two distinct ecdysteroid-responsive gene expression constructs in Drosophila S2 cells.

Inducible expression systems have proven to be of major interest when analysing the function of specific genes or when expressing cytotoxic proteins. In an effort to develop inducible switches allowing for flexible fine-tuning of gene expression levels in insect cells, we have compared the induction capacities of two Drosophila minimal promoters when linked to four consecutive ecdysone response elements. These minimal promoters, either containing a TATA-box or a downstream promoter element, drove the expression of a luciferase reporter gene. Potent induction capacities were observed with the insect moulting hormone, 20-hydroxyecdysone, and with ponasterone A, a plant ecdysteroid. The developed inducible switches further expand the repertoire of molecular tools for functional expression of proteins of interest in insect cells. In addition, the combination of an ecdysone switch with promoters that possess different structural elements can provide novel insights into ecdysteroid-induced transcription in an insect cell line.

Animals↗

Towards abolition of immunogenic structures in insect cells: characterization of a honey-bee (Apis mellifera) multi-gene family reveals both an allergy-related core alpha1,3-fucosyltransferase and the first insect Lewis-histo-blood-group-related antigen-synthesizing enzyme.

Glycoproteins from honey-bee (Apis mellifera), such as phospholipase A2 and hyaluronidase, are well-known major bee-venom allergens. They carry N-linked oligosaccharide structures with two types of alpha1,3-fucosylation: the modification by alpha1,3-fucose of the innermost core GlcNAc, which constitutes an epitope recognized by IgE from some bee-venom-allergic patients, and an antennal Lewis-like GalNAcbeta1,4(Fucalpha1,3)GlcNAc moiety. We now report the cloning and expression of two cDNAs encoding the relevant active alpha1,3-FucTs (alpha1,3-fucosyltransferases). The first sequence, closest to that of fruitfly (Drosophila melanogaster) FucTA, was found to be a core alpha1,3-FucT (EC 2.4.1.214), as judged by several enzyme and biochemical assays. The second cDNA encoded an enzyme, most related to Drosophila FucTC, that was shown to be capable of generating the Le(x) [Galbeta1-4(Fucalpha1-3)GlcNAc] epitope in vitro and is the first Lewis-type alpha1,3-FucT (EC 2.4.1.152) to be described in insects. The transcription levels of these two genes in various tissues were examined: FucTA was found to be predominantly expressed in the brain tissue and venom glands, whereas FucTC transcripts were detected at highest levels in venom and hypopharyngeal glands. Very low expression of a third homologue of unknown function, FucTB, was also observed in various tissues. The characterization of these honey-bee gene products not only accounts for the observed alpha1,3-fucosylation of bee-venom glycoproteins, but is expected to aid the identification and subsequent down-regulation of the FucTs in insect cell lines of biotechnological importance.

Allergens↗

Persistent expression of a newly characterized Hyposoter didymator polydnavirus gene in long-term infected lepidopteran cell lines.

An Hyposoter didymator ichnovirus (HdIV) gene was stably maintained and efficiently transcribed in lepidopteran cell lines more than 3 years after HdIV infection. This K-gene had two introns and the fully spliced cDNA, named K19, comprised a short open reading frame and a long 3'-untranslated region with 13 imperfectly repeated sequences (44 to 102 nt). Transcripts related to the K-gene were detected in several long-term infected cell lines (Sf9, Spodoptera littoralis haemocytes, Trichoplusia ni). Conversely, no transcripts related to seven other viral cDNAs were detected, suggesting that the K-related DNA is selectively retained in long-term infected Sf9 cells. The function of the K-gene product and its association with stably transformed insect cell lines remains to be investigated.

Amino Acid Sequence↗

Cytotoxicity against insect cells of entomopathogenic fungi of the genera Hypocrella (anamorph Aschersonia): possible agents for biological control.

Extracts from entomopathogenic fungi of the genus Hypocrella (7 species) and its anamorph Aschersonia (11 species) were screened for cytotoxicity to Sf9 and C6/36 insect cells and L929, BHK(21)C13 and HepG2 mammalian cells. Cytotoxic extracts to insect cells (ID50's < or = 10 microg ml(-1)) but not to mammalian cells (ID50's > or = 10 microg ml(-1)) conformed to the criteria of the project and were considered 'lead' extracts for further investigation. 'Leads' were found in two of the Hypocrella species: H. discoidea, and H. tamurai and in three of the Aschersonia species: A. samoensis, A. badia, and A. tamurai. Bioassay-guided fractionation of the cell extract of the fungus A. samoensis BCC 1393 led to the identification of two known anthraquinone dimers, (+)rugulosin (1) and skyrin (2) which showed selective toxicity towards insect cells. (+)Rugulosin (1) and skyrin (2) exhibited strong cytotoxic activity against the insect cell line Sf9 with respective ID50 values of 1.2 and 9.6 microg ml(-1), but showed weak activity toward mammalian cells. This first report of (+)rugulosin (1) and skyrin (2) in A. samoensis is confirmed and demonstrated in another four strains of A. samoensis isolated in Thailand. The preferential cytotoxicity against Sf9 insect cells gives evidence that these insect-pathogenic fungi of the Hypocrella/Aschersonia group might be useful as an agent for pest control.

Aedes↗

Multiplication of tomato spotted wilt virus in primary cell cultures derived from two thrips species.

Primary cell cultures prepared from embryos of the thrips species Frankliniella occidentalis and Thrips tabaci were tested for their potential to support replication of tomato spotted wilt virus (TSWV). Using polyclonal antibodies against the viral nucleocapsid protein (N) and indirect immunofluorescent staining, discrete spots with strong signals were observed in the cytoplasm at 48 h post-inoculation in the cell cultures of a F. occidentalis, and a T. tabaci population which failed to transmit the virus. The infection was found in approximately 40% of the monolayer cells. Using antibodies against a nonstructural protein (NSs) of TSWV, uniform and more diffused staining was observed throughout the cytoplasm of these cells, underlying active genome replication. The NSs protein accumulated slower than the N protein in the cells of both thrips species. No multiplication of TSWV was observed in a heterologous insect cell line, i.e. from Spodoptera frugiperda, suggesting the existence of specific host factors in the thrips-derived cells.

Animals↗

Baculovirus inhibitor of apoptosis functions at or upstream of the apoptotic suppressor P35 to prevent programmed cell death.

Members of the inhibitor of apoptosis (iap) gene family prevent programmed cell death induced by multiple signals in diverse organisms, suggesting that they act at a conserved step in the apoptotic pathway. To investigate the molecular mechanism of iap function, we expressed epitope-tagged Op-iap, the prototype viral iap from Orgyia pseudotsugata nuclear polyhedrosis virus, by using novel baculovirus recombinants and stably transfected insect cell lines. Epitope-tagged Op-iap blocked both virus- and UV radiation-induced apoptosis. With or without apoptotic stimuli, Op-IAP protein (31 kDa) cofractionated with cellular membranes and the cytosol, suggesting a cytoplasmic site of action. To identify the step(s) at which Op-iap blocks apoptosis, we monitored the effect of Op-iap expression on in vivo activation of the insect CED-3/ICE death proteases (caspases). Op-iap prevented in vivo caspase-mediated cleavage of the baculovirus substrate inhibitor P35 and blocked caspase activity upon viral infection or UV irradiation. However, unlike the stoichiometric inhibitor P35, Op-IAP failed to affect activated caspase as determined by in vitro protease assays. These findings provide the first biochemical evidence that Op-iap blocks activation of the host caspase or inhibits its activity by a mechanism distinct from P35. Moreover, as suggested by the capacity of Op-iap to block apoptosis induced by diverse signals, including virus infection and UV radiation, iap functions at a central point at or upstream from steps involving the death proteases.

Animals↗

Cell growth arrest by nucleotides, nucleosides and bases as a tool for improved production of recombinant proteins.

Arresting cell growth and thus decreasing cell division potentially lessens the chance for genetic drift in the cell population; this would be of utmost importance for the consistent production of biopharmaceuticals during long periods. The drawback of the addition of well-known synchronizing agents, such as chemotherapeutics, is that they cause a disproportionate accumulation of cellular constituents, leading to cell death. The use of compounds that are naturally synthesized by the cell, as is the case of nucleotides, nucleosides, and bases (Nt/Ns/B), is shown in this work to be a promising tool. The addition of purines and pyrimidines was tested using a CHO cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP). From the chemical alternatives tested, AMP was the most promising compound for protein production improvement; it reduced cell growth and maintained the culture with high cell viability for long periods, while increasing SEAP specific productivity 3-fold. The use of CHO and BHK mammalian cells producing Factor VII and the use of a insect cell line (Sf9) showed that the effect of AMP addition seems to be independent of the r-protein and cell line. With the addition of AMP, accumulation of cells at the S phase was accompanied by an increase of the protein specific productivity. Addition of known synchronizing drugs (aphidicolin and doxorubicin) and application of environmental cell growth arrest strategies (depletion of nutrients and byproduct accumulation) showed also to effectively arrest CHO cell growth. A careful look onto cell cycle distribution in the different scenarios created, shows whether it is important to consider r-protein expression dependency upon cell cycle in process optimization and operation strategies.

Alkaline Phosphatase↗

In vitro expression of bovine opsin using recombinant baculovirus: the role of glutamic acid (134) in opsin biosynthesis and glycosylation.

Expression levels of functional bovine opsin in the insect cell line IPLB-Sf9 using recombinant baculovirus were shown not to depend on the use of novel transfer vectors (pAcRP23, pAcDZ1) that were reported to improve biosynthesis levels of other proteins in this system. A production of 5 micrograms opsin per 10(6) cells (approx. 1.5% of total cell protein) was achieved by batch fermentation of infected cells in spinner cultures. Infection of the cells in the presence of the glycosyltransferase inhibitor tunicamycin led to the synthesis of the complete protein, which, however, now migrated with a substantially lower Mr. This demonstrates that opsin in insect cells also undergoes N-linked glycosylation and allowed partial purification (10-fold) of the resulting rhodopsin by affinity chromatography over Concanavalin A-Sepharose. Through site-directed mutagenesis (rhod)opsin mutants have been obtained allowing dissection of functional domains of opsin. Amino acid substitutions that involved Glu-134 and/or Arg-135 affected the normal biosynthetic process leading in part to nonglycosylated, to a small extent even incomplete, protein. A number of mutations, that involve other charged residues within the second and third transmembrane domain of the protein, had no effect on the biosynthetic processing of the protein. We therefore suggest that the charge-pair Glu-134-Arg-135 is part of an important internal signal sequence and that alterations in this region may result in incorrect membrane translocation and/or folding of the protein.

Animals↗

Beauvericin cytotoxicity to the invertebrate cell line SF-9.

The cyclic hexadepsipeptide beauvericin, initially known as a secondary metabolite produced by the entomopathogenic fungus Beauveria bassiana and toxic to Artemia salina larvae, has been more recently recognized as an important mycotoxin synthesized by a number of Fusarium strains, which parasite maize, wheat and rice. Therefore, this mycotoxin may enter the food chain, causing yet unknown effects to the health of both domestic animals and humans. The cytotoxic effects of beauvericin on mammalian cells have been studied. We investigated the cytotoxicity of this compound in an in vitro invertebrate model, viz. the insect cell line SF-9 (immortalized pupal ovarian cells of the lepidopter Spodoptera frugiperda). Cultures of SF-9 cells in the stationary phase were exposed to beauvericin at concentrations ranging from 100 nM to 300 microM, for different periods of time (from 30' to 120 h). The effects on cell viability were assessed by the trypan blue exclusion method. After 4 h of incubation no significant decrease in cell viability was recorded in SF-9 cell cultures exposed to low concentrations of beauvericin, i.e. 100 nM and 300 nM. However, a slight decrease in viability (3.9%) was seen already in cells exposed to the mycotoxin at the 1 microM concentration. This effect became gradually more evident at higher concentrations (approximately equal to 28% at 30 microM, approximately equal to 50% at 100 microM, approximately equal to 68% at 300 microM). An even more pronounced reduction in cell viability was observed after a 24 h exposure. Under these conditions, 1 microM beauvericin caused an approx. 10% decrease in the number of viable cells, which became more significant at higher concentrations approximately equal to 23% at 3 microM, approximately equal to 47% at 10 microM, approximately equal to 65% at 30 microM, approximately equal to 90% at 100 microM, approximately equal to 99% at 300 microM). Therefore, the 50% cytotoxic concentrations (CC50) at 4 h and 24 h could be estimated as 85 microM and 10 microM, respectively. In time-course experiments, no effect of beauvericin (30 microM) on cell viability could be seen after exposure for periods of time as long as 30', 1 h and 2 h, respectively. In contrast, when SF-9 cells were exposed to the mycotoxin for longer periods of time, from 8 h to 120 h, we recorded a strong cytotoxic effect already in the low micromolar concentration range. Thus, the CC50 after both 72 h and 120 h exposure times was assessed as 2.5 microM. Higher concentrations caused a virtually 100% cell death. The data collected suggest that beauvericin exerts a substantial dose- and time-dependent cytotoxic effect on invertebrate cells, comparable to the effects described in mammalian cells.

Animals↗

Extrachromosomal transposition of the transposable element Minos in embryos of the cricket Gryllus bimaculatus.

Effective germline transformation of insects has been shown to depend on the right choice of transposon system and selection marker. In this study the promoter region of a Gryllus cytoplasmic actin (GbA3/4) gene was isolated and characterized, and was used to drive the expression of Minos transposase in embryos of the cricket Gryllus bimaculatus. Active Minos transposase was produced in these embryos as monitored through established transposon excision and interplasmid transposition assays. In contrast, Drosophila melanogaster hsp70 promoter, previously used to express Minos transposase in a number of insect species and insect cell lines, failed to produce any detectable Minos transposase activity, as recorded by using the very sensitive transposon excision assay. In addition, the GbA3/4 promoter was found to drive expression of enhanced green fluorescent protein (eGFP) predominantly in vitellophages of the developing Gryllus eggs when a plasmid carrying a GbA3/4 promoter-eGFP fusion gene was transiently injected into embryos. These results strongly support the use of Minos transposons marked with the GbA3/4 promoter-eGFP for the genetic transformation of this emerging model insect species.

Actins↗

Overexpression, purification, and characterization of third component of complement.

The third component of complement (C3) plays a critical role in both pathways of complement activation by interacting with numerous other complement proteins. To elucidate the molecular features of C3 that relate to the functional activities of the molecule, we expressed the cDNA of human complement component C3 in cultured insect cells using a baculovirus expression vector system derived from the baculovirus Autographa california nuclear polyhedrosis virus (AcNPV). The expression of C3 was controlled by the promoter of the polyhedrin gene and, when recombinant baculovirus infected insect cells were cultured in serum-free medium, C3 was detected at a level of 10 micrograms/ml of culture medium. Characterization of the recombinant C3 (rC3) by SDS-PAGE revealed that the C3 gene product was translated as a 188 kDa protein comprised of two chains of 115 kDa and 73 kDa analogous to the alpha and beta chains of serum-derived human C3 (sC3). An analysis of the glycosylation pattern of purified rC3 revealed that, whereas both the alpha and beta chains were glycosylated as in sC3, the proC3 moiety of rC3 also was glycosylated. When rC3 was produced in the High Five cell line of insect cells and evaluated for reactivity with a panel of anti-C3 monoclonal antibodies (MoAb), the results suggested that the conformation of the baculovirus expressed C3 was similar to that of native C3. When the rC3 was purified by anion exchange column chromatography, it was able to react with several C3-binding proteins (CR1, P and H), reconstitute C3-deficient serum and support the activation of both complement pathways thus demonstrating that a baculovirus-expressed C3 can participate in the formation of and can be cleaved by both the classical and alternative pathway convertases. Incubation of rC3 with factor I and H revealed that both C3 and proC3 are susceptible to cleavage by factor I.

Animals↗

Expression of a pheromone-binding protein in insect cells using a baculovirus vector.

A cDNA encoding a pheromone-binding protein from the male silkmoth Antheraea pernyi has been integrated into the genome of the Autographa californica multiple nuclear polyhydrosis virus such that the transcription was under the control of the strong polyhedrin promoter. Recombinant pheromone-binding protein was expressed in a baculovirus-infected insect cell line (Sf9) and secreted from the cells into the culture medium. Using a two-step protocol, recombinant pheromone-binding protein has been isolated and purified to homogeneity. Pheromone binding of recombinant protein has been demonstrated using a tritiated analog of (E,Z)-6,11-hexadecadienyl acetate.

Affinity Labels↗

Synchronization of Aedes albopictus mosquito cells using hydroxyurea.

We have established conditions for use of hydroxyurea, a reversible inhibitor of DNA synthesis, to synchronize the division cycle of a continuous cell line from the mosquito, Aedes albopictus. In the range of 0.15-0.25 mM hydroxyurea, an 18 h treatment, followed by removal of the drug, results in effective synchronization. When combined with the partial synchronization that occurs within 10 h of dilution and plating, more than 80% of cells treated with hydroxyurea could be recovered in the synthesis (S) phase of the cell cycle during the 4 h period after removal of the drug. The degree of synchrony was enhanced when cells were exposed to two consecutive hydroxyurea treatments spaced 10 h apart. Synchronized cells expressed maximal levels of a reporter gene when transfected immediately after removal of hydroxyurea. This is the first description of effective chemical synchronization of an insect cell line using hydroxyurea.

Aedes↗

Molecular cloning and expression of feline CD3epsilon.

The cDNA of feline CD3epsilon, one of the T-cell receptor components, was cloned from a feline T-lymphoblastoid cell line (MYA-1 cells) and peripheral blood mononuclear cells and thymocytes of cats by polymerase chain reaction. Sequencing analysis revealed that the open reading frame of feline CD3epsilon consists of 606 base pairs encoding a predicted molecular mass of 25 kDa transmembrane protein which lacks N-glycosylation site. Comparison of the predicted amino acid sequence of feline CD3epsilon with those of other mammalians' homologues revealed that a relatively low homology was present in the extracellular domain. However, the cytoplasmic domain contained several characteristic motifs highly conserved across the species. These motifs were known to be important for signal transduction upon T-cell activation or endoplasmic reticulum retention. In addition, the feline CD3epsilon protein was expressed in an insect cell line (Sf9) by a baculovirus expression system. The expression was confirmed by indirect immunofluorescence assay and immunoblotting analysis using an anti-human CD3epsilon polyclonal antibody. These results will provide additional information for understanding the feline immune system.

Amino Acid Sequence↗

Update on viral accommodation, a model for host-viral interaction in shrimp and other arthropods.

Comparatively little published information is available on the mechanistic response of shrimp and other arthropods to viral pathogens. Much of the literature has been focused on the use of viruses for biological control of insect pests or disease vectors and on the use of baculoviruses as a means of heterologous protein expression in insect cell lines. The situation changed dramatically with the rapid global increase in cultivation of penaeid shrimp and the massive farm losses that have occurred due to viral pathogens. Urgency to solve these problems has led to a closer examination of the shrimp response to viral pathogens in the hope of finding new methods of disease control. Field observations and results of laboratory experiments in the past decade indicate that shrimp may be capable of a specific, adaptive response to viral pathogens that cannot be explained by current knowledge and understanding of their cellular and humoral defenses. Hallmarks of this response are specific tolerance to single and multiple viral infections without gross or histological signs of disease, a phenomenon common to crustaceans and insects. The concept of viral accommodation was introduced in 1998 as a simple testable hypothesis to explain these phenomena. Key elements of the hypothesis were an unknown mechanism for specific memory of pathogens and the role of this memory in dampening viral triggered apoptosis. Recent field and research results have supported predictions of the viral accommodation hypothesis and suggest that memory may be provided by the viral pathogens themselves in persistent infections that result in reduced severity of disease. The well-known phenomenon of defective interfering viral particles may play an important role in this process, but it cannot explain cross protection that has recently been described for heterologous viral infections. The major conclusion is that homologous and heterologous reduction in disease severity resulting from persistent viral infections (i.e., accommodated viral infections) may be a key process that has evolved from host viral interaction in the arthropod line.

Animals↗

Functional expression of insecticide-resistant GABA receptors from the mosquito Aedes aegypti.

We are interested in cloning insecticide resistance genes from vector mosquitos for use as selectable markers in their genetic transformation. As a first step towards this goal, we here report the functional homomultimeric expression of a gamma-aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin (Rdl), from the yellow fever mosquito Aedes aegypti in baculovirus-infected insect cell lines. Replacement of alanine296 with a serine leads to approximately 100-fold insensitivity to picrotoxin as previously observed in Drosophila. This shows not only that the mosquito GABA receptor cDNA is functional but also that it can be simply mutated to resistance. Strategies for incorporation of this cDNA into a minigene for the genetic transformation of mosquitoes are discussed.

Aedes↗

Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein.

The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.

Alkaline Phosphatase↗