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At least 811 records · Page 45Linked to original sources
PubMed1995

Production of vampire bat plasminogen activator DSPA alpha 1 in CHO and insect cells.

Salivary plasminogen activator from the vampire bat Desmodus rotundus (DSPA alpha 1) is a promising new thrombolytic agent. Continuous growth of a stably transfected, methotrexate amplified, dhfr- CHO cell line yields up to 60 mg l-1 of DSPA alpha 1. Utilizing an engineered baculovirus 10 mg l-1 were produced in batches of Sf9 insect cells. Recombinant DSPA alpha 1 is purified from both sources using a one-step purification protocol. Although differences in glycosylation were detected, enzymatic activity and fibrin cofactor dependency are unaffected when DSPA alpha 1 derived from the two expression systems is compared.

Animals↗
PubMed1996

Isolation and characterization of a dihydrofolate reductase gene mutation in methotrexate-resistant Drosophila cells.

Stepwise increases in methotrexate (MTX) concentration over a 4-year period led to the selection of a highly drug-resistant (2 x 10(-4) M MTX) Drosophila cell line. Uptake experiments with [3H]MTX showed a slightly lower level of intracellular MTX in the resistant S3Mtx cells than in the susceptible S3 parental cell line. Southern blot analysis demonstrated that the gene for the MTX target, dihydrofolate reductase (DHFR), was not significantly amplified in the resistant line. To determine the molecular basis for resistance, the DHFR cDNA sequence was amplified by polymerase chain reaction from both the resistant and susceptible cells. Sequence comparison revealed a single T to A base change at nucleotide 89, which resulted in the substitution of Gln for Leu at residue 30 in S3Mtx cells. Expression and purification of the wild-type and mutant DHFR from E. coli cells showed that the S3Mtx enzyme had a reduced binding affinity for the antifolates, MTX and trimethoprim, with 15-fold higher K[d] and K[i] values than those from the wild-type enzyme. Molecular modeling confirmed that the replacement of the hydrophobic Leu by the more polar Gln was in the substrate binding site and thus would decrease the binding of MTX. These results suggest that the high level of MTX resistance in the selected cell line can be attributed to the mutation in the DHFR gene and also provides a model for pesticide resistance in insects.

Animals↗
PubMed1994

First isolations of arboviruses from phlebotomine sand flies in West Africa.

For the first time in West Africa, arboviruses were isolated from phlebotomine sand fly pools. One strain of Chandipura virus (a Vesiculovirus), four strains of Saboya virus (a Flavivirus), and one strain of a not yet identified virus were isolated. Three hundred twenty-two pools were established from a population of 33,917 sand flies caught in CO2 light traps in the Ferlo Sahelian region of Senegal from November 1991 to December 1992. This is the first isolation of Chandipura virus from any arthropod in Africa. Saboya virus has already been isolated from small rodents in Senegal; thus, its transmission cycle probably involves rodentophilic sand flies. No strain of Rift Valley fever phlebovirus, which caused an epizootic in this region in 1987, was isolated. During the same time at the same site, 11 sand fly species were identified from 4,191 specimens caught on sticky traps, including Phlebotomus duboscqi, a leishmaniasis vector.

Animals↗
PubMed1993

Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation.

3T3 Cells↗
PubMed2005

Crystal lattice as biological phenotype for insect viruses.

Many insect viruses survive for long periods by occlusion within robust crystalline polyhedra composed primarily of a single polyhedrin protein. We show that two different virus families form polyhedra which, despite lack of sequence similarity in the virally encoded polyhedrin protein, have identical cell constants and a body-centered cubic lattice. It is almost inconceivable that this could have arisen by chance, suggesting that the crystal lattice has been preserved because it is particularly well-suited to its function of packaging and protecting viruses.

Animals↗
PubMed1989

Cell lines from Culicoides variipennis (Diptera: Ceratopogonidae) support replication of bluetongue virus.

Cell lines have been developed from 2-day-old embryos of the biting midge, Culicoides variipennis (Diptera: Ceratopogonidae). In North America C. variipennis is the primary insect vector of bluetongue virus (BTV), an orbivirus that causes disease of ruminants. The C. variipennis (CuVa) cells, grown in Schneider's Drosophila medium, consist primarily of a fibroblast-like cell type. CuVa cells are very hardy. They can grow over a wide range of temperature and pH and adapt to growth in minimal essential medium. BTV replicates to high titer (7.5-8.0 log10 50% tissue culture infectious doses/ml) in CuVa cells over a wide range of temperatures (19 degrees to 37 degrees C) without inducing any significant cytopathic effects. The highest BTV titers were obtained in CuVa cells grown at 25 degrees and 32 degrees C. Cells from C. variipennis can be useful for many diverse investigations.

Animals↗
PubMed2001

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides.

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.

Amino Acid Sequence↗
PubMed2005

Identification of mosquito sterol carrier protein-2 inhibitors.

A mosquito sterol carrier protein-2, AeSCP-2, has been shown to aid in the uptake of cholesterol in mosquito cells. The discovery of chemical inhibitors of AeSCP-2 is reported here. AeSCP-2 inhibitors (SCPIs) belong to several chemotypes of hydrophobic compounds. Those inhibitors competed with cholesterol for AeSCP-2, binding with relatively high binding affinities. In cultured insect cells, SCPIs reduced cholesterol uptake by as much as 30% at 1-5 microM concentrations. SCPIs were potent larvicides to the yellow fever mosquito, Aedes aegypti, and to the tobacco hornworm, Manduca sexta, with 50% lethal doses (LD50s) of 5-21 microM and 0.013-15 ng/mg diet, respectively. The results indicate that sterol carrier protein-2 has functional similarity in two different insect species.

Animals↗
PubMed2004

Synchronization of Plodia interpunctella lepidopteran cells and effects of 20-hydroxyecdysone.

We have investigated the molecular and cellular mechanisms involved in the control of insect cell cycle by 20-hydroxyecdysone (20E) using the IAL-PID2 cell line established from imaginal wing discs of Plodia interpunctella. We first defined conditions for use of hydroxyurea, a reversible inhibitor of DNA synthesis, in order to synchronize the IAL-PID2 cells in their division cycle. A high degree of synchrony was reached when cells were exposed to two consecutive hydroxyurea treatments at 1 mm for 36 h spaced 16 h apart. Under these conditions, flow cytometry analysis demonstrated that 20E at 10(-6) m induced an inhibition of cell growth by an arrest of 90% of the cells in G2/M phase. Using cDNA probes specifically designed from E75 and HR3 nuclear receptors of Plodia interpunctella, we showed that PiE75 and PHR3 were highly induced by 20E through S and G2 phases with maximal enhancement just before the G2/M arrest of cells. These findings suggest that PiE75 and PHR3 could be involved in a 20E-induced genetic cascade leading to G2/M arrest.

Amino Acid Sequence↗
PubMed2006

Growth and production of the dengue virus in C6/36 cells and identification of a laminin-binding protein as a candidate serotype 3 and 4 receptor protein.

OBJECTIVES: Although dengue is one of the most common mosquito-borne viral diseases, few studies have investigated the relationship between the dengue virus and mosquito cells, and this study sought to describe the binding and propagation of the dengue viruses in C6/36 cells. METHODS: The internalization and production of the dengue virus was assayed by standard plaque assay methodologies, while dengue virus receptor proteins were examined by a virus overlay protein-binding assay and candidate gene analysis coupled with virus inhibition studies. RESULTS: All four serotypes were internalized linearly, and de novo virus production occurred 14-19 h postinfection. Virus overlay protein-binding assay identified a band of 50 kDa for dengue serotypes 2, 3 and 4 which comigrated with a protein that reacts with antibodies directed against the human 37/67-kDa high-affinity laminin receptor. Both antibodies directed against the human 37/67-kDa high-affinity laminin receptor protein and soluble laminin inhibited the binding and internalization of serotypes 3 and 4, but not serotypes 1 and 2. CONCLUSIONS: The results suggest that multiple receptors may be used by the dengue virus to enter into insect cells, and that one of these proteins may be a laminin-binding protein.

Animals↗
PubMed1989

Insect virus: assays for toxic effects and transformation potential in mammalian cells.

The nuclear polyhedrosis virus of Autographa californica (AcNPV) was evaluated by using in vitro test systems for toxicity and transforming potential in mammalian cells. Mass cell cultures of CV-1 and WI38 cells appeared unaffected by AcNPV at a multiplicity of infection of 5. Human foreskin cells grew more slowly after inoculation but eventually produced healthy monolayers. The sensitivities of the inhibition of reproductive survivability assays were greater and demonstrated slight AcNPV toxicity to CV-1, WI38, and human foreskin cells. Toxicity was not ameliorated when gradient-purified or psoralen-inactivated virus was used, suggesting that the toxic component of the preparation is part of the virion or copurifies with it. AcNPV was not toxic to and did not transform BALB/c 3T3 cells or primary cell cultures derived from Syrian hamster embryo cells (SHE). Unlike the BALB/c 3T3 transformation assay, the SHE assay detected no spontaneous transformants. The SHE transformation assay can employ simian adenovirus 7 as a positive control. SHE are transformed by numerous viruses and so are useful in assessment protocols. This study suggests that in vitro assessment of viral pesticide toxicity should employ the inhibition of reproductive survivability assay and that transformation assessment is best done with the SHE-simian adenovirus 7 procedure.

Animals↗
PubMed1986

Bidirectional transcription from a solo long terminal repeat of the retrotransposon TED: symmetrical RNA start sites.

A single copy of the retrotransposon TED was found integrated within the DNA genome of the insect baculovirus, Autographa californica nuclear polyhedrosis virus. After excision of the element from the viral genome, a single long terminal repeat (LTR) remained behind. We have examined the effect of this solo TED LTR on the local pattern of viral transcription. Most prominent was the transcription of two sets of abundant RNAs; both originated within the LTR but extended in opposite directions into flanking viral genes. By promoting symmetric transcription of adjacent genes, the solo LTR has the capacity to activate or repress gene expression in two directions. Primer extension analysis demonstrated that the divergent LTR transcripts were initiated near the same point within a 22-base-pair sequence having hyphenated twofold symmetry. Analogous symmetries at the initiation sites of other retrotransposon LTRs, including copia and Ty, suggested that these sequences serve to establish the precise start for transcription.

Animals↗
PubMed1987

Identification of the N and NSS proteins coded by the ambisense S RNA of Punta Toro phlebovirus using monospecific antisera raised to baculovirus expressed N and NSS proteins.

An essentially complete DNA copy of the ambisense S RNA species of Punta Toro (PT) phlebovirus (T. Ihara, H. Akashi, and D.H.L. Bishop, 1984, Virology 136, 293-306) has been inserted in either orientation into Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the 5' coding region of the AcNPV polyhedrin gene (G.E. Smith, M.D. Summers, and M.J. Fraser, 1983, Mol. Cell. Biol. 3, 2156-2165). The two types of recombinant viruses were used to infect Spodoptera frugiperda cells and the expressed PT viral proteins characterized. Recombinant AcNPV having the S DNA in one orientation expressed PT virus N protein in amounts estimated to represent some 50% of the infected cell extracts, whereas recombinants with the S DNA in the other orientation expressed the putative PT virus NSS protein in lower quantities. Antisera that were monospecific with respect to each of the two PT proteins virus were raised in mice using the corresponding S. frugiperda infected cell extracts and were employed to identify N and NSS proteins in PT virus-infected Vero cells.

Animals↗
PubMed1975

hnRNA size and processing as related to different DNA content in two dipterans: Drosophila and Aedes.

The size of hnRNA transcripts and the fraction of hnRNA that is converted to mRNA in cell lines of Drosophila melanogaster and Aedes albopictus are compared. Both insects belong to the order Diptera, but Aedes has a 5-6 fold larger genome than does Drosophila. The Aedes line produces significantly (2-2.5 fold) larger hnRNA than does the Drosophila line, even though the two cell lines grow under similar conditions and produce mRNA of the same size and sequence complexity. These data suggest that within a given taxonomic order, the size of hnRNA increases with increasing genome size. The fraction of hnRNA converted to mRNA [cytoplasmic poly(A)+ RNA] has been measured for the two cell types by comparing initial rates of labeling of hnRNA with initial rates of appearance of labeled mRNA in the cytoplasm. While 20of the Drosophila hnRNA is converted to mRNA, only 3.3% of the Aedes hn %RNA is converted to mrRNA. The poly(A) content of the hnRNA from the two species is also different; Drosophila hnRNA has approximately three times as much poly(A) as does Aedes hnRNA. The data show-at least for these two species-that the average amount of hnRNA transcribed relative to the amount of mRNA formed increases as genome size increases. The data are consistent with the interpretation that more DNA is transcribed into hnRNA in Aedes, the organism with the larger genome, than in Drosophila.

Adenine Nucleotides↗
PubMed1990

A baculovirus polyhedron envelope protein: immunogold localization in infected cells and mature polyhedra.

A polyclonal antiserum against a trpE fusion protein containing the complete open reading frame of the polyhedron envelope (PE) protein from the nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was used for immunogold staining and electron microscopic examination of polyhedra, isolated polyhedron envelopes, and infected insect cells at selected times postinfection. The antiserum specifically stained the peripheral envelope of mature polyhedra and also stained the envelope structure which remained after polyhedra were dissolved in dilute alkaline solutions. In OpMNPV-infected Lymantria dispar cells, the PE protein was detected by 48 hr postinfection (hr p.i.) but specific localization and staining of developing polyhedra were not evident. However, by 72 hr p.i. substantial and preferential staining of the periphery of developing polyhedra was evident even though a distinct polyhedron envelope was not yet observed. In addition, the periphery of fibrillar structures was stained by the PE antiserum. By 96 hr p.i., mature envelopes surrounded polyhedra and these polyhedron envelopes were stained with the PE antibody. The progression of PE protein staining during polyhedron morphogenesis indicates that the PE protein accumulates and becomes associated with developing polyhedra in the nucleus between 48 and 72 hr p.i. Very late in infection the mature polyhedron envelope forms on the polyhedron surface. The apparent affinity of the PE protein for the surface of maturing polyhedra suggests that it may be a major component of the polyhedron envelope or may form the matrix for the deposition of other components which contribute to the mature envelope. Immunogold staining and protease digestion experiments indicate that protein is an essential component of the polyhedron envelope.

Animals↗
PubMed2004

Morphological and molecular effects of 20-hydroxyecdysone and its agonist tebufenozide on CF-203, a midgut-derived cell line from the spruce budworm, Choristoneura fumiferana.

The morphological and molecular responses of a midgut-derived cell line of the spruce budworm, Choristoneura fumiferana, to 20-hydroxyecdysone (20E) and the nonsteroidal ecdysone agonist, tebufenozide (RH-5992), were investigated. The cells responded to these compounds by clumping, generating filamentous extensions, increased mortality and expression of the transcription factor, Choristoneura hormone receptor 3 (CHR3). This cell line can be used as a model system to study the mode of action of ecdysone and its agonists. With subsequent passaging in ecdysteroid-containing medium, the degree of clumping increased and the clumping could not be reversed by subculturing in ecdysteroid-free medium. Cell numbers of the adapted cell lines in 20E and RH-5992 containing media were not significantly decreased, compared to the control, but both cell lines accumulated less (14)C-labeled RH-5992 and lost the capability of expressing CHR3 in response to these compounds. Taken together, the cell lines appeared to develop a mechanism to adapt to the toxic effects of these compounds. Arch. Insect Biochem. Physiol. 55:68-78, 2004.

Animals↗
PubMed1993

High level expression and phosphorylation of hepatitis B virus polymerase in insect cells with recombinant baculoviruses.

The hepatitis B virus polymerase open reading frame, as well as various subdomains of polymerase, was expressed in insect cells using the recombinant baculovirus expression system. Full-length polymerase was expressed at very low levels in a Spodoptera frugiperda cell line, the amino-terminal domain of polymerase was expressed at high levels, and other constructs were expressed at intermediate levels. Infections of a Trichoplusia ni cell line with the same recombinant baculoviruses resulted in high levels of protein production for all polymerase constructs. Each of the polymerase polypeptides was phosphorylated in insect cells. Since polypeptides with non-overlapping sequences were phosphorylated, polymerase must be phosphorylated at a minimum of two sites.

Animals↗