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PubMedDate not supplied

Production of rotenone-inactivating substance(s) by rotenone-resistant insect cell line.

In a previous paper, we showed that a cell line derived from hemocytes of the cabbage armyworm, Mamestra brassicae (R-cell) was a thousand times as resistant to rotenone as that from ovaries of the same species (S-cell). The S-cells were killed by rotenone at concentrations higher than 10(-9) M, while R-cells at higher than 10(-6) M. When the R-cells were cultured in the medium containing 10(-9) M rotenone, the ability of rotenone to kill the S-cells was lost in the used medium. Also, when rotenone was incubated in the medium conditioned with R-cells, it lost its cell killing activity. It became evident that rotenone-inactivating substance(s) were produced in cells and stored in water-soluble form or liberated into the medium. The substance(s) were inactivated by heat treatment.

Animals↗
PubMed2003

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Animals↗
PubMed1998

N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines.

The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5b 1-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of alpha 1-3-linked mannose.

Alkaline Phosphatase↗
PubMed1995

Endogenous K+/H+ exchange activity in the Sf9 insect cell line.

The fluorescent pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to investigate changes in the intracellular pH (pHi) of individual Sf9 cells in response to changes in the composition of the external medium. Under standard conditions, the resting pHi was 0.2-0.3 unit lower than the extracellular pH (6.5). The extracellular concentration of K+ had a major influence on pHi. Removal of K+ from the medium resulted in a rapid but reversible acidification of the cells. The buffer capacity of the cells was a U-shaped function of pHi with a minimum at about pHi = 6.2. In the presence of K+, a change in the pH of the medium was followed by an equivalent change in pHi. In its absence, however, changes in the external pH had little effect on the pH of the cells. Following removal of K+ from the medium, the cells realkalinized at an initial rate which increased with increasing concentration of added K+. This cation was about 30 times more effective in promoting realkalinization of the cells than Li+ Na+, Rb+, and Cs+. The apparent Km for K(+)-dependent H+ efflux was about 12 mM and was slightly modulated by extracellular pH. These results strongly suggest that, in Sf9 cells, a K+/H+ antiporter plays a key role in the movement of protons across the cell membrane.

Animals↗
PubMed1994

Glycosylation of recombinant prorenin in insect cells: the insect cell line Sf9 does not express the mannose 6-phosphate recognition signal.

Sf9 cells infected with a recombinant baculovirus containing the gene for human prorenin were cultured in the presence of [3H]mannose. In vivo labeled prorenin was isolated by immunoprecipitation from the culture medium and digested with Pronase. The oligosaccharide structures on the resulting glycopeptides were analyzed by a combination of lectin, ion-exchange, paper, and high-pressure liquid chromatography. Of the N-linked oligosaccharides isolated from the Sf9-produced prorenin, 98% were of a truncated (trimannosyl) high-mannose type, approximately two-thirds of which contained a fucose residue linked to the reducing N-acetylglucosamine. The remaining 2% constituted a mixture of high-mannose-type structures containing six, seven, or eight mannose residues; none of these structures were core-fucosylated. None of the oligosaccharide structures recovered from recombinant prorenin synthesized by Sf9 cells were phosphorylated or contained any other form of charge. Furthermore, assays for UDP-GlcNAc-lysosomal-enzyme N-acetylglucosamine phosphotransferase demonstrated no activity above background in lysates prepared from Sf9 cells. Blotting of Sf9 cell lysates with an 125I-labeled, soluble form of the cation-independent mannose 6-phosphate receptor failed to detect any proteins carrying the mannose 6-phosphate recognition signal. Taken together, the data suggest that Sf9 cells do not synthesize high-mannose-type oligosaccharides containing mannose 6-phosphate, and consequently it appears unlikely that these cells utilize the mannose 6-phosphate receptor mediated pathway for targeting of lysosomal enzymes.

Animals↗
PubMedDate not supplied

Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines.

Recombinant Autographa california baculoviruses expressing genes for pseudorabies virus glycoprotein (gp50T), human plasminogen (HPg), and beta-galactosidase (beta-gal) were used to infect 23 cell lines or strains. The objectives were to compare amounts of recombinant proteins expressed in the cell lines, compare yields from clones and parent lines, investigate the effects of long-term culture in serum-free medium on production, and determine if some lines yield gp50T with different glycosylation patterns. For HPg, IZD-MB0503 had the highest yield and four other lines (IPLB-TN-R2, IPLB-SF-1254, IPLB-LdEIta, and CM-1) had levels above that of SF-9 cells. For gp50T, four lines (IPLB-HvT1, IPLB-SF21AE, IPLB-SF21AE-15, and IPLB-SF-1254) had higher amounts than SF-9 cells. Some lines yielded gp50T with molecular mass about 1000 daltons larger than that from SF-9 cells, which suggests increased oligosaccharide processing. Equally high levels of beta-gal were expressed in three lines (SF-9, IZD-MB0503, and BCIRL-PX2-HNV3). The major conclusion is that no single cell line produced highest yields for all three recombinant proteins. Four lines were cultured in serum-free medium for 31-34 passages and then infected with the three recombinant viruses. For most cell line-recombinant combinations, the yields in serum-free medium were equal to or better than those in serum-supplemented medium. Medium composition had a much stronger effect on foreign gene expression than on susceptibility of cells to wild-type virus.

Animals↗
PubMedDate not supplied

Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture.

Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPLBeta-Sf21-AE) and Trichoplusia ni (Tn 5Beta-1-4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150-160 rpm were necessary for maximum growth of suspended Tn 5Beta-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 x 10(5) cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for beta-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Beta-1-4 cells are 2-4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of beta-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5Beta-1-4 cells produced 2.6-4.4 and 2.7-3 times more beta-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the beta-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of beta-galactosidase by Tn 5Beta-1-4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production.

Alkaline Phosphatase↗
PubMed2000

Transfection of Lymantria dispar insect cell lines.

Lepidopteran cell lines derived from the gypsy moth, Lymantria dispar, have not been widely used in protein expression studies or systems because they are weakly adherent, have specific growth requirements and characteristics, and are generally difficult to transfect. Using lipid-mediated transfection of a reporter plasmid, we modify the standard method for transfection of L. dispar-derived embryonic cell lines IPLB-LdEp and -LdEIta, obtaining transfection efficiencies of 34% and 30%, respectively, as determined by image analysis assays. Using the standard lipid-mediated method, we obtain transfection efficiencies for L. dispar-derived cell line IPLB-Ld652Y of at least 40% with high mean expression levels, indicating the IPLB-Ld652Y cell line may be a superior choice for expression studies or systems requiring L. dispar-derived cells.

Animals↗
PubMed2003

Induction of apoptosis in an insect cell line, IPLB-Ld652Y, infected with nucleopolyhedroviruses.

Ld652Y cells derived from the gypsy moth, Lymantria dispar, were infected with seven different nucleopolyhedroviruses (NPVs) including those from Autographa californica, Bombyx mori (BmNPV), Hyphantria cunea (HycuNPV), Spodoptera exigua (SeMNPV), L. dispar, Orgyia pseudotsugata (OpMNPV) and Spodoptera litura (SpltMNPV). The results showed that Ld652Y cells infected with BmNPV, HycuNPV, SeMNPV, OpMNPV and SpltMNPV underwent apoptosis, displaying apoptotic bodies, characteristic DNA fragmentation and increased caspase-3-like protease activity; HycuNPV induced the most severe apoptosis. In HycuNPV-infected Ld652Y cells, a considerable amount of viral DNA was synthesized although there was no detectable yield of budded virions and polyhedrin. Northern blot and immunoblot analyses revealed that HycuNPV inhibitor of apoptosis 3 (IAP3), which has been shown to function in Sf9 cells, was expressed in HycuNPV-infected Ld652Y cells at a level higher than or comparable with that in HycuNPV-infected SpIm cells, which produced a high titre of progeny virions without any apoptotic response. These results imply that the relative ease of apoptosis induction in NPV-infected Ld652Y cells is largely dependent on inherent cellular properties rather than functions of the respective NPVs, and indicate that the defect in progeny virion production is not merely due to the virus-induced apoptosis in HycuNPV-infected Ld652Y cells.

Animals↗