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PubMed1984

Characterization of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line CHE).

The monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K+] through both a furosemide-inhibitable and a vanadate-inhibitable transport system. Intracellular exchangeable [Na+] was slightly lower than the extracellular [Na+] and was maintained at this level through a vanadate-sensitive transport system. Na+ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide-sensitive Na+ uptake when compared with furosemide-sensitive K+ uptake indicated that these cations are not cotransported. 4,4'-Diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) inhibited Na+, K+, and Cl- uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na+, K+, Cl-, H+ and HCO3-fluxes are regulated in these cells.

Animals↗
PubMed2002

Molecular cloning and induction of nuclear receptors from insect cell lines.

Fragments of EcR and USP were cloned from two insect cell lines, Sf21 and High Five cells (derived respectively from Spodoptera frugiperda and Trichoplusia ni), using a PCR-based approach employing degenerate primers designed on the basis of conserved regions of nuclear receptors, together with 5'- and 3'-RACE. An additional orphan nuclear receptor, HR4 fragment, was cloned from High Five cells. Comparison of these fragments with Manduca sexta counterparts showed that the cloned SfEcR [ecdysone receptor (EcR) from Sf21 cells] had high similarity to MsEcR-B1, whereas the cloned SfUSP [ultraspiracle (USP) from Sf21 cells] and TnUSP (USP from High Five cells) matched more closely to MsUSP-2 than to MsUSP-1. The TnHR4 showed most similarity to a recently cloned Bombyx mori GRF. While EcR and USP were constitutively expressed in both cell lines, HR4 was barely detectable by Northern blot analysis in High Five cells. Treatment with 20-hydroxyecdysone (20E) and agonist RH-5992 enhanced transcription of EcR in both cell lines, while the transcription of USP was suppressed in High Five cells. Such suppressed USP transcription was not observed in Sf21 cells. Transcription of TnEcR could also be enhanced by ecdysone and 3-dehydroecdysone, whereas transcription of SfEcR was unchanged with these two ecdysteroid compounds. Induction of HR4 transcription was also observed with 20E, RH-5992, ecdysone and 3-dehydroecdysone. The protein synthesis inhibitor, cycloheximide, superinduced expression of EcR and HR4 and restored the 20E/RH-5992-suppressed expression of TnUSP in the cells. Northern blot analysis also revealed that PCR, using degenerate USP primers, was able to amplify some other orphan nuclear receptors and their expression was inducible by 20E and RH-5992 and some of them were superinducible by cycloheximide.

Amino Acid Sequence↗
PubMed1997

Engineering the folding pathway of insect cells: generation of a stably transformed insect cell line showing improved folding of a recombinant membrane protein.

The baculovirus-insect cell expression system has proven to be a valuable tool for the high level production of a multitude of recombinant proteins. However, production of membrane proteins in infected insect cells is often hampered by incorrect folding and processing which results in the accumulation of non-functional protein. Here, we report the construction of a Sf9 insect cell line stably transformed with the ninaA gene from D. melanogaster (Sfn cell line). The ninaA protein is a membrane bound cyclophilin which acts as a peptidyl-prolyl cis/trans isomerase during the folding process of rhodopsin 1 in D. melanogaster rhabdomere. Engineered Sfn insect cells infected with a recombinant baculovirus bearing the human dopamine transporter gene under the control of the polyhedrin promoter showed a > or = 5 times enhanced uptake of [3H]dopamine in comparison to similarly infected Sf9 cells. This increase in specific transport activity was not due to an altered Km value in the Sfn cell line. The uptake in infected Sfn cells was blocked by the peptidyl-prolyl cis/trans isomerase inhibitor cyclosporin A which had no effect on infected Sf9 cells. From these results we conclude that the prolyl-cis/trans isomerase activity of the ninaA in the stably transformed Sfn cell line was responsible, directly or indirectly, for the improved folding of the heterologously produced human dopamine transporter.

Animals↗
PubMed1993

Partial characterization of protein kinase C from an insect cell line.

The characteristics of protein kinase C (EC 2.7.1.37) from an insect cell line (Choristoneura fumiferana) have been described. DEAE-cellulose chromatography produced a major peak of activity which eluted at 0.04-0.055 M NaCl. The enzyme was sensitive to phosphatidylserine in the presence of calcium. Phorbol 12-myristate 13-acetate (PMA) in nanomolar concentrations stimulated protein kinase C activity 8-fold over basal levels and reduced the enzymes requirement for Ca2+. The enzyme had a Ka of 10 nM for PMA. Diacylglycerols tested included diolein, dilinolein, diarachidonin, oleoyl-acetyl-glycerol, dioctonoyl-sn-glycerol, dipalmitin and distearin. A 2.5- to 3-fold activation was obtained in the presence of 26 microM diolein, 40 microM oleoyl-acetyl-glycerol and 46 microM dioctonoyl-sn-glycerol. The enzyme activity was sensitive to the inhibitor H-7 and 50% inhibition was achieved at a concentration of 52 microM H-7. Phosphatidylinositol enhanced enzyme activity in the absence of phosphatidylserine but phosphatidylethanolamine had no effect.

Animals↗
PubMed1988

Modulation of octopamine-mediated production of cyclic AMP by phorbol-ester-sensitive protein kinase C in an insect cell line.

The presence of protein kinase C (EC 2.7.1.37) in an insect cell line has been demonstrated. Phorbol 12-myristate 13-acetate (PMA), in micromolar concentrations, activated protein kinase C with a translocation of the enzyme from the cytosol to the particulate fraction. Cyclic AMP production in the presence of PMA, octopamine and a combination of both increased in a dose-dependent and time-dependent fashion. The biologically inactive 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity or on octopamine-mediated cyclic AMP production. Pretreatment of the cells with pertussis toxin had no effect on the response of cells to octopamine or PMA. However, pretreatment with cholera toxin resulted in increased cyclic AMP production which was further enhanced when both cholera toxin and PMA were used in combination. Our data indicate that the octopamine-mediated cyclic AMP production is modulated by protein kinase C.

Animals↗
PubMed1986

[Infection of insect cell lines by the HIV virus, an agent of AIDS, and a demonstration of insects of African origin infected by this virus].

The etiological agent of AIDS known as HIV has been shown to bind on different insect cell lines including Drosophila, Mosquito, Ceratitis; and his DNA to be integrated in the cellular genome, but no expression of the viral genome was detected in those cells. None of the human lymphocytes markers is expressed at the surface of the insect cells. HIV proviral DNA has been also found in various insects from Central Africa (Zaïre and Central Africa Republic) but not similar insects from the Paris area. These data suggest that insects could be a reservoir or a vector for the AIDS virus.

Acquired Immunodeficiency Syndrome↗
PubMed1999

O-glycosylation potential of lepidopteran insect cell lines.

The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line.

Animals↗
PubMedDate not supplied

[Identification of insect cell lines].

Identification of 10 cell lines of 6 insect species received by the cell culture collection of Molecular Biology Institute from different research institutions of the USSR was carried out by karyological analysis and determinations of the spectrum of isoforms of the following enzymes: glucose-6-phosphate dehydrogenase (G-6-PDH), lactate dehydrogenase (LDH), isocitrate dehydrogenase (ICDH), and superoxide dismutase (SOD). Nine out of the 10 cell lines examined were shown to be identical to those of gypsy moth. Possible ways of cell contamination with other cells in long-term cultivation and preparation of new lines were determined.

Animals↗
PubMedDate not supplied

Application of inter-simple sequence repeats to insect cell lines: identification at the clonal and tissue-specific level.

Inter-simple sequence repeat (ISSR) primers designed to anneal to microsatellites were used to obtain deoxyribonucleic acid (DNA) fingerprint profiles to distinguish among 16 established insect cell lines derived from an assortment of lepidopteran, dipteran, and coleopteran species. Three different levels of cell line comparison were made: (1) between parents and their clones, (2) among cell lines derived from different tissues from the same species, and (3) among cell lines derived from different insect species. Of the 16 repeat oligonucleotide primers used in this study, nine primers generated several unique markers to distinguish between parental cell lines and their clones. Four of the 16 primers also generated DNA profiles with a number of unique bands, enabling the distinction among cell lines derived from specific tissues from the same species. In addition, ISSR-generated DNA profiles provided the greatest number of unique markers to distinguish easily among insect cell lines derived from different species.

Animals↗
PubMed2003

Comparative susceptibilities of insect cell lines to infection by the occlusion-body derived phenotype of baculoviruses.

Twelve insect cell lines from six species were tested for susceptibility to baculovirus infection by occlusion-derived virus (ODV) phenotype through the use of a typical endpoint assay procedure. ODV from three nucleopolyhedroviruses were prepared by alkali treatment (sodium carbonate) of occlusion bodies (OBs) and the virus preparations were titered on various cell lines. More than a four-log difference was realized for each of theses viruses between the various cell lines. The TN368 line from Trichoplusia ni was only marginally susceptible to ODV from each virus, showing only 3-6 infectious units (IU) per million OBs while the gypsy moth line, LdEp was most susceptible, realizing more than 100,000 IU/million OBs. The other lines tested showed various levels of susceptibility between these two extremes and also varied between the three viruses tested. In additional tests, the ODV were treated with trypsin prior to application to the cells. With most cell lines, this treatment increased the infectivity of each virus by 2-10-fold. Exceptions to this trend included the gypsy moth LdEp line, on which the trypsinized ODV from two of the viruses were slightly less infectious than each virus without trypsin, and the TN-368 line, on which the trypsinized ODV was 5,000-75,000 times more infectious. The variable results of trypsinized virus on the different lines are probably due to the levels of endogenous protease activity in the various lines, but the mode of action of the trypsin has not been elucidated. Ultimately, the variable response of cell lines to ODV of different viruses, and the variable effects of trypsin on the ODV may lead to an improved understanding of the infection process of this virus phenotype as well as factors relating to baculovirus host range.

Animals↗
PubMed1999

Transformation of lepidopteran and coleopteran insect cell lines by Glyptapanteles indiensis polydnavirus DNA.

Recently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R2, IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.

Aedes↗
PubMed2003

Characterization and authentication of insect cell lines using RAPD markers.

Random amplified polymorphic DNA (RAPD) analysis was used to characterize 11 insect cell lines, including six from lepidoptera (five species), one from diptera and four from coleoptera (one species: Leptinotarsa decemlineata). Whatever the order and even when comparing two closely related species from the same genus (Spodoptera), the DNA fingerprints are very different from one species or from one primer to the other. On the other hand, two independently isolated cell lines from the lepidopteran Phthorimaea operculella produce nearly identical profiles with only minor differences. Finally, a statistical analysis based on Nei's similarity coefficient was performed on the fingerprints of four independent cell lines from the Colorado potato beetle, L. decemlineata. Each possesses a common recognizable pattern also found in field-collected insects, while also showing a series of polymorphic markers which allow one to distinguish each cell line from the three others. RAPD fingerprinting, together with the use of appropriate statistics, thus constitutes a highly specific method both for the authentication of the species from which a cell line was developed and for the individual characterization of each cell line from a given species.

Animals↗
PubMed1992

Cytoplasmic bridges and gap junctions in an insect cell line (Aedes albopictus).

Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used study simultaneously the diffusional and electrical properties of intercellular junctions. Diffusion studies involved injection of fluorescent molecules into one cell of a cell pair and visual inspection of their intercellular redistribution. Electrical measurements involved a dual voltage clamp method and whole-cell recording with patch pipette. The voltage clamp protocol was aimed at examining the dependency of the junctional conductance, gj, on membrane potential, Vm. Cell pairs exhibiting a voltage-dependent gj were found to allow intercellular diffusion of Lucifer Yellow CH (molecular mass, 443 Da), but not of FITC-dextran (molecular mass, 4,400 Da). This response pattern is consistent with the presence of gap junctions in the intercellular junctions. Cell pairs showing no voltage dependence of gj were found to permit intercellular diffusion of both Lucifer Yellow CH and FITC-dextran (dextran labelled with fluorescein isothiocyanate). This behaviour is compatible with the presence of cytoplasmic bridges connecting the two adjacent cells. Hence, in culture the cells investigated express two kinds of intercellular structures, gap junctions and cytoplasmic bridges.

Aedes↗
PubMedDate not supplied

Expression of functional human muscarinic M2 receptors in different insect cell lines.

Human M2 receptors were expressed using the baculovirus expression system in three different insect cell lines: Sf9, Sf21 and High5. The level of expression was slightly increased in Sf21 cells versus Sf9 cells. In contrast, High5 cells were not able to produce more recombinant protein than Sf9. We also show that in both Spodoptera frugiperda cell lines a peak of expression was reached after 6 days of infection, whereas in High5 cells, the maximum of expression occurred after 3 days. Immunodetection of m2 muscarinic receptor clearly shows that the expressed protein undergoes significant proteolysis in both the Sf9 and High5 cells, whereas in the Sf21 cells this phenomenon was less detectable. Additionally, we show that in all three cell lines, the expressed recombinant receptor was functional in that it was able to stimulate GTP gamma S binding in the presence of exogenous G-proteins. Analysis of the population of G-proteins (G alpha i, G alpha o and G beta common) in Sf21 and High5 cells is provided.

Animals↗
PubMed2005

Aphid-symbiotic bacteria cultured in insect cell lines.

The cells and tissues of many aphids contain bacteria known as "secondary symbionts," which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names "Candidatus Consessoris aphidicola" for T type and "Candidatus Adiaceo aphidicola" for U type.

Animals↗
PubMed2000

Endogenous glutathione-binding proteins of insect cell lines: characterization and removal from glutathione S-transferase (GST) fusion proteins.

After affinity purification on immobilized glutathione, insect-cell-derived glutathione S-transferase (GST) fusion proteins contain variable amounts of protein contaminants of about 23-24 kDa. We have isolated these glutathione-binding proteins from the widely used Sf9 and Hi5 insect cell lines and characterized them by LC-MS and N-terminal sequencing. Based on the observation that these proteins have higher affinity for glutathione than GST fusions, we have found that by using differential elution conditions the amount of such contaminants in GST fusion preparations can be strongly reduced directly during the affinity purification step. The main interest of these results is that they are not restricted to a specific construct, but rather they seem to apply to various insect-cell-derived GST fusions.

Animals↗
PubMedDate not supplied

Phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities in Md 66 insect cell line.

Phosphatidylinositol kinase (PI kinase) and phosphatidylinositol 4-phosphate kinase (PIP kinase) activities in an insect cell line (Md 66) were studied by measuring the incorporation of 32P from [gamma-32P] ATP into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) respectively. The apparent Km values of phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase for ATP were 78.5 microM and 71.7 microM respectively. PIP kinase activity was enhanced by GTP analogue guanosine 5-O(thio)triphosphate (GTP gamma S).

1-Phosphatidylinositol 4-Kinase↗