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At least 127 records · Page 7Linked to original sources
PubMed2002

Resistance of the insect cell line IPLB-LdFB to salsolinol-induced apoptosis.

Apoptosis is a form of cell death that is manifested in Parkinson's disease (PD) and certain other neurodegenerative disorders. Metabolites of salsolinol (SAL), an intraneuronal, dopamine-derived tetrahydroisoquinoline (TIQ), have been shown to induce apoptosis in human dopaminergic neuroblastoma cells, implicating these molecules as causative or contributory factors in the selective killing of nigrostriatal dopaminergic neurons, a cardinal manifestation of Parkinson's disease. Since insects employ dopamine and related catecholamines in a variety of processes including cuticular sclerotization and cellular immune reactions, it was of interest to know how insect cells metabolized exogenous SAL. Propidium iodide staining combined with flow cytometry showed that IPLB-LdFB cells from Lymantria dispar exhibited no significant (P < 0.05) increase in apoptosis when incubated for 48 h with concentrations of SAL ranging from 10 microM to 1 mM. A significant increase in apoptosis (P < 0.05) was observed in cell cultures containing the highest concentration of SAL tested (5 mM), but only 12.4% of the cells manifested this form of cell death. High pressure liquid chromatography with electrochemical detection (HPLC-ED) was used to document the production of two potentially cytotoxic quinonoids generated during the autoxidation of SAL, a reaction that was found to be significantly (P < 0.05) enhanced by peroxidase. The resistance of IPLB-LdFB cells to SAL-induced apoptosis is attributed to the ability of these insect cells to metabolize and/or detoxify such dopamine-derived catecholic TIQs. Thus, the biochemical pathways employed by insect cells in these processes may be of considerable interest to individuals investigating certain neurodegenerative disorders.

Animals↗
PubMed1976

Utilization of uridine for RNA synthesis in the insect cell line CP-1268 derived from the codling moth, Laspeyresia pomonella.

The utilization of [3H]-5-uridine by CP-1268 cells was studied. Uridine was rapidly transported into these cells by a concentration dependent, saturable process. Exogenous uridine rapidly equilibrated with cellular nucleotide pools and virtually all of the uridine transported into the cells was phosphorylated. Uridine incorporation into RNA was studied by continuous and pulse-labeling techniques in the prescence or absence of actinomycin D and cordycepin. These studies have shown that the pattern of unstable RNA precursor and relatively stable RNA product relationship known to exist in mammalian cells similarly exists in insect cells in vitro. This pattern varied markedly with pulse-labeling time and required the addition of RNA inhibitors to block reincorporation of intracellular labeled metabolites during the chase.

Biological Transport↗
PubMed1975

Two very different components of messenger RNA in an insect cell line.

The messenger RNA lifetimes have been measured in a cell line derived from an invertebrate source, the mosquito Aedes albopictus. The experiments were made possible by a new technique for obtaining undegraded cytoplasmic RNA from cells with high endogenous nuclease levels. There are two components to the decay kinetics of Aedes mRNA. The major fraction of the steady state message population has a half-life of 20 hr which is, as in mammalian cells, comparable to the cell generation time. The short-lived component turns over very rapidly with a half-life estimated to be about 1.2 hr. The difference in lifetime between the short and long-lived components is about 15 fold in these cells, compared to 3-4 fold in mammalian cells. This may reflect the need for a more responsive mRNA regulating system in poikilothermic organisms. The great disparity between the principle messenger lifetimes permits a more definite assignment of a two component behavior to message decay. The data in the case of mammalian cells could not rule out a family of intermediate lifetimes. The long-lived mRNA has a much smaller average sedimentation value than the short-lived material. The effect is similar to, but much larger than, that seen in mammalian cells. Although the lifetime difference is much greater in the insect cells than in human (HeLa) cells, the fast and slow components comprise about the same proportion of the steady state mRNA population: 30 percent and 70 percent, respectively.

Aedes↗
PubMed1981

Replication of Heliothis zea baculovirus in an insect cell line.

The baculovirus, Heliothis zea nuclear polyhedrosis virus (HzSNPV), was grown in the H. zea cell line, IPLB-1075. Light microscopy observations showed that 95% of inoculated cells had CPE by 6 days postinoculation (p.i.). Growth curves of intracellular and extracellular virus showed that maximum titers (TCID50 units) were reached 3-4 days p.i. Studies on HzSNPV morphogenesis revealed that ultrastructural events were typical of baculovirus replication in vitro. EcoRI restriction enzyme analyses of viral DNA produced in larvae and in cell culture were identical and indicated that this virus was HzSNPV.

Animals↗
PubMed1992

Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line.

Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.

Amino Acid Sequence↗
PubMed1996

Establishment and characterization of Japanese B encephalitis virus persistent infection in the Sf9 insect cell line.

The Sf9 cell line, commonly used for gene expression by recombinant baculoviruses, can be productively infected by Japanese B encephalitis virus (JEV). Two wild-type JEV strains (P3 and SA14) caused a cytopathic effect (CPE) in the Sf9 cells, while no apparent CPE was caused by an attenuated strain (SA14-14-2). The JEV viral antigens were expressed in the infected Sf9 cells and intracellular virus particles were found by electron microscopy as a result of infection with all three strains. Titres of cell-associated and cell-free supernatant virus remained stable for relatively long periods of cultivation, suggesting that both wild-type and attenuated JEV strains established productive and persistent infections of Sf9 cells. The JEV produced by the Sf9 cells could be neutralized by anti-JEV reference serum, but relatively smaller plaques were formed in BHK21 cells infected with JEV that had been cultivated long term in Sf9 cells. This system for virus propagation has a number of potentially important uses for enhancing progress in JEV study and control.

Animals↗
PubMed1999

Presence of IL-1- and TNF-like molecules in Galleria mellonella (Lepidoptera) haemocytes and in an insect cell line Fromestigmene acraea (Lepidoptera).

In this study the authors give immunocytochemical evidence for the presence of interleukin (IL)-1alpha- and tumour necrosis factor (TNF) alpha-like molecules in the haemocytes of last instar larvae from the greater wax moth Galleria mellonella. Similar results are demonstrated in a continuous haemocyte line (BTI-EA-1174-A) from the salt marsh caterpillar Estigmene acraea. In Galleria mellonella larvae granular cells show a strong positive reaction with both primary antibodies, whereas plasmatocytes are stained to a lesser extent. Cell line haemocytes also react positively with both antibodies. After activating the cells with lipopolysaccharide (LPS) staining of Estigmene acraea cells is decreased, whereas Galleria mellonella haemocytes show no visible reaction in comparison to non-activated cells.

Animals↗
PubMed1998

Uptake of HgCl2 and MeHgCl in an insect cell line (Aedes albopictus C6/36).

We studied the uptake mechanism of mercuric chloride (Hg) and methylmercuric chloride (MeHg) in Aedes albopictus C6/36 cells. The uptake kinetics, together with the effect of temperature and a metabolic inhibitor (2, 4-dinitrophenol) on the mercury accumulation, were examined. Both amounts of internalized Hg and MeHg increased linearly with the extracellular concentration. Initially, the influx rate was high for both metal species but MeHg was found to accumulate seven times faster than Hg. At longer exposure times it leveled off for Hg, while for MeHg, the intracellular concentration decreased. Hg toxicity was not significantly influenced by elevated temperatures; in contrast there was a marked decrease of the LC50/24h value for MeHg. On the other hand, Hg accumulation was temperature dependent but MeHg was not. The different toxicity and uptake rate of both mercury compounds can be explained in terms of membrane permeability and target site. For Hg the main target seems to be the plasma membrane, while MeHg readily crosses this barrier and reacts with intracellular targets. 2, 4-Dinitrophenol had no effect on the accumulation of Hg but that of MeHg was doubled. This increased MeHg accumulation might be the result of the inhibition of an active MeHg efflux mechanism; this is in agreement with the MeHg influx kinetics. Despite these differences between Hg and MeHg, which probably result from their physicochemical properties, our experiments indicate that, for both mercury species, simple diffusion is probably the main way to entrance in Aedes cells.

2,4-Dinitrophenol↗
PubMed1985

Replication pattern of double minutes derived from an insect cell line.

The DNA replication pattern of double minutes derived from an established cell line of Aedes albopictus is described. Although the vast majority of double minutes replicate semiconservatively once during the S phase, some double minutes appear to exhibit different pattern(s). Two theories are suggested as possible explanations of our findings.

Aedes↗
PubMed2001

Establishment and characterization of insect cell lines from 10 lepidopteran species.

Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer, Ostrinia nubilalis, a plutellid, the diamondback moth, Plutella xylostella, as well as eight noctuids: the black cutworm, Agrotis ipsilon, the celery looper, Anagrapha falcifera, the velvetbean caterpillar, Anticarsia gemmatalis, the corn earworm, Helicoverpa zea, the tobacco budworm, Heliothis virescens, the beet armyworm, Spodoptera exigua, the fall armyworm, Spodoptera frugiperda, and the cabbage looper, Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid amplification fingerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines from A. ipsilon and H. virescens being adapted to suspension culture using shaker flasks. The potential use for these cell lines in baculovirus production is discussed.

Animals↗
PubMed1977

Development of an attached strain from a continuous insect cell line.

A continuous attached cell strain has been developed from the IPRI-CF-124 line of the spruce budworm, Choristoneura fumiferana. This was done by discarding suspended cells at each passage, rinsing attached cells with 0.05% trypsin and using only the strongly attached cells for subculturing. The method is very effective in that the proportion of attached cells increased from 6% in the parent cell line to 97% in the new cell strain after 20 passages. The attachment and growth properties are stable after storage of cells in liquid nitrogen. The new cell strain is designated IPRI-CF-124T and has a population doubling time comparable to that of the parent cell line.

Cell Adhesion↗
PubMed1983

Characterization of the respiratory activity of mitochondria isolated from an insect cell line CP-1268 Laspeyresia pomonella.

Mitochondria have been isolated from the codling moth Laspeyresia pomonella, CP-1268 cell line. The mitochondrial fraction was isolated from pooled 4 d, exponential growth phase, cultures. The mitochondria were determined to be intact based on the demonstration of respiratory control, the effects of 2,4 dinitrophenol and oligomycin on respiration, the inability to oxidize NADH, and the inability of cytochrome c to enhance respiration. The isolated mitochondria were able to oxidize succinate, pyruvate, malate, alpha-ketoglutarate, and alpha-glycerophosphate efficiently. Of the substrates tested, the CP-1268 mitochondria oxidized succinate most efficiently. The respiratory control ratios ranged from a high of 4.6 for pyruvate to a low of 1.7 with alpha-glycerophosphate. These findings confirm that the mitochondria were tightly coupled. The data also confirm the presence of three sites of oxidative phosphorylation because NAD-linked substrates had ADP-to-O ratios approaching 3 and flavoprotein linked substrates had values approaching 2.

Animals↗