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At least 379 records · Page 21Linked to original sources

PubMed1985

Primary culture of ant venom gland cells.

Venom from the ant Pseudomyrmex triplarinus reduces the symptoms and swelling of rheumatoid arthritis. The cells that produce the venom were dissected from larval and pupal ants and culture conditions studied. Cell dissociation, with minimal amount of damage, was done with 0.25% trypsin at 4 degrees C with subsequent use of soybean trypsin inhibitor. A new medium was formulated and epidermal growth factor, fibroblast growth factor, insulin, cAMP, cGMP, and isoproterenol were beneficial. The optimum osmotic pressure was a relatively high 500 mOSM. Conditioning the medium with an established insect cell line was essential for long-term cell survival. Under these culture conditions the structural and metabolic integrity of the cells were maintained for up to 12 mo.

Animals↗

PubMed1976

A cytotoxic substance in insect cell culture spent media.

Spent media from five different insect cell lines when inoculated into Trichoplusia ni (TN-368) cultures produced cytotoxicity resulting in rounding and detachment of cells. The substance in spent medium from the established cellline Carpocapsa pomonella (CP-169) is believed to be a toxin, based on the failure to serially passage the agent, the early appearance of the cytotoxic effect, and the inability to detect microbes by culturing techniques as well as by electron microscopy. The ability to extract the toxic substance from CP-169 cells indicates that it is cell associated. Biophysical and biochemical properties of the CP-169 cytotoxin are presented.

Cell Line↗

PubMed2000

Kinetic properties of the channels formed by the bacillus thuringiensis insecticidal crystal protein Cry1C in the plasma membrane of Sf9 cells.

Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K(+) and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K(+). These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K(+) efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K(+) efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line.

Animals↗

PubMed1998

Long-chain n-alkanols and arachidonic acid interfere with the Vm-sensitive gating mechanism of gap junction channels.

Experiments were carried out on preformed cell pairs and induced cell pairs of an insect cell line (mosquito Aedes albopictus, clone C6/36). The coupling conductance, gj, was determined with the dual voltage-clamp method. Exposure of preformed cell pairs to lipophilic agents, such as long-chain n-alkanols (n-hexanol, n-heptanol, n-octanol, n-nonanol, n-decanol) or arachidonic acid, provoked a decrease in gj. Hyperpolarization of both cells led to a recovery of gj. Systematic studies revealed that this phenomenon is caused by a shift of the sigmoidal relationship gj(ss) = f(Vm) towards more negative values of Vm (where gj(ss) = conductance at steady-state; Vm = membrane potential). The shift was dose dependent, it developed with time and was reversible. The longer the hydrocarbon chain of n-alkanols, the lower was the concentration required to produce a given shift. Besides shifting the function gj(ss) = f(Vm), arachidonic acid decreased the maximal conductance, gj(max). Single-channel records gained from induced cell pairs revealed that the lipophilic agents interfere with the Vm-sensitive slow channel gating mechanism. Application provoked slow current transitions (transition time: 5-40 ms) between an open state of the channel (i.e. main state or residual state) and the closed state; subsequently, fast channel transitions (transition time: < 2 ms) involving the main state and the residual state ceased completely. Hyperpolarization of Vm or washout of the lipophilic agents gave rise to the inverse sequence of events. The single-channel conductances gammaj(main state) and gammaj(residual state) were not affected by n-heptanol. We conclude that long-chain n-alkanols and arachidonic acid interact with the Vm-sensitive gating mechanism.

1-Octanol↗

PubMed2002

Cell culture of mechanoreceptor neurons innervating proleg sensory hairs in Manduca sexta larvae, and co-culture with target motoneurons.

The tip of each proleg in Manduca sexta larvae bears a dense array of mechanosensory hairs termed planta hairs (PHs), each innervated by a single sensory neuron (termed a PH-SN) located in the underlying epidermis. In the CNS, axon terminals of PH-SNs make direct, excitatory, nicotinic cholinergic synapses with proleg retractor motoneurons including the accessory planta retractor (APR). These synapses mediate a proleg withdrawal reflex, exhibit multiple forms of activity-dependent plasticity and weaken during the prepupal peak of ecdysteroids. In the present study we developed methods to dissociate PH-SNs from the epidermis and culture them alone or with APRs. The PH-SNs were fluorescently labeled in situ by introducing dye through the cut hair shaft or by retrograde axonal staining. Alternatively, unlabeled PH-SNs were utilized. The epidermis beneath the planta hair array was separated from the cuticle, enzymatically treated and mechanically dissociated into single cells. PH-SNs were cultured on glass coverslips coated with concanavalin A and laminin, in modified Leibovitz's IL-15 medium. Supplementation with medium conditioned by an insect cell line produced the best results. Dissociated PH-SNs had somatic diameters of ~10 micro m and typically bore a stout dendrite consisting of the inner and, occasionally, the outer dendritic segment. An axonal stump was sometimes retained. Viable PH-SNs typically extended new processes and often survived for 2-4 weeks. When co-cultured, PH-SNs and APRs exhibited robust growth and made close anatomical appositions. This culture system provides convenient experimental access to PH-SNs and may potentially permit sensorimotor synapses to be investigated in vitro.

Animals↗

PubMed2005

Galatosylation and sialylation of mammalian glycoproteins produced by baculovirus-madiated gene expression in insect cells.

The baculovirus expression vector system (BEVS) is used extensively for the production of proteins from exogenous cDNAs. However, BEVS is not ideal for pharmaceutical production of glycoproteins owing to the properties of the N-glycans in the expressed products and that insect cells lack several of the enzymes required for mammalian-type N-glycan synthesis. This study describes the effective mammalian-like production of glycoproteins, such as beta-1,4-galactosyltransferase and alpha-2,6-sialyltransferase, in the insect cell line Sf9.

Animals↗

PubMed1993

Biosynthesis and initial processing of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

Based on the deduced amino-acid sequence of the cardiac Na(+)-Ca2+ exchanger, there are six potential N-linked glycosylation sites and a potential cleaved signal sequence. To study the post-translational modifications of the exchanger, in vitro translation was examined in the presence and absence of canine pancreatic microsomes. Glycosylation, detected as endoglycosidase H induced shifts in molecular size, was examined for proteins having different numbers of potential N-linked glycosylation sites by using full and partial length RNA transcripts. In the presence of microsomes, the molecular mass of the full-length clone increased from 110 to 113 kDa. Endoglycosidase H treatment led to a reduction to 108 kDa, indicating that glycosylation increases the molecular mass by approx. 5 kDa and a signal sequence of approx. 2 kDa is cleaved during processing. Analysis of molecular-mass shifts obtained with partial transcripts suggested that glycosylation occurs at position N-9. This was confirmed by site-directed mutagenesis studies. A molecular mass of approx. 120 kDa was measured for Western blots of cardiac sarcolemmal membrane or oocytes expressing the wild-type exchanger. The molecular mass was reduced by approx. 10 kDa for the N9Y mutant or from exchanger obtained from a baculovirus-infected insect cell line where glycosylation does not occur. The giant excised patch technique was used to determine the functional consequences of glycosylation. Na(+)-Ca2+ exchange current was examined in patches from oocytes expressing either the wild-type or N9Y mutant. The non-glycosylated mutant exhibited the same properties as the native exchanger with respect to voltage, sodium dependence, and the effects of chymotrypsin. The results indicate that glycosylation does not affect exchanger function in Xenopus oocytes and help to define exchanger topology.

Amino Acid Sequence↗

PubMed1993

Expression of a Drosophila GABA receptor in a baculovirus insect cell system. Functional expression of insecticide susceptible and resistant GABA receptors from the cyclodiene resistance gene Rdl.

Recombinant baculoviruses containing two alternative splice forms of the Drosophila Rdl GABA receptor gene were constructed. Spodoptera frugiperda (Sf21) cells infected with either splice form expressed a transcript of expected size (2.5 kb). Western blotting of cell membrane extracts and immunoprecipitation experiments with an anti-Rdl antiserum recognized a protein of the expected size of approximately 65 kDa. Whole cell patch clamp analysis of cells infected with either splice form revealed functional expression of GABA gated chloride ion channels which were blocked by application of 1 microM picrotoxinin. Following replacement of alanine 302 with a serine, a mutation associated with resistance to picrotoxinin and cyclodiene insecticides, mutant channels showed similar levels of insensitivity to picrotoxinin (approximately 100-fold) as those observed in recordings from cultured Drosophila neurons. The significance of the expression of an insect GABA receptor in an insect cell line and the similarity of the results from these functional expression studies to recordings from cultured neurons is discussed.

Alternative Splicing↗

PubMed1993

Characterization of glucose transport system in Drosophila Kc cells.

We have characterized the glucose transport system in the Kc cells of Drosophila melanogaster. The transporter is stereospecific and can be inhibited by both cytochalasin B and phloretin, with ki values of 4 microM and 30 microM for cytochalasin B and phloretin, respectively. Moreover, phlordzin does not affect uptake of glucose by Kc cells. Thus, on the basis of inhibitor studies, it appears that this insect cell line contains glucose transporters similar to those of the mammalian cells. However, probes prepared from rat transporter cDNAs showed no cross-hybridization with Drosophila RNA under moderately stringent conditions. Sugar selectively has also been investigated by competition assay. The km values for D-glucose, 2-deoxy-D-glucose, D-fructose and D-galactose are 2 mM, 6 mM, 17 mM and 16 mM, respectively. The results indicate that this transporter prefers D-glucose and that it does not discriminate between D-galactose and D-fructose.

Animals↗

PubMed1996

Functional expression of the plant K+ channel KAT1 in insect cells.

Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1, and KST1 have recently been identified on the molecular level. Among them, we focussed on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K+-selective (impermeable to Na+ and even NH4+) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as -60 to -80mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-traditional modification and assembly of a green inward rectifier.

Animals↗

PubMed1994

Biochemical and functional characterization of soluble form of IL-5 receptor alpha (sIL-5R alpha). Development of ELISA system for detection of sIL-5R alpha.

Interleukin-5 (IL-5) mediates pleiotropic functions in various types of cells through its specific receptor (IL-5R) which is composed of two distinct subunits, alpha and beta. In mice, the alpha subunit (IL-5R alpha) specifically binds IL-5 with low affinity. The beta subunit (IL-5R beta) does not bind IL-5 by itself, but constructs the high affinity receptor with IL-5R alpha. We have isolated cDNA encoding the soluble form of IL-5R alpha (sIL-5R alpha). To elucidate the biochemical and functional properties of sIL-5R alpha, we developed an expression system for sIL-5R alpha cDNA in insect cell line Sf21 using baculovirus expression vector and obtained conditioned medium containing large quantities of mouse sIL-5R alpha. Mouse sIL-5R alpha was purified from the conditioned medium by using anti-IL-5R alpha mAb-coupled beads. Immunoaffinity-purified sIL-5R alpha with an approximate molecular mass of 42 kDa inhibited the binding of 125I-labeled IL-5 to IL-5R. By using purified sIL-5R alpha, we prepared rabbit anti-sIL-5R alpha antibody and developed a sandwich ELISA for detection of sIL-5R alpha. Significant amounts of sIL-5R alpha were detected in sera and ascitic fluids of mice bearing tumors (BCL1 and MOPC104E) that responded to IL-5 for DNA synthesis, but not in sera of normal mice. Interestingly, elevated levels of serum sIL-5R alpha were observed in NZB and NZW mice. The sIL-5R alpha may, therefore, have an immunoregulatory role in vivo.

Animals↗

PubMed1988

Cytotoxic effect of T-2 mycotoxin on cells in culture as determined by a rapid colorimetric bioassay.

We developed a colorimetric assay for determining metabolic activity (viability) of cells exposed to toxic agents. This system is based on the ability of mitochondrial enzymes in viable cells to modify a tetrazolium salt into a blue formazan product that can be detected spectrophotometrically at 570 nm. The assay works equally well for mammalian and insect cell lines and at 48 hr color formation is linear over a cell input range of 1.56-50 X 10(4) cells/ml. The inhibitory effects of T-2 mycotoxin on tetrazolium cleavage in L929 cells is comparable to that observed for protein and DNA synthesis (50% inhibition = 6-8 ng/ml). Using this system to analyze the lethal effect of T-2 toxin on cells from various animal species, it was found that bovine cells were the most sensitive (50% inhibition at 2.2 ng/ml) while hamster cells were the most resistant (50% inhibition at 26.2 ng/ml). Murine cells exhibited intermediate sensitivity (50% inhibition at 10.9 ng/ml). Variable toxin susceptibility was also observed among different cell types. Lymphocytes were 3-fold more sensitive to the T-2 inhibitory effects than comparable tissue culture cell lines. These data indicate that the colorimetric assay system could have broad applications in toxicological studies. Further, the observed differences in species sensitivity may provide insight into the primary mechanism of the T-2 toxin-cell interaction that ultimately leads to cell death.

Animals↗

PubMed1990

Semipermissive replication of Tipula iridescent virus in Aedes albopictus C6/36 cells.

Comparative studies were carried out using two different insect cell lines, Aedes albopictus and Estigmene acrea, for Tipula iridescent virus (TIV) propagation. Light microscope autoradiography showed viral DNA present in viroplasmic centers (VCs) and an inhibition of nuclear DNA synthesis. These VCs appeared to be morphologically similar in both cell lines when examined by light and electron microscopy. Radiolabeled cDNA was synthesized from RNA samples obtained from infected cells at different times after infection and hybridized to TIV DNA digested with various restriction endonucleases. The results indicated that the pattern of transcription and the kinetics of TIV infection were qualitatively similar in both cell lines. The major TIV DNA components, L (greater than 174 kbp) and S1 (10.8 kbp) that are found in virions in approximately equivalent amounts, were made in both infected cell lines. However, the infected cell lines produced S1 DNA at higher levels relative to L than in virions. The cDNA hybridization studies also revealed that the S1 DNA has sequences that are transcribed and are TIV specific. While VC morphology, levels of L and S1 DNA synthesis, transcription, and capsid protein synthesis were similar in both cell lines, time course electron microscope studies revealed that progeny virions were detected only in the VCs of E. acrea cells and not in the VCs of A. albopictus cells, even by 96 hr p.i. These data suggest that the A. albopictus C6/36 cell line is semipermissive for TIV replication.

Aedes↗

PubMed1991

High-level expression of the Epstein-Barr virus alkaline deoxyribonuclease using a recombinant baculovirus: application to the diagnosis of nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). Infection of the insect cell line Spodoptera frugiperda (SF9) with the recombinant virus led to the expression of an enzymatically active alkaline DNase. The recombinant EBV alkaline DNase was highly soluble, and the recombinant baculovirus produced approximately 10-20 mg of EBV DNase per 1 X 10(9) cells. The recombinant enzyme activity was neutralized by specific antisera to the EBV DNase and was recognized by these sera in Western blot analysis and immunofluorescence tests. The recombinant EBV DNase was neutralized by these sera from patients with nasopharyngeal carcinoma and chronic infectious mononucleosis. Western blot analysis using these patients' sera showed that IgG and IgA antibodies to the EBV DNase could be readily detected.

Animals↗

PubMed1992

Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR).

Circumstantial evidence has accumulated suggesting that CFTR is a regulated low-conductance Cl- channel. To test this postulate directly, we have purified to homogeneity a recombinant CFTR protein from a high-level baculovirus-infected insect cell line. Evidence of purity included one- and two-dimensional gel electrophoresis, N-terminal peptide sequence, and quantitative amino acid analysis. Reconstitution into proteoliposomes at less than one molecule per vesicle was accomplished by established procedures. Nystatin and ergosterol were included in these vesicles, so that nystatin conductance could serve as a quantitative marker of vesicle fusion with a planar lipid bilayer. Upon incorporation, purified CFTR exhibited regulated chloride channel activity, providing evidence that the protein itself is the channel. This activity exhibited the basic biophysical and regulatory properties of the type of Cl- channel found exclusively in CFTR-expressing cell types and believed to underlie cAMP-evoked secretion in epithelial cells.

Animals↗

PubMed1993

Functional analysis of the human alpha 2C-C4 adrenergic receptor in insect cells expressed by a luciferase-based baculovirus vector.

A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human alpha 2-adrenergic receptor (alpha 2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-alpha 2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the alpha 2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of alpha 2-receptors at a level of 1.370 pmol/mg protein was obtained at 48 h after infection as determined by ligand-binding experiments using the alpha 2-receptor antagonist, [3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [3H]rauwolscine binding with Ki values 12.3 +/- 1.54 microM and 1.23 +/- 0.11 microM, respectively. The recombinant receptors were functionally intact since the agonists inhibited forskolin-stimulated cAMP production. Here, however, the maximal inhibition was obtained at 36 h after the infection. The results presented here, suggest that the baculovirus expression vector system (BEVS) provides a simple method for abundant expression of functional alpha 2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.

Adrenergic alpha-Antagonists↗

PubMed1991

DNA-binding proteins of baculovirus-infected cells during permissive and semipermissive replication.

Certain insect cell lines have been shown to be permissive (TN-368) or semipermissive (IPLB-LD-652Y) for Autographa californica nuclear polyhedrosis virus (AcMNPV) replication (McClintock et al., 1986b). In this report DNA-binding proteins were identified in such cell:virus systems by hybridizing Western blots containing uninfected and infected cell proteins with AcMNPV or host DNA probes. In the AcMNPV-infected TN-368 permissive cell system, 8 virus-induced DNA-binding proteins with molecular weights ranging from 67.5K to 18.75K were observed under the highest conditions of stringency. When these DNA-binding proteins were compared to structural proteins of AcMNPV, several appeared to be similar to those observed in SDS-PAGE protein profiles of nonoccluded virus (NOV) and occlusion body (PIBs) preparations. Using an AcMNPV occlusion negative mutant (L1GP-gal3) and an anti-AcMNPV-polyhedrin monoclonal antibody, a major DNA-binding protein (33.0K), observed in the permissive system, corresponded to polyhedrin and to a comigrating virus-induced DNA-binding protein. In the AcMNPV-infected IPLB-LD-652Y semipermissive cell system, no virus-induced DNA-binding proteins were detected. However, several host DNA-binding proteins were present but their ability to bind DNA decreased significantly following infection.

Animals↗

PubMed1995

Synthesis and processing of the rubella virus p110 polyprotein precursor in baculovirus-infected Spodoptera frugiperda cells.

In order to study the processing of rubella virus (RV) structural proteins (capsid protein, of 33 kDa; E2 of 42-47 kDa; and E1 of 58 kDa) in Spodoptera frugiperda (fall armyworm) cells, a 24S cDNA encoding the polyprotein precursor, p110, was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during viral infection. By immunoblot analysis using antibodies directed against whole RV and the individual structural proteins, evidence is presented that polypeptides similar to those synthesized in RV-infected B-Vero cells are expressed in this lepidopteran insect cell line infected with the recombinant baculovirus, VL1392-RV24S. The identity of the recombinant proteins was further confirmed using human convalescent sera. By expressing the recombinant proteins in the presence and absence of tunicamycin, we have further demonstrated that the 24S transcription-translation unit of RV, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared to B-Vero cells.

Animals↗