| expression and characterization of functionally active recombinant perforin produced in insect cells. | a key cytolytic mediator used by killer lymphocytes, perforin (also known as pore-forming protein or cytolysin), has been shown to be capable of undergoing polymerization to form pores in cell membranes and cause osmotic lysis of target cells. although perforin has been purified from killer lymphocytes and the coding gene has been cloned and sequenced, information concerning the domain structure of the perforin molecule has remained scarce. to overcome the difficulty in obtaining sufficient amou ... | 1996 | 8617952 |
| functional expression of the aequorea victoria green fluorescent protein in insect cells using the baculovirus expression system. | a dna fragment encoding the green fluorescent protein (gfp) was isolated via pcr from a jellyfish aequorea victoria cdna, cloned and sequenced. subsequently, a recombinant baculovirus bearing the coding region of the gfp under the transcriptional control of the autographa californica nuclear polyhedrosis virus (acmnpv) polyhedrin gene promoter was constructed and isolated. high-level expression of gfp could be easily monitored in spodoptera frugiperda (sf9) insect cells after infection with reco ... | 1996 | 8619796 |
| characterization of the interaction between rhogdi and cdc42hs using fluorescence spectroscopy. | the gdp-dissociation-inhibitor (gdi) for rho-like gtp-binding proteins is capable of three different biochemical activities. these are the inhibition of gdp dissociation, the inhibition of gtp hydrolysis, and the stimulation of the release of gtp-binding proteins from membranes. in order to better understand how gdi interactions with rho-like proteins mediate these different effects, we have set out to develop a direct fluorescence spectroscopic assay for the binding of the gdi to the rho-like p ... | 1996 | 8626553 |
| identification of a unique vp4 serotype that is shared by a human rotavirus (69m strain) and an equine rotavirus (h-2 strain). | a cdna clone representing the vp4-encoding gene of human rotavirus strain 69m(vp7 serotype 8) was constructed and inserted into a baculovirus expression vector. baculovirus recombinants that expressed the 69 m vp4 protein in spodoptera frugiperda (sf9) cells were screened by immunofluorescence with hyperimmune antiserum to the 69m strain and purified by terminal dilution. the expressed vp4 was detected by coomassie blue staining of page-separated proteins. the antigenic relationships between the ... | 1996 | 8629943 |
| expression and characterization of recombinant alpha-galactosidase in baculovirus-infected insect cells. | a cdna encoding coffee bean alpha-galactosidase was subcloned into baculovirus expression vectors, pvl-1393 and pac-gp67b, for intracellular and extracellular expression in spodoptera frugiperda (sf9) insect cells, respectively. the expressed protein (recombinant alpha-galactosidase) was immunologically reactive with antisera raised against its native counterpart isolated from coffee beans and was biologically active towards the substrate p-nitrophenyl alpha-galactopyranoside. the subcellular di ... | 1996 | 8631350 |
| differential coupling of rat d2 dopamine receptor isoforms expressed in spodoptera frugiperda insect cells. | by using a baculovirus expression system, the two isoforms of the rat d2 dopamine receptor were expressed at densities ranging up to 15 pmol/mg of protein. d2l and d2s dopamine receptors expressed in aline of spodoptera frugiperda (sf9) insect cells sf9cells, displayed high affinity for the antagonists spiroperidol and (+)-butaclamol and the agonist n-propylnorapomorphine. antisera raised against the d2 receptor immunoprecipitated binding sites for a radiolabeled d2 antagonist from solubilized e ... | 1996 | 8632351 |
| identification of phosphorylation sites of human 85-kda cytosolic phospholipase a2 expressed in insect cells and present in human monocytes. | the phosphorylation sites on the human, 85-kda cytosolic phospholipase a2 (cpla2) were identified using recombinant cpla2 expressed in spodoptera frugiperda (sf9) cells. analysis by high performance liquid chromatography of tryptic digests of 32p-labeled recombinant cpla2 showed four major peaks of radiolabeled phosphopeptides. the phosphorylated residues were identified as ser-437, ser-454, ser-505, and ser-727 using mass spectrometry and automated edman sequencing. sf9 cells infected with reco ... | 1996 | 8636128 |
| functional high level expression of cytochrome p450 cyp2d6 using baculoviral expression systems. | cytochrome p-450 cyp2d6 plays a central role in the metabolism of many widely used therapeutic drugs including beta-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. recombinant baculoviruses have been constructed containing the full-length human cyp2d6 cdna and used to express cyp2d6 in spodoptera frugiperda (sf9) cells. high levels of recombinant protein have been produced using either polyhedrin or basic protein promoters (0.05-0.20 nmol/mg cell protein; 0.05-0.15 nmol/l ... | 1996 | 8638923 |
| human bleomycin hydrolase: molecular cloning, sequencing, functional expression, and enzymatic characterization. | we have cloned the cdna of human bleomycin hydrolase (hbh), a protease which is thought to be involved in the metabolic inactivation of the antineoplastic drug bleomycin. the open reading frame consists of 1365 base pairs and is predicted to encode a 52 kda protein. the protein shares 40% identity with yeast bleomycin hydrolase and contains the conserved active site residues (cys, his, asn) characteristic for cysteine proteases of the papain superfamily. human bleomycin hydrolase has been functi ... | 1996 | 8639621 |
| expression of l protein of vesicular stomatitis virus indiana serotype from recombinant baculovirus in insect cells: requirement of a host factor(s) for its biological activity in vitro. | the 241-kda large (l) protein of vesicular stomatitis virus (vsv) indiana serotype, a multifunctional catalytic subunit of the viral rna polymerase, has been expressed in spodoptera frugiperda cells infected with recombinant baculovirus bacpak6-l containing the l gene under the control of a polyhedrin promoter. the recombinant l protein was biologically active and supported viral mrna synthesis in vitro. when the expressed l protein was purified by phosphocellulose column chromatography, it elut ... | 1996 | 8642651 |
| vip3a, a novel bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. | a novel vegetative insecticidal gene, vip3a(a), whose gene product shows activity against lepidopteran insect larvae including black cutworm (agrotis ipsilon), fall armyworm (spodoptera frugiperda), beet armyworm (spodoptera exigua), tobacco budworm (heliothis virescens), and corn earworm (helicoverpa zea) has been isolated from bacillus thuringiensis strain ab88. vip3-insecticidal gene homologues have been detected in approximately 15% of bacillus strains analyzed. the sequence of the vip3a(b) ... | 1996 | 8643585 |
| pharmacological and molecular characterization of the neurotensin receptor expressed in sf9 cells. | the rat neurotensin receptor was expressed in spodoptera frugiperda insect (sf9) cells using infection with a recombinant baculovirus. immunoblot experiments performed with an antibody raised against the c-terminus of the receptor showed major bands at 47 (corresponding to the unglycosylated receptor protein) and 50 kda, and minor bands at 65 and 36 kda. the expressed receptor bound 125i-neurotensin with high affinity, was coupled to endogenous g-proteins, and agonist-induced inositol phosphate ... | 1996 | 8645349 |
| production, purification and characterization of non-myristylated human t-cell protein tyrosine kinase in a baculovirus expression system. | a non-myristylated form (lck m) of the human t-lymphocyte-specific protein tyrosine kinase (lck) was produced at high levels in a baculovirus expression system (bves) using two strategies. first, lck m was produced by direct expression of a gly2 --> ala mutant of lck. second, lck was produced as a glutathione s-transferase (gst) fusion, and lck m was derived from the fusion protein by cleavage with thrombin. both recombinant proteins (re-proteins) were produced at 5% of the total protein of infe ... | 1996 | 8647461 |
| n-acetyl-beta-glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expressed in insect cells from a baculovirus vector. | the hemagglutinin of fowl plague virus has been expressed in spodoptera frugiperda (sf9) cells and in estigmene acrea cells by using a baculovirus vector. structural analysis revealed that the endo-h-resistant n-glycans of ha from sf9 cells were predominantly trimannosyl core oligosaccharides, whereas in e. acrea cells most of these cores were elongated by at least one terminal n-acetylglucosamine residue. to understand the difference in carbohydrate structures, enzymes involved in n-glycan proc ... | 1996 | 8648750 |
| expression and initial characterization of recombinant mouse thrombospondin 1 and thrombospondin 3. | to analyze the function of tsp family members, we have expressed and purified mouse tsp1 and tsp3 encoded by recombinant baculoviruses in spodoptera frugiperda cells and compared these tsps to mouse tsp2 prepared in a similar way. yields of purified tsp1 and tsp3 were 5-15 and 2-4 micrograms, respectively, per ml of conditioned medium. mature, secreted mouse t41 and tsp3 had the previously predicted nh2-terminal sequences of dhvkdtsfdlfsi, and sqdlqvidllt, respectively. analysis by sds-page and ... | 1996 | 8654563 |
| secretion of biologically active human proapolipoprotein a-i in a baculovirus-insect cell system: protection from degradation by protease inhibitors. | studies of the structure and function of apolipoprotein a-i (apoa-i) often require its purification by delipidation of high density lipoprotein isolated from large quantities of human plasma and separation of apoa-i from other plasma apolipoproteins. to reduce the need for extensive purification procedures, we have developed an insect cell/baculovirus expression system for the production and secretion of human proapoa-i. the recombinant baculovirus containing full-length human apoa-i cdna, when ... | 1995 | 8656073 |
| metabolic engineering of animal cells. | substrate-limited fed-batch cultures were used to study growth and overflow metabolism in hybridoma and insect cells. in hybridoma cells a glucose-limited fed-batch culture decreased lactate formation but increased glutamine consumption and ammonium formation. glutamine limitation decreased ammonium and alanine formation but did not enhance glucose consumption. instead lactate formation was reduced, indicating that glucose was used more efficiently. the formation of lactate, alanine, and ammoniu ... | 1996 | 8659912 |
| purification, cloning, and bacterial expression of retinol dehydratase from spodoptera frugiperda. | anhydroretinol and 14-hydroxy-4,14-retro-retinol, retro-retinoids endogenous to both mammals and insects, act as agonist and antagonist, respectively, in controlling proliferation in lymphoblasts and other retinol-dependent cells. we describe here the identification, purification, cloning, and bacterial expression of the enzyme retinol dehydratase, which converts retinol to anhydroretinol in spodoptera frugiperda. retinol dehydratase has nanomolar affinity for its substrate and is, therefore, th ... | 1996 | 8663216 |
| baculovirus-directed expression of human prostatic steroid 5 alpha-reductase 1 in an active form. | in the human prostate, the enzyme steroid 5 alpha-reductase (h5 alpha r) catalyses the conversion of testosterone into the more potent androgen, dihydrotestosterone. two distinct cdnas coding for h 5 alpha r in the human prostate have been previously characterized. enzyme h5 alpha r1 shows a maximum activity at basic ph whereas h5 alpha r2 has an acidic ph optimum activity. we report here the expression of the human steroid h5 alpha r1 in a eukaryotic expression system: the baculovirus-directed- ... | 1993 | 8664165 |
| semiautomated quantification of cytotoxic damage induced in cultured insect cells exposed to commercial bacillus thuringiensis biopesticides. | a convenient in vitro bioassay based on semiautomated quantification of live-cell reduction of tetrazolium dyes--3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt) and 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2h-tetrazolium-5-corb oxanilide sodium salt (xtt)--to formazan was developed and used to evaluate cytotoxic effects of two commercial insecticides (bt) derived from bacillus thuringiensis subsp. kurstaki (btk). comparison of two target insect cell lines mg1 (trichoplusia ... | 1995 | 8666719 |
| wild type and mutant human heart (r)-3-hydroxybutyrate dehydrogenase expressed in insect cells. | (r)-3-hydroxybutyrate dehydrogenase (bdh) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (pc) for function. pc is an allosteric activator that enhances nad(h) binding to bdh. the enzyme serves as a paradigm to study specific lipid-protein interactions in membranes. analysis of the primary sequence of bdh, as determined by molecular cloning, predicts that lipid binding and substrate specificity are contributed by the c-terminal third of the protein [m ... | 1996 | 8679568 |
| human 25-hydroxyvitamin d3-24-hydroxylase, a multicatalytic enzyme. | human 25-hydroxyvitamin d-24-hydroxylase has been expressed in spodoptera frugiperda (sf21) insect cells using the previously cloned cdna in baculovirus (acnpv-p450cc24). the activity of recombinant h-p450cc24 required adrenodoxin, adrenodoxin reductase, and nadph. incubation of this reconstituted system with 25-oh-[26,27-(3)h]d3 substrate produced several metabolites that were resolved on a normal-phase cyano hplc system. these products exactly comigrated with authentic standards for 24-oxo-25- ... | 1996 | 8679605 |
| isoform-specific intracellular vesicle formation by recombinant annexin xi-a in sf9 cells. | annexins are a group of structurally related proteins that bind phospholipids in a ca2(+)-dependent manner and have the ability to self-aggregate and to promote vesicle aggregation and membrane fusion. two isoforms of annexin xi, termed xi-a and xi-b, were previously identified by screening a bovine chondrocyte cdna library. but little is known about differences in their biological function. in the present study, we therefore examined the results of expression of the two proteins in spodoptera f ... | 1996 | 8687461 |
| soluble adenylyl cyclase from spodoptera frugiperda (sf9) cells. purification and biochemical characterization. | an insect ovarian cell, spodoptera frugiperda (sf9), has been widely used to express recombinant proteins, including adenylyl cyclase, as a host cell in the baculovirus expression system. we report the presence and characterization of a soluble adenylyl cyclase (sac) distinct from a membrane-bound form of adenylyl cyclase (mac) that is also present in sf9 cells. sac was purified 3,500-fold to near homogeneity; a single band at 25 kda on sds-polyacrylamide gel electrophoresis correlated well with ... | 1996 | 8702736 |
| a molecular basis for affinity modulation of fab ligand binding to integrin alphaiib beta3. | the arg-gly-asp (rgd) sequence within the third complementarity-determining region (cdr3) of the heavy chain (h3) is responsible for the binding of the recombinant murine fab molecules, ap7 and pac1.1, to the platelet integrin alphaiibbeta3. ap7 binding is minimally influenced by the conformational state of this receptor, whereas pac1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. to study the molecular basis for this functional difference, we replace ... | 1996 | 8702765 |
| the ribosome-inactivating protein restrictocin deters insect feeding on aspergillus restrictus. | the fungus-feeding beetle, carpophilus freemani, consumed equal quantities of young mycelia, fewer phialides bearing mature spores and much fewer phialides bearing developing spores of aspergillus restrictus compared to those of aspergillus nidulans when tested in diet choice assays. the degree to which specific fungal structures were consumed was inversely related to the localization of high levels of restrictocin, a ribosome-inactivating protein, to those structures. pure restrictocin added to ... | 1996 | 8704996 |
| abortive infection of the baculovirus autographa californica nuclear polyhedrosis virus in sf-9 cells after mutation of the putative dna helicase gene. | homologous recombination between the autographa californica nuclear polyhedrosis virus (acnpv) genome and a 0.6-kbp-long dna fragment derived from the putative dna helicase gene of bombyx mori nuclear polyhedrosis virus generates eh2-acnpv, an expanded-host-range acnpv mutant (s. maeda, s.g. kamita, and a. kondo, j. virol. 67:6234-6238, 1993). after inoculation at a high multiplicity of infection (moi), eh2-acnpv replicates efficiently in both the sf-9 (acnpv-permissive) and bmn (non-acnpv-permi ... | 1996 | 8709251 |
| glycosylation in lepidopteran insect cells: identification of a beta 1-->4-n-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains. | the choice for a heterologous expression system to produce glycoprotein therapeutics highly depends on its potential to perform mammalian-like posttranslational modifications such as glycosylation. to gain more insight into the glycosylation potential of the baculovirus mediated insect cell expression system, we have studied the expression of glycosyltransferases involved in complex-type n-glycosylation. lepidopteran insect cell lines derived from trichoplusia ni, spodoptera frugiperda, and mame ... | 1996 | 8727788 |
| elongation of the n-glycans of fowl plague virus hemagglutinin expressed in spodoptera frugiperda (sf9) cells by coexpression of human beta 1,2-n-acetylglucosaminyltransferase i. | spodoptera frugiperda (sf9)-cells differ markedly in their protein glycosylation capacities from vertebrate cells in that they are not able to generate complex type oligosaccharide side chains. in order to improve the oligosaccharide processing properties of these cells we have used baculovirus vectors for expression of human (beta 1,2-n-acetylglucosaminyltransferase i (hgnt-i), the enzyme catalysing the crucial step in the pathway leading to complex type n-glycans in vertebrate cells. one vecto ... | 1996 | 8727789 |
| double-site ricin b chain mutants retain galactose binding. | three distinct double-site and two single-site ricin b chain (rtb) mutants were expressed in spodoptera frugiperda insect cells and purified from infected cell supernatants. the yields of recombinant proteins were 0.01-0.2 mg/l. the purity after monoclonal antibody affinity chromatography was 1-20%. the mutant proteins were soluble, immunoreactive with monoclonal antibodies and polyclonal antibodies to rtb and demonstrated molecular weights of 32 kda, similar to plant rtb. all three double-site ... | 1996 | 8738213 |
| sex-specific and hormone-controlled expression of a vitellogenin-encoding gene in the gypsy moth. | microvitellogenin and vitellogenin cdna from manduca sexta (tobacco hornworm) were tested for use as molecular probes to investigate the expression of genes coding for vitellogenins in spodoptera frugiperda (fall armyworm) and lymantria dispar (gypsy moth). cross-hybridization was not observed between the m. sexta cdnas and s. frugiperda dna and mrna. vitellogenin cdna from m. sexta did not hybridize to l. dispar dna or mrna. however, the 834 bp microvitellogenin cdna from m. sexta hybridized to ... | 1996 | 8742824 |
| stat-like dna-binding activity in spodoptera frugiperda cells. | nuclear extracts from spodoptera frugiperda (sf9) cells were shown to contain a factor binding to dna elements with gamma interferon activated site-like sequences. the dna-binding activity was shown to be dependent on tyrosine phosphorylation. hydrodynamic characterization of this entity revealed a stokes radius of 8.4 nm and a sedimentation coefficient of 5.9 s. the molecular weight was calculated to 209,000. estimated frictional (f/to) and axial (a/b) ratios indicated an elongated structure of ... | 1996 | 8753801 |
| species-specific effects of the hcf-1 gene on baculovirus virulence. | the host cell-specific factor 1 gene (hcf-1) of the baculovirus autographa californica nuclear polyhedrosis virus (acmnpv) is required for the efficient replication and/or stability of reporter plasmids carrying an acmnpv-derived origin of dna replication in a cell-specific manner; hcf-1 is required for reporter plasmid replication or stability in tn-368 cells, a cell line derived from the cabbage looper trichoplusia ni, but not in iplb-sf-21 (sf-21) cells, a cell line derived from the fall army ... | 1996 | 8764020 |
| excision of the piggybac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase. | the piggybac lepidopteran transposable element moves from the cellular genome into infecting baculovirus genomes during passage of the virus in cultured tn-368 cells. we have constructed genetically tagged piggybac elements that permit analysis of excision when transiently introduced on plasmids into the piggybac-deficient spodoptera frugiperda iplb-sf21ae cell line. precise excision of the element from these plasmids occurs at a higher frequency in the presence of a helper plasmid that presumab ... | 1996 | 8765680 |
| a common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in acmnpv-infected spodoptera frugiperda larvae. | the assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. we have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or pep), to follow envelope assembly in acmnpv-infected tissues of spodoptera frugiperda larvae. we show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers ... | 1996 | 8774685 |
| production of a polyhydroxyalkanoate biopolymer in insect cells with a modified eucaryotic fatty acid synthase. | a novel pathway for the synthesis of poly-3-hydroxybutyrate has been engineered by simultaneous delivery of two genes into insect cells (spodoptera frugiperda) by use of individual baculovirus vectors. this system includes expression of a dehydrase-domain mutant rat fatty acid synthase cdna and the phbc gene encoding polyhydroxyalkanoate synthase from alcaligenes eutrophus. the dehydrase-deficient fatty acid synthase provides de novo synthesis of r-(-)-3-hydroxybutyryl-coenzyme a as a premature ... | 1996 | 8779593 |
| isolation of highly purified, functional carboxy-terminally truncated hepatitis b virus middle surface protein activators from eucaryotic expression systems. | carboxy-terminally truncated hepatitis b virus (hbv) middle surface proteins (mhbst) show a transcriptional activator function. two different subtypes of mhbst activators can be distinguished: an er-localized type, represented here by mhbst76 (truncated at amino acid 76), and a cytosol-localized type, represented here by mhbst63. to characterize the mhbst activator on the protein level and to analyze posttranslational modifications, we established recombinant baculoviruses encoding for fusion pr ... | 1996 | 8781314 |
| endogenous chloride channels of insect sf9 cells. evidence for coordinated activity of small elementary channel units. | the endogenous cl- conductance of spodoptera frugiperda (sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-gal cells). with the cation tris+ in the pipette and na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing cl- equilibrium potential and could be fitted by the goldman-hodgkin-katz equation with similar permeabilities for uninfected an ... | 1996 | 8783071 |
| quantification of systemic and local immune responses to individual rotavirus proteins during rotavirus infection in mice. | the purpose of the present study was to develop a quantitative assay that could be used to measure the local and systemic immune responses to specific rotavirus proteins following rotavirus infection of adult mice. to measure these responses, we used an immunocytochemical staining assay of spodoptera frugiperda (sf-9) cells which were infected with recombinant baculovirus expressing selected rotavirus proteins. the specificity of the assay was documented by using a series of monoclonal antibodie ... | 1996 | 8784572 |
| green fluorescent protein as a tool for screening recombinant baculoviruses. | the gene encoding the green fluorescent protein (gfp) from the jellyfish aequorea victoria, ligated to the honeybee melittin signal peptide-encoding sequence, was inserted under transcriptional control of the polyhedrin promoter of the autographa californica nuclear polyhedrosis virus and expressed in the spodoptera frugiperda insect cell line sf9 during viral infection. the recombinant green fluorescent protein was identified by sds-page gel electrophoresis followed by coomassie blue staining o ... | 1996 | 8793839 |
| attachment and entry of recombinant norwalk virus capsids to cultured human and animal cell lines. | norwalk virus (nv) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. while these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant norwalk virus-like particles (rnv vlps) that are morphologically and antigenically similar to native nv. we have used these rnv vlps to examine virus-cell interactions. binding and ... | 1996 | 8794293 |
| identification of insect cell lines by dna amplification fingerprinting (daf). | fingerprint profiles were generated from twenty insect cell lines spanning the orders, lepidoptera, diptera, coleoptera and homoptera employing dna amplification fingerprinting (daf) with arbitrarily selected primers. the fingerprint pattern is a stable characteristic of the cell line because high and low passages generated the same profile. in addition, insect hosts and homologous cell lines generated similar profiles. all cell lines could be distinguished from each other with the following exc ... | 1996 | 8799737 |
| characterization of normal and point-mutated human androgen receptors expressed in the baculovirus system. | the baculovirus system is able to generate large amounts of a protein, permitting detailed analysis of structure-function relations. we have used this system to overexpress and characterize normal human androgen receptors (har) and mutant hars from humans with complete or partial androgen insensitivity. maximum specific binding of [3h]mibolerone (mb) in recombinant baculovirus-infected spodoptera frugiperda (sf9) cells varied from 15 to 40 pmol/mg protein, about 1000-fold higher than in genital ... | 1995 | 8800637 |
| cfmnpv blocks acmnpv-induced apoptosis in a continuous midgut cell line. | morphological and molecular changes produced by autographa californica nuclear polyhedrosis virus (acmnpv) infection in a permissive cell line, iplb-sf-21ae (sf-21), of spodoptera frugiperda and a nonpermissive cell line, fpmi-cf-203 (cf-203), of choristoneura fumiferana are described. cf-203 cells inoculated with acmnpv showed a dna ladder and morphological changes such as plasma membrane granulation, blebbing, and nuclear fragmentation, which are characteristic of apoptosis. typical virus repl ... | 1996 | 8806500 |
| the mode of action of hirsutellin a on eukaryotic cells. | a 16-kda protein toxin was purified from hirsutella thompsonii var thompsonii and named hirsutellin a (hta). at 0.5 and 5.0 microm concentrations, hta caused detectable cytopathic effects on spodoptera frugiperda cells (sf-9) within 2-4 hr and completely inhibited sf-9 cell growth at 4 days posttreatment. electron microscope data showed that the hta treated sf-9 cells became hypotrophied and internal organelles and cell membranes were disrupted. at the same concentration, hta effectively inhibit ... | 1996 | 8812603 |
| purification of recombinant insect transferrin from large volumes of cell culture medium using high capacity ni(2+)-dipicolylamine gel. | we report the purification of secreted recombinant manduca sexta transferrin from spodoptera frugiperda (sf9) cell culture medium in a single step using high capacity ni(2+)-dipicolylamine (dpa)-novarose gel. although the original sample was highly diluted (approximately 10 micrograms transferrin/ml medium) and the cell culture medium contained 10% surfactant (pluronic f68) and a lipid emulsion, we were able to recover the recombinant transferrin (1 mg protein/100 ml) under gentle elution condit ... | 1996 | 8812846 |
| purification of a soluble hepatitis e open reading frame 2-derived protein with unique antigenic properties. | the second open reading frame (orf2) of hepatitis e virus (hev) is predicted to encode a 73-kda capsid protein (1). when full-length orf2 was expressed in insect cells (spodoptera frugiperda (sf9)) using a recombinant baculovirus, two distinct hev polypeptides were observed: a full-length insoluble 73-kda protein, and a soluble 56.5-kda protein. following purification and sequence analysis, it was determined that the 56.5-kda protein was derived from endoproteolytic cleavage site that was betwee ... | 1996 | 8812876 |
| secretion of green fluorescent protein from recombinant baculovirus-infected insect cells. | trichoplusia ni (high five) and spodoptera frugiperda (sf21) cells were engineered for expression of epitope (flag)-tagged signal peptide-green fluorescent protein (gfp) fusions to examine the suitability of gfp as a secretory marker. the recombinant baculovirus-infected cells became fluorescent, and the high five cells but not sf21 cells secreted gfp in the culture medium as detected by the presence in the culture supernatant of a flag-immunoreactive 30-kda species and the characteristic 510-nm ... | 1996 | 8831686 |
| immunogenic properties of rabbit haemorrhagic disease virus structural protein vp60 expressed by a recombinant baculovirus: an efficient vaccine. | we have constructed a recombinant baculovirus containing the gene encoding the structural protein vp60 from the spanish field isolate ast/89 of rabbit haemorrhagic disease virus (rhdv). infection of cultured spodoptera frugiperda sf9 cells with this recombinant virus resulted in the production of high yields of vp60 protein which did not seem to assemble to form virus like particles, but was antigenically similar to the corresponding viral protein obtained from purified virions. a vp60-dose stud ... | 1995 | 8837879 |
| the p1a alloantigen system is a sensitive indicator of the structural integrity of the amino-terminal domain of the human integrin beta 3 subunit. | within the native integrin alpha pi b beta 3, the conformation of the amino-terminal domain of the beta 3 subunit has a significant influence on the availability of the leu33/pro33 polymorphism that defines the a1 and a2 alleles, respectively, of the p1a alloantigen system. the majority of anti-p1a1 igg antibodies, affinity-purified by adsorption to either (a1/a1)-platelets or purified alpha pi b(a1) beta 3, fail to bind to the leu33 polymorphic loop within cys26-cys38 in native beta 3 unless th ... | 1995 | 8846042 |
| baculovirus-mediated high level expression of a human thiopurine methyl transferase. | we have expressed the human thiopurine methyltransferase cdna in a baculovirus vector in sf21 (spodoptera frugiperda) cells. this system expresses the enzyme at levels such that the thiopurine methyltransferase enzyme may be readily visualised by coomassie blue stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis. the expressed enzyme catalysed the methylation of 6-mercaptopurine with an apparent km of 892 microm, similar to that observed in human liver cytosol ie. 657 microm howev ... | 1996 | 8850531 |
| juvenile and mature map2 isoforms induce distinct patterns of process outgrowth. | microtubule-associated protein-2 (map2) is the most abundant map in neurons, where its distribution is restricted to the somatodendritic compartment. this molecule undergoes developmentally regulated alternative splicing, resulting in at least two isoforms, a juvenile isoform (termed map2c) and a mature isoform (map2), with greatly different molecular masses. spodoptera frugiperda (sf9) cell expression of the juvenile versus the mature map2 isoform generates two distinct patterns of process outg ... | 1996 | 8868472 |
| purification, biochemistry and molecular cloning of an insect glycosylasparaginase from spodoptera frugiperda. | glycosylasparaginase (ec 3.5.1.26) from sf9 cells (spodoptera frugiperda) was purified to homogeneity with a specific activity of 2.1 unit/mg. the enzyme is composed of two non-identical alpha/beta subunits joined by strong non-covalent forces and has one glycosylation site located in the alpha subunit. molecular masses of the subunits were determined to be 28 kda and 17 kda by sds-page. native enzyme existed in quaternary structures of either heterodimer (alpha beta) or heterotetramer (alpha 2 ... | 1996 | 8877373 |
| dengue virus envelope glycoprotein can be secreted from insect cells as a fusion with the maltose-binding protein. | the maltose-binding protein (male) contains a signal sequence which allows its translocation in the periplasm of prokaryotic microorganisms. in this study, male was produced in spodoptera frugiperda (sf9) lepidopterian cells using the baculovirus expression system. the secretion of male, following cleavage of its signal sequence, to the supernatant fluid of recombinant baculovirus-infected sf9 cells and its affinity for maltodextrin polymers allowed recovery of significant amounts (> or = 10 mic ... | 1996 | 8882648 |
| expression and purification of the seven nonstructural proteins of the flavivirus kunjin in the e. coli and the baculovirus expression systems. | all seven nonstructural (ns) proteins of the flavivirus kunjin (kun) ranging from ns1 to ns5 were expressed either alone or as fusion proteins with glutathione-s-transferase (gst). high level expression of recombinant proteins was achieved in spodoptera frugiperda (sf9) cells using the baculovirus expression system in contrast to the low level of expression in e. coli. the order of the level of expression of the recombinant fusion proteins per 4 x 10(7) sf9 cells was: gst-ns5 (yields approximate ... | 1996 | 8882936 |
| polycistronic (tri- or bicistronic) phytoreoviral segments translatable in both plant and insect cells. | genomic segment s12 of rice dwarf virus and segment s9 of wound tumor virus, both members of the genus phytoreovirus, have small out-of-phase overlapping open reading frames (orfs). western blot (immunoblot) analysis revealed that rice dwarf virus s12 mrna specified translation products from the large orf and two overlapping small orfs both in rice plant hosts and in spodoptera frugiperda insect cells. these results provide the first example of a tricistronic mrna for a segmented double-stranded ... | 1996 | 8892945 |
| molecular and immunological characterization of soluble aggregated a/victoria/3/75 (h3n2) influenza haemagglutinin expressed in insect cells. | a/victoria/3/75 (h3n2)-derived cdna coding for a secreted haemagglutinin (ha0s) was cloned into the polyhedrin promoter-based pvl1392 transfer vector, and a recombinant baculovirus was isolated. 5 to 10 micrograms/ml of secreted ha were obtained following infection of spodoptera frugiperda-9 cells. gel filtration revealed the presence in the cell supernatant of immunoreactive ha molecules with varying m(r). the high m(r) fraction (aha0s) could be purified by matrex cellufine sulphate and lentil- ... | 1996 | 8893793 |
| expression and coupling of human cytochrome p450 2e1 and nadph-cytochrome p450 oxidoreductase in dual expression and co-infection systems with baculovirus in insect cells. | in order to study the interaction between human cytochrome p450 2e1 (h2e1) and nadph-p450 oxidoreductase (hor) in a native membrane environment, we used two approaches to express both h2e1 and hor in a baculovirus expression system. for a dual-expression system, h2e1 and hor were coexpressed in spodoptera frugiperda (sf9) insect cells using a recombinant baculovirus carrying both h2e1 and hor cdnas (v-h2e1-hor). the h2e1 cdna was expressed under the control of the polyhedrin promoter p(polh), wh ... | 1996 | 8900414 |
| the production of a stably transformed insect cell line expressing an invertebrate gabaa receptor beta-subunit. | we have produced a stable insect cell line derived from spodoptera frugiperda (sf9) cells expressing a cdna encoding a beta-subunit of the lymnaea stagnalis gabaa receptor. the cdna was randomly integrated into the insect cell genome under the control of a baculovirus immediate early gene (ie-1) promoter. stable cell lines were established by transformation of sf9 cells with the expression vector piek1. lgbeta1 together with a plasmid encoding a selectable marker which confers neomycin (g418) re ... | 1995 | 8903929 |
| reconstitution of atp-dependent leukotriene c4 transport by co-expression of both half-molecules of human multidrug resistance protein in insect cells. | multidrug resistance protein (mrp) confers a multidrug resistance phenotype similar to that associated with overexpression of p-glycoprotein. unlike p-glycoprotein, mrp has also been shown to be a primary active atp-dependent transporter of conjugated organic anions. the mechanism(s) by which mrp transports these compounds and increases resistance to natural product drugs is unknown. to facilitate studies on the structure and function of mrp, we have determined whether a baculovirus expression s ... | 1996 | 8910374 |
| a baculovirus vector derived from immediately early gene promoter of autographa californica nuclear polyhedrosis virus. | a transfer vector was constructed in which the neomycin resistance (neo) gene was under the control of a copy of autographa californica nuclear polyhedrosis virus (acmnpv) ie1 gene promoter at the p10 locus. after cotransfection of spodoptera frugiperda (sf9) cells with the transfer vector and infectious acmnpv dna, the recombinant virus-containing neo gene was selected by serial passage of the mixed progenies from cotransfection. this was done at low moi in the presence of g418 in growth medium ... | 1996 | 8910650 |
| expression and loading of recombinant heavy and light chain homopolymers of rat liver ferritin. | the full-length genes for the heavy (h) and light (l) chains of ferritin isolated from a rat liver cdna library were amplified using polymerase chain reaction. each was inserted at the unique bglii site downstream of the p10 promoter of the baculovirus transfer vector pacuw21. the genes were transferred separately to infectious autographa californica nuclear polyhedrosis virus (acnpv) expression vectors after in vivo homologous recombination. ferritin homopolymers of either h or l chain were exp ... | 1996 | 8914851 |
| interactions of bacillus thuringiensis crystal proteins with the midgut epithelial cells of spodoptera frugiperda (lepidoptera: noctuidae). | binding of different bacillus thuringiensis insecticidal crystal proteins (icps) to the midgut epithelium of spodoptera frugiperda larvae was characterized by binding experiments with midgut tissue sections and isolated brush border membrane vesicles. our results show that icps interact with the microvilli of epithelial cells of s. frugiperda in two different ways. the first is typical of highly toxic proteins (like cryic and cryid); this interaction is saturable and specific. in contrast, some ... | 1996 | 8931361 |
| high-level expression of recombinant immunoreactive thyroid peroxidase in the high five insect cell line. | expression of a major thyroid autoantigen, thyroid peroxidase (tpo) was studied using the baculovirus-insect cell expression system. human tpo cdna modified so as to code for the extracellular fragment of the protein was placed under the control of the strong polyhedrin promoter in baculovirus transfer vector pbluebaciii and cotransfected with linearized acmnpv viral dna. expression in two insect cell lines spodoptera frugiperda (sf9) and tricoplusia ni (high five) was investigated and levels of ... | 1996 | 8938592 |
| the synaptic protein unc-18 is phosphorylated by protein kinase c. | the c. elegans unc-18 encoded protein unc-18 is implicated in the interactions between synaptic vesicles and presynaptic plasma membrane. to further characterize the neural protein, we investigated the phosphorylation in vitro of the protein expressed in spodoptera frugiperda sf21 cells. the unc-18 protein is selectively phosphorylated by protein kinase c (pkc) but not by casein kinase ii and cyclic amp-dependent protein kinase. the presumed phosphorylation sites determined by manual edman degra ... | 1996 | 8939464 |
| expression, purification and characterization of recombinant c. elegans unc-18. | the caenorhabditis elegans unc-18-encoded protein (unc-18) is implicated in the processes of vesicle targeting, docking, and/or fusion. to further characterize the properties of this important neural protein, we expressed it at a high level in spodoptera frugiperda sf21 cells using a baculovirus expressing system. a cdna containing the coding sequence for unc-18 was inserted into the transfer vector pbluebac to yield the recombinant virus pacnpv/unc-18. at maximal expression, the recombinant vir ... | 1996 | 8939465 |
| rat placental lactogen-i variant (rpl-iv), product of an unique gene, is biologically different from rpl-i. | the rat placenta expresses two rat placental lactogen-i (rpl-i) proteins: the normal rpl-i in the first half of pregnancy and a variant (rpl-iv) in the second half of pregnancy. they are 70% identical at the amino acid level but arise from different cell types: rpl-i from giant cells and rpl-iv from cytotrophoblasts. to assess whether rpl-iv originates from alternative splicing of the rpl-i gene or is the product of a separate gene, genomic clones of rpl-i and rpl-iv were isolated from a lambda ... | 1996 | 8940341 |
| glycosylation, palmitoylation, and localization of the human d2s receptor in baculovirus-infected insect cells. | in order to evaluate the baculovirus expression system as a means for high-yield production of homogeneous d2s receptor, we have expressed various d2s receptor constructs in two spodoptera frugiperda cell lines, a trichoplusia ni and a mammestra brassicae cell line. to improve expression yield, the environment of the polyhedrin gene translational initiation site was retained by fusing the first 12 codons of the polyhedrin gene to the 5'-end of the d2s receptor coding sequence. the pharmacologica ... | 1996 | 8952462 |
| expression and characterization of a truncated, soluble, low-density lipoprotein receptor. | the low-density lipoprotein (ldl) receptor mediates the clearance of apolipoprotein b- and e-containing lipoproteins from the bloodstream. in the current study, we characterized the binding properties of the amino terminus of the ldl receptor. we produced a recombinant baculovirus that encoded the first 354 amino acids, including the endogenous signal sequence, of the human ldl receptor. this truncated receptor protein (ldl-r354) was secreted from recombinant baculovirus-infected spodoptera frug ... | 1996 | 8954898 |
| interleukin-1 receptor antagonist: characterisation of its gene expression in rabbit tissues and large-scale expression in eucaryotic cells using a baculovirus expression system. | the gene expression of rabbit interleukin-1 receptor antagonist (rbil-lra) was examined in rabbit tissues. rna was isolated from heart, lung, kidney, muscle, liver, spleen, brain, and peripheral blood monocytes (pbms), and rbil-lra mrna was identified as a single species by northern analysis using a rbil-lra probe. rbil-lra was abundantly expressed in lung, brain, heart, and liver, expressed at low levels in spleen, and undetectable in kidney and unstimulated pbms. expression of large scale reco ... | 1996 | 8960095 |
| selective inhibition of cyclooxygenase-1 and -2 using intact insect cell assays. | we have utilized the baculovirus expression system to develop an in vitro intact cell assay for screening nonsteroidal anti-inflammatory drug (nsaid) inhibition of the two isozymes of human cyclooxygenase (prostaglandin endoperoxidase synthase, ec 1.14.99.1). infected spodoptera frugiperda (sf9) cells expressing either human cyclooxygenase-1 (hcox-1) or human cyclooxygenase-2 (hcox-2) were harvested 24 hr postinfection, a time point where all cells are viable and hcox-1 or hcox-2 are correctly p ... | 1996 | 8986141 |
| synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the e1 protein. | the two envelope glycoproteins of rubella virus (rv), e1 of 58 kda and e2 of 42-47 kda, were individually expressed in lepidopteran spodoptera frugiperda as well as in trichoplusia ni insect cells using baculovirus vectors. the authentic signal sequences of e1 and e2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. in addition, the hydrophobic transmembrane anchors at the carboxyl termini of e1 and e2 proteins we ... | 1996 | 8987625 |
| role of early and late replication events in induction of apoptosis by baculoviruses. | autographa californica nuclear polyhedrosis virus (acmnpv) mutants that lack the apoptotic suppressor gene p35 cause apoptosis in spodoptera frugiperda sf21 cells. to identify a viral signal(s) that induces programmed cell death, we first defined the timing of apoptotic events during infection. activation of a p35-inhibitable caspase, intracellular fragmentation of host and acmnpv dna, and cell membrane blebbing coincided with the initiation of viral dna synthesis between 9 and 12 h after infect ... | 1997 | 8995679 |
| spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin fkbp46, is the target of the baculovirus antiapoptotic protein p35. | employing the degenerate primer-dependent polymerase chain reaction approach used recently to clone human mch2, we have identified and cloned the insect spodoptera frugiperda target of the baculovirus antiapoptotic protein p35. this protein named sf caspase-1 belongs to the family of caspases and is highly related to human mch3 and cpp32 in sequence and specific activity. the proenzyme of sf caspase-1 is 299 amino acids in length and can undergo autocatalytic processing in escherichia coli to an ... | 1997 | 8999805 |
| reconstitution of receptors and gtp-binding regulatory proteins (g proteins) in sf9 cells. a direct evaluation of selectivity in receptor.g protein coupling. | the selectivity in coupling of various receptors to gtp-binding regulatory proteins (g proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in spodoptera frugiperda (sf9) cells. activation of g proteins was monitored in membranes prepared from sf9 cells co-expressing selected pairs of receptors and g proteins (i.e. alpha, beta1, and gamma2 subunits). membranes were incubated with [35s]guanosine 5'-(3-o-thio)triphosphate (gtpgammas) +/- an agonist, and the ... | 1997 | 8999927 |
| role of diacylglycerol-regulated protein kinase c isotypes in growth factor activation of the raf-1 protein kinase. | the raf protein kinases function downstream of ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. interaction with ras recruits raf to the plasma membrane, but the subsequent mechanism of raf activation has not been established. previous studies implicated hydrolysis of phosphatidylcholine (pc) in raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase c (pkc), which is stimulated by pc-derived diacylglyce ... | 1997 | 9001227 |
| processing in vitro of pronapin, the 2s storage-protein precursor of brassica napus produced in a baculovirus expression system. | the maturation of the 2s albumin, napin, in brassica napus l. involves removal of an amino-terminal and an internal propeptide. pulse-chase experiments with b. napus embryos showed that intermediates are detectable during the pronapin processing. intact pronapin was expressed by baculovirus in spodoptera frugiperda insect cells in order to obtain substrate for studying the processing event. processing of pronapin with a crude b. napus embryo protein extract resulted in several fragments of simil ... | 1996 | 9004547 |
| insecticidal activity of a recombinant baculovirus containing an antisense c-myc fragment. | attempts to develop baculovirus-based insecticides by insertion of genes encoding enzyme inhibitors, neuropeptides or toxins have met with some success. however, it is often difficult to ensure correct processing or secretion of the encoded peptides. here we tested a simpler strategy by insertion of an antisense fragment of a host gene to block translation of a protein essential for larval growth and development. we selected the c-myc gene for two main reasons: (i) its protein is known to be wel ... | 1997 | 9010314 |
| transposable elements and gene transformation in non-drosophilid insects. | this review summarizes recent data on the development of non-drosophilid insect transformation systems. the discussion focuses on one particular approach to developing transformation systems that relies on the use of short inverted repeat-type transposable elements analogous to that employed for drosophila melanogaster transformation. representatives from four families of short inverted repeat-type transposable elements have been shown to either act as non-drosophilid gene vectors or to have the ... | 1996 | 9014324 |
| detection and identification of multiple baculoviruses using the polymerase chain reaction (pcr) and restriction endonuclease analysis. | a technique using the polymerase chain reaction (pcr) and restriction analysis was developed for the simultaneous detection of eight baculoviruses. the baculoviruses detected by this technique were autographa californica multiple-embedded nuclear polyhedrosis virus (mnpv). anticarsia gemmatalis mnpv, bombyx mori mnpv, orgyia pseudotsugata mnpv. spodoptera frugiperda mnpv, s. exigua mnpv, anagrapha falcifera mnpv, heliothis zea single-embedded nuclear polyhedrosis virus (snpv). a highly conserved ... | 1997 | 9015292 |
| the bacillus thuringiensis vegetative insecticidal protein vip3a lyses midgut epithelium cells of susceptible insects. | the vip3a protein is a member of a newly discovered class of vegetative insecticidal proteins with activity against a broad spectrum of lepidopteran insects. histopathological observations indicate that vip3a ingestion by susceptible insects such as the black cutworm (agrotis ipsilon) and fall armyworm (spodoptera frugiperda) causes gut paralysis at concentrations as low as 4 ng/cm2 of diet and complete lysis of gut epithelium cells resulting in larval death at concentrations above 40 ng/cm2. th ... | 1997 | 9023933 |
| replication of anticarsia gemmatalis nuclear polyhedrosis virus in four lepidopteran cell lines. | anticarsia gemmatalis nuclear polyhedrosis virus (agnpv; family baculoviridae) is pathogenic for larvae of the velvetbean caterpillar, anticarsia gemmatalis hübner-an important pest of soybean. agnpv is a viable alternative to chemical control of a. gemmatalis in brazil, where its use as a pesticide has brought significant economic and environmental benefits. although a significant amount of information is available on the ecological and biological control aspects of agnpv, very little is known ... | 1997 | 9028926 |
| inactivation of conidia of paecilomyces fumosoroseus by near-ultraviolet (uvb and uva) and visible radiation. | the detrimental effects of solar radiation, especially the ultraviolet waveband, on quiescent conidia of paecilomyces fumosoroseus were investigated. conidia were irradiated by a high-intensity source, which emitted a continuous spectrum from 270 to 1100 nm and which was equipped with long-pass filters to block short wavelengths below 280, 295, 320, or 400 nm. after irradiation, conidia were tested for germinability, survival, and infectivity toward spodoptera frugiperda larvae. it was demonstra ... | 1997 | 9028931 |
| 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. immunodetec ... | 1997 | 9029498 |
| expression of the mouse interleukin-2 receptor gamma chain in insect cells using a baculovirus expression vector--comparison with the human common gamma chain. | the gene encoding the gamma-chain of the mouse interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. the corresponding gene was inserted under the polyhedrin promoter of the autographa californica nuclear polyhedrosis virus and expressed in the spodoptera frugiperda insect cell line sf9 during viral infection. the recombinant receptor protein was identified by immunoblotting in cell lysates prepared from insect cells infected with the p ... | 1997 | 9042425 |
| the human immunodeficiency virus type 1 nef protein functions as a protein kinase c substrate in vitro. | the human immunodeficiency virus type 1 nef protein was expressed in escherichia coli as a c-terminal fusion with glutathione s-transferase (gst). the ability of gst-nef to act as a substrate for cellular kinases in vitro was examined by incubation of purified gst-nef fusion proteins, immobilized on glutathione-agarose beads, with cytoplasmic extracts from a number of human cell lines. in the presence of [gamma32p]atp, phosphorylation of nef occurred predominantly on serine residues. studies wit ... | 1995 | 9049329 |
| maturation of the dengue-2 virus ns1 protein in insect cells: effects of downstream ns2a sequences on baculovirus-expressed gene constructs. | a series of recombinant baculoviruses was constructed in order to study the influence of downstream ns2a sequences on the processing of the dengue virus ns1 glycoprotein in insect cells. ns1 alone was expressed at a high level in its native dimeric form and processed efficiently through the spodoptera frugiperda (sf) cell secretory pathway. recombinant ns1 was found associated with the plasma membrane and was also secreted into the extracellular medium. although both intra- and extracellular ns1 ... | 1995 | 9049346 |
| translational properties of the untranslated regions of the p10 messenger rna of autographa californica multicapsid nucleopolyhedrovirus. | protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mrnas. therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mrnas of autographa californica multicapsid nucleopolyhedrovirus. the untranslated regions (utrs) of the p10 mrna of this baculovirus were studied by in vitro translation and after transfection into spodoptera frugiperda insect cells. lysates from ... | 1997 | 9049423 |
| a novel potassium channel blocking toxin from the scorpion pandinus imperator: a 1h nmr analysis using a nano-nmr probe. | the three-dimensional solution structure of a novel peptide, pi 1, purified from the venom of the scorpion pandinus imperator and specific for potassium channels was determined by homonuclear proton nmr methods at 500 mhz from nanomole amounts of compound. p. imperator toxin is a voltage-dependent potassium channel specific peptide capable of blocking the shaker b k+ channels expressed in sf9 cells in culture (spodoptera frugiperda cell line no. 9) and displacing labeled noxiustoxin from rat bra ... | 1997 | 9054572 |
| characterization of human lactoferrin produced in the baculovirus expression system. | lactoferrin, an iron-binding 80-kda glycoprotein, is a major component of human milk whose structure is now well defined. the binding site of lactoferrin to the membrane receptor of lymphocyte has been located in the region 4-52, but the amino acids directly involved in the interaction have not been identified yet. to gain further insights into the structure-function relationships of the lactoferrin binding site, we first expressed the cdna encoding human lactoferrin in the lepidoptera spodopter ... | 1997 | 9056485 |
| purification and characterization of full-length mammalian poly(a) polymerase. | bovine poly(a) polymerase was purified from overexpressing strains of escherichia coli and from spodoptera frugiperda sf21 cells infected with a recombinant baculovirus. the e. coli-expressed enzyme had an apparent molecular mass of 85 kda in sds gels, as anticipated from the cdna sequence. poly(a) polymerase from insect cells consisted of several species with higher apparent molecular weights due to phosphorylation. the two preparations showed minor differences in their catalytic properties. th ... | 1997 | 9061026 |
| analysis of the glycine binding domain of the nmda receptor channel zeta 1 subunit. | in an attempt to examine glycine binding domain of the zeta 1 subunit of the mouse n-methyl-d-aspartate (nmda) receptor channel, we constructed n-terminal or c-terminal deletion mutants of the zeta 1 subunit cdna and expressed them in spodoptera frugiperda cells using a baculovirus system. analysis of binding of a glycine binding site antagonist, 5,7-[3(-3)h]dichlorokynurenate ([3h]dcka) to the deleted zeta 1 mutants provided the first direct experimental evidence that the regions of n-terminal ... | 1997 | 9080426 |
| infection of a spodoptera frugiperda cell line with bombyx mori nucleopolyhedrovirus. | the interactions of bombyx mori nucleopolyhedrovirus (bmnpv) with spodoptera frugiperda cells (sf9) were investigated. s. frugiperda cells are usually considered nonpermissive for bmnpv. however, in the present study, bmnpv dna replication was observed and an increasing infectious titre, reaching 10(4) tcid50/ml on b. mori permissive cells by 6 days post-transfection, developed in the supernatant of infected sf9 cells. infection of sf9 cells by bmnpv did not induce a discernible shutoff of cellu ... | 1997 | 9085549 |
| functional properties of cyp2d6 1 (wild-type) and cyp2d6 7 (his324pro) expressed by recombinant baculovirus in insect cells. | the debrisoquine/sparteine or cyp2d6 genetic polymorphism of drug oxidation is a common cause for interindividual variability in drug response. we recently identified a mutant allele, designated cyp2d6-e or cyp2d6*7, which is associated with the poor metabolizer phenotype and occurs in caucasian populations with a frequency of about 1%. in contrast to other loss-of-function alleles, a full length protein with a single amino acid substitution. his324pro, is encoded by the cyp2d6*7 allele. to func ... | 1997 | 9089660 |
| expression of rat steroid 5 alpha-reductase (isozyme-1) in spodoptera frugiperda, sf21, insect cells: expression of rat steroid 5 alpha-reductase. | the enzyme steroid 5 alpha-reductase (5 alpha r) catalyzes the reduction of testosterone (t) to 5 alpha-dihydrotestosterone (dht). in this study, the baculovirus expression system was used to overexpress rat 5 alpha r type i isozyme (r5 alpha r 1). the full length of r5 alpha r1 cdna was inserted into the autographa californica nuclear polyhedrosis virus (ac-mnpv) genome and expressed in spodoptera frugiperda, sf 21, insect cells. the expressed recombinant r5 alpha-r1 showed maximal enzymatic ac ... | 1997 | 9090798 |
| the second intracellular loop of the alpha2-adrenergic receptors determines subtype-specific coupling to camp production. | the alpha2-adrenergic receptors (alpha2-ars), which primarily couple to inhibition of camp production, have been reported to have a stimulating effect on adenylyl cyclase activity in certain cases. when expressed in spodoptera frugiperda sf9 cells the alpha2a subtype showed only inhibition of forskolin-stimulated camp production when activated by norepinephrine (ne), whereas the alpha2b subtype displayed a biphasic dose-response curve with inhibition at low concentrations of ne and a potentiatio ... | 1997 | 9092501 |
| identification of recombinant baculoviruses using green fluorescent protein as a selectable marker. | a rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the aquorea victoria green fluorescent protein (gfp). expression of the gfp cdna (without signal peptide sequence) in spodoptera frugiperda cells resulted in the synthesis of a 30-kda protein, which was confirmed as gfp by western blotting and by the emission of green fluorescence when illuminated with longwave uv light (495 or 365 nm). to use gfp as a mar ... | 1997 | 9105619 |
| variant cdna sequences of human atp:citrate lyase: cloning, expression, and purification from baculovirus-infected insect cells. | atp:citrate lyase (acl) is a major generator of cytosolic acetyl-coenzymea, which is required for both fatty acid and cholesterol biosynthesis. the human acl (hacl) cdna was cloned by rt-pcr, and our results indicate the existence of previously unknown sequence variations in hacl. expression of the hacl cdna in spodoptera frugiperda 9 insect cells resulted in the production of high levels of soluble, active enzyme. the recombinant protein (re-hacl) was purified to homogeneity from the soluble ly ... | 1997 | 9116495 |
| the production and purification of functional decorin in a baculovirus system. | human decorin was expressed in spodoptera frugiperda 21 (sf21) insect cells. a full-length cdna encoding preprodecorin of 359 amino acids from a human fibroblast library was cloned into baculovirus transfer vector pvl1392, and transfected into sf21 insect cells. the infected cells secreted the mature decorin into the culture medium. the secreted decorin lacked glycosaminoglycan but was n-glycosylated, whereas the unmodified decorin was present in the cell lysates, suggesting that n-glycosylation ... | 1997 | 9125159 |
| biophysical properties of heterotypic gap junctions newly formed between two types of insect cells. | 1. cell pairs of the insect cell line sf9 (spodoptera frugiperda) were chosen to examine the electrical properties of gap junction channels. the dual voltage-clamp method was used to control the membrane potential of each cell (vm,1 and vm,2) and hence the junctional voltage gradient (vj), and to measure intercellular current. 2. studies with preformed pairs revealed that the gap junction conductance (gj) is controlled by a vj- and a vm-sensitive gate. at steady state, gj = f(vj) was bell shaped ... | 1997 | 9130166 |