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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 ...19968738213
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 ...19968742824
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 ...19968753801
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 ...19968764020
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 ...19968765680
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 ...19968774685
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 ...19968779593
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 ...19968781314
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 ...19968783071
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 ...19968784572
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 ...19968793839
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 ...19968794293
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 ...19968799737
comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells.glucose and glutamine metabolism in several cultured mammalian cell lines (bhk, cho, and hybridoma cell lines) were investigated by correlating specific utilization and formation rates with specific maximum activities of regulatory enzymes involved in glycolysis and glutaminolysis. results were compared with data from two insect cell lines and primary liver cells. flux distribution was measured in a representative mammalian (bhk) and an insect (spodoptera frugiperda) cell line using radioactive ...19968557765
identification of a short domain within the non-structural protein ns2 of epizootic haemorrhagic disease virus that is important for single strand rna-binding activity.the role that a conserved amino acid motif, found in the non-structural protein ns2 of orbiviruses, plays in the interaction of this protein with single stranded (ss) rna was investigated by mutation analysis of the ns2 of epizootic haemorrhagic disease virus. an ns2 mutant in which this motif (amino acids 75 to 83) was deleted was expressed in spodoptera frugiperda cells by a recombinant baculovirus and found to be unable to bind to poly(u)-sepharose. the deletion mutant also differed from wild ...19968558121
the dna polymerase and helicase genes of a baculovirus of orgyia pseudosugata.regions of the genome of the orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (opmnpv) containing the dna polymerase and helicase genes were sequenced. the dna polymerase and helicase genes encode predicted proteins of 985 (112.6 kda) and 1223 (140.5 kda) amino acids and exhibited 63 % and 59 % amino acid identity, respectively, with their homologues in the autographa californica mnpv (acmnpv). the influence of sequence variation between the opmnpv and acmnpv dna polymerase and ...19968609478
induction of specific allograft immunity by soluble class i mhc heavy chain protein produced in a baculovirus expression system.spodoptera frugiperda (sf9) insect cells secreted a class i mhc rt1.aa heavy chain protein when infected with baculovirus that bore a construct that contained a honeybee melittin secretion (ms) signal attached to rt1.aa cdna. the rt1.aa heavy chain protein in the culture supernatant and cell lysate immunoprecipitated in the presence of 5 individual anti-rt1.aa-specific mab. as was revealed by densitometric analysis, the ms signal increased the production (7- to 17-fold) and secretion (20- to 47- ...19968610360
studies of choristoneura fumiferana nuclear polyhedrosis virus gene expression in insect cells.to investigate the mechanisms regulating baculovirus virulence and host range we have begun to study choristoneura fumiferana nuclear polyhedrosis virus (cfmnpv) and its gene expression in permissive and nonpermissive cells. we have identified and mapped three genes on the cfmnpv genome. the polyhedrin gene is located from 0.0 to 2.0 m.u. and two other genes, dnapol and p143, both of which are essential for baculovirus dna replication, are located from 35.3 to 40.9 m.u. and 55.5 to 63.4 m.u., re ...19968610448
purification of a virus-induced rna polymerase from autographa californica nuclear polyhedrosis virus-infected spodoptera frugiperda cells that accurately initiates late and very late transcription in vitro.the virus-induced rna polymerase from autographa californica nuclear polyhedrosis virus-infected spodoptera frugiperda cells was separated from the three host nuclear rna polymerases by deae-sephadex chromatography and then purified through two more steps: heparin-agarose chromatography and glycerol gradient ultracentrifugation. fractions from each of these purification steps have been assayed in vitro for the ability to perform accurate initiation of transcription on a late (p6.9) and a very la ...19968614978
trans-activation of a cell housekeeping gene promoter by the ie1 gene product of baculoviruses.protein ie1 is the product of a baculovirus gene, ie1, that is activated immediately upon entrance of the viral genome into the cell nucleus. this protein was previously shown to be a trans-regulator of viral genes whose products are required for initiation of the infectious cycle including viral dna replication. to test whether the ie1 protein is also capable of trans-regulating nuclear genes of the host in vitro and in vivo, we transfected the ie1 gene of bombyx mori nuclear polyhedrosis virus ...19968615012
the role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity.the microsomal triacylglycerol transfer protein (mtp), an alpha beta dimer, is obligatory for the assembly of apob-containing lipoproteins in liver and intestinal cells. the beta subunit is identical with protein disulphide isomerase, a 58 kda endoplasmic reticulum luminal protein involved in ensuring correct disulphide bond formation of newly synthesized proteins. we report here the expression of the human mtp subunits in spodoptera frugiperda cells. when the alpha subunit was expressed alone, ...19968615825
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 ...19968617952
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 ...19968619796
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 ...19968626553
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 ...19968629943
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 ...19968631350
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 ...19968632351
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 ...19968636128
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 ...19968638923
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 ...19968639621
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 ...19968642651
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) ...19968643585
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 ...19968645349
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 ...19968647461
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 ...19968648750
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 ...19968654563
a viral movement protein as a nuclear shuttle. the geminivirus br1 movement protein contains domains essential for interaction with bl1 and nuclear localization.for the nuclear replicating bipartite geminiviruses such as squash leaf curl to systemically infect the host requires the active participation of two virus-encoded movement proteins, br1 and bl1. these act in a cooperative manner to transport the viral single-stranded dna genome from its site of replication in the nucleus to the cell periphery (a.a. sanderfoot, s.g. lazarowitz [1995] plant cell 7: 1185-1194). we have proposed that br1 functions as a nuclear shuttle protein, transporting the vira ...19968587985
isolation of the full-length murine erythropoietin receptor using a baculovirus expression system.the full-length murine erythropoietin receptor was expressed in spodoptera frugiperda (sf9) cells using a recombinant baculovirus vector. erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kd. erythropoietin receptors produced in sf9 cells could be solubilized using chaps in a form capable of binding erythropoietin, and the solubilized receptor bound to im ...19968562964
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 ...19979049423
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 ...19979054572
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 ...19979056485
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 ...19979061026
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 ...19979080426
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 ...19979085549
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 ...19979089660
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 ...19979090798
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 ...19979092501
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 ...19979105619
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 ...19979116495
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 ...19979125159
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 ...19979130166
in vitro assembly of sv40 virions and pseudovirions: vector development for gene therapy.sv40 is an attractive potential vector with high-efficiency gene transfer into a wide variety of human tissues, including the bone marrow, a critical target organ for the cure of many diseases. in the present study, the three sv40 capsid proteins, vp1, vp2, and vp3, were produced in spodoptera frugiperda (sf9) insect cells. their co-production led to spontaneous assembly of sv40-like particles. nuclear extracts containing the three proteins were allowed to interact with purified sv40 dna, or wit ...19979143910
expression of a fungal cellobiohydrolase in insect cells.the gene for trichoderma reesei cellobiohydrolase i (cbhi) was expressed with a recombinant baculovirus and high levels of secreted protein were produced in spodoptera frugiperda and trichoplusia ni insect cells. electrophoretic analysis indicated that the recombinant cbhi (rcbhi) was similar in apparent molecular weight to the native form and immunoblotting with anti-cbhi monoclonal antibodies confirmed its identity. the rcbhi was easily purified by affinity and hydrophobic interaction chromato ...19979144389
cloning and characterization of a metabotropic glutamate receptor, mglur4b.an alternative spliced variant of metabotropic glutamate receptor subtype mglur4a, termed mglur4b was isolated from a rat cdna library. subtype mglur4b was identical to the previously described mglur4a, except for the last 64 amino acids in the c-terminal region in which were replaced by 135 new amino acids in mglur4b. recombinant baculoviruses coding for mglur4a and mglur4b were expressed in spodoptera frugiperda, sf-9, insect cells and characterized pharmacologically by measuring [3h]-l-2-amin ...19979144638
virus-like particle-induced fusion from without in tissue culture cells: role of outer-layer proteins vp4 and vp7.we recently described an assay that measures fusion from without induced in tissue culture cells by rotavirus, a nonenveloped, triple-protein-layered member of the reoviridae family (m. m. falconer, j. m. gilbert, a. m. roper, h. b. greenberg, and j. s. gavora, j. virol. 69:5582-5591, 1995). the conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection of host cells, as the presence of the outer-layer protei ...19979151849
cytolytic activity of bacillus thuringiensis cryic and cryiac toxins to spodoptera sp. midgut epithelial cells in vitro.a sensitive lactate dehydrogenase (ldh) assay was modified to determine the cytolytic activity of bacillus thuringiensis cryic and cryiac delta endotoxins to viable collagenase-dissociated midgut epithelial cells (mec) from larvae of spodoptera frugiperda and spodoptera exigua. the mec preparations from these spodoptera sp. consisted predominantly of columnar cells (65-75%) and goblet cells (25-35%). time course microscopy experiments indicated that only the columnar cells became swollen during ...19979156349
agonist-induced [35s]gtpgammas binding in the membranes of spodoptera frugiperda insect cells expressing the human d3 dopamine receptor.in the membranes of spodoptera frugiperda (sf-9) insect cells heterologously expressing the human d3 dopamine receptor, agonists selective for the receptor, but not antagonists, robustly enhanced [35s]gtpgammas binding. quinpirole, for instance, dose-dependently enhanced [35s]gtpgammas binding with a half-maximal concentration of 2.3 +/- 0.2 nm. its action was absent in the cells infected with wild type viruses, and competitively blocked by an antagonist, ym-09151-2. a number of known agonists e ...19979159497
membrane lipid metabolism and phospholipase activity in insect spodoptera frugiperda 9 ovarian cells.although there is increasing use of insect ovarian sf9 cells for the production of recombinant proteins, namely, via the baculovirus vector expression system, little is known about the lipids in the cell membrane and whether endogenous phospholipases are present for regulation of the cell membrane lipids. in this study, analysis of membrane lipids of sf9 cells indicated the presence of phosphatidylethanolamine (pe) (diacyl type) and phosphatidylcholine as major phospholipids, followed by phospha ...19979168454
characterization of the human multidrug resistance protein containing mutations in the atp-binding cassette signature region.a number of mutants with single amino acid replacements were generated in the highly conserved atp-binding cassette (abc)-signature region (amino acids 531-543) of the n-terminal half of the human multidrug resistance (mdr1) protein. the cdna variants were inserted into recombinant baculoviruses and the mdr1 proteins were expressed in spodoptera frugiperda (sf9) insect cells. the level of expression and membrane insertion of the mdr1 variants was examined by immunostaining, and mdr1 function was ...19979169612
localization of cryptic tolerogenic epitopes in the alpha1-helical region of the rt1.au alloantigen.transplantation tolerance is induced by perioperative administration of host class i major histocompatibility complex proteins bearing donor-type amino acid (a.a.) epitopes substituted for native residues. herein we demonstrate that two cryptic tolerogenic a.a. epitopes are localized in the alpha1-helical region of the rat rt1.au class i major histocompatibility complex alloantigen.19979175796
multiplication of tomato spotted wilt virus in primary cell cultures derived from two thrips species.primary cell cultures prepared from embryos of the thrips species frankliniella occidentalis and thrips tabaci were tested for their potential to support replication of tomato spotted wilt virus (tswv). using polyclonal antibodies against the viral nucleocapsid protein (n) and indirect immunofluorescent staining, discrete spots with strong signals were observed in the cytoplasm at 48 h post-inoculation in the cell cultures of a f. occidentalis, and a t. tabaci population which failed to transmit ...19979178497
optimal ligand binding by the recombinant human glucocorticoid receptor and assembly of the receptor complex with heat shock protein 90 correlate with high intracellular atp levels in spodoptera frugiperda cells.the full-length human glucocorticoid receptor (hgr), overexpressed in spodoptera frugiperda (sf9) cells, associates with heat shock protein 90 (hsp90) and hsp70 and binds dexamethasone with high affinity. baculovirus infection of sf9 cells grown in tnm-fh medium results in the rapid depletion of glucose from the medium within 24 h. noting a discrepancy between hgr protein levels and ligand binding capacity in such cultures, we hypothesized that the depletion of glucose from the medium could resu ...19979182852
the prothoracicotropic hormone bombyxin has specific receptors on insect ovarian cells.bombyxin ii, a product of the brain of the adult silkmoth, bombyx mori, binds to ovarian cells of three different species of lepidoptera, i.e. b. mori (silkmoth), samia cynthia ricini (ailanthus moth), and an ovarian cell line of spodoptera frugiperda (sf9) (fall armyworm). crude sf9 cell membrane preparations were used to show that the purported bombyxin receptor binds its ligand in a specific, saturable, and reversible manner. the dissociation constant of the bombyxin-receptor complex is 260+/ ...19979183018
new structure and function in plant k+ channels: kco1, an outward rectifier with a steep ca2+ dependency.potassium (k+) channels mediating important physiological functions are characterized by a common pore-forming (p) domain. we report the cloning and functional analysis of the first higher plant outward rectifying k+ channel (kco1) from arabidopsis thaliana. kco1 belongs to a new class of 'two-pore' k+ channels recently described in human and yeast. kco1 has four putative transmembrane segments and tandem calcium-binding ef-hand motifs. heterologous expression of kco1 in baculovirus-infected ins ...19979184204
purification and characterization of secreted human leptin produced in baculovirus-infected insect cells.the product of the human ob (obesity) gene, leptin, appears to function in the maintenance of body weight in vivo. when injected into mice, this hormone reduces food consumption and causes weight loss. this work has been done with recombinant leptin (re-leptin) purified and renatured from inclusion bodies in escherichia coli. we have expressed the human obesity gene encoding the predicted full-length leptin in spodoptera frugiperda (sf-9) cells by infection with the recombinant baculovirus syste ...19979185858
expression of lacz reporter gene under the control of the polyhedrin promoter of spodoptera litura nuclear polyhedrosis virus.promoter function of the putative polyhedrin-encoding gene (polh) of spodoptera litura nuclear polyhedrosis virus (s1mnpv) was determined by transferring it to the autographa californica nuclear polyhedrosis virus (acmnpv) through the acnpv polh based vector, pvl1393. three transfer vectors pcbt2, pcbt3 and pcbt4 were constructed by substituting the promoter and the neighbouring sequences of acnpv in pvl1393 by that of s1npv. the escherichia coli lacz gene was placed downstream from the s1npv po ...19979185860
functional co-expression of the canine cardiac ca2+ pump and phospholamban in spodoptera frugiperda (sf21) cells reveals new insights on atpase regulation.the utility of the baculovirus cell expression system for investigating ca2+-atpase and phospholamban regulatory interactions was examined. cdna encoding the canine cardiac sarco(endo)plasmic ca2+-atpase pump (serca2a) was cloned for the first time and expressed in the presence and absence of phospholamban in spodoptera frugiperda (sf21) insect cells. the recombinant ca2+ pump was produced in high yield, contributing 20% of the total membrane protein in sf21 microsomes. at least 70% of the expre ...19979188486
identification and functional analysis of a non-hr origin of dna replication in the genome of spodoptera exigua multicapsid nucleopolyhedrovirus.the genome of spodoptera exigua multicapsid nucleopolyhedrovirus (semnpv) was screened for the presence of putative origins of dna replication (oris). using a transient dna replication assay, several fragments were identified that underwent semnpv-dependent dna replication in spodoptera frugiperda cells (sf-ae-21). preliminary sequence data revealed the presence of multiple copies of homologous repeats (hrs). restriction fragment xbal-f2 showed a distinct sequence reminiscent of autographa calif ...19979191948
baculovirus-directed expression of rabbit udp-glucuronosyltransferases in spodoptera frugiperda cells.the rabbit liver udp-glucuronosyltransferase (ugt) cdnas that encode the 4-hydroxybiphenyl ugt2b13 and 4-nitrophenol ugt1a6 have been cloned into baculovirus. spodoptera frugiperda (sf-9) cells infected with the ugt recombinant baculovirus produced significant amounts of protein, which was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, and pulse chase with 35s-amino acids. the expression of the ugt proteins in sf-9 cells were detected at approximately ...19979193877
cultivation of fall armyworm ovary cells in simulated microgravity.a methodology is presented to culture fall armyworm ovary cells in simulated micrograviy using a novel bioreactor developed by nasa, the high-aspect ratio vessel. in this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture. specifically, stationary phase in the nasa vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 x 10(7) viable cells/ml and 90%, respectiv ...19979196890
characterization of p91, a protein associated with virions of an orgyia pseudotsugata baculovirus.polyclonal antiserum produced against preoccluded virions from the orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (opmnpv) was used to screen an opmnpv lambda gt11 expression library. the insert from one of the immunoreactive phage isolates hybridized to opmnpv orf86 (p91), a 2460-bp (819 amino acids) open reading frame that encodes a predicted protein of 91 kda. antibodies generated against a maltose binding protein-p91 fusion detected a band of approximately 91 kda on wester ...19979201231
differential requirements of two insect cell lines for growth in serum-free medium.the development of a serum-free medium that supports the growth of cells from a spodoptera frugiperda and a lymantria dispar cell line is reported. a yeast hydrolysate provided the b-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. supplemental cystine and methionine were required to achieve maximum cell growth. the serum or serum replacements used in earlier formulations were replaced with commercial lipid prep ...19979201517
obtaining a functional recombinant anti-rhesus (d) antibody using the baculovirus-insect cell expression system.the cloning and production of a human anti-rhesus (rh) d monoclonal antibody (mab) using the baculovirus-insect cell expression system is described. this monoclonal recombinant antibody r.d7c2 derived from a human parental igm lambda immunoglobulin was obtained after immortalization of lymphocytes by epstein-barr virus (ebv). the human heavy (vh) and light (vl) variable regions were cloned from the parental cell line and genetically fused to the human constant igg1 heavy (h) and light (l) chain ...19979203960
baculovirus expression of the respiratory syncytial virus fusion protein using trichoplusia ni insect cells.respiratory syncytial virus (rsv) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. the rsv fusion (f) protein is highly conserved among rsv subgroups a and b and is the major protective immunogen. a genetically-engineered version of the rsv f protein was produced in insect cells using the baculovirus expression system. to express a secreted form of this protein, the transmembrane domain was eliminated by removing the regio ...19979208456
contact inhibition-induced inactivation of the cyclin d-dependent kinase in rat fibroblast cell line, 3y1.inhibitory proteins for cdks (ckis) are involved in cell cycle arrest induced by anti-mitotic factors, chemicals, or dna damage in mammalian cells. high cell density also induces cell cycle arrest with unreplicated genomic dna even in the presence of mitotic dose of the growth factors, termed contact inhibition. although rat fibroblast cell line, 3y1, arrested in quiescence by contact inhibition, cdk4 bound its regulatory subunit, cyclin d1 or d3. however, these complexes were enzymatically inac ...19979209390
purification and properties of alpha-mannosidase ii from golgi-like membranes of baculovirus-infected spodoptera frugiperda (iplb-sf-21ae) cells.an alpha-mannosidase ii-like activity was identified in baculovirus-infected spodoptera frugiperda (iplb-sf21-ae) cells. the enzyme responsible was purified from golgi-type membranes to apparent homogeneity by using a combination of steps including deae-cellulose, hydroxyapatite, concanavalin a-sepharose and gel filtration chromatography. the molecular mass of this purified protein was approx. 120 kda by sds/page under reducing conditions and approx. 240 kda under non-reducing conditions, indica ...19979210421
characterization of the malonyl-/acetyltransacylase domain of the multifunctional animal fatty acid synthase by expression in escherichia coli and refolding in vitro.cdnas of various lengths encoding the second domain of the multifunctional fatty acid synthase (fas) have been expressed in escherichia coli and the recombinant proteins refolded in vitro to catalytically active monomeric malonyl-/acetyltransacylases. fas residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, can be omitted from the recombinant enzyme with no loss in catalytic activity. this shortened transacylase, consisting of fas residues 488- ...19979215574
expression, characterization, and immunoreactivities of a soluble hepatitis e virus putative capsid protein species expressed in insect cells.the hepatitis e virus (hev) open reading frame-2 (orf-2) is predicted to encode a 71-kda putative capsid protein involved in virus particle formation. when insect spodoptera frugiperda (sf9) cells were infected with a recombinant baculovirus containing the entire orf-2 sequence, two types of recombinant proteins were produced; an insoluble protein of 73 kda and a soluble protein of 62 kda. the 62-kda species was shown to be a proteolytic cleavage product of the 73-kda protein. n-terminal sequenc ...19979220158
dna helicase activity in werner's syndrome gene product synthesized in a baculovirus system.the gene responsible for werner's syndrome (wrn) contains a region homologous to the escherichia coli recq type dna helicase and was thought to code for a dna helicase belonging to this helicase family. however, no evidence has been shown before to substantiate this prediction. here, we show data that the product of the wrn gene is indeed a dna helicase. the gene product, a polypeptide with a relative molecular mass of 170 kda, expressed in the insect spodoptera frugiperda (sf21) cell and purifi ...19979224595
expression of stable human o-glycan core 2 beta-1,6-n-acetylglucosaminyltransferase in sf9 insect cells.udp-glcnac:galbeta1-3galnac-r (glcnac to galnac) beta-1, 6-n-acetylglucosaminyltransferase (c2gnt) catalyses the formation of o-glycan core 2. purification and characterization of c2gnt from natural sources has been hampered by the instability of this enzyme. we have been able to prepare a stable partly purified recombinant human c2gnt by expression of a truncated form of the enzyme in the baculovirus/spodoptera frugiperda 9 (sf9) insect cell system. c2gnt activity was secreted into the sf9 cult ...19979224630
expression, purification, and characterization of an activated cytokine-suppressive anti-inflammatory drug-binding protein 2 (csbp2) kinase from baculovirus-infected insect cells.an activated form of the human cytokine-suppressive anti-inflammatory drug-binding protein 2 (csbp2) kinase was expressed in spodoptera frugiperda (sf9) cells from a baculovirus vector. to maximize expression and to facilitate purification of the recombinant protein, csbp2 kinase was expressed as a carboxy-terminal fusion protein to glutathione s-transferase (gst). under optimal conditions, 2-3 mg of gst-csbp2 could be obtained per liter of infected cell culture. the fusion protein was easily pu ...19979226723
differential effects of subunit interactions on protein kinase a- and c-mediated phosphorylation of l-type calcium channels.we have expressed the pore-forming alpha1s (skeletal muscle isoform) and alpha1c (cardiac/brain isoform) subunits, as well as the accessory beta2a (cardiac/brain isoform) and alpha2/delta subunits of the l-type, dihydropyridine-sensitive calcium (ca) channels in spodoptera frugiperda insect cells (sf9 cells) by infection with recombinant baculoviruses in order to facilitate biochemical studies of these rare, heteromultimeric membrane proteins. since the l-type channels are believed to be regulat ...19979236007
characterization of delta, kappa, and mu human opioid receptors overexpressed in baculovirus-infected insect cells.the cdnas encoding human delta (hdor), kappa (hkor) and micro (hmor) opioid receptors were cloned in the baculovirus autographa californica (acmnpv) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag. expression levels were optimized in spodoptera frugiperda (sf9) cells and were in the following order hmor > hdor > hkor. the receptors bound antagonists with affinity values similar to those published previously for the receptors expressed in mammalian ...19979242668
the influence of various insect cell lines, p10 and polyhedrin promoters in the production of secreted insulin-like growth factor-interleukin-3 chimeras in the baculovirus expression system.a technique for the optimal synthesis of secreted fusion proteins between insulin-like growth factors (igfs) and cytokines is described. the cdna of bomigf, a fusion protein between the insect insulin-like peptide bombyxin and igf ii, has been linked to the gene of interleukin-3. the bomigf-interleukin 3 fusion gene was cloned downstream of the promoter regions of the p10 and polyhedrin proteins within baculovirus transfer vectors. a third, dual transfer vector was constructed with the gene inse ...19979246791
a line-like transposable element in drosophila, the i factor, encodes a protein with properties similar to those of retroviral nucleocapsids.i factors are members of the line-like family of transposable elements and move by reverse transcription of an rna intermediate. complete i factors contain two open reading frames. the amino acid sequence encoded by the first of these, orf1, includes the motif cx2cx4hx4c that is characteristic of the nucleocapsid domain of retroviral gag polypeptides followed by a copy of the slightly different sequences cx2cx4hx6c and cx2cx9hx6c. the function of this protein is unknown. we have expressed this p ...19979250689
baculovirus expression provides direct evidence for heteromeric assembly of p2x2 and p2x3 receptors.p2x2 and p2x3 are subunits of p2x receptors, cation channels opened by binding extracellular atp. cdnas encoding p2x2 and p2x3 receptor subunits, each with one of two c-terminal epitope tags, were cloned into baculovirus. virally infected insect cells (spodoptera frugiperda) expressed moderate to high levels of the corresponding proteins, as detected by western blotting, by the specific binding of [35s]atp and by whole-cell recordings of membrane current evoked by atp or alphabetamethylene-atp. ...19979254665
characterization of [125i]s(-)5-oh-pipat binding to dopamine d2-like receptors expressed in cell lines.recently, [125i]s(-)5-oh-pipat [5-hydroxy-2-(n-n-propyl-n-3'-iodo-2'-propenyl)aminotetralin], a derivative of s(-)5-oh-dpat (5-hydroxy-n,n-di-n-propyl-aminotetralin), was reported to be a better radioiodinated dopamine d2-like receptor ligand than the previously reported iodinated ligand, [125i]r(+)7-oh-pipat. therefore, in the present study, the binding profile of [125i]s(-)5-oh-pipat to d2-like receptors expressed in cell lines was established. high binding affinity (kd = 0.3-0.4 nm) and nacl ...19979257944
regulation of infected-cell-specific protein synthesis in sf iplb-21 cells productively infected with spodoptera frugiperda multicapsid nuclear polyhedrosis virus.in spodoptera frugiperda (sf iplb-21) cells productively infected with s. frugiperda multicapsid nuclear polyhedrosis virus (sfmnpv), we observed the synthesis of infected-cell-specific polypeptides (icsps) using the protein synthesis inhibitor cycloheximide (cx) and the dan synthesis inhibitor cytosine arabinoside (ara-c) in an attempt to assess whether a temporal cascade and viral dna synthesis are involved in the gene expression program of sfmnpv. inhibition of protein synthesis with cx at 0 ...19979268770
bombyxin: an insect neurohormone targets the ovaries in lepidoptera.bombyxin, an insect hormone structurally related to insulin, was chemically synthesized and defined radioactively labeled probes were generated in order to detect and characterize bombyxin receptors in insect tissue. in all species tested, bombyx mori (silkmoth), samia cynthia ricini (ailanthus moth). manduca sexta (tobacco hornworm) and spodoptera frugiperda (fall armyworm), bombyxin receptors were found in ovaries. a radioactive photolabeled bombyxin analog was crosslinked to the potential bom ...19979274060
improved heterologous expression of the white-rot fungal ligninase h8 by crossover linker mutagenesis.using the crossover-linker mutagenesis method, the 5' noncoding region of the lambda ml-1 cdna, which encodes the ligninase h8 isozyme of the white-rot fungus, phanerochaete chrysosporium, was deleted with the simultaneous insertion of the putative spodoptera frugiperda ribosome-binding sequence (rbs) (tataaat) directly in front of the translation-initiation codon of this gene. a recombinant baculovirus, pvl-mu-h8, carrying the ligninase-h8 gene was successfully constructed, as determined by bot ...19979276925
baculovirus expression of a human g2-specific, neutralizing igg monoclonal antibody to puumala virus.we amplified by polymerase chain reaction the heavy- and light-chain antibody genes of a human hybridoma secreting a neutralizing, igg monoclonal antibody to the g2 protein of puumala virus. the heavy- and light-chain genes were cloned and sequenced and the deduced amino acids were aligned with those of other human antibodies to identify the constant and variable regions. the genes were cloned into the baculovirus plasmid transfer vector pacuw51 such that the heavy-chain and light-chain genes we ...19979281505
stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis.we have constructed several plasmid expression vectors to express foreign genes in stably transformed insect cells. unlike baculovirus-based expression vectors by which genes of interest are expressed transiently before lysis of the virus-infected cells, genes can be expressed continuously over many passages in a stable cell line. furthermore, the function of a gene or genes expressed in a stable cell line from an insect-specific promoter that is constitutively expressed can be studied in the ab ...19979282319
phosphorylation site mutations in the human multidrug transporter modulate its drug-stimulated atpase activity.in the human multidrug transporter (mdr1), three serine residues located in the "linker" region of the protein are targets of in vivo phosphorylation. these three serines, or all eight serines and threonines in the linker, were substituted by alanines (mutants 3a and 8a) or with glutamic acids (mutants 3e and 8e). the wild-type and mutant proteins were expressed in baculovirus-infected spodoptera frugiperda (sf9) ovarian insect cells, and the vanadate-sensitive, drug-stimulated atpase activity w ...19979287320
efficient photoaffinity labeling of the rat v1a vasopressin receptor using a linear azidopeptidic antagonist.we have synthesized and fully characterized by fast-atom-bombardment-mass, nmr and ultraviolet spectroscopies the vasopressin antagonist 3-azidophenylpropionyl-d-tyr(me)-phe-gln-asn-arg-pro-arg-tyr(3i )-nh2. easily radioiodinatable just before use, it has a high affinity for the natural rat liver v1a receptor [dissociation constant (kd) = 54 +/- 20 pm; carnazzi, e., aumelas, a., barberis, c., guillon, g. & seyer, r. (1994) j. med. chem. 37, 1841-1849] and for both the rat vasopressin v1a recepto ...19979288914
ligand specificity and affinity of bt-r1, the bacillus thuringiensis cry1a toxin receptor from manduca sexta, expressed in mammalian and insect cell cultures.the manduca sexta receptor for the bacillus thuringiensis cry1aa, cry1ab, and cry1ac toxins, bt-r1, has been expressed in heterologous cell culture, and its ligand binding characteristics have been determined. when transfected with the bt-r1 cdna, insect and mammalian cell cultures produce a binding protein of approximately 195 kda, in contrast to natural bt-r1 from m. sexia, which has an apparent molecular weight of 210 kda. transfection of cultured spodoptera frugiperda cells with the bt-r1 cd ...19979292994
ricin fusion toxin targeted to the human granulocyte-macrophage colony stimulating factor receptor is selectively toxic to acute myeloid leukemia cells.treatment failure of patients with acute myelogenous leukemia (aml) is frequently due to the development of multidrug resistance phenotype blasts. we have expressed a fusion protein consisting of human granulocyte-macrophage colony stimulating factor (gmcsf) fused to the n-terminus of a lectin-deficient ricin toxin b chain (rtb) in spodoptera frugiperda insect cells. the fusion protein was purified by immunoaffinity chromatography and reassociated with chemically deglycosylated ricin toxin a cha ...19979301690
characterization of the polyhedrin gene of spodoptera litura nuclear polyhedrosis virus.the polyhedrin gene (polh) of a characteristically distinct spodoptera litura nuclear polyhedrosis virus isolate (simnpv) is identified in the hindiii-f fragment of the viral dna. the nucleotide sequence of the 1057 base pair (bp) region of this fragment contains an open reading frame (orf) without any intervening sequence for coding a polypeptide of 246 amino acids. analysis of the nucleotide sequence and deduced amino acid sequence indicate that this has more than 70% sequence identity to know ...19979311561
herpes simplex virus 1 alpha regulatory protein icp0 interacts with and stabilizes the cell cycle regulator cyclin d3.the herpes simplex virus 1 (hsv-1) infected-cell protein 0 (icp0) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection. to identify cellular proteins interacting with icp0, we used a domain of exon ii of icp0 that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen. our results were as follows. (i) a cdna in a positive yeast colony was found to encode cyclin d3, a cell cycle regulator ...19979311810
identification of the early actin-rearrangement-inducing factor gene, arif-1, from autographa californica multicapsid nuclear polyhedrosis virus.in response to autographa californica multicapsid nuclear polyhedrosis virus (acmnpv) infection, a sequential rearrangement of the actin cytoskeleton occurs. previous studies suggest that the penetration of nucleocapsids induces early actin cables followed by further changes of the actin cytoskeleton which depend on early viral gene expression. by transfection of a plasmid library into trichoplusia ni tn-368 cells, we have identified an early viral gene, designated arif-1, that is able to induce ...19979311884
biochemical characterization of a smaller form of recombinant norwalk virus capsids assembled in insect cells.the expression of the single capsid protein of norwalk virus (nv) in spodoptera frugiperda (sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (vlps) of two sizes, the predominant 38-nm, or virion-size vlps, and smaller, 23-nm vlps. here we describe the purification and biochemical characterization of the 23-nm vlps. the 23-nm vlps were purified to 95% homogeneity from the medium of sf9 cultures by isopycnic cscl gradient centrifugation follow ...19979311906
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