Publications

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human acid beta-glucosidase: glycosylation is required for catalytic activity.the role of oligosaccharide modification in human acid beta-glucosidase function was investigated. this lysosomal enzyme has five putative n-glycosylation sites, four of which are occupied. the unglycosylated human protein was stable when expressed in bacteria or in spodoptera frugiperda cells in the presence of tunicamycin but lacked catalytic activity. deglycosylation of purified acid beta-glucosidase from human placenta with n-glycanase under native conditions resulted in the removal of an ac ...19902110456
cloning and expression of human aldose reductase.the complete amino acid sequence of human retina and muscle aldose reductase was determined by nucleotide analysis of cdna clones isolated using synthetic oligonucleotide probes based on partial amino acid sequences of purified human psoas muscle aldose reductase. the cdna sequence differs substantially in the noncoding and coding regions of recently published sequences of this enzyme. the mrna for aldose reductase was abundantly expressed in hela cells, but only scarcely in a neuroblastoma cell ...19902112546
co-expression of the human hla-b27 class i antigen and the e3/19k protein of adenovirus-2 in insect cells using a baculovirus vector.we have expressed the human mhc class i hla-b27 antigen, human beta 2-microglobulin, and the e3/19k protein of adenovirus-2 in spodoptera frugiperda insect cells (sf9) by using the autographa californica nuclear polyhedrosis virus. all genes were inserted under the strong polyhedrin promoter in the vector pvl941. the proteins were expressed at high levels, ranging from 1 to 8 mg protein per 3 x 10(9) cells. both a full-length and a truncated form of hla-b27 were expressed. the latter was termina ...19902150332
identification of bluetongue virus vp6 protein as a nucleic acid-binding protein and the localization of vp6 in virus-infected vertebrate cells.recently the insect baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has been effectively adapted as a highly efficient vector in insect cells for the expression of various genes. a cdna sequence of rna segment 9 of bluetongue virus serotype 10 (btv-10, an orbivirus member of the reoviridae family) encoding a minor core protein (vp6) has been inserted into the bamhi site of the pacym1 transfer vector derived from acnpv. spodoptera frugiperda cells were cotransfected with the ...19902152806
identification of a viral gene encoding a ubiquitin-like protein.the baculovirus autographa californica nuclear polyhedrosis virus (acmnpv, which is representative of the mnpv subtype in which the virions may contain many nucleocapsids within a single viral envelope) encodes a protein, v-ubi, that has 76% identity with the eukaryotic protein ubiquitin. transcriptional mapping indicated that the gene for v-ubi was transcribed during the late phase of viral infection. two transcriptional start sites potentially encoding v-ubi were identified. both sites were co ...19902153300
purification and characterization of the recombinant extracellular domain of human nerve growth factor receptor expressed in a baculovirus system.to obtain the large quantities of the extracellular domain of the nerve growth factor receptor (ngf-r) necessary for structural analyses, we produced this protein in the baculovirus expression system. a cdna coding for the extracellular domain of the human ngf-r was first introduced into transfer vector pvl941. recombinant baculovirus was produced by cotransfecting spodoptera frugiperda cells with the transfer vector and dna of autographa californica nuclear polyhedrosis virus. recombinant viral ...19902155239
high level expression of the two outer capsid proteins of bluetongue virus serotype 10: their relationship with the neutralization of virus infection.dna representing rna segments 2 and 5 of bluetongue virus (btv) serotype 10, corresponding to the genes that code for the outer capsid proteins vp2 and vp5, have been inserted into a baculovirus transfer vector in lieu of the coding region of the polyhedrin gene of autographa californica nuclear polyhedrosis virus (acnpv). after co-transfection of spodoptera frugiperda cells with wild-type acnpv dna in the presence of the recombinant transfer vector dnas polyhedrin-negative recombinant baculovir ...19902160763
expression of pseudomonas phosphotriesterase activity in the fall armyworm confers resistance to insecticides.the gene encoding for the phosphotriesterase (opd) from pseudomonas diminuta has been subcloned into a baculovirus expression system. functional enzyme is produced when the recombinant baculovirus is used to infect either cultured spodoptera frugiperda sf9 cells or the larval stage of the fall armyworm. the ld50 for paraoxon toxicity was found to increase 280-fold in the larvae after infection with the recombinant baculovirus and expression of the functional phosphotriesterase.19902164956
biophysical studies on the morphology of baculovirus-expressed bluetongue virus tubules.bluetongue virus tubules were purified from spodoptera frugiperda cells infected with a recombinant baculovirus containing the ns1 gene from bluetongue virus serotype 10, and expressed under control of the autographa californica nuclear polyhedrosis virus polyhedrin promoter. these tubules were subjected to a variety of chemical and physical treatments and the resulting effects on tubule morphology were examined by electron microscopy. a number of morphological similarities were noted between bl ...19902167940
synthesis of bluetongue virus-encoded phosphoprotein and formation of inclusion bodies by recombinant baculovirus in insect cells: it binds the single-stranded rna species.a dna clone of rna segment 8 (s8) of bluetongue virus type 10 (btv-10), an orbivirus member of the reoviridae family has been expressed to high levels (20 mg/1 x 10(9) cells) using an autographa californica nuclear polyhedrosis virus expression vector (pa-cym1). the expressed protein is similar to the authentic btv phosphoprotein ns2, in its size, antigenicity, and also the manner of phosphorylation (e.g. same peptides and residues). both mammalian and insect cell-derived ns2 proteins are phosph ...19902170574
characterization of the basement membrane glycoprotein entactin synthesized in a baculovirus expression system.a cdna clone encoding the entire open reading frame of mouse entactin was constructed. this cdna was inserted into autographa californica multiple nuclear polyhedrosis virus and expressed in spodoptera frugiperda, sf9, insect cells. the recombinant entactin was produced in large quantities under the control of the polyhedrin promoter. amino terminus sequence analysis indicated that the signal peptide in the preprotein was correctly cleaved. polyclonal antibodies prepared against either the natur ...19902180961
efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.human adenosine deaminase (ec 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in spodoptera frugiperda cells and in trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. the coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular ...19902181448
antigenic parvovirus b19 coat proteins vp1 and vp2 produced in large quantities in a baculovirus expression system.two baculovirus expression vectors derived from autographica californica nuclear polyhedrosis virus (acnpv) were prepared containing the complete 2.5 kb coding region for parvovirus b19 coat protein vp1 (acb19vp1l) and the 1.8 kb coding region for vp2 (acb19vp2l), placed under the control of the polyhedrin promoter. the recombinant viruses were used to infect spodoptera frugiperda cells and the proteins expressed were analysed using appropriate antibodies. acb19vp1l-infected cells produced b19 v ...19902188463
autographa californica m nuclear polyhedrosis virus: microtubules and replication.progressive reorganization and depolymerization of microtubules corresponded with virus-induced rounding of autographa californica m nuclear polyhedrosis virus (acmnpv)-infected spodoptera frugiperda iplb-sf-21 cells, suggesting that microtubules were instrumental in maintaining the normal shape of these cells. depolymerization of all cortical and most of the paranuclear microtubules with colchicine also resulted in cell rounding, confirming this hypothesis. studies with aphidicolin and cyclohex ...19902408230
characterization of the da26 gene in a hypervariable region of the autographa californica nuclear polyhedrosis virus genome.a region of the baculovirus autographa californica nuclear polyhedrosis virus genome that is frequently found to be altered after serial passage of the virus in cell culture was characterized. sequence analysis of this region of the genome in wild-type and mutant viruses revealed that some of the mutations affected a 675 bp open reading frame, designated da26. the da26 gene was disrupted both by deletion and by insertion of sequences that resembled transposable elements. northern blot analysis o ...19902189022
rabies diagnostic reagents prepared from a rabies n gene recombinant expressed in baculovirus.a gene encoding the nucleoprotein (n) of rabies virus was inserted into the genome of the baculovirus autographa californica nuclear polyhedrosis virus. recombinant gene expression was controlled by the strong polyhedrin gene promoter. insect cells (spodoptera frugiperda) infected by a baculovirus recombinant containing the rabies virus n gene produced abundant amounts of a novel 55-kilodalton protein of a size comparable to that of the rabies virus n protein, as demonstrated by polyacrylamide g ...19902191008
human acidic fibroblast growth factor overexpressed in insect cells is not secreted into the medium.we have overexpressed human acidic fibroblast growth factor (afgf) in spodoptera frugiperda sf9 cells from a cdna clone under the control of the promoter of the polyhedrin gene of the baculovirus autographa californica nuclear polyhedrosis virus. a 16.5-kd product was made in recombinant virus-infected cells that specifically reacted in immunoblots with various antibodies prepared against afgf. recombinant afgf was mitogenic for bhk21 cells and its activity was stimulated by heparin. the mechani ...19902200451
secretion of functional papain precursor from insect cells. requirement for n-glycosylation of the pro-region.the synthetic gene coding for the precursor of the cysteine protease papain (ec 3.4.22.2) has been expressed using the baculovirus/insect cell system. the prepropapain gene was cloned into the transfer vector ipdc125 behind the polyhedrin promoter. the recombinant construct was then incorporated by homologous recombination into the autographa californiaca nuclear polyhedrosis virus genome. the host spodoptera frugiperda sf9 cells infected with the recombinant baculovirus secrete an enzymatically ...19902204628
expression of the lassa virus nucleocapsid protein in insect cells infected with a recombinant baculovirus: application to diagnostic assays for lassa virus infection.the coding region of the gene for the nucleocapsid protein of lassa virus has been inserted into the genome of autographa californica nuclear polyhedrosis virus (acnpv) using the transfer vector pacym1, so that expression of the foreign dna is under the control of the promoter of the acnpv polyhedrin gene. infection of cultured spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of a protein that was indistinguishable from the authentic lassa virus protein ...19902406367
the oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector.the hemagglutinin of fowl plague virus has been expressed in spodoptera frugiperda (sf) cell cultures using a baculovirus vector. to elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase h and glycopeptidase f. sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures man5-9glcnac2, and the truncated ol ...19902407026
sparged animal cell bioreactors: mechanism of cell damage and pluronic f-68 protection.pluronic f-68 is a widely used protective agent in sparged animal cell bioreactors. in this study, the attachment-independent spodoptera frugiperda sf9 insect cell line was used to explore the mechanism of this protective effect and the nature of cell damage in sparged bioreactors. first, bubble incorporation via cavitation or vortexing was induced by increasing the agitation rate in a surface-aerated bioreactor; insect cells were rapidly killed under these conditions of the absence of polyols. ...19901366875
continuous production of baculovirus in a cascade of insect-cell reactors.insect cells (spodoptera frugiperda) were cultured in a continuous stirred-tank reactor. the effluent was led to a cascade of another two reactors, each containing half the volume of the cell-growth reactor, where the cells were infected with autographa californica nuclear polyhedrosis virus. for about 10 days production of 10(7) polyhedra (virus particles embedded in a protein capsule) per cm3 was achieved. this short production time compared to previous experiments involving an analogous syste ...19901366563
protective effect of methylcellulose and other polymers on insect cells subjected to laminar shear stress.the relative sensitivity of two insect cell lines to laminar shear stress was determined, and the protective effect of polymers added to the growth media of two insect cell lines, trichoplusia ni (tn-368) and spodoptera frugiperda (sf-9), was evaluated. tn-368 and sf-9 cells were found to be equally sensitive to laminar shear stress. methylcellulose [0.5% (w/v) dow e4m methocel] and dextran [4.5% (w/v)] increased the resistance of suspended cells to lysis due to laminar shear stress by factors o ...19901370016
dna distribution and respiratory activity of spodoptera frugiperda populations infected with wild-type and recombinant autographa californica nuclear polyhedrosis virus.spodoptera frugiperda cells were infected with a wild-type autographa californica nuclear polyhedrosis virus and with a recombinant autographa californica nuclear polyhedrosis virus. the recombinant virus was derived from the wild-type virus and produced beta-galactosidase instead of polyhedrin. the changes in cell size, cell growth, viability, dna distribution, and respiratory activity were followed through the time course of the infection. the dna content as measured by flow cytometry of infec ...19901367465
multistage production of autographa californica nuclear polyhedrosis virus in insect cell cultures.the aim of our study was to establish an efficient system for the in vitro production of the insect pathogenic autographa californica nuclear polyhedrosis virus in a spodoptera frugiperda cell line. we optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. in order to provide sufficient nutrients during virus synthesis filtration and p ...19901366992
analysis of the s spike (peplomer) glycoprotein of bovine coronavirus synthesized in insect cells.the bovine coronavirus (bcv) spike glycoprotein precursor (s, formerly termed peplomer) and its two subunit polypeptides (s1 and s2) were individually expressed in spodoptera frugiperda (sf9) insect cells. each recombinant baculovirus expressed both glycosylated (s, 170k; s1, 95k; s2, 80k) and unglycosylated (s0, 140k; s10, 75k; and s20, 65k) forms of bcv spike polypeptides in sf9 cells. the mature 95k s1 polypeptide was secreted whereas the s and s2 polypeptides remained cell-associated. the s ...19901699351
categorizing some early and late transcripts directed by the autographa californica nuclear polyhedrosis virus.using an s1 mapping assay on rna from spodoptera frugiperda cells infected by the autographa californica nuclear polyhedrosis virus in the presence and absence of cycloheximide and aphidicolin, we can distinguish three classes of transcripts. first, there are those whose synthesis is blocked by the dna synthesis inhibitor aphidicolin and which are therefore late transcripts. these include the late transcript of the 39k gene and a late leftward transcript across the xhoi site in the hindiii-f reg ...19901698929
characterization of baculovirus-expressed rift valley fever virus glycoproteins synthesized in insect cells.a cdna corresponding to the complete coding region of the m rna of the m12 mutant of rift valley fever virus (rvfv) strain zh548 (k. takehara, m-k. min, j.k. battles, k. sugiyama, v.c. emery, j.m. dalrymple, and d.h.l. bishop, virology, 169, 452-457, 1989) has been inserted into the baculovirus transfer vector pacym1. by comparison with the parent rvfv, the m rna of the m12 mutant has a new small open reading frame (orf1) upstream of the one that initiates the precursor of the viral glycoprotein ...19902077783
multispecies trapping ofhelicoverpa (heliothis) zea, spodoptera frugiperda, pseudaletia unipuncta, andagrotis ipsilon (lepidoptera: noctuidae).multispecies sex pheromone trapping (trapping of more than one species in the same trap) for the bollworm,helicoverpa (heliothis) zea (boddie); fall armyworm,spodoptera frugiperda (j.e. smith); armyworm,pseudaletia unipuncta (haworth); and the black cutworm,agrotis ipsilon (hufnagel) was evaluated. baiting of individual traps with all possible combinations of the four species taken two at a time caused a reduction in the catches of at least one of any two species involved when compared to traps ...199024263443
high-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system.in order to develop an efficient process for large-scale production of recombinant protein, various factors were studied which affect the productivity of sf-9 (spodoptera frugiperda) insect cells when using the baculovirus expression system. it was shown that upon infection with the bac-brv6l recombinant baculovirus, the level per cell of vp6 (a bovine rotavirus nucleocapsid protein) would drop 10-fold when host cell density at the time of infection increased from 2 x 10(6) to 3 x 10(6) cells/ml ...199018595054
insecticidal activity of a bacterial crystal protein expressed by a recombinant baculovirus in insect cells.baculoviruses are insect pathogens with a relatively slow speed of action, and this has limited their use as control agents of insect pests. introduction into baculoviruses of genes which code for proteins interfering specifically with insect metabolism or metamorphosis, such as toxins, hormones, and enzymes, may enhance the pathogenicity of these viruses. the complete insecticidal crystal protein gene cryia(b) of bacillus thuringiensis subsp. aizawai 7.21 was engineered into the nuclear polyhed ...199016348284
co-occlusion and persistence of a baculovirus mutant lacking the polyhedrin gene.a co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. coinfection of spodoptera frugiperda (iplb-sf-21) tissue culture cells with autographa californica nuclear polyhedrosis virus (acmnpv) and an acmnpv mutant (ac-e10) lacking the polyhedrin gene resulted in occlusion of both virus types within polyhedra. the amount of occluded ac-e10 virions in progeny polyhedra populations during serial ...199016348313
assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus.spodoptera frugiperda insect cells were grown in sf-900 serum-free medium and two kinds of serum-supplemented media (ipl -41 and grace's). the specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees c. the ipl -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/ml) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/ml with the grace's and serum-free media, respectively. in temperature shifdown experiments with a temperat ...199118600874
culture of insect cells in helical ribbon impeller bioreactor.an 11-l helical ribbon impeller (hri) bioreactor was tested for the culture of spodoptera frugiperda (sf-9) cells. this impeller and surface baffling ensured homogeneous mixing and high oxygen transfer through surface aeration and surface-induced babble generation. serum-supplemented and serum-free cultures, using tnmfh and ipl/41 media, respectively, grew a similar specific growth rates(0.031 and 0.028 h(-1)) to maximum cell densities of 5.5 x 10(6)-6.0 x 10(6) cells. ml(-1) with viability exce ...199118604880
modulation of trehalase activity in two insect cell lines by virus infection and trehalose.trehalase (ec 3.2.1.28), an important glycosidase involved in regulating trehalose levels and metabolic energy in insects, was measured in cell lines from fall army worm, spodoptera frugiperda and salt marsh caterpillar, estigmene acrea, treated with either glucose or trehalose in the presence or absence of tipula iridescent virus (tiv), a cytoplasmic deoxyribovirus. in medium containing 15-35 mm trehalose, both of these cells increased their trehalase activities by 4.5 to 8x the basal levels fr ...19911953800
high level expression of dna polymerases from herpesviruses.the dna polymerase genes of human cytomegalovirus (hcmv) and varicella-zoster virus (vzv) were inserted separately into the polyhedrin gene of autographa californica nuclear polyhedrosis virus (acnpv) by cotransfection of spodoptera frugiperda (sf9) cells with baculovirus transfer vectors carrying the genes and acnpv infectious dna. infection of sf9 cells with the recombinant viruses resulted in expression from the polyhedrin promoter of proteins of the expected mrs. these proteins possessed dna ...19911649906
purification and characterization of human immunodeficiency virus type 1 nef gene product expressed by a recombinant baculovirus.we have constructed the recombinant baculovirus which expresses the human immunodeficiency virus type 1 negative factor (nef) gene. spodoptera frugiperda cells infected with the recombinant virus produced a 27-kda protein which reacted with rabbit antisera raised against a carboxy-terminal synthetic peptide of the nef protein by immunoblot analysis. labeling experiment showed that the recombinant nef protein was myristoylated. the recombinant nef protein was purified to near homogeneity by deae- ...19911909480
enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products.five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of autographa californica nuclear polyhedrosis virus by homologous recombination. the first cassette polf contains the full-length pol open reading frame; the second cassette pol100 starts with the first aug codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire prot ...19911711203
molecular basis of thyroid hormone regulation of myelin basic protein gene expression in rodent brain.regulation of myelin basic protein (mbp) gene expression by thyroid hormone has been investigated in rodent brain. quantitation of the 4 major alternatively spliced transcripts by rnase protection assay showed that the individual mrnas, corresponding to mbp isoforms 21.5, 18.5, 17, and 14 kda, were decreased from 2- to 17-fold at all ages studied (4-60 days) in hypothyroid animals when compared to euthyroid, but the timing of onset of expression was not altered. mbp mrna was also reduced in youn ...19911720778
construction, purification and characterization of a recombinant baculovirus containing the gene for alpha subunit of human chorionic gonadotropin.a cdna encoding the alpha subunit of human chorionic gonadotropin, a placental glycoprotein hormone, was cloned downstream to the viral polyhedrin gene promoter of autographa california nuclear polyhedrosis virus and the recombinant transfer vector was used to co-transfect spodoptera frugiperda cells growing in culture. recombinant baculovirus carrying the alpha hcg gene was detected and isolated after dot hybridization using supernatant from co-transfected cells. recombinant vac alpha hcg havin ...19911721605
expression of hepatitis b virus surface antigen gene in insect baculovirus vector systems.the gene coding for the hepatitis b virus surface antigen (hbsag) under the control of autographa californicanuclear polyhedrosis virus polyhedrin promoter was successfully inserted into the genome of the trichoplusia ni nuclear polyhdrosis virus. infection of spodoptera frugiperda cells with this recombinant virus produced a significant amount of hbsag protein and secreted 22 nm particles containing the hbsag. the expression of hbsag gene was also obtained both in trichoplusia ni larvae and in ...19911773016
characterization of yellow fever virus proteins e and ns1 expressed in vero and spodoptera frugiperda cells.the cdna encoding the e and ns1 proteins of the yellow fever virus (yfv) was expressed in spodoptera frugiperda cells via the recombinant baculovirus ac-e. ns1 as a gp100 precursor which was cleaved to generate the recombinant proteins e and ns1 similar in size, folding and antigenicity to the authentic ones. recombinant protein e exhibited immunodominant epitopes as judged by its reactivity with yfv-neutralizing mabs. using the triton x-114 phase separation system, authentic and recombinant e p ...19911710649
17d yellow fever vaccine virus envelope protein expressed by recombinant baculovirus is antigenically indistinguishable from authentic viral protein.we have constructed a recombinant baculovirus containing cloned dna encoding the membrane and envelope (e) proteins of 17d yellow fever vaccine virus. spodoptera frugiperda cells infected with this recombinant baculovirus produced a 66k protein which corresponded to the estimated size of the protein encoded by the cloned inserted dna, and a 54k protein with the same molecular size as that of the authentic 17d yellow fever virus e protein. this recombinant 54k protein was labile, producing e prot ...19911710651
plasmodium falciparum: recombinant baculoviruses direct the expression of circumsporozoite proteins in spodoptera frugiperda cell cultures.the dna coding for the circumsporozoite protein (cps) of plasmodium falciparum has been cloned into the baculovirus expression vector pacym1 and expressed in spodoptera frugiperda (sf9) insect cells. three dna constructs have been made: the first one directs the synthesis of the complete csp (aa 1-412), the second leads to the production of a species devoid of the anchor domain (aa 1-391) and the third one to a molecule lacking both signal and membrane anchor sequences (aa 18-391). all three rec ...19911749376
immunological characterization of the epstein-barr virus phosphoprotein pp58 and deoxyribonuclease expressed in the baculovirus expression system.the open reading frames of the phosphoprotein pp58 (bmrfi) and the deoxyribonuclease (bglf5) of the epstein-barr-virus (ebv) strain m-aba were cloned in the baculovirus expression vectors pac373 and pac360 and expressed in the spodoptera frugiperda (sf158) insect cells. the recombinant phosphoprotein pp58 expressed in sf158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a trpe-bmrfi fusion protein expressed in e. coli. dna-cellulose chromat ...19911650330
overproduction of rat 1,25-dihydroxyvitamin d3 receptor in insect cells using the baculovirus expression system.the rat 1,25-dihydroxyvitamin d3 [1,25-(oh)2d3] receptor has been expressed at elevated levels in spodoptera frugiperda cells using the baculovirus expression vector system. the recombinant 1,25-(oh)2d3 receptor is full-length, binds 1,25-(oh)2d3, and is recognized by a monoclonal antibody specific for 1,25-(oh)2d3 receptor. densitometric scanning of coomassie brilliant blue-stained sds/polyacrylamide gels indicated a recombinant receptor protein level comprising 5% of the total soluble protein ...19911650474
functional expression of the human growth factor activatable na+/h+ antiporter (nhe-1) in baculovirus-infected cells.we constructed a recombinant baculovirus, based on autographa californica nuclear polyhedrosis virus, containing the human na+/h+ antiporter cdna under control of the polyhedrin promoter. when infected with this recombinant baculovirus, the sf9 cell line, derived from spodoptera frugiperda, expresses a fully functional na+/h+ antiporter as measured by the generation of an amiloride-sensitive na+ influx in response to an acid load. the na+/h(+)-exchange activity, not detectable in sf9 cells, emer ...19911654101
recombinant baculoviruses expressing yellow fever virus e and ns1 proteins elicit protective immunity in mice.recently, we showed that yellow fever virus (yfv) e and ns1 proteins in spodoptera frugiperda cells infected with a recombinant baculovirus are similar, if not identical to those produced during yfv infection. to study the role of e and ns1 in the induction of protective immunity against fatal yfv challenge, these viral antigens were expressed either alone or in tandem via recombinant baculoviruses ac-e. ns1, ac-e1 and ac-ns1. swiss mice were immunized with lysates of insect cells infected with ...19911834798
overexpression and characterization of the human mineralocorticoid receptor.the full-length human renal mineralocorticoid receptor (hmr) has been overproduced in spodoptera frugiperda (sf9) insect cells using baculovirus-mediated expression. the overproduced hmr binds aldosterone with high affinity (kd = 1.36 nm) and has high affinity for cortisol, cortexolone, and progesterone. immunoprecipitation and immunoblot analysis of the recombinant hmr with mr-specific antibodies reveal three major protein bands with molecular masses of 115, 119, and 125 kda. hmr isoforms show ...19911655735
expression of bovine herpesvirus 1 glycoprotein giv by recombinant baculovirus and analysis of its immunogenic properties.the gene encoding the giv glycoprotein of bovine herpesvirus 1 has been inserted into the genome of autographa californica baculovirus in lieu of the coding region of the a. californica baculovirus polyhedrin gene. recombinant protein was identified by its reactivity with giv-specific monoclonal antibodies and expressed at high levels (about 85 micrograms per 2.5 x 10(6) cells) in spodoptera frugiperda (sf9) cells. the recombinant glycoprotein had an apparent molecular mass of 63 kda, indicating ...19911845887
baculovirus-mediated expression of the human vitamin d receptor. functional characterization, vitamin d response element interactions, and evidence for a receptor auxiliary factor.a baculovirus expression vector system (bevs) was used to overproduce the full-length human vitamin d receptor (hvdr) in spodoptera frugiperda ovarian cells. hvdr was expressed to a level of 0.5% of the total soluble protein in this system. western analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologic properties equivalent to those of hvdr expressed in mammalian cells. the bevs-derived receptor displayed specificity and high affinity (apparent kd = 0.7 ...19911655763
high-level expression of the epstein-barr virus alkaline deoxyribonuclease using a recombinant baculovirus: application to the diagnosis of nasopharyngeal carcinoma.the epstein-barr virus (ebv) alkaline deoxyribonuclease (dnase) was inserted into the baculovirus autographa californica nuclear polyhedrosis virus (acmnpv). infection of the insect cell line spodoptera frugiperda (sf9) with the recombinant virus led to the expression of an enzymatically active alkaline dnase. the recombinant ebv alkaline dnase was highly soluble, and the recombinant baculovirus produced approximately 10-20 mg of ebv dnase per 1 x 10(9) cells. the recombinant enzyme activity was ...19911847261
evidence for dissimilar properties of comoviral and picornaviral rna polymerases.the poliovirus rna polymerase has been synthesized in spodoptera frugiperda cells by using the baculovirus expression system. crude sonicates of these cells exhibited an rna-elongating activity of a synthetic oligo(u) primer with poly(a) or cowpea mosaic virus (cpmv) rna as a template. a similar polymerase activity was found in extracts of insect cells in which foot-and-mouth disease virus (fmdv) proteins, including the putative polymerase, were produced. the analogous cpmv 87k protein and sever ...19911848591
immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein d.the dna sequence encoding the complete herpes simplex virus type 1 (hsv-1) glycoprotein d (gd) was inserted into a baculovirus transfer vector under control of the polyhedrin gene promoter of the baculovirus autographa california nuclear polyhedrosis virus (acnpv). after co-transfection of spodoptera frugiperda (sf9) insect cells with wild-type acnpv dna and the recombinant transfer vector dna, polyhedrin-negative recombinants that expressed high levels of hsv-1 gd were isolated using immunoaffi ...19911662037
molecular cloning of a human basic fibroblast growth factor receptor cdna and expression of a biologically active extracellular domain in a baculovirus system.a cdna clone encoding a human fibroblast growth factor (fgf) receptor was isolated from a hepatoma cell line cdna library. the cdna encodes a three immunoglobulinlike-domain fgf receptor that is similar to a human placental fgf receptor cdna but lacks two amino acids. the variation observed at these two amino acids, also seen in the two immunoglobulinlike-domain fgf-receptors, can be explained by an alternate splicing mechanism. we have used a baculovirus expression system to produce high levels ...19911662973
lepidopteran cell variants resistant to 5-bromodeoxyuridine and their use for transfection of the hsv-tk gene.following mutagenesis of cultured lepidopteran cells (spodoptera frugiperda) by ethylmethanesulfonate, three variants resistant to 5-bromodeoxyuridine (brdurd) were isolated. these clones were 100- to 200-fold more resistant to brdurd than the parental cells and were shown to be deficient in thymidine kinase (tk). the drug-resistant phenotype was stable for up to three years of culture under nonselective conditions. it was also found that the s. frugiperda cell line was highly resistant to amino ...19911665497
dna restriction polymorphism in wild isolates of spodoptera frugiperda nuclear polyhedrosis virus.restriction endonuclease analysis was used to examine variation in dna of 22 wild isolates of spodoptera frugiperda nuclear polyhedrosis virus (sfnpv). eleven of the 15 isolated from louisiana were distinguishable based on restriction fragment profiles from the enzymes bamhi, hindiii, and ecori. there was significant genetic variation in sfnpv isolates within single agricultural fields. nucleotide sequence divergence values, based on restriction fragment profiles, indicated that genetic variatio ...19911679452
identification of putative insect brush border membrane-binding molecules specific to bacillus thuringiensis delta-endotoxin by protein blot analysis.binding sites for insecticidal toxins of bacillus thuringiensis are located in the brush border membranes of insect midguts. two approaches were used to investigate the interactions of b. thuringiensis subsp. kurstaki hd-73 cryia(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125i-toxin-vesicle binding assays and protein blots probed with 125i-cryia(c) toxin. in bioassays, manduca sexta and heliothis virescens larvae were highly sensitive, helicoverp ...19911746942
expression and characterization of recombinant mouse beta 2-microglobulin type a in insect cells infected with recombinant baculoviruses.the murine beta 2-microglobulina cdna was cloned into pac373 and pvl941 transfer vectors and introduced via homologous recombination into the genome of autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. both types of recombinant baculoviruses were isolated and used to infect spodoptera frugiperda (sf9) lepidopteran cells. beta 2m was synthesized at a substantially higher rate in cells infected with the pvl941-derived virus than when the pac373-based virus wa ...19911754712
a baculovirus dual expression vector derived from the autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.a baculovirus transfer vector, pacuw3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (ha) and neuraminidase (na) membrane glycoproteins, into the autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. the na gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the ha gene was placed under the control of a copy of the p10 promoter at a ...19911765769
structural comparison of the autographa californica nuclear polyhedrosis virus-induced rna polymerase and the three nuclear rna polymerases from the host, spodoptera frugiperda.a partial subunit structure has been determined for the novel rna polymerase that is induced in fall armyworm (spodoptera frugiperda) cells upon infection with the autographa californica nuclear polyhedrosis virus (acnpv). the putative structure includes nine polypeptides; the complexity of this structure is in accord with the high sedimentation coefficient (15s) estimated for this enzyme. a comparison of the putative structure of the virus-induced polymerase with those of the three host nuclear ...19911767583
diagnostic potential of baculovirus-expressed rubella virus envelope proteins.the envelope glycoproteins e1 and e2 of rubella virus were abundantly expressed in spodoptera frugiperda sf9 insect cells by using a baculovirus expression vector. the recombinant protein products were purified by immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (eia). the purified recombinant antigen consisted of the envelope polypeptides, corresponding to the viral e1 and e2 proteins, and a poly ...19911774311
characterization and purification of human beta-galactosidase overexpressed in recombinant baculovirus-infected spodoptera frugiperda cells. 19911779628
characterization and functional properties of the a and b forms of human progesterone receptors synthesized in a baculovirus system.human progesterone receptors (pr) were overexpressed in spodoptera frugiperda (sf9) insect cells using a recombinant baculovirus system. recombinant viruses were constructed that produced either full-length a (94k) or b (120k) forms of human pr, and each was expressed as a functional protein. steroid and dna binding activities were found to be indistinguishable from that of endogenous human pr in t47d breast cancer cells. moreover, as analyzed by gel-mobility shift, recombinant pr-a and pr-b eac ...19911779977
immunolocalization of alzheimer beta-amyloid peptide precursor to cellular membranes in baculovirus expression system.one characteristic of alzheimer's disease (a beta disease) is the accumulation of amyloid deposits within the extracellular space of the brain and meninges. a 40 amino acid peptide called beta-peptide or a4 protein is the subunit of the amyloid fibrils found in these deposits. the sequence of beta-peptide is contained within those of a family of larger proteins called the alzheimer beta-amyloid peptide precursor (app). these apps contain, in addition to a signal sequence, a hydrophobic sequence ...19911787542
purification and biochemical characterization of a soluble mouse interferon-gamma receptor produced in insect cells.the extracellular domain of the mouse interferon gamma receptor comprising amino acids 17-243 of the protein was produced in spodoptera frugiperda cells infected with a recombinant baculovirus. the receptor was mainly secreted into the culture medium and was purified to homogeneity in several hundred milligram amounts. the purification procedure involved four chromatography steps and delivered a soluble and active receptor with an overall recovery of 30%. from each purification run, two pools of ...19911828230
expression of the tobacco mosaic virus movement protein using a baculovirus expression vector.a cdna clone of the tobacco mosaic virus 30k movement protein (mp) gene was constructed and introduced into an autographa californica nuclear polyhedrosis baculovirus expression vector. infection of spodoptera frugiperda cells with the vector resulted in the synthesis of low levels of mp, which was detected by anti-mp serum as two closely related species of mr approximately 34k and a third species of 32k. the authenticity of the recombinant mp was confirmed by comparison of the protein, on the b ...19911940871
secretion of single-chain urokinase-type plasminogen activator from insect cells.a cdna encoding human urokinase-type plasminogen activator was inserted downstream from the polyhedrin promoter of the baculovirus autographa californica nuclear polyhedrosis virus. a protein of similar mr to urokinase (uk) was synthesized and approx. 90% was secreted from recombinant virus-infected spodoptera frugiperda cells. zymography and western blotting analysis of the insect-derived protein demonstrated that it was comprised solely of the high-mr form of uk. no low-mr uk was detected. ami ...19911834526
identification of spliced baculovirus rnas expressed late in infection.previous to this study, the autographa californica multicapsid nuclear polyhedrosis virus (acmnpv) was known to express only one spliced rna (spliced ie1 or ie0). we have conducted an analysis of rna expressed during infection of spodoptera frugiperda cells with acmnpv and have identified a set of five additional spliced rnas expressed late in infection. a reverse transcription-polymerase chain reaction analysis was used to confirm the identification of the ls (late, spliced) rnas. s1 nuclease a ...19911962442
heterologous recombination between autographa californica nuclear polyhedrosis virus dna and foreign dna in non-polyhedrin segments of the viral genome.we used the expression vector system of autographa californica nuclear polyhedrosis virus (acnpv) and spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. we concentrated on the isolation and analysis of heterologous recombinants. the e1 region of human adenovirus type 2 (ad2) was inserted into regions of the acnpv genome which lacked apparent homologies to the polyhedrin region. out of a total of 122 recombinant acnpv plaques, which hybridized to ad2 dna in p ...19911962504
overexpression and purification of transcriptionally competent creb from a recombinant baculovirus.signal transduction and viral stimulatory pathways converge ultimately at the level of transcriptional activation to influence the expression of a variety of cellular genes in response to environmental stimuli and developmental signals. recent studies have implicated the cyclic amp-responsive element-binding protein (creb) to be involved in mediating transcriptional activation in response to multiple varied stimuli, including (1) stimulation of the protein kinase a signal transduction pathway; ( ...19911840397
expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11.the nucleotide sequence of rotavirus genome segment 11 shows that this gene contains three potential open reading frames. we used several approaches to determine whether any polypeptides other than ns26, the primary protein product, are expressed. in particular, we sought to determine whether the strong out-of-phase start codon present at nucleotides 80-82, which would encode a protein of 92 amino acids, is used in vivo or in cell-free systems. several modifications of gene 11 were made and foun ...19911847258
expression of rotavirus vp2 produces empty corelike particles.the complete vp2 gene of bovine rotavirus strain rf has been inserted into the baculovirus transfer vector pvl941 under the control of the polyhedrin promoter. cotransfection of spodoptera frugiperda 9 cells with wild-type baculovirus dna and transfer vector dna led to the formation of recombinant baculoviruses which contain bovine rotavirus gene 2. infection of s. frugiperda cells with this recombinant virus resulted in the production of a protein similar in size and antigenic properties to the ...19911851866
expression of biologically active human corticosteroid binding globulin by insect cells: acquisition of function requires glycosylation and transport.human corticosteroid binding globulin (hcbg) is a 50- to 55-kda serum glycoprotein that binds cortisol and progesterone with high affinity. to map the steroid-binding domain and to investigate the folding pathways of hcbg, we have established an expression system based on infection of insect cells with a recombinant baculovirus encoding hcbg. infected spodoptera frugiperda (sf9) cells secrete immunoreactive hcbg at high levels (16-24 pmol per 10(6) cells per 40 h), and the recombinant protein bi ...19911862072
dengue-1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicits virus-neutralizing antibody in mice and protects them from virus challenge.in order to test the feasibility of baculovirus (autographa californica nuclear polyhedrosis virus, acnpv) expression vectors for making immunogens against dengue-1 (den-1) virus, a portion of the envelope (e) glycoprotein gene of den-1 virus was cloned and expressed. the recombinant baculovirus contains 107 nucleotides from the 3' terminus of the den-1 matrix (m) gene, which encodes a hydrophobic signal peptide and extends through the first 1, 245 nucleotides of e, terminating 243 nucleotides b ...19911877710
cysteine-86 is not needed for the enzymic activity of glutathione s-transferase 3-3.recombinant glutathione s-transferase 3-3 expressed in spodoptera frugiperda (sf9) cells with the use of a baculovirus expression system was modified with 1 mm-iodoacetamide. amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit. the s-carbaminomethylated protein retains the gsh-conjugating activity. glutathione s-transferase 3-3 modified with iodo[14c]acetamide was digested with achromobacter proteinase i and the resulting peptides were separated by h ...19911883338
identification of the very early transcribed baculovirus gene pe-38.we have started to identify early viral rnas that are transcribed at 1 h after inoculation to investigate the mechanism involved in the regulation of early gene expression of autographa californica nuclear polyhedrosis virus (acnpv). cloned viral dna fragments were hybridized to northern (rna) blots of polyadenylated rna isolated from spodoptera frugiperda cells at 1, 2, and 6 h postinfection to localize very early transcripts. subsequently we prepared a cdna library of polyadenylated rna transc ...19911987375
identification and characterization of the ie-1 gene of orgyia pseudotsugata multicapsid nuclear polyhedrosis virus.the ie-1 gene of orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (opmnpv) was mapped between 95.7 and 97.1 map units on the viral genome. sequence analysis of the opmnpv ie-1 gene (opie-1) identified an open reading frame that coded for a predicted protein of 560 amino acids with a molecular weight of 64,775. transcriptional analysis of opmnpv-infected lymantria dispar (ld652y) cells identified two rnas homologous to the opie-1 open reading frame that were 1.7 and 1.9 kb in size. the ...19911989381
expression of the outer capsid protein vp5 of two bluetongue viruses, and synthesis of chimeric double-shelled virus-like particles using combinations of recombinant baculoviruses.we have previously reported the assembly of virus-like particles (vlps), consisting of the four major structural proteins of bluetongue virus (btv), in spodoptera frugiperda cells coinfected with recombinant baculoviruses (french et al. (1990). j. virol. 64, 5695-5700). in this paper we report further studies using this system to assemble heterologous vlps containing the outer capsid proteins (vp2 and vp5) of a range of different btv serotypes. s. frugiperda cells were coinfected with three reco ...19911850928
biological function of recombinant il-6 expressed in a baculovirus system.the cdna of human interleukin-6 (il-6) was cloned into baculovirus dna. insect cells (spodoptera frugiperda cells) infected with the recombinant baculovirus secreted a large amount of 22k protein into the culture medium. this culture fluid contained high biological activity of growth stimulation of a mouse myeloid cell line (mh-60). the il-6 was purified by a one-step procedure employing immunoaffinity chromatography of monoclonal antibody to il-6. the specific activities (bsf-2 reference units/ ...19911883915
characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal antibody specific for human fibrin fragment d-dimer.a recombinant single-chain molecule, scfv-k12g0, containing the variable domains of the monoclonal antibody ma-15c5, specific for fragment d-dimer of human cross-linked fibrin, was constructed and expressed in spodoptera frugiperda, sf9, insect cells. the arg108 carboxyl-terminal amino acid of the variable domain of the light-chain of the antibody was connected through a synthetic ala-gly-gln-gly-ser-ser-val peptide linker with the gln1 amino-terminal amino acid of the variable domain of its hea ...19911885569
synthesis of the virus-specified tubules of epizootic haemorrhagic disease virus using a baculovirus expression system.the formation of virus-specific tubules is one of the most characteristic features in the orbivirus infection cycle, yet little is known about their role in virus replication. the tubuli are composed of a major nonstructural protein, ns1. we have investigated the expression of the ns1-encoding gene of epizootic haemorrhagic disease virus serotype 2 (alberta-strain) by producing a recombinant autographa californica nuclear polyhedrosis virus (acnpv). prior to cloning in the baculovirus transfer v ...19911891959
purification and characterization of the recombinant human aldose reductase expressed in baculovirus system.large quantities of recombinant human aldose reductase were produced using spodoptera frugiperda cells and properties of the enzyme were characterized. direct purification of the recombinant aldose reductase by affinity column chromatography using matrex gel orange a yielded a single 36 kda band, similar in size to the purified human muscle aldose reductase, on a sodium dodecyl sulfate-polyacrylamide gel after silver staining. the isoelectric point of the recombinant enzyme was 5.85 which is ide ...19911905957
synthesis of newcastle disease virus (ndv)-like envelopes in insect cells infected with a recombinant baculovirus expressing the haemagglutinin-neuraminidase of ndv.electron microscopical examination of negatively stained extracellular fluids (ecf) from spodoptera frugiperda cell cultures infected with a recombinant baculovirus expressing the newcastle disease virus (ndv) haemagglutinin-neuraminidase (hn) revealed ndv-like envelopes which resembled the envelopes of authentic ndv. immunogold staining with anti-ndv hn monoclonal antibodies demonstrated hn antigen in spikes on the ndv-like envelopes. the ecf from the recombinant-infected cultures also containe ...19912005440
characterization of an active, non-myristylated, cytoplasmic form of the lymphoid protein tyrosine kinase pp56lck.pp56lck is a member of the src family of tyrosine kinases mainly expressed in t lymphocytes. src tyrosine kinases have been implicated in the control of cell growth and differentiation in different cell types, but the mechanism of regulation of these enzymes is poorly understood. in order to characterize the distinct species of pp56lck, we have produced high yields of enzymatically active wild type pp56lck using the eukaryotic baculovirus expression system in spodoptera frugiperda insect cells ( ...19911911540
baculovirus-directed high level expression of the hepatitis delta antigen in spodoptera frugiperda cells.the hepatitis delta antigen (hdag) is a multifunctional protein. it forms the core-like structure of the hepatitis delta virus (hdv) but also enhances replication of hdv in the nucleus of the hepatocyte. a cdna fragment encoding hdag was inserted adjacent to the polyhedrin promoter of autographa californica nuclear polyhedrosis virus present in the baculovirus transfer vector pvl941. after transfection of spodoptera frugiperda (sf9) cells a recombinant baculovirus ac delta 1 was isolated and pur ...19912016595
expression and characterization of a rabbit liver cytochrome p450 belonging to p450iib subfamily with the aid of the baculovirus expression vector system.using baculovirus a cdna for cytochrome p450 (p450f1) belonging to rabbit p450iib subfamily was expressed in spodoptera frugiperda cells, where p450f1 was located on electron-dense structures (derived from the endoplasmic reticulum) present in both the cytoplasm and nucleus. partially purified p450f1 exhibited absorption spectra similar to those of p450(1), the major phenobarbital-inducible form in rabbit liver. like p450(1), p450f1 could oxidize aminopyrine, benzphetamine, 7-ethoxycoumarin, 1-n ...19912018490
alpha-mannosidase-catalyzed trimming of high-mannose glycans in noninfected and baculovirus-infected spodoptera frugiperda cells (iplb-sf-21ae). a possible contributing regulatory mechanism for assembly of complex-type oligosaccharides in infected cells.incubation of a spodoptera frugiperda (iplb-sf-21ae) cell extract with the oligosaccharide man9glcnac2, the aglucosyl derivative of the glycan that is normally transferred from the dolichol carrier to the relevant asn residue in the nascent protein, results in its trimming to man6glcnac2, an intermediate that is relatively stable to further alpha-d-mannosidase action in these cells. on the other hand, incubation of a similar extract from cells that had been infected for various times with a wild ...19911911772
characterization of a chimeric plasminogen activator consisting of a single-chain fv fragment derived from a fibrin fragment d-dimer-specific antibody and a truncated single-chain urokinase.an mr 57,000 single-chain chimeric plasminogen activator, k12g0s32, consisting of a variable region fragment (fv) derived from the fibrin fragment d-dimer-specific monoclonal antibody ma-15c5 and of a 33-kda (amino acids ala132 to leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-pa-33k) was studied. k12g0s32, secreted by infected spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography a ...19911918077
transcriptional analyses of baculovirus polyhedrin and foreign gene expression relative to baculovirus p10 mrna levels.comparisons have been made between the p10 and polyhedrin mrna levels recovered from spodoptera frugiperda cells infected with autographa californica nuclear polyhedrosis virus (acnpv). in molar terms and from 18 h post-infection (p.i.), the polyhedrin mrna species increased to levels one and a half times to twice as high as the p10 levels. the influence of the polyhedrin leader sequence on the expression of a foreign gene under the control of the polyhedrin promoter was investigated using a ser ...19911919531
baculovirus gp64 gene expression: analysis of sequences modulating early transcription and transactivation by ie1.expression of the baculovirus major envelope glycoprotein gene (gp64) is regulated by transcription from both early and late promoters. to characterize the early promoter and identify sequences involved in the regulation of gp64 early transcription, promoter-reporter gene fusions were generated from the orygia pseudotsugata nuclear polyhedrosis virus gp64 promoter and were analyzed by transient expression in uninfected insect cells. for these analyses, 5' deletion mutations were constructed in t ...19911920618
inhibition of human immunodeficiency virus type 1-induced cell fusion by recombinant human interferons.pretreatment of hela t4 cells with recombinant alpha, beta, or gamma interferon (ifn) was found to significantly inhibit syncytium formation induced by the human immunodeficiency virus type 1 (hiv-1) envelope glycoprotein. all three ifns were found to be potent inhibitors of fusion in a system in which spodoptera frugiperda cells, infected with a baculovirus recombinant expressing the hiv-1 envelope protein, were cocultivated with hela t4 cells. in addition, these ifns were also found to block h ...19911920634
characterization of haemagglutinin-neuraminidase glycoprotein of newcastle disease virus expressed by a recombinant baculovirus.a recombinant baculovirus containing a cdna which encodes haemagglutinin-neuraminidase (hn) of newcastle disease virus (ndv) was constructed. spodoptera frugiperda cells infected with this recombinant virus produced a large amount of hn glycoprotein similar to the authentic hn in size. the recombinant hn glycoprotein was localized on the surface of the infected cells and conserved its haemadsorption and neuraminidase activities. the antigenic properties of the recombinant hn glycoprotein seemed ...19911927050
the solubility of inclusion proteins from bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects.bacillus thuringiensis subsp. aizawai hd133 is one of several strains particularly effective against plodia interpunctella selected for resistance to b. thuringiensis subsp. kurstaki hd1 (dipel). b. thuringiensis subsp. aizawai hd133 produces inclusions containing three protoxins, cryia(b), cryic, and cryid, and the cryic protoxin has been shown to be active on resistant p. interpunctella as well as on spodoptera larvae. the cryia(b) protoxin is very similar to the major one in b. thuringiensis ...19912059054
the single cysteine residue on an alpha family chick liver glutathione s-transferase cl 3-3 is not functionally important.chick liver glutathione s-transferase cl 3-3, expressed using a baculovirus system in spodoptera frugiperda (sf9) cells, contains a single cysteine residue per subunit. this enzyme was modified with iodoacetamide. amino acid analysis indicates that 0.85 +/- 0.10 cysteine residue was modified per enzyme subunit. gst cl 3-3 modified with iodo[14c]acetamide was further digested with trypsin and the isotope-labelled fragments were isolated. the fragment containing the cysteine residue accounts for 5 ...19911930229
generation of recombinant baculovirus via liposome-mediated transfection.baculovirus expression vectors have become a popular method of producing recombinant proteins. production of recombinant virus requires the transfection of both the native viral dna and a transfer plasmid into insect cells where recombination takes place. while several methods of transfecting insect cells exist, we have found liposome-mediated transfection to be highly efficient. here we detail the protocols and medium needed for efficient, simple transfection of spodoptera frugiperda cells.19911931027
high-level secretion of the extracellular domain of the human growth hormone receptor using a baculovirus system.a chemically synthesized gene (hghr-ed) coding for the extracellular domain (ed) of the human growth hormone (hgh) receptor (hghr) was inserted into the genome of autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. spodoptera frugiperda cells infected with the recombinant virus secreted a protein with hgh-binding activity into the medium. the secreted 35-kda protein was purified to near homogeneity. the purified protein exhibited a high binding affinity (kd = 0 ...19911937047
identification of the human platelet gtpase activating protein for the cdc42hs protein.the cdc42hs protein appears to be an isoform of the ras-related gtp-binding protein g25k and is an apparent human homolog of the saccharomyces cerevisiae cell-division-cycle protein, cdc42sc. in this study, we report the identification of a gtpase-activating protein (gap) for cdc42hs from human platelets (designated from here on as cdc42hs-gap). the cdc42hs-gap activity was solubilized from platelet membranes, recovered through successive chromatography steps (the final step being mono-q chromat ...19911939135
evaluation of the role of conserved his and met residues among lipoxygenases by site-directed mutagenesis of recombinant human 5-lipoxygenase.the 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form his-(x)4-his-(x)4-his-(x)17-his-(x)8-his which represents a potential binding site for non heme iron to the protein. the importance of selected amino acids within this his cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems. after single mutation of each of the 5 his residues at positions 363, 368, 373, 391, and 400 by ser ...19911939225
microscopic visualization of insect cell-bubble interactions. ii: the bubble film and bubble rupture.in this paper, the second in the series, the use of a microscopic, high-speed video system to study the interactions of two suspended insect cells strains, trichoplusia ni (tn-368) and spodoptera frugiperda (sf-9), with rupturing bubbles is reported. events such as the adsorption of cells onto the bubble film and the mechanism of bubble rupture were observed. on the basis of these observations and the experimental and theoretical work of other researchers on bubble rupture and cell death as a re ...19911367170
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