| reconstitution of the recombinant 70-kda subunit of the clathrin-coated vesicle h+ atpase. | vacuolar-type proton pumps are complex heterooligomers. when dissociated into subcomplexes and subunits, the partial reactions of atp hydrolysis and transmembranous proton flow can be assigned to isolated domains. data suggest that the molecular site of atp hydrolysis resides within the 70-kda subunit but that atpase activity likely requires at least three additional subunits of 58, 40, and 33 kda (xie, x.-s., and stone, d. k. (1988) j. biol. chem. 263, 9859-9867). we have now cloned and sequenc ... | 1994 | 7961699 |
| comparative morphology of gag protein structures produced by mutants of the gag gene of human immunodeficiency virus type 1. | six mutants that differ in the extent of their carboxyterminal sequences and two deletion mutants of the gag gene of hiv-1 have been characterized morphologically following their expression in spodoptera frugiperda cells using recombinant baculoviruses. electron microscopy has revealed distinct morphological forms of the gag protein that can be classified as either (i) particulate, three-dimensional, spherical or tubular shells or (ii) non-particulate, two-dimensional, flat, curved or convoluted ... | 1994 | 7964608 |
| crystallization and x-ray crystallographic analysis of recombinant chicken poly(adp-ribose) polymerase catalytic domain produced in sf9 insect cells. | poly (adp-ribose) polymerase (parp) participates in the immediate response in mammalian cells exposed to dna-damaging agents. recombinant baculovirus harboring the cdna of the chicken parp catalytic domain (40 kda) have been used to infect spodoptera frugiperda (sf9) insect cells. the recombinant polypeptide (30 mg per 1 x 10(9) cells) was purified to homogeneity by 3-aminobenzamide affinity chromatography. the enzymatic properties of the recombinant domain were similar to those of the native fr ... | 1994 | 7966315 |
| suppression of apoptosis in insect cells stably transfected with baculovirus p35: dominant interference by n-terminal sequences p35(1-76). | expression of p35 from the dna genome of autographa californica nuclear polyhedrosis virus (acmnpv) suppresses virus-induced apoptosis and promotes virus replication in spodoptera frugiperda (sf21) cells. to examine the molecular mechanism by which p35 prevents apoptosis in insects, sf21 cells were stably transfected with p35. neomycin-resistant cell lines that synthesized protein p35 were identified. stable transfection with p35 protected sf21 cells from apoptosis induced by actinomycin d conce ... | 1994 | 7966562 |
| functional characterization of partially purified epstein-barr virus dna polymerase expressed in the baculovirus system. | the dna polymerase gene of epstein-barr virus (ebv) was cloned into baculovirus transfer vector (pbluebac). the recombinant baculovirus (acebp-15) was obtained by cotransfection of spodoptera frugiperda (sf9) cells with infectious dna from autographa californica multiple nuclear polyhedrin virus (acmnpv) and pbluebac plasmid carrying ebv polymerase gene. infection of sf9 cells with the recombinant virus produced substantial quantities of the ebv dna polymerase protein of the expected size (110 k ... | 1994 | 7975269 |
| expression of ricin b chain in spodoptera frugiperda. | dna encoding ricin b chain was derived from preproricin genomic dna and ligated into the baculovirus transfer vector, pacgp67a. co-transfection of spodoptera frugiperda sf9 cells with baculogold dna was followed by limiting dilution purification of recombinant baculovirus. infection of sf9 cells at a multiplicity of infection of 1 in the presence of 25 mm lactose produced 3 mg/l of soluble, glycosylated, 34 kda protein immunoreactive with monoclonal and polyclonal antibodies to ricin b chain. th ... | 1994 | 7980448 |
| production, purification, and characterization of recombinant maspin proteins. | maspin, a novel mammary serine protease inhibitor, was shown to have tumor suppressing activity (zou, z., anisowicz, a., hendrix, m. j. c., thor, a., neveu, m., sheng, s., rafidi, k., seftor, e., and sager, r. (1994) science 263, 526-529). in this paper, we report the production of recombinant glutathione s-transferase-maspin fusion protein, expressed in the bacterium escherichia coli, and recombinant maspin, expressed in the insect spodoptera frugiperda cells. the fusion protein was purified by ... | 1994 | 7983035 |
| early transcription of the ie-1 transregulator gene of autographa californica nuclear polyhedrosis virus is regulated by dna sequences within its 5' noncoding leader region. | the ie-1 gene of autographa californica nuclear polyhedrosis virus (acmnpv) encodes a transregulatory protein (ie1) which accelerates the expression of early and late virus genes. transcription of ie-1 occurs immediately upon infection from a conserved cagt motif and continues into the late phases. to examine the mechanisms by which ie-1 expression is regulated, cis-acting control elements within the ie-1 promoter were identified by constructing hybrid early promoters and by using site-directed ... | 1995 | 7983706 |
| metallopanstimulin gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to dna. | the protein product of the human metallopanstimulin (mps-1) gene was produced in the insect cell line spodoptera frugiperda (sf9) using the baculovirus expression vector system autographa californica nuclear polyhedrosis virus (acnpv). when a cloned mps-1 complementary dna sequence was inserted into the acnpv viral genome downstream from the promoter of the polyhedrin gene, a polypeptide with an apparent molecular weight of approximately 10,000 was observed in extracts of infected sf9 cells. thi ... | 1994 | 7986747 |
| molecular characterization of a strain-specific repeated dna sequence in the fall armyworm spodoptera frugiperda (lepidoptera: noctuidae). | the fall armyworm moth, spodoptera frugiperda, is a migratory species composed of sympatric corn and rice strains. the strains are indistinguishable in morphology but can be recognized by molecular markers. we have cloned and characterized seven monomer units of a repeated dna sequence, called fr, which is found exclusively in the genome of the rice strain individuals. the 189 bp fr units are tandemly organized in arrays longer than 30 kb. female individuals possess over 100-fold more of the fr ... | 1994 | 7987522 |
| baculovirus expression of non-structural protein ns2 and core protein vp7 of african horsesickness virus serotype 3 and their use as antigens in an indirect elisa. | non-structural protein ns2 and core protein vp7 of african horsesickness virus serotype 3 (ahsv3) were expressed in spodoptera frugiperda cells by recombinant baculoviruses containing the relevant genes. these proteins were purified and analysed by polyacrylamide gel electrophoresis and western blot. ns2 and vp7 were used separately as antigens in an indirect elisa for the detection of ahsv antibodies. both antigens cross-reacted with hyperimmune guinea-pig antisera to infected cell lysates of a ... | 1994 | 7989441 |
| iodinated 2-aminotetralins and 3-amino-1-benzopyrans: ligands for dopamine d2 and d3 receptors. | in developing selective ligands for dopamine d2 and d3 receptors, several iodinated 2-aminotetralins and 3-amino-1-benzopyrans, trans-7-hydroxy-2-[n-(3'-iodo-2'- propenyl)amino]tetralin (1), trans-monohydroxy-2-[n-propyl-n-(3'-iodo-2'-propenyl)amino]tetralin (7-, 5-, and 6-oh-pipat) (2, 3, and 4), and trans-monohydroxy-3,4-dihydro-3-[n-propyl-n-(3'-iodo-2'- propenyl)-amino]-2h-1-benzopyran (6- and 8-oh-benzopyrans) (5 and 6), were prepared. these compounds were evaluated for their binding profil ... | 1994 | 7990123 |
| cloning and production of functional active recombinant hepatitis b virus surface antigen binding protein. | endonexin ii present on the surface of human hepatocytes has recently been identified as a hepatitis b virus surface antigen (hbsag) binding protein. a full-length cdna clone encoding human endonexin ii was isolated from a human liver cdna library and was placed under the control of the polyhedrin promoter of autographa californica nuclear polyhedrosis virus (acnpv). infection of spodoptera frugiperda cells with recombinant virus resulted in the production of high amounts of recombinant protein. ... | 1994 | 7999073 |
| baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the kozak consensus and comparison with bacterial expression. | synthetic genes encoding the 146 and 155 amino acid forms of human basic fibroblast growth factor (bfgf) were constructed with codon usage biased towards the polyhedrin-encoding gene of autographa californica nuclear polyhedrosis virus (acnpv). expression of both bfgf genes in spodoptera frugiperda (sf-21) suspension cell culture using a recombinant baculovirus yielded approximately 2.5 mg of mitogenically fully active protein per 10(9) cells following heparin-affinity chromatography. to improve ... | 1995 | 7999789 |
| glycoform heterogeneity of porcine interferon-gamma expressed in sf9 cells. | porcine interferon-gamma (sfpoifn-gamma) was expressed with high efficiency in spodoptera frugiperda (sf9) cells by means of the baculovirus expression system. up to 10(5) u/ml of antivirally active sfpoifn-gamma could be tracked down in the culture medium at 64 h postinfection. three proteins (17, 19, and 21 kda), which under nondenaturing conditions primarily exist as mutual-dimeric combinations, were purified by immunoaffinity chromatography. carbohydrate labeling and kinetic deglycosylation ... | 1994 | 7999924 |
| autographa californica nuclear polyhedrosis virus, pdv, and ecv viral envelopes and nucleocapsids: structural proteins, antigens, lipid and fatty acid profiles. | autographa californica nuclear polyhedrosis virus (acmnpv) infection results in the production of two types of infectious, enveloped viruses. as both of these viral forms play significantly different roles in the virus life cycle, the different functional characteristics in the roles of the virus may be explained, in part, by the protein and lipid composition and source of the viral envelopes. both viruses utilize different maturation and envelopment strategies: extracellular virus (ecv) obtains ... | 1994 | 8009843 |
| engineering the assembly pathway of the baculovirus-insect cell expression system. | the synthesis of complex biological structures such as antibodies using recombinant dna technology is a major biotechnological advance. active murine antibody (igg) oligomers, composed of two heavy (h) and two light (l) polypeptide chains, have been expressed and secreted by the baculovirus-insect cell expression system. unfortunately, expression of the functional antibodies is accompanied by the formation of abnormal protein complexes and aggregates in which the polypeptide chains are bound tog ... | 1994 | 8010671 |
| expression of aleutian mink disease parvovirus capsid proteins in a baculovirus expression system for potential diagnostic use. | a 2.3-kb cdna clone encoding aleutian mink disease parvovirus (adv) structural proteins vp1 and vp2 was inserted into the polyhedron gene of autographa californica nuclear polyhedrosis virus (acnpv) and expressed by the recombinant virus, acadv-1, in spodoptera frugiperda-9 cells. sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (wia) indicated that synthesis of both vp1 and vp2 was being directed by acadv-1. fluorescence microscopic examination of acadv- ... | 1994 | 8011776 |
| processing of the precursors of the human thyroid-stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected l cells and baculovirus-infected insect cells). | the complementary dna for human thyroid-stimulating hormone (tsh) receptor encodes a single protein with a deduced molecular mass of 84.5 kda. this protein is cleaved during its maturation in the human thyroid since the receptor protein has been shown to be composed of two subunits (alpha subunit of approximately 53 kda and beta subunit of approximately 38 kda) held together by disulfide bridges [loosfelt, h., pichon, c., jolivet, a., misrahi, m., caillou, b., jamous, m., vannier, b. & miligrom, ... | 1994 | 8020509 |
| analysis of c-terminally truncated dengue 2 and dengue 3 virus envelope glycoproteins: processing in insect cells and immunogenic properties in mice. | we constructed two recombinant autographa californica nuclear polyhedrosis baculoviruses. spodoptera frugiperda (sf9) cells containing these constructs produce carboxy-terminally truncated envelope e proteins representing dengue (den) virus serotypes 2 and 3. the two recombinant proteins contained their homologous signal sequences at the n terminus and were truncated by 71 and 74 amino acids at the c terminus, respectively. this allowed the translocation of the recombinant proteins to the endopl ... | 1994 | 8021588 |
| cloning and expression of the cervine interleukin 4 gene. | interleukin 4 (il-4), a cytokine produced by the t helper 2 subset of t lymphocytes is involved in up regulating antibody responses. a source of recombinant cervine il-4 would be useful for studying the immune response of deer to tuberculosis. we report here the cloning, sequencing and expression of recombinant cervine il-4. to achieve this mrna was isolated from red deer (cervus elaphus) mononuclear leucocytes. first strand cdna was synthesized from the mrna and the il-4 cdna was amplified, clo ... | 1994 | 8029646 |
| the autographa californica nuclear polyhedrosis virus ie2 gene encodes a transcriptional regulator. | the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) expresses several immediate early genes in infected cells. one of these, ie2 (formerly called ien), was mapped and identified by its ability to augment ie1-mediated transactivation of the delayed early 39k gene. ie2 also stimulated the expression of reporter genes linked to the ie1 promoter. to test whether the increase in expression was due to increased mrna, primer extension analyses were performed. spodoptera frugiperda ... | 1994 | 8030237 |
| expression of porcine leukocyte 12-lipoxygenase in a baculovirus/insect cell system and its characterization. | arachidonate 12-lipoxygenase (12-lo) from porcine leukocytes was expressed in insect cells using a baculovirus expression vector. the recombinant 12-lo was expressed as an n-terminal fusion protein with a 31-amino acid polypeptide carrying a six-histidine tag and an enterokinase cleavage site. maximal intracellular enzyme activity and protein levels were observed 48 h after infection of spodoptera frugiperda cells with the recombinant virus. cells were lysed and the recombinant protein was purif ... | 1994 | 8031130 |
| expression and post-translational processing of a broad-spectrum organophosphorus-neurotoxin-degrading enzyme in insect tissue culture. | a recombinant baculovirus, autographa californica nuclear polyhedrosis virus (acnpv), has been utilized to express the opd (organophosphate-degrading) gene from pseudomonas diminuta in insect tissue-culture cells (sf9) of the fall armyworm (spodoptera frugiperda). the broad-spectrum organophosphate hydrolase (ec 3.1.8.1) encoded by this gene is a member of a general class of enzymes [organophosphate (op) anhydrolases] that include parathion hydrolases, di-isopropyl-fluorophosphatases (dfpases), ... | 1994 | 8031504 |
| production of active human interleukin-1 beta-converting enzyme in a baculovirus expression system. | the cdna coding for the precursor form of human interleukin-1 beta-converting enzyme (proice) was expressed in spodoptera frugiperda (sf9) insect cells using a baculovirus expression system. the 45-kda recombinant protein was further processed to several smaller forms of 32, 24, 20, 13 and 10 kda. active recombinant ice derived from the baculovirus expression system (bvice) was found to be present in soluble lysates of insect cells as an associated heterodimer consisting of 10- and 20-kda subuni ... | 1994 | 8056342 |
| the geminivirus br1 movement protein binds single-stranded dna and localizes to the cell nucleus. | plant viruses encode movement proteins that are essential for infection of the host but are not required for viral replication or encapsidation. squash leaf curl virus (sqlcv), a bipartite geminivirus with a single-stranded dna genome, encodes two movement proteins, br1 and bl1, that have been implicated in separate functions in viral movement. to further elucidate these functions, we have investigated the nucleic acid binding properties and cellular localization of br1 and bl1. in this study, w ... | 1994 | 8069108 |
| baculovirus infection does not alter n-glycosylation in spodoptera frugiperda cells. | spodoptera frugiperda cells are the standard host system for baculovirus-vector-mediated expression of recombinant glycoproteins. in an attempt to explore their ability to produce complex n-glycans containing terminal neuraminic acid, we tested both mock- and acmnpv-infected sf9 cells. to elucidate the structures of the carbohydrate chains of cellular glycoproteins, radiolabeled oligosaccharides were liberated by treatment with endo-h and glycopeptidase f. when the endo-h resistant material was ... | 1994 | 8074803 |
| a rapid one-step method to purify baculovirus-expressed human estrogen receptor to be used in the analysis of the oxytocin promoter. | we have produced a truncated form of the human estrogen receptor (her) as a fusion protein with glutathione s-transferase (gst) in spodoptera frugiperda (sf) cells using the baculovirus expression vector (bev) system. the protein is correctly produced and can be purified from crude whole-cell extracts by a single-step, batch-wise affinity-purification procedure. we show that this gst-her fusion protein binds at its dna-binding site specifically and in a hormone-inducible manner. furthermore, we ... | 1994 | 8076833 |
| serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles. | the l1 major capsid protein-coding sequences of human papillomavirus (hpv) types 11, 16 and 18 were expressed in the baculovirus system. virus-like particles (vlps) were purified from recombinant-infected spodoptera frugiperda sf9 cells and cell-free culture supernatants. rabbits immunized with purified vlps developed antibodies that reacted only with the specific vlp type used as the immunogen. in addition, rabbit antibodies raised against infectious hpv-11 virions only reacted with hpv-11 l1 v ... | 1994 | 8077946 |
| subcore- and core-like particles of broadhaven virus (brdv), a tick-borne orbivirus, synthesized from baculovirus expressed vp2 and vp7, the major core proteins of brdv. | the genes encoding the two major core proteins (vp2 and vp7) of broadhaven (brd) virus, a tick-borne orbivirus, were inserted into the genome of autographa californica nuclear polyhedrosis virus (acnpv) under the control of copies of the acnpv polyhedrin promoter to produce two recombinant baculoviruses. infection of spodoptera frugiperda (sf) cells with a recombinant acnpv that synthesized brdv vp2 produced large numbers of brdv subcore-like particles. co-infection of cells with the two recombi ... | 1994 | 8079519 |
| functional expression and purification of the abc transporter complex associated with antigen processing (tap) in insect cells. | using the baculovirus expression system the gene products of human tap1 and tap2 were over-expressed as wild-type as well as oligohistidine fusion proteins in spodoptera frugiperda (sf9) insect cells. both gene products were co-expressed within the same cells and were found enriched in microsomal membranes. immunoprecipitation and immobilized metal affinity chromatography revealed complex formation between tap1 and tap2. the expressed tap complex was shown to be functional by peptide translocati ... | 1994 | 8082812 |
| influence of infection route on the infectivity of baculovirus mutants lacking the apoptosis-inhibiting gene p35 and the adjacent gene p94. | the infectivity of autographa californica nuclear polyhedrosis virus mutants lacking the apoptosis-inhibiting gene p35 is decreased 1,000-fold or more in larvae of the insect spodoptera frugiperda if the budded form of the virus is administered by hemocoelic injection; this decrease is correlated with the antiviral effects of apoptosis (r. j. clem and l. k. miller, j. virol. 67:3730-3738, 1993). we have extended this correlation by showing that the infectivity of p35 mutant budded virus is resto ... | 1994 | 8084009 |
| transcription, translation, and cellular localization of pdv-e66: a structural protein of the pdv envelope of autographa californica nuclear polyhedrosis virus. | a late gene encoding a 66-kda structural protein of autographa californica nuclear polyhedrosis virus was mapped and sequenced (26.9-29.7 mu). transcription initiates from two conserved taag motifs (-15 and -37) with transcripts detected from 12 to 72 hr pi. the protein is detected in infected spodoptera frugiperda (sf9) cells from 24 to 72 hr pi. western-blot and immunoelectron microscopic data identify this protein as specific for the polyhedra-derived virus (pdv) envelope. this protein has be ... | 1994 | 8091653 |
| control of expression, glycosylation, and secretion of hiv-1 gp120 by homologous and heterologous signal sequences. | the hiv-1 gp120 signal sequence of 30 amino acids is longer than most glycoprotein signal sequences and contains an average of 5 positively charged amino acids. the hiv-1 gp120 gene with its natural signal sequence expressed in any prokaryotic or eukaryotic expression systems showed extremely low levels of synthesis and secretion. however, deletion of the hiv-1 gp120 signal sequence results in production of large quantities of a nonglycosylated form of gp120 in spodoptera frugiperda cells. subst ... | 1994 | 8091657 |
| mutagenesis of two surface-exposed loops of the bacillus thuringiensis cryic delta-endotoxin affects insecticidal specificity. | site-directed mutagenesis was used to determine the role of two surface-exposed loops (gly-317-phe-320 and gln-374-pro-377) in the insecticidal specificity of the bacillus thuringiensis cryic delta-endotoxin. mutant toxins were generated by pcr using degenerate oligonucleotide primers, and expressed in escherichia coli. more than 50 mutant toxins were screened for toxicity to the lepidopteran spodoptera frugiperda sf9 cell line using an in vitro lawn assay. a panel of these mutant toxins, which ... | 1994 | 8093015 |
| baculovirus gene me53, which contains a putative zinc finger motif, is one of the major early-transcribed genes. | we have been investigating the first viral genes to be transcribed in spodoptera frugiperda cells after the infection with autographa california nuclear polyhedrosis virus in order to identify regulatory proteins required for the activation of early and/or late gene transcription. by screening a cdna library from polyadenylated rna transcribed at 1 h postinfection, we identified a gene which is designated me53. this newly identified gene is located upstream of the late p74 gene and forms a diver ... | 1993 | 8093490 |
| human papillomavirus type 16 e6, e7 and l1 and type 18 e7 proteins produced by recombinant baculoviruses. | proteins derived from the e6, e7 and l1 orfs of hpv16 and the e7 orf of hpv18 were produced in insect cells using a baculovirus expression system. hpv orfs were inserted into baculovirus transfer vectors pacym1 or pvl1393/2, and recombinant baculoviruses isolated using a combination of limiting dilution and plaque assay. using hpv-specific antisera and monoclonal antibodies hpv proteins were identified in lysates of spodoptera frugiperda (sf-21) cells infected with hpv-recombinant baculovirus. i ... | 1993 | 8106603 |
| crystallization and initial x-ray crystallographic characterization of recombinant bovine inositol polyphosphate 1-phosphatase produced in spodoptera frugiperda cells. | bovine inositol polyphosphate 1-phosphatase, a monomeric protein with a molecular mass of 44,000 da, hydrolyzes the 1-position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. the low abundance of inositol polyphosphate 1-phosphatase in tissues has precluded structural studies requiring large quantities of enzyme. we used recombinant baculovirus harboring the cdna of bovine inositol polyphosphate 1-phosphatase to infect spodoptera frugiperda (sf9) insect cells. recombin ... | 1994 | 8107142 |
| characterization of [125i](r)-trans-7-hydroxy-2-[n-propyl-n-(3'-iodo-2'-propenyl)amino] tetralin binding to dopamine d3 receptors in rat olfactory tubercle. | [125i](r,s)-trans-7-hydroxy-2-[n-propyl-n-(3'-iodo-2'-propenyl)- amino]tetralin ([125](r,s)-trans-7-oh-pipat) has been shown to bind with high affinity to dopamine d3 receptors expressed in spodoptera frugiperda cells. no specific binding was seen in spodoptera frugiperda cells expressing a high density of d2 receptors. it was therefore, suggested that [125i] (r,s)-trans-7-oh-pipat selectively labels d3 receptors. in the present study, saturation binding of [125i](r)-trans-7-oh-pipat to membrane ... | 1994 | 8114008 |
| location of agonist-dependent-phosphorylation sites in the third intracellular loop of muscarinic acetylcholine receptors (m2 subtype). | muscarinic acetylcholine receptors (machr, human m2 subtype) expressed in sf9 (spodoptera frugiperda) cells using the baculovirus system were purified and subjected to phosphorylation by a machr kinase, which was partially purified from porcine cerebrum. two bands with apparent molecular masses of 59 kda and 39 kda as determined by sds/page were found to be phosphorylated in an agonist-dependent manner. both bands were labeled by the irreversible muscarinic ligand [3h]propylbenzilylcholine musta ... | 1994 | 8119296 |
| complementation of human immunodeficiency virus (hiv-1) gag particle formation. | the human immunodeficiency virus gag precursor protein pr55gag exhibits the ability of particle assembly when expressed using recombinant baculoviruses. in order to delineate the sequences required for particle formation, two mutants of gag (d1 and d2) were constructed in which 10 amino acids within the ca domain were deleted. both mutants yielded stable high levels of gag antigen following expression in spodoptera frugiperda insect cells. electron microscopy of sections through infected cells r ... | 1994 | 8122369 |
| development of a recombinant enzyme-linked immunosorbent assay for detection of antibodies against epstein-barr virus nuclear antigens 2a and 2b. | the baculovirus expression system was used to produce full-length epstein-barr virus nuclear antigens (ebnas) 2a and 2b. recombinant baculoviruses that contained the ebna-2a- and ebna-2b-encoding sequences were constructed. the proteins were expressed in spodoptera frugiperda sf-9 cells infected with the recombinant viruses and were characterized by using monoclonal and human polyclonal antibodies by immunoblotting and immunofluorescence techniques. partially purified extracts of the ebna-2a- an ... | 1994 | 8126164 |
| processing of asparagine-linked oligosaccharides in insect cells. n-acetylglucosaminyltransferase i and ii activities in cultured lepidopteran cells. | the levels of beta 1,2-n-acetylglucosaminyltransferase (glcnac-t) i and ii activities in cultured cells from bombyx mori (bm-n), mamestra brassicae (izd-mb-0503) and spodoptera frugiperda (sf-9 and sf-21) were investigated. apart from initial experiments with man alpha-3(man alpha 1-6)-man beta 1-o(ch2)8cooh3 and 3h-labelled udp-glcnac as substrates, glcnac-t i activity was measured with a non-radioactive hplc method using pyridylaminated man3-glcnac2 and man5glcnac2 as acceptor oligosaccharides ... | 1993 | 8130393 |
| association of peroxisome proliferator-activated receptor and hsp72. | in an effort to understand the relationship between a 72-kda heat shock protein (hsp72) and peroxisome proliferator-activated receptors (ppars), we have characterized their interaction using clofibric acid-sepharose chromatography and co-immunoprecipitation with antisera raised against either rat ppar (rppar) or hsp72. first, we observed that both rppar and hsp72 elute in a clofibrate-dependent manner from the clofibric acid-sepharose matrix. second, we found that immunoprecipitation of either p ... | 1994 | 8132576 |
| expression of recombinant human thyroid peroxidase by the baculovirus system and its use in elisa screening for diagnosis of autoimmune thyroid disease. | the cdnas coding for human full-length and soluble thyroid peroxidase (tpo) were constructed, cloned into a baculovirus transfer vector and used for infection of spodoptera frugiperda (sf9) cells. the soluble tpo lacking 87 amino acids of the c-terminal transmembrane and intracisternal domains was designed as a fusion protein with a histidine-hexapeptide as an affinity ligand at its c-terminus. whereas the recombinant full-length tpo was expressed mainly in an insoluble form in sf9 cells, the re ... | 1993 | 8136455 |
| an apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with cys/his sequence motifs. | two different baculovirus genes are known to be able to block apoptosis triggered upon infection of spodoptera frugiperda cells with p35 mutants of the insect baculovirus autographa californica nuclear polyhedrosis virus (acmnpv):p35 (p35-encoding gene) of acmnpv (r. j. clem, m. fechheimer, and l. k. miller, science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of cydia pomonella granulosis virus (cpgv) (n. e. crook, r. j. clem, and l. k. miller, j. virol. 67:2168-2174, 1993). using ... | 1994 | 8139034 |
| the porcine thromboxane synthase-encoding cdna: sequence, mrna expression and enzyme production in sf9 insect cells. | a full-length cdna encoding porcine thromboxane synthase (ts) was isolated and sequenced. the open reading frame encodes a 534-amino acid (aa) protein (m(r) 60,451) which shares more than 75% identity with ts from other species and is 30% homologous to several enzymes of the cytochrome p-450 iii family. sequence comparison among porcine (p), human (h), and murine (m) ts indicated conservation of eight cys residues and one putative n-glycosylation site. several highly conserved regions were ident ... | 1994 | 8144035 |
| human monocyte chemoattractant protein-1 expressed in a baculovirus system. | human monocyte chemoattractant protein-1 (hmcp-1) was produced using a baculovirus system. the hmcp-1 cdna was inserted into the genomic dna of autographa californica nuclear polyhedrosis virus (acnpv) using a transfer vector, pjvp10z. spodoptera frugiperda insect cells, which were infected with this recombinant virus, secreted recombinant hmcp-1 (re-hmcp-1) at the level of 10-20 micrograms/ml of culture medium. this product was shown to chemoattract monocytes. three distinct bands of 11, 11.5 a ... | 1994 | 8144036 |
| dengue type-2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge. | the gene coding for the envelope (e) glycoprotein of dengue-2 virus was cloned into baculovirus (autographa californica nuclear polyhedrosis virus). the recombinant virus contained the entire e protein gene, preceded by 38 nucleotides from the end of the prematrix glycoprotein gene and followed by the first 83 nucleotides of nonstructural protein 1. when expressed in spodoptera frugiperda (sf9) cells, approximately 1 mg of recombinant e antigen was made per 10(9) cells. this antigen reacted with ... | 1994 | 8147490 |
| properties of a recombinant kainate receptor expressed in baculovirus-infected insect cells. | glur6 is a glutamate receptor of the kainate subtype that is expressed in the mammalian central nervous system. the cdna coding for the rat brain receptor was subcloned into a baculovirus expression vector and the purified recombinant virus was used to infect spodoptera frugiperda (sf9) insect cells. the pharmacological profile and the status of several post-translational modifications of the glur6 protein were analyzed. saturation analyses of [3h]kainic acid binding demonstrated that glur6 expr ... | 1994 | 8157071 |
| reconstitution of recombinant 33-kda subunit of the clathrin-coated vesicle h(+)-atpase. | evidence suggests that the atp hydrolytic sector of the clathrin-coated vesicle proton-translocating atpase is composed of four subunits of molecular masses of 70, 58, 40, and 33 kda (xie, x. s., and stone, d. k. (1988) j. biol. chem. 263, 9859-9867). we have now expressed recombinant 33-kda polypeptide in escherichia coli and in spodoptera frugiperda (sf9) cells. this subunit, renatured and purified from both sources, lacks intrinsic atpase activity. co-reconstitution of these recombinant 33-kd ... | 1994 | 8157666 |
| amphibian allantoinase. molecular cloning, tissue distribution, and functional expression. | the chain of enzymes necessary to convert uric acid to its metabolic products urea and glyoxylic acid in vertebrates is truncated through the successive loss of allantoicase, allantoinase, and urate oxidase during phylogenetic evolution. previous studies have assigned the localization of both urate oxidase and allantinase to the peroxisome in the amphibian liver. this study reports the cloning of a cdna encoding bullfrog (rana catesbeiana) allantoinase, an enzyme that converts allantoin to allan ... | 1994 | 8163532 |
| accurate processing and secretion in the baculovirus expression system of an erythroid-cell-stimulating factor consisting of a chimaera of insulin-like growth factor ii and an insect insulin-like peptide. | a synthetic gene encoding the signal peptide and the n-terminal sequence of bombyxin, an insect insulin-like peptide, and the 58 amino acids of the c-terminal sequence of human insulin-like growth factor ii (igf ii) has been expressed using the baculovirus system. this synthetic chimaera was obtained by amplification of four overlapping oligonucleotides using taq polymerase and cloning into the transfer vector pbluebac. the construct was integrated by homologous recombination into the autographa ... | 1994 | 8166627 |
| functional expression of rat peroxisomal acyl-coa oxidase in spodoptera frugiperda cells. | a cdna coding the 661-residue rat peroxisomal fatty acyl-coa oxidase (acox) has been constructed and expressed as catalytically active protein in spodoptera frugiperda (sf9) insect cells using baculovirus expression system. recombinant viral clones were purified and the expressed protein was identified by immunoblotting and catalytic activity. in the rat liver, acox consists of three polypeptide components a, b and c, with relative molecular mass of 72 kda, 51 kda and 21 kda, respectively. in sf ... | 1994 | 8166685 |
| comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein. | the processing of the n-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (seap) expressed with a recombinant autographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established from spodoptera frugiperda, trichoplusia ni, and mamestra brassicae. studies with endoglycosidase h (endo h), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular seap produced in the m. brassicae-derived mb0503 cell line was endo h r ... | 1993 | 8167899 |
| cellular toxicities and membrane binding characteristics of insecticidal crystal proteins from bacillus thuringiensis toward cultured insect cells. | the pattern of in vitro toxicity of activated toxins from several classes of entomocidal inclusion genes from bacillus thuringiensis was measured using eight established cell lines from lepidopteran insects. protoxins representing cryia(b), cryia(c), and a mixture of all three cryia toxins (subtypes a, b, and c; from b. thuringiensis subsp. kurstaki hd-1) were compared with the protoxin representing cryic in a bioassay which measured the viability of cultured insect cells upon exposure to entomo ... | 1994 | 8176242 |
| synthesis of recombinant baculoviruses expressing the outer capsid protein vp2 of five btv serotypes and the induction of neutralizing antibodies to homologous and heterologous btv serotypes. | dna representing rna segment l2 of 5 different bluetongue virus (btv) serotypes (btv-1, -2, -11, -13 and -17) corresponding to the gene that codes for the btv neutralization antigen vp2, have been inserted individually into baculovirus transfer vectors in lieu of the 5' coding region of the polyhedrin gene of autographa california nuclear polyhedrosis virus (acnpv). after co-transfection of spodoptera frugiperda cells with wild-type acnpv dna in the presence of the derived transfer vector dnas, ... | 1994 | 8178571 |
| expression and ligand binding characterization of the beta-subunit (p75) ectodomain of the interleukin-2 receptor. | the baculovirus-mediated eukaryotic insect cell expression system was used to prepare large quantities of the beta-subunit ectodomain of the high-affinity interleukin-2 receptor (il-2r beta x). we describe the expression, purification, and biophysical characterization of this ligand binding domain. the human cdna encoding il-2r beta x was inserted into baculovirus transfer vectors. high titer recombinant baculovirus was produced in spodoptera frugiperda (sf9) insect cells, and the viral supernat ... | 1994 | 8180212 |
| expression and characterization of human dopamine d2 receptor in baculovirus-infected insect cells. | multiple types of dopamine d2-like receptors (d2, d3, d4) have been identified. differences in pharmacology among these receptors may have profound clinical ramifications for the treatment of psychosis. analysis of the structure and function of their binding sites requires a source of large amounts of receptor, uncontaminated by the other types of d2-like receptor. we engineered a recombinant baculovirus containing the human d2 receptor cdna (drd2) to express this receptor in insect cells. spodo ... | 1994 | 8182575 |
| flow cytometric analysis of bioluminescence emitted by recombinant baculovirus-infected insect cells. | five recombinant baculoviruses, each containing a different insect luciferase gene encoding a protein with characteristic light emission properties, namely, luc gr (546 nm), luc ff (556 nm), luc yg (560 nm), luc ye (578 nm), and luc or (593 nm) were constructed. all genes were inserted under the transcriptional control of the polyhedrin gene promoter of the autographa californica nuclear polyhedrosis virus (acnpv) and expressed in spodoptera frugiperda insect cells during viral infection. the bi ... | 1994 | 8187580 |
| spodoptera frugiperda sf-9 cells nuclear factor binds to a specific sequence on the baculovirus polyhedrin promoter. | the promoter of the baculovirus polyhedrin gene is used for the heterologous expression of proteins in the baculovirus expression system. the polyhedrin gene of baculovirus is a very late gene that is hyper-expressed 18-24 h post-infection (p.i). we analyzed the dna-binding activity of the polyhedrin promoter using gel mobility shift analysis, dnasei footprint analysis and uv cross-linking experiments. specific dna-binding activity was found in uninfected cells. specific binding activity was als ... | 1994 | 8191787 |
| detection of a putative 30-kda ligand of the cluster-2 antigen. | the cluster-2 antigen, also called egp-2, is a 38-kda transmembrane glycoprotein with a distribution that is largely confined to human epithelial cells and their derived carcinomas. monoclonal antibodies (mabs) directed against egp-2 have been extensively studied as anti-tumor agents, yet the function of the antigen is not known. in the present study we used a biotinylated recombinant soluble derivative of the egp-2 (segpbio) as a probe to detect a possible egp-2 ligand, using various carcinoma ... | 1994 | 8194899 |
| eukaryotic expression of recombinant human centromere autoantigen and its use in a novel elisa for diagnosis of crest syndrome. | the cdna coding for the human full-length centromere protein b (cenp-b) was isolated from a liver cdna library by oligonucleotide screening and extended at the 5' and 3' ends by linker addition. the cdna was inserted into a modified baculovirus transfer vector which mediated high-level expression of recombinant human cenp-b with a histidine-hexapeptide as affinity ligand at its n-terminus in infected spodoptera frugiperda (sf9) insect cells. based on the histidine-hexapeptide moiety, the recombi ... | 1994 | 8198697 |
| biologically active recombinant human complement factor h: synthesis and secretion by the baculovirus system. | a complete cdna clone (4.3 kb) encoding human complement factor h (hcfh; 155 kda) has been cloned into the pvl1393 baculovirus expression vector and transfected into spodoptera frugiperda sf9 insect cells. a biologically active (92 +/- 15%) 140-kda protein was secreted into the medium with a yield of more than 5 mg/liter. this is the first report of synthesis of biologically active recombinant hcfh in a heterologous system. | 1994 | 8206393 |
| penetration of autographa californica nuclear polyhedrosis virus nucleocapsids into iplb sf 21 cells induces actin cable formation. | the budded form of autographa californica nuclear polyhedrosis virus functionally enters cells by adsorptive endocytosis. during the period of virus uptake into spodoptera frugiperda iplb sf21 cells (1 to 4 hr postinfection), filamentous f-actin cables, visualized by fluorescence microscopy, were formed within the cytoplasm of infected cells. cable formation appeared to be a direct effect of viral inoculum in that the numbers of observed cables increased with an increase in multiplicity of infec ... | 1993 | 8212560 |
| characterization of productive and non-productive acmnpv infection in selected insect cell lines. | we have investigated the process of productive and non-productive acmnpv infection in a variety of cultured insect cells including lines derived from spodoptera frugiperda (iplb-sf21), mamestra brassicae (ses-mabr-3), choristoneura fumiferana (iprl-cf-1), bombyx mori (bmn-4), lymantria dispar (iplb-ld652y), helicoverpa zea (hz1b3), and drosophila melanogaster (dm). in each cell line, we have examined viral utilization of an early, a late, and a very late promoter, the replication of viral dna an ... | 1993 | 8212569 |
| characterization of poliovirus conformational alteration mediated by soluble cell receptors. | soluble extracts of spodoptera frugiperda cells expressing the poliovirus receptor (pvr) induce the native poliovirus (pv) to "a" particle conformational change (j. virol. 64, 4697-4702). we describe the variables that regulate this passage and study the suitability of solubilized pvr both for use as an in vitro system to characterize the receptor-mediated conformational alteration and for the production of large amounts of altered virus for structural analysis. pvr seems to function in a stoich ... | 1993 | 8212594 |
| the 85-kda, arachidonic acid-specific phospholipase a2 is expressed as an activated phosphoprotein in sf9 cells. | a human, 85-kda arachidonoyl-specific, cytosolic phospholipase a2 was expressed using the baculovirus-insect cell expression system. expression resulted in the production of an active protein which consisted of approximately 3% of the total protein in the host spodoptera frugiperda (sf9) cells at 67 h after infection. the phospholipase a2 was purified to apparent homogeneity and exhibited calcium-dependent phospholipase a2 activity with a specific activity of 8 mumol/min/mg protein, as well as c ... | 1993 | 8215460 |
| expression of the human follicle-stimulating hormone receptor in the baculovirus system. | human follitropin receptor (hfshr) cdna was inserted into the genome of autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. spodoptera frugiperda (sf9) insect cells expressed the hfshr at the membrane level, 2-3 days after infection. ligand binding analysis demonstrated saturable, high-affinity binding of 125i-labeled recombinant hfsh (kd = 0.17 nm) as well as binding specificity for both human and equine fsh. interestingly, a highly purified preparati ... | 1993 | 8216320 |
| expression of human interferon omega 1 in sf9 cells. no evidence for complex-type n-linked glycosylation or sialylation. | human interferon omega 1 (ifn-omega 1) was expressed in spodoptera frugiperda sf9 insect cells using the baculovirus expression system. half of the protein purified by immunoaffinity chromatography was shown to be n-glycosylated at the same site as the natural ifn-omega 1. the degree of glycosylation was independent of the expression rate. while natural ifn-omega 1 was shown to carry complex-type oligosaccharides [adolf, g. r., maurer-fogy, i., kalsner, i. & cantell, k. (1990) j. biol. chem. 265 ... | 1993 | 8223648 |
| expression of human interferon-alpha 2 in sf9 cells. characterization of o-linked glycosylation and protein heterogeneities. | human interferon alpha 2 (ifn-alpha 2) was expressed in spodoptera frugiperda sf9 insect cells using the baculovirus expression system. the protein purified by immunoaffinity chromatography exhibited biological activity identical to that of leukocyte-derived 'natural' ifn-alpha 2. however, the protein was found to be heterogeneously glycosylated, partially truncated by proteolysis and partially lacking a disulfide bridge. the major product was shown to be o-glycosylated at the same position as n ... | 1993 | 8223649 |
| phosphorylation of synthetic peptides containing tyr-met-x-met motifs by nonreceptor tyrosine kinases in vitro. | several tyrosine phosphorylation sites in the insulin receptor kinase substrate irs-1 are predicted to be within tyr-met-x-met (ymxm) motifs, and synthetic peptides corresponding to these sequences are excellent substrates for the insulin receptor kinase in vitro (shoelson, s. e., chatterjee, s., chaudhuri, m., and white, m. f. (1992) proc. natl. acad. sci. u. s. a. 89, 2027-2031). in this study, ymxm-containing peptides are shown to act as substrates for two members of the nonreceptor subfamily ... | 1993 | 8227078 |
| the antimicrosporidial activity of albendazole. | the antimicrosporidial activity of albendazole was tested on nosema bombycis in vitro in spodoptera frugiperda cells and in vivo in heliocoverpa zea larvae and pupae. significant reductions in the percentage of infected s. frugiperda cells were obtained using a concentration of 5.3 micrograms/ml albendazole in tissue culture medium but recrudescence occurred after the drug was withdrawn from the cultures. significant reductions in the number of spores harvested from 6th-instar larvae or pupae we ... | 1993 | 8228321 |
| superinfection-induced apoptosis and its correlation with the reduction of viral progeny in cells persistently infected with hz-1 baculovirus. | differential induction of necrosis or apoptosis was found upon challenge of cells of the insect spodoptera frugiperda productively or persistently infected with hz-1 baculovirus, respectively. unlike parental sf9 cells, which were essentially all killed by virally induced necrosis, persistently infected cells underwent a process of massive cell death by apoptosis; cells which were not killed by apoptosis then reestablished a cell monolayer. upon viral challenge, the yield of viral progeny was re ... | 1993 | 8230422 |
| expression of two bovine rotavirus non-structural proteins (nsp2, nsp3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins. | studies on rotavirus non-structural proteins have been hampered in the past by difficulties in obtaining monospecific reagents. to make such reagents available, we have expressed in the baculovirus system nsp2 and nsp3 (formerly called ns35 and ns34, respectively) of the bovine rotavirus rf and produced hybridomas against these proteins. full-length dna copies of rna segments 7 (coding for nsp3) and 8 (coding for nsp2) of the virus strain rf were cloned and sequenced. each cdna was inserted in t ... | 1993 | 8240020 |
| expression of two different forms of cdna for thromboxane synthase in insect cells and site-directed mutagenesis of a critical cysteine residue. | cdna coding for human placental thromboxane synthase (ec 5.3.99.5) was amplified by pcr from a human placental cdna library and sequenced. this cdna and a shorter cdna isolated from a human lung cdna library with a deletion of 163 bp near the 3' end were expressed in spodoptera frugiperda (sf9) insect cells using a baculovirus expression system. the cdna from human placenta was expressed as an active enzyme (60 kda) with a specific activity higher than those reported from other cell types, where ... | 1993 | 8240243 |
| synthesis of functional gabaa receptors in stable insect cell lines. | we have synthesised the beta 1-subunit of the bovine gabaa receptor in stable, continuous insect (spodoptera frugiperda) cell lines. a cdna was integrated randomly into the insect cell genome under control of a baculovirus immediate early (ie-1) gene promoter. transformed cells were obtained by co-transfection of the insect cells with piek1.gr beta 1, encoding the beta 1 subunit cdna, and piek1.neo, encoding the neomycin resistance gene. g-418-resistant clones were selected and expanded into con ... | 1993 | 8243667 |
| er-localization and functional expression of the hbv transactivator mhbst. | in two recently reported cases, integrated hepatitis b virus (hbv) dnas cloned from hepatocellular carcinoma were found to express a transcriptional transactivator from 3'-terminally truncated hbv surface (pres/s) genes. in this study, we characterized the transactivator at the protein level. expression of a 3'-truncated pres2/s gene in spodoptera frugiperda (sf9) insect cells resulted in a c-terminally truncated middle surface protein of 76 amino acids (mhbst76), which was found to be associate ... | 1993 | 8247538 |
| identification and characterization of a putative origin of dna replication in the genome of a baculovirus pathogenic for orgyia pseudotsugata. | a four-kilobase (kb) region (hindiii-n, map units 7.0-11.3) of the orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (opmnpv) genome was found to contain sequences that conferred upon plasmids the ability to undergo infection-dependent replication. the plasmid dna appeared to be replicated to form high molecular weight multimers. plasmids with deletions of up to 1.8 kb from either end of the hindiii-n region were replication competent. however, a discrete sequence, contained with ... | 1993 | 8249294 |
| construction of a cdna encoding the multifunctional animal fatty acid synthase and expression in spodoptera frugiperda cells using baculoviral vectors. | a cdna encoding the 2505-residue multifunctional rat fatty acid synthase has been constructed and expressed as a catalytically active protein in spodoptera frugiperda (sf9) cells using autographa californica nuclear polyhedrosis virus (baculovirus). the 7.5 kb cdna was engineered by the amplification and sequential splicing together of seven fragments contained in overlapping cdnas that collectively spanned the entire rat fatty acid synthase coding sequence. the full-length cdna was cloned into ... | 1993 | 8250834 |
| high-level expression and purification of human leukotriene a4 hydrolase from insect cells infected with a baculovirus vector. | leukotrienes constitute a group of bioactive compounds derived from arachidonic acid which play important roles in immediate hypersensitivity and inflammation. leukotriene a4 hydrolase (lta4h) is an epoxide hydrolase, catalyzing the hydration of lta4 to ltb4, and also acts an aminopeptidase, with the ability to cleave amides of p-nitroaniline. the cdna for lta4h was cloned using oligonucleotide-directed amplification of the cdna sequence by polymerase chain reaction and by oligonucleotide-based ... | 1993 | 8251746 |
| analysis of the tyrosine protein kinase p56lck expressed as a glutathione s-transferase fusion protein in spodoptera frugiperda cells. | a baculovirus vector system that expresses cloned dna sequences as glutathione s-transferase fusion proteins was developed. this system was used to express and purify the lymphocyte-specific tyrosine kinase p56lck. this recombinant p56lck was purified to homogeneity in a single chromatography step using glutathione resin. by sds gel analysis recombinant p56lck was found to migrate as two species with molecular masses of approximately 56,000 da. p56lck purified in this manner retained a high leve ... | 1993 | 8251750 |
| characterization of the human oxysterol receptor overexpressed in the baculovirus system. | oxysterols are potent regulators of enzymes of the de novo cholesterol biosynthetic pathway and do not require the ldl (low density lipoprotein):ldl receptor system for their regulatory actions. the search for an alternate transduction system led to the identification of an oxysterol binding protein. this cytosolic protein has been extensively characterized, purified, and cloned. although it fulfills the pharmacologic criteria for an oxysterol receptor by binding to oxysterols with affinities co ... | 1993 | 8251955 |
| the nonstructural glycoprotein of rotavirus affects intracellular calcium levels. | rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. this increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. we tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect spodoptera frugiperda (sf9) cells. insect cells were infected with wild-type baculovirus or baculovirus recombinants that conta ... | 1994 | 8254736 |
| cloning and expression of the nucleocapsid gene of virulent kabete o strain of rinderpest virus in baculovirus: use in differential diagnosis between vaccinated and infected animals. | rinderpest (rp) is a viral disease of ruminants with > 95% morbidity and mortality. we have cloned the cdna of the nucleocapsid (n) gene of the virulent kabete o strain of rinderpest virus (rpvk) and compared its nucleotide and deduced amino acid sequences with those of the n genes of the lapinized strain of rinderpest virus (rpvl), measles virus (mv), and canine distemper virus (cdv). the gene has an open reading frame of 1575 nucleotides and encodes a protein of 525 amino acids with a molecula ... | 1994 | 8259649 |
| expression of a drosophila gaba receptor in a baculovirus insect cell system. functional expression of insecticide susceptible and resistant gaba receptors from the cyclodiene resistance gene rdl. | recombinant baculoviruses containing two alternative splice forms of the drosophila rdl gaba receptor gene were constructed. spodoptera frugiperda (sf21) cells infected with either splice form expressed a transcript of expected size (2.5 kb). western blotting of cell membrane extracts and immunoprecipitation experiments with an anti-rdl antiserum recognized a protein of the expected size of approximately 65 kda. whole cell patch clamp analysis of cells infected with either splice form revealed f ... | 1993 | 8262177 |
| synthesis and optical resolution of (r)- and (s)-trans-7-hydroxy-2-[n-propyl-n-(3'-iodo-2'-propenyl)amino]tetralin: a new d3 dopamine receptor ligand. | an improved method for synthesis and resolution of (r,s)-trans-7-hydroxy-2-[n-propyl-n-(3'-iodo-2'-propenyl)amino]tetralin (trans-7-oh-plpat,5), a new d3 dopamine receptor ligand, was reported. both isomers, (r)-(+)- and (s)-(-)-5, were prepared and characterized. hplc retention times obtained on a chiral column for these isomers were consistent with those observed for [125i]-(r)-(+)- and (s)-(-)-5. direct radioiodination of an optically resolved tin precursor, (r)-(+)-7, yielded the desired [12 ... | 1993 | 8277513 |
| expression of human placental cytochrome p450 aromatase (cyp19) cdna in insect cells using a luciferase based baculovirus vector. | the cdna encoding human placental cytochrome p450 aromatase (cyp19) was expressed in spodoptera frugiperda (sf9) insect cells using the baculovirus expression vector system. the recombinant protein product was characterized by northern and western blot analyses as well as by direct measurement of aromatase activity. the expressed enzyme proved to be both catalytically active in the presence of p450 reductase and immunologically reactive with polyclonal antibodies raised against human placental a ... | 1993 | 8280169 |
| in vivo and in vitro analyses of recombinant baculoviruses lacking a functional cg30 gene. | the cg30 gene of autographa californica nuclear polyhedrosis virus (acmnpv) encodes two sequence motifs, a zinc finger-like motif and a leucine zipper, found in other polypeptides known to be involved in gene regulation. to gain insight into the function of the cg30 product, cg30, we constructed and characterized recombinant viruses lacking a functional cg30 gene. we found that cg30 mutants had no striking phenotype in cell lines derived from spodoptera frugiperda or trichoplusia ni or in t. ni ... | 1994 | 8289348 |
| high-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gd synthesized by baculovirus-infected insect cells. | two forms of herpes simplex virus glycoprotein gd were recombined into autographa californica nuclear polyhedrosis virus (baculovirus) and expressed in infected spodoptera frugiperda (sf9) cells. each protein was truncated at residue 306 of mature gd. one form, gd-1(306t), contains the coding sequence of patton strain herpes simplex virus type 1 gd; the other, gd-1(qaat), contains three mutations which eliminate all signals for addition of n-linked oligosaccharides. prior to recombination, each ... | 1994 | 8289380 |
| functional mapping of regions of the autographa californica nuclear polyhedrosis viral genome required for dna replication. | previous results showed that plasmids containing one of the eight putative origins (ori's) of autographa californica nuclear polyhedrosis virus (acmnpv) are replicated after transfection into spodoptera frugiperda cells if essential trans-acting factors are supplied by acmnpv infection (kool et al., virology, 192, 94-101, 1993a; kool et al., j. gen. virol., in press, 1993b; leisy and rohrmann, virology, 196, 722-730, 1993). in this report a transient complementation assay is described in which f ... | 1994 | 8291249 |
| analysis of human acid beta-glucosidase by site-directed mutagenesis and heterologous expression. | structure/function relationships of acid beta-glucosidase, the enzyme deficient in gaucher disease, were evaluated by characterizing the proteins expressed from cdnas encoding normal and mutant enzymes. twenty-two gaucher disease mutations or created mutations were expressed in spodoptera frugiperda (sf9) cells and analyzed for catalytic properties, stability, inhibitor binding, and modifier interactions. many gaucher disease mutations encoded highly disruptive amino acid substitutions (e.g. p28 ... | 1994 | 8294487 |
| production and characterization of recombinant rat brain-derived neurotrophic factor and neurotrophin-3 from insect cells. | rat brain-derived neurotrophic factor (bdnf) and neurotrophin-3 (nt-3) were engineered for expression in a baculovirus-infected spodoptera frugiperda insect cell system. the bdnf and nt-3 from the culture supernatants were purified by ion-exchange and reverse-phase chromatography to apparent homogeneity. the purification procedure yielded approximately 2 mg of pure rat bdnf or nt-3 per liter of culture supernatant. a single n-terminus only was found for either secreted molecule and was analogous ... | 1994 | 8294909 |
| glycosylation and high-level secretion of human tumour necrosis factor-beta in recombinant baculovirus-infected insect cells. | human tumour necrosis factor-beta (tnf-beta) was produced in eukaryotic cells using the insect baculovirus cloning and expression system. a novel insect signal sequence, the honey-bee (apis mellifera) prepromelittin secretory sequence, was used to aid in the post-translational modifications, glycosylation and secretion of recombinant human tnf-beta. human tnf-beta cdna was cloned using the insect baculovirus vector pacc4s. expression of the human tnf-beta was regulated by the insect autographa c ... | 1993 | 8297505 |
| an unusual 30-kda protein binding to the polyhedrin gene promoter of autographa californica nuclear polyhedrosis virus. | gel retardation assays using nuclear extracts from the autographa californica nuclear polyhedrosis virus-infected sf21 (spodoptera frugiperda) insect cell line revealed a host factor (polyhedrin promoter-binding protein or ppbp) that binds to the polyhedrin gene promoter. a hexanucleotide sequence (aataaa) within the promoter is important for binding in association with neighboring elements, some of which are contributed by the sequence taagtatt present at the transcription start point. ppbp was ... | 1994 | 8300607 |
| structures of the n-linked oligosaccharides of the membrane glycoproteins from three lepidopteran cell lines (sf-21, izd-mb-0503, bm-n). | the primary structures of the asn-linked carbohydrate chains isolated from membrane glycoproteins of the three insect cell lines mamestra brassicae (mb-0503), bombyx mori (bm-n), and spodoptera frugiperda (sf-21) have been determined. tryptic glycopeptides derived from the membrane fraction were digested with peptide-n-glycanase a. the resulting oligosaccharides were reductively aminated with 2-aminopyridine and identified by two-dimensional hplc mapping in combination with exoglycosidase digest ... | 1994 | 8311447 |
| comparative recombinant protein production of eight insect cell lines. | a recombinant autographa californica baculovirus expressing secreted alkaline phosphatase (seap) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived from spodoptera frugiperda, trichoplusia ni, mamestra brassicae and estigmene acrea. because cell density was found to influence protein production, seap production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. on a per cell basis, the t. ni- ... | 1993 | 8314732 |
| identification and immunocharacterization of a 25k structural protein in autographa californica nuclear polyhedrosis virus (acmnpv). | a 25k protein was a major component of the triton x-100 solubilized fraction prepared for the isolation of nuclear matrices from spodoptera frugiperda cells infected with autographa californica nuclear polyhedrosis virus (acmnpv). polyclonal antisera to p25 were produced in rabbits and used in the characterization of p25. western blots of proteins probed with the p25 antiserum demonstrated the presence of the p25 in both the polyhedral-derived and extracellular viral phenotypes. using an indirec ... | 1993 | 8317143 |
| non-homologous recombination between adenovirus and acnpv dna fragments in cell-free extracts from insect spodoptera frugiperda nuclei. | in previous work, we have developed a cell-free system from nuclear extracts of hamster cells to study the mechanism of integrative recombination between adenovirus type 12 (ad12) dna and hamster cell dna (jessberger et al., 1989; tatzelt et al., 1992). we have also demonstrated that in insect cells the left terminal fragment of ad2 dna can insert by non-homologous recombination into the 32.6 to 34.5 map unit (ecori-o) fragment and into other segments of autographa californica nuclear polyhedros ... | 1993 | 8317144 |
| purification and characterization of recombinant histidine-tagged human platelet 12-lipoxygenase expressed in a baculovirus/insect cell system. | a baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cdna, under control of the polyhedrin promoter, was constructed. recombinant 12-lipoxygenase baculoviruses were used to infect spodoptera frugiperda insect cells (sf9). at 54 h post-infection, maximal 12-lipoxygenase activity and protein levels were achieved; the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in w ... | 1993 | 8319693 |