| in vitro assembly of sv40 virions and pseudovirions: vector development for gene therapy. | sv40 is an attractive potential vector with high-efficiency gene transfer into a wide variety of human tissues, including the bone marrow, a critical target organ for the cure of many diseases. in the present study, the three sv40 capsid proteins, vp1, vp2, and vp3, were produced in spodoptera frugiperda (sf9) insect cells. their co-production led to spontaneous assembly of sv40-like particles. nuclear extracts containing the three proteins were allowed to interact with purified sv40 dna, or wit ... | 1997 | 9143910 |
| expression of a fungal cellobiohydrolase in insect cells. | the gene for trichoderma reesei cellobiohydrolase i (cbhi) was expressed with a recombinant baculovirus and high levels of secreted protein were produced in spodoptera frugiperda and trichoplusia ni insect cells. electrophoretic analysis indicated that the recombinant cbhi (rcbhi) was similar in apparent molecular weight to the native form and immunoblotting with anti-cbhi monoclonal antibodies confirmed its identity. the rcbhi was easily purified by affinity and hydrophobic interaction chromato ... | 1997 | 9144389 |
| cloning and characterization of a metabotropic glutamate receptor, mglur4b. | an alternative spliced variant of metabotropic glutamate receptor subtype mglur4a, termed mglur4b was isolated from a rat cdna library. subtype mglur4b was identical to the previously described mglur4a, except for the last 64 amino acids in the c-terminal region in which were replaced by 135 new amino acids in mglur4b. recombinant baculoviruses coding for mglur4a and mglur4b were expressed in spodoptera frugiperda, sf-9, insect cells and characterized pharmacologically by measuring [3h]-l-2-amin ... | 1997 | 9144638 |
| virus-like particle-induced fusion from without in tissue culture cells: role of outer-layer proteins vp4 and vp7. | we recently described an assay that measures fusion from without induced in tissue culture cells by rotavirus, a nonenveloped, triple-protein-layered member of the reoviridae family (m. m. falconer, j. m. gilbert, a. m. roper, h. b. greenberg, and j. s. gavora, j. virol. 69:5582-5591, 1995). the conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection of host cells, as the presence of the outer-layer protei ... | 1997 | 9151849 |
| cytolytic activity of bacillus thuringiensis cryic and cryiac toxins to spodoptera sp. midgut epithelial cells in vitro. | a sensitive lactate dehydrogenase (ldh) assay was modified to determine the cytolytic activity of bacillus thuringiensis cryic and cryiac delta endotoxins to viable collagenase-dissociated midgut epithelial cells (mec) from larvae of spodoptera frugiperda and spodoptera exigua. the mec preparations from these spodoptera sp. consisted predominantly of columnar cells (65-75%) and goblet cells (25-35%). time course microscopy experiments indicated that only the columnar cells became swollen during ... | 1997 | 9156349 |
| agonist-induced [35s]gtpgammas binding in the membranes of spodoptera frugiperda insect cells expressing the human d3 dopamine receptor. | in the membranes of spodoptera frugiperda (sf-9) insect cells heterologously expressing the human d3 dopamine receptor, agonists selective for the receptor, but not antagonists, robustly enhanced [35s]gtpgammas binding. quinpirole, for instance, dose-dependently enhanced [35s]gtpgammas binding with a half-maximal concentration of 2.3 +/- 0.2 nm. its action was absent in the cells infected with wild type viruses, and competitively blocked by an antagonist, ym-09151-2. a number of known agonists e ... | 1997 | 9159497 |
| membrane lipid metabolism and phospholipase activity in insect spodoptera frugiperda 9 ovarian cells. | although there is increasing use of insect ovarian sf9 cells for the production of recombinant proteins, namely, via the baculovirus vector expression system, little is known about the lipids in the cell membrane and whether endogenous phospholipases are present for regulation of the cell membrane lipids. in this study, analysis of membrane lipids of sf9 cells indicated the presence of phosphatidylethanolamine (pe) (diacyl type) and phosphatidylcholine as major phospholipids, followed by phospha ... | 1997 | 9168454 |
| characterization of the human multidrug resistance protein containing mutations in the atp-binding cassette signature region. | a number of mutants with single amino acid replacements were generated in the highly conserved atp-binding cassette (abc)-signature region (amino acids 531-543) of the n-terminal half of the human multidrug resistance (mdr1) protein. the cdna variants were inserted into recombinant baculoviruses and the mdr1 proteins were expressed in spodoptera frugiperda (sf9) insect cells. the level of expression and membrane insertion of the mdr1 variants was examined by immunostaining, and mdr1 function was ... | 1997 | 9169612 |
| localization of cryptic tolerogenic epitopes in the alpha1-helical region of the rt1.au alloantigen. | transplantation tolerance is induced by perioperative administration of host class i major histocompatibility complex proteins bearing donor-type amino acid (a.a.) epitopes substituted for native residues. herein we demonstrate that two cryptic tolerogenic a.a. epitopes are localized in the alpha1-helical region of the rat rt1.au class i major histocompatibility complex alloantigen. | 1997 | 9175796 |
| multiplication of tomato spotted wilt virus in primary cell cultures derived from two thrips species. | primary cell cultures prepared from embryos of the thrips species frankliniella occidentalis and thrips tabaci were tested for their potential to support replication of tomato spotted wilt virus (tswv). using polyclonal antibodies against the viral nucleocapsid protein (n) and indirect immunofluorescent staining, discrete spots with strong signals were observed in the cytoplasm at 48 h post-inoculation in the cell cultures of a f. occidentalis, and a t. tabaci population which failed to transmit ... | 1997 | 9178497 |
| optimal ligand binding by the recombinant human glucocorticoid receptor and assembly of the receptor complex with heat shock protein 90 correlate with high intracellular atp levels in spodoptera frugiperda cells. | the full-length human glucocorticoid receptor (hgr), overexpressed in spodoptera frugiperda (sf9) cells, associates with heat shock protein 90 (hsp90) and hsp70 and binds dexamethasone with high affinity. baculovirus infection of sf9 cells grown in tnm-fh medium results in the rapid depletion of glucose from the medium within 24 h. noting a discrepancy between hgr protein levels and ligand binding capacity in such cultures, we hypothesized that the depletion of glucose from the medium could resu ... | 1997 | 9182852 |
| the prothoracicotropic hormone bombyxin has specific receptors on insect ovarian cells. | bombyxin ii, a product of the brain of the adult silkmoth, bombyx mori, binds to ovarian cells of three different species of lepidoptera, i.e. b. mori (silkmoth), samia cynthia ricini (ailanthus moth), and an ovarian cell line of spodoptera frugiperda (sf9) (fall armyworm). crude sf9 cell membrane preparations were used to show that the purported bombyxin receptor binds its ligand in a specific, saturable, and reversible manner. the dissociation constant of the bombyxin-receptor complex is 260+/ ... | 1997 | 9183018 |
| new structure and function in plant k+ channels: kco1, an outward rectifier with a steep ca2+ dependency. | potassium (k+) channels mediating important physiological functions are characterized by a common pore-forming (p) domain. we report the cloning and functional analysis of the first higher plant outward rectifying k+ channel (kco1) from arabidopsis thaliana. kco1 belongs to a new class of 'two-pore' k+ channels recently described in human and yeast. kco1 has four putative transmembrane segments and tandem calcium-binding ef-hand motifs. heterologous expression of kco1 in baculovirus-infected ins ... | 1997 | 9184204 |
| purification and characterization of secreted human leptin produced in baculovirus-infected insect cells. | the product of the human ob (obesity) gene, leptin, appears to function in the maintenance of body weight in vivo. when injected into mice, this hormone reduces food consumption and causes weight loss. this work has been done with recombinant leptin (re-leptin) purified and renatured from inclusion bodies in escherichia coli. we have expressed the human obesity gene encoding the predicted full-length leptin in spodoptera frugiperda (sf-9) cells by infection with the recombinant baculovirus syste ... | 1997 | 9185858 |
| expression of lacz reporter gene under the control of the polyhedrin promoter of spodoptera litura nuclear polyhedrosis virus. | promoter function of the putative polyhedrin-encoding gene (polh) of spodoptera litura nuclear polyhedrosis virus (s1mnpv) was determined by transferring it to the autographa californica nuclear polyhedrosis virus (acmnpv) through the acnpv polh based vector, pvl1393. three transfer vectors pcbt2, pcbt3 and pcbt4 were constructed by substituting the promoter and the neighbouring sequences of acnpv in pvl1393 by that of s1npv. the escherichia coli lacz gene was placed downstream from the s1npv po ... | 1997 | 9185860 |
| functional co-expression of the canine cardiac ca2+ pump and phospholamban in spodoptera frugiperda (sf21) cells reveals new insights on atpase regulation. | the utility of the baculovirus cell expression system for investigating ca2+-atpase and phospholamban regulatory interactions was examined. cdna encoding the canine cardiac sarco(endo)plasmic ca2+-atpase pump (serca2a) was cloned for the first time and expressed in the presence and absence of phospholamban in spodoptera frugiperda (sf21) insect cells. the recombinant ca2+ pump was produced in high yield, contributing 20% of the total membrane protein in sf21 microsomes. at least 70% of the expre ... | 1997 | 9188486 |
| identification and functional analysis of a non-hr origin of dna replication in the genome of spodoptera exigua multicapsid nucleopolyhedrovirus. | the genome of spodoptera exigua multicapsid nucleopolyhedrovirus (semnpv) was screened for the presence of putative origins of dna replication (oris). using a transient dna replication assay, several fragments were identified that underwent semnpv-dependent dna replication in spodoptera frugiperda cells (sf-ae-21). preliminary sequence data revealed the presence of multiple copies of homologous repeats (hrs). restriction fragment xbal-f2 showed a distinct sequence reminiscent of autographa calif ... | 1997 | 9191948 |
| baculovirus-directed expression of rabbit udp-glucuronosyltransferases in spodoptera frugiperda cells. | the rabbit liver udp-glucuronosyltransferase (ugt) cdnas that encode the 4-hydroxybiphenyl ugt2b13 and 4-nitrophenol ugt1a6 have been cloned into baculovirus. spodoptera frugiperda (sf-9) cells infected with the ugt recombinant baculovirus produced significant amounts of protein, which was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, and pulse chase with 35s-amino acids. the expression of the ugt proteins in sf-9 cells were detected at approximately ... | 1997 | 9193877 |
| cultivation of fall armyworm ovary cells in simulated microgravity. | a methodology is presented to culture fall armyworm ovary cells in simulated micrograviy using a novel bioreactor developed by nasa, the high-aspect ratio vessel. in this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture. specifically, stationary phase in the nasa vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 x 10(7) viable cells/ml and 90%, respectiv ... | 1997 | 9196890 |
| characterization of p91, a protein associated with virions of an orgyia pseudotsugata baculovirus. | polyclonal antiserum produced against preoccluded virions from the orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (opmnpv) was used to screen an opmnpv lambda gt11 expression library. the insert from one of the immunoreactive phage isolates hybridized to opmnpv orf86 (p91), a 2460-bp (819 amino acids) open reading frame that encodes a predicted protein of 91 kda. antibodies generated against a maltose binding protein-p91 fusion detected a band of approximately 91 kda on wester ... | 1997 | 9201231 |
| differential requirements of two insect cell lines for growth in serum-free medium. | the development of a serum-free medium that supports the growth of cells from a spodoptera frugiperda and a lymantria dispar cell line is reported. a yeast hydrolysate provided the b-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. supplemental cystine and methionine were required to achieve maximum cell growth. the serum or serum replacements used in earlier formulations were replaced with commercial lipid prep ... | 1997 | 9201517 |
| obtaining a functional recombinant anti-rhesus (d) antibody using the baculovirus-insect cell expression system. | the cloning and production of a human anti-rhesus (rh) d monoclonal antibody (mab) using the baculovirus-insect cell expression system is described. this monoclonal recombinant antibody r.d7c2 derived from a human parental igm lambda immunoglobulin was obtained after immortalization of lymphocytes by epstein-barr virus (ebv). the human heavy (vh) and light (vl) variable regions were cloned from the parental cell line and genetically fused to the human constant igg1 heavy (h) and light (l) chain ... | 1997 | 9203960 |
| baculovirus expression of the respiratory syncytial virus fusion protein using trichoplusia ni insect cells. | respiratory syncytial virus (rsv) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. the rsv fusion (f) protein is highly conserved among rsv subgroups a and b and is the major protective immunogen. a genetically-engineered version of the rsv f protein was produced in insect cells using the baculovirus expression system. to express a secreted form of this protein, the transmembrane domain was eliminated by removing the regio ... | 1997 | 9208456 |
| contact inhibition-induced inactivation of the cyclin d-dependent kinase in rat fibroblast cell line, 3y1. | inhibitory proteins for cdks (ckis) are involved in cell cycle arrest induced by anti-mitotic factors, chemicals, or dna damage in mammalian cells. high cell density also induces cell cycle arrest with unreplicated genomic dna even in the presence of mitotic dose of the growth factors, termed contact inhibition. although rat fibroblast cell line, 3y1, arrested in quiescence by contact inhibition, cdk4 bound its regulatory subunit, cyclin d1 or d3. however, these complexes were enzymatically inac ... | 1997 | 9209390 |
| purification and properties of alpha-mannosidase ii from golgi-like membranes of baculovirus-infected spodoptera frugiperda (iplb-sf-21ae) cells. | an alpha-mannosidase ii-like activity was identified in baculovirus-infected spodoptera frugiperda (iplb-sf21-ae) cells. the enzyme responsible was purified from golgi-type membranes to apparent homogeneity by using a combination of steps including deae-cellulose, hydroxyapatite, concanavalin a-sepharose and gel filtration chromatography. the molecular mass of this purified protein was approx. 120 kda by sds/page under reducing conditions and approx. 240 kda under non-reducing conditions, indica ... | 1997 | 9210421 |
| characterization of the malonyl-/acetyltransacylase domain of the multifunctional animal fatty acid synthase by expression in escherichia coli and refolding in vitro. | cdnas of various lengths encoding the second domain of the multifunctional fatty acid synthase (fas) have been expressed in escherichia coli and the recombinant proteins refolded in vitro to catalytically active monomeric malonyl-/acetyltransacylases. fas residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, can be omitted from the recombinant enzyme with no loss in catalytic activity. this shortened transacylase, consisting of fas residues 488- ... | 1997 | 9215574 |
| expression, characterization, and immunoreactivities of a soluble hepatitis e virus putative capsid protein species expressed in insect cells. | the hepatitis e virus (hev) open reading frame-2 (orf-2) is predicted to encode a 71-kda putative capsid protein involved in virus particle formation. when insect spodoptera frugiperda (sf9) cells were infected with a recombinant baculovirus containing the entire orf-2 sequence, two types of recombinant proteins were produced; an insoluble protein of 73 kda and a soluble protein of 62 kda. the 62-kda species was shown to be a proteolytic cleavage product of the 73-kda protein. n-terminal sequenc ... | 1997 | 9220158 |
| dna helicase activity in werner's syndrome gene product synthesized in a baculovirus system. | the gene responsible for werner's syndrome (wrn) contains a region homologous to the escherichia coli recq type dna helicase and was thought to code for a dna helicase belonging to this helicase family. however, no evidence has been shown before to substantiate this prediction. here, we show data that the product of the wrn gene is indeed a dna helicase. the gene product, a polypeptide with a relative molecular mass of 170 kda, expressed in the insect spodoptera frugiperda (sf21) cell and purifi ... | 1997 | 9224595 |
| expression of stable human o-glycan core 2 beta-1,6-n-acetylglucosaminyltransferase in sf9 insect cells. | udp-glcnac:galbeta1-3galnac-r (glcnac to galnac) beta-1, 6-n-acetylglucosaminyltransferase (c2gnt) catalyses the formation of o-glycan core 2. purification and characterization of c2gnt from natural sources has been hampered by the instability of this enzyme. we have been able to prepare a stable partly purified recombinant human c2gnt by expression of a truncated form of the enzyme in the baculovirus/spodoptera frugiperda 9 (sf9) insect cell system. c2gnt activity was secreted into the sf9 cult ... | 1997 | 9224630 |
| expression, purification, and characterization of an activated cytokine-suppressive anti-inflammatory drug-binding protein 2 (csbp2) kinase from baculovirus-infected insect cells. | an activated form of the human cytokine-suppressive anti-inflammatory drug-binding protein 2 (csbp2) kinase was expressed in spodoptera frugiperda (sf9) cells from a baculovirus vector. to maximize expression and to facilitate purification of the recombinant protein, csbp2 kinase was expressed as a carboxy-terminal fusion protein to glutathione s-transferase (gst). under optimal conditions, 2-3 mg of gst-csbp2 could be obtained per liter of infected cell culture. the fusion protein was easily pu ... | 1997 | 9226723 |
| differential effects of subunit interactions on protein kinase a- and c-mediated phosphorylation of l-type calcium channels. | we have expressed the pore-forming alpha1s (skeletal muscle isoform) and alpha1c (cardiac/brain isoform) subunits, as well as the accessory beta2a (cardiac/brain isoform) and alpha2/delta subunits of the l-type, dihydropyridine-sensitive calcium (ca) channels in spodoptera frugiperda insect cells (sf9 cells) by infection with recombinant baculoviruses in order to facilitate biochemical studies of these rare, heteromultimeric membrane proteins. since the l-type channels are believed to be regulat ... | 1997 | 9236007 |
| gaucher disease: functional expression of the normal glucocerebrosidase and gaucher t1366g and g1604a alleles in baculovirus-transfected spodoptera frugiperda cells. | gaucher disease is an inherited sphingolipidosis resulting from deficient glucocerebrosidase activity. three clinical forms of gaucher disease have been described: type 1 as non-neuronopathic, type 2 as acute neuronopathic, and type 3 as subacute neuronopathic. we recently identified a rare mutation (g-->a at glucocerebrosidase cdna nucleotide position 1604) [choy et al., 1994a, am j med genet 51:156-160] and a novel mutation (t-->g at glucocerebrosidase cdna nucleotide position 1366) in two typ ... | 1996 | 9240741 |
| characterization of delta, kappa, and mu human opioid receptors overexpressed in baculovirus-infected insect cells. | the cdnas encoding human delta (hdor), kappa (hkor) and micro (hmor) opioid receptors were cloned in the baculovirus autographa californica (acmnpv) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag. expression levels were optimized in spodoptera frugiperda (sf9) cells and were in the following order hmor > hdor > hkor. the receptors bound antagonists with affinity values similar to those published previously for the receptors expressed in mammalian ... | 1997 | 9242668 |
| the influence of various insect cell lines, p10 and polyhedrin promoters in the production of secreted insulin-like growth factor-interleukin-3 chimeras in the baculovirus expression system. | a technique for the optimal synthesis of secreted fusion proteins between insulin-like growth factors (igfs) and cytokines is described. the cdna of bomigf, a fusion protein between the insect insulin-like peptide bombyxin and igf ii, has been linked to the gene of interleukin-3. the bomigf-interleukin 3 fusion gene was cloned downstream of the promoter regions of the p10 and polyhedrin proteins within baculovirus transfer vectors. a third, dual transfer vector was constructed with the gene inse ... | 1997 | 9246791 |
| a line-like transposable element in drosophila, the i factor, encodes a protein with properties similar to those of retroviral nucleocapsids. | i factors are members of the line-like family of transposable elements and move by reverse transcription of an rna intermediate. complete i factors contain two open reading frames. the amino acid sequence encoded by the first of these, orf1, includes the motif cx2cx4hx4c that is characteristic of the nucleocapsid domain of retroviral gag polypeptides followed by a copy of the slightly different sequences cx2cx4hx6c and cx2cx9hx6c. the function of this protein is unknown. we have expressed this p ... | 1997 | 9250689 |
| baculovirus expression provides direct evidence for heteromeric assembly of p2x2 and p2x3 receptors. | p2x2 and p2x3 are subunits of p2x receptors, cation channels opened by binding extracellular atp. cdnas encoding p2x2 and p2x3 receptor subunits, each with one of two c-terminal epitope tags, were cloned into baculovirus. virally infected insect cells (spodoptera frugiperda) expressed moderate to high levels of the corresponding proteins, as detected by western blotting, by the specific binding of [35s]atp and by whole-cell recordings of membrane current evoked by atp or alphabetamethylene-atp. ... | 1997 | 9254665 |
| characterization of [125i]s(-)5-oh-pipat binding to dopamine d2-like receptors expressed in cell lines. | recently, [125i]s(-)5-oh-pipat [5-hydroxy-2-(n-n-propyl-n-3'-iodo-2'-propenyl)aminotetralin], a derivative of s(-)5-oh-dpat (5-hydroxy-n,n-di-n-propyl-aminotetralin), was reported to be a better radioiodinated dopamine d2-like receptor ligand than the previously reported iodinated ligand, [125i]r(+)7-oh-pipat. therefore, in the present study, the binding profile of [125i]s(-)5-oh-pipat to d2-like receptors expressed in cell lines was established. high binding affinity (kd = 0.3-0.4 nm) and nacl ... | 1997 | 9257944 |
| regulation of infected-cell-specific protein synthesis in sf iplb-21 cells productively infected with spodoptera frugiperda multicapsid nuclear polyhedrosis virus. | in spodoptera frugiperda (sf iplb-21) cells productively infected with s. frugiperda multicapsid nuclear polyhedrosis virus (sfmnpv), we observed the synthesis of infected-cell-specific polypeptides (icsps) using the protein synthesis inhibitor cycloheximide (cx) and the dan synthesis inhibitor cytosine arabinoside (ara-c) in an attempt to assess whether a temporal cascade and viral dna synthesis are involved in the gene expression program of sfmnpv. inhibition of protein synthesis with cx at 0 ... | 1997 | 9268770 |
| bombyxin: an insect neurohormone targets the ovaries in lepidoptera. | bombyxin, an insect hormone structurally related to insulin, was chemically synthesized and defined radioactively labeled probes were generated in order to detect and characterize bombyxin receptors in insect tissue. in all species tested, bombyx mori (silkmoth), samia cynthia ricini (ailanthus moth). manduca sexta (tobacco hornworm) and spodoptera frugiperda (fall armyworm), bombyxin receptors were found in ovaries. a radioactive photolabeled bombyxin analog was crosslinked to the potential bom ... | 1997 | 9274060 |
| improved heterologous expression of the white-rot fungal ligninase h8 by crossover linker mutagenesis. | using the crossover-linker mutagenesis method, the 5' noncoding region of the lambda ml-1 cdna, which encodes the ligninase h8 isozyme of the white-rot fungus, phanerochaete chrysosporium, was deleted with the simultaneous insertion of the putative spodoptera frugiperda ribosome-binding sequence (rbs) (tataaat) directly in front of the translation-initiation codon of this gene. a recombinant baculovirus, pvl-mu-h8, carrying the ligninase-h8 gene was successfully constructed, as determined by bot ... | 1997 | 9276925 |
| baculovirus expression of a human g2-specific, neutralizing igg monoclonal antibody to puumala virus. | we amplified by polymerase chain reaction the heavy- and light-chain antibody genes of a human hybridoma secreting a neutralizing, igg monoclonal antibody to the g2 protein of puumala virus. the heavy- and light-chain genes were cloned and sequenced and the deduced amino acids were aligned with those of other human antibodies to identify the constant and variable regions. the genes were cloned into the baculovirus plasmid transfer vector pacuw51 such that the heavy-chain and light-chain genes we ... | 1997 | 9281505 |
| stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis. | we have constructed several plasmid expression vectors to express foreign genes in stably transformed insect cells. unlike baculovirus-based expression vectors by which genes of interest are expressed transiently before lysis of the virus-infected cells, genes can be expressed continuously over many passages in a stable cell line. furthermore, the function of a gene or genes expressed in a stable cell line from an insect-specific promoter that is constitutively expressed can be studied in the ab ... | 1997 | 9282319 |
| phosphorylation site mutations in the human multidrug transporter modulate its drug-stimulated atpase activity. | in the human multidrug transporter (mdr1), three serine residues located in the "linker" region of the protein are targets of in vivo phosphorylation. these three serines, or all eight serines and threonines in the linker, were substituted by alanines (mutants 3a and 8a) or with glutamic acids (mutants 3e and 8e). the wild-type and mutant proteins were expressed in baculovirus-infected spodoptera frugiperda (sf9) ovarian insect cells, and the vanadate-sensitive, drug-stimulated atpase activity w ... | 1997 | 9287320 |
| efficient photoaffinity labeling of the rat v1a vasopressin receptor using a linear azidopeptidic antagonist. | we have synthesized and fully characterized by fast-atom-bombardment-mass, nmr and ultraviolet spectroscopies the vasopressin antagonist 3-azidophenylpropionyl-d-tyr(me)-phe-gln-asn-arg-pro-arg-tyr(3i )-nh2. easily radioiodinatable just before use, it has a high affinity for the natural rat liver v1a receptor [dissociation constant (kd) = 54 +/- 20 pm; carnazzi, e., aumelas, a., barberis, c., guillon, g. & seyer, r. (1994) j. med. chem. 37, 1841-1849] and for both the rat vasopressin v1a recepto ... | 1997 | 9288914 |
| ligand specificity and affinity of bt-r1, the bacillus thuringiensis cry1a toxin receptor from manduca sexta, expressed in mammalian and insect cell cultures. | the manduca sexta receptor for the bacillus thuringiensis cry1aa, cry1ab, and cry1ac toxins, bt-r1, has been expressed in heterologous cell culture, and its ligand binding characteristics have been determined. when transfected with the bt-r1 cdna, insect and mammalian cell cultures produce a binding protein of approximately 195 kda, in contrast to natural bt-r1 from m. sexia, which has an apparent molecular weight of 210 kda. transfection of cultured spodoptera frugiperda cells with the bt-r1 cd ... | 1997 | 9292994 |
| ricin fusion toxin targeted to the human granulocyte-macrophage colony stimulating factor receptor is selectively toxic to acute myeloid leukemia cells. | treatment failure of patients with acute myelogenous leukemia (aml) is frequently due to the development of multidrug resistance phenotype blasts. we have expressed a fusion protein consisting of human granulocyte-macrophage colony stimulating factor (gmcsf) fused to the n-terminus of a lectin-deficient ricin toxin b chain (rtb) in spodoptera frugiperda insect cells. the fusion protein was purified by immunoaffinity chromatography and reassociated with chemically deglycosylated ricin toxin a cha ... | 1997 | 9301690 |
| characterization of the polyhedrin gene of spodoptera litura nuclear polyhedrosis virus. | the polyhedrin gene (polh) of a characteristically distinct spodoptera litura nuclear polyhedrosis virus isolate (simnpv) is identified in the hindiii-f fragment of the viral dna. the nucleotide sequence of the 1057 base pair (bp) region of this fragment contains an open reading frame (orf) without any intervening sequence for coding a polypeptide of 246 amino acids. analysis of the nucleotide sequence and deduced amino acid sequence indicate that this has more than 70% sequence identity to know ... | 1997 | 9311561 |
| herpes simplex virus 1 alpha regulatory protein icp0 interacts with and stabilizes the cell cycle regulator cyclin d3. | the herpes simplex virus 1 (hsv-1) infected-cell protein 0 (icp0) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection. to identify cellular proteins interacting with icp0, we used a domain of exon ii of icp0 that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen. our results were as follows. (i) a cdna in a positive yeast colony was found to encode cyclin d3, a cell cycle regulator ... | 1997 | 9311810 |
| identification of the early actin-rearrangement-inducing factor gene, arif-1, from autographa californica multicapsid nuclear polyhedrosis virus. | in response to autographa californica multicapsid nuclear polyhedrosis virus (acmnpv) infection, a sequential rearrangement of the actin cytoskeleton occurs. previous studies suggest that the penetration of nucleocapsids induces early actin cables followed by further changes of the actin cytoskeleton which depend on early viral gene expression. by transfection of a plasmid library into trichoplusia ni tn-368 cells, we have identified an early viral gene, designated arif-1, that is able to induce ... | 1997 | 9311884 |
| biochemical characterization of a smaller form of recombinant norwalk virus capsids assembled in insect cells. | the expression of the single capsid protein of norwalk virus (nv) in spodoptera frugiperda (sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (vlps) of two sizes, the predominant 38-nm, or virion-size vlps, and smaller, 23-nm vlps. here we describe the purification and biochemical characterization of the 23-nm vlps. the 23-nm vlps were purified to 95% homogeneity from the medium of sf9 cultures by isopycnic cscl gradient centrifugation follow ... | 1997 | 9311906 |
| peptide mapping of the [125i]iodoazidoketanserin and [125i]2-n-[(3'-iodo-4'-azidophenyl)propionyl]tetrabenazine binding sites for the synaptic vesicle monoamine transporter. | the full-length cdna for the rat recombinant synaptic vesicle monoamine transporter (rvmat2) containing a cooh-terminal polyhistidine epitope was engineered into baculovirus dna for expression in spodoptera frugiperda (sf9) cells. using this recombinant baculovirus and cultured sf9 cells, rvmat2 has been expressed to high levels and purified to >95% homogeneity using immobilized ni2+-affinity chromatography followed by lectin (concanavalin a) chromatography. purified transporter was photolabeled ... | 1997 | 9325342 |
| interaction of phosphorylated tryptophan hydroxylase with 14-3-3 proteins. | rabbit brain tryptophan hydroxylase (tph) has been expressed in insect cells (spodoptera frugiperda) as a histidine-tagged enzyme. the specific activity of the purified fusion enzyme is 80 nmol of 5-hydroxytryptophan/min/mg. multifunctional regulatory 14-3-3 proteins were purified from fresh bovine brain. phosphorylation and 14-3-3 proteins play important roles in the regulation of tph activity. we have found that phosphorylation of tph by camp-dependent protein kinase increased the activity of ... | 1997 | 9334190 |
| production of recombinant goldfish growth hormone i in a baculovirus expression system. | the cdna sequence encoding the growth hormone (gh) i gene of goldfish (gf gh-i) was cloned into the psxivvi+ x3 baculovirus transfer vector. this transfer vector, without the initiation codon (atg) and using a synxiv promoter to activate transcription, was constructed for a baculovirus expression system. the recombinant transfer vector with the gf gh-i insert was used to produce the recombinant baculovirus tnnpv-sx+ gf gh-i 21a. both in vivo and in vitro approaches were used to test the expressi ... | 1997 | 9343707 |
| application of recombinant bovine viral diarrhea virus proteins in the diagnosis of bovine viral diarrhea infection in cattle. | the national animal disease laboratory (nadl) vaccine strain of bovine viral diarrhea virus (bvdv) genes for gp48 and p80 were expressed in escherichia coli. the bvdv-nadl gene for gp62 was integrated into a baculovirus genome for expression in spodoptera frugiperda (sf-9) insect ovarian cells. the antigenicity of baculovirus expressed bvdv protein was detected by anti-bvdv specific antibodies in an enzyme-linked immunosorbent assay (elisa), indirect immunofluorescent assay (ifa) and radio-immun ... | 1997 | 9355247 |
| production of recombinant herpes simplex virus protease in 10-l stirred vessels using a baculovirus-insect cell expression system. | a gene expression system using recombinant autographa california nuclear polyhedrosis virus (baculovirus) and sf-9 cells has been scaled up to the 10-l tank level and shown to be capable of producing herpes simplex virus (hsv) protease in serum-free media. high densities of spodoptera frugiperda (sf-9) cells were achieved by modifying two 10-l biolafitte fermenters specifically for insect cell growth. the existing rushton impellers were replaced by marine impellers to reduce shear and the aerati ... | 1997 | 9366090 |
| the mast cell function-associated antigen exhibits saccharide binding capacity. | the mast cell function-associated antigen (mafa) is a membrane glycoprotein first identified on rat mucosal type mast cells (line rbl-2h3) and known to inhibit the fc epsilon ri-mediated secretory response. in its extracellular domain, an amino acid stretch homologous to the carbohydrate binding domain of calcium-dependent animal lectins has been found. to investigate its carbohydrate binding capacity, the mafa has been expressed in the spodoptera frugiperda insect cell line (sf9) using the bacu ... | 1997 | 9368610 |
| baculovirus inhibitors of apoptosis (iaps) block activation of sf-caspase-1. | we have investigated the ability of sf-caspase-1 and two mammalian caspases, caspase-1 and caspase-3, to induce apoptosis in spodoptera frugiperda sf-21 insect cells. while the transient expression of the pro-sf-caspase-1 did not induce apoptosis, expression of the pro-domain deleted form, p31, or coexpression of the two subunits of mature sf-caspase-1, p19 and p12, induced apoptosis in sf-21 cells. the behavior of sf-caspase-1 resembled that of the closely related mammalian caspase, caspase-3, ... | 1997 | 9391073 |
| induction of tolerance toward rat cardiac allografts by treatment with allochimeric class i mhc antigen and fty720. | the combination of fty 720, a novel immunosuppressant, and allochimeric class i mhc proteins bearing donor-type amino acid (aa) epitope substitutions for host-type sequences induces tolerance of wistar furth (wf; rt1.au) heart allografts in aci (rt1.aa) recipients. | 1997 | 9392303 |
| the unliganded mineralocorticoid receptor is associated with heat shock proteins 70 and 90 and the immunophilin fkbp-52. | the human mineralocorticoid receptor (mr) is a member of the steroid-thyroid hormone receptor superfamily, which includes receptors for retinoic acid, vitamin d, and other steroids, such as the glucocorticoids (which bind the glucocorticoid receptor, gr). mr and gr, the corticosteroid receptors, share significant homology and are activated by steroid binding, resulting in a conformational change, nuclear translocation, and dna binding. despite these similarities with gr, the mr remains less well ... | 1997 | 9392437 |
| binding and fusion of autographa californica nucleopolyhedrovirus to cultured insect cells. | binding of baculoviruses to insect cells and fusion of the virus envelope to cell membranes are early events suggested to be affected by baculovirus enhancins. the binding of autographa californica nucleopolyhedrovirus (acmnpv) to the spodoptera frugiperda cell line sf21 and the fusion of the virus envelope to cell membranes were characterized. virus binding assays demonstrated that acmnpv budded virus (bv) bound to specific binding sites on sf21 cells with an avidity of 2.3 x 10(10) m(-1). the ... | 1997 | 9400956 |
| expression of human immunodeficiency virus genes using baculovirus expression system. | the structural protein genes of hiv-1 and hiv-2 have been expressed in spodoptera frugiperda (sf) cells using baculovirus expression system. the noncoding flanking sequences of hiv structural genes were removed and a putative ribosome binding site was placed in front of the open reading frame of each gene by using crossover linker mutagenesis. the coding sequences of the gag, pol, env, and vif proteins were inserted into autographa californica nuclear polyhedrosis virus (acnpv) so that hiv genes ... | 1997 | 9406188 |
| agonist-independent activation of gz by the 5-hydroxytryptamine1a receptor co-expressed in spodoptera frugiperda cells. distinguishing inverse agonists from neutral antagonists. | the human 5-hydroxytryptamine1a receptor, when expressed in spodoptera frugiperda (sf9) cells, facilitates the binding of [35s]gtpgammas to a co-expressed gtp-binding regulatory protein, gz, consistent with constitutive activity. the antagonists 4-(2'-methoxyphenyl)-1-[2'(n-2"-pyridinyl)-p-iodobenzamido]ethyl-p ipe razine (p-mppi) and the related fluorobenzamido analogue p-mppf had little (p-mppi) or no (p-mppf) effect on this activity. in contrast, a third antagonist, the neuroleptic spiperone, ... | 1997 | 9407078 |
| characterization of canine herpesvirus glycoprotein d (hemagglutinin). | glycoprotein d (gd) of canine herpesvirus (chv) yp2 strain was expressed in cos-7 and insect (spodoptera frugiperda; sf9) cells. the gds expressed in cos-7 and sf9 cells reacted with a panel of monoclonal antibodies (mabs) against chv gd (hemagglutinin) and an mab 25c9 against feline herpesvirus type 1 (fhv-1) gd by indirect immunofluorescence assay, and possessed a molecular weight (mw) of approximately 51-55 and 41-46 kilodalton (kda), respectively, when examined by immunoblot analysis. after ... | 1997 | 9409515 |
| affinity-purified dengue-2 virus envelope glycoprotein induces neutralizing antibodies and protective immunity in mice. | we constructed a recombinant baculovirus which produces a dengue (den)-2 virus envelope (e) protein containing a six-histidine (h6) tag in place of the last 100 amino acids at its c-terminus. the recombinant protein was purified from the supernatant of baculovirus-infected spodoptera frugiperda insect cell cultures to apparent homogeneity by cation-chelation chromatography (talon) in which the h6-tagged e-protein was eluted under non-denaturing conditions with 100 mm imidazole at ph 8.0. mice va ... | 1997 | 9413106 |
| role of baculovirus ie2 and its ring finger in cell cycle arrest. | the ie2 gene of autographa californica nuclear polyhedrosis virus (acmnpv) is known to transactivate transient expression from viral promoters in a host cell-specific manner. we report that transfection of spodoptera frugiperda (sf-21) cells with ie2 was sufficient to arrest the cell cycle, resulting in the accumulation of enlarged cells with abnormally high dna contents. by 72 h posttransfection, more than 50% of ie2-transfected cells had dna contents greater than 4n. there was no evidence of m ... | 1998 | 9420274 |
| cloning and heterologous expression of nadph-cytochrome p450 reductases from the papaveraceae. | cytochrome p450 reductase was purified to homogeneity from cell suspension cultures of the opium poppy papaver somniferum, the enzyme was characterized (k(m) cytochrome c, 8.3 microm; k(m) nadph, 4.2 microm; ph optimum, 8.0; m(r), 80 kda), and the amino acid sequence of internal peptides was determined. partial cdna clones from p. somniferum and from eschscholzia californica (california poppy) were then generated using the polymerase chain reaction and were used as hybridization probes to isolat ... | 1997 | 9434750 |
| suppression of cell growth by heavy chain ferritin. | while producing recombinant rat liver h and l chain ferritin homopolymers using the baculovirus expression system, we noticed that rat liver h chain ferritin, but not l chain ferritin, had a suppressive effect on the growth of spodoptera frugiperda (sf-21) cells. suppression was observed immediately after infection with recombinant h chain ferritin baculovirus prepared from lysed infected cells. immediate suppression was observed when purified with either recombinant h chain apoferritin or vario ... | 1998 | 9439606 |
| neuronal cell surface molecules mediate specific binding to rabies virus glycoprotein expressed by a recombinant baculovirus on the surfaces of lepidopteran cells. | the existence of specific rabies virus (rv) glycoprotein (g) binding sites on the surfaces of neuroblastoma cells is demonstrated. spodoptera frugiperda (sf21) cells expressing g of the rv strain cvs (gcvs-sf21 cells) bind specifically to neuroblastoma cells of different species but not to any other cell type (fibroblast, myoblast, epithelial, or glioma). attachment to mouse neuroblastoma ng108-15 cells is abolished by previous treatment of gcvs-sf2 cells with anti-g antibody. substitutions for ... | 1998 | 9445003 |
| role of one n-linked oligosaccharide chain on canine herpesvirus gd in its biological activity. | the yp11mu strain of a plaque-selected canine herpesvirus (chv) encoded a smaller molecular weight (mw) of gd than those of other strains including yp2 strain (xuan et al., 1990). when nucleotide sequence of the mutated gd of yp11mu strain (gd(yp11mu)) was compared with that of gds of other chv strains, gd(yp11mu) lacked 12 nucleotides encoding 4 amino acids, nkti, including one predicted potential n-linked glycosylation site and no other change was found in other regions. when the gd(yp11mu) an ... | 1997 | 9450242 |
| video imaging analysis of the plasma membrane permeabilizing effects of bacillus thuringiensis insecticidal toxins in sf9 cells. | the size and ionic selectivity of the pores formed by the insecticidal crystal protein cry1c from bacillus thuringiensis in the plasma membrane of sf9 cells, an established cell line derived from the fall armyworm spodoptera frugiperda, were analyzed with a video imaging technique. changes in the permeability of the membrane were estimated from the rate of osmotic swelling of the cells. in the presence of cry1c, which is toxic to sf9 cells, the permeability of the cell membrane to kcl and glucos ... | 1998 | 9459581 |
| high-efficiency expression and characterization of the synaptic-vesicle monoamine transporter from baculovirus-infected insect cells. | the full-length cdna for the rat synaptic-vesicle monoamine transporter (vmat2) containing a c-terminal polyhistidine epitope has been engineered into baculovirus dna for expression in spodoptera frugiperda (sf9) insect cells. using this recombinant baculovirus and cultured sf9 cells, rvmat2 has been expressed at levels of 7.8x10(6) transporters per cell, as assessed by [3h]dihydrotetrabenazine binding. a 1l culture of infected cells produced approx. 15 nmol (900 microg) of transporter. rvmat2 e ... | 1998 | 9480916 |
| physiology of cultured animal cells. | the physiology of cultured animal cells, in particular hybridoma, myeloma and insect cells, with respect to growth and proliferation, amino acid metabolism, energy metabolism and cellular responses to environmental stress is discussed in this paper. the rate of proliferation of hybridoma cells in serum-containing media is limited by growth factors at a surprisingly early stage of growth. to maintain exponential growth in a batch culture, it is necessary to stimulate cell proliferation with repea ... | 1997 | 9487719 |
| in vivo kinetics as a sensitive method for testing physiologically intact human recombinant apolipoprotein a-i: comparison of three different expression systems. | in order to assess the structural and functional integrity of recombinant human apoa-i, we expressed apoa-i using three different expression systems: baculovirus transfected spodoptera frugiperda (sf9) cells, stably transfected chinese hamster ovary (cho) cells, and transformed escherichia coli (e. coli). purified apoa-i from the three expression systems was radioiodinated and their catabolism was compared in normolipemic rabbits. the kinetic turnover studies of radiolabelled apoa-i in normolipe ... | 1997 | 9495570 |
| toxicity and binding properties of the bacillus thuringiensis delta-endotoxin cry1c to cultured insect cells. | a better understanding of the mode of action of bacillus thuringiensis delta-endotoxins is needed to develop strategies which may prevent or slow down selection for resistance. we studied the effect of cry1c on several different cultured insect cell lines by means of toxicity assays, ligand blotting, and toxin binding studies. a clear difference in sensitivity toward cry1c between the insect cell lines was observed. spodoptera frugiperda cell line sf9 was most sensitive, whereas spodoptera exigu ... | 1998 | 9500946 |
| characterization of skt1, an inwardly rectifying potassium channel from potato, by heterologous expression in insect cells. | a cdna encoding a novel, inwardly rectifying k+ (k+in) channel protein, skt1, was cloned from potato (solanum tuberosum l.). skt1 is related to members of the akt family of k+in channels previously identified in arabidopsis thaliana and potato. skt1 mrna is most strongly expressed in leaf epidermal fragments and in roots. in electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (spodoptera frugiperda) cells, skt1 was identified as a k+in channel that ... | 1998 | 9501121 |
| expression of biologically active human calpastatin in baculovirus-infected insect cells and in escherichia coli. | calpastatin, an endogeneous inhibitor protein acting on calpain (ca(2+)-dependent cysteine proteinase), is widely distributed in animal tissues and cells. two different expression systems, baculovirus-infected spodoptera frugiperda (sf9) insect cells and escherichia coli, were used for overexpression of the human calpastatin tagged with n-terminal hexahistidine peptide. recombinant calpastatin was purified to homogeneity by nickel ion affinity chromatography and gel filtration separation. purifi ... | 1998 | 9501525 |
| dirofilaria immitis: molecular cloning and expression of a cdna encoding a selenium-independent secreted glutathione peroxidase. | a cdna clone, di29, encoding a homolog of glutathione peroxidase, was isolated from a dirofilaria immitis adult female cdna expression library by a combination of polymerase chain reaction amplification with primers designed from the brugia pahangi glutathione peroxidase gene sequence and hybridization screening of d. immitis cdna libraries. the di29 nucleotide and deduced amino acid sequences were very similar to those described for lymphatic filariae and predicted a secreted form of glutathion ... | 1998 | 9501847 |
| molecular basis of the dark agouti rat drug oxidation polymorphism: importance of cyp2d1 and cyp2d2. | the dark agouti rat has been proposed as a model for the human debrisoquine 4-hydroxylase polymorphism. earlier studies suggested that the poor metabolizer phenotype in the dark agouti rat is caused by the absence of the expression of cyp2d1 mrna. although cyp2d1 is the major enzyme catalyzing debrisoquine 4-hydroxylation, other reports have indicated the involvement of a cyp2d, purified from rat hepatic microsomes and presumed to be cyp2d2, which also exhibits this activity. the levels of cyp2d ... | 1998 | 9511184 |
| assembly of amsacta moorei entomopoxvirus spheroidin into spheroids following synthesis in insect cells using a baculovirus vector. | the gene encoding the major occlusion body protein, spheroidin, of amsacta moorei entomopoxvirus (amepv) was introduced into a baculovirus vector under control of the polyhedrin gene promoter. a recombinant virus produced large, ovoid occlusion body-like structures in both spodoptera frugiperda and trichoplusia ni cells. these structures resembled the spheroids found in amepv-infected lymantria dispar cells, except they were devoid of virus particles and were not surrounded by a membrane- or env ... | 1998 | 9519843 |
| the pnk/pnl gene (orf 86) of autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene. | autographa californica nucleopolyhedrovirus (acmnpv) orf 86, located within the hindiii c fragment, potentially encodes a protein which shares sequence similarity with two t4 bacteriophage gene products, rna ligase and polynucleotide kinase. this acmnpv gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. it has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mrna transcripts were detectable fr ... | 1998 | 9519844 |
| baculovirus-mediated expression of cytochrome p4502c8 and human nadph-cytochrome p450 reductase: optimization of protein expression. | 1. high expression levels of cytochrome p450 (cyp) 2c8 and nadph-cytochrome p450 oxidoreductase (oxr) in spodoptera frugiperda (sf21) cells have been achieved using the baculovirus expression system. 2. the baculovirus dual expression plasmid, pacuw31, was used to insert cyp2c8 and oxr cdnas downstream of the polyhedrin (polh) or p10 promoters, either separately or together, generating four recombinant baculoviruses; two expressing single proteins (cyp2c8 driven by the p10 promoter, bvp10.2c8 or ... | 1998 | 9522439 |
| development and validation of a competitive enzyme-linked immunosorbent assay for detection of type a influenza antibodies in avian sera. | serologic screening of avian sera for group-specific antibodies to type a influenza is currently accomplished by using the avian influenza (ai) agar gel immunodiffusion (agid) test. a competitive enzyme-linked immunosorbent assay (celisa) was developed using a baculovirus vector, autographa californica nuclear polyhedrosis virus, expressing the nucleoprotein (np) gene of a/ann arbor/6/60 influenza virus. the recombinant np was obtained by inoculation of spodoptera frugiperda (sf9) insect cells o ... | 1998 | 9533078 |
| baculovirus-mediated large-scale expression and purification of a polyhistidine-tagged rubella virus capsid protein. | the capsid protein of rubella virus was produced in baculovirus-infected spodoptera frugiperda insect cells, with a polyhistidine affinity tag at the carboxy terminus. the rv capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal-ion affinity chromatography. immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight. the final yield was 5 mg of purif ... | 1998 | 9535699 |
| characterization of a nuclear polyhedrosis virus of spodoptera frugiperda from argentina. | | 1998 | 9538035 |
| n-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines. | the capacity of two trichoplusia ni (tn-368 and bti-tn-5b 1-4) and a spodoptera frugiperda (iplb-sf-21a) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. the alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. the n-linked oligosaccharides were released from the purified protein with pngase f and analyzed by fluorophore-assisted carbohydrate electrophoresis. the ... | 1998 | 9542646 |
| expression of fas-fas ligand system associated with atresia in murine ovary. | we detected that fas receptor (fas) was expressed at transcriptional levels in oocytes/eggs and some granulosa cells of murine ovary, whereas, fas ligand (fasl) was found to be strongly expressed in granulosa cells by means of in situ hybridisation. these results were supported by an indirect immunofluorescence (iif) test with anti-fas monoclonal antibody (mab)/fasl ab. the lysates from granulosa cells were precipitated by anti-fasl ab, exhibiting a specific band at 40 kda. when zona pellucida ( ... | 1997 | 9563680 |
| superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with escherichia coli. | hyaluronidase (hya) is one of several allergens in honeybee venom. its cdna sequence was recently described. | 1998 | 9600508 |
| adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance-associated protein mrp2 expressed in insect cells. | the multidrug resistance-associated protein mrp2 is expressed in liver, kidney, and small intestine and mediates atp-dependent transport of conjugated organic anions across the apical membrane of epithelial cells. we recently cloned a rabbit cdna encoding a protein that on basis of highest amino acid homology and tissue distribution was considered to be the rabbit homolog of rat mrp2. to investigate whether rabbit mrp2 mediates atp-dependent transport similar to rat mrp2, we expressed rabbit mrp ... | 1998 | 9614209 |
| cloning, sequencing, and expression of the bombyx mori receptor for bacillus thuringiensis insecticidal cryia(a) toxin. | bacillus thuringiensis strains produce insect-specific bt toxins. bt cryia(a) toxin binds to a 175-kda glycoprotein (btr175) on the microvillus membranes of columnar cells in the bombyx mori midgut and causes lysis of the cells. btr175 was purified, and its cdna was cloned. the cdna encodes a newly identified 193.3-kda preproprotein form of btr175 that includes nine extracellular cadherin repeats, a 23.5-kda membrane-proximal domain, a membrane-spanning region, and a 13.6-kda cytoplasmic domain. ... | 1998 | 9614703 |
| n-linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells. | the potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. a comparison was made of the n-linked glycans attached to secreted alkaline phosphatase (seap) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. these data survey n-linked oligosaccharides produced in four families and s ... | 1998 | 9621115 |
| expression and purification of the human thrombin receptor. | the human thrombin receptor has been overexpressed in sf9 (spodoptera frugiperda) insect cells using a baculovirus vector. cell surface expression of the receptor was confirmed by immunocytochemistry with polyclonal antibodies raised against the extracellular domain of the receptor. the expressed receptor was functional; both thrombin and the thrombin receptor agonist peptide produced increases in intracellular calcium in transfected cells. the concentration of thrombin causing the half-maximal ... | 1998 | 9631508 |
| the single-nucleocapsid nucleopolyhedrovirus of buzura suppressaria encodes a p10 protein. | the p10 gene of buzura suppressaria single-nucleocapsid nucleopolyhedrovirus (busunpv) was identified by virtue of its localization downstream from the autographa californica (ac) mnpv p26 homologue. the busunpv p10 gene encodes a protein of 94 amino acids. the amino acid sequence contains domains characteristic of baculovirus p10 proteins, e.g. a coiled-coil domain, a proline-rich motif and a positively charged c terminus. the highest amino acid homologies were found with the spodoptera littora ... | 1998 | 9634101 |
| eukaryotic virus display: engineering the major surface glycoprotein of the autographa californica nuclear polyhedrosis virus (acnpv) for the presentation of foreign proteins on the virus surface. | we describe the development of the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) as a vector for the display of distinct proteins on the viral surface in a manner that is analogous to the established bacterial "phage display" systems. as a model system, the marker gene encoding the 26kda protein glutathione-s-transferase (gst) was used to construct several fusions with the major baculovirus glycoprotein gp64 gene. following expression in spodoptera frugiperda (sf9) cells, ... | 1995 | 9636281 |
| expression of active, secreted human prostate-specific antigen by recombinant baculovirus-infected insect cells on a pilot-scale. | we have expressed human prostate-specific antigen (psa) on a pilot-scale in spodoptera frugiperda sf9 insect cells using recombinant baculovirus system. infected cells secreted psa into culture medium at a concentration of 2-4 mg per liter. psa was expressed both in active and inactive forms which were separated in a final purification step using cation-exchange chromatography eluted with a low salt gradient. the n-terminus of active psa was correctly cleaved; two amino acids of the propeptide r ... | 1995 | 9636298 |
| recombinant chicken interferon-gamma-mediated inhibition of eimeria tenella development in vitro and reduction of oocyst production and body weight loss following eimeria acervulina challenge infection. | recombinant chicken interferon-gamma (chifn-gamma) was produced in cho-k1 or spodoptera frugiperda (sf9) insect cells by transfection with a pcdna vector or recombinant baculovirus (sf9-interferon-gamma [ifn-gamma] carrying the chifn-gamma gene. a rabbit antibody against a synthetic peptide corresponding to an immunogenic portion of chifn-gamma recognized a 22-23-kda band in sf9-ifn-gamma cell extracts by western blot analysis. biological activity of recombinant chifn-gamma was shown by its inhi ... | 1998 | 9645322 |
| evaluation of baculovirus-expressed bovine herpesvirus-1 (bhv-1) glycoproteins for detection and analysis of bhv-1-specific antibody responses. | baculovirus (autographa californica nuclear polyhedrosis)-expressed bovine herpesvirus-1 (bhv-1) glycoproteins b (gb), gc, and gd were developed and characterized. the recombinant proteins retained their antigenic properties as determined by immunoblotting against monoclonal antibodies. the proteins were examined as antigens for detection of bhv-1 infection and for the analysis of antibody responses to the individual viral proteins. a total of 115 bovine serum samples were tested for their react ... | 1998 | 9646475 |
| expression of functionally active atp-sensitive k-channels in insect cells using baculovirus. | we have expressed active atp-sensitive k-channels (k(atp) channels) in spodoptera frugiperda (sf9) cells using a baculovirus vector. a high yield of active channels was obtained on co-infection with sur1 and kir6.2 engineered to contain n- and/or c-terminal tags to permit detection by western blotting. channel activity was sensitive to atp, glibenclamide and diazoxide. channel activity was also obtained on expression of a c-terminally truncated kir6.2 (kir6.2 deltac26): these channels were block ... | 1998 | 9662455 |
| molecular cloning and functional heterologous expression of two alleles encoding (s)-n-methylcoclaurine 3'-hydroxylase (cyp80b1), a new methyl jasmonate-inducible cytochrome p-450-dependent mono-oxygenase of benzylisoquinoline alkaloid biosynthesis. | alkaloids derived from the tetrahydrobenzylisoquinoline alkaloid (s)-n-methylcoclaurine represent a vast and varied structural array of physiologically active molecules. these compounds range from the dimeric bisbenzylisoquinolines, such as the muscle relaxant (+)-tubocurarine, to the powerful anaesthetic opiate morphine, the antimicrobial berberine and the anti-microbial benzo[c]-phenanthridine sanguinarine. the 3'-hydroxylation of (s)-n-methylcoclaurine is a branch point that is the penultimat ... | 1998 | 9681018 |
| expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase. | mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, ec 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight ... | 1998 | 9693067 |
| the host factor polyhedrin promoter binding protein (ppbp) is involved in transcription from the baculovirus polyhedrin gene promoter. | hypertranscription and temporal expression from the autographa californica nuclear polyhedrosis (acnpv) baculovirus polyhedrin promoter involves an alpha-amanitin-resistant rna polymerase and requires a trans-acting viral factor(s). we previously reported that a 30-kda host factor, polyhedrin promoter binding protein (ppbp), binds with unusual affinity, specificity, and stability to the transcriptionally important motif aataaataagtatt within the polyhedrin (polh) initiator promoter and also disp ... | 1998 | 9696845 |