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molecular and biochemical evidence for the involvement of the asp-333-his-523 pair in the catalytic mechanism of soluble epoxide hydrolase.in order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. our results are consistent with the involvement of asp-333 and his-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. mutation of his-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. when his-3 ...19957713895
molecular cloning and expression of catrocollastatin, a snake-venom protein from crotalus atrox (western diamondback rattlesnake) which inhibits platelet adhesion to collagen.a 50 kda protein that inhibits platelet adhesion to collagen has been isolated from snake venom of crotalus atrox (western diamondback rattlesnake) and has been named 'catrocollastatin'. the cdna cloning of catrocollastatin has been accomplished. a full-length cdna of 2310 bp with an open reading frame between nucleotides 51 and 1880 was obtained. the deduced amino acid sequence consists of 609 amino acids. the cdna-predicted amino acid sequence is highly similar to that of haemorrhagic metallop ...19957733877
overexpression of bombyx mori prothoracicotropic hormone using baculovirus vectors.recombinant baculoviruses were constructed that express the cdna encoding the prothoracicotropic hormone (ptth) of bombyx mori. this hormone stimulates the production of ecdysteroids by the insect's prothoracic glands. two groups of viruses were constructed, expressing either the entire cdna encoding prepro-ptth, or a synthetic chimeric gene encoding a signal peptide fused to the mature ptth subunit. in both cases, the genes were expressed in wild-type autographa californica nuclear polyhedrosis ...19957742834
functional expression of a diuretic hormone receptor in baculovirus-infected insect cells: evidence suggesting that the n-terminal region of diuretic hormone is associated with receptor activation.a recombinant baculovirus containing the diuretic hormone receptor cdna from manduca sexta was constructed. when spodoptera frugiperda (sf9) cells were infected with the virus, a time-dependent expression of the receptor appeared. the expressed receptor displayed high affinity for diuretic hormone, which was similar to the affinity observed in malpighian tubules and transfected cos-7 cells. the receptor expression level was 77 pmol/mg protein, as compared to 3.1 pmol/mg protein in malpighian tub ...19957742837
histidine tagging both allows convenient single-step purification of bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry.for rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3'-end by a short sequence encoding six histidine residues. recombinant baculovirus-infected spodoptera frugiperda cells produce bovine opsin carrying a c-terminal histidine tag (v-opshis6x). the presence of this tag was confirmed by immunoblot analysis. incubation with 11-cis-retinal produced a photosensitive pigment (v-rhohis6x ...19957744755
the autographa californica nuclear polyhedrosis virus homologous region 1a: identical sequences are essential for dna replication activity and transcriptional enhancer function.hr1a consists of two 30-bp imperfect palindrome sequences separated by 58 bp and each palindrome contains a naturally occurring ecori site at its core. plasmid subclones of the hr1a-containing acmnpv hindiii-n fragment were examined for their ability to replicate in virus-infected (spodoptera frugiperda) sf9 cells, and to stimulate transcription when linked in cis with a 39k gene promoter-beta-glucuronidase fusion and cotransfected into cells along with a plasmid (ple-1) containing the gene enco ...19957747446
cloning and expression of the nucleoprotein of peste des petits ruminants virus in baculovirus for use in serological diagnosis.peste des petits ruminants (ppr) is a viral disease of goats and sheep characterized by erosive stomatitis, enteritis, and pneumonia. the virus is a member of the family paramyxoviridae and the genus morbillivirus. the disease has high morbidity and mortality rates and has a substantial economic impact in developing countries. we have cloned and sequenced the cdna of the nucleocapsid (n) gene of the nigeria 75/1 strain of ppr virus (pprv). a comparison of its nucleotide and deduced amino acid se ...19957747450
synthesis and processing of the rubella virus p110 polyprotein precursor in baculovirus-infected spodoptera frugiperda cells.in order to study the processing of rubella virus (rv) structural proteins (capsid protein, of 33 kda; e2 of 42-47 kda; and e1 of 58 kda) in spodoptera frugiperda (fall armyworm) cells, a 24s cdna encoding the polyprotein precursor, p110, was inserted under the transcriptional regulation of the polyhedrin gene promoter of the autographa californica nuclear polyhedrosis virus (acnpv) and expressed during viral infection. by immunoblot analysis using antibodies directed against whole rv and the in ...19957754676
expression of trypanosoma congolense antigens in spodoptera frugiperda insect cells.transcripts which encode two metacyclic-form-specific variable surface glycoproteins (mvsgs) of trypanosoma congolense il3000 have been cloned into baculovirus expression vectors using a novel transfer vector, pacl11. one of the recombinant baculoviruses (acvsg1) expressed a mvsg as a glycoprotein with a signal peptide which was cleaved in this expression system, whereas the other one (acvsg2) expressed an unprocessed protein. from 1 liter of culture containing 10(9) spodoptera frugiperda cells ...19957758544
protection of guinea-pigs from experimental rocky mountain spotted fever by immunization with baculovirus-expressed rickettsia rickettsii rompa protein.baculovirus recombinants that express the rickettsia rickettsii rompa protein were constructed. monoclonal antibodies (mabs) against the rompa protein reacted with recombinant-infected spodoptera frugiperda (sf9) cells in indirect immunofluorescence assays. sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of infected sf9 cell lysates with a mab against rompa showed that the recombinant-expressed rompa protein migrated slightly below rompa extracted from r. rickettsii. ...19957762273
expression of marek's disease virus (mdv) serotype 2 gene which has partial homology with mdv serotype 1 pp38 gene.we constructed the recombinant baculovirus expressing the gene of non-pathogenic marek's disease virus (mdv) serotype 2 (mdv2) which encodes a polypeptide with partial homology to mdv serotype 1 (mdv1) pp38, an antigen associated with transformed cells. the recombinant mdv2 protein was detected as a band of 32 kda in immunoblot analysis with mdv2-infected chicken serum. mouse serum against insect spodoptera frugiperda cells infected with the recombinant baculovirus immunoprecipitated a 38 kda mo ...19957762295
cyp3a4 expressed by insect cells infected with a recombinant baculovirus containing both cyp3a4 and human nadph-cytochrome p450 reductase is catalytically similar to human liver microsomal cyp3a4.human cytochrome cyp3a4 is the most abundant of all the p450s in human liver and is involved in the metabolism of many environmental toxicants and drugs. kinetic studies with cyp3a4 have been hampered due to low activity of this enzyme obtained from recombinant gene expression systems or difficulty in reconstituting activity with the native enzyme purified from human liver. to overcome these obstacles, we have expressed high levels of catalytically active cyp3a4 and human nadph-cytochrome p450 r ...19957771780
n-glycosylated polypeptides synthesized in the early phase of acnpv infected insect cells.infected cell specific polypeptides (icsp) in autographa californica nuclear polyhedrosis virus (acnpv) infected spodoptera frugiperda cell line, sf9, were radiolabelled with [14c] mannose and the labelled proteins were monitored at various time intervals. a major glycoprotein of molecular size of 64 kda appears as early as 3h post infection (p.i.) which peaks at 12h p.i., but in presence of tunicamycin this protein is not labelled. two other glycoproteins of molecular size 67 kda and 63 kda app ...19957773189
identification and analysis of a putative origin of dna replication in the choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus genome.a recombinant plasmid containing the choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus (cfmnpv) hindiii r fragment (m.u. 2.2-3.9) was shown to undergo cfmnpv infection-dependent dna replication in cf-124t cells. replication of this dna sequence was detectable by 24 hr p.i. and did not appear to have resulted as a consequence of recombination with the virus genome. replication was inhibited by mimosine, an inhibitor of eukaryotic dna replication. these data suggest that hindii ...19957778276
the wild-type autographa californica nuclear polyhedrosis virus induces apoptosis of spodoptera littoralis cells.spodoptera littoralis cells infected with the autographa californica multiple nuclear polyhedrosis virus (acmnpv) yielded significantly lower budded virus titers than spodoptera frugiperda-infected cells and produced very low levels of polyhedrin. relative to acmnpv-infected s. frugiperda sf9 cells viral dna replication was severely reduced in spodoptera littoralis sl2 cells. microscopic examination of sl2-infected cells revealed progressive cell blebbing starting at 6-8 hr postinfection and cul ...19957778284
nucleotide sequence and transcriptional analysis of the gp41 gene of spodoptera frugiperda nuclear polyhedrosis virus.the spodoptera frugiperda multiple nucleocapsid nuclear polyhedrosis virus (sfmnpv) gp41 structural protein gene was located in the 1.9 kbp ecori-s fragment and sequenced. an open reading frame (orf) of 999 nucleotides was detected that encoded a protein of 332 amino acids. the gp41 gene transcript was detected after 12 h post-infection (p.i.) and remained detectable at 48 h p.i. two major mrnas, about 1.6 and 2.8 kb in length, were determined by northern blot analysis. primer extension analysis ...19957782772
mutations in the autographa californica multinucleocapsid nuclear polyhedrosis virus 25 kda protein gene result in reduced virion occlusion, altered intranuclear envelopment and enhanced virus production.serial passage of nuclear polyhedrosis viruses (npvs) through cultured cell lines results in the appearance of mutants with a complex phenotype referred to as the 'few polyhedra' (fp) phenotype. the altered plaque morphology and reduced occlusion production associated with the fp phenotype have been observed in autographa californica multinucleocapsid nuclear polyhedrosis virus (acmnpv) bearing mutations in the gene encoding the 25 kda protein (25k gene). in this study, we sequenced the 25k gene ...19957782773
potassium channel structure and function as reported by a single glycosylation sequon.inwardly rectifying k+ channels (irks) are highly k(+)-selective, integral membrane proteins that help maintain resting the membrane potential and cell volume. integral membrane proteins as a class are frequently n-glycosylated with the attached carbohydrate being extracellular and perhaps modulating function. however, dynamic effects of glycosylation have yet to be demonstrated at the molecular level. romk1, a member of the irk family is particularly suited to the study of glycosylation because ...19957797521
physiological and environmental factors affecting the growth of insect cells and infection with baculovirus.insect cell growth can be significantly improved by close attention to the conditions used in the inoculum stages. initial cell concentration, spent medium carry over and inoculum phase withdrawal significantly influenced the growth kinetics of spodoptera frugiperda (sf9) cells. the percentage of cells infected with wild and recombinant baculovirus acnpv and (in the later case) the beta-galactosidase yield in fresh medium was appreciably affected by the stage of the growth curve that cells were ...19957765874
ionic permeabilities induced by bacillus thuringiensis in sf9 cells.the effect of bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular ph (phi) of individual sf9 cells of the lepidopteran species spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (pbfi) and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (bcecf). the sequential removal of k+ and na+ from the medium, in the presence of cryic, a toxin which is highly active against sf9 cells, caused s ...19958558602
two human alpha 2-adrenoceptor subtypes alpha 2a-c10 and alpha 2b-c2 expressed in sf9 cells couple to transduction pathway resulting in opposite effects on camp production.the baculovirus expression vector system utilizing the strong polyhedrin gene promoter of the autographa californica nuclear polyhedrosis virus (acnpv) was used for high level expression of the two alpha 2-adrenoceptor subtypes alpha 2a-c10 and alpha 2b-c2 in spodoptera frugiperda (sf-9) insect cells. for rapid screening of recombinant viruses the luciferase gene was expressed under the early etl-promoter (early transcript large) in the same plasmid. both receptor subtypes showed the same rank o ...19958575536
preparation of monoclonal antibodies against marek's disease virus serotype 1 glycoprotein d expressed by a recombinant baculovirus.a recombinant baculovirus, the genome of which contains dna encoding marek's disease virus serotype 1 (mdv1) homolog of glycoprotein d (gd) of herpes simplex virus under the polyhedrin promoter was constructed and designated racmdv1gd. five monoclonal antibodies (mabs) which recognize the mdv1 homolog of gd (mdv1 gd) in spodoptera frugiperda cells infected with racmdv1gd were prepared. the mabs reacted with proteins ranging from 52 to 49 kda in racmdv1gd-infected cell lysates by immunoblot analy ...19958578860
replication of a mamestra brassicae nuclear polyhedrosis virus in a newly established mamestra brassicae cell line.a continuous cell line, designated mbl-3, was newly established from minced neonate larvae of the cabbage armyworm, mamestra brassicae. this new cell line is heteroploid, containing triploid cells predominantly at the frequency of about 50%. doubling time of the cell population is 33 hrs. the akutsu isolate of a mamestra brassicae nuclear polyhedrosis virus (mbnpv) was examined for replication in 16 continuous cell lines, including the cell line mbl-3, from seven lepidoptera species: mamestra br ...19958578995
improved yields of the extracellular domain of the epidermal growth factor receptor produced using the baculovirus expression system by medium replacement following infection.the extracellular domain of the epidermal growth factor receptor (egfr) was expressed using the baculovirus expression vector system. the maximum level of the egfr extracellular domain secreted into the medium in sf-9 (spodoptera frugiperda or fall army-worm) cell batch culture was approximately 2.5 micrograms ml-1. in order to increase this yield, a process was developed that included the following sequence of steps: batch growth to maximum cell density, infection of the cells with recombinant ...19958579836
purification of posttranslationally modified and unmodified rab5 protein expressed in spodoptera frugiperda cells. 19958583943
a diagnostic immunoassay for newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus.a recombinant baculovirus containing a cdna clone encoding the nucleocapsid (np) protein of newcastle disease virus (strain ulster 2c) has been used to infect insect cells (spodoptera frugiperda). high levels of overexpressed np protein were observed, comprising up to 40% of total cellular protein, which were subsequently shown to be antigenic. nucleoprotein derived from the crude soluble lysate of infected insect cells has been used in an indirect elisa to detect the presence of anti-ndv antibo ...19958609201
purification of recombinant glur-d glutamate receptor produced in sf21 insect cells.glur-d glutamate receptors carrying flag and polyhis affinity tags at the n-terminus and c-terminus, respectively, were expressed in recombinant baculovirus-infected spodoptera frugiperda sf21 cells. affinity-tagged receptors displayed ligand-binding affinity (kd = 40 nm) and an expression level (bmax 10-30 pmol/mg protein) similar to that of insect-cell-expressed wild-type glur-d, as determined by [3h]-alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid ([3h]ampa) binding. the receptor wa ...19958521834
expression of functional ricin b chain using the baculovirus system.the ricin b chain (rtb) was expressed using a baculovirus expression system. the rtb coding sequence downstream of the preproricin signal sequence was inserted in the baculovirus transfer vector pm34t. after cotransfection of spodoptera frugiperda sf9 cells with linearized baculovirus dna, recombinant viruses were selected, cloned and amplified. upon infection of sf9 cells with these recombinant baculoviruses, rtb production was revealed by immunoblotting. rtb expression using this system was op ...19958521841
real-time measurement of cell permeabilization with low-molecular-weight membranolytic agents.a new method for studying the action of membranolytic agents by simple measurement of light emitted from cells is described. it is based on the expression of the click beetle (pyrophorus plagiophthalamus) luciferase gene (lucgr) in escherichia coli, bacillus subtilis and spodoptera frugiperda cells in order to make them bioluminescent. the diffusion of the substrate for luciferase enzyme through the cell membranes is very low at physiological ph, and therefore a change in membrane permeability i ...19958522460
the protoxin composition of bacillus thuringiensis insecticidal inclusions affects solubility and toxicity.most bacillus thuringiensis strains producing toxins active on lepidoptera contain several plasmid-encoded delta-endotoxin genes and package related protoxins into a single inclusion. it was previously found that in b. thuringiensis subsp. aizawai hd133, which produces an inclusion comprising the cryiab, cryic, and cryid protoxins, there is a spontaneous loss in about 1% of the cells of a 45-mda plasmid containing the cryiab gene. as a result, inclusions produced by the cured strain were less re ...19958526519
characterization of human dna ligase i expressed in a baculovirus-insect cell system.the baculovirus expression system was used to overexpress human dna ligase i (hlig i). approx. 2 mg of recombinant hlig i was produced per 10(8) spodoptera frugiperda sf9 insect cells infected with the recombinant baculovirus. the optimum time point for the production of biologically active recombinant hlig i was 48 h post-infection. lig i activity was demonstrated by auto-adenylating, polynucleotide joining and dna relaxation assays. the baculovirus system has the advantage over previously desc ...19958526875
purification and renaturation of japanese encephalitis virus nonstructural glycoprotein ns1 overproduced by insect cells.the nonstructural protein ns1 of japanese encephalitis virus is a major immunogen produced during flavivirus infection. however, the function of this protein has not been identified. to analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in spodoptera frugiperda insect cells (sf9), using a recombinant baculovirus, and purified. as described previously by m. flamand, v. deubel, and m. girard (1992, virology 191, 826-836), a smal ...19958527939
convenient desktop-scale production of the extracellular domain of the human growth hormone receptor by an insect-baculovirus secretion system using a protein-free culture.expression of a gene encoding the extracellular domain of the human growth hormone receptor (hghr-ed) inserted into the genome of autographa californica nuclear polyhedrosis virus was done using a desktop-scale spinner culture. spodoptera frugiperda 9 (sf9) cells infected with the recombinant virus secreted a protein with hgh-binding activity into the medium. oxygen supplementation was required for high level secretion of the product. the highest cell production capability was estimated at more ...19958534995
expression of a 32 kilodalton theileria sergenti piroplasm surface protein by recombinant baculoviruses.previous studies detected a single amino acid substitution (ala196 to gly196) between cdna clones encoding a 32 kda antigen (p32) of theileria sergenti (chitose stock) obtained from a persistently infected calf. in this study, 2 different recombinant baculoviruses (pac/p32-ala196 and pac/p32-gly196) were constructed for the expression of p32. molecular masses of the polypeptides produced in spodoptera frugiperda cells infected with the recombinant baculoviruses were the same as that of authentic ...19958550294
the nonstructural nsm protein of tomato spotted wilt virus induces tubular structures in plant and insect cells.the expression and subcellular location of the 33.6-kda nonstructural protein nsm of tomato spotted wilt virus (tswv) was analyzed in nicotiana rustica plants and protoplasts as a function of time. immunofluorescent studies in protoplasts isolated from tswv-infected n. rustica leaves showed that this protein could first be detected close to the periphery of the cell, near the plasmamembrane, and later in tubular structures emerging from the cell surface. in situ, these tubules appeared specifica ...19958553550
agonist-induced desensitization of dopamine d1 receptor-stimulated adenylyl cyclase activity is temporally and biochemically separated from d1 receptor internalization.the regulation of the dopamine d1 receptor was investigated by using c-myc epitope-tagged d1 receptors expressed in sf9 (fall armyworm ovary) cells. treatment of d1 receptors with 10 microm dopamine for 15 min led to a loss of the dopamine-detected high-affinity state of the receptor accompanying a 40% reduction in the ability of the receptor to mediate maximal dopamine stimulation of adenylyl cyclase activity. after 60 min of agonist exposure, 45 min after the occurrence of desensitization, 28% ...19957479745
immune protection conferred by the baculovirus-related glycoprotein of thogoto virus (orthomyxoviridae).the coding region of segment 4 of thogoto (tho) virus, a tick-borne member of the orthomyxoviridae, was expressed in a baculovirus system under the control of the polyhedrin promoter. this construct expressed authentic envelope glycoprotein as determined by size and antigenic reactivity with a panel of monoclonal antibodies (mabs). immunization of hamsters with spodoptera frugiperda (sf21) cells infected with the recombinant baculovirus induced neutralizing and protective antibodies against viru ...19957483270
high level production and localization of bovine rod cgmp-gated cation channel subunit 1 in baculovirus-infected insect cells and saccharomyces cerevisiae.the cgmp-gated cation channel is responsible for the last step in the vertebrate phototransduction cascade which couples light activation of rhodopsin to a change in membrane permeability. two different expression systems, baculovirus-infected spodoptera frugiperda (sf9) insect cells and saccharomyces cerevisiae, were used for the overproduction of the cgmp-gated cation channel subunit 1 of bovine rod cells. presence of recombinant channel protein was monitored by sds-page and western-blot analy ...19957488067
modulation of cd4 lateral mobility in intact cells by an intracellularly applied antibody.this study shows that the lateral mobility of cd4, an important plasma-membrane immune receptor, can be modulated by intracellular application of an anti-cd4 antibody. for this purpose, (i) full-length cd4 and a truncated cd4 mutant, lacking a 32-residue-long c-terminal intracellularly exposed domain, were expressed in spodoptera frugiperda (sf9) insect cells, (ii) a monoclonal antibody, c6, with specificity for the c-terminal domain was generated, and (iii) a versatile apparatus for fluorescenc ...19957492321
semiautomated quantification of cytotoxic damage induced in cultured insect cells exposed to commercial bacillus thuringiensis biopesticides.a convenient in vitro bioassay based on semiautomated quantification of live-cell reduction of tetrazolium dyes--3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt) and 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2h-tetrazolium-5-corb oxanilide sodium salt (xtt)--to formazan was developed and used to evaluate cytotoxic effects of two commercial insecticides (bt) derived from bacillus thuringiensis subsp. kurstaki (btk). comparison of two target insect cell lines mg1 (trichoplusia ...19958666719
induction of a metabolic switch in insect cells by substrate-limited fed batch cultures.insect cell metabolism was studied in substrate-limited fed batch cultures of spodoptera frugiperda (sf-9) cells. results from a glucose-limited culture, a glutamine-limited culture, a culture limited in both glucose and glutamine, and a batch culture were compared. a stringent relation between glucose excess and alanine formation was found. in contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. simultaneous glucose and g ...19958590651
intrinsic glycosylation potentials of insect cell cultures and insect larvae.the glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines and insect larvae were compared. the spodoptera frugiperda (sf21) and trichoplusia ni (tn-368 and bti-tn-5b1-4) cell lines possessed several intrinsic glycoproteins that are modified with both n- and o-linked oligosaccharides. the n-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing sialic acid) types. similarly, the t. ni l ...19958564076
secretion of biologically active human proapolipoprotein a-i in a baculovirus-insect cell system: protection from degradation by protease inhibitors.studies of the structure and function of apolipoprotein a-i (apoa-i) often require its purification by delipidation of high density lipoprotein isolated from large quantities of human plasma and separation of apoa-i from other plasma apolipoproteins. to reduce the need for extensive purification procedures, we have developed an insect cell/baculovirus expression system for the production and secretion of human proapoa-i. the recombinant baculovirus containing full-length human apoa-i cdna, when ...19958656073
immunogenic properties of rabbit haemorrhagic disease virus structural protein vp60 expressed by a recombinant baculovirus: an efficient vaccine.we have constructed a recombinant baculovirus containing the gene encoding the structural protein vp60 from the spanish field isolate ast/89 of rabbit haemorrhagic disease virus (rhdv). infection of cultured spodoptera frugiperda sf9 cells with this recombinant virus resulted in the production of high yields of vp60 protein which did not seem to assemble to form virus like particles, but was antigenically similar to the corresponding viral protein obtained from purified virions. a vp60-dose stud ...19958837879
the p1a alloantigen system is a sensitive indicator of the structural integrity of the amino-terminal domain of the human integrin beta 3 subunit.within the native integrin alpha pi b beta 3, the conformation of the amino-terminal domain of the beta 3 subunit has a significant influence on the availability of the leu33/pro33 polymorphism that defines the a1 and a2 alleles, respectively, of the p1a alloantigen system. the majority of anti-p1a1 igg antibodies, affinity-purified by adsorption to either (a1/a1)-platelets or purified alpha pi b(a1) beta 3, fail to bind to the leu33 polymorphic loop within cys26-cys38 in native beta 3 unless th ...19958846042
the human immunodeficiency virus type 1 nef protein functions as a protein kinase c substrate in vitro.the human immunodeficiency virus type 1 nef protein was expressed in escherichia coli as a c-terminal fusion with glutathione s-transferase (gst). the ability of gst-nef to act as a substrate for cellular kinases in vitro was examined by incubation of purified gst-nef fusion proteins, immobilized on glutathione-agarose beads, with cytoplasmic extracts from a number of human cell lines. in the presence of [gamma32p]atp, phosphorylation of nef occurred predominantly on serine residues. studies wit ...19959049329
maturation of the dengue-2 virus ns1 protein in insect cells: effects of downstream ns2a sequences on baculovirus-expressed gene constructs.a series of recombinant baculoviruses was constructed in order to study the influence of downstream ns2a sequences on the processing of the dengue virus ns1 glycoprotein in insect cells. ns1 alone was expressed at a high level in its native dimeric form and processed efficiently through the spodoptera frugiperda (sf) cell secretory pathway. recombinant ns1 was found associated with the plasma membrane and was also secreted into the extracellular medium. although both intra- and extracellular ns1 ...19959049346
the production of a stably transformed insect cell line expressing an invertebrate gabaa receptor beta-subunit.we have produced a stable insect cell line derived from spodoptera frugiperda (sf9) cells expressing a cdna encoding a beta-subunit of the lymnaea stagnalis gabaa receptor. the cdna was randomly integrated into the insect cell genome under the control of a baculovirus immediate early gene (ie-1) promoter. stable cell lines were established by transformation of sf9 cells with the expression vector piek1. lgbeta1 together with a plasmid encoding a selectable marker which confers neomycin (g418) re ...19958903929
characterization of normal and point-mutated human androgen receptors expressed in the baculovirus system.the baculovirus system is able to generate large amounts of a protein, permitting detailed analysis of structure-function relations. we have used this system to overexpress and characterize normal human androgen receptors (har) and mutant hars from humans with complete or partial androgen insensitivity. maximum specific binding of [3h]mibolerone (mb) in recombinant baculovirus-infected spodoptera frugiperda (sf9) cells varied from 15 to 40 pmol/mg protein, about 1000-fold higher than in genital ...19958800637
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, ...19959636281
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 ...19959636298
rat monoclonal antibodies differentiating between the epstein-barr virus nuclear antigens 2a (ebna2a) and 2b (ebna2b).rat monoclonal antibodies were produced against the c-terminus of epstein-barr virus nuclear antigens 2a (ebna2a) and 2b (ebna2b) expressed as bacterial trpe fusion proteins. the initial screening was performed using a soluble bacterial extract containing the fusion proteins. positive hybridomas were confirmed by immunofluorescence on sf158 (spodoptera frugiperda) insect cells infected with recombinant baculovirus (autographa californica nuclear polyhedrosis virus) and expressing the complete eb ...199511831716
the effect of oscillating dissolved oxygen concentrations on the metabolism of a spodoptera frugiperda iplb-sf21-ae clonal isolate.the effect of oscillating dissolved oxygen (do) concentration on the metabolism of a clonal isolate of the spodoptera frugiperda iplb-sf21-ae insect cell line was investigated. specifically, the effect on cell growth, re- combinant protein synthesis, glucose and glutamine consumption, and lactate accumulation was determined. prior to conducting the oscillating do experiments, it was found that the do concentration could be reduced to 15% air saturation without adversely affecting the growth rate ...199518623445
on-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures.respiration rates in spodoptera frugiperda (sf-9) cell bioreactor cultures were successfully measured on-line using two methods: the o(2) uptake rate (our) was determined using gas phase po(2) values imposed by a dissolved oxygen controller and the co(2) evolution rate (cer) was measured using an infrared detector. the measurement methods were accurate, reliable, and relatively inexpensive. the cer was routinely determined in bioreactor cultures used for the production of several recombinant pro ...199618626897
ectoparasitic acugutturid nematodes of adult lepidoptera.noctuidonema guyaneme is an interesting ectoparasite of adult lepidoptera that feeds on hosts from at least five families with its long stylet. noctuidonema guyanense spends its entire life on the adult moth and is sustained as it is passed from moth to moth during host mating. overlapping host generations are essential for parasite survival. this nematode occurs throughout tropical and subtropical america and is transported by at least one of its hosts, spodoptera frugiperda, during migration t ...199619277339
processing in vitro of pronapin, the 2s storage-protein precursor of brassica napus produced in a baculovirus expression system.the maturation of the 2s albumin, napin, in brassica napus l. involves removal of an amino-terminal and an internal propeptide. pulse-chase experiments with b. napus embryos showed that intermediates are detectable during the pronapin processing. intact pronapin was expressed by baculovirus in spodoptera frugiperda insect cells in order to obtain substrate for studying the processing event. processing of pronapin with a crude b. napus embryo protein extract resulted in several fragments of simil ...19969004547
cfmnpv blocks acmnpv-induced apoptosis in a continuous midgut cell line.morphological and molecular changes produced by autographa californica nuclear polyhedrosis virus (acmnpv) infection in a permissive cell line, iplb-sf-21ae (sf-21), of spodoptera frugiperda and a nonpermissive cell line, fpmi-cf-203 (cf-203), of choristoneura fumiferana are described. cf-203 cells inoculated with acmnpv showed a dna ladder and morphological changes such as plasma membrane granulation, blebbing, and nuclear fragmentation, which are characteristic of apoptosis. typical virus repl ...19968806500
the mode of action of hirsutellin a on eukaryotic cells.a 16-kda protein toxin was purified from hirsutella thompsonii var thompsonii and named hirsutellin a (hta). at 0.5 and 5.0 microm concentrations, hta caused detectable cytopathic effects on spodoptera frugiperda cells (sf-9) within 2-4 hr and completely inhibited sf-9 cell growth at 4 days posttreatment. electron microscope data showed that the hta treated sf-9 cells became hypotrophied and internal organelles and cell membranes were disrupted. at the same concentration, hta effectively inhibit ...19968812603
purification of recombinant insect transferrin from large volumes of cell culture medium using high capacity ni(2+)-dipicolylamine gel.we report the purification of secreted recombinant manduca sexta transferrin from spodoptera frugiperda (sf9) cell culture medium in a single step using high capacity ni(2+)-dipicolylamine (dpa)-novarose gel. although the original sample was highly diluted (approximately 10 micrograms transferrin/ml medium) and the cell culture medium contained 10% surfactant (pluronic f68) and a lipid emulsion, we were able to recover the recombinant transferrin (1 mg protein/100 ml) under gentle elution condit ...19968812846
purification of a soluble hepatitis e open reading frame 2-derived protein with unique antigenic properties.the second open reading frame (orf2) of hepatitis e virus (hev) is predicted to encode a 73-kda capsid protein (1). when full-length orf2 was expressed in insect cells (spodoptera frugiperda (sf9)) using a recombinant baculovirus, two distinct hev polypeptides were observed: a full-length insoluble 73-kda protein, and a soluble 56.5-kda protein. following purification and sequence analysis, it was determined that the 56.5-kda protein was derived from endoproteolytic cleavage site that was betwee ...19968812876
secretion of green fluorescent protein from recombinant baculovirus-infected insect cells.trichoplusia ni (high five) and spodoptera frugiperda (sf21) cells were engineered for expression of epitope (flag)-tagged signal peptide-green fluorescent protein (gfp) fusions to examine the suitability of gfp as a secretory marker. the recombinant baculovirus-infected cells became fluorescent, and the high five cells but not sf21 cells secreted gfp in the culture medium as detected by the presence in the culture supernatant of a flag-immunoreactive 30-kda species and the characteristic 510-nm ...19968831686
transposable elements and gene transformation in non-drosophilid insects.this review summarizes recent data on the development of non-drosophilid insect transformation systems. the discussion focuses on one particular approach to developing transformation systems that relies on the use of short inverted repeat-type transposable elements analogous to that employed for drosophila melanogaster transformation. representatives from four families of short inverted repeat-type transposable elements have been shown to either act as non-drosophilid gene vectors or to have the ...19969014324
reconstitution of atp-dependent leukotriene c4 transport by co-expression of both half-molecules of human multidrug resistance protein in insect cells.multidrug resistance protein (mrp) confers a multidrug resistance phenotype similar to that associated with overexpression of p-glycoprotein. unlike p-glycoprotein, mrp has also been shown to be a primary active atp-dependent transporter of conjugated organic anions. the mechanism(s) by which mrp transports these compounds and increases resistance to natural product drugs is unknown. to facilitate studies on the structure and function of mrp, we have determined whether a baculovirus expression s ...19968910374
a baculovirus vector derived from immediately early gene promoter of autographa californica nuclear polyhedrosis virus.a transfer vector was constructed in which the neomycin resistance (neo) gene was under the control of a copy of autographa californica nuclear polyhedrosis virus (acmnpv) ie1 gene promoter at the p10 locus. after cotransfection of spodoptera frugiperda (sf9) cells with the transfer vector and infectious acmnpv dna, the recombinant virus-containing neo gene was selected by serial passage of the mixed progenies from cotransfection. this was done at low moi in the presence of g418 in growth medium ...19968910650
expression and loading of recombinant heavy and light chain homopolymers of rat liver ferritin.the full-length genes for the heavy (h) and light (l) chains of ferritin isolated from a rat liver cdna library were amplified using polymerase chain reaction. each was inserted at the unique bglii site downstream of the p10 promoter of the baculovirus transfer vector pacuw21. the genes were transferred separately to infectious autographa californica nuclear polyhedrosis virus (acnpv) expression vectors after in vivo homologous recombination. ferritin homopolymers of either h or l chain were exp ...19968914851
interactions of bacillus thuringiensis crystal proteins with the midgut epithelial cells of spodoptera frugiperda (lepidoptera: noctuidae).binding of different bacillus thuringiensis insecticidal crystal proteins (icps) to the midgut epithelium of spodoptera frugiperda larvae was characterized by binding experiments with midgut tissue sections and isolated brush border membrane vesicles. our results show that icps interact with the microvilli of epithelial cells of s. frugiperda in two different ways. the first is typical of highly toxic proteins (like cryic and cryid); this interaction is saturable and specific. in contrast, some ...19968931361
high-level expression of recombinant immunoreactive thyroid peroxidase in the high five insect cell line.expression of a major thyroid autoantigen, thyroid peroxidase (tpo) was studied using the baculovirus-insect cell expression system. human tpo cdna modified so as to code for the extracellular fragment of the protein was placed under the control of the strong polyhedrin promoter in baculovirus transfer vector pbluebaciii and cotransfected with linearized acmnpv viral dna. expression in two insect cell lines spodoptera frugiperda (sf9) and tricoplusia ni (high five) was investigated and levels of ...19968938592
the synaptic protein unc-18 is phosphorylated by protein kinase c.the c. elegans unc-18 encoded protein unc-18 is implicated in the interactions between synaptic vesicles and presynaptic plasma membrane. to further characterize the neural protein, we investigated the phosphorylation in vitro of the protein expressed in spodoptera frugiperda sf21 cells. the unc-18 protein is selectively phosphorylated by protein kinase c (pkc) but not by casein kinase ii and cyclic amp-dependent protein kinase. the presumed phosphorylation sites determined by manual edman degra ...19968939464
expression, purification and characterization of recombinant c. elegans unc-18.the caenorhabditis elegans unc-18-encoded protein (unc-18) is implicated in the processes of vesicle targeting, docking, and/or fusion. to further characterize the properties of this important neural protein, we expressed it at a high level in spodoptera frugiperda sf21 cells using a baculovirus expressing system. a cdna containing the coding sequence for unc-18 was inserted into the transfer vector pbluebac to yield the recombinant virus pacnpv/unc-18. at maximal expression, the recombinant vir ...19968939465
rat placental lactogen-i variant (rpl-iv), product of an unique gene, is biologically different from rpl-i.the rat placenta expresses two rat placental lactogen-i (rpl-i) proteins: the normal rpl-i in the first half of pregnancy and a variant (rpl-iv) in the second half of pregnancy. they are 70% identical at the amino acid level but arise from different cell types: rpl-i from giant cells and rpl-iv from cytotrophoblasts. to assess whether rpl-iv originates from alternative splicing of the rpl-i gene or is the product of a separate gene, genomic clones of rpl-i and rpl-iv were isolated from a lambda ...19968940341
glycosylation, palmitoylation, and localization of the human d2s receptor in baculovirus-infected insect cells.in order to evaluate the baculovirus expression system as a means for high-yield production of homogeneous d2s receptor, we have expressed various d2s receptor constructs in two spodoptera frugiperda cell lines, a trichoplusia ni and a mammestra brassicae cell line. to improve expression yield, the environment of the polyhedrin gene translational initiation site was retained by fusing the first 12 codons of the polyhedrin gene to the 5'-end of the d2s receptor coding sequence. the pharmacologica ...19968952462
expression and characterization of a truncated, soluble, low-density lipoprotein receptor.the low-density lipoprotein (ldl) receptor mediates the clearance of apolipoprotein b- and e-containing lipoproteins from the bloodstream. in the current study, we characterized the binding properties of the amino terminus of the ldl receptor. we produced a recombinant baculovirus that encoded the first 354 amino acids, including the endogenous signal sequence, of the human ldl receptor. this truncated receptor protein (ldl-r354) was secreted from recombinant baculovirus-infected spodoptera frug ...19968954898
interleukin-1 receptor antagonist: characterisation of its gene expression in rabbit tissues and large-scale expression in eucaryotic cells using a baculovirus expression system.the gene expression of rabbit interleukin-1 receptor antagonist (rbil-lra) was examined in rabbit tissues. rna was isolated from heart, lung, kidney, muscle, liver, spleen, brain, and peripheral blood monocytes (pbms), and rbil-lra mrna was identified as a single species by northern analysis using a rbil-lra probe. rbil-lra was abundantly expressed in lung, brain, heart, and liver, expressed at low levels in spleen, and undetectable in kidney and unstimulated pbms. expression of large scale reco ...19968960095
selective inhibition of cyclooxygenase-1 and -2 using intact insect cell assays.we have utilized the baculovirus expression system to develop an in vitro intact cell assay for screening nonsteroidal anti-inflammatory drug (nsaid) inhibition of the two isozymes of human cyclooxygenase (prostaglandin endoperoxidase synthase, ec 1.14.99.1). infected spodoptera frugiperda (sf9) cells expressing either human cyclooxygenase-1 (hcox-1) or human cyclooxygenase-2 (hcox-2) were harvested 24 hr postinfection, a time point where all cells are viable and hcox-1 or hcox-2 are correctly p ...19968986141
synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the e1 protein.the two envelope glycoproteins of rubella virus (rv), e1 of 58 kda and e2 of 42-47 kda, were individually expressed in lepidopteran spodoptera frugiperda as well as in trichoplusia ni insect cells using baculovirus vectors. the authentic signal sequences of e1 and e2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. in addition, the hydrophobic transmembrane anchors at the carboxyl termini of e1 and e2 proteins we ...19968987625
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 ...19969240741
baculovirus-mediated high level expression of a human thiopurine methyl transferase.we have expressed the human thiopurine methyltransferase cdna in a baculovirus vector in sf21 (spodoptera frugiperda) cells. this system expresses the enzyme at levels such that the thiopurine methyltransferase enzyme may be readily visualised by coomassie blue stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis. the expressed enzyme catalysed the methylation of 6-mercaptopurine with an apparent km of 892 microm, similar to that observed in human liver cytosol ie. 657 microm howev ...19968850531
juvenile and mature map2 isoforms induce distinct patterns of process outgrowth.microtubule-associated protein-2 (map2) is the most abundant map in neurons, where its distribution is restricted to the somatodendritic compartment. this molecule undergoes developmentally regulated alternative splicing, resulting in at least two isoforms, a juvenile isoform (termed map2c) and a mature isoform (map2), with greatly different molecular masses. spodoptera frugiperda (sf9) cell expression of the juvenile versus the mature map2 isoform generates two distinct patterns of process outg ...19968868472
purification, biochemistry and molecular cloning of an insect glycosylasparaginase from spodoptera frugiperda.glycosylasparaginase (ec 3.5.1.26) from sf9 cells (spodoptera frugiperda) was purified to homogeneity with a specific activity of 2.1 unit/mg. the enzyme is composed of two non-identical alpha/beta subunits joined by strong non-covalent forces and has one glycosylation site located in the alpha subunit. molecular masses of the subunits were determined to be 28 kda and 17 kda by sds-page. native enzyme existed in quaternary structures of either heterodimer (alpha beta) or heterotetramer (alpha 2 ...19968877373
dengue virus envelope glycoprotein can be secreted from insect cells as a fusion with the maltose-binding protein.the maltose-binding protein (male) contains a signal sequence which allows its translocation in the periplasm of prokaryotic microorganisms. in this study, male was produced in spodoptera frugiperda (sf9) lepidopterian cells using the baculovirus expression system. the secretion of male, following cleavage of its signal sequence, to the supernatant fluid of recombinant baculovirus-infected sf9 cells and its affinity for maltodextrin polymers allowed recovery of significant amounts (> or = 10 mic ...19968882648
expression and purification of the seven nonstructural proteins of the flavivirus kunjin in the e. coli and the baculovirus expression systems.all seven nonstructural (ns) proteins of the flavivirus kunjin (kun) ranging from ns1 to ns5 were expressed either alone or as fusion proteins with glutathione-s-transferase (gst). high level expression of recombinant proteins was achieved in spodoptera frugiperda (sf9) cells using the baculovirus expression system in contrast to the low level of expression in e. coli. the order of the level of expression of the recombinant fusion proteins per 4 x 10(7) sf9 cells was: gst-ns5 (yields approximate ...19968882936
polycistronic (tri- or bicistronic) phytoreoviral segments translatable in both plant and insect cells.genomic segment s12 of rice dwarf virus and segment s9 of wound tumor virus, both members of the genus phytoreovirus, have small out-of-phase overlapping open reading frames (orfs). western blot (immunoblot) analysis revealed that rice dwarf virus s12 mrna specified translation products from the large orf and two overlapping small orfs both in rice plant hosts and in spodoptera frugiperda insect cells. these results provide the first example of a tricistronic mrna for a segmented double-stranded ...19968892945
molecular and immunological characterization of soluble aggregated a/victoria/3/75 (h3n2) influenza haemagglutinin expressed in insect cells.a/victoria/3/75 (h3n2)-derived cdna coding for a secreted haemagglutinin (ha0s) was cloned into the polyhedrin promoter-based pvl1392 transfer vector, and a recombinant baculovirus was isolated. 5 to 10 micrograms/ml of secreted ha were obtained following infection of spodoptera frugiperda-9 cells. gel filtration revealed the presence in the cell supernatant of immunoreactive ha molecules with varying m(r). the high m(r) fraction (aha0s) could be purified by matrex cellufine sulphate and lentil- ...19968893793
expression and coupling of human cytochrome p450 2e1 and nadph-cytochrome p450 oxidoreductase in dual expression and co-infection systems with baculovirus in insect cells.in order to study the interaction between human cytochrome p450 2e1 (h2e1) and nadph-p450 oxidoreductase (hor) in a native membrane environment, we used two approaches to express both h2e1 and hor in a baculovirus expression system. for a dual-expression system, h2e1 and hor were coexpressed in spodoptera frugiperda (sf9) insect cells using a recombinant baculovirus carrying both h2e1 and hor cdnas (v-h2e1-hor). the h2e1 cdna was expressed under the control of the polyhedrin promoter p(polh), wh ...19968900414
metabolic engineering of animal cells.substrate-limited fed-batch cultures were used to study growth and overflow metabolism in hybridoma and insect cells. in hybridoma cells a glucose-limited fed-batch culture decreased lactate formation but increased glutamine consumption and ammonium formation. glutamine limitation decreased ammonium and alanine formation but did not enhance glucose consumption. instead lactate formation was reduced, indicating that glucose was used more efficiently. the formation of lactate, alanine, and ammoniu ...19968659912
purification, cloning, and bacterial expression of retinol dehydratase from spodoptera frugiperda.anhydroretinol and 14-hydroxy-4,14-retro-retinol, retro-retinoids endogenous to both mammals and insects, act as agonist and antagonist, respectively, in controlling proliferation in lymphoblasts and other retinol-dependent cells. we describe here the identification, purification, cloning, and bacterial expression of the enzyme retinol dehydratase, which converts retinol to anhydroretinol in spodoptera frugiperda. retinol dehydratase has nanomolar affinity for its substrate and is, therefore, th ...19968663216
human cathepsin o2, a matrix protein-degrading cysteine protease expressed in osteoclasts. functional expression of human cathepsin o2 in spodoptera frugiperda and characterization of the enzyme.cathepsin o2, a human cysteine protease predominantly present in osteoclasts, has been functionally expressed in spodoptera frugiperda sf9 cells using the autographa californica nuclear polyhedrosis virus. following in vitro activation at ph 4.0 with pepsin, active enzyme with an apparent molecular weight of 29,000 was obtained. n-terminal sequencing revealed the typical processing site for cysteine proteases of the papain family with a proline in the position adjacent to the n-terminal alanine ...19968567669
mosquitocidal activity of the cryic delta-endotoxin from bacillus thuringiensis subsp. aizawai.the cloned 135-kda cryic delta-endotoxin from bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against spodoptera litoralis and spodoptera frugiperda larvae and in vitro against the s. frugiperda sf9 cell line. here, we report that the cryic delta-endotoxin cloned from b. thuringienesis subsp. aizawai hd-229 and expressed in an acrystalliferous b. thuringiensis strain is also toxic to aedes aegypti, anophles gambiae, and culex quinquefasciatus mosquito larv ...19968593070
evidence for a proprotein intermediate during maturation of type ii antifreeze protein in sea raven, hemitripterus americanus.the circulating type ii antifreeze protein (afp) in sea raven is 129 amino acids (aa) long (14 kda) and is derived from an initial 163 aa translation product that is synthesised in the liver. signal peptide cleavage algorithms, as well as transgenic expression studies in fall armyworm cells, predict the formation of a 146 aa (16 kda) proprotein intermediate. a protein of this size that cross-reacted with anti-sea raven afp antibody was detected in sea raven serum using phosphate/urea sds-page, a ...19968597578
human beta2-glycoprotein i as an anticardiolipin cofactor determined using mutants expressed by a baculovirus system.beta2-glycoprotein i (beta2-gpi) consists of five repeats of a homologous domain. we designed a series of human beta2-gpi mutant genes, ie, three mutant genes lacking the domain(s) present in the nh2-terminal region and two of those present in the cooh-terminal region. these mutant genes were expressed in spodoptera frugiperda insect cells (sf9) infected with recombinant baculoviruses and the mutant proteins were secreted into the culture medium. the molecular mass of the purified mutant protein ...19968605342
actin binding and proteolysis by the baculovirus acmnpv: the role of virion-associated v-cath.infection of larvae by autographa californica m nuclear polyhedrosis virus (acmnpv) results in liquefaction of susceptible hosts, presumably due to the breakdown of cells and extracellular matrices. in spodoptera frugiperda tissue culture cells, infection leads to dramatic rearrangement and eventual destruction of the actin cytoskeleton. the first of these rearrangements is the formation of actin cables in the cytoplasm of the cell. cable formation requires release of the budded virus (bv) nucle ...19968607267
wild type and mutant human heart (r)-3-hydroxybutyrate dehydrogenase expressed in insect cells.(r)-3-hydroxybutyrate dehydrogenase (bdh) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (pc) for function. pc is an allosteric activator that enhances nad(h) binding to bdh. the enzyme serves as a paradigm to study specific lipid-protein interactions in membranes. analysis of the primary sequence of bdh, as determined by molecular cloning, predicts that lipid binding and substrate specificity are contributed by the c-terminal third of the protein [m ...19968679568
human 25-hydroxyvitamin d3-24-hydroxylase, a multicatalytic enzyme.human 25-hydroxyvitamin d-24-hydroxylase has been expressed in spodoptera frugiperda (sf21) insect cells using the previously cloned cdna in baculovirus (acnpv-p450cc24). the activity of recombinant h-p450cc24 required adrenodoxin, adrenodoxin reductase, and nadph. incubation of this reconstituted system with 25-oh-[26,27-(3)h]d3 substrate produced several metabolites that were resolved on a normal-phase cyano hplc system. these products exactly comigrated with authentic standards for 24-oxo-25- ...19968679605
isoform-specific intracellular vesicle formation by recombinant annexin xi-a in sf9 cells.annexins are a group of structurally related proteins that bind phospholipids in a ca2(+)-dependent manner and have the ability to self-aggregate and to promote vesicle aggregation and membrane fusion. two isoforms of annexin xi, termed xi-a and xi-b, were previously identified by screening a bovine chondrocyte cdna library. but little is known about differences in their biological function. in the present study, we therefore examined the results of expression of the two proteins in spodoptera f ...19968687461
soluble adenylyl cyclase from spodoptera frugiperda (sf9) cells. purification and biochemical characterization.an insect ovarian cell, spodoptera frugiperda (sf9), has been widely used to express recombinant proteins, including adenylyl cyclase, as a host cell in the baculovirus expression system. we report the presence and characterization of a soluble adenylyl cyclase (sac) distinct from a membrane-bound form of adenylyl cyclase (mac) that is also present in sf9 cells. sac was purified 3,500-fold to near homogeneity; a single band at 25 kda on sds-polyacrylamide gel electrophoresis correlated well with ...19968702736
a molecular basis for affinity modulation of fab ligand binding to integrin alphaiib beta3.the arg-gly-asp (rgd) sequence within the third complementarity-determining region (cdr3) of the heavy chain (h3) is responsible for the binding of the recombinant murine fab molecules, ap7 and pac1.1, to the platelet integrin alphaiibbeta3. ap7 binding is minimally influenced by the conformational state of this receptor, whereas pac1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. to study the molecular basis for this functional difference, we replace ...19968702765
the ribosome-inactivating protein restrictocin deters insect feeding on aspergillus restrictus.the fungus-feeding beetle, carpophilus freemani, consumed equal quantities of young mycelia, fewer phialides bearing mature spores and much fewer phialides bearing developing spores of aspergillus restrictus compared to those of aspergillus nidulans when tested in diet choice assays. the degree to which specific fungal structures were consumed was inversely related to the localization of high levels of restrictocin, a ribosome-inactivating protein, to those structures. pure restrictocin added to ...19968704996
abortive infection of the baculovirus autographa californica nuclear polyhedrosis virus in sf-9 cells after mutation of the putative dna helicase gene.homologous recombination between the autographa californica nuclear polyhedrosis virus (acnpv) genome and a 0.6-kbp-long dna fragment derived from the putative dna helicase gene of bombyx mori nuclear polyhedrosis virus generates eh2-acnpv, an expanded-host-range acnpv mutant (s. maeda, s.g. kamita, and a. kondo, j. virol. 67:6234-6238, 1993). after inoculation at a high multiplicity of infection (moi), eh2-acnpv replicates efficiently in both the sf-9 (acnpv-permissive) and bmn (non-acnpv-permi ...19968709251
glycosylation in lepidopteran insect cells: identification of a beta 1-->4-n-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains.the choice for a heterologous expression system to produce glycoprotein therapeutics highly depends on its potential to perform mammalian-like posttranslational modifications such as glycosylation. to gain more insight into the glycosylation potential of the baculovirus mediated insect cell expression system, we have studied the expression of glycosyltransferases involved in complex-type n-glycosylation. lepidopteran insect cell lines derived from trichoplusia ni, spodoptera frugiperda, and mame ...19968727788
elongation of the n-glycans of fowl plague virus hemagglutinin expressed in spodoptera frugiperda (sf9) cells by coexpression of human beta 1,2-n-acetylglucosaminyltransferase i.spodoptera frugiperda (sf9)-cells differ markedly in their protein glycosylation capacities from vertebrate cells in that they are not able to generate complex type oligosaccharide side chains. in order to improve the oligosaccharide processing properties of these cells we have used baculovirus vectors for expression of human (beta 1,2-n-acetylglucosaminyltransferase i (hgnt-i), the enzyme catalysing the crucial step in the pathway leading to complex type n-glycans in vertebrate cells. one vecto ...19968727789
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