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Results for “Insect cell lines”

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PubMed1977

Effect of polyene macrolide antibiotics on invertebrate tissue culture cells.

The effect of amphotericin B (AB) and amphotericin B methyl ester (AME) on viability and DNA synthesis in three insect cell lines, Trichoplusia ni (Tn) Carpocapsa pomonella 169 (Cp) and Aedes aegypti (Aa), has been evaluated. In all cases AME was less toxic than AB and inhibited DNA synthesis to a lesser degree than AB. However, the three cell lines differed in their response to the two polyene macrolide antibiotic preparations. Tn and Aa cell responded in a similar manner when exposed to either AME or AB, whereas Cp cells were more sensitive to both antibiotics than Tn or Aa cells.

Aedes↗

PubMed1999

Cytochrome P-450-mediated metabolism of the individual enantiomers of the antidepressant agent reboxetine in human liver microsomes.

In vitro studies were conducted to identify the hepatic cytochrome P-450 (CYP) enzymes responsible for the oxidative metabolism of the individual enantiomers of reboxetine. In human liver microsomes, each reboxetine enantiomer was metabolized to one primary metabolite, O-desethylreboxetine, and three minor metabolites, two arising via oxidation of the ethoxy aromatic ring and a third yet unidentified metabolite. Over a concentration range of 2 to 200 microM, the rate O-desethylreboxetine formation for either enantiomer conformed to monophasic Michaelis-Menten kinetics. Evidence for a principal role of CYP3A in the formation of O-desethylreboxetine for (S, S)-reboxetine and (R,R)-reboxetine was based on the results from the following studies: 1) inhibition of CYP3A activity by ketoconazole markedly decreased the formation of O-desethylreboxetine, whereas inhibitors selective for other CYP enzymes did not inhibit reboxetine metabolism, 2) formation of O-desethylreboxetine correlated (r(2) = 0.99; p <.001) with CYP3A-selective testosterone 6-beta-hydroxylase activity across a population of human livers (n = 14). Consistent with inhibition and correlation data, O-desethylreboxetine formation was only detectable in incubations using microsomes prepared from a Baculovirus-insect cell line expressing CYP3A4. Furthermore, the apparent K(M) for the O-desethylation of reboxetine in cDNA CYP3A4 microsomes was similar to the affinity constants determined in human liver microsomes. In addition, (S,S)-reboxetine and (R,R)-reboxetine were found to be competitive inhibitors of CYP2D6 and CYP3A4 (K(i) = 2.5 and 11 microM, respectively). Based on the results of the study, it is concluded that the metabolism of both reboxetine enantiomers in humans is principally mediated via CYP3A.

Antidepressive Agents↗

PubMed2000

Oxidation of the novel oxazolidinone antibiotic linezolid in human liver microsomes.

In vitro studies were conducted to identify the hepatic enzyme(s) responsible for the oxidative metabolism of linezolid. In human liver microsomes, linezolid was oxidized to a single metabolite, hydroxylinezolid (M1). Formation of M1 was determined to be dependent upon microsomal protein and NADPH. Over a concentration range of 2 to 700 microM, the rate of M1 formation conformed to first-order (nonsaturable) kinetics. Application of conventional in vitro techniques were unable to identify the molecular origin of M1 based on the following experiments: a) inhibitor/substrates for various cytochrome P-450 (CYP) enzymes were unable to inhibit M1 formation; b) formation of M1 did not correlate (r(2) < 0.23) with any of the measured catalytic activities across a population of human livers (n = 14); c) M1 formation was not detectable in incubations using microsomes prepared from a baculovirus insect cell line expressing CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 4A11. In addition, results obtained from an in vitro P-450 inhibition screen revealed that linezolid was devoid of any inhibitory activity toward the following CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4). Additional in vitro studies excluded the possibility of flavin-containing monooxygenase and monoamine oxidase as potential enzymes responsible for metabolite formation. However, metabolite formation was found to be optimal under basic (pH 9.0) conditions, which suggests the potential involvement of either an uncharacterized P-450 enzyme or an alternative microsomal mediated oxidative pathway.

Acetamides↗

PubMed2002

O-glycosylation of the nuclear forms of Pax-6 products in quail neuroretina cells.

Many transcription factors are demonstrated as being glycosylated with O-N-acetylglucosamine (GlcNAc) residue in transfected insect cell lines, but rarely in the original cells. For the first time, we demonstrate the O-GlcNAc modification of the p48/p46 Pax-6 gene (a developmental control gene involved in the eye morphogenesis) products in the quail neuroretina (QNR). In conjunction with a systematic PNGase F treatment, we used wheat germ agglutinin (WGA) binding, in vitro labeling with bovine galactosyltransferase, and labeling of cultured QNR with [14C]GlcNH2. Glycosylated forms of Pax-6 proteins were found in the nucleus of the neuroretina cells. WGA-selected Pax-6 proteins produced in the reticulocyte lysate were able to bind a DNA target, as well as to the unglycosylated form. The O-GlcNAc may, however, modulate protein interactions, mainly with other factors involved in the transcription process. Characterization of products released after reductive alkaline treatment of the proteins clearly demonstrates that N-acetylglucosamine is directly linked to serine or threonine residues. Examination of Pax-6 primary sequence allowed us to determine potential O-GlcNAc attachment sites. Most of these expected glycosylation sites appear to be located on the two DNA binding domains and on the carboxyterminal transactivation domain, while experimental evidence taken from WGA-selected proteins experiment points in favor of a main localization on the paired-box domain.

Acetylglucosamine↗

PubMed2001

[Production of HPV 16 major capsid protein L1 with baculovirus expression system].

BACKGROUND: To obtain human papillomavirus type 16(HPV 16) major capsid protein L1 with baculovirus expression system. METHODS: Using pFb1 as a vector,a recombinant baculovirus expressing plasmid,which contains HPV16L1 gene sequence,was generated. The constructed virus was infected into an insect cell line Sf9. RESULTS: After incubating at 27 degrees for 72 hours, the infected cells were collected and total cellular poteins were extracted. SDS-PAGE assay revealed a roughly 56 000 expressed protein and Western blot confirmed that the expressed protein arose from HPV16L1. CONCLUSIONS: HPV16 later protein L1 could be efficiently expressed with baculovirus expression system,and the expressed L1 protein remains to have good immunoreactivity. This study may supply a basic work for preparing virus-like particle and prophylactic HPV subunit vaccines.

Animals↗

PubMed2002

[Expression of full-length hepatitis E virus structural gene using baculovirus expression system].

OBJECTIVE: To obtain recombinant baculovirus containing full-length structural gene of HEV and to express HEV structural protein. METHODS: Full-length structural gene of HEV (5147-7126 nt) was inserted into baculovirus expression vector pAcUW51. The recombinant plasmid and Baculo Gold DNA were co-transfected into insect cell line Sf9. Single plaque was picked and amplified and expression of HEV structural protein was tested by SDS-PAGE, Western blot and immunofluorescence methods. RESULTS: SDS-PAGE analysis showed HEV structural protein was highly expressed; Western blot and immunofluorescence assay showed that the expression product could specifically react with HEV positive sera, confirming the protein possessing HEV specific antigenicity. CONCLUSIONS: HEV structural protein was successfully expressed using baculovirus expression system.

Animals↗

PubMed2003

[Cloning and expression of a novel ubiquitin fusion gene Uba256].

Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene is the only Ub-gp37 fusion gene in the genome of insect viruses. With the specific primers designed for Uba256 gene that was reported recently, the coding regions of Uba256, N-terminal ubiquitin and C-terminal GP37 that lacking the signal sequence, were amplified from SpltMNPV genomic DNA by PCR. The Uba256 coding region was expressed using the expression vector pBV220, and a band of 38 kD was detected with Western blot analysis, indicating that ubiquitin-GP37 fusion protein did not undergo post-transcriptional processing in E. coli. The ubiquitin and GP37 coding region were highly expressed, respectively, using pQE30 expression vector. Antibody to the purified ubiquitin reacted not only to the recombinant ubiquitin but also to bovine ubiquitin, and an antibody to the purified GP37 also reacted to the recombinant GP37. The results indicated that the purified ubiquitin and GP37 retained their antigenicity. Western blot analysis results of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed in this insect cell line to yield free ubiquitin and GP37 protein.

Animals↗

PubMed2001

[Gene cloning and expression of human soluble interleukin-6 receptor in insect cells].

OBJECTIVE: To clone the human soluble interleukin-6 receptor(hsIL-6R) gene and expression in insect cell line. METHODS: The hsIL-6R gene was cloned into plasmid pAcGP67B. After co-transfection of recombinant plasmid and wild type AcNPV DNA, the rAcNPV was confirmed by the end point dilution assay and dot blot. Then it was purified by plaque assay. RESULTS: SDS-PAGE showed molecular weight of the expressed product was about 47,000. The expressed recombinant protein was confirmed to be specific and capable of binding its ligand IL-6 by Western blot and ligand-receptor binding assays. CONCLUSIONS: Secretory expression of hsIL-6R gene in baculovirus expression system was indicated. The expressed product had immunological and biological activities.

Animals↗

PubMed1992

Localization of O-GlcNAc modification on the serum response transcription factor.

A unique form of nucleoplasmic and cytoplasmic protein glycosylation, O-linked GlcNAc, has previously been detected, using Gal transferase labeling techniques, on a myriad of proteins (for review see Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G. (1989a) Annu. Rev. Biochem. 58, 841-874), including many RNA polymerase II transcription factors (Jackson, S. P., and Tjian, R. (1988) Cell 55, 125-133). However, virtually nothing is known about the degree of glycosylation at individual sites, or, indeed, the actual sites of attachment of O-GlcNAc on transcription factors. In this paper we provide rigorous evidence for the occurrence and locations of O-GlcNAc on the c-fos transcription factor, serum response factor (SRF), expressed in an insect cell line. Fast atom bombardment mass spectrometry (FAB-MS) of proteolytic digests of SRF provides evidence for the presence of a single substoichiometric O-GlcNAc residue on each of four peptides isolated after sequential cyanogen bromide, tryptic, and proline specific enzyme digestion: these peptides are 306VSASVSP312, 274GTTSTIQTAP283, 313SAVSSADGTVLK324, and 374DSSTDLTQTSSSGTVTLP391. Using an array of techniques, including manual Edman degradation, aminopeptidase, and elastase digestion, together with FAB-MS, the major sites of O-GlcNAc attachment were shown to be serine residues within short tandem repeat regions. The highest level of glycosylation was found on the SSS tandem repeat of peptide (374-391) which is situated within the transcriptional activation domain of SRF. The other glycosylation sites observed in SRF are located in the region of the protein between the DNA binding domain and the transcriptional activation domain. Glycosylation of peptides (274-283) and (313-324) was found to occur on the serine in the TTST tandem repeat and on serine 316 in the SS repeat, respectively. The lowest level of glycosylation was recovered in peptide (306-312) which lacks tandem repeats. All the glycosylation sites identified in SRF are situated in a relatively short region of the primary sequence close to or within the transcriptional activation domain which is distant from the major sites of phosphorylation catalyzed by casein kinase II.

Acetylglucosamine↗

PubMed1992

The NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase is not expressed in Spodoptera frugiperda cells.

The insect cell line derived from Spodoptera frugiperda (Sf9) does not express the activities of the trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. The lack of synthetase activity was confirmed by the inability to incorporate radiolabeled formate into nucleotides. The cells express, instead, a Mg2+ and NAD-dependent bifunctional methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase with properties similar to the enzyme found in the mitochondria of transformed mammalian cells. In contrast, the enzyme in Sf9 cells is localized in the cytoplasm. Nutritional studies in defined medium with dialyzed serum demonstrated that the Sf9 cell does not required added purines or pyrimidines for growth. It is auxotrophic for cysteine and glycine; this latter requirement is probably due to the absence of mitochondrial serine hydroxymethyltransferase. Incorporation of labeled glycine and serine into DNA indicates that only serine is a source of one-carbon units. These results suggest that the mitochondria in Sf9 cells do not play a major role in folate-mediated metabolism.

Aminohydrolases↗

PubMedDate not supplied

[Expression of genes for orthopoxviral TNF-binding proteins and study resulted recombinant proteins].

Genes for TNF-binding proteins (CrmBs) of variola virus (VARV), monkeypox virus (MPXV) or cowpox (CPXV) were isolated with PCR from viral genomes and expressed within baculovirus DNAs in Sf21 insect cell line. Properties of resulted recombinant proteins were studied with physical-chemical and immunological methods. It was shown with solid phase enzyme-linked immunoassay that viral proteins inhibited hTNF binding with polyclonal hTNF-antibodies. The strongest inhibitor was VARV-CrmB, the less one was MPXV-CrmB. Biological activity of recombinant protein preparations was studied in the test of neutralization of TNF cytotoxicity for L929 murine fibroblast cells. It was shown that recombinant CrmBs neutralized cytotoxicity of hTNF, mTNF or rTNF in species-specific manner. It was shown also that effectiveness of hTNF cytotoxicity inhibition in vitro with VARV-CrmB exceeded the same effect of polyclonal hTNF-antibody. A possibility of the elaboration of new therapeutics for anti-TNF therapy on the base of CrmB-like proteins is discussed.

Animals↗

PubMed2005

[Eukaryotic expression and tumor-targeting modification of human mutated-IL-18 fusion gene].

Interleukin-18 (IL-18) is a proinflammatory cytokinin. This protein has a role in regulating immune responses and exhibits significant anti- tumor activities. Epidermal growth factor (EGF) is an important growth factor that plays a central role in the regulation of cell cycle and differentiation. It was proposed that a targeted delivery of IL-18 by generation of IL-18-EGF fusion protein might decrease adverse effects and result in enhancing cytotoxic and antitumor activities. In the present study, a fusion protein, consisting of EGFR binding domain fused to human IL18 mature peptide via a linker peptide of (Gly4ser) 3, was constructed and expressed in the insect cell line Sf9 using Bac-to-Bac baculovirus expression system. We showed that the purified recombinant fusion protein induced similar levels of IFN-gamma to that of native IL-18 protein in human PBMC in the presence of ConA. Furthermore, EGF Receptor competitive test in human epithelial cancer A431 cell line showed that EGF- IL18 fusion protein can specifically bind with EGFR by competing with native EGF protein. The results suggested that EGF-IL18 fusion protein could specifically be targeted on tumor cells. The work may provide a new insight into the cytokinin for the tumor treatment.

Animals↗

PubMed1986

Enhanced survival by photoreactivation and liquid holding following UV damage of TN-368 insect cells.

These studies demonstrate that the TN-368 lepidopteran insect cell line, which is extremely resistant to the lethal effects of ionizing radiation, is also quite resistant to 254-nm ultraviolet light. While resistance to ionizing radiation in TN-368 cells has been associated with superior DNA repair processes, previous findings have indicated no correlation between survival ability and amount of unscheduled DNA synthesis in response to ultraviolet light. The present studies were undertaken to define the TN-368 ultraviolet light survival response, the ability of the cells to repair UV-induced damage by photoreactivation, the capacity of the cells to undergo UV repair during liquid holding in the dark, and the relationship between photoreactivation and liquid-holding recovery. Survival was assayed by colony formation. 254-nm irradiations were performed using germicidal lamps and photoreactivation was accomplished using black lights. Photoreactivable sectors of UV damage at 50 and 10% survival are 0.65 and 0.68, respectively. Survival responses, both with and without photoreactivation, have a small initial shoulder followed by an exponential region, and finally the curves continue to decrease but with decreasing slope. F0, Fq, and extrapolation number for the exponential portion of the curves are 77.5 J/m2, 16.8 J/m2, and 1.7 for non-photoreactivated cells and 234 J/m2, 56.1 J/m2, and 1.7 for those exposed to photoreactivating light. In the primarily exponential survival region, the fluences required to produce equivalent levels of survival in photoreactivated cells range from approximately 10.8 to 23.3 times as great as cells receiving UV light alone. The maximum survival enhancement of cells maintained under liquid-holding conditions over cells plated immediately following 100-400 J/m2 irradiations appears to be about 2-fold and occurs at 3-6 h of holding. Photoreactivation alone has a greater enhancement of survival than when photoreactivation follows liquid holding, but when liquid holding follows photoreactivation, the enhancement surpasses that of photoreactivation alone.

Animals↗

PubMed1994

Purification of recombinant 38-kDa phosphorylated protein of Marek's disease virus from insect cells through an affinity column.

The 38-kDa phosphorylated protein (pp38) of Marek's disease virus (MDV) expressed in insect cell line Sf9 cells infected with recombinant baculovirus BP38II was purified through an affinity column made of CNBr-Sepharose 4B linked with monoclonal antibody (Mab) H19 specific to serotype I MDV. The result of SDS-PAGE showed a main band of 38 kDa in the lane loaded with the purified pp38, and this band was specifically recognized by MAb H19 in Western blot. The serum from mice immunized with the purified recombinant pp38 reacted only to Sf9 cells infected with the recombinant baculovirus BP38II but not with wild baculovirus. It also gave a titer of 1:128 in indirect fluorescence antibody test to MDV-infected chick embryo fibroblast cells. These results indicated that the purification procedure was effective and that it would be useful for investigating the biological functions of MDV pp38.

Animals↗

PubMed1993

Expression of rat liver phenylalanine hydroxylase in insect cells and site-directed mutagenesis of putative non-heme iron-binding sites.

Rat liver phenylalanine hydroxylase was expressed in both Escherichia coli and the Spodoptera frugiperda insect cell line, Sf9. Recombinant enzyme from E. coli was inactive and contained less than 0.1 iron atom/subunit. In contrast, recombinant enzyme expressed in Sf9 cells using a baculovirus vector was active and identical in several properties to phenylalanine hydroxylase from rat liver: the Km for 6-methyltetrahydropterin was 39 microM (compared with 35 microM for the rat liver enzyme), 1 atom of iron was "associated" per enzyme subunit, and electron paramagnetic resonance spectra showed that iron was distributed within two distinct environments. Putative iron-binding sites of phenylalanine hydroxylase were studied by mutating either histidine 284 or 289 to serine and expressing these mutant enzymes (PAH-H284S and PAH-H289S) in Sf9 cells. Mutants were expressed at levels similar to wild-type PAH, but contained < or = 0.1 iron/subunit and were inactive. Thus, both His284 and His289 apparently are required for iron binding and hydroxylation activity.

Amino Acid Sequence↗

PubMed1998

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 six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8-aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.

Alkaline Phosphatase↗

PubMed2001

Two isoforms of trimming glucosidase II exist in mammalian tissues and cell lines but not in yeast and insect cells.

We previously cloned glucosidase II and provided in vivo evidence for its involvement in protein folding quality control. DNA-sequencing of different clones demonstrated the existence of two isoforms of glucosidase II which differed by 66 nucleotides due to alternative splicing. The existence of two enzyme isoforms in various organs of pig and rat as well as human, bovine, rat, and mouse cell lines could be demonstrated by RT-PCR and Western blotting. Furthermore, the two isoforms of glucosidase II could be detected in embryonic and postnatal rat kidney and liver. In yeast, Saccharomyces cerevisiae, and in insects, Drosophila S2 cells, only one isoforms of the enzyme was detectable. The ubiquitous occurrence of the two glucosidase II isoforms in mammalian tissues and cell lines might be indicative of a special function of each isoform.

Animals↗

PubMed2005

Development of a novel serum-free freezing medium for mammalian cells using the silk protein sericin.

Cryopreservation is a pivotal process in cellular engineering for creating a continuous source of generated functional cell lines and for the convenience of various medical treatments that involve cell culture. FBS (fetal bovine serum) supplemented with 10% (v/v) DMSO is extensively used as a freezing medium for mammalian cells using conventional methods. However, FBS should ideally be avoided, owing to serious concerns regarding bovine spongiform encephalopathy and other infections such as viruses, and an alternative to FBS is eagerly awaited. Furthermore, bio-medicines and cells for transplantation should not be infectious. The present study aimed to develop a novel serum-free freezing medium. For this purpose, we focused on using the silk protein sericin as a cryoprotectant for storage and developed a novel serum-free freezing medium consisting of PBS, 1% (v/w) sericin, 0.5% (v/w) maltose, 0.3% (v/w) proline, 0.3% (v/w) glutamine and 10% DMSO. This novel freezing medium was compared with the conventional FBS supplemented with DMSO and also with three purchased freezing media with respect to cryopreservation of the P 3 U1 myeloma cell line and Chinese-hamster ovary cells. As a result, the constructed medium containing sericin successfully cryopreserved both cell types as efficiently as the conventional medium of FBS containing 10% DMSO and was superior to all three of the purchased media. The constructed medium containing sericin also cryopreserved normal human dermal fibroblasts, human epidermal keratinocytes, the rat phaeochromocytoma cell line PC12 and insect (Spodoptera frugiperda) cell line S f 9 as effectively as the conventional medium of FBS and DMSO.

Animals↗