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PubMed1999

High prevalence of antibodies against HERV-K10 in patients with testicular cancer but not with AIDS.

Human endogenous retrovirus K10 (HERV-K10) env and gag expression has been detected in placenta, embryonic tissue, and cell lines. By transfection, these sequences have been expressed in insect cells and developed into serological assays, revealing HERV-K10 antibodies in patients with testicular cancer. Patients with AIDS are at an increased risk for testicular cancer and frequently reactivate latent infections. We postulated that HERV-K10 seroprevalence might be increased with HIV infection or AIDS. Stored, frozen serum samples from 52 patients with testicular cancer (8 patients with HIV and 30 patients with samples near the time of diagnosis) and 84 controls (40 patients with HIV) were diluted 1:40 and tested by immunofluorescence against SF158 cells transfected with HERV-K10 env [ENV1.9(+)] or gag (pACGAG). Seroprevalence rates were compared cross-sectionally in cases and controls, excluding those with indeterminate results (3 of 30 cases and 7 of 84 controls), and also were examined longitudinally in the cases before or after diagnosis of testicular cancer. Seroprevalence to HERV-K10 Env or Gag was 17 of 27 testicular cancer patients (63%) around the time of diagnosis, compared to 4 of 77 controls (5%; P < 0.0001). Seroprevalence was similar (50% to 60%) with seminoma, teratocarcinoma, or embryonal carcinoma, and it was not increased with HIV infection in either cases (33%) or controls (3%). HERV-K10 antibodies were detected in 12 of 19 cases (63%) more than 6 months before seminoma diagnosis, as well as in four cases with residual or recurrent malignancy more than 1 month after initial diagnosis. Thus, HERV-K10 antibodies are detected frequently with testicular cancer and seem to resolve rapidly with effective therapy of the malignancy. Antibody reactivity also occurs in approximately 5% of controls, perhaps because of nonspecific or cross-reactive epitopes. HIV and AIDS were not associated with HERV-K10 antibodies, thus, leaving their higher risk of testicular cancer unexplained.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Morphogenesis of Sindbis virus in three subclones of Aedes albopictus (mosquito) cells.

The morphogenesis of Sindbis virus in three Aedes albopictus subcloned cell lines was examined. Each line was distinguishable with respect to morphology, cytopathic response to infection, and progeny yield. C7-10 cells, which produced the highest titers of virus and exhibited the most severe cytopathic response, were characterized ultrastructurally by the presence of budding particles at the cell surface and at the membranes of internal vesicles. C6/36 cells, which displayed a moderate cytotoxic response, manifested similar features in response to Sindbis virus infection. Both cell types also produced a structure composed of an electron-dense matrix in which nucleocapsids were embedded. Internally matured virions were released by exocytosis from these cells. In addition to a lack of cytopathic effect, u4.4 cells also failed to exhibit obvious morphogenetic changes upon infection. Virus particles were occasionally seen within vesicles, but budding at the cell surface was not detected. The mechanism of release of internally matured virions was not apparent. These studies provide further evidence that these three subcloned mosquito cell lines represent different tissues in the larval or adult insect.

Aedes↗
PubMed1990

Wild-type, but not mutant, human p53 proteins inhibit the replication activities of simian virus 40 large tumor antigen.

Murine p53 blocks many of the replication activities of simian virus 40 (SV40) large tumor antigen (T antigen) in vitro. As murine cells do not replicate SV40 DNA, it was of interest to determine how p53 from permissive human cells functions. Recombinant baculoviruses encoding either the wild-type form of human p53 or a mutant p53 cloned from a human tumor cell line were constructed, and p53 proteins were purified from infected insect cells. Surprisingly, we found that wild-type human p53 was as inhibitory to the ability of T antigen to mediate replication of an SV40 origin-containing (ori DNA) plasmid in vitro as was murine p53. Wild-type human p53 also blocked the DNA unwinding activity of T antigen, as did its murine counterpart. In contrast to murine and wild-type human p53, the mutant human p53 did not block ori DNA replication or DNA unwinding. Murine p53 formed a complex with mutant human p53 in vivo. Furthermore, mutant human p53 reduced the inhibition of SV40 ori DNA replication by murine p53 in vitro. These results provide a model for the way in which mutant p53 proteins can affect normal functions of p53.

Animals↗
PubMed2006

Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells.

L1 is a human cell adhesion glycoprotein involved in the development of the central nervous system that comprises six immunoglobulin-like domains (Ig1-Ig6), five fibronectin-type III (FN1-FN5) domains, a single transmembrane region and a cytoplasmic domain. It contains 20 potential N-glycosylation sites and is heavily glycosylated in a variety of cell types. In this work, seven truncated soluble forms including L1 ectodomain (L1/ECD) and Ig domains 5-6 (L1/Ig5-6) have been constructed by PCR and have been cloned, as well as the full-length form (L1), in the stable expression vector for insect cells pMIB/V5-His-TOPO. Spodoptera frugiperda Sf9 cell lines expressing the truncated forms have been obtained, and all proteins were successfully secreted. L1/ECD and L1/Ig5-6 were produced in shake flasks with productions of 3 and 32 mg/L on the third and fourth day of culture, respectively. When L1/Ig5-6 was produced for four days in 2L bioreactor 200 mg/L protein were recovered from the supernatants on the fourth day of culture. Affinity-purified L1/ECD and L1/Ig5-6 were immobilized on poly-d-lysine coated coverslips, and were shown to be active in inducing neurite outgrowth from human NT2N neurons. Therefore, correctly folded and functional truncated forms of human L1 have been produced in high amounts from insect cells using a stable expression system.

Animals↗
PubMed1989

Expression of Sindbis virus 26S cDNA in Spodoptera frugiperda (Sf9) cells, using a baculovirus expression vector.

To study protein processing in an insect Spodoptera frugiperda (fall armyworm; Sf9) cell line, a 26S cDNA encoding the sequence of Sindbis virus structural proteins (capsid protein, of 30 kilodaltons [kDa]; p62 [the precursor of E3 and E2], of 62 kDa; a 6-kDa peptide; and the E1 protein, of 56 kDa) was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. By immunoblot analysis with antisera directed against whole Sindbis virus and the individual structural proteins (capsid, E2, and E1), we have shown that polypeptides similar in size and antigenicity to those synthesized in Sindbis virus-infected BHK cells are expressed in Sf9 cells infected with the recombinant baculovirus Ac373-SV26. By pulse-chase labeling in the presence or absence of tunicamycin, by endo-beta-N-acetylglucosaminidase H (endo-H) treatment of the recombinant glycoproteins, and by N-terminal sequence analysis of the E1 envelope glycoprotein, we have further shown that the 26S transcription translation unit of Sindbis virus, although normally encoded by nonnuclear RNA, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared with BHK cells when a baculovirus expression vector is used.

Animals↗
PubMed2003

High-level expression of human glycosyltransferases in insect cells as biochemically active form.

cDNAs, encoding human beta1,4-galactosyltransferase (hGalT I, EC 2.4.1.22), human Galbeta1,3(4)-GlcNAc alpha2,3-sialyltransferase (hST3GalIII, EC 2.4.99), and human Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (hST6Gal I, EC 2.4.99.1), were cloned from human cell lines. In order to express these glycosyltransferases as secreted form in insect cells, cDNAs were inserted into a novel baculovirus transfer vector equipped with the mouse IgM signal peptide and IgG binding domain of the Staphylococcus aureus protein A as an N-terminal fusion partner. About 14 mg hGalT I, 8 mg hST3GalIII, and 6.4 mg hST6Gal I were purified from 1 liter of recombinant baculovirus infected insect cell culture media. The specific activities of recombinant hGalT I and hST6Gal I were determined as 0.65 and 1.6 U/mg protein, respectively. These results indicated that the recombinant hGalT I and hST6Gal I retained enzyme activities at similar level to those of the authentic one although they were fused with the IgG binding domain at the N-terminus. Taken together, the mouse IgM signal peptide and IgG binding domain of the protein A could be efficiently used as an N-terminus fusion partner for the over-expression of heterologous proteins in insect cells.

Amino Acid Sequence↗
PubMed1996

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 larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active delta-endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran delta-endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC delta-endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC delta-endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active delta-endotoxin.

Aedes↗
PubMed2005

Pharmacological profiles of recombinant and native insect nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are targets for insect-selective neonicotinoid insecticides exemplified by imidacloprid (IMI) and mammalian-selective nicotinoids including nicotine and epibatidine (EPI). Despite their importance, insect nAChRs are poorly understood compared with their vertebrate counterparts. This study characterizes the [(3)H]IMI, [(3)H]EPI, and [(3)H]alpha-bungarotoxin (alpha-BGT) binding sites in hybrid nAChRs consisting of Drosophila melanogaster (fruit fly) or Myzus persicae (peach-potato aphid) alpha2 coassembled with rat beta2 subunits (Dalpha2/Rbeta2 and Mpalpha2/Rbeta2) and compares them with native insect and vertebrate alpha4beta2nAChRs. [(3)H]IMI and [(3)H]EPI bind to Dalpha2/Rbeta2 and Mpalpha2/Rbeta2 hybrids but [(3)H]alpha-BGT does not. In native Drosophila receptors, [(3)H]EPI has a single high-affinity binding site that is independent from that for [(3)H]IMI and, interestingly, overlaps the [(3)H]alpha-BGT site. In the Mpalpha2/Rbeta2 hybrid, [(3)H]IMI and [(3)H]EPI bind to the same site and have similar pharmacological profiles. On considering both neonicotinoids and nicotinoids, the Dalpha2/Rbeta2 and Mpalpha2/Rbeta2 receptors display intermediate pharmacological profiles between those of native insect and vertebrate alpha4beta2 receptors, limiting the use of these hybrid receptors for predictive toxicology. These findings are consistent with the agonist binding site being located at the nAChR subunit interface and indicate that both alpha and beta subunits influence the pharmacological properties of insect nAChRs.

Animals↗
PubMed2004

Acoustic cell filter: a proven cell retention technology for perfusion of animal cell cultures.

This article is a review highlighting the application of the acoustic filter as a reliable cell retention device during the long-term perfusion of animal cell cultures. Critical operating parameters such as duty cycle, perfusion and re-circulation flow rates, acoustic power and backflush frequency are discussed with regard to influence on the separation efficiency and optimal operating ranges have been identified. Perfusion data gathered from the literature have been complemented with original data from a series of perfusion experiments carried out in the context of industrial projects for industrially relevant cell lines including NS0, HEK-293, SP2-derived hybridoma and insect cells in different serum-supplemented and serum-free media at different perfusion rates and acoustic chamber volumes. Finally, scale-up potential of the acoustic filter for large-scale industrial applications is discussed.

Acoustics↗
PubMed2001

Effect of selected pesticides and their ozonation by-products on gap junctional intercellular communication using rat liver epithelial cell lines.

The non-genotoxic effects of two commonly used pesticides, 1,1-bis (p-chlorophenyl)-2,2,2-trichloroethane (DDT) and malathion, and one widely used commercial insect repellent N,N-diethy-m-toluamide (DEET) on gap junction intercellular communication (GJIC) were determined using a rat liver epithelial cell line. Malathion and DDT reversibly inhibited GJIC in a treatment time- and dose-dependent manner at non-cytotoxic doses, whereas, DEET did not inhibit GJIC. Malathion was very reactive with ozone, while DEET and DDT did not react to any appreciable extent with ozone. The mixtures of ozonation products from malathion and DEET did not inhibit GJIC. The mixtures of ozonation by-products formed from DDT inhibited GJIC, but to a lesser extent than did DDT, itself. These results suggest that ozone can effectively remove malathion from solution without forming GJIC-toxic products, but is less effective in eliminating DEET and DDT from solution.

Animals↗
PubMed1998

The G11 gene located in the major histocompatibility complex encodes a novel nuclear serine/threonine protein kinase.

Protein kinases are involved in signal transduction pathways and play fundamental roles in the regulation of cell functions. Here we report that the gene G11 located in the human major histocompatibility complex encodes a novel Ser/Thr protein kinase. The G11 gene products of 41.5 and 30 kDa were expressed in insect cells using the baculovirus system and transiently in the mammalian cell line COS-7. It was found that after immunoprecipitation of the G11 polypeptides from recombinant baculovirus-infected insect cell lysates or transfected COS-7 cell lysates the immunoprecipitates contained a Mn2+-dependent protein kinase activity that phosphorylated alpha-casein at Ser/Thr residues and histone at Ser residues. Furthermore, mutation of the ATP-binding site by converting the invariant lysine in the catalytic domain (amino acid 317) to a proline resulted in the complete ablation of the enzyme activity. This was consistent with the observation that the G11 polypeptide can be covalently modified by the reactive ATP analogue 5'-p-fluorosulfonylbenzoyladenosine in the absence of ATP, and that this modification is prevented in the presence of 1 mM ATP, indicating that the kinase domain of the G11 polypeptide is capable of binding ATP. Immunofluorescence staining of transfected COS-7 cells transiently expressing G11 revealed that this novel Ser/Thr protein kinase is localized predominantly in the nucleus.

Adenosine↗
PubMed2001

Expression in Spodoptera frugiperda (Sf21) insect cells of BT-R(1), a cadherin-related receptor from Manduca sexta for Bacillus thuringiensis Cry1Ab toxin.

The cadherin-related receptor of Manduca sexta, BT-R(1), for the Cry1A family of Bacillus thuringiensis insecticidal toxins, was expressed in cultured Spodoptera frugiperda (Sf21) insect cells utilizing the expression vector deltaOp-gp64. Recombinant BT-R(1) was released by the Sf21 cells in soluble form into the culture medium and represents approximately 58% of total BT-R(1) produced by the cells. The soluble protein was purified by affinity chromatography using Cry1Ab toxin coupled to Sepharose 4B. The apparent molecular mass of purified soluble recombinant BT-R(1) is 195 kDa. Radiolabeled toxin bound to purified soluble BT-R(1) with a K(d) value of 1.1 nM, which is similar to that of both membrane-bound BT-R(1) in Sf21 cells and natural BT-R(1) from M. sexta larval midgut tissue. Binding of radiolabeled toxin to soluble BT-R(1) was competitively inhibited by unlabeled Cry1Ab toxin but not by other Cry toxins as was observed also for membrane-bound BT-R(1). The recombinant soluble protein was stable in culture medium for at least 3 days at 27 degrees C and for 7 days at 4 degrees C and exhibited toxin-binding properties similar to the natural protein. Apparently, neither membrane association nor the extent of glycosylation influences the binding affinity and specificity of BT-R(1). Approximately 1 mg of purified BT-R(1) was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) Sf21 cells.

Animals↗
PubMed1995

Production of functional human bone morphogenetic protein-2 using a baculovirus/Sf-9 insect cell system.

A clone of a human Bone Morphogenetic Protein-2 (hBMP-2) cDNA was obtained from a cDNA library established from human dental pulp cells. After subcloning hBMP-2 cDNA into Autographa californica nuclear polyhedrosis virus, the recombinant baculovirus was transfected to Sf-9 cells. Immuno-reactive recombinant hBMP-2 (rhBMP-2) was detected by a polyclonal antibody against Xenopus BMP-2 in the transfected insect cells but not in the culture media. Three days after treatment with the lysate of the transfected Sf-9 cells, increase in alkaline phosphatase activity of a murine stromal cell line, ST2, was detected. Subcutaneous implantation of rhBMP-2 produced in the insect cells induced formation of cartilage, bone and bone marrow in the rats. The present data indicated that the rhBMP-2 preparation produced in the insect Sf-9 cells had a comparable activity to that produced in mammalian cells.

Alkaline Phosphatase↗
PubMed1991

Host-dependent variation of bluetongue virus neutralization.

To determine if neutralizing epitopes of Bluetongue virus (BTV) 17 are host dependent, e.g., that monoclonal antibodies (mAb) to Bluetongue virus 17 (BTV 17) differ in their ability to neutralize BTV infectivity in insect versus mammalian cells, a panel of neutralizing mAb was developed. The relative neutralizing titer of eight mAb for BTV 17 infectivity in mammalian versus insect target cells was determined. Four mAb differed in their relative neutralization titer when assayed on mammalian target cells as compared to insect target cells. These findings suggest that different epitopes involved in neutralization might be important in virus infectivity and neutralization in mammalian versus insect target cells. To determine which viral protein(s) these mAb bind, the specificity of the mAb was determined by radioimmunoprecipitations. Five BTV 17 neutralizing mAb bound to the major outer coat protein P2, a 98-kDa protein, whereas the BTV protein(s) bound by the other three neutralizing mAb could not be determined. The potential role of the two BTV outer coat proteins in infection of mammalian and insect host cells is discussed.

Animals↗
PubMed2000

Bioreactor-scale production and one-step purification of Epstein-Barr nuclear antigen 1 expressed in baculovirus-infected insect cells.

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated malignancies and is essential for EBV-genome maintenance. Antibodies to EBNA1 are abundantly detected in serum of most EBV carriers but EBNA1 escapes recognition by effector T-lymphocytes. To further study the functional and immunological characteristics of EBNA1 it is important to have sufficient quantities of purified EBNA1 available. This paper describes a simple, reproducible method for the production and purification of EBV-encoded EBNA1 expressed in insect cells (bEBNA1). For quantification of EBNA1 expression levels in cell lines and for monitoring bEBNA1 purification and overall yields we developed a quantitative and EBNA1-specific capture ELISA. We observed that EBV-positive cell lines express EBNA1 at different levels, with the B cell lymphoblastoid cell line X50/7 having the highest production. However, much larger quantities (380-fold) were obtained by expressing bEBNA1 in recombinant-baculovirus-infected Sf9 insect cells. Scaling-up experiments revealed that bEBNA1 expression kinetics and protein stability are identical in 1-liter stirred bioreactors when compared to expression in stationary culture flasks. Optimal expression was reached after 72 h following inoculation at 1 pfu/cell, when insect cell viability was about 50%. For purification the nuclear fraction containing most of the bEBNA1 (>95%) was isolated. Solubilized bEBNA1 was purified by a one-step oriP DNA-Sepharose affinity purification procedure, using biotinylated PCR-amplified family of repeats (FR)-domain products immobilized onto streptavidin agarose. A >200-fold specific enrichment was reached and yields of bEBNA1 with an estimated purity of >95%.

Animals↗