Applications of insect cell gene expression in pharmaceutical research.
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Animals↗
Biomedical subjects
A L Meyer
Publications and source records attributed to A L Meyer.
Detection of ovine lentivirus in seronegative sheep by in situ hybridization, PCR, and cocultivation with susceptible cells.
Serological surveys for ovine lentivirus (OvLV), a worldwide cause of pneumonia and chronic debilitation in sheep, have demonstrated a wide range of seroprevalence rates. This study analyzed OvLV infection in a purebred sheep flock with a history of OvLV disease (flock 1), and compared the prevalence with that of a flock lacking previous OvLV-associated disease (flock 2). Serological tests (ELISA and Western blot assay) indicated that 25% of sheep of all ages in flock 1 (Group A) and 33% of animals of all ages in flock 2 (Group B) had antibodies to OvLV. In situ hybridization, however, detected viral RNA in a much larger proportion of sheep (72 and 67%, respectively). Animals less than 1 year of age rarely had antibodies to OvLV, although most harbored viral RNA. Twenty animals in this age group from flock 1 (Group C) were therefore studied more closely for infection. These yearling animals were tested serologically by ELISA and their peripheral blood-derived macrophages were cultured for 14 days to amplify any infection in these target cells. The macrophages were then tested by in situ hybridization, PCR, and cocultivation with susceptible target cells. The results of these tests showed that while only 10% of animals in Group C were seropositive, 70% were positive by in situ hybridization, PCR, and cocultivation. These data suggest that latent OvLV infection is common in sheep and that infection is frequently undetected by serological tests.
Age Factors↗
The use of feline herpesvirus and baculovirus as vaccine vectors for the gag and env genes of feline leukaemia virus.
The env and gag genes from feline leukaemia virus were expressed in a thymidine kinase-negative feline herpes-virus and a baculovirus. Cats were vaccinated with various combinations of these recombinant viruses and 100% protection against feline leukaemia virus challenge was achieved using an immunization schedule which utilized both env and gag products delivered at both a mucosal and systemic site.
Animals↗
Expression of feline leukaemia virus gp85 and gag proteins and assembly into virus-like particles using the baculovirus expression vector system.
In order to test components of feline leukaemia virus (FeLV) as subunit vaccines, we have constructed recombinant baculoviruses that express the FeLV envelope glycoprotein gp85 [Autographa californica nuclear polyhedrosis virus (AcNPV)-gp85] and the structural protein, gag (AcNPVgag). The gag protein is expressed and shed into the medium of infected cells as particles which have a buoyant density on sucrose gradients and appearance by electron microscopy similar to those of authentic FeLV virions. The gag precursor protein within the particles is not fully processed and appears to be a result of partial cleavage of the gag polypeptide. Insect cells that are coinfected with AcNPVgag and AcNPVgp85 shed particles that contain both the gag protein and the gp85 glycoprotein.
Baculoviridae↗
Cloning and sequence of an infectious bovine rhinotracheitis virus (BHV-1) gene homologous to glycoprotein H of herpes simplex virus.
A homologue to the glycoprotein H (gH) gene of herpes simplex virus (HSV) has been identified in the genome of infectious bovine rhinotracheitis virus (IBR, BHV-1). The gene is located immediately downstream from the thymidine kinase gene, and codes for an open reading frame (orf) of 842 amino acids. The orf has the characteristics of a membrane glycoprotein, including an N-terminal hydrophobic region resembling a signal sequence, a C-terminal region which is probably a transmembrane domain, and six potential sites for N-linked glycosylation. This orf shows significant homology to the gH sequences of both HSV and pseudorabies virus (PRV). We conclude that this gene encodes BHV-1 gH.
Base Sequence↗
Immune response in pigs to Aujeszky's disease viruses defective in glycoprotein g1 or gX.
Two Aujeszky's disease virus glycoprotein genes, gX and g1, have been used to produce deletion mutants which have then been developed into vaccines. These deletions then allow differentiation between pigs infected with wild type virus and those given the vaccine. It is not clear whether the glycoproteins encoded for by these genes are needed to induce a full protective immune response, in which case deletion mutants would suffer from lack of potency. To test this, commercially available Aujeszky's virus vaccines which lacked either gX or g1 were compared and isogenic constructs were made which differed only in the absence or presence of gX and, or, g1. These constructs and vaccines were used to vaccinate the natural host of Aujeszky's disease, the pig, and potency was measured using challenge with wild type virus. In all cases vaccines which lacked g1 performed significantly less well than those in which g1 was present, whereas deletions of gX had no significant effect on vaccine performance.
Animals↗
Cytotoxic T cell activity against the 22-kDa protein of human respiratory syncytial virus (RSV) is associated with a significant reduction in pulmonary RSV replication.
Recombinant vaccinia viruses expressing the RSV F glycoprotein (Vac-F), or a previously described chimeric protein consisting of the extracellular domains of the F and G glycoproteins (Vac-FG), or the 22-kDa membrane protein (Vac-22 kDa) were evaluated for their ability to protect BALB/c mice against infection by RSV subgroup A or subgroup B viruses and for their ability to induce a humoral immune response or a cytolytic T lymphocyte (CTL) response. Immunization with Vac-F or Vac-FG fully protected mice against challenge with RSV of subgroup A or B and induced high levels of both humoral and CTL-mediated immunity. Immunization with Vac-22 kDa partially to fully protected mice against challenge with RSV of subgroup A or B, depending on the immunization and challenge conditions, and induced a potent CTL response in the apparent absence of a significant humoral response. These vectors fortuitously allowed us to evaluate the contribution of a protein-specific memory CTL response to subgroup-specific and subgroup-cross-reactive reductions in pulmonary RSV replication independently from a humoral response. Our data suggest that 22-kDa-specific CTL contribute significantly to the reduction of RSV within the lung, but that complete protection also requires a humoral component.
Animals↗
Molecular dosimetry of DNA adducts in Chinese hamster ovary cells and C3H10T1/2 mouse embryo fibroblast cells treated with benzo[a]pyrene.
Chromatographic profiles of DNA adducts formed following treatment of Chinese hamster ovary (CHO) cells or C3H10T1/2 mouse embryo fibroblast cells with benzo[a]pyrene (BP), in the presence or absence of rat liver S9 fraction, have been obtained by both direct labelling ([3H]BP) and 32P-post-labelling methods. The principal adduct formed in both cell lines was (+)-N2-(7R,8S,9R-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene- 10S-yl) 2'-deoxyguanosine (detected as its 3',5'-bisphosphate by 32P-post-labelling). In both cell lines significantly higher levels of adducts were observed in the presence of S9 fraction. The results of these studies provide useful reference data on the genotoxic metabolism of BP in these widely used cell culture systems and allow comparisons with data obtained in other experimental mammalian systems. Valid comparisons of metabolism in experimental systems used as extrapolative models for man are particularly important where multiple bioactivation pathways are possible, such as in the case of polycyclic aromatic hydrocarbons.
Animals↗
Genetic engineering of the pseudorabies virus genome to construct live vaccines.
Pseudorabies virus (PRV) is a herpesvirus of pigs. Homologous recombination with plasmids offers a method to engineer precise changes in the PRV genome to produce advantageous live vaccines. Safety can be ensured by using a non-reverting deletion to inactivate the thymidine kinase gene. One particularly important feature of new PRV vaccines is deletion of an antigen, so that vaccinated pigs are serologically distinguishable from infected pigs. We have constructed a live vaccine strain with deletions in the thymidine kinase gene and in the gene for a glycoprotein, gX. Molecular engineering techniques made it possible to choose deletion of gX, which has no known immunological significance, over deletion of other glycoproteins that contribute to protective immunity. Extensive experiments in pigs with isogenic virus pairs show that deletion of gX does not compromise efficacy of a vaccine as gI deletions do. Deletion of gX also suggests a site for replacement with antigens from other pathogens. In addition to molecular engineering of a live vaccine strain, research on PRV glycoproteins has led to the discovery that expression of the glycoprotein gp50 makes cells resistant to PRV infection. Perhaps this observation could be extrapolated to the level of a whole animal to allow engineering of pigs to become an alternative to engineered vaccines.
Animals↗
Monoclonal antibody-based immunoaffinity chromatography for purifying corn and squash NADH: nitrate reductases. Evidence for an interchain disulfide bond in nitrate reductase.
NADH: nitrate reductase (EC 1.6.6.1) (NR) is present in small amounts in plant tissues and its polypeptide in inherently labile. Consequently, NR is difficult to purify. We have generated 20 monoclonal antibodies (McAb) for corn and squash NR and selected two for use in immunoaffinity chromatography. Squash McAb CM 15(11) and corn McAb ZM 2(69)9, which both bind corn and squash NR, were covalently coupled to Sepharose and used for purification of NR with elution of the purified enzyme by a pH 11 buffer. Although this procedure yielded highly purified NR, its activity was diminished by the pH 11 treatment. When corn leaf crude extract was applied to McAb CM 15(11)-Sepharose, NR bound and could be eluted in homogeneous form by its substrate, NADH. Corn leaf NR prepared by substrate elution retained a high level of NADH: NR activity. Immunoaffinity-purified corn and squash NR were shown to have an interchain disulfide bond as well as a reactive thiol group. These results are discussed in relation to the recently obtained sequences of NR clones and suggestions made for site-directed mutagenesis experiments to aid in identifying the cysteine residues of NR associated with these features of the enzyme.
Antibodies, Monoclonal↗
The genetic toxicology of some hydrocarbon and oxygenated solvents.
Two hydrocarbon solvents (heptane and Special Boiling Point Spirit 100/140) and eight oxygenated solvents [methyl ethyl ketone, methyl isobutyl ketone, diacetone alcohol, di-isobutyl ketone, isopropyl ether, hexylene glycol, secondary butyl alcohol and ME 6K (pentoxone)] have been tested for genotoxic activity. The solvents were tested in bacterial mutation assays, a yeast assay for mitotic gene conversion and in cultured mammalian cells (either rat liver or Chinese hamster ovary) for structural chromosome damage. All of the solvents gave a negative response in the bacterial mutation assays and the yeast mitotic gene conversion assay. In the rat liver chromosome assay, diacetone alcohol evoked a weak positive response, the remaining solvents gave a negative response.
Alcohols↗
Reduced yield of infectious pseudorabies virus and herpes simplex virus from cell lines producing viral glycoprotein gp50.
Pseudorabies virus (PRV) glycoprotein gp50 is the homolog of herpes simplex virus (HSV) glycoprotein D. Several cell lines that constitutively synthesize gp50 were constructed. Vero cells, HeLa cells, and pig kidney (MVPK) cells that produce gp50 all gave reduced yields of PRV and HSV progeny viruses when compared with the parent cell line or the same cell line transfected to produce a different protein. The reduction in virus yield was greatest at low multiplicities of infection. The Vero and HeLa cells that produce gp50 showed an even greater reduction in HSV yield than in PRV yield. This phenomenon may be an example in a herpesvirus of the interference observed in retroviruses or cross-protection in plant virus systems.
Animals↗
Molecular phenotyping of non-transformed and chemically transformed C3H10T1/2 mouse fibroblasts.
A systematic molecular phenotyping approach based on two-dimensional gel electrophoresis is being applied in an attempt to identify protein changes associated with malignant transformation. Using the C3H10T1/2 mouse cell line, two-dimensional polypeptide maps of the non-transformed cell line, several chemically transformed lines and a tumour cell line were compared. Although there is a large degree of similarity between the protein profiles of all cell lines, clear differences are evident. Initial results are consistent with the view that many of the protein changes are incidental to malignant transformation. Changes induced by 3-methylcholanthrene are retained after transplantation of the cells into nude mice.
Animals↗
A framework for assessing performance problems.
How do we as managers address problems of nonperformance ? Robert Mager and Peter Pipe provide a framework for identifying the real problem, deciding whether or not there is a skill deficiency, and whether performance is punishing or whether nonperformance is rewarding. If performance really matters, are there obstacles to performing? This framework helps us to analyze performance deficiencies systematically and to move toward an appropriate solution.
Clinical Competence↗
In vitro transformation assays for chemical carcinogens.
A variety of in vitro mammalian cell assays, designed specifically for the identification of carcinogenic compounds, have been in operation for more than a decade. Although no individual transformation system has won universal acceptance during this time, recent advances have led to the improved reliability and sensitivity of a number of these short-term tests. The underlying problems associated with the most widely used assays are identified and new developments in this rapidly expanding field are noted and discussed.
Animals↗
Genotoxicity studies with a blend of zinc dialkyldithiophosphate lubricant additives.
The mutagenic activity of a blend of primary zinc dialkyldithiophosphate lubricant additives suspended in process oils and a blend of the process oils alone was investigated in agar layer cultures of Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100, both with and without the incorporation of a rat liver microsomal activation system (S9). Zinc dimethyldithiocarbamate was tested as a structurally-related model bacterial mutagen, and, in additional control experiments, the mutagenic activity of zinc dimethyldithiocarbamate and benzo[a]pyrene was investigated in combination with the blend of process oils in selected bacterial tester strains. Transformation frequencies of BHK cells were determined by colony growth in soft agar culture following treatment with a blend of the zinc dialkyldithiophosphate lubricant additives suspended in process oils, a blend of the process oils, 7,12-dimethylbenzanthracene, both in the presence and in the absence of a blend of the process oils, or zinc dimethyldithiocarbamate. All experiments incorporated a rat liver microsomal activation system (S9). Application of a blend of the zinc dialkyldithiophosphate additives or a blend of the process oils used in their manufacture did not increase the reverse mutation frequencies of Salmonella typhimurium TA1535, TA1537, TA1538, TA98 or TA100, or significantly increase the transformation frequency of BHK cells under the experimental conditions described. Zinc dimethyldithiocarbamate increased the reverse mutation frequency in some bacterial tester strains, but did not significantly increase the transformation frequency of BHK cells under the described experimental conditions. The addition of the blend of the process oils in combination with the control materials, zinc dimethyldithiocarbamate or benzo[a]pyrene had an inhibitory effect on the mutagenic activity at high doses of each in the bacterial assays, and in the BHK assay the transforming ability of 7,12-dimethylbenzanthracene was suppressed in the presence of the blend of the process oils. Thus, the additive materials showed no evidence of genotoxic activity in the bacterial mutation assays, or in the BHK transformation assay under the experimental conditions described.
Animals↗
Induction of sister-chromatid exchanges in a rat-liver cell line with chemical carcinogens.
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9,10-Dimethyl-1,2-benzanthracene↗
Mapping of the G1 phase of a mammalian cell cycle.
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Cell Division↗