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PubMed1989

Maintenance of DNA methylation level in SV40-infected human fibroblasts during their in vitro limited proliferative life span.

Methylation level as expressed by the molar ratio of 5-methylcytosine content to the combined content of cytosine and 5-methylcytosine was determined by HPLC and uv adsorption of cellular DNA extracted from SV40-infected and pretransformed MRC-5 human diploid fibroblasts (HDFs) during their limited in vitro life span. The level decreased slightly during early passages, and then was maintained within a certain range in the subsequent pretransformed stage of serial passages. When HDFs were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) at an effective concentration shortly after the SV40 infection, the level decreased and then increased or was maintained again within a certain range in the subsequent pretransformed state. The proliferative life span potential of SV40-infected HDFs was not significantly decreased by the 5-aza-CdR treatment. These results are in contrast to the established observations for uninfected HDFs, that methylation level decreases during serial passages, and that, after treatment with 5-aza-CdR, the level, as well as the proliferative life span, is decreased in comparison to untreated populations. These results show that SV40-infected pretransformed HDFs are in an intermediate state between normal finite growth and an established permanent line, in that they retain limited in vitro cell proliferation, while acquiring the ability to maintain methylation levels.

5-Methylcytosine↗

PubMed1995

Immune responses induced by administration of encapsidated poliovirus replicons which express HIV-1 gag and envelope proteins.

Several viruses have been exploited for the development of recombinant vaccine vectors in which to express foreign proteins. Recently, we have described a system utilizing the RNA virus, poliovirus. We have constructed poliovirus genomes in which regions of the capsid have been substituted with gene fragments of the HIV gag and env genes. A complementation system has been designed to encapsidate defective genomes by providing the capsid protein in trans from a recombinant vaccinia virus (VV-P1). Serial passage in the presence of VV-P1 resulted in the generation of stocks of these encapsidated replicons. Infection of cells with these encapsidated replicons resulted in the expression of the recombinant protein as a fusion protein with the poliovirus capsid proteins VP4 and VP1. In this study, we have utilized encapsidated replicons which express the HIV-1-gag capsid protein (p24) as well as 1.5 kb of the HIV-1 env gene. Stocks of these encapsidated replicons were obtained by 20 serial passages in the presence of VV-P1. In addition, passage of the encapsidated replicons in the presence of poliovirus type 2 Lansing resulted in the encapsidation of the replicons by the capsid proteins provided by poliovirus. The administration of the type 2 Lansing/encapsidated replicons expressing HIV-1 gag in BALB/c mice by intramuscular, intrarectal, or intragastric routes resulted in the generation of antibodies in the serum and secretions against both poliovirus and HIV-1 gag. To prove that the replicons alone are immunogenic, we administered replicons expressing either HIV-1 gag or env to transgenic mice which expressed the receptor for poliovirus type 1. Immunization of these mice by the intramuscular route resulted in the generation of serum antibodies specific for poliovirus as well as for HIV-1 antigens. The results obtained led us to the conclusion that the replicons are immunogenic when given alone or in the presence of poliovirus. These results are important for the use of the poliovirus replicons as a recombinant vaccine vector.

AIDS Vaccines↗

PubMed1992

Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations.

Glucose toxicity of the pancreatic beta cell is considered to play a secondary role in the pathogenesis of type II diabetes mellitus. To gain insights into possible mechanisms of action of glucose toxicity, we designed studies to assess whether the loss of insulin secretion associated with serial passages of HIT-T15 cells might be caused by chronic exposure to high glucose levels since these cells are routinely cultured in media containing supramaximal stimulatory concentrations of glucose. We found that late passages of HIT cells serially cultured in media containing 11.1 mM glucose lost insulin responsivity and had greatly diminished levels of insulin content and insulin mRNA. In marked contrast, late passages of HIT cells cultured serially in media containing 0.8 mM glucose retained insulin mRNA, insulin content, and insulin responsivity to glucose in static incubations and during perifusion with glucose. No insulin gene mutation or alteration of levels of GLUT-2 were found in late passages of HIT cells cultured with media containing 11.1 mM glucose. These data uniquely indicate that loss of beta cell function in HIT cells passed serially under high glucose conditions is caused by loss of insulin mRNA, insulin content, and insulin secretion and is preventable by culturing HIT cells under low glucose conditions. This strongly suggests potential genetic mechanisms of action for glucose toxicity of beta cells.

Animals↗

PubMed1994

Comparison of the genomes of attenuated equine herpesvirus-1 strains with their parent virulent strain.

To elucidate the virulence factors of equine herpesvirus-1 (EHV-1), we compared the genomic architecture of the HH1 strain, a Japanese isolate of EHV-1, with that of its four descendant strains obtained by serial passage in bovine kidney (BK) cells, i.e. BK77, BK161, BK271, and BK343. In preliminary studies high-passaged BK271 and BK343 lost virulence in colts, and in the present study digestion with restriction endonuclease BamHl showed that the DNAs of BK271 and BK343 differed markedly from the DNA profiles of HH1, BK77, and BK161. Employing all the molecularly cloned BamHl fragments of the HH1 strain as probes, Southern blot analysis revealed seven variable fragments. All BamHl fragments of BK343 were subsequently cloned into plasmid pUC18 and on mapping to the HH1 DNA maps, two additional variable fragments were disclosed. Finally, the precise variable regions identified in these nine fragments were fine mapped on the viral prototype genome, and among the open reading frames (ORFs) located in the variable fragments, three (ORFs 1,24, and 71) were found to have changed via serial passage in the BK cells. Thus these three ORFs are thought to play an important role on the pathogenicity of EHV-1.

Animals↗

PubMed1989

Characterization of cell lines derived from a multiply aneuploid human bladder transitional-cell carcinoma, UCRU-BL-13.

A series of cultured cell lines (designated UCRU-BL-13) has been established from different serial passages of a multiply aneuploid human bladder transitional-cell carcinoma xenografted in nude mice. Serial passage of the xenografts in vivo and of the cell lines in vitro was accompanied by shifts in the tumor ploidy, with dominance of different major peaks. Despite this, the expression of tumor markers remained constant, and consistent chromosomal markers were observed both in the 8th xenograft passage and in a subline in tissue culture established over a year apart. Chromosomal numbers reflected the predominant aneuploid peaks observed; consistent numerical and structural changes included a marker derived from chromosome 1, 8p-, -10, 11q+, and 17q+. The cell line derived from the initial xenograft comprised a mixture of transitional, adenocarcinoma and squamous carcinoma cells in early passage, but adenocarcinoma cells were absent from later passages. The lines expressed the B-blood-group antigen, histocompatibility antigens, receptors for transferrin and EGF, and reacted with a series of monoclonal antibodies (MAbs) directed to malignant human epithelial cell lines. These lines provide a model for studying the evolution of tumor heterogeneity and drug resistance in bladder carcinoma exhibiting multiple aneuploidy.

Aneuploidy↗

PubMed1990

Regulation of expression of a baculovirus ecdysteroid UDPglucosyltransferase gene.

The Autographa californica nuclear polyhedrosis virus egt gene encodes an ecdysteroid UDPglucosyltransferase which catalyzes the transfer of glucose from UDPglucose to ecdysteroid insect molting hormones. Expression of this gene allows the virus to block molting and pupation of infected insect larvae. In this study, we present the nucleotide sequence of the A. californica nuclear polyhedrosis virus egt gene and characterize egt gene expression at the transcriptional and translational levels. egt was transcribed as two 5'-coterminal mRNAs early in infection. Transferase activity was detected in infected cells and in the extracellular fluid by 3 h after infection. The majority of the activity accumulated in the extracellular fluid. We show that the egt gene product is a 60-kilodalton protein which is secreted from the infected cell. The egt gene is located in a region of the A. californica nuclear polyhedrosis virus genome which exhibited hypervariability in serially passaged virus stocks. Nucleotide sequence analysis revealed that the most common deletion occurring in these serially passaged virus isolates is located in the egt gene.

Amino Acid Sequence↗

PubMed2006

Rapid adaptation of a recombinant vesicular stomatitis virus to a targeted cell line.

Vesicular stomatitis virus (VSV) is being developed for cancer therapy. We created a recombinant replicating VSV (rrVSV) that preferentially infected Her2/neu-expressing breast cancer cells. This rrVSV did not express the native VSV-G glycoprotein (gp). Instead, it expressed a chimeric Sindbis gp which included a single-chain antibody (SCA) directed to the human Her2/neu receptor. The virus infected mouse mammary carcinoma cells (D2F2/E2) expressing Her2/neu 23-fold better than the parent cells (D2F2). However, viral growth in cultured D2F2/E2 cells was curtailed after several cycles, and viral yield was very poor at 2 x 10(4) infectious doses (ID)/ml. We performed in vitro serial passage in D2F2/E2 cells to evolve a virus with improved growth that could be used for preclinical therapy trials in mice. Fifteen passes generated an adapted virus that progressed through multiple cycles in cultured D2F2/E2 cells until all cells were infected and had a viral yield of 1 x 10(8) ID/ml. Sequencing of the entire viral genomes found only 2 mutations in the adapted virus. Both mutations occurred in the gp gene segment coding for the SCA. An additional N-glycosylation site was created by one of the mutations. The adapted virus showed higher density of gp on the viral envelope, improved infectivity, much greater stability, higher burst size, and decreased induction of cellular interferon. The specificity for cells expressing the Her2/neu receptor was unchanged. These studies demonstrate that serial passage can be used to rapidly evolve a VSV genome encoding an improved chimeric glycoprotein.

Adaptation, Physiological↗

PubMed2001

Studies on the development and stability of resistance of Helicobacter pylori to metronidazole and clarithromycin.

Thirty-one strains of Helicobacter pylori, susceptible to metronidazole and clarithromycin, were isolated from 31 biopsy samples from patients with various gastric pathologies. We investigated the possibility of selecting resistant strains and the stability of resistance by exposing isolates to increasing subinhibitory concentrations of metronidazole and clarithromycin using a serial passages technique. Resistance to metronidazole was obtained in 100% of the isolates, while 32.2% displayed resistance to clarithromycin. Resistance to metronidazole was stable in 93.5% (29 of 31) of the isolates, whereas 100% (10 of 10) of the strains were stably resistant to clarithromycin. The stability of the resistance that occurred after three passages on medium containing the two drugs was statistically significant (P <0.001). Thus, the technique of serial passages in vitro could be useful as a first screening in selecting drugs for use in therapeutic protocols for clinical trials.

Anti-Bacterial Agents↗

PubMed1990

Molecular and biological properties of c-mil transducing retroviruses generated during passage of Rous-associated virus type 1 in chicken neuroretina cells.

IC1, IC2, and IC3 are novel c-mil transducing retroviruses generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina cells. They were isolated by their ability to induce proliferation of these nondividing cells. IC2 and IC3 were generated during early passages of RAV-1 in neuroretina cells, whereas IC1 was isolated after six consecutive passages of virus supernatants. We sequenced the transduced genes and the mil-RAV-1 junctions of the three viruses. The 5' RAV-1-mil junction of IC2 and IC3 was formed by a splicing process between the RAV-1 leader sequence and exon 8 of the c-mil gene. The 5' end of IC1 resulted from homologous recombination between gag and mil sequences. Reconstitution experiments showed that serial passaging of IC2 in neuroretina cells also led to the formation of a gag-mil-containing retrovirus. Therefore, constitution of a U5-leader-delta c-mil-delta RAV-1-U3 virus represents early steps in c-mil transduction by RAV-1. This virus further recombined with RAV-1 to generate a gag-mil-containing virus. The three IC viruses transduced the serine/threonine kinase domain of the cellular gene. Hence, amino-terminal truncation is sufficient to activate the mitogenic property of c-mil. Comparison of the transforming properties of IC2 and IC1 showed that the transduced mil gene, expressed as a unique protein independent of gag sequences, was weakly transforming in avian cells. Acquisition of gag sequences by IC1 not only increased the rate of virus replication but also enhanced the transforming capacity of the virus.

Animals↗

PubMed1994

Natural occurrence of drug resistance mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates.

Reverse transcriptase-associated amino acid substitutions related to ddC, d4T, and nevirapine resistance have been found in isolates of human immunodeficiency virus type 1 (HIV-1) from patients treated with AZT only. Sequence analysis of 23 isolates documented the presence of 4 unexpected mutations at amino acid residues related to drug resistance. Two isolates contained an aspartic residue in codon 69 associated with ddC resistance, and another a change in codon 75 associated with resistance to d4T. The Y-to-C alteration in codon 181 associated with nevirapine resistance was observed in another isolate after serial passage in cell culture in the absence of drug. Changes in substitution patterns were also noted after serial passage of four AZT resistant isolates in cell culture without inhibitors. One of the strains showed changes in codons 67 and 70 to wild-type residues. Clonal analysis showed that this alteration occurred by the selection during cell culture passage of the wild-type genotype, which was present as a minority subpopulation in the initially resistant virus stock, rather than to genetic reversion. In summary, we present evidence documenting the presence of mutations associated with drug resistance in the absence of drug treatment and supporting the role played by gentic variability in the emergence of HIV-1 antiviral resistance.

Amino Acid Sequence↗

PubMed1994

Individual, culture-specific alterations in the human endothelial glutathione system: relationships to oxidant toxicity.

In this communication we report on the effects of serial passage of human umbilical vein endothelial cells on the levels of GSH and activities of selenium-dependent GSH peroxidase (SeGSHpx) and GSSG reductase (GSSGred) in confluent monolayers of human umbilical vein endothelial cells derived from 4 individual donors. The mean levels of GSH in confluent monolayers of individual human umbilical vein endothelial cells batches fell from an average of 35 nmol/mg protein to 16 nmol/mg protein from primary culture to the third passage, with little interindividual variation noted. Simultaneously, the mean activities of SeGSHpx and GSSGred remained unaltered during serial culture, again with little interindividual variation noted. However, serial passage of the cells from different individuals did not affect the sensitivity of the cells to sublethal injury induced by H2O2 (10 or 100 microM), as judged by the stimulated release of (3H)-deoxyglucose. When these GSH-dependent parameters were assayed during the coarse of the growth of an individual passage of cells, GSH levels declined following trypsinisation, but the activities of SeGSHpx and GSSGred remained essentially unaltered, when the data were standardised to cell number. On the other hand, standardisation of these data to cell protein revealed decreases in all three parameters following trypsinisation, indicating the introduction of artefacts into the data and the unsuitability of this standardisation procedure. For the first four hours following plating, all of the GSH-dependent parameters remained essentially unaltered, but began increasing thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗

PubMed2003

Attenuation of itraconazole fungicidal activity following preexposure of Aspergillus fumigatus to fluconazole.

Fluconazole (FLC), a triazole with limited activity against Aspergillus species, is frequently used as prophylaxis in leukemia patients and bone marrow transplant recipients. Prior FLC use has been associated with an increasing incidence of invasive aspergillosis in these patients. We hypothesized that prior exposure of Aspergillus fumigatus to FLC could result in altered in vitro susceptibility of this fungus to other, more active triazoles. Thus, we performed serial passages of conidia of 10 clinical isolates of A. fumigatus (all itraconazole [ITC] susceptible) on FLC-containing yeast agar glucose plates. The MICs and minimal fungicidal concentrations (MFCs) of amphotericin B, FLC, ITC, and voriconazole (VRC) for A. fumigatus conidia were measured following four passages on FLC-containing medium according to the National Committee for Clinical Laboratory Standards microdilution method. Serial passages on FLC-containing plates resulted in a fourfold increase in the MFCs (but not the MICs) of ITC for nine isolates. The attenuated ITC fungicidal activity against A. fumigatus following FLC preexposure was medium independent and was also observed against FLC-preexposed A. fumigatus hyphae with the viability staining FUN-1 dye. Moreover, FLC preexposure of A. fumigatus conidia resulted in an analogous increase in the MFCs (but not the MICs) of VRC. Our findings suggest that preexposure of A. fumigatus to FLC attenuates the in vitro fungicidal activity of subsequent ITC use against it. This phenotypic adaptation is not captured by a routine MIC determination but requires MFC measurement. The in vivo significance of this in vitro phenomenon requires further investigation.

Antifungal Agents↗

PubMed1978

Pathogenicity of equine herpesvirus: in vivo persistence in equine tissue macrophages of herpesviuus type 2 detected in monolayer macrophage cell culture.

Equine macrophages from the mammary glands of a yearling filly and an 18-year-old barren nonlactatind mare formed cell monolayers in continuous cultures. There was absence of viral cytopathic effect (CPE) in early cell culture passages. The cells from the early cell culture passages having no CPE failed to show evidence of virus or viral antigen by electron microscopic and immunofluorescence studies. Foci of CPE first appeared in the monolayer cell cultures from the filly and the mare in the 3rd and the 4th serial passages respectively, and the CPE increased on subsequent serial passages. Equine herpesvirus type 2 (EHV 2) cultures showing CPE. The macrophage monolayer culture established from the mare produced CPE foci more consistently than did the culture from the filly, and they were more numerous. Conversely, the peripheral blood mononuclear cells from the filly on cocultivation with an equine embryo kidney monolayer cell culture produced more EHV 2 CPE foci than did those from the mare. This study indicated that tissue macrophages may be a site for latent and persistent herpesvirus infections in horses.

Age Factors↗

PubMed1984

Cell fusion responsible for horizontal oncogenesis by human tumors in nude mice.

Human tumor xenotransplants derived from 4 different carcinomas were established in athymic mice and maintained by serial passage. In vivo human/mouse cell fusion and induction of neoplastic growth of adjacent mouse tissue was investigated in serial passages by isoenzyme analysis, by chromosome analysis and by cloning of induced tumorigenic mouse cells. In 2 xenotransplants human/murine hybrid isoenzyme complexes of pyruvate-glutamate transaminase, glucosephosphate isomerase and mitochondrial malate dehydrogenase were detected. Metaphases containing human and murine chromosomes were found in the first in vitro passage of excised xenotransplants. Cloning of induced mouse tumor cells yielded tumor cell lines tumorigenic in athymic as well as in immunocompetent mice.

Animals↗

PubMed2005

In vitro selection of resistance in Pseudomonas aeruginosa and Acinetobacter spp. by levofloxacin and ciprofloxacin alone and in combination with beta-lactams and amikacin.

OBJECTIVES: The aim of this study was to evaluate the ability of levofloxacin and ciprofloxacin alone and in combination with either ceftazidime, cefepime, imipenem, piperacillin-tazobactam or amikacin to select for antibiotic-resistant mutants of Pseudomonas aeruginosa and Acinetobacter spp. METHODS: Clinical strains of P. aeruginosa (n = 5) and Acinetobacter spp. (n = 5) susceptible to all the drugs used in the study were assayed. Development of resistance was determined by multi-step and single-step methodologies. For multi-step studies, MICs were determined after five serial passages on antibiotic-gradient plates containing each antibiotic alone or in combination with levofloxacin or ciprofloxacin. Acquisition of resistance was defined as an increase of >or=4-fold from the starting MIC. In single-step studies, the frequency of spontaneous mutations was calculated after a passage on plates containing antibiotics alone and in combinations at concentrations equal to the highest NCCLS breakpoints. RESULTS: Serial passages on medium containing single antibiotics resulted in increased MICs for each antibiotic; MIC increases were limited by antibiotics in combination. A decrease in the number of strains with MICs above the NCCLS breakpoints occurred when fluoroquinolones were combined with a second antibiotic for both P. aeruginosa and Acinetobacter spp. isolates. Frequencies of mutation were higher for antibiotics alone than for combinations. CONCLUSIONS: Use of combinations of fluoroquinolones with beta-lactams and amikacin reduces the risk for in vitro selection of resistant P. aeruginosa and Acinetobacter spp.

Acinetobacter↗

PubMed1985

Oncogenes: their presence and significance in squamous cell cancer of the head and neck.

DNA extracted from squamous cell carcinomas of the larynx and tongue has been shown to contain cellular transforming genes characterized by their ability to transform mouse fibroblasts into malignant foci of cells which, when subsequently cloned and grown to volume, have been found to contain human DNA sequences. This DNA has been serially passaged through subsequent populations of NIH/3T3 mouse fibroblasts. Higher malignant transformation efficiencies have been observed and reported with serial passage. Of greater significance is the repeated identification of oncogenes of identical characteristics on electrophoretic radioisotope analysis.

Carcinoma, Squamous Cell↗

PubMed1989

[Establishment and characterization of a human malignant fibrous histiocytoma line serially transplanted in nude mice].

Serial heterotransplantation of human malignant fibrous histiocytoma (MFH) derived from tibia was attempted in BALB/c nu/nu mice, and HKMFH-nu transplantable tumor line was established. This line had the following biological properties. (1) Eighteen serial passages were carried out in 41 months. (2) Morphological changes of the grafts occurred in nude mice with serial passages: During the first 6 passages, histiological picture was consistent with the common type of MFH similar to that of the original tumor, then after the 7th passage, the myxoid type coexisted with the common type, and finally the myxoid type occupied the entire grafts to form large cysts. (3) The common type grafts grew more rapidly than the myxoid type grafts. (4) Granulocytosis (neutrophilia) was observed in mice bearing the common type tumor, but not in mice bearing the myxoid type tumor.

Animals↗

PubMed1989

Genetic and phenotypic studies on Ross River virus variants of enhanced virulence selected during mouse passage.

We have passaged Ross River virus (RRV) in mice to generate variants with increased mouse virulence and attempted to relate changes in virulence to genome sequence changes. RRV NBO (zero passage in mice) is a plaque-purified clone of the mouse-avirulent strain RRV NB5092, and is of low virulence for day-old mice. During RRV NBO replication in infant mice, its virulence for day-old mice increased markedly with time. By 7 days postinfection the LD50 value of harvested virus (passage level one) was congruent to 10(4)-fold less than that of the parental virus. No further decrease in LD50 followed 10 serial passages in infant mice. However, 10th passage level virus showed increased clinical effects in week-old mice by comparison with virus from passage levels one and two. The growth kinetics of RRV variants in mice suggested that the rate and extent of RRV replication in the brain tissue determined the enhanced mouse virulence of serially passaged virus. Seven out of eight independently passaged, 10th passage level variants had changes in the E2 gene leading to one or two amino acid substitutions. The changes were at residues 212, 232, 234, 251, 341, 27 and 172, and 72 and 134 in these variants; all changes except two were nonconservative. Residues 212, 234, and 251 form part of a neutralization determinant in RRV. Changes in epitope b2 (which includes amino acids 246, 248, and 251) alter the kinetics of RRV entry into cells (P. Kerr, R. C. Weir, and L. Dalgarno, unpublished data). First and second passage level virus of enhanced virulence was unchanged in E2 or E1 gene sequences from RRV NBO. However, 1st, 2nd, and 10th passage level virus induced higher levels of virus-specific RNA synthesis than did RRV NBO in cultured BHK cells. We propose a model for the mechanism of virulence enhancement on passaging RRV NBO in mice.

Alphavirus↗