SEARCH · PubMed Health

Results for “Serial Passage”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,189 records · Page 66Linked to original sources

PubMed1993

Studies on acute myelomonocytic leukemia in LBF1 rats.

Granulocytic leukemia was induced in Long-Evans (LE) rats by using the Huggins and Sugiyama method. After serial passage the cells became transformed. The newly transformed cells could be transplanted to LBF1 hybrid rats and observed more readily. A quantity of 10(8) cells/100 g body weight was injected intravenously and after 2-3 weeks myelomonocytic leukemia developed. By examining the bone marrow, spleen and lymph nodes, cytochemical tests verified this transformation. Transplanting 10(2)-10(4) cells under the renal capsule, a quickly growing solid tumor was observed, which caused metastasis to the parathymical lymph nodes and peritoneum. The investigation of oncogene expression for the myc and ras families revealed the presence of myc p62 and ras p21 oncoproteins in the tumor cells by using monoclonal antibodies in immunohistochemical tests. LBF1 rats proved to be good models in obtaining solid tumor growth and myelomonocytic leukemias, equivalent to human M4-M5 type leukemia.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1986

Eimeria tenella: experimental studies on the development of resistance to halofuginone.

Resistance to halofuginone has been developed by serially passaging the Houghton strain of Eimeria tenella in chickens medicated with progressively greater concentrations of drug. Attempts to develop resistance to 3 ppm (as recommended for use by the manufacturer) or higher concentrations, by the method of Weppelman et al., were unsuccessful.

Animals↗

PubMed1991

SV40 recombinants carrying a d(CT.GA)22 sequence show increased genomic instability.

Repetitive d(CT.GA)n sequences are commonly found in eukaryotic genomic DNA. They are frequently located in sites involved in genetic recombination or in promoter regions. To test for their possible biological function, a d(CT.GA)22 synthetic sequence was introduced into the genome of SV40, since it constitutes an appropriate model system for eukaryotic chromatin. When SV40 infects permissive cells, it proliferates in the form of a minichromosome. The simple repetitive sequence indicated above was inserted at the unique HpaII site of SV40 (at nt 346), and the genomic stability of SV40 recombinants carrying the d(CT.GA)22 sequence (SV/CT22 viruses) was analyzed. Upon serial passage through permissive CV1 cells, SV/CT22 recombinants show an increased production of defective viruses. Generation of SV/CT22 variants is likely to take place via recombination between and within viral molecules. The enhancement of the rate of recombination induced by the repetitive sequence is likely to be related to its known propensity to form triple-stranded structures. Many different variants coexist in the same viral population indicating that the mechanism by which they are produced is not unique. One variant (SV/X), showing a replicative advantage, was characterized in detail. Variant SV/X accounts for a large proportion of the total viral population. Its genomic organization corresponds to a tandem duplication of an early SV40 DNA fragment spanning from approx. nt 3200-nt 160. Variant SV/X contains a duplicated SV40 ori.

Base Sequence↗

PubMed1985

Comparison of Mycoplasma ovipneumoniae isolates using bacterial restriction endonuclease DNA analysis and SDS-PAGE.

Sixteen isolates of Mycoplasma ovipneumoniae recovered from the nasal tract or lungs of sheep from different flocks in New Zealand were examined by bacterial restriction endonuclease DNA analysis (BRENDA) using EcoR1 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). All isolates gave BRENDA patterns which differed entirely from one another. Following 20 serial passages (corresponding to approximately 67 generations) of an isolate, no change was detected in the BRENDA pattern. When eight isolates were examined by SDS-PAGE most bands were common but, nevertheless, each isolate was unique in the sense that they differed from one another in one or more bands. The marked heterogeneity of patterns observed when strains of M. ovipneumoniae are compared by BRENDA, together with the stability of such patterns over many generations, will enable this approach to be used to study the epidemiology of individual strains of M. ovipneumoniae within a flock.

Animals↗

PubMed1987

Relative rates of Aujeszky's disease virus attenuation, as assessed in mice.

Field strains of Aujeszky's disease virus (ADV) were attenuated by heat treatment and serial passage at sub-optimal growth temperatures in chicken embryo fibroblasts (CEF). At chosen passage levels, virus was titrated in cell culture and in mice. For each strain, the pathogenicity was expressed as a mouse lethal index (MLI), defined as the inverse of the log10 (CCID50:LD50). MLIs determined for field strains displayed a wide range of comparatively high values. The attenuation of field strains was accompanied by a rapid fall in MLI values, particularly in the initial stages. Heat-treated ADV attenuated faster than untreated ADV, when passaged at 30 degrees C. Passage at 27 degrees C resulted in considerably accelerated attenuation compared to passage at 30 degrees C, in the case of both untreated and heat-treated ADV. MLIs were determined for attenuated ADV strains that had been tested in 6-day-old piglets. Low MLI values were found to correlate with low virulence in piglets and high MLI values with virulence.

Animals↗

PubMed1986

Changes in activity and amount of active elongation factor 1 alpha in aging and immortal human fibroblast cultures.

Stoichiometrically estimated amounts of active elongation factor, EF-1 alpha, remain constant in serially passaged Phase II cultures of human fibroblasts, MRC-5, but decrease by 45% towards the end (Phase III) of their lifespan. Catalytic activity of EF-1 alpha is also reduced by 35% in Phase III old cells. The SV40 transformed immortal cell line MRC-5V2 has 30% higher levels of active EF-1 alpha without significant increase in its catalytic activity. Low-serum-associated G1 arrest of normal and transformed cells reduces amounts of active EF-1 alpha by 35% and 20%, respectively. Catalytic activity, however, is reduced rapidly only in G1 arrested normal cells and not in transformed cells. Even though the cell cycle-related changes are reversible both in normal and transformed cells, the age-related decline in amounts of active EF-1 alpha and its activity are irreversible and, most probably, crucial.

Catalysis↗

PubMed1989

Levels of DNA methylation in diploid and SV40 transformed human fibroblasts.

Total 5-methylcytosine (5mC) has been measured in the DNA of cultured human cells by HPLC chromatography. In agreement with other reports, the level of 5mC declines continuously during the serial passaging of diploid fibroblasts, strain MRC-5. On the contrary, fibroblasts infected with SV40, which have acquired most of the properties of transformed cells, maintain a constant level of 5mC, prior to dying out in the crisis period. MRC-5 and Werner's syndrome fibroblasts fully transformed and immortalised by SV40, also maintain a constant level of 5mC. These results indicate that the ability of cells to maintain DNA methylation is a necessary but not a sufficient step for immortalisation.

Cell Survival↗

PubMed1989

Reversible dedifferentiation and redifferentiation of a melanized cell line from a goldfish tumor.

We reported previously the isolation of a melanized cell line that can undergo reversible dedifferentiation and redifferentiation. A heavily pigmented cell line, designated as P15, originally isolated by fish serum-induced melanization of some GEM 81 cells, cloned and serially passaged in fish serum medium, became noticeably less pigmented after several months in fetal calf serum medium and completely unpigmented after another year in the same medium. Addition of fish serum to the medium of this dedifferentiated cell line, designated P15D, induced pigmentation within a week. This re-induced pigmented cell line, designated as P15DI, became unpigmented when cultured in fetal calf serum medium for one month. We report here that the dedifferentiation of P15 occurs in two stages. One week after withdrawal of fish serum, the specific activity of tyrosinase of the culture dropped by approximately 70% and remained at this reduced level for at least one month. After one year, the specific activity of tyrosinase had dropped to a barely detectable level and the culture became completely unpigmented (P15D). Electron microscopic studies showed that the P15D cells have no melanosomes, probably no large vesicles for melanosome formation, but some dopa-positive trans-Golgi network (TGN). Addition of fish serum to the growth medium of P15 cultures led to a steady increase in the specific activity of tyrosinase, detectable after one day. There was also an increase in the amount of dopa-positive TGN within one day. Melanosomes first appeared after three days and became numerous after one week. Upon removal of fish serum, these re-induced cells (P15D1) underwent a rapid decrease in the specific activity of tyrosinase, reaching, after eight days, the basal level seen in P15D cells. We also report that a protein designated as p75 (Mr approximately 75,000), previously shown to be associated with melanosomes in two melanized cell types of goldfish origin, is present in all melanized cell lines, including P15 and P15DI but absent in unmelanized cell lines, including P15D.

Animals↗

PubMed2003

Reversible stepwise negative regulation of CYP1A1 in cultured rat epidermal cells.

When serially passaged, rat epidermal keratinocytes lose the inducibility of CYP1A1 gene expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. In present experiments, loss of CYP1A1 inducibility occurred in a stepwise fashion, with some keratinocyte lines progressing through a transiently inducible state before becoming completely uninducible. The negative regulation occurred at the level of transcription, but the aryl hydrocarbon receptor (AhR) pathway appeared fully functional. Transient and stable transfection of uninducible cells with reporter constructs containing up to 4.2kb of the CYP1A1 5'-flanking region resulted in a TCDD-inducible increase in luciferase activity, despite no induction of the endogenous gene. Co-treatment with protein synthesis inhibitors and TCDD restored responsiveness of the endogenous CYP1A1 gene, indicating that the negative regulation was reversible and likely mediated by a labile protein. Together, these results demonstrate a novel mechanism of CYP1A1 transcriptional repression that does not involve any previously reported negative regulatory elements for CYP1A1.

Adaptation, Physiological↗

PubMed2004

Comparison of mitochondrial cytochrome oxidase 1 DNA sequences from Necator americanus hookworms maintained for 100 generations in golden hamsters (Mesocricetus auratus) and hookworms from natural human infections.

The human hookworm Necator americanus was maintained through one hundred generations in the golden hamsters. The strain is now routinely maintained in laboratory hamsters through serial passage, and is the laboratory strain of choice for vaccine studies. Comparison of the mitochondrial cytochrome oxidase 1 (cox-1) sequences was shown previously to be useful for comparing the genetic structure of populations of N. americanus in China. Cytochrome oxidase 1 genes were amplified by the polymerase chain reaction, and the sequences compared to those of N. americanus recovered from infected humans from several regions in China. Sequence comparison revealed little difference between the laboratory strain and the field isolates at the cox-1 locus, but also indicated that the laboratory strain is represented by a single cox-1 haplotype. These results suggest that the laboratory strain of N. americanus has undergone a severe genetic bottleneck, and that the genetic diversity in other genes, including potential vaccine antigens, could be similarly limited.

Animals↗

PubMed2004

No cross-resistance or selection of HIV-1 resistant mutants in vitro to the antiretroviral tripeptide glycyl-prolyl-glycine-amide.

The chemically modified tripeptide glycyl-prolyl-glycine-amide (GPG-NH(2)) inhibits replication of HIV-1 in vitro, probably by interfering with capsid formation. This study was aimed at determining cross-resistance between antiretroviral drugs and GPG-NH(2), and whether resistance to GPG-NH(2) can be induced in vitro. Fifty-five clinical HIV-1 isolates with different resistance-related mutations were tested for susceptibility to GPG-NH(2). No correlation between NRTI-, NNRTI- or PI-resistance and efficacy of GPG-NH(2) was found, indicating the lack of cross-resistance. Serial passages were performed with GPG-NH(2), and with lamivudine, and genotypic or phenotypic changes were determined. Resistance to lamivudine was detected after six passages. No resistance to GPG-NH(2) was generated after 30 passages in two parallel series. However, one mutation (T107I) in the p24 gene was detected in both series, but this mutation was not associated with decreased sensitivity to GPG-NH(2).

Anti-HIV Agents↗

PubMed2004

In vitro selection of drug-resistant varicella-zoster virus (VZV) mutants (OKA strain): differences between acyclovir and penciclovir?

Varicella-zoster virus (VZV) mutants were isolated under the pressure of different classes of antiviral compounds: (i) drugs that depend on the viral thymidine kinase (TK) for their activation, i.e. acyclovir (ACV), brivudin (BVDU), penciclovir (PCV) and sorivudine (BVaraU); (ii) drugs that are independent of the viral TK for their activation, i.e. 2-phosphonylmethoxyethyl (PME) derivatives of adenine (PMEA, adefovir) and 2,6-diaminopurine (PMEDAP); and (iii) drugs that do not require any metabolism to inhibit the viral DNA polymerase, i.e. foscarnet (PFA). Drug-resistant virus strains were obtained by serial passage of the OKA strain in human embryonic lung (HEL) fibroblasts and the different drug-resistant mutants were subsequently evaluated for their in vitro susceptibility to a broad range of antiviral drugs. Virus strains emerging under the pressure of ACV, BVDU and BVaraU were cross-resistant to all drugs that depend on the viral TK for activation, but remained susceptible to the acyclic nucleoside phosphonates (i.e. PMEA, PMEDAP and the 3-hydroxy-2-phosphonylmethoxypropyl derivatives of adenine (HPMPA) and cytosine (HPMPC, cidofovir)) and PFA. In contrast, the virus strains selected under pressure of PCV were resistant to PCV, ACV, PMEA and PFA; but not BVDU, BVaraU, GCV, HPMPC or HPMPA. Similar patterns of drug susceptibility were noted for the virus strains selected under the pressure of PMEA or PFA, pointing to an alteration in the viral DNA polymerase as basis for the resistant phenotype selected by PCV, as well as PMEA and PFA. In contrast, the resistant phenotype selected by ACV as well as BVDU and BVaraU may be attributed primarily to mutations in the viral TK gene. Our data thus indicate that ACV and PCV select in vitro for different drug-resistant VZV phenotypes; whether this is also the situation in vivo remains to be investigated.

2-Aminopurine↗

PubMed2004

Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase-primase.

The aminothiazolylphenyl-containing compounds BILS 179 BS and BILS 45 BS are novel inhibitors of the herpes simplex virus helicase-primase with antiviral activity in vitro and in animal models of HSV disease. To verify the mechanism of antiviral action, resistant viruses were selected by serial passage or by single-step plaque selection of HSV-1 KOS in the presence of inhibitors. Three resistant isolates K138r3, K22r5, and K22r1 were found to be 38-, 316-, and 2500-fold resistant to BILS 22 BS, a potent analog of BILS 45 BS. All three viruses had growth properties in vitro similar to wild-type HSV-1 KOS but they were sensitive to acyclovir. Cutaneous and intra-cerebral inoculation of mice with K22r1 or K22r5 resulted in pathogenicity equivalent to that of HSV-1 KOS. Both isolates were fully competent for reactivation from latency following corneal inoculation. Helicase-primase purified from cells infected with resistant viruses showed decreased inhibition in an in vitro DNA-dependent ATPase assay that correlated well with antiviral resistance. Marker transfer experiments and DNA sequence analysis identified single base pair mutations clustered in the N-terminus of the UL5 gene that resulted in single amino acid changes in the UL5 protein. Taken together, the results indicate that helicase-primase inhibitors prevent HSV growth by inhibiting HSV helicase-primase through specific interaction with the UL5 protein.

Animals↗

PubMed2004

A novel model of HPV infection in meshed human foreskin grafts.

The present study describes a novel meshing procedure that provided successful low-risk papillomavirus propagation and reproducible wart induction in human foreskin xenografts. The initial HPV6 and/or 11 inocula were collected from clinically excised human wart tissues and confirmed to be free of HPV16, 18 and 31 by PCR analysis. Human foreskin grafts were collected from a circumcision clinic, and pre-inoculated with HPV virions by scarification. Meshing was carried out with a Zimmer Skin Grafter Mesher. Grafts were cut to appropriate size (1cm x 1cm or 5mm x 5mm) for cutaneous or subcutaneous grafting to NIH-nu-bg-xid mice under halothane anesthesia. Cutaneous xenografts were dressed with antibiotics and protective band-aids for 3 weeks. In the paralleled experiment using the same viral stock containing both HPV6 and 11, and matched grafts, no visible papillomas were observed in non-meshed cutaneous xenografts (n = 4 up to 6 months). In comparison, six of eight cutaneous xenografts treated with the meshing procedure formed visible papillomas within 4 months. This high frequency of distinct papilloma induction over the surface of meshed xenografts were reproduced in subsequent experiments with viral stocks containing both HPV11 and 6 (8 out of 10 grafts), or with a single-type HPV11 inoculum (80-100%). In contrast, an initial viral stock of single-type HPV6 provided lower frequency and more delayed papilloma induction. Serial passage of HPV6 in the meshed xenograft appeared to improve both the induction frequency and growth rate up to the 3rd generation. Histology, in situ hybridization, and immunohistochemical analysis revealed similarity of xenograft warts to those observed in the clinic. The highly reproducible papilloma induction rate and successful viral stock propagation associated with the meshing procedure provide a novel feature in the HPV xenograft model.

Animals↗

PubMed2005

KP-1212/1461, a nucleoside designed for the treatment of HIV by viral mutagenesis.

We report the activities of a novel nucleoside analog against HIV. This nucleoside (KP-1212) is not a chain terminator but exerts its antiviral effects via mutagenesis of the viral genome. Serial passaging of HIV in the presence of KP-1212 causes an increase in the mutation rate of the virus leading to viral ablation. HIV strains resistant to KP-1212 have not yet been isolated. Quite to the contrary, virus treated with KP-1212 exhibited an increased sensitivity not only to KP-1212 but also to another nucleoside reverse transcriptase inhibitor (NRTI), zidovudine. HIV strains resistant to other NRTIs (e.g. zidovudine, lamivudine, stavudine, abacavir, etc.) exhibited no cross-resistance towards KP-1212. Multiple assays confirmed that KP-1212 has a favorable (low) genotoxicity profile when compared to some approved antiviral nucleosides. In addition, KP-1212 is not toxic to mitochondria nor does it exhibit any inhibitory effects on mitochondrial DNA synthesis.

Animals↗

PubMed2005

Polymer-bound 6' sialyl-N-acetyllactosamine protects mice infected by influenza virus.

To develop a mouse model for testing receptor attachment inhibitors of human influenza viruses, the human clinical virus isolate in MDCK cells A/NIB/23/89M (H1N1) was adapted to mice by serial passaging through mouse lungs. The adaptation enhanced the viral pathogenicity for mice, but preserved the virus receptor binding phenotype, preferential binding to 2-6-linked sialic acid receptors and low affinity for 2-3-linked receptors. Sequencing of the HA gene of the mouse-adapted virus A/NIB/23/89-MA revealed a loss of the glycosylation sites in positions 94 and 163 of HA1 and substitutions 275Asp-->Gly in HA1 and 145Asn-->Asp in HA2. The four mouse strains tested differed significantly in their sensitivity to A/NIB/23/89-MA with the sensitivity increasing in the order of BALB/cJCitMoise, C57BL/6LacSto, CBA/CaLacSto and A/SnJCitMoise strains. Testing of protective efficacy of the polyacrylamide conjugate bearing Neu5Acalpha2-6Galbeta1-4GlcNAc trisaccharide under conditions of lethal or sublethal virus infection demonstrated a strong protective effect of this preparation. In particular, aerosol treatment of mice with the polymeric attachment inhibitor on 24-110 h after infection completely prevented mortality in sensitive animals and lessened disease symptoms in more resistant mouse strains.

Amino Sugars↗

PubMed2006

In vitro selection of mutations in human immunodeficiency virus type 1 reverse transcriptase that confer resistance to capravirine, a novel nonnucleoside reverse transcriptase inhibitor.

Capravirine (CPV; formerly AG1549 and S-1153) is a novel, nonnucleoside reverse transcriptase inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1) that has demonstrated potent in vitro antiviral activity against several HIV-1 laboratory strains and clinical isolates with EC50 values ranging from 0.7 to 10.3 nM. In this study, we evaluated the resistance and cross-resistance profiles of CPV through selection of resistant HIV-1 variants from in vitro serial passage of HIV-1 NL4-3 and HIV-1 IIIB and by performing susceptibility assays on HIV-1 variants constructed to contain CPV-specific amino acid substitutions in reverse transcriptase (RT). Results demonstrate that HIV-1 variants selected at increasing CPV concentrations contained multiple substitutions in diverse patterns including L100I, Y181C, G190E and/or L234I in various combinations with K101R/E, K103T, V106A/I, V108I, E138K, T139K, A158T, V179D/I/G, Y188D, V189I, G190A, F227C, W229R, L234F, M230I/L and P236H/T. Interestingly, HIV-1 variants constructed to contain the T215Y zidovudine (AZT)-resistance associated substitution with CPV-resistance associated substitutions V106A, Y181C, F227C, F227L, L234I or V106A/F227L demonstrated 2.4-5.4-fold increased susceptibility to CPV. Results also demonstrate that the CPV-resistance associated substitutions Y181C, F227C, F227L and L234I reverse the phenotypic resistance to AZT conferred by the T215Y substitution.

Anti-HIV Agents↗

PubMed2006

Phenotype properties of a novel spontaneously immortalized odontoblast-lineage cell line.

Here we report on the spontaneous immortalization upon serial passages of mouse fetal dental papilla cells, which present odontoblast phenotype features. The cells named odontoblast-lineage cell (OLC) produced dentin extracellular matrix proteins, such as DSP and DMP1, and maintained transcripts of various matrix components as osteopontin, BMP-4, procollagen-1, and MEPE. The addition of osteogenic differentiation medium with beta-glycerophosphate and ascorbic acid was effective for inducing calcification and mineralization in vitro in cell cultures for up to 28 days. For the first time, we investigated the expression of Lhx6 and Lhx7 genes during induced biomineralization, since these new members of LIM homeodomain proteins have been recently proposed tracking odontoblastic phenotypes. Our results indicate that beta-glycerophosphate treatment of OLC cultures decreases Lhx6 transcript levels in vitro. Our findings proved odontoblast phenotype-specificity, which demonstrates that this novel odontoblast-lineage cell line is a valuable tool for future experiments in odontology.

Animals↗