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Results for “Serial Passage”

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PubMed1990

Glycosaminoglycan synthesis by smooth muscle cells of differing phenotype and their response to endothelial cell conditioned medium.

The synthesis of glycosaminoglycans (GAG) by contractile and irreversible synthetic phenotypes of vascular smooth muscle cells (SMC), and their response to endothelial cell conditioned medium (ECCM), has been investigated. Contractile SMC, (with a high volume fraction of myofilaments) were obtained by culturing freshly isolated rabbit aortic SMC for 3 days in primary culture. Irreversible synthetic SMC (with a low volume fraction of myofilaments) were obtained by serially passaging SMC to achieve more than 5 cumulative population doublings. In fresh medium both phenotypes produced significant amounts of GAG, but irreversible synthetic cells were more than twice as active on a per cell and cell volume basis. The proportions of individual GAG also changed with change in phenotype. Hyaluronic acid (HA) was the predominant GAG (78%) synthesised by contractile SMC but was significantly reduced (47%) in the irreversible synthetic cells with a corresponding increase in sulphated GAG (SGAG). The changed levels in GAG synthesis were independent of SMC growth. Both phenotypes responded to ECCM from bovine endothelial cells (EC) and significantly increased their synthesis of GAG and by the same relative amounts (50-100%). This response was density dependent, with ECCM from low and high density cultures of EC producing maximal responses and EC of intermediate densities producing minimal increases. Furthermore, dense cultures of EC preferentially stimulated SGAG. These findings show that an increase in synthesis of SMC GAG, and especially sulphated GAG as is found in atherosclerosis, may occur either through a change in phenotype or through endothelial mediated stimulation of GAG synthesis by either phenotype.

Animals↗

PubMed1992

Morphological characteristics of a transplantable nephroblastoma (NB-Y) in F344 rats and the relation of tumour growth to hyper-reninaemia in NB-Y-bearing rats.

A transplantable tumour, designated NB-Y, was established from a spontaneous nephroblastoma in an F344 rat. NB-Y was serially passaged in syngeneic rats by subcutaneous implantation up to the 49th generation. The transplants grew into nodules with an average diameter of 5 cm and average weight of 92.9 g 4 weeks after implantation. The primary tumour and NB-Y consisted mainly of sheets or clusters of undifferentiated blastemal cells, which reacted immunohistochemically for vimentin but not for keratin. Renin-containing cells were observed in the small blood vessel walls within the primary tumour, but neoplastic cells of both primary tumour and NB-Y failed to stain for renin. Plasma renin activity was significantly higher (40.7 ng per ml per h) in transplanted rats 4 weeks after implantation compared with non-transplanted controls (28.0 ng per ml per h). Hyperplastic juxtaglomerular cells were often observed in rats bearing NB-Y. Sinusoidal dilatation was present in the liver, adrenal glands, pituitary gland and bone marrow of recipients, suggesting abnormal blood flow provoked via the renin-angiotensin system. The present study revealed the development of hyper-reninaemia in NB-Y-bearing rats, but its pathogenesis remains unknown.

Anemia↗

PubMed1977

Electron-microscopic appearance of the DA virus, a demyelinating murine virus.

The DA virus is a neurotropic murine virus which can induce acute encephalomyelitis in suckling mice and a chronic myelopathy in weanlings. The agent has been attenuated by serial passage in baby hamster kidney (BHK-21) cells. When attenuated virus is inoculated in 8-week-old C3HeJ mice a myelopathy of delayed onset with prominent demyelination of lateral and anterior columns occurs. The DA virus is believed to be related to the Theiler murine encephalomyelitis (TME) viruses because of the similar clinical and pathological conditions which it causes, and because neutralization tests indicate shared antigens between it and GD7, a TME virus. This paper reports electron-microscopic studies of BHK-21 cells infected with DA virus. The cells were prepared 24 and 48 hr after inoculation. Cytopathic effects were observed and infected cells contained plaques consisting of numerous 25 nm virus particles in crystalline array. The virions were exclusively intracytoplasmic and were morphologically indistinguishable from human poliomyelitis virus. These observations appear to establish DA as a picorna virus, related to the TME virus group. The chronic myelopathy caused by DA may prove relevant to chronic demyelinative myelopathies in man, such as multiple sclerosis, and also to amyotrophic lateral sclerosis.

Animals↗

PubMed1994

Culture from human leptomeninges of cells containing neurofilament protein and neuron-specific enolase.

Actively dividing cells cultured from human leptomeninges obtained at autopsy and from human pia obtained at neurosurgery expressed two antigens characteristic of a mature neuronal phenotype: the 160 kDa neurofilament protein (NF-M) and neuron-specific enolase (NSE). The cells lacked typical glial, epithelial and endothelial markers but did contain vimentin, a protein normally associated with but not limited to mesenchymally derived cell types. Immunocytochemical results using redundant antibodies were consistent over serial passage and were confirmed by Western blot analysis. Morphologically the cells were pleiomorphic with frequent long, NF-M + processes. The antigenic characteristics of the cultured leptomeningeal and pial cells were similar to those exhibited by human autopsy and biopsy brain-derived cultures described previously, suggesting that the latter may have originated from pial cells adherent to the autopsy and biopsy brain tissue specimens.

Adult↗

PubMed1991

Skeletal alkaline phosphatase specific activity is an index of the osteoblastic phenotype in subpopulations of the human osteosarcoma cell line SaOS-2.

During continuous culture with serial passage, the human osteosarcoma cell line SaOS-2 showed a time-dependent decrease in skeletal alkaline phosphatase (ALP) activity. Because this was indicative of heterogeneity, subpopulations of SaOS-2 cells were isolated from replicate low-density cultures. The subpopulations were less heterogeneous and more stable (with respect to ALP) than the parent population. ALP specific activity in the subpopulations ranged from 0.05 to 2.3 U/mg protein, and cytochemical analyses indicated multiple steady-state levels of ALP activity per cell. The amount of ALP activity in SaOS-2 subpopulations was proportional to collagen production ([3H]proline incorporation into collagenase-digestible protein; r = .84, P less than .005), and to parathyroid hormone (PTH)-linked synthesis of cyclic adenosine monophosphate (cAMP) (r = .88, P less than .01). From these data, we inferred that ALP activity in SaOS-2 cells can provide a useful index of the osteoblastic phenotype, and that ALP activity, collagen production, and PTH-linked adenylate cyclase were coordinately regulated in these osteoblast-like osteosarcoma cells (ie, selection of subpopulations for ALP activity coselected for collagen synthesis and PTH-linked synthesis of cAMP). Further comparative studies showed that micromolar fluoride concentrations stimulated cell proliferation ([3H]thymidine incorporation into DNA) in low-ALP SaOS-2 subpopulations, but not in high-ALP cells (P less than .001), and that this differential sensitivity to fluoride was associated with an inverse correlation between fluoride-sensitive acid phosphatase and ALP activities (r = -.91, P less than .001).

Alkaline Phosphatase↗

PubMed1986

Synthesis of basement membrane-specific macromolecules by cultured human microvascular endothelial cells isolated from skin of diabetic and nondiabetic subjects.

Microvascular endothelial cells isolated from abdominal skin of diabetic and nondiabetic adults were maintained in culture by serial passage. Both cell types showed typical endothelial cell morphology, expressed factor VII-associated antigen, contained Weibel-Palade bodies, and produced an extensive subendothelial extracellular matrix containing type IV (basement membrane) procollagen. Biosynthetic studies using radioactive amino acids indicated that under the conditions of cell culture the matrix proteins newly synthesized by MEC of both cell types were similar in type and amount. Both cell types produced type IV procollagen, laminin, and fibronectin, which were deposited in the matrix. Electron microscopy showed that the matrices of both cell types had a similar multilayered, discontinuous, filamentous ultrastructure. Immunoperoxidase staining showed type IV collagen to be distributed similarly, in a fibrillar meshwork, in both matrices. The extractability of individual matrix macromolecules from both matrices was identical; 4 M urea or guanidine-HCl partially removed fibronectin and thrombospondin, but reducing agent was required to solubilize type IV procollagen. The results suggest that diabetic microangiopathy is not due to an inherent defect in the endothelium, and that this in vitro system may be useful for examining environmental factors possibly involved in its development.

Aged↗

PubMed1982

Tunicamycin-resistant mutations in mouse FM3A cells.

Tunicamycin is an antibiotic that inhibits the oligosaccharide synthesis of glycoproteins. It greatly suppressed the growth of cultured mouse mammary carcinoma FM3A cells, when added to growth medium at concentrations of more than 0.1 microgram/ml. We have developed a single-step selection system for quantitatively detecting mutations resistant to the antibiotic in FM3A cells. Mutant colonies resistant to 1-1.2 micrograms tunicamycin per ml (the optimal concentration of the selecting agent) appeared at a frequency of 10(-4) to 10(-5) in an unmutagenized population, but they increased over 50-fold in the population mutagenized with 0.5 microgram N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) per ml for 2 h and selected under optimal conditions for the time of mutation expression and cell density in selective medium. Fluctuation analysis, by the method of Luria and Delbrück, revealed that tunicamycin-resistant mutations occurred at random during proliferation in normal medium at a rate of 1.2 x 10(-6) per cell per generation. So far 45 spontaneous and MNNG-induced mutant lines have been isolated and serially passaged in the absence of tunicamycin. These mutant lines all inherited their resistance for more than 60 generations. The mutants examined in detail were 12- to 26-fold more resistant than wild-type cells in terms of the D10 value, the concentration of tunicamycin reducing the plating efficiency to 10% of the control. In the hybrids between wild-type and mutant cells the tunicamycin resistance behaved in a co-dominant manner. Tunicamycin inhibited the incorporation of [3H]mannose into the acid-insoluble cell fraction; in this respect, mutant cells were over 30-fold more resistant than wild-type cells. Possible mechanisms of tunicamycin resistance are discussed.

Animals↗

PubMed1982

Reduced pathogenicity associated with a small plaque variant of the Egyptian strain of Rift Valley fever virus (ZH501).

Variants of Rift Valley fever virus producing plaques in CER cells of four different sizes are described. A plaque-forming unit (PFU) variant forming minute plaques was isolated and purified. Virus derived from this variant was not pathogenic to adult Swiss albino mice by the intraperitoneal (i.p.) route and was less pathogenic than the parent strain (ZH501) to adult Sprague Dawley rats by i.p. route, but produced typical severe liver necrosis in adult Syrian hamsters with intranuclear and intracytoplasmic eosinophilic inclusions. Antigen and antiserum to the minute variant prepared in mice reciprocally cross-reacted with antisera and antigens of the original strain (ZH501) in the complement fixation test. Plaque size of the minute variant remained constant after serial passages in cell culture and in suckling mouse brain. When the minute plaque variant was passaged i.p. in hamsters, virus which formed large plaques in CER cells was recovered from the hamster sera.

Animals↗

PubMed1983

Isolation of a unique retrovirus, MNV-1, from Macaca nemestrina.

Cell cultures established from the spleen of a Macaca nemestrina with enzootic retroperitoneal fibromatosis (ERF) spontaneously released a unique retrovirus. Throughout 14 serial passages, the spleen cell cultures remained fibroblastic and no cytopathic effect was evident. The virus incorporates [3H]uridine, contains an RNA-dependent DNA polymerase (RDDP), has a buoyant density of 1.15 g/cm3 in sucrose, and was designated MNV-1. Virion-associated reverse transcriptase showed no preference for either Mg2+ or Mn2+ in standard RDDP assays. Complementary DNA (cDNA) transcribed from polyadenylated MNV-1 RNA hybridized to genomic DNA and RNA extracted from diseased tissues but not to nucleic acids from normal tissues of a healthy Macaca nemestrina or a Macaca mulatta. MNV-1 is therefore exogenous to these species. MNV-1 had no detectable homology to the endogenous macaque virus isolates MAC-1 and MMC-1. Liquid hybridization of MNV-1 cDNA to viral RNA derived from exogenous and endogenous subhuman primate retroviruses (SiSV(SSAV), GALV-SF, BaEV-M7, and BILN) did not reveal any significant sequence homologies. In addition, MNV-1 does not share homology with bovine leukemia virus or Mason-Pfizer monkey virus as determined by Southern blot hybridization. We conclude that MNV-1 is a unique retrovirus which has not previously been described. As the ultrastructure of virions in in vitro cell cultures, as well as disease involved tissue, show some particles with type C morphology and others with type D morphology, MNV-1 may be comprised of more than one component.

Animals↗

PubMed1983

High-titer SV40 replication in human fibroblast cell lines derived from patients with Wiskott-Aldrich syndrome.

Human fibroblast cell lines established from skin biopsies of patients with Wiskott-Aldrich syndrome (WAS), a sex-linked immunodeficiency disorder, were found to have unusually high sensitivity to SV40 infection. When examined by immunofluorescence, two of the cell lines showed almost 100% positive staining for tumor and viral antigens 4 days after infection, while the third cell line showed 75% positive cells for both antigens. Marked cytopathic changes were seen in infected cultures and viral yields of 10(8) pfu/ml were obtained. After six serial passages in WAS cells, the viral DNA was examined by restriction endonuclease analysis and found to have HindIII cleavage pattern similar to that of DNA from SV40 grown in monkey cells.

Antigens, Viral↗

PubMed1984

A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2.

Following serial passage of Sindbis virus (SV) on Aedes albopictus mosquito cells a mutant (SVap15/21) was isolated which in chick cells produced small plaques and was temperature sensitive (ts). At 34.5 degrees this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SVap15/21 was RNA+ at both 34.5 and 40 degrees and on the basis of complementation tests carried out at 40 degrees, was assigned to complementation group E. The block in the replication of this mutant, like that of ts20, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SVap15/21 were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SVstd). Analysis of revertants of SVap15/21 suggests a causal relationship between PE2 and E2 hyperglycosylation and the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range.

Aedes↗

PubMed1985

Sindbis virus mutants able to replicate in methionine-deprived Aedes albopictus cells.

Previous work from this laboratory has shown that the replication of Sindbis virus (SV) in Aedes albopictus cells is sensitive to methionine withdrawal. This sensitivity is thought to reflect a diminished concentration of S-adenosylmethionine (Ado Met) resulting from methionine starvation. Serial passage of SV on Ae. albopictus cells maintained in low concentrations of methionine gave rise to a population of mutants whose replication in mosquito cells was resistant to methionine starvation. In vertebrate cells, these mutants were also resistant to inhibition by cycloleucine. We favor the hypothesis that the adaptation to low methionine reflects the accumulation of mutations resulting in a viral RNA "cap" methyltransferase with an increased affinity for Ado Met.

Aedes↗

PubMed1985

The herpes simplex virus amplicon. IV. Efficient expression of a chimeric chicken ovalbumin gene amplified within defective virus genomes.

cDNA sequences of the chicken ovalbumin gene were fused to an alpha (immediate early) promoter of herpes simplex virus and to genomic ovalbumin 3'-flanking sequences. The chimeric alpha-ovalbumin gene was introduced into defective virus genomes which were stably propagated in serially passaged virus stocks in the presence of helper virus. Analyses of polypeptides synthesized in cells infected with the resultant defective virus stocks revealed the abundant expression of the chimeric alpha-ovalbumin gene. The presence of introns was not essential for this expression.

Carcinoma, Squamous Cell↗

PubMed1986

A herpes simplex virus mutant is temperature sensitive for reactivation from the latent state: evidence for selective restriction in neuronal cells.

When the replicative defect in the HSV-1 temperature sensitive mutant tsI was repaired, the agent derived (RI-1) was found to possess an additional temperature sensitive lesion limiting its reactivation from the latent state. Thus, when spinal ganglia from latently infected mice were scored for reactivation by cocultivating them with indicator cells in vitro, significantly more were found to be positive at 31 degrees than at 38.5 degrees. To assess a possible relationship between reactivation and replication in neurons, the replication of RI-1 in murine C1300 neuroblastoma cells was studied. In these cells, RI-1 was severely restricted, and viral replication was delayed at 38.5 degrees. Serial passage of RI-1 in neuroblastoma cells at the restrictive temperature resulted in selection of an agent which gained both the capacity to replicate efficiently in neuroblastoma cells and reactivate from the latent state at 38.5 degrees. However, the replication pattern of this neuron adapted virus in mouse embryo fibroblasts remained unchanged from the parental RI-1. Taken together, these results indicate that RI-1 possesses a neuron specific temperature sensitive replicative lesion which is also manifest during reactivation from the latent state.

Animals↗

PubMed1986

Butyrate-induced reversal of herpes simplex virus restriction in neuroblastoma cells.

The synthesis of herpes simplex virus (HSV) in mouse neuroblastoma cells (NB, clone 41A3) is restricted. There was a disappearance of infectious virus upon serial passage of infected cells. NB cells treated with sodium-n-butyrate for 24 hr before infection synthesized 200-2000 times more HSV than untreated cells. Infectious center assays demonstrated that the number of cells capable of producing HSV was increased as a result of butyrate pretreatment. Although host protein synthesis was inhibited by HSV infection, viral-induced protein and DNA syntheses were not detected in the absence of butyrate. Cycloheximide blocked the induction of permissiveness by butyrate suggesting that a protein(s) was responsible for allowing HSV synthesis in NB cells. Regulatable host factors involved in HSV replication in neural cells can be studied in the system described.

Animals↗

PubMed1986

Friend murine leukemia virus and spleen focus-forming virus expression in highly malignant interferon-sensitive and interferon-resistant Friend leukemia cells.

Analysis of expression of the Friend murine leukemia virus (F-MuLV) and of the spleen focus forming virus (SFFV) has been undertaken in highly malignant interferon (IFN)-sensitive (745) and IFN-resistant (3Cl-8) Friend leukemia cells (FLC), serially passaged intraperitoneally in DBA/2 mice. In vivo passaged 745 cells, as well as the clones derived thereof, did not release Friend virus (FV). Western blot analysis of the plasma membrane fractions of the virus nonproducer 745 cells revealed the lack of gp69/70 glycoprotein expression. At least 10 intraperitoneal passages of virus producer in vitro passaged of virus producer in vitro passaged 745 cells were necessary to obtain the selection of the virus nonproducer phenotype. In contrast in vivo passaged 3Cl-8 cells continued to produce FV even after 100 in vivo passages and expressed gp69/70 antigens to a similar extent as the original in vitro passaged FLC. The expression of F-MuLV and SFFV RNAs in virus producer and virus nonproducer FLC clones has been investigated by means of Northern blot technique using probes specific for either F-MuLV or SFFV. No F-MuLV specific RNA sequences were detected in virus nonproducer 745 clones. SFFV specific RNA transcripts and gp52/55 glycoprotein production could be revealed in all the FLC tested. Southern blot analysis showed the presence of F-MuLV specific sequences in the cellular DNA of virus nonproducer 745 clones. As both in vivo passaged F-MuLV producer 3Cl-8 and F-MuLV nonproducer 745 cells were equally barely immunogenic and highly malignant when injected into syngeneic DBA/2 mice, these results indicate that F-MuLV expression does not result per se in a high immunogenic potential of tumor cells. For the time being, as a specific property of 3Cl-8 versus 745 cells is the interferon-resistant phenotype, it is tempting to speculate that the selection of virus nonproducer cell variants after in vivo passages of interferon-sensitive 745 cells could depend on the presence of low levels of endogenous interferon in normal young mice.

Animals↗

PubMed1987

Late coding region sequences required for competition by SV40 defectives.

SV40 defectives containing the complete early coding region (E-SV40) or the complete late region (L-SV40) were separately transfected into green monkey cells. They were analyzed for their ability to compete with wtSV40 (introduced by infection) or to undergo replication in the presence of constitutively produced SV40 T-antigen. L-SV40 competed very strongly. It appeared rapidly in infected cells, overgrowing wt genomes by at least 10:1. In addition, it slowed the growth of wt virus and reduced its ability to kill cells. L-SV40 DNA, as expected, replicated continuously in Cosl cells. E-SV40 genomes were poor competitors. They appeared slowly and by themselves did not overgrow wtSV40. When transfected into Cosl cells, E-SV40 genomes replicated efficiently for the first few days and disappeared within a week. Deletion or insertion mutations were introduced into a molecular clone of L-SV40, within the Vp1 gene or the Vp2 gene. All mutants were unable to form infectious virus in two different assays. The mutants were then assayed for competition against wtSV40 and for replication in Cosl cells. The Vp1 mutants competed very poorly with wt genomes and were rapidly lost from coinfected cells. These mutants, like E-SV40, replicated for only a few days in Cosl cells. In contrast, the Vp2 mutant competed with wtSV40 nearly as well as L-SV40. It also replicated continuously rather than transiently in Cosl cells. Next, we determined whether L-SV40 could effectively compete with other evolved SV40 defectives, not containing the late region but containing up to nine SV40 origin regions. We have shown that within five serial passages, L-SV40 became the predominant viral DNA species and the other defectives were lost. Although the Vp1 mutants and E-SV40 were weak competitors, they were shown to recombine with wtSV40 genomes to generate new L-SV40 genomes which again became the predominant species of viral DNA. These results demonstrate that L-SV40 is a potent competitor and that the Vp1 gene or a part of Vp1 plays an important role in this extraordinary competition. We suggest that the Vp1 gene functions to allow L-SV40 genomes to persist rather than generating a product which directly interferes with wtSV40 replication.

Animals↗

PubMed1987

Sindbis virus mutants resistant to mycophenolic acid and ribavirin.

Previous work from this laboratory has demonstrated a correlation between the inhibition by ribavirin (Rbv), mycophenolic acid (MPA), or 2-amino thiadiazole (TDA) of Sindbis virus replication in Aedes albopictus mosquito cells and a reduction in cellular GTP levels. This reduction in GTP results from the inhibition by these drugs of inosine monophosphate dehydrogenase (IMPDH), the first enzyme specific for the de novo synthesis of GMP. By serial passage of SV in A. albopictus cells in the presence of 25 microM MPA, we have now isolated viral mutants which are highly resistant not only to MPA but also to Rbv and TDA. For example, whereas 500 microM Rbv reduced the plaquing efficiency of SVSTD by at least 10(6)-fold, the same concentration of Rbv reduced the plaquing efficiency of the MPA-resistant mutants less than 5-fold. This is the first example of a viral mutant resistant to the antiviral compound Rbv.

Aedes↗