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At least 55 records · Page 3Linked to original sources
PubMed1999

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.

3T3 Cells↗
PubMed2003

E/NS1 modifications of dengue 2 virus after serial passages in mammalian and/or mosquito cells.

OBJECTIVE: Dengue viruses are routinely maintained in nature by transmission cycles involving the passage of virus between humans and AEDES mosquitoes. The number of dengue virus lineages has been increasing over time. The aim of this study was to identify the genetic diversity of dengue 2 virus serially transferred in mammalian and/or mosquito cells. METHODS: The E/NS1 gene of dengue 2 virus variants derived from serial passages in Vero or C6/36 cells, or alternately in both cell systems, was amplified and sequenced in order to observe gene modification after serial passages. RESULTS: Three nucleotides (two in E and one in NS1) or two amino acids (one each in E and NS1) changed in the virus that was continuously cultured in Vero cells for 20 passages, whereas four nucleotides (two each in E and NS1) or three amino acids (one in E and two in NS1) changed in the virus cultured for 30 passages. The genome of dengue 2 virus remained stable even when the virus was serially transferred in C6/36 cells for 30 generations. However, there was one amino acid substitution (E46 I-->V) resulting from a single nucleotide change in the E region of dengue 2 virus alternately transferred in C6/36 and Vero cells for either 20 or 30 passages. In addition, dengue 2 virus obtained from serially cultured Vero cells usually replicated better when it reinfected Vero cells, reflecting its high adaptation fitness to the host cell. CONCLUSIONS: It is concluded that genetic changes of dengue 2 virus are constrained in Vero (mammalian) cells, resulting in a variety of genome-related quasispecies populations. Some populations of the virus are subsequently selected by and genetically (at least in the E/NS1 portion of the viral genome) maintained in C6/36 (mosquito) cells during replicative competition.

Aedes↗
PubMed2003

Serially passaged human nasal epithelial cell monolayer for in vitro drug transport studies.

PURPOSE: To evaluate the feasibility of using a serially passaged culture of human nasal epithelial cell monolayers on a permeable support for in vitro drug transport studies. The optimum conditions for passaged culture as well as the correlation between the transepithelial electrical resistance (TEER) value and drug permeability (Papp) were evaluated. METHODS: Fresh human nasal epithelial cells were collected from normal inferior turbinates and were subcultured repeatedly in serum-free bronchial epithelial cell growth media (BEGM) in petri dishes. The subcultured cells of each passage were seeded onto permeable supports at 5 x 10(5) cells/cm2 and grown in Dulbecco's modified Eagle medium (DMEM). Morphologic characteristics were observed by scanning electron microscopy (SEM). To verify the formation of tight junctions, actin staining and transmission electron microscopy (TEM) studies were conducted. In the drug transport study, [14C]mannitol and budesonide were selected as the paracellular and the transcellular route markers, respectively. RESULTS: Serially passaged cells were successfully cultured on a permeable support and showed significantly high TEER values up to passage 4. After 14 days of seeding, SEM showed microvilli, and protrusions of cilia and mucin granules were detected by TEM. The paracellular marker [14C]mannitol showed a nearly constant permeability coefficient (Papp) when the TEER value exceeded 500 omega x cm2 regardless of the passage number. However, as expected, budesonide showed a higher permeability coefficient compared to [14C]mannitol and was less affected by the TEER value. CONCLUSIONS: Human nasal epithelial cell monolayers were successfully subcultured on a permeable support up to passage 4. These cell culture methods may be useful in high-throughput screening of in vitro nasal transport studies of various drugs.

Biological Transport↗
PubMed2005

Biological properties of Roakin strain of NDV and TK900 strain of ADV after serial passages in CECC in the presence of methisoprinol and KLP-602.

Twenty serial passages of the TK900 strain of Aujeszky's disease virus (ADV) and the Roakin strain of Newcastle disease virus (NDV) were made in a chicken embryo cell culture (CECC), in the presence of two antiviral agents: Methisoprinol and KLP-602. The physicochemical properties of passaged viruses were determined. The results obtained suggest that Methisoprinol causes changes in the structure of viral proteins, whereas KLP-602 affects the envelope-dependent properties of the virus. It was also found that the alternations observed in passaged viruses were temporary phenotypic changes only, and not a consequence of permanent transformations of their genotypes.

Animals↗
PubMed1986

Restriction endonuclease cleavage patterns of commercial and serially passaged isolates of Heliothis baculovirus.

The restriction endonuclease patterns of three Baculovirus isolates (Br, Vh, El) from Heliothis zea having different passages and production histories were compared. Digestion of the ds DNA genomes of the three isolates with EcoRI, HindIII, and XhoI showed no major difference in the cleavage patterns, although submolar fragments were detected. One of the commercial isolates (Vh) was serially passaged 20 times through larvae of H. zea and a substitute host, H. virescens. EcoRI cleavage profiles of the ds DNA showed the absence of a band in the 3-megadalton region that was present in the original isolates. In addition, the cleavage pattern of the DNA from Vh passed in H. zea showed additional submolar fragments in the 15- and 6-megadalton regions. Differences also were observed in the HindIII and XhoI restriction patterns. No significant differences in virulence between the original isolate and passage isolates were detected after 20 serial passages in larvae of either H. zea or H. virescens.

Animals↗
PubMed2003

Biologic properties of dengue viruses following serial passage in primary dog kidney cells: studies at the University of Hawaii.

Serial passage at low dilution of seven different wild-type dengue (DEN) viruses into primary dog kidney (PDK) cell cultures placed selective pressure that resulted in the following changes from parental phenotype: smaller plaques in LLC-MK2 cells, absent plaque formation in green monkey kidney cells, lack of a cytopathic effect in LLC-MK2 cells, shut-off of virus replication at high temperatures (temperature sensitivity), reduced virulence for rhesus monkeys manifested by reduced or absent viremia and/or absence of a secondary-type antibody response following homotypic challenge, and progressive increase in the mean day of death following intracerebral inoculation of sucking mice. Two DEN-1 strains showed most of these changes by the 15th PDK passage. Only one of two DEN-2 strains studied was carried to the 50th PDK passage at the University of Hawaii. For the latter strain, both the temperature of viral replicative shutoff and mouse neurovirulence were reduced. Three DEN-4 strains showed similar late-passage biologic marker changes. The observations made, although not exhaustive, provide laboratory correlates for virus strains that have shown reduced virulence but retained immunogenicity in humans. Candidate human vaccines have been prepared from five of the studied strains: DEN-1 (16007) at PDK 13, DEN-2 (16681 and S-16803) at PDK 50 or above, and DEN-4 (1036 and 341750) at PDK 48 and 20, respectively.

Animals↗
PubMed2003

Determinants of increased replicative capacity of serially passaged simian immunodeficiency virus with nef deleted in rhesus monkeys.

Most rhesus macaques infected with simian immunodeficiency virus SIVmac239 with nef deleted (either Delta nef or Delta nef Delta vpr Delta US [Delta 3]) control viral replication and do not progress to AIDS. Some monkeys, however, develop moderate viral load set points and progress to AIDS. When simian immunodeficiency viruses (SIVs) recovered from two such animals (one Delta nef and the other Delta 3) were serially passaged in rhesus monkeys, the SIVs derived from both lineages were found to consistently induce moderate viral loads and disease progression. Analysis of viral sequences in the serially passaged derivatives revealed interesting changes in three regions: (i) an unusually high number of predicted amino acid changes (12 to 14) in the cytoplasmic domain of gp41, most of which were in regions that are usually conserved; these changes were observed in both lineages; (ii) an extreme shortening of nef sequences in the region of overlap with U3; these changes were observed in both lineages; and (iii) duplication of the NF-kappa B binding site in one lineage only. Neither the polymorphic gp41 changes alone nor the U3 deletion alone appeared to be responsible for increased replicative capacity because recombinant SIVmac239 Delta nef, engineered to contain either of these changes, induced moderate viral loads in only one of six monkeys. However, five of six monkeys infected with recombinant SIVmac239 Delta nef containing both TM and U3 changes did develop persisting moderate viral loads. These genetic changes did not increase lymphoid cell-activating properties in the monkey interleukin-2-dependent T-cell line 221, but the gp41 changes did increase the fusogenic activity of the SIV envelope two- to threefold. These results delineate sequence changes in SIV that can compensate for the loss of the nef gene to partially restore replicative and pathogenic potential in rhesus monkeys.

Amino Acid Sequence↗
PubMed2006

MAP kinases and heat shock-induced hormesis in human fibroblasts during serial passaging in vitro.

Adult human skin fibroblasts were exposed repeatedly to 41 degrees C or 42 degrees C heat shock (HS) for 1 h twice a week during serial passaging throughout their replicative life span. On the basis of longevity curves, cell size, and morphology, we observed that repeated mild heat shock (RMHS) at 41, degrees C had strong anti-aging hormetic effects, including 20% extension of cellular longevity. The basal levels of the MAP kinases JNK1, JNK2, and p38 increased during serial passaging, while that of ERK2 decreased. RMHS further exaggerated these effects, which suggests that age-related changes in MAP kinases may be an adaptive response for better cell survival.

Adult↗
PubMed1995

Mutation analyses of molecularly cloned satellite tobacco mosaic virus during serial passage in plants: evidence for hotspots of genetic change.

The high level of genetic diversity and rapid evolution of viral RNA genomes are well documented, but few studies have characterized the rate and nature of ongoing genetic change over time under controlled experimental conditions, especially in plant hosts. The RNA genome of satellite tobacco mosaic virus (STMV) was used as an effective model for such studies because of advantageous features of its genome structure and because the extant genetic heterogeneity of STMV has been characterized previously. In the present study, the process of genetic change over time was studied by monitoring multiple serial passage lines of STMV populations for changes in their consensus sequences. A total of 42 passage lines were initiated by inoculation of tobacco plants with a helper tobamovirus and one of four STMV RNA inocula that were transcribed from full-length infectious STMV clones or extracted from purified STMV type strain virions. Ten serial passages were carried out for each line and the consensus genotypes of progeny STMV populations were assessed for genetic change by RNase protection analyses of the entire 1,059-nt STMV genome. Three different types of genetic change were observed, including the fixation of novel mutations in 9 of 42 lines, mutation at the major heterogeneity site near nt 751 in 5 of the 19 lines inoculated with a single genotype, and selection of a single major genotype in 6 of the 23 lines inoculated with mixed genotypes. Sequence analyses showed that the majority of mutations were single base substitutions. The distribution of mutation sites included three clusters in which mutations occurred at or very near the same site, suggesting hot spots of genetic change in the STMV genome. The diversity of genetic changes in sibling lines is clear evidence for the important role of chance and random sampling events in the process of genetic diversification of STMV virus populations.

Biological Evolution↗
PubMed2002

Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture.

Rapid accumulation of few polyhedra (FP) mutants was detected during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in cell culture. 100% FP infected cells were observed by passage 6. The specific yield decreased from 178 polyhedra per cell at passage 2 to two polyhedra per cell at passage 6. The polyhedra at passage 6 were not biologically active, with a 28-fold reduction in potency compared to passage 3. Electron microscopy studies revealed that very few polyhedra were produced in an FP infected cell (<10 polyhedra per section) and in most cases these polyhedra contained no virions. A specific failure in the intranuclear nucleocapsid envelopment process in the FP infected cells, leading to the accumulation of naked nucleocapsids, was observed. Genomic restriction endonuclease digestion profiles of budded virus DNA from all passages did not indicate any large DNA insertions or deletions that are often associated with such FP phenotypes for the extensively studied Autographa californica nucleopolyhedrovirus and Galleria mellonella nucleopolyhedrovirus. Within an HaSNPV 25K FP gene homologue, a single base-pair insertion (an adenine residue) within a region of repetitive sequences (seven adenine residues) was identified in one plaque-purified HaSNPV FP mutant. Furthermore, the sequences obtained from individual clones of the 25K FP gene PCR products of a late passage revealed point mutations or single base-pair insertions occurring throughout the gene. The mechanism of FP mutation in HaSNPV is likely similar to that seen for Lymantria dispar nucleopolyhedrovirus, involving point mutations or small insertions/deletions of the 25K FP gene.

Amino Acid Sequence↗
PubMed2001

Effect of serial passage on gene expression in MC3T3-E1 preosteoblastic cells: a microarray study.

The osteoblastic function of mouse preosteoblastic MC3T3-E1 cells, as measured by alkaline phosphatase activity and osteocalcin secretion, decreases after serial passage. To uncover genes responsible for decreased osteoblastic function in high-passage cells, we have studied passage-dependent change of gene expression in MC3T3-E1 cells. Changes in the expression pattern of 2000 selected genes were examined simultaneously by comparing mRNA levels between MC3T3-E1 cells at passage 20 and passage 60 using the cDNA microarray analysis. Significant changes in the steady-state abundance of 27 mRNAs were observed in response to different passage numbers, including 17 known genes, 4 ESTs with homology to known genes, and 6 genes with no previously described function or homology. Northern blot analysis was used to verify and quantify the expression of selected genes, and revealed a significant higher level of up- and down-regulation compared to microarray data. These results indicate the existence of a significant change in gene expression in osteoblastic cells undergoing serial passages. Such changes might be responsible for a reduction in bone regeneration in older osteoblasts. Potential roles of selected genes in bone aging are discussed.

Aging↗
PubMed1981

Propagation of human hepatitis A virus in African green monkey kidney cell culture: primary isolation and serial passage.

Human hepatitis A virus (HAV) was propagated in primary African Green Monkey (Cercopithecus aethiops) kidney (AGMK) cell cultures. Three strains of HAV were used: MS-1, SD-11, and HM-175. Cells were inoculated with marmoset-passaged material or human clinical specimens and were stained by direct immunofluorescence to establish the identity of the virus. Both clinical samples and marmoset-passaged material produced immunofluorescence. HAV antigen was found scattered throughout the cytoplasm of inoculated cultures. The HM-175 strain produced the most intense immunofluorescence. This strain of HAV had been serially passaged in cell culture seven times. Blocking experiments with paired human sera from naturally acquired HAV infections and hyperimmune chimpanzee serum from an experimentally infected animal established that the immunofluorescence was specific. The viral antigen was found to be exclusively intracellular. The interval to maximum HAV antigen expression was decreased by serial passage. The HAV strain described herein, which was recovered directly from the stool specimen of a patient with HAV in primary AGMK cell culture, may prove useful as a source of antigen for serological tests and as a candidate vaccine strain.

Animals↗
PubMed1984

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. III. Reversion to virulence by passage of cloned virus in fetal rhesus lung cells.

Two strains of primary dog kidney-passaged dengue (DEN) 4 (H-241) virus cloned by terminal dilution (PDK 24-TD3 and 35-TD3) were propagated in fetal rhesus lung (FRhL) cells to produce candidate vaccine virus seeds. Both serial passage and prolonged replication of PDK 24-TD3 in FRhL resulted in appearance of medium and large plaques in LLC-MK2 assays. When picked, these plaques proved to contain temperature-resistant, monkey-virulent revertants. Serial passage and prolonged replication of PDK 24-TD3 in LLC-MK2 cells did not result in reversion; but, prolonged replication in PDK cells did. Passage of PDK 35-TD3 in FRhL cells resulted in appearance of medium size plaques which, when picked, yielded temperature sensitive (ts) (38.5 degrees C) viruses of low monkey-virulence. Because of its stability in monkeys and FRhL cells, reduced monkey virulence and ts property. PDK 35-TD3 is a promising candidate for trial in man.

Animals↗
PubMed2003

[Decrease of HIV-1 replicative capacity after serial passage in MT-4 cells under conditions of the combined antiretroviral therapy].

The replicative activity of HIV-1/IIIB was determined in MT-4 cells under conditions of combined HIV reverse transcriptase and protease inhibitors in the presence of four different drug cocktails: (1) phosphazide, didanosine, nevirapine; (2) stavudine, didanosine, nevirapine; (3) phosphazide, didanosine, nevirapine, indinavir; (4) stavudine, didanosine, nevirapine, indinavir. The concentration of every inhibitor was 10 times higher than the 50% effective concentration. Alpha interferon was used as a natural antiretroviral agent in addition. The virus was subjected to 5 serial passages in the presence of the drug cocktails and then to 5 serial passages without the agents using cocultivation of infected and uninfected cells at ratio 1:5 to increase virus activity. Virus replication in the presence of all the drug combinations resulted in the appearance of HIV-1 variants with low replicative activity that was insignificantly increased during further passages even without the antiretroviral agents. If extrapolated to the clinical practice, these results indicate that all the drug cocktails were effective inhibitors of HIV-1 replication because the virus variants with high replicative activity did not emerge. Moreover, the results showed that the clinical use of the drug cocktails was promising.

Anti-HIV Agents↗
PubMedDate not supplied

Enhancement of Campylobacter jejuni virulence by serial passage in chicks.

Four isolates of Campylobacter jejuni were studied to determine changes in virulence following six serial passages in chicks. Chicks that received invasive isolates exhibited diarrhea and depressed weight gain. Immature mice were used to assess virulence of the passaged isolates of C. jejuni. Nine-day-old mice infected with passaged isolates showed lethargy, dehydration, depression, decreased weight gain, and occult blood in feces. Mouse pups inoculated with the third and sixth chick passage levels of an invasive isolate showed significant depression in mean daily weight gain and elevated mortality compared with controls and subjects inoculated with unpassaged isolates. This study demonstrated enhancement of virulence in a C. jejuni isolate following chick passage. In contrast, three other passaged isolates failed to show any consistent increase in virulence.

Animals↗
PubMed2001

Reduced levels of oxidized and glycoxidized proteins in human fibroblasts exposed to repeated mild heat shock during serial passaging in vitro.

Repeated mild heat shock (RMHS) has beneficial hormesis-like effects on various characteristics of human skin fibroblasts undergoing replicative senescence in vitro. We have tested whether RMHS could reduce the accumulation of oxidized and glycoxidized proteins, which is a major age-related change. Levels of carbonylated proteins, furosine, N(epsilon)-carboxymethyl-lysine-rich proteins and advanced glycation end products increased during serial passaging of fibroblasts in culture. However, the extent of accumulation of oxidized and glycoxidized proteins was significantly reduced in RMHS cells. The basal concentration of reduced glutathione was higher and that of oxidized glutathione was lower in RMHS cells. Whereas the basal level of heat shock protein HSP27 decreased in both RMHS and control cells during serial passaging, the increase of the basal level of HSP70 with increasing passage level was significantly higher in RMHS cells. These results show that the slower accumulation of damaged proteins in fibroblasts exposed to RMHS results partly from the increased ability of these cells to cope with oxidative stress, and to synthesize HSP responsible for protein capping and refolding.

Cell Division↗
PubMed1996

Serial passage of tobacco rattle virus under different selection conditions results in deletion of structural and nonstructural genes in RNA 2.

The RNA genome of tobacco rattle virus (TRV) is bipartite. RNA 2 of the nematode-transmissible TRV isolate PPK20 encodes the viral coat protein (cp) and proteins with molecular weights of 29,400 and 32,800 (29.4K and 32.8K proteins). When this isolate was serially passaged in tobacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of these DI RNAs were cloned. Six DI RNAs had single internal deletions in RNA 2 that removed most of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. The borders of the deletions in these DI RNAs were found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs. Two DI RNAs were found to be recombinants containing a 5' sequence derived from RNA 2 and a 3' sequence derived from RNA 1. When serial passage of TRV isolate PPK20 was carried out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) with a functional cp gene was observed. The deletion in D7 covered the 3' end of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK20 isolate. These results may explain the observation that many laboratory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.

Culture Media↗