Low Passage Levels Research Articles

Lipid peroxidation-derived 4-hydroxynonenal-modified proteins accumulate in human facial skin fibroblasts during ageing in vitro

The reactive aldehyde, 4-hydroxynonenal (HNE), is recognized as a product of lipid peroxidation, which binds to macromolecules, in particular proteins. HNE-modified proteins (HNE-MP) have been shown to accumulate during ageing, generally by using polyclonal antibodies, which increase the possibility of detecting false positives. Therefore, we have used a genuine monoclonal antibody specific for HNE-His adducts of proteins/peptides, which were revealed by immunoblotting method for whole-cell HNE-MP measurements in serially passaged human facial skin fibroblasts undergoing ageing in vitro. There was a significant increase in the levels of HNE-MP in serially passaged cells approaching a near senescent state at high passage level (P-61), as compared with low passage level (P-11) young and middle-aged (P-27) cells. However, if the cells were analyzed soon after re-initiation from the frozen samples with little further passaging, the amount of HNE-MP was low even in relatively high passage level (P-37) cells, which is an indication of selective elimination of cells with high molecular damage during the process of thawing and re-initiation in culture. This pilot study on normal human facial skin fibroblasts shows that HNE-MP detection by monoclonal antibody-based dot blot method can be used as a marker for age-related accumulation of lipid peroxidative molecular damage, and could be useful for testing and monitoring the effects of potential skin care products on ageing parameters.

Characterization of a variant vlhA gene of Mycoplasma synoviae, strain WVU 1853, with a highly divergent haemagglutinin region

BackgroundIn Mycoplasma synoviae, type strain WVU 1853, a single member of the haemaglutinin vlhA gene family has been previously shown to be expressed. Variants of vlhA are expressed from the same unique vlhA promoter by recruiting pseudogene sequences via site-specific recombination events, thus generating antigenic variability. Using a bacterial stock of M. synoviae WVU 1853 that had been colony purified thrice and maintained in our laboratory at low passage level, we previously identified a vlhA gene-related partial coding sequence, referred to as MS2/28.1. The E. coli-expressed product of this partial coding sequence was found to be immunodominant, suggesting that it might be expressed.ResultsReverse transcription-PCR amplification (RT-PCR), using a sense primer located at the 5'-end region of the expected vlhA transcript and a reverse primer located at the 3' end of MS2/28.1 coding sequence, yielded a consistent amplification product showing that MS2/28.1 was indeed transcribed. Nucleotide sequence analysis of the RT-PCR product identified an 1815-nucleotide full-length open reading frame (ORF), immediately preceded by a nucleotide sequence identical to that previously reported for expressed vlhA genes. PCR amplifications using genomic DNA isolated from single colonies further confirmed that the full-length ORF of MS2/28.1 was located downstream of the unique vlhA promoter sequence. The deduced 604-amino acid (aa) sequence showed a perfect sequence identity to the previously reported vlhA expressed genes along the first 224 residues, then highly diverged with only 37.6% aa identity. Despite the fact that this M. synoviae clone expressed a highly divergent and considerably shorter C-terminal haemagglutinin product, it was found to be expressed at the surface of the bacterium and was able to haemagglutinate chicken erythrocytes. Importantly, the E. coli-expressed C-terminal highly divergent 60 residues of MS2/28.1 proved haemagglutination competent.ConclusionsIn contrast to the previously characterized vlhA expressedvariants, MS2/28.1 displayed a highly divergent sequence, while still able to haemagglutinate erythrocytes. Overall, the data provide an indication as to which extent the M. synoviae vlhA gene could vary its antigenic repertoire.

Evaluation of an Ovine Testis Cell Line (OA3.Ts) for Propagation of Capripoxvirus Isolates and Development of an Immunostaining Technique for Viral Plaque Visualization

An ovine testis cell line (OA3.Ts) was evaluated and compared with primary lamb kidney (LK) cells for its utility in capripoxvirus propagation and titration. A comparison of OA3.Ts cell growth kinetics and morphology at low (<33) and high (34-36) passage levels indicated a difference in both characteristics. However, viral titers determined in low and high passage OA3.Ts cells were comparable with those obtained using LK cells. Capripoxvirus infection of OA3.Ts and LK cells resulted in a similar cytopathic effect, which allowed for the detection of discrete viral plaques following immunostaining with capripoxvirus-specific antiserum.

Increased susceptibility of human respiratory syncytial virus to neutralization by anti‐fusion protein antibodies on adaptation to replication in cell culture

Subgroup A respiratory syncytial viruses present in respiratory secretions and low passage level cell culture isolates were found to be markedly less susceptible to neutralization with monoclonal antibodies (MAbs) to the F glycoprotein than the cell culture adapted A2 virus strain. Low passage virus isolates collected over a 20 year period and belonging to several sub-group A lineages were refractory to neutralization with antibodies recognizing two major neutralizing antigenic sites located sub-terminally at opposite ends of the F(1) glycoprotein sub-unit. On further passage in cell culture, virus isolates exhibited both increased infectivity titers and increased susceptibility to neutralization by antibodies to both antigenic sites. The consensus nucleotide sequence of the membrane associated proteins M and of the SH, G and F glycoprotein genes, and their intergenic regions were compared for neutralization resistant and susceptible stocks of one virus strain, R17532. No changes were observed in the known monoclonal antibody epitopes on the F glycoprotein. In line with this, the increase in susceptibility was not found to be associated with any increased binding of monoclonal antibody to isolated F glycoprotein in a BIAcore assay, thus excluding the possibility that passage in cell culture selected for viruses with mutations in the antibody binding sites. M and SH genes were conserved but a number of sites in the G and F glycoprotein genes were found to vary on adaptation to cell culture suggesting that change in susceptibility to neutralization was associated with a change in the prevalent quasispecies present in the virus population. The genetic basis of phenotypic change in susceptibility remains to be determined.

Matrix metalloproteinase and cytokine production by bone marrow adherent cells from multiple myeloma patients

Cultures of bone marrow stromal cells derived from the bone marrow of multiple myeloma (MM) patients were shown to exhibit several abnormalities compared with control cultures from healthy subjects. The aim of the study was to examine whether cultures of bone marrow adherent cells, at low passage level, exhibit differences in matrix metalloproteinases (MMPs) and cytokine production compared with cultures from normal donors. MMP production was evaluated by gel zymography and by ELISA in supernatants of serum-free cultures of bone marrow adherent cells derived from 20 MM patients and 23 healthy controls. Spontaneous and lipopolysaccharide (LPS)- or Newcastle disease virus (NDV)-induced cytokine release was assessed in the supernatants of the cultures by the ELISA method. Both cultures produced MMP-1, -2, -3, and -9 under serum-free conditions; however, the levels of MMP-1 and MMP-2 were significantly higher in cultures derived from MM patients, while MMP-3 was significantly higher in control cultures. The level of MMP-9 was comparable in the cultures derived from MM patients and controls. All cultures produced interleukin (IL)-10 and IL-11 spontaneously, but after LPS or NDV induction the levels of IL-10, IL-11, interferon alpha, and tumor necrosis factor alpha, were significantly higher in the cultures derived from MM patients than in control cultures. The results indicate that both the abnormalities in MMP production and the overproduction of cytokines (in the presence of LPS or virus, which mimic inflammatory conditions) may be involved in bone destruction and tumor spread in multiple myeloma.

Aberrant cell cycle checkpoint function in transformed hepatocytes and WB-F344 hepatic epithelial stem-like cells.

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.

Telomere shortening, telomerase expression, and chromosome instability in rat hepatic epithelial stem-like cells.

Telomeres, which are specialized structures consisting of T2AG3 repeats and proteins at the ends of chromosomes, may be essential for genomic stability. To test whether telomere length maintenance preserves genomic stability in rats (Rattus rattus and Fischer 344), we assayed telomerase activity and telomere length in the rat hepatic epithelial stem-like cell line WB-F344 during aging in vitro and in tumor-derived lines. Telomerase activity in the parental WB-F344 line was repressed at low and intermediate passage levels in vitro and reexpressed at high passages. Southern blot hybridization and quantitative fluorescence in situ hybridization analyses demonstrated that telomeres were significantly eroded at intermediate passage levels, when telomerase was repressed, and at high passage levels, when telomerase was expressed. Fluorescence in situ hybridization analysis also revealed interstitial telomeric sequences in rat chromosomes. Tumor-derived WB-F344 cell lines that express telomerase had variably shortened telomeres. Cytogenetic analyses performed on WB-F344 cells at low, intermediate, and high passages demonstrated that chromosome instability was most severe in the intermediate passage cells. These data suggest that telomere shortening during aging of rat hepatic epithelial stem-like WB-F344 cells in vitro and during selection of tumorigenic lines in vivo may destabilize chromosomes. Expression of telomerase in high passage cells appeared to partially stabilize chromosomes.

Expression of telomerase in normal and malignant rat hepatic epithelia.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA repeats onto the ends of chromosomes. More than 85% of human cancers express telomerase activity and a large proportion of human hepatocellular carcinomas are positive. To study the role of telomerase expression in rat hepatocarcinogenesis, telomerase activity was assayed in various rat tissues and in two types of liver epithelial cells: hepatocytes and hepatic epithelial stem-like cells. In the present study, we demonstrate that telomerase activity in rats is tissue-specific and stable with animal aging. Liver and testis were found to be telomerase positive, spleen had low or no activity, and kidney was negative. Telomerase activity did not change significantly in 18 month-old rats compared to 2 month-old rats, but was moderately (twofold) increased during liver regeneration induced by a 2/3's partial hepatectomy. Telomerase activity was detected in isolated rat hepatocytes and low passage hepatic epithelial stem-like cells (WB-F344). Telomerase activity displayed significant variations in a propagable clone of WB-F344 cells. At low passage levels after establishment in vitro (passages 4-9) non-tumorigenic WB-F344 cells expressed telomerase activity. During further in vitro passaging these cells lost expression of telomerase. Expression of telomerase in the tumor-derived lines of WB-F344 cells but not in the selectively cycled, parental lineages of these cells suggests that there may be a role for telomerase in hepatocarcinogenesis.

A preliminary study on the attenuation of Tunisian schizont-infected cell lines of Theileria annulata.

Four Theileria annulata cell lines were characterised at low passage levels using two polymorphic markers and then used to infect calves. Their virulence seemed to be related to the number of genotypes present within the cell line. In all, 3 of the 4 cell lines were cultured up to passage 100 or 200 and inoculated into calves. Their characterisation using the same markers indicated that the attenuation was related to a reduction in the parasite polymorphism down to a single genotype. The immunogenicity of the three attenuated cell lines was assessed in calves using two types of challenge. Optimal protection was observed against homologous challenges. The level of immunity to heterologous challenges appeared to decrease with attenuation and seemed to depend on the cell line used.

Effects of the developmental colour mutations silver and recessive spotting on proliferation of diploid and immortal mouse melanocytes in culture

The developmental mouse coat-colour mutations silver (si, chromosome 10) and recessive spotting (rs, chromosome 5, mapping very close to the dominant white spotting or W/c-kit locus), appear to reduce the numbers of functional melanocytes in the skin. They were studied at the cellular level by melanocyte culture. Cellular morphology, differentiation and survival appeared normal. However, both mutations were found to reduce the melanocyte proliferation rate in primary cultures, as measured by [3H]thymidine labelling indices. Two immortal si/si melanocyte lines (designated melan-si1 and melan-si2) and one rs/rs line (melan-rs) were established. Melan-si1 and melan-rs were cloned. All three immortal lines at low passage levels had doubling times significantly greater than those of our other melanocyte lines melan-a, melan-b and melan-c. Thus they retained the phenotype of slow proliferation.

Comparison of hemagglutinating, receptor-destroying, and acetylesterase activities of avirulent and virulent bovine coronavirus strains.

SummaryHemagglutinating and acetylesterase functions as well as the 124 kDa glycoprotein were present in the highly cell-culture adapted, avirulent bovine coronavirus strain BCV-L9, in the Norden vaccine strain derived from it, and in 5 wild-type, virulent strains that multiplied in HRT-18 cells but were restricted in several types of cultured bovine cells. The BCV-L9 and the wild-type strain BCV-LY-138 agglutinated chicken and mouse erythrocytes. The acetylesterase facilitated break-down of the BCV-erythrocyte complex with chicken but only to a minimal extent with mouse erythrocytes in the receptor-destroying enzyme test. Purified preparations of the vaccine and the wild-type strains agglutinated chicken erythrocytes at low titers and mouse erythrocytes at 128 to 256 times higher titers whereas receptor destroying enzyme activity was detectable only with chicken erythrocytes. When wild-type strains were propagated in HRT cells at low passage levels, they produced 5×105 to 4.5×106 plaque forming units per 50 µl which agglutinated erythrocytes from mice but not from chickens. Diisopropylfluoro-phosphate moderately increased the hemagglutination titers, but completely inhibited the receptor destroying enzyme of purified virus of all strains. It had virtually no influence on the plaque-forming infectivity of the different BCV strains. The acetylesterase of strain BCV-L9 reacting in the receptor-destroying enzyme test was stable for 3h at 37 and 42°C. It was inactivated within 30 min at 56°C while the hemagglutinin function of this strain was stable for 3 h at 37, 42, and 56°C, but it was inactivated at 65°C within 1 h.

Free and integrated plasmid DNA in spiroplasmas

Spiroplasma citri was found to carry an 8.0 kb plasmid that differed from previously describedS. citri plasmids in its restriction map. It was also clonable in pBR322. The plasmid, named pRA1, was found in large quantities as free plasmid inS. citri (R8A2, Maroc) subclones of low passage level. In subclones of higher passage levels, free plasmid was replaced by plasmid sequences integrated into the spiroplasma chromosome. Significant quantities of integrated plasmid sequences were also observed in the corn stunt spiroplasma,S. kunkelii, while small quantities of free and/or integrated plasmid DNA could be detected in some spiroplasmas serologically and genotypically remote fromS. citri. Integrated plasmid sequences were cloned into theEscherichia coli plasmid pUC13. Hybridization tests and restriction maps of these clones indicated that the integrated plasmid sequences consisted of fragments, rather than entire plasmid DNA, inserted into specific sites in the spiroplasma chromosome. Although the biological role of the pRA1 plasmid remains unclear, theS. citri subclones containing large quantities of free plasmid exhibited slower growth rates and a tendency to lyse.

Properties of an Australian isolate of bovine parvovirus type 1

An Australian bovine parvovirus isolate (BPV 267) was found to haemagglutinate human, guinea-pig, rat and dog erythrocytes, out of a range of 16 species of erythrocyte tested. The haemagglutinating activity was generally found to be both pH and temperature dependent. The virus was found to replicate best in intestinal epithelium, macrophage and lung cells, out of 9 bovine cell types tested. Highest yields of virus were obtained by the use of selected cell strains at low-passage levels which were maintained near neutral pH under conditions of high rates of cell growth. Studies of the rates of thermal inactivation with time showed the virus to be relatively stable at 37°C, 56°C and 70°C, the incorporation of serum proteins, 1 M MgCl 2 and 2 M NaCl in the medium having no influence on stability at 56°C. The virus was resistant to the action of CHCl 3, ether and 1% trypsin, and its replication was inhibited by BUDR, this effect being reversed by thymidine. Actinomycin D was found to inhibit virus replication, but only when applied during the first 8 h post-infection. Density gradient studies showed infective virus to have a density of 1.41 g cm −; haemagglutinating non-infective virus with defective morphology having a density of 1.31 g cm −3. In addition, a proportion of morphologically-complete haemagglutinating, but non-infective virus particles was found at a density of 1.36 g cm −3. The virus proved to have a mean diameter of 22 nm.

Identification of Field Isolates of Infectious Bronchitis Virus by Interference with the La Sota Strain of Newcastle Disease Virus

The interference phenomenon of infectious bronchitis virus (IBV) with growth of Newcastle disease virus (NDV) in embryonating chicken eggs (ECE) was used as a diagnostic method. Fifteen field isolates obtained from presumptively infectious-bronchitis-affected chickens were analyzed by the IBV-NDV interference test. Eight isolates were capable of interfering with the growth of the La Sota strain of NDV, as measured by hemagglutination (HA) activity when IBV was inoculated 10 hr before NDV into ECE. The interference was considered specific for IBV, because it could be eliminated by adding homologous anti-IBV serum. The sensibility of this method could be demonstrated, because in some cases low-passage levels of IBV isolates showing HA interference ability were not capable of producing lesions in ECE. Furthermore, serologically negative IBV samples did not interfere with NDV growth. From these results, the IBV-NDV interference test appears to be a potential diagnostic alternative for identifying IBV field isolates.

Transformation-Associated Increase of Adhesion, Cellular Fibronectin, and Stress Fiber Development in a Liver Epithelial Cell Line

Cytoskeletal and adhesion characteristics of DL-ethionine (CAS: 67-21-0)-transformed rat liver epithelial cells (ETC) were compared with those of nontumorigenic, untreated cells of the same cell line both at the same passage level as ETC and at an early low passage level. ETC and high-passage-level cells (HPC) showed increased cell spreading and prominent actin stress fibers compared to low-passage-level cells (LPC). The number of adhesion plaques per unit cell area was higher for ETC than for LPC. At confluence, fibronectin expression was high for ETC, moderate for HPC, and low for LPC. The observed increases in cell spreading and in actin and fibronectin expression appeared to be associated with transformation of this cell line rather than being specific responses to ethionine treatment. This conclusion is suggested by the fact that HPC, which display preneoplastic markers, are similar in many respects to ETC.

The vector competence of Australian Culex annulirostris with Murray Valley encephalitis and Kunjin viruses.

Australian populations of Culex annulirostris Skuse from 10 localities, Brisbane, Townsville, Cairns, Charleville, Kowanyama (Queensland), Darwin (Northern Territory), Mildura (Victoria) and Port Hedland, Karratha and Mt. Tom Price (Western Australia) showed considerable heterogeneity in response to oral infection with low passage level Murray Valley encephalitis (MVE) and Kunjin (KUN) viruses. However, there was no consistent pattern of either high or low vector efficiency for populations collected from different regions. Overall, Cx annulirostris was a more competent vector of MVE virus than KUN virus, both in oral susceptibility (suckling mouse intracerebral ID50 of 10(1.7)-10(3.9) compared to 10(2.7)-10(4.8)/mosquito, respectively) and in ability to transmit virus to chicks after 10 d extrinsic incubation (50-89% compared to 0-55%). High or low susceptibility with one virus did not correlate with susceptibility for the other. Although these date were probably subject to some seasonal variation, they generally support the view that Cx annulirostris is the principal vector of MVE in Australia. Poor to moderate susceptibility with KUN suggests that (1) it is only a minor vector in some localities, (2) vertebrate hosts have high viraemias or (3) the strain used may have been atypical.

Factors affecting syncytial development in Aedes pseudoscutellaris cells by dengue viruses.

Several factors that were suspected of affecting the development of syncytia in cultured Aedes pseudoscutellaris cells inoculated with dengue viruses were studied. The results indicated that fresh media (less than 1 week old), low cell density at inoculation (2.8 x 10(5) cells/cm2), and low cell passage level (less than 52 passages) favored the development of syncytia. All three types (1, 2, and 3) of dengue viruses tested could be isolated from human sera by Aedes pseudoscutellaris cell culture by using syncytial development as an indicator, but the isolation rate was lower than that obtained by using intrathoracically inoculated Aedes aegypti mosquitoes.

Properties of an established cell line from the atlantic croaker (Micropogon undulatus).

AbstractA fibroblast-like cell line, CrF, has been developed from muscle tissue of the marine teleost fish Micropogon undulatus (the Atlantic croaker). The line was initiated by subculture of cells from explanted tissue of a single male fish. The growth medium employed contained L-15 basal medium supplemented with 15% fetal calf serum, 1 to 5% human serum, and antibiotic-antimycotic mixture. The line was characterized fully and carried through 85 subcultures. Karyological analysis showed the line to be heteroploid, with a modal chromosome number of 48 at low passage levels and 49 at high levels. All chromosomes are small and telocentric. Growth characteristics were carried out under varying conditions of incubation. Optimal growth occurred at 26 to 32° in growth medium adjusted to a NaCl molarity of 0.147 to 0.177 M (although a wider range was tolerated). A plating efficiency of up to 30% and freeze viability rates of 65 to 70% were observed. The line was shown to be susceptible to lymphocystis virus, but...

An experimental inactivated virus vaccine against bovine ephemeral fever 2. Do neutralizing antibodies protect against infection?

In order to develop a safe vaccine against bovine ephemeral fever (BEF) which could be used in areas normally free of the disease, studies were carried out on inactivated virus vaccines. Initial experiments were carried out in cattle using virus vaccines that had been inactivated with β-propiolactone or formalin and then made-up in aluminium phosphate gel or Freund's incomplete adjuvant. A minimum inactivated virus dose of 10 6 PFU was necessary to stimulate a serum neutralizing antibody response in cattle. β-propiolactone inactivated BEF virus vaccines in Freund's incomplete adjuvant gave the best serum neutralizing antibody responses, producing high levels of neutralizing antibody with both high and low passage level virus. However, the magnitude of the antibody response bore little relationship to resistance of vaccinated animals to challenge with virulent BEF virus. A number of animals with high neutralizing antibody titres to BEF virus did not resist challenge. Using 500-fold less live virus at equivalent passage level to the low passage inactivated vaccine, similar or slightly lower antibody levels were attained, but most of the animals resisted challenge. It is suggested that the nature of the immune response and resistance to BEF infection may be complex and that reliance on serum neutralizing antibody as an indicator of resistance may give misleading results.

In vitro isolation of stable rat sarcoma viruses

A Sprague-Dawley (SD-1) rat embryo culture, at low passage level, released an endogenous ecotropic type C virus (SD-RaLV) and after about 20 further passages it underwent spontaneous transformation. The SD-RaLV, released from the transformed cells, did not cause rapid transformation of other rat embryo cells. However, when the transformed cells were repeatedly cocultivated with three different chemically transformed and serially transplanted rat tumor cell lines (sarcoma, carcinoma, and hepatoma), rapidly fibroblast-transforming "sarcoma" viruses (RaSV) were recovered after each attempt. RaSV was not recovered from one of these tumor cell lines before transplantation, nor could focus-forming virus be rescued from these same tumor cells by cocultivation with other cells releasing heterologous type C viruses. Foci were induced on normal rat kidney and several other rat embryo cell strains within 7-15 days and both productive and nonproductive NRK clones were derived. The productive clones were positive for rat specific p30 antigen and the RaSVs released were serially transmitted to other rat embryo cells. RaSV genome was rescued from the nonproductive clones by superinfection with SD-RaLV, wild rat type C virus, and several heterologous type C viruses. These observations appear to represent naturally occurring transformation-specific (src) genes being recovered in vitro in the form of stable "sarcoma" viruses. These viruses differ from the Kirsten and Harvey strains of murine sarcoma virus in that they apparently contain no MuLV sequences and are of purely rat origin.