Telomerase expression prevents replicative senescence but does not fully reset mRNA expression patterns in Werner syndrome cell strains.

Reduced replicative capacity is a consistent characteristic of cells derived from patients with Werner syndrome. This premature senescence is phenotypically similar to replicative senescence observed in normal cell strains and includes altered cell morphology and gene expression patterns. Telomeres shorten with in vitro passaging of both WRN and normal cell strains; however, the rate of shortening has been reported to be faster in WRN cell strains, and the length of telomeres in senescent WRN cells appears to be longer than that observed in normal strains, leading to the suggestion that senescence in WRN cell strains may not be exclusively associated with telomere effects. We report here that the telomere restriction fragment length in senescent WRN fibroblasts cultures is within the size range observed for normal fibroblasts strains and that the expression of a telomerase transgene in WRN cell strains results in lengthened telomeres and replicative immortalization, thus indicating that telomere effects are the predominant trigger of premature senescence in WRN cells. Microarray analyses showed that mRNA expression patterns induced in senescent WRN cells appeared similar to those in normal strains and that hTERT expression could prevent the induction of most of these genes. However, substantial differences in expression were seen in comparisons of early-passage and telomerase-immortalized derivative lines, indicating that telomerase expression does not prevent the phenotypic drift, or destabilized genotype, resulting from the WRN defect.     

hRev7, putative subunit of hPolzeta, plays a critical role in survival, induction of mutations, and progression through S-phase, of UV((254nm))-irradiated human fibroblasts

Translesion synthesis (TLS) refers to mechanisms by which specialized DNA polymerases incorporate nucleotides opposite fork-blocking lesions and extend replication until standard replicative polymerases take over. The first eukaryotic TLS polymerase discovered, S. cerevisiae Polzeta, consists of catalytic subunit Rev3 and non-catalytic subunit Rev7. Human homologs of these two proteins have been identified. Studies by Lawrence, Maher, and colleagues comparing UV((254nm))-irradiated human fibroblast cell strains expressing high levels of hRev3 antisense to their normal parental strains demonstrated that there was no difference in cell survival, but that the frequency of UV-induced mutations in the derivative strains was 10-fold lower than that of the parental strains, indicating that hRev3 plays a critical role in such mutagenesis. To examine the role of hRev7 in TLS, we generated human fibroblasts expressing hRev7 siRNA, identified two derivative cell strains with significantly reduced levels of hRev7, and compared them to their parental strain and a vector control for cell survival, induction of mutations, and ability to traverse the cell cycle following exposure to UV radiation. Cells with reduced hRev7 were approximately 2-times more sensitive to UV-induced cytotoxicity than the controls, indicating that unlike hRev3, hRev7 plays a protective role for cells exposed to UV radiation. When these cell strains were assayed for the frequency of mutations induced by UV in their HPRT gene, cell stains with reduced hRev7 were 5-times less sensitive to UV-induced mutagenesis than control strains. In addition, when these four strains were synchronized at the G1/S border, released from the block, UV-irradiated, and allowed to traverse the cell cycle, the rate of progression through S-phase of the cell strains with reduced hRev7 was significantly slower than that of the control strains. These data strongly support the hypothesis that hRev7 is required for TLS past UV-photoproducts, and together with hRev3, comprise hPolzeta.     

Susceptibility of Human Cell Strains to Transformation by Simian Virus 40 and Simian Virus 40 Deoxyribonucleic Acid

Marked differences were found in the susceptibility of human fibroblasts to transformation by simian virus 40 (SV40). Highly susceptible cell strains were derived from patients with diseases associated with chromosomal abnormalities and a high incidence of tumors. In the present study, SV40 transformation-susceptible cell strains were not found to have a generalized increase in viral sensitivity. The differences in transformation frequency among cell strains with whole virus are eliminated by the use of isolated SV40 deoxyribonucleic acid, suggesting that the relative resistance of most cell strains to transformation by whole virus is due to a block at an early step in infection.     

Demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism

Some strains of mouse hepatitis virus (MHV) can induce chronic inflammatory demyelination in mice that mimics certain pathological features of multiple sclerosis. We have examined neural cell tropism of demyelinating and nondemyelinating strains of MHV in order to determine whether central nervous system (CNS) cell tropism plays a role in demyelination. Previous studies demonstrated that recombinant MHV strains, isogenic other than for the spike gene, differ in the extent of neurovirulence and the ability to induce demyelination. Here we demonstrate that these strains also differ in their abilities to infect a particular cell type(s) in the brain. Furthermore, there is a correlation between the differential localization of viral antigen in spinal cord gray matter and that in white matter during acute infection and the ability to induce demyelination later on. Viral antigen from demyelinating strains is detected initially in both gray and white matter, with subsequent localization to white matter of the spinal cord, whereas viral antigen localization of nondemyelinating strains is restricted mainly to gray matter. This observation suggests that the localization of viral antigen to white matter during the acute stage of infection is essential for the induction of chronic demyelination. Overall, these observations suggest that isogenic demyelinating and nondemyelinating strains of MHV, differing in the spike protein expressed, infect neurons and glial cells in different proportions and that differential tropism to a particular CNS cell type may play a significant role in mediating the onset and mechanisms of demyelination.     

Evidence that the PBP 5 synthesis repressor (psr) of Enterococcus hirae is also involved in the regulation of cell wall composition and other cell wall-related properties.

psr has been reported by M. Ligozzi, F. Pittaluga, and R. Fontana, (J. Bacteriol. 175:2046-2051, 1993) to be a genetic element located just upstream of the structural gene for the low-affinity penicillin-binding protein 5 (PBP 5) in the chromosome of Enterococcus hirae ATCC 9790 and to be involved in the repression of PBP 5 synthesis. By comparing properties of strains of E. hirae that contain a full-length, functional psr with those of strains that possess a truncated form of the gene, we have obtained data that indicate that psr is involved in the regulation of several additional surface-related properties. We observed that cells of strains that possessed a truncated psr were more sensitive to lysozyme-catalyzed protoplast formation, autolyzed more rapidly in 10 mM sodium phosphate (pH 6.8), and, in contrast to strains that possess a functional psr, retained these characteristics after the cultures entered the stationary growth phase. Cellular lytic properties did not correlate with differences in the cellular contents of muramidase-1 or muramidase-2, with the levels of PBP 5 produced, or with the penicillin susceptibilities of the strains. However, a strong correlation was observed with the amounts of rhamnose present in the cell walls of the various strains. All of the strains examined that possessed a truncated form of psr also possessed approximately one-half of the rhamnose content present in the walls of strains that possessed a functional psr. These data suggest that psr is also involved in the regulation of the synthesis of, or covalent linkage to the cell wall peptidoglycan of, a rhamnose-rich polysaccharide. These differences in cell wall composition could be responsible for the observed phenotypic differences. However, the multiple effects of psr suggest that it is part of a global regulatory system that, perhaps independently, affects several cell surface-related properties.     

Siderophores and outer membrane proteins of antagonistic, plant-growth-stimulating, root-colonizing Pseudomonas spp

As an approach to understanding the molecular basis of the reduction in plant yield depression by root-colonizing Pseudomonas spp. and especially of the role of the bacterial cell surfaces in this process, we characterized 30 plant-root-colonizing Pseudomonas spp. with respect to siderophore production, antagonistic activity, plasmid content, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of their cell envelope proteins. The results showed that all strains produce hydroxamate-type siderophores which, because of the correlation with Fe3+ limitation, are thought to be the major factor responsible for antagonistic activity. Siderophore-negative mutants of two strains had a strongly decreased antagonistic activity. Five strains maintained their antagonistic activity under conditions of iron excess. Analysis of cell envelope protein patterns of cells grown in excess Fe3+ showed that most strains differed from each other, although two classes of similar or identical strains were found. In one case such a class was subdivided on the basis of the patterns of proteins derepressed by iron limitation. Small plasmids were not detected in any of the strains, and only one of the four tested strains contained a large plasmid. Therefore, it is unlikely that the Fe3+ uptake system of the antagonistic strains is usually plasmid encoded.     

Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.     

Biogenesis of mitochondria

Summary The proportion of total cell DNA which is mitochondrial DNA was measured in haploid, diploid and tetraploid strains of S. cerevisiae grown under a standard set of conditions. For all strains tested the mitochondrial DNA level was in the range 16%–25% of total cell DNA. Repeated measurements of the cellular level of mitochondrial DNA in two haploid strains showed that these strains have measurably different cellular mitochondrial DNA levels (17% and 24% of total DNA, respectively) under our conditions. These two grande strains were used to investigate the role of the mitochondrial and nuclear genomes in the regulation of the mitochondrial DNA level. We have shown by genetic analysis that the difference between these two strains is determined by at least two nuclear genes. The mitochondrial genome is not involved in the regulation of cellular mitochondrial DNA levels.A number of purified petite clones derived from independent spontaneous petite isolates of the grande strain which contained 24% mitochondrial DNA were also studied. The mitochondrial DNA levels in all but one of these petites fell in the range 20–25% of total cell DNA. From these results we conclude that, in general, the mitochondrial DNA level in petite strains is controlled by the same mechanism as operates in grande strains.We propose a general model for the control of the cellular mitochondrial DNA level, in which the amount of mitochondrial DNA per cell is determined by regulation of the number of mitochondrial DNA molecules per cell. This regulation is mediated through the availability of a set of nuclear coded components, possibly a mitochondrial membrane site, which are required for the replication of mitochondrial DNA.     

Growth capacity in response to auxin of coleoptile segments of normal and dwarf rice strains

Auxin-induced growth, epidermal cell length, cellular osmotic potential, and cell wall composition of coleoptile segments excised from one normal and two dwarf rice strains were studied 2, 3, 4, and 5 days after soaking. The auxin-induced growth was higher at the early stages of coleoptile growth and decreased with age, being always higher in normal than in the two dwarf strains. A good correlation between auxin-induced growth and auxin-induced decrease in the minimum stress-relaxation time has been found, suggesting that the different growth capacity in response to auxin among the three different strains is due to differences in the structure of their cell walls. In fact, cell wall analysis revealed that (1) the relative α-cellulose content of the cell walls was higher in the two dwarf strains than in the normal one, and (2) the auxin-induced decrease in noncellulosic glucose was high, compared with dwarf strains, in the normal strain, which showed the higher auxin-induced growth, showing a highly significant correlation between the decrease in noncellulosic glucose and the growth in response to auxin. Thus, the different growth between normal and dwarf strains might be attributed to their different capacity to degrade β-glucan of their cell walls.     

Radioprotective effect of cysteamine in glutathione synthetase-deficient cells

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect.     

Efficient and quantitative incorporation of diaminopimelic acid into the peptidoglycan of Salmonella typhimurium

Abstract Diaminopimelic acid is incorporated into the peptidoglycan of Salmonella typhimurium in an efficient and quantitative manner. The amount of DAP incorporated is similar to the number of molecules estimated to exist in the Salmonella cell wall. In contrast, strains of E. coli , including those most used for studies of cell wall synthesis, are much less efficient in the incorporation of diaminopimelic acid. The lysine-requiring strains of E. coli appear to excrete diaminopimelic acid related material during growth and this accounts, in part, for the inefficient incorporation of radioactive diaminopimelic acid into Escherichia strains. In addition, the Escherichia strains are much less permeable to DAP than Salmonella strains. Cysteine and cystine inhibit the incorporation of DAP into the cell and this result suggests that Salmonella uses the cystine uptake system to allow DAP into the cell.     

Effects of Bacillus anthracis hydrophobicity and induction of host cell death on sample collection from environmental surfaces

The objective of this study is to determine whether DNA signature recovery of Bacillus anthracis strains from different environmental substrates correlates with pathogen cell surface hydrophobicity and induction of host cell death. We compared recovery of DNA signatures from a panel of B. anthracis strains collected from two environmental substrates, non-porous surfaces and soil, using real-time qPCR. We further assessed both cell surface hydrophobicity of the B. anthracis strains by contact angle measurements and host cell viability in response to B. anthracis infection in a mouse macrophage cell model system. Our studies demonstrated correlation between reduced B. anthracis sample recovery from environmental substrates and increased cell surface hydrophobicity. Surprisingly, the most hydrophilic strain, K4596, which exhibited the highest level of recovery from the environmental surfaces, induced the highest level of host cell cytotoxicity compared to more hydrophobic B. anthracis strains in the panel. Our results suggest that cell surface hydrophobicity may play a leading role in mediating pathogen adherence to environmental surfaces. These findings can contribute to the optimization of pathogen detection efforts by understanding how bacterial parameters such as hydrophobicity and induction of host cell death affect bacterial adherence to environmental surfaces.     

Effect of amino acid deprivation and chloramphenicol treatment on cell sizes of rel+ and relA- strains of Escherichia coli.

The effects of inhibition of protein synthesis on the cell size distributions of rel+ and relA- derivatives of Escherichia coli K-12 were determined. Amino acid deprivation resulted in a reduction in the cell sizes of rel+ strains but not of relA- strains. Treatment with chloramphenicol (CAM) did not alter the size distributions of either rel+ or relA- strains except when they were rel+ dap-. CAM treatment of rel+ dap- strains resulted in an increase in cell size. It is proposed that these results reflect differences in the structures of the cell envelopes of rel+ and relA- bacteria.     

The response of normal and ataxia-telangiectasia human fibroblasts to the lethal effects of far, mid and near ultraviolet radiations

The responses of two ataxia-telangiectasia (A-T) cell strains to the lethal effects of monochromatic far, mid and near ultraviolet radiations have been determined and compared with the responses of three normal human cell strains. Our results confirm a previous observation that the A-T cell strain AT4BI is abnormally sensitive to the lethal effects of mid u.v. (313 nm) radiation. After far u.v. (254 nm) radiation the strain AT4BI exhibits a small but statistically significant increase in sensitivity compared to the normal strains. Of most interest, in terms of a mechanistic interpretation of the sensitivity of A-T strains, the survival responses of neither A-T strain tested to near u.v. (365 nm) radiation differed significantly from the mean response of the normal strains, although it is of interest that one normal strain (48BR) was found to be significantly more resistant to near u.v. radiation than any of the other strains tested. The results are discussed in terms of the possible induction of radiogenic lesions in DNA by ultraviolet radiations and the possible mechanisms of radiation sensitivity in ataxia-telangiectasia.     

High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain

During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases. Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in degP prc spr was necessary for the cells to produce high levels of the desired recombinant antibody fragment. In order to eliminate the possible effects of mutations in other genes, we constructed E. coli strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations. Extensive light chain proteolysis persisted in degP strains. However, light chain proteolysis was substantially decreased in prc and prc spr strains, and was further decreased with the introduction of a degP mutation in prc and prc spr mutant strains. These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in E. coli, and that DegP (HtrA) makes a minor contribution to this degradation as well. The results also show that spr, a suppressor of growth defects in prc strains, is required for a prc mutant to survive throughout high cell density fermentations.     

Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry

A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits. Mol. Reprod. Dev. 67: 91-100, 2004.     

Fusion and hybridization of marsupial and eutherian cells. V. Development of selective systems

The availability of systems which permit the selective elimination of marsupial cells from fused cultures is an essential requirement for the production of marsupial X eutherian somatic cell hybrids. Such hybrids have particular advantages for genetic studies of mammalian cells. We describe the isolation and characterization of several drug-resistant marsupial cell strains. We have selected strains resistant to concentrations of 10 micrograms/ml of the purine analogues 8-azaguanine and 6-thioguanine. Several of these strains were found to be deficient in the enzyme hypoxanthine phosphoribosyltransferase and consequently sensitive to hypoxanthine-aminopterin-thymidine (HAT) selective medium. We have also isolated marsupial cell strains resistant to concentrations of 22 micrograms/ml of the thymidine analogue 5-bromodeoxyuridine. These strains were thymidine kinase deficient and HAT sensitive. Drug resistance was a stable characteristic maintained for many generations in the absence of the drug. However, inhibition of growth of these drug-resistant strains was strongly density dependent, a factor that caused difficulties in the selection of hybrids. We have also developed selective systems which exploit differences between marsupial and eutherian cells in sensitivity to growth in ouabain, and in adhesiveness and other growth properties. Marsupial cells were found to be naturally much more sensitive to ouabain than rodent cells, a phenomenon that should be useful in the selection of marsupial X rodent cellular hybrids. We discuss a number of difficulties associated with the derivation and use of variant marsupial cell strains.     

Variation in Galr1 expression determines susceptibility to exocitotoxin-induced cell death in mice

Inbred strains of mice differ in their susceptibility to excitotoxin-induced cell death, but the genetic basis of individual variation in differential susceptibility is unknown. Previously, we identified a highly significant quantitative trait locus (QTL) on chromosome 18 that influenced susceptibility to kainic acid-induced cell death ( Sicd1 ). Comparison of susceptibility to seizure-induced cell death between reciprocal congenic lines for Sicd1 and parental background mice indicates that genes influencing this trait were captured in both strains. Two positional gene candidates, Galr1 and Mbp , map to 55 cM, where the Sicd1 QTL had been previously mapped. Thus, this study was undertaken to determine if Galr1 and/or Mbp could be considered as candidate genes. Genomic sequence comparison of these two functional candidate genes from the C57BL/6J (resistant at Sicd1 ) and the FVB/NJ (susceptible at Sicd1 ) strains showed no single-nucleotide polymorphisms. However, expression studies confirmed that Galr1 shows significant differential expression in the congenic and parental inbred strains. Galr1 expression was downregulated in the hippocampus of C57BL/6J mice and FVB.B6- Sicd1 congenic mice when compared with FVB/NJ or B6.FVB- Sicd1 congenic mice. A survey of Galr1 expression among other inbred strains showed a significant effect such that 'susceptible' strains showed a reduction in Galr1 expression as compared with 'resistant' strains. In contrast, no differences in Mbp expression were observed. In summary, these results suggest that differential expression of Galr1 may contribute to the differences in susceptibility to seizure-induced cell death between cell death-resistant and cell death-susceptible strains.     

Cultivation of formerly noncultivable strains ofMycoplasma hyorhinis

Growth on axenic agar medium is one of several characters by which mycoplasmas are defined. In apparent contradiction of the definition, DBS 1050 and other noncultivable strains ofMycoplasma hyorhinis do not grow on axenic medium but grow in cell culture. Our results show that BHK-21 cell extracts support DBS 1050 growth in appropriate medium. An inhibition assay, based on a virus neutralization format, shows that a variety of common medium ingredients inhibit DBS 1050 growth. The most potent activity was found in yeast extract. All other noncultivable strains ofM. hyorhinis tested have a yeast extract sensitivity, while cultivable strains do not. The apparent cell dependence of DBS 1050 can be attributed to growth inhibition due to factors present in a wide variety of peptones and extracts commonly used in medium; preferential growth in cell cultures is due to the absence of effective levels of these factors. Data are not available to determine if cell cultures provide growth factors not found in standard medium. The infraspecific taxon,M. hyorhinis cultivar α, is proposed for formerly noncultivable strains ofM. hyorhinis.