Serial Subcultivations Research Articles

Potential probiotic lactobacilli strains isolated from artisanal Mexican Cocido cheese: evidence-based biosafety and probiotic action-related traits on in vitro tests.

The biosafety of four potentially probiotic lactobacilli strains, isolated from artisanal Mexican Cocido cheese, was assessed through in vitro tests aimed to determine (1) the antibiotic susceptibility profile by broth microdilution, (2) the transferability of antibiotic resistance determinants by filter-mating, and (3) the phenotypic and genotypic stability during serial batch sub-culture (100-day period) by evaluating physiological and probiotic features and RAPD-PCR fingerprinting. Lactobacilli strains exhibited multidrug-resistance; however, resistance determinants were not transferred in the filter-mating assay. Significant (p < 0.05) differences were observed in bacterial morphology and some functional and technological properties when strains were serially sub-cultured over 50 generations (G50), compared to the initial cultures (G0). Conversely, the strains did not show mucinolytic and hemolytic activities either at G0 or after 100 generations (G100). Genetic polymorphism and genomic template instability on selected strains were detected, which suggest possible evolutionary arrangements that may occur when these bacteria are largely cultured. Our findings suggest that the assessed strains did not raise in vitro biosafety concerns; however, complementary studies are still needed to establish the safe potential applications in humans and animals.

Overproduction of efflux pumps caused reduced susceptibility to carbapenem under consecutive imipenem-selected stress in Acinetobacter baumannii.

PurposeAcinetobacter baumannii is an important pathogen in the nosocomial infections worldwide. Combining with carbapenemases, efflux pumps and outer membrane proteins (OMPs) have been thought to affect the development of carbapenem resistance in A. baumannii. This study aimed to investigate the contributions of different efflux pumps and OMPs in developing carbapenem resistance in a clinical isolate of A. baumannii and reveal the possible mechanism of overproduction of main efflux pumps.Patients and methodsIn this study, an imipenem-susceptible clinical isolate was identified as A. baumannii and named SZE. Several common carbapenemases were detected by polymerase chain reaction (PCR). Imipenem-selected mutants were selected from SZE by serial subcultivations on Mueller–Hinton agar, and the minimum inhibitory concentration (MIC) was detected. Gene expressions of four families of efflux pumps, five OMPs, and blaOXA-51 were determined by reverse transcription quantitative PCR, and comparisons were made between SZE strain and the imipenem-selected mutants. The adeRS system in SZE and its mutant was sequenced and aligned.ResultsUnder consecutive imipenem-selected stress, the MIC to imipenem increased gradually from 0.125 μg/mL to 8 μg/mL. The effect of resistance inducement was almost neutralized when treated with an efflux pump inhibitor. The expression of efflux pumps, adeB, adeG, and adeJ, was increased by 6.9-, 4.0-, and 2.1-fold in mutants, respectively, compared to SZE. A single mutation (G to A) at position 58 was detected in the regulatory adeRS system and possibly upregulated the adeB expression, and then affected the carbapenem resistance in A. baumannii strains.ConclusionIn conclusion, under consecutive imipenem-selected stress in vitro, A. baumannii strain evolved the ability to reduce susceptibility to a variety of antimicrobials by overproduction of efflux pumps. Especially, the resistance-nodulation-cell division super family and a nucleotide mutant in adeRS regulating system caused the overexpression of adeABC.

Genome Characteristics of Two Novel Type I Methanotrophs Enriched from North Sea Sediments Containing Exclusively a Lanthanide-Dependent XoxF5-Type Methanol Dehydrogenase.

Microbial methane oxidizers play a crucial role in the oxidation of methane in marine ecosystems, as such preventing the escape of excessive methane to the atmosphere. Despite the important role of methanotrophs in marine ecosystems, only a limited number of isolates are described, with only four genomes available. Here, we report on two genomes of gammaproteobacterial methanotroph cultures, affiliated with the deep-sea cluster 2, obtained from North Sea sediment. Initial enrichments using methane as sole source of carbon and energy and mimicking the in situ conditions followed by serial subcultivations and multiple extinction culturing events over a period of 3years resulted in a highly enriched culture. The draft genomes of the methane oxidizer in both cultures showed the presence of genes typically found in type I methanotrophs, including genes encoding particulate methane monooxygenase (pmoCAB), genes for tetrahydromethanopterin (H4MPT)- and tetrahydrofolate (H4F)-dependent C1-transfer pathways, and genes of the ribulose monophosphate (RuMP) pathway. The most distinctive feature, when compared to other available gammaproteobacterial genomes, is the absence of a calcium-dependent methanol dehydrogenase. Both genomes reported here only have a xoxF gene encoding a lanthanide-dependent XoxF5-type methanol dehydrogenase. Thus, these genomes offer novel insight in the genomic landscape of uncultured diversity of marine methanotrophs.

Increased expression of IL-1 receptors in response to IL-1β may produce more IL-6, IL-8, VEGF, and PGE2 in senescent synovial cells induced in vitro than in presenescent cells

Primary synovial cells with high passage number demonstrate increased production of proinflammatory mediators in response to inflammatory stimuli compared with cells with low passage number. This study used synovial cells to learn how different numbers of serial subculture passages affect the production of proinflammatory mediators in response to interleukin (IL)-1β. Synovial cells were serially subcultured in flasks until passage 7. During cell passage, synovial cells were treated with IL-1β for 24 h. Levels of proinflammatory mediators were analyzed by ELISA, real-time PCR, and Western blot. Synovial cells at passage 7 had elongated morphology and higher activity of β-galactosidase, a marker of senescence, than those at passage 3. Production of IL-6, IL-8, vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE(2)) in response to IL-1β was significantly increased in cells at passage 7 compared with passage 3. To evaluate the mechanism of this different response, IL-1 receptor expression was studied in cells stimulated with IL-1β. Compared with cells at passage 3, cells at passage 7 had stronger expression of IL-1 receptors 1 and 2, as well as significantly increased expression of both receptors, in response to IL-1β. This finding suggests that differential production in response to inflammatory stimuli associated with cell passages should be considered when in vitro experiments are used to evaluate the production levels of proinflammatory mediators.

Nicotinamide phosphoribosyltransferase imparts human endothelial cells with extended replicative lifespan and enhanced angiogenic capacity in a high glucose environment

Endothelial dysfunction is a characteristic of aging-related vascular disease and is worsened during diabetes. High glucose can impair endothelial cell (EC) function through cellular accumulation of reactive oxygen species, an insult that can also limit replicative lifespan. Nicotinamide phosphoribosyltransferase (Nampt), also known as PBEF and visfatin, is rate-limiting for NAD+ salvage from nicotinamide and confers resistance to oxidative stress via SIRT1. We therefore sought to determine if Nampt expression could resist the detrimental effects of high glucose and confer a survival advantage to human vascular EC in this pathologic environment. Human aortic EC were infected with retrovirus encoding eGFP or eGFP-Nampt, and FACS-selected to yield populations with similar, modest transgene expression. Using a chronic glucose exposure model we tracked EC populations to senescence, assessed cellular metabolism, and determined in vitro angiogenic function. Overexpression of Nampt increased proliferation and extended replicative lifespan, and did so preferentially during glucose overload. Nampt expression delayed markers of senescence and limited reactive oxygen species accumulation in high glucose through a modest increase in aerobic glycolysis. Furthermore, tube networks formed by Nampt-overexpressing EC were more extensive and glucose-resistant, in accordance with SIRT1-mediated repression of the anti-angiogenic transcription factor, FoxO1. We conclude that Nampt enables proliferating human EC to resist the oxidative stress of aging and of high glucose, and to productively use excess glucose to support replicative longevity and angiogenic activity. Enhancing endothelial Nampt activity may thus be beneficial in scenarios requiring EC-based vascular repair and regeneration during aging and hyperglycemia, such as atherosclerosis and diabetes-related vascular disease.

Susceptibility of chimeric mice with livers repopulated by serially subcultured human hepatocytes to hepatitis B virus

We previously identified a small population of replicative hepatocytes in long-term cultures of human adult parenchymal hepatocytes (PHs) at a frequency of 0.01%-0.09%. These hepatocytes were able to grow continuously through serial subcultures as colony-forming parenchymal hepatocytes (CFPHs). In the present study, we generated gene expression profiles for cultured CFPHs and found that they expressed cytokeratin 19, CD90 (Thy-1), and CD44, but not mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) and glucose-6-phosphatase (G6P), confirming that these cells are hepatic progenitor-like cells. The cultured CFPHs were resistant to infection with human hepatitis B virus (HBV). To examine the growth and differentiation capacity of the cells in vivo, serially subcultured CFPHs were transplanted into the progeny of a cross between albumin promoter/enhancer-driven urokinase plasminogen activator-transgenic mice and severe combined immunodeficient (SCID) mice. The cells were engrafted into the liver and were able to grow for at least 10 weeks, ultimately reaching a maximum occupancy rate of 27%. The CFPHs in the host liver expressed differentiation markers such as TO, G6P, and cytochrome P450 subtypes and could be infected with HBV. CFPH-chimeric mice with a relatively high replacement rate exhibited viremia and had high serum levels of hepatitis B surface antigen. Serially subcultured human hepatic progenitor-like cells from postnatal livers successfully repopulated injured livers and exhibited several phenotypes of mature hepatocytes, including susceptibility to HBV. In vitro-expanded CFPHs can be used to characterize the differentiation state of human hepatic progenitor-like cells.

Age‐dependent telomere‐shortening is repressed by phosphorylated α‐tocopherol together with cellular longevity and intracellular oxidative‐stress reduction in human brain microvascular endotheliocytes

Cellular life-span of neonatal human brain microvascular endotheliocytes (HBME) was estimated by population doubling levels (PDLs) for serial subcultivations until spontaneous proliferation stoppage, and was 2.4-fold longer for continuous administration with the 6-O-phosphorylated derivative (TocP) of alpha-tocopherol (Toc), being bio-available owing to its water-solubility, or TocP plus 2-O-phosphorylated ascorbate (Asc2P), and 1.3-fold longer with Asc2P, at a dose of 150 microM, than for the non-administered control. Enlarged cell diameters indicative of cellular aging were repressed for TocP-administered cells as analyzed with a channelizer. Age-dependent shortening of telomeric DNA length (291 bp/PDL) was slowed markedly for TocP (165 bp/PDL) or TocP plus Asc2P, but slightly for Asc2P. Telomerase activity as assessed by the PCR-based TRAP method was detectable slightly at younger ages but no longer at middle ages for the non-administered cells, but, for TocP-administered cells, was intensely detected at younger ages and appreciably until middle ages. Intracellular TocP amounts were not changed age-dependently in contrast to a marked decrease in Toc which accrued from TocP esterolysis. This may be partly attributed to age-dependent changes in the lipid peroxidation product acrolein (ACR), which was abundant at older ages in non-administered cells, but scarcely in TocP-administered cells. Furthermore, intracellular reactive oxygen species (ROS) such as H(2)O(2) and hydroperoxides as detected using the redox indicator CDCFH-DA was less abundant in TocP-administered cells than in non-administered cells. Thus the telomeric-DNA retention, concurrently with retained telomerase activity, was shown to be correlated with cellular longevity, and may be supported by diminished oxidative stress, in hydrophobic microenvironment, which can be achieved by TocP rather than AscP.

Common genes responsible for differentiation and senescence of human mucosal and epidermal keratinocytes

Serial subcultures of normal human oral keratinocytes (NHOKs) and normal human epidermal keratinocytes (NHEKs) to the postmitotic stage result in terminal differentiation and replicative senescence. In order to investigate the common mode of differentiation and/or senescence between mucosal and epidermal keratinocytes, gene expression profiling on both NHOKs and NHEKs was performed by a cDNA microarray analysis. Primary NHOKs and NHEKs were serially subcultured, and the expression level of 3,063 genes was compared between the exponentially growing and senescent cultures. The senescent NHOKs and NHEKs highly expressed 55 and 37 genes, respectively. Among these genes, 16 genes were common in both NHOKs and NHEKs while the other genes were upregulated either in the NHOKs or in the NHEKs. Furthermore, the expression levels of the common genes did not change in the human diploid fibroblasts during the subcultures. These results suggest that subculture-induced differentiation and/or replicative senescence in NHOKs and NHEKs has similar characteristics, but that the pathways leading to these processes are distinct and keratinocyte specific.

Relation of the slow growth phenotype to neoplastic transformation: possible significance for human cancer.

Deletions are widely distributed over the genome in the most frequently occurring human cancers and are the most abundant genetic lesion found there. Deletions are highly correlated with the slow growth phenotype of mutated animal and human cells and result in chromosomal transposition when the retained ends are joined. Transpositions are only a minor source of mutation in rapidly multiplying bacteria but are a major cause of mutations in stationary bacteria. The NIH 3T3 line of mouse cells undergoes neoplastic transformation during prolonged incubation in a stationary state and expresses the slow growth phenotype on serial subculture at low density, suggesting a relation between transformation and chromosomal deletions. To further explore the relation between neoplastic transformation and the slow growth phenotype as a surrogate for deletions, two sublines of the NIH 3T3 cells with differing competence for transformation were serially subcultured in the stationary state at confluence and tested at each subculture for transformation and growth rate. Cell death in a fraction of the population and a heritable slowdown in proliferation of most of the survivors became increasingly pronounced with successive rounds of confluence. The reduction in growth rate was not proportional to the degree of transformation of the cultures, but all of the transformed cultures were slow growers at low density. All of the discrete colonies from cloning transformed cultures developed at a lower initial rate than control colonies under optimal conditions for growth, but they continued to grow at later stages, forming multilayered colonies under conditions that inhibited the further growth of the control colonies. The results suggest that prolonged incubation of NIH 3T3 cells in the stationary state results in growth-impairing deletions over a wide range of sites in the genome, but more restricted subsets of such lesions are responsible for neoplastic transformation. These findings provide dynamic, functional support in culture for the histopathological evidence that the quiescent state of cells associated with atrophy and fibrosis plays a significant role in the origin of some cancers in experimental animals and human beings.

Restoration of differentiated functions in multicellular aggregates of a human liver epithelial cell line

Whether or not the differentiated functions of a SV40 T antigen-immortalized human liver epithelial cell line (THLE-5b), most of which were lost after serial subcultivations, are restored was examined by culturing it in a three-dimensional system using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE). The morphology and differentiated functions of THLE-5b cells were influenced by the modification of the ePTFE surface. THLE-5b cells formed anchored multicellular aggregates on a PVA-coated ePTFE surface 3 to 4 days in culture, but formed cell monolayers on a PVA-uncoated ePTFE surface as did the cells on a type I collagen-coated polystyrene surface. Albumin and transferrin were expressed in multicellular aggregates, but was negative in the corresponding monolayer cultures. However, α1-antitrypsin was not expressed in multicellular aggregates as well as monolayer cultures. Although cytokeratin 18 was found both in monolayer cultures and in multicellular aggregates, cytokeratin 19 and γ-glutamyltranspeptidase were negative in monolayers and in multicellular aggregates. The present data suggest that some of the differentiated functions of THLE-5b cells are restored by three-dimensional cell–cell interaction in multicellular aggregates and also suggest that the multicellular aggregates of THLE-5b, as immobilized on a PVA-coated ePTFE surface, will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver.

Cellular characterization and successful transfection of serially subcultured normal human esophageal keratinocytes.

In vitro cell culture models can provide unique insights into squamous epithelial proliferation, differentiation, and neoplastic transformation. Cultures of human esophageal keratinocytes could be advantageous for the study of these processes. Normal human esophageal keratinocytes were cultivated on 3T3 fibroblast feeder layers in vitro and expanded through four serial subcultivations. Confluent tertiary cultures were analyzed by morphological and immunohistochemical techniques to define their basic properties. The ability to transiently transfect cultured esophageal epithelium was tested using a Rous sarcoma virus-luciferase reporter gene by the calcium phosphate and lipofection methods. Postconfluent cultures displayed a predominantly basal cell phenotype with limited stratification, widespread expression of keratins 5 and 14, and production of attachment specialization proteins such as alpha6beta4 integrin and collagen VII. Terminal differentiation markers (involucrin and transglutaminase) were prematurely expressed. The cells expressed growth factors important in proliferation and differentiation, such as transforming growth factor-beta and interleukin-1beta. Tertiary cultures were successfully transiently transfected with a Rous sarcoma virus-luciferase reporter gene construct. Normal human esophageal cells can be serially passaged through extended numbers of cell generations and transfected by standard methods. This in vitro system may be useful in the study of fundamental cellular processes governing proliferation and differentiation in the esophageal epithelium.

Cellular characterization and successful transfection of serially subcultured normal human esophageal keratinocytes

Background: In vitro cell culture models can provide unique insights into squamous epithelial proliferation, differentiation, and neoplastic transformation. Cultures of human esophageal keratinocytes could be advantageous for the study of these processes. Methods: Normal human esophageal keratinocytes were cultivated on 3T3 fibroblast feeder layers in vitro and expanded through four serial subcultivations. Confluent tertiary cultures were analyzed by morphological and immunohistochemical techniques to define their basic properties. The ability to transiently transfect cultured esophageal epithelium was tested using a Rous sarcoma virus-luciferase reporter gene by the calcium phosphate and lipofection methods. Results: Postconfluent cultures displayed a predominantly basal cell phenotype with limited stratification, widespread expression of keratins 5 and 14, and production of attachment specialization proteins such as a6b4 integrin and collagen VII. Terminal differentiation markers (involucrin and transglutaminase) were prematurely expressed. The cells expressed growth factors important in proliferation and differentiation, such as transforming growth factor-b and interleukin-1b. Tertiary cultures were successfully transiently transfected with a Rous sarcoma virus-luciferase reporter gene construct. Conclusion: Normal human esophageal cells can be serially passaged through extended numbers of cell generations and transfected by standard methods. This in vitro system may be useful in the study of fundamental cellular processes governing proliferation and differentiation in the esophageal epithelium. J. Cell. Physiol. 177:274‐281, 1998. q 1998 Wiley-Liss, Inc.

Unmasking large and persistent reductions in proliferation rate of aging cells.

We have reported that nontransformed sublines of NIH 3T3 cells that are incubated under the growth constraint of confluence for 10 d or longer exhibit heritable reductions of growth rate upon serial subculture at low density, which simulate the effects of aging in vivo on cell growth. There is also a marked increase in the likelihood of neoplastic transformation. After switching to a new batch of calf serum (CS), we found the reduced growth rate was no longer produced within the previously established timeframe. However, substitution of fetal bovine serum (FBS) for CS during the period of recovery from confluence or the following tests of growth rate resulted in profound inhibition of growth in cells serially subcultured from confluent cultures. In some cases, fewer than one in a thousand cells from subcultures of confluent cultures formed colonies in FBS although they cloned at relatively high efficiency in CS. The reduced growth in FBS was retained in the postconfluent subcultures after many generations of multiplication at low density in CS. Generally, similar results with individual variations were obtained with three other batches of FBS. The numbers of cells per 3-d colony initiated from subcultures of confluent cultures were lower than those of control cultures that had never been confluent. Supplementation of FBS-containing medium with CS fully restored the growth of the postconfluent subcultures to the rate in CS medium, indicating that there is a deficiency of growth factor(s) in FBS rather than the presence of an inhibitor. The results show that prolonged incubation at confluence induces a populationwide heritable increase in requirement for growth factor(s) in short supply in FBS. Because clonal studies have shown that the reduction in growth rate is irreversible and varies in degree from clone to clone, we propose it arises from damage to DNA at any of many different genetic loci or from chromosome aberrations. Such genetic damage is also consistent with the increased tendency for neoplastic transformation in subcultures from the long-term confluent cultures.

Cell aging in vivo and in vitro

It has become a staple assumption of biology that there is an intrinsic fixed limit to the number of divisions that normal vertebrate cells can undergo before they senesce, and this limit is in some way related to aging of the organism. The notion of such a limited replicative lifespan arose from the often repeated observation that diploid fibroblasts cannot proliferate indefinitely in monolayer culture, and that the number of divisions before senescence is directly related to the in vivo lifespan of different species. The in vitro evidence is countered by estimates that the number of cell divisions in some organs of rodents and man are one or more orders of magnitude higher than the in vitro limit, with no indication of the degenerative changes seen in culture. Serial transplantation experiments in animals also exhibit many more cell divisions than the in vitro studies, with some indicating an indefinite replicative lifespan. I present evidence that vertebrate cells are severely stressed by enzymatic dispersion and sustain cumulative damage during serial subcultivations. The evidence includes large increases in cell size and its heterogeneity, reductions in replicative efficiency at low seeding densities, appearance of abnormal structures in the cytoplasm, changes in metabolism to a common cell culture type, continuous loss of methyl groups and reiterated sequences from DNA, and a constant rate of decline of growth rate with passage. This evidence is complemented by the reduction induced in the replicative life span of diploid cells by a large array of treatments which have different primary targets in the cells. The most consistent and general observation of cell behavior in aging animals, with only a few exceptions, is a reduction in the rate of cell proliferation. This reduction is perpetuated when the cells are grown in culture, indicating it is an enduring and intrinsic property of the cells rather than a systemic effect of the aging organism. A similar heritable reduction in growth rate can be induced in established cell lines by prolonged incubation at quiescence. The reduction can be exaggerated by subculturing the quiescent cells under suboptimal conditions, just as the effects of age are exaggerated under stress. The constant decline of growth rate that occurs during serial passage of diploid cells may represent a similar decay of cell function. I propose that the limit on replicative lifespan is an artifact that reflects the failure of diploid cells to adapt to the trauma of dissociation and the radically foreign environment of cell culture. It is, however, a useful artifact that has given us much information about cell behavior under stressful conditions. The overall evidence indicates cells in vivo accumulate damage over a lifetime that results in gradual loss of differentiated function and growth rate accompanied by an increased probability for the development of cancer. Such changes are normally held to a minimum by the organized state of the tissues and homeostatic regulation of the organism. The rejection of an intrinsic limit on the number of cell divisions eliminates the need for a cellular clock, such as telomere length, that counts mitoses. I offer a heuristic explanation for the gradual reduction of cell function and growth capacity with age based on a cumulative discoordination of interacting pathways within and between cells and tissues. I also make a case for the use of established cell lines as model systems for studying heritable damage to cell populations that simulates the effects of aging in vivo, and represents a relatively unexplored area of cell biology.

Ubiquitous, heritable damage in cell populations that survive treatment with methotrexate.

A permanent line of mouse embryo fibroblasts was treated with concentrations of the anticancer drug methotrexate (MTX) that left 20-50% surviving colonies. The surviving population initially multiplied at a much slower rate than controls after subculture in the absence of the drug, and required 9-12 days of serial subculture, with selective growth of the faster growing cells, to approximate the control rate. To determine the distribution of growth rates of cells in the original posttreatment populations, many single cells were isolated in multiwell plates immediately after the treatment period, and the resulting clones were serially subcultured. Most of the control clones underwent about 2 population doublings per day (PD/D). Almost all the survivors of MTX treatment multiplied at heterogeneously reduced rates, ranging from 0.6 PD/D to as high as control rates for a very few clones. They maintained the reduced rates through many subcultivations. The heritability of the reduced growth rates indicates that most cells that retain proliferative capacity after treatment with MTX carry random genetic damage that is perpetuated through many divisions of their progeny. Similar results have been described for cells that survive x-irradiation, and suggest random genetic damage is a common occurrence among cells in rapidly growing tissues that survive cytotoxic treatment. It also occurs in serial subcultures of cells that had been held under the constraint of confluence for extended periods, which suggests that the accumulation of random genetic damage to somatic cells during aging of mammals underlies the reduction of growth rate and function of the cells that characterizes the aging process.

Identification and elimination of mycoplasmas in fish cell line cultures

AbstractMycoplasma contamination was identified in 11 cell lines derived from both coldwater and warmwater fish using DNA staining, microbiological culture and ELISA test detection methods. Acholeplasma laidlawii and Mycoplasma arginini were present as single or combined infections. Treatment of cells over three to six serial subcultivations with the addition of 0.5 μg m−1 mycoplasma removal agent (MRA) or 10 μg m−1 ciprofloxacin to culture growth medium effectively eliminated all contaminants except for one instance of M. arginini infection.

Telomere elongation observed in immortalized human fibroblasts by treatment with 60Co gamma rays or 4-nitroquinoline 1-oxide

Telomeres are the tandemly repeated (TTAGGG)n sequences that make up the structural and functional ends of all chromosomes in mammals. Many lines of evidence indicate that telomeres stabilize chromosomes, prevent aberrant recombination, and direct chromosome attachment to the nuclear membrane. Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' DNA synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this end-replication problem. To overcome this problem, most eukaryotic cells use telomerase, an enzyme that elongates telomeres. However, this enzyme has not been detected in normal human cells, and these cells lose telomeres with cell division. Cellular senescence might be the result of this loss. Thus, activation of telomerase seems to be critical for the immortalization of human cell lines. In addition, substantial evidence indicates that immortalization in itself is a rate-limiting step for the malignant transformation of human cells. We have treated normal human fibroblasts (AD387, KMS-6, and OUMS-24 lines) intermittently with either 60Co gamma rays or 4-nitroquinoline 1-oxide (4NQO) during serial subcultivations, and have obtained three immortalized cell lines, SUSM-1, KMST-6, and OUMS-24F. In KMS-6 and OUMS-24, the mean terminal restriction fragment length significantly decreased as the population-doubling level increased. The rate of telomere loss was 40 and 50 bp/population doubling in the KMS-6 and OUMS-24 cell lines, respectively. Once these normal cell lines were immortalized, their telomeres became elongated. Similar data were obtained for AD387 cells and their immortalized SUSM-1 cells. These results suggest that telomeres play a critical role in cellular senescence and in the immortalization processes of human cells.

Adherence of Haemophilus influenzae to human buccal and pharyngeal epithelial cells: relationship to pilation.

An improved understanding of the role of pili in adherence of Haemophilus influenzae type b to human epithelial cells (EC) would enhance knowledge of the pathogenesis of H. influenzae b infections. In this study a highly sensitive in-vitro assay allowed the quantitative assessment of H. influenzae b adherence to EC. The degree of adherence was influenced by incubation time, temperature, bacteria/EC ratio, EC type and the growth phase of the bacteria. Most serially subcultured (SC) capsular type-b strains originally isolated from cerebrospinal fluid, blood, nasopharynx or throat gave similar low degrees of adherence, as did representative single strains of capsular types a, c, d, e and f. SC non-capsulated H. influenzae strains adhered in significantly greater numbers than most SC capsulated strains (p less than 0.001). One SC type-b strain isolated from a throat, with stable piliation, adhered in very high numbers despite capsulation. Piliated subpopulations selected from type-b capsulated strains adhered in greater numbers than did their parent strains. These data suggest that capsulation of H. influenzae is a deterrent to adherence of the bacteria to EC. However, the presence of pili may allow type-b organisms to overcome the effects of capsulation.

Glucocorticoids enhance the mitogenic action of insulin in serum‐free cultures of chick embryo cells

AbstractAddition of insulin to nonproliferating serum‐free cultures of secondary chicken embryo (CE) cells caused a 30% to 50% increase in cell number. Addition of any one of several glucocorticoids (dexamethasone, cortisol, or corticosterone) to the cultures two days before insulin addition increased the mitogenic effect of insulin by about twofold at each insulin concentration tested. This glucocorticoid stimulation of cell proliferation was “permissive” because in the absence of insulin glucocorticoids caused little increase in cell number (usually less than 15%). Glucocorticoids were maximally active at low concentrations (e.g., 10−10 M dexamethasone). Steroids without glucocorticoid activity were inactive over a wide range of concentrations. Glucocorticoids increased the mitogenic response to insulin largely by increasing the percentage of cells that insulin stimulated to synthesize DNA.The maximum mitogenic effect of insulin upon CE cells rapidly decreased after the cells were serially subcultured. After only nine population doublings (4 passages) in culture, the response to insulin was diminished by about 70%. The mitogenic effect of insulin plus dexamethasone declined similarly during serial subculture, and was always about twofold greater than the effect of insulin alone. The cells maintained their mitogenic responsiveness to serum as these responses decreased.In contrast to the growth promoting influence of glucocorticoids in the presence of insulin, glucocorticoids inhibited the mitogenic response of CE cells to serum. This result may resolve our above findings with reports that glucocorticoids inhibit the proliferation of CE cells.

Rhinovirus type 14 persistence in hela cells studied by use of guanidine

AbstractGuanidine at 2 mM completely prevents replication of rhinovirus type 14 (RV14) in HeLa cells and spares these cells from RV14‐induced cytopathic effects. Three serial subcultivations of these cells in the continued presence of guanidine reduced infectious virus titers in cell lysates to undetectable levels. However, the capacity of RV14‐infected guanidine‐cultured cells to form infectious centers when cultured in the absence of guanidine was not completely abolished until the fifth serial subcultivation (14 days post‐inoculation). Production of defective‐interfering particles of RV 14 in HeLa cells was not observed. Thermal stability and transmission of uncoated RV 14 to daughter cells in the presence of guanidine can account for the persistence. Cells inoculated with RV14 and cultured in the presence of guanidine synthesized genomic RNA associated with a structure of 1.394 gm/ml density in CsCI, and these structures exhibited low infectivity by standard plaque assay. Nascent RV14 RNA did not appear in virions in cells treated with guanidine. HeLa cell lines (HeLaV+G), established following six serial subcultures of cells initially infected with RV 14 and continuously cultured in the presence of guanidine, were negative for RV 14 antigens by specific immunofluorescence tests. HeLaV+G cells were as efficient as normal HeLa cells in production of interferon; replication of RV 14 and mengovirus; and plaque assay of RV 14, rhinovirus type 2, and coxsackievirus B3.