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PubMed1990

Transformation and immortalization of diploid xeroderma pigmentosum fibroblasts.

Diploid xeroderma pigmentosum (XP) skin fibroblast strains from various XP-complementation groups (B, C, G, and H) were transformed with an origin-defective SV40 early region or with the pSV3 gpt plasmid. In the latter case, transfected cells were selected for their ability to express the dominant xgpt gene. Immortalized cell lines were obtained, from XP-complementation groups C (8CA, 3MA, and 20MA; XP3MA and XP20MA were formerly considered to belong to complementation group I), G (2BI and 3BR), and H (2CS). No immortalized cells could be isolated from complementation group B (11BE). The immortalization frequency of wild-type diploid fibroblasts and diploid cultures from XP patients was not significantly increased by cotransfection with the SV40 early region plus several selected viral and cellular oncogenes. In fact, co-transfection with some of the oncogenes caused a marked decrease of the transformation frequency. The observed immortalization occurred at a frequency of approximately 5 x 10(-8).

Cell Line, Transformed↗

PubMed1977

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. I. Cell cycle inhibition and aberration induction in diploid and Down's fibroblasts.

The effect of N-methyl-N-nitrosourea (MNU) on the cell cycle, DNA synthesis and chromosomal sensitivity of cultivated diploid fibroblasts and fibroblasts with trisomy 21 was investigated in vitro. With the exception of the inhibition of G2, Down's cells proved to be more sensitive than diploid cells with respect to the decrease of the mitotic and labelling index, the inhibition of the progression of cells through the early and middle S and the frequency of induced chromosomal aberrations. The chromosomal sensitivity was dependent on the position of cells in the cell cycle during treatment with MNU. If treated during late S no differences concerning the S block and aberration frequencies were found between diploid and Down's cells. However, if MNU treatment took place in the middle and early S, Down's cells were more sensitive. The higher aberration frequencies in Down's cells resulted from elevated levels of chromatid breaks, multiple fragmentations and chromatid translocations. Possible reasons for the increased sensitivity of Down's cells are discussed.

Cell Division↗

PubMed1977

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. II. Non-random distribution of chromatid aberrations in diploid and Down's cells.

Chromatid gaps, breaks and aberrations involved in interchanges induced by N-methyl-N-nitrosourea (MNU) were found non-randomly distributed on individual chromosomes and chromosome segments (G bands) both in human diploid fibroblasts with trisomy 21 cultured in vitro. Aberration events were located exclusively in pale G bands. Considering cells in the first post-treatment mitosis, the pattern of aberration distribution, as revealed by the position of hot spots, varied with recovery time and was different in diploid and Down's cells. In comparison with diploid cells, the X chromosomes of Down's cells were not involved in aberrations. Despite the higher aberration frequencies of Down's cells, the number of hot spots and the proportion of aberrations located in hot spots were not increased in this cell type. Therefore, the increased chromosomal sensitivity to MNU of Down's cells does not reflect an increased sensitivity of special chromosomes or chromosome sites.

Cell Division↗

PubMed1980

The induction and detection of diploid spermatozoa in Gallus domesticus.

Colcemid at the dose level of 0.37 mg/kg/day was injected intraperitoneally to 3 sexually active chicken males for 3 consecutive days. 10-12 days after the first colcemid injection, 14-25% of the sperm population in the semen samples from the treated males was found to be diploid in DNA content by flow microfluorometric analysis. Cytogeneic and developmental analyses on early embryos indicate that, during the process of spermatogenesis, the male germ cells are most susceptible to colcemid treatment 10-12 days prior to the maturation of the spermatozoa which is equivalent to the primary through secondary spermatocyte stages in chicken males. By the application of an extremely unequal chromosomal translocation as a cytological marker of parentage, it is confirmed that the diploid sperm induced are capable of uniting with a normal haploid or diploid egg to produce a triploid or tetraploid zygote.

Animals↗

PubMed1981

Further studies on effects of X-irradiation on prespermatid stages of the Northern vole Microtus oeconomus: low induction of sex-chromosomal nondisjunction and very high induction of diploid spermatids.

Microtus males have been irradiated with X-ray doses of 25, 50, 100 and 200 rad and early spermatids were then analyzed for evidence of induction of sex-chromosomal nondisjunction and diploid spermatids at 1, 4, 6, 7, 9 and 12 days after treatment. In contrast to earlier findings, there was no induction of nondisjunction above control levels. A possible explanation for the differences in results of old and new experiments might be that genetic changes have taken place in the Microtus colony that was initiated with animals trapped in the wild, but which has now become highly inbred. In the present experiment, diploid spermatids were frequently induced. The dose--effect relationships at the different time intervals were linear, but the slopes were different, indicating stage-specific differences in sensitivity. The average doubling dose is of the order of 12 rad with a range of 5-30 rad for the individual time intervals. When diploid spermatozoa in man are also inducible by such low doses of X-rays, the consequence would be an increase of triploid abortions which would constitute an undesirable form of personal or family hardship.

Animals↗

PubMed1987

Hydatidiform mole: genetic markers in diploid abortuses with macroscopic villous enlargement.

In a series of diploid abortion specimens with gross villous enlargement the parental origin was determined by chromosomal heteromorphisms, HLA typing, and enzyme analysis. Diploid androgenesis was the mechanism in 33 cases; in 28 cases the most likely origin was by duplication of a haploid sperm after fertilization of an anucleated ovum, whereas heteromorphisms in five cases indicated a dispermic origin. In one macroscopically complete molar specimen all marker techniques applied indicated a normal conception. In two homozygous specimens a second cell line with both maternal and paternal contributions indicated a twin gestation, whereas, in four conceptuses twinning was suggested solely by HLA determination or ultrasound scan. The observation of a heterogeneous origin of diploid moles, as indicated by three marker systems studied simultaneously, emphasizes that additional information about the frequency of different types of molar conceptions may be obtained by this approach.

Abortion, Induced↗

PubMed1985

Diploid and tetraploid grey treefrogs (Hyla chrysoscelis and Hyla versicolor) have similar metabolic rates.

We measured oxygen consumption of the diploid frog Hyla chrysoscelis and its recently evolved tetraploid cryptic species Hyla versicolor at rest and during forced locomotory exercise. We also measured whole-body lactic acid concentrations of individuals of the two species at rest and following 4 min of exercise. Contrary to predictions based on tissue-culture experiments, rates of organismal metabolism are not different in this diploid/tetraploid species pair: the diploid and tetraploid species did not differ in rates of oxygen consumption at rest or during exercise. Furthermore, whole-body lactate concentrations of frogs at rest and following forced locomotion were the same in the diploid and tetraploid species. For these species, polyploidy does not appear to be associated with lower maintenance costs at the level of the organism. However, polyploidy also does not appear to have metabolic consequences that would limit the behavioral or ecological repertoire of an anuran.

Aerobiosis↗

PubMed1979

The biosynthesis of nicotinamide adenine dinucleotide during early stages of frog embryonic development of haploid and diploid embryos.

1. Concentration of NAD during embryonic development of haploid and diploid embryos of frog was followed. NAD content in haploid embryonic forms is twice that in diploid embryos. 2. The variation of the NMN adenylyltransferase activity in the oocytes and during the first states of embryonic development as surveyed in the nuclear soluble fraction and the nuclear insoluble fraction (chromatin). 3. The enzyme activity in the soluble fraction is low during embryonic development and shows higher values in haploid embryos. 4. In the nonfertilized mature oocytes, the NMN adenylyltransferase activity is sixfold higher in the insoluble chromatin fraction than in the soluble fraction. 5. The evolution of the NMN adenylyltransferase in the insoluble chromatin fraction also shows higher values in haploid embryos, as compared with diploid forms.

Animals↗

PubMed1992

Differential radioprotection of cultured human diploid fibroblasts and fibrosarcoma cells by WR1065.

The present studies were performed to determine whether WR1065, the dephosphorylated, free-thiol active metabolite of WR2721, could provide differential radioprotection of normal and tumor cell lines in vitro and secondly to investigate potential mechanisms for the selective nature of the radioprotection at the cellular and molecular level. When 4 mM WR1065 was administered 30 min prior to and during irradiation, a protection factor of 1.9 was obtained in clonogenic assays performed with normal human diploid fibroblasts (AG1522) while no protection of fibrosarcoma cells (HT1080) was observed. Some radioprotection of fibrosarcoma cells was observed with higher drug concentrations (10-40 mM), but the increase in survival was considerably less than the plateau level reached with the diploid fibroblasts (3-fold vs 24-fold at 6 Gy). The observation of such a selective effect in vitro with WR1065 indicates that differences in tissue-specific variables such as blood flow, pH, pO2, and drug dephosphorylation cannot solely account for the selective nature of the radioprotection afforded by WR2721 in vivo. Incubation of nucleoids with increasing concentrations of the DNA intercalating dye propidium iodide was used to titrate the ability of DNA to undergo supercoiling changes. The relaxation and rewinding of supercoiled DNA loops in isolated nucleoids serves as an indicator of both the presence of DNA damage and inherent differences in DNA loop characteristics. Fibrosarcoma cells had a much larger propidium iodide-relaxable DNA loop size than fibroblasts. The rewinding phase of the DNA supercoiling response is impaired by the presence of radiation-induced DNA strand breaks. Four mM WR1065 resulted in a significant reduction in the amount of rewinding inhibition observed after a dose of 10 Gy in diploid fibroblasts (protection factor = 1.43) but did not alter the response of irradiated fibrosarcoma cells. These results, indicating that WR1065 had a preferential radioprotective effect in vitro on both survival and the manifestation of DNA damage at the nucleoid level, are consistent with the hypothesis that cell type differences in chromatin organization and DNA-drug associations could play a role in the selective radioprotection.

Cell Survival↗

PubMed1978

Transformation of normal diploid cells by isolated metaphase chromosomes of virus-transformed or spontaneous tumor cells.

Normal diploid human cells with a limited life-span in culture, as well as primary or secondary cell cultures of mouse or rat embryos, can be transformed in vitro (i.e. grow in soft-agar or low-serum medium) after a single exposure to metaphase chromosomes from SV40-transformed human or rat cells, Ad5-transformed human cells and several spontaneous human or mouse tumor cells. Chromosomes from normal diploid cells do not show any such transforming activity. As judged from the number of colonies formed in selective medium, the efficiency of transformation is, with some exceptions, of the order of 10(-5)--10(-6) and is generally higher for homologous than for heterologous transfers. A fraction of the colonies demonstrate abortive transformation. Nevertheless, using chromosomes from all but one donor cell population, at least one transferent cell line expressing a stable transformed phenotype has been established. Our results demonstrate that transformation of normal diploid cells by a presumptive chromosome-mediated gene transfer can be obtained with a variety of donor and recipient cells.

Adenoviridae↗

PubMed1990

Long-term growth of diploid human fibroblasts in low serum media.

Hayflick and Moorhead demonstrated that diploid human fibroblasts have a limited life span when grown in media containing 10% bovine calf sera. Recent experiments have suggested that antigrowth factors in serum may be a potential contributor to the limited proliferative capacity of normal diploid cells. To reduce the concentration of inhibitory serum factors 10-fold, MRC-5 diploid fibroblasts were cultured in media with only 1% serum. Long-term culture in 1% serum requires the addition of purified growth factors to sustain proliferation. Although there are dramatic changes in cell morphology, we find that the long-term division potential of MRC-5 cells cultured in media containing 1% serum and growth factors differs little from that found with cells cultured in 10% serum. In contrast, MRC-5 cells cultured in 10% serum and added growth factors have a somewhat extended life span. These results suggest that negative growth factors are not responsible for the limited proliferative capacity of in vitro cultured human fibroblasts. Moreover, the evidence that human fibroblasts can undergo major changes in cell morphology and still retain a normal life span raises questions about the validity of using morphological changes as indicators of cellular senescence.

Aging↗

PubMedDate not supplied

Changes in the cell surface of human diploid fibroblasts during cellular aging.

The electrophoretic mobility of 13 human diploid cell strains, TIG-1, TIG-2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, TIG-1H, TIG-1L, TIG-2M, TIG-2B, and TIG-3S, which were established from different tissues of human embryos, was studied at different passages. The net negative surface charge of the cells was characteristic for each cell strain and decreased significantly during the in vitro aging of the cells. The decrease in the net negative charge of the cells correlated well with the decrease in cell density throughout the life span of the cells. A strict linear correlation between the electrophoretic mobility and the number of cells harvested at each passage was obtained for all the human diploid cell strains. Moreover, almost the same linear regression coefficient of the cells was obtained among these cell strains. Therefore, the net negative surface charge of human diploid cell strains could serve as a cell surface marker for in vitro cellular aging.

Animals↗

PubMed2006

The activity of several components of the innate immune system in diploid and triploid turbot.

The use of triploid fish may be of interest in research, e.g. study of how this condition affects the size and activity of cells. In addition, triploid fish are sterile and production of triploids in fish species that are marketed after reaching sexual maturity may be of economic interest. In the present study, the effects of triploidy on the activity of several components of the innate immune system of turbot (Psetta maxima L) were determined. Triploid turbot had bigger cells (erythrocytes and neutrophils) but the number of blood erythrocytes, leucocytes and thrombocytes was lower than in diploid fish. The differential cell count was similar in both types of fish. The respiratory burst and the phagocytic activities were higher in neutrophils of triploid turbot. However, because the number of neutrophils was higher in diploids, the total respiratory burst activity and the phagocytosis per microliter of blood was similar in both types of fish. No differences were found in serum complement, lysozyme or bactericidal activities. The results indicate that the activities of the humoral components of the innate immune system tested are similar in diploid and triploid fish and that the lower leucocyte number found in triploids is compensated for by higher cell activity.

Animals↗

PubMed2005

Molecular epidemiology of clonal diploids: a quick overview and a short DIY (do it yourself) notice.

In this short review we report the basic notions needed for understanding the population genetics of clonal diploids. We focus on the consequences of clonality on the distribution of genetic diversity within individuals, between individuals and between populations. We then summarise how to detect clonality in mainly sexual populations, conversely, how to detect sexuality in mainly clonal populations and also how genetic differentiation between populations is affected by clonality in diploids. This information is then used for building recipes on how to analyse and interpret genetic polymorphism data in molecular epidemiology studies of clonal diploids.

Clone Cells↗

PubMed2000

Partial hydatidiform mole with diploid karyotype in a live fetus.

Even though most instances of partial mole are triploid, only a few cases of diploid partial moles have been reported. Prognosis of partial mole is usually better than the complete mole as few cases of partial moles progress to persistent trophoblastic disease. However, the nature and the risks of diploid partial moles are not well established and they seem to be a distinct clinical entity. Here we report a case of partial mole presenting with a 22 weeks live fetus and preeclampsia. Fetal blood sampling was performed for cytogenetic analysis which revealed a diploid (46XY) karyotype. No progression to malignant gestational trophoblastic disease occurred.

Adult↗

PubMed1996

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer.

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post coïtum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.

Journal Article↗

PubMed1999

Electroactivation of rabbit oocytes in an hypotonic pulsing medium and parthenogenetic in vitro development without cytochalasin B-diploidizing pretreatment.

We investigated the electroactivation frequencies, type of activation and in vitro development of rabbit oocytes. In Experiment 1, activation (8 pulses, 12 min apart, 60 microsec, 0.6 kVcm(-1)) was performed by altering osmolarity (190 vs. 320 mOsm kg(-1)) and Ca++ concentration (10, 60 or 100 microM) in mannitol pulsing media. More oocytes were activated in hypotonic pulsing medium, regardless of Ca++ concentration (96 to 100%). Both haploid and diploid parthenogenetic embryos developed to compacted morulae (57 to 92% and 63 to 100%, respectively) regardless of the activation treatment; however, the blastocyst rates were more variable (0 to 74% and 0 to 73%, respectively). In Experiment 2, the effects of pulse duration (30 or 60 microsec) and number of applied pulses (4, 8 or 12) under hypotonic conditions were studied. Activation frequencies were the lowest after four 30 microsec-pulses (58 vs. 88 to 100%, respectively). A lower haploid frequency was obtained when more than four 30 or 60 microsec-pulses were applied (from 67 to 25% and 83 to 0%, respectively). Increasing the number of 60-microsec pulses improved the compacted morula rate of haploid and diploid oocytes (47 to 83% and 57 to 96%, respectively). Overall, haploid development to morulae and blastocysts was lower than diploid development to these stages (69 and 25% vs. 74 and 44%, respectively).

Animals↗

PubMed2001

Relationships between growth and gas exchange characteristics in some salt-tolerant amphidiploid Brassica species in relation to their diploid parents.

Relationships between growth and different gas exchange characteristics of two amphidiploid salt tolerant species, Brassica napus, and B. carinata with respect to their salt sensitive parents, B. oleracea, and B. nigra were investigated. Twenty three-day old plants of these four species along with those of another amphidiploid moderately salt tolerant B. juncea (developed by hybridization of diploids, B. campestris and B. nigra), and a diploid moderately salt tolerant, B. campestris, were subjected for 28 days to salinized sand culture containing 0, 100 or 200 mol NaCl m(-3) in Hoagland's nutrient solution. The species B. napus and B. carinata produced significantly greater shoot fresh and dry matters than their parents under saline conditions. A close association was found between growth, and assimilation rate for all species differing in degree of salt tolerance. Stomatal conductance (g(s)) was reduced due to salt stress in all species but this variable had no significant correlation with assimilation rate (A). However, the amphidiploid salt tolerant species, B. napus and B. carinata had significantly greater photosynthetic rate, water use efficiency (A/E), intrinsic water use efficiency (A/g(s)) than those of their diploid parents. In conclusion, high salt tolerance of the two amphidiploid species, B. napus and B. carinata was associated with a high assimilation rate, water use efficiency and intrinsic water use efficiency but there was little association of the tolerance of these species with stomatal conductance, leaf water potential or transpiration rate (E).

Journal Article↗