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PubMed1992

Characterization of an immortalized human cell line derived from neonatal foreskin diploid fibroblasts.

A new human skin cell line, designated as CCFS-1/KMC, immortalized from human neonatal foreskin diploid fibroblast cells, has been subcultured successfully in vitro for more than 500 passages. This anchorage-dependent cell line possesses many common features of transformation such as morphological and cytoskeletal changes, hypotriploidy, infinite lifespan, increasing plating efficiency and saturation density, and decreasing serum requirement and population doubling time. Human papillomavirus (HPV) type 18 DNA was detected in the cell line before and after immortalization by the polymerase chain reaction (PCR) method. Tumorigenicity, however, was not demonstrated in vivo. The isoenzyme activity of the cell line shows activation of a placental form of alkaline phosphatase and a changing lactate dehydrogenase isoenzyme pattern that is different from transformation by carcinogens. Class I HLA and class II HLA antigens are constitutively expressed on this skin cell line. Here we report that these immortalized human fibroblasts derived from neonatal HPV-18-DNA-contained diploid fibroblasts possess double minute chromosomes (DMs), a karyotypic aberration usually found in cancer cells.

Cell Division↗
PubMed1984

Separation of diploid from aneuploid cells in acute lymphoblastic leukaemia.

A new method for the separation of diploid and aneuploid cells from blood of patients with leukaemia is described. Density gradient separation using a modified silica particle gradient was combined with analysis of cellular DNA content by flow cytometry. Submicroscopic levels of diploid or aneuploid cells were enriched up to 90% purity. Three patients with acute lymphoblastic leukaemia and no evidence of aneuploid cells after separation are in continuing complete remission after 24.5-47 months. Two patients with aneuploid cells detected after separation expired in relapse 0.5 and 3 months later. Cell separation prior to flow cytometric analysis might be a useful tool to detect submicroscopic levels of aneuploid leukaemic cells in blood and bone marrow, and to predict impending relapse.

Aneuploidy↗
PubMed1976

The 'transition probability model' and the regulation of proliferation of human diploid cell cultures during aging.

Genealogies derived from time-lapse cinemicrophotographic studies of aging human diploid cell cultures were analysed in terms of the 'transition probability' model. It was found that the distribution of intermitotic times obtained from middle passage cells deviated only slightly from that predicted by the model. In contrast, the plot for late passage cultures did not fit the predicted pattern and appeared to be composed of multiple curves. These changes are discussed in reference to cellular senescence as expressed by normal human diploid cells in vitro.

Cell Division↗
PubMed2006

Comparative genomics and repetitive sequence divergence in the species of diploid Nicotiana section Alatae.

Combining phylogenetic reconstructions of species relationships with comparative genomic approaches is a powerful way to decipher evolutionary events associated with genome divergence. Here, we reconstruct the history of karyotype and tandem repeat evolution in species of diploid Nicotiana section Alatae. By analysis of plastid DNA, we resolved two clades with high bootstrap support, one containing N. alata, N. langsdorffii, N. forgetiana and N. bonariensis (called the n = 9 group) and another containing N. plumbaginifolia and N. longiflora (called the n = 10 group). Despite little plastid DNA sequence divergence, we observed, via fluorescent in situ hybridization, substantial chromosomal repatterning, including altered chromosome numbers, structure and distribution of repeats. Effort was focussed on 35S and 5S nuclear ribosomal DNA (rDNA) and the HRS60 satellite family of tandem repeats comprising the elements HRS60, NP3R and NP4R. We compared divergence of these repeats in diploids and polyploids of Nicotiana. There are dramatic shifts in the distribution of the satellite repeats and complete replacement of intergenic spacers (IGSs) of 35S rDNA associated with divergence of the species in section Alatae. We suggest that sequence homogenization has replaced HRS60 family repeats at sub-telomeric regions, but that this process may not occur, or occurs more slowly, when the repeats are found at intercalary locations. Sequence homogenization acts more rapidly (at least two orders of magnitude) on 35S rDNA than 5S rDNA and sub-telomeric satellite sequences. This rapid rate of divergence is analogous to that found in polyploid species, and is therefore, in plants, not only associated with polyploidy.

Chromosomes, Plant↗
PubMed1997

Comparison of concentrations of Cry j 1 and Cry j 2 in diploid and triploid Japanese cedar (Cryptomeria japonica) pollen extracts.

Japanese cedar (Cryptomeria japonica) possibilities has been a serious allergic disease in Japan. There are two kinds of Japanese cedar trees; the popular one is diploid, the less popular is triploid. These trees are not very different morphologically. However, the relative allergenicity of their pollens is unknown, although both major allergens, Cry j 1 and Cry j 22, have been purified and cloned from the diploid line. Triploid trees are sterile and the allergenicity of their pollen may differ. Using Japanese-cedar-allergic patient sera, we compared the concentration of these two major allergens in both kinds of pollen. Pollen collected from different years and regions was also studied. The results indicate that triploid tree pollen extract has lower concentrations of both major allergens; therefore, the pollen may be less allergenic.

Allergens↗
PubMed1975

A diploid-triploid human mosaic with cytogenetic evidence of double fertilization.

The karyotype 46,XX/69,XXY was found in a 13-year-old mentally subnormal patient with club feet, strabismus, eunuchoid habitus, small penis, midscrotal urethrovaginal opening, small descended left testis, and small undescended right testis; no ovarian tissue could be found at laparotomy. Triploid:diploid cell ratios were 60:40 and 4:96 in skin fibroblasts and curculating lymphocytes, respectively. In the triploid line, two of the no. 13 chromosomes had unusually large satellites and one of the no. 22 chromosomes had a brightly fluorescent zone on its short arms. The patient's father was heterozygous for both these autosomal markers; the mother carried neither marker. This, together with the single Y, indicated that the extra haploid set was derived from the father. Of several possible mechanisms, we favor the suggestion that double fertilization occurred; one sperm nucleus immediately fused with the egg nucleus producing the diploid line; the second sperm nucleus was incorporated later into one of the two cells resulting from the first division of the zygote, producing the triploid line.

Adolescent↗
PubMed2006

Abortive second meiosis detected in cytochalasin-treated eggs in androgenetic diploid Corbicula fluminea.

The hermaphroditic diploid clam Corbicula fluminea reproduces by androgenesis. In the control (androgenetic development), all maternal chromosomes and maternal centrosomes at the meiotic poles were extruded as two first polar bodies and subsequently second meiosis did not occur. In eggs treated with cytochalasin D (CD) to inhibit the polar body extrusion, the second meiosis was abortive. After the first meiosis, two centrosomes at the spindle poles remained in the cytoplasm because of the effect of CD. The chromosomes divided into two groups at anaphase-I as observed in the control eggs. Two centrosomes divided into four just after the first meiosis but did not separate completely. The microtubules from the centrosomes were rather short. So at the second meiosis, two monoasters or tetrapolar spindles were formed. The fluorescence signal from microtubules of the monoaster or tetrapolar spindle was weak compared with the spindle at the first meiosis. The maternal chromosomes on the monoaster or tetrapolar spindle did not move, and became large female pronuclei. The pronuclei became the metaphase chromosomes on the spindle for the first cleavage. The present study suggests that second meiosis regulating factors may be abortive in androgenetic diploid C. fluminea.

Animals↗
PubMed2006

Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblasts.

Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3',5'-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs alpha/beta both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gialpha in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC alpha/beta and AC4/6.

A Kinase Anchor Proteins↗
PubMed1990

Effect of sodium fluoride on growth of human diploid cells in culture.

Cytotoxicity of sodium fluoride (NaF) on human diploid cells in exponential growth was investigated using a) Flow 1000 cells, passage No. 13, obtained from skin and muscle tissues of male black foetus, b) IMR-90 cells, passage No. 22, derived from lung tissue of female Caucasian foetus and c) primary fibroblast-like cell cultures from 5 Japanese whole foetuses. Diploid cells did not survive at 20 p.p.m. of ionic fluoride (F-) concentration. However, the cells were capable of proliferation with no significant impairment of growth up to 0.2 p.p.m. F-, a level which is much higher than the plasma concentration in human subjects from areas with highly fluoridated water. The growth of the cells was markedly affected by F- concentrations greater than 2 p.p.m.

Aneuploidy↗
PubMed2000

ITS regions in diploids of Aegilops (Poaceae) and their phylogenetic implications.

The nucleotide sequences of the internal transcribed spacer (ITS) of nuclear ribosomal DNA in nine diploid species representing six sections of Aegilops were determined by direct sequencing of PCR-amplified DNA fragments. These sequences were aligned with two ITS sequences of additional species from Genbank. Sequence divergences were estimated using Kimura two-parameter model, and the phylogenetic analyses were performed using the maximum parsimony (MP) and the neighbor-joining (NJ) methods with PAUP and PHYLIP, respectively. The sequence divergences between the diploid species varied from 0.5% to 4.68%. The resulting MP tree and NJ tree showed relatively congruent phylogenetic relationships among these species, except Ae. caudata. Particularly, Ae. speltoides was basal within the two trees. The paraphyletic relationships between Ae. speltoides and two species of Sect. Sitopsis, and between Ae. uniaristata and two species of Sect. Comopyrum were supported strongly. The ITS data suggest that currently recognized sections within Aegilops should be reconsidered.

Base Composition↗
PubMed2004

Further cytogenetical studies on diploid and polyploid species of Eryngium L. (Saniculoideae, Apiaceae) from Argentina.

Meiotic studies are carried out in 7 species of Eryngium L. (Saniculoideae, Apiaceae), belonging to both sections Foetida and Panniculata. The chromosome number of E. dorae Norm. (n=8) (Foetida) is reported for the first time, while the gametic chromosome number of E. nudicaule Lam. (n=7) (Foetida) and E. eburneum Decne. (n=8), E. horridum Malme (n=8), E. megapotamicum Malme (n=16), E. mesopotamicum Pedersen (n=24), and E. pandanifolium Cham. et Schlechtd. (n=24) (all belonging to Panniculata) is confirmed in several natural populations. Whereas in section Foetida all species are diploids and two basic chromosome numbers are present (x=8 and x=7), in section Panniculata all species are x=8 but there are three different ploidy levels (diploid, tetraploid, hexaploid). This study reveals that meiosis in all species is normal, with regular bivalent formation in all studied cells. Furthermore, the pollen stainability is above 80% in all cases. These data, together with the previous karyotype analyses, will contribute to the clarification of the relationships between members of both sections, where different mechanisms of speciation have been postulated.

Argentina↗
PubMed1967

Genomic exclusion: a rapid means for inducing homozygous diploid lines in Tetrahymena pyriformis, syngen 1.

Genomic exclusion is an abnormal form of conjugation occurring between cells with defective micronuclei and normal cells with diploid micronuclei. The progeny are heterocaryons; each cell has an old macronucleus but a new diploid micronucleus derived from one meiotic product of the normal mate. Such cells express genes found in the old macronucleus, are sexually mature, and can be specifically selected. When inbred, they give rise to lines genetically homozygous at all known loci.

Genes↗
PubMed1967

Chlamydomonas reinhardi: heterozygous diploid strains.

Zygotes of the unicellular green alga Chlamydomonas reinhardi occasionally divide mitotically to give rise to stable diploid vegetative strains. As well as by their mode of origin, these strains are distinguished from haploids by cell and nuclear size, DNA content per nucleus, and chromosome number. Diploid strains heterozygous for a variety of mutant genes are phenotypically wild type and mating-type minus. Thus these mutant genes are recessive to their wild-type alleles, and the mating-type-minus is dominant over the mating-type-plus allele.

Cell Nucleus↗
PubMed1993

Elements of the yeast pheromone response pathway required for filamentous growth of diploids.

Transmission of an external signal from receptors to downstream targets is often mediated by a conserved set of protein kinases that act in sequence (a kinase cascade). In haploid strains of Saccharomyces cerevisiae, a signal initiated by peptide pheromones is transmitted through this kinase cascade to a transcription factor STE12, which is required for the expression of many mating-specific genes. Here it was shown that in diploids some of the same kinases and STE12 are required for filamentous growth, but the pheromone receptors and guanosine triphosphate-binding protein are not required for filament formation. Thus, a similar kinase cascade is activated by different signals in haploids and diploids and mediates different developmental outcomes in the two cell types.

Fungal Proteins↗
PubMed1976

Polyploid amphibians: three more diploid-tetraploid cryptic species of frogs.

The nominal African species Pyxicephalus delalandii and Dicroglossus occipitalis have diploid and tetraploid populations. There are also cryptic tetraploid and diploid species similar to Bufo kerinyagae. These represent the first bisexual polyploid "species" so far encountered in the major frog families Ranidae and Bufonidae. The contention that polyploidy is a widespread and important evolutionary phenomenon in anuran amphibians is supported.

Africa↗
PubMed2002

Isolation and characterization of a freeze-tolerant diploid derivative of an industrial baker's yeast strain and its use in frozen doughs.

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.

Bread↗