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PubMed1980

Increase in glutaminase activity during the growth cycle of cultured human diploid fibroblasts.

The activity of glutaminase per milligram of protein increased threefold after cultured human diploid fibroblasts were subcultured in fresh medium. The maximum activity was reached after 2 days of growth and decreased once the cells reached confluency. The increase of glutaminase activity was independent of the glutamine concentration between 0.2 and 2.0 mmol/l. In contrast, the specific activity of glutamate dehydrogenase was independent both of the glutamine concentration and the growth phase of the cultured cells. These results indicate that glutaminase, the first enzyme involved in the ultilization of glutamine as an energy source, is elevated in rapidly dividing human diploid fibroblasts.

Cell Cycle↗

PubMed1995

Collection of peripheral-blood diploid cells from chronic myelogenous leukemia patients early in the recovery phase from myelosuppression induced by intensive-dose chemotherapy.

PURPOSE: To evaluate whether intensive chemotherapy followed by peripheral stem-cell (PSC) collections during early hematopoietic recovery results in a higher percentage of diploid cell collections in patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML). PATIENTS AND METHODS: Fifty-five adults with Ph-positive CML received intensive chemotherapy with daunorubicin and high-dose cytarabine (ara-C) (DAUNO-HDAC; 26 patients) or fludarabine, high-dose ara-C, and mitoxantrone (FAM; 29 patients). Collections of the peripheral mononuclear cells were initiated when the WBC count was > or = 0.8 x 10(3)/microL. Simultaneous peripheral and marrow samples were subjected to cytogenetic studies. RESULTS: Thirty-eight of 55 patients (69%) were able to undergo the PSC collections. The rate of collection was higher in chronic phase (26 of 30 patients; 87%) than in accelerated (11 of 17; 65%) and blastic phases (1 of 8; 12%). Among the 30 patients in chronic phase, cytogenetic analyses of PSC showed cytogenetic responses (Ph-positive < 95%) in 60%, which were major (Ph < 35%) in 43% and complete (Ph = 0%) in 27%. Seven of 19 patients with simultaneous studies (37%; 23% of total) had a significantly lower percentage of Ph-positive cells in the peripheral collection compared with the marrow collection; one had the reverse phenomenon (5%; 3% of total). Cytogenetic responses were modest in both peripheral and marrow collections in CML accelerated and blastic phases. Myelosuppression-associated complications were frequent, resulting in febrile episodes in 76% of patients. CONCLUSION: PSC collection during early hematopoietic recovery from intensive chemotherapy allowed the collection of diploid-rich stem cells, mostly in chronic-phase CML. The approach could be used for in vivo purging before autologous stem-cell transplantation (ASCT).

Adolescent↗

PubMed2003

Chromatin of endoreduplicated pavement cells has greater range of movement than that of diploid guard cells in Arabidopsis thaliana.

In the current model of chromatin organization in the interphase cell nucleus, chromosomes are organized into territories. Although constrained diffusion of chromatin in interphase cells has been confirmed in all cell types examined, little is known about chromatin dynamics in plant interphase cells. In this work, we measured for the first time interphase chromatin dynamics in plants using the green-fluorescent-protein-mediated chromatin-tagging system. Moreover, we compared the dynamics of diploid guard cells and endoreduplicated pavement cells. The movement of tagged loci in live seedlings shows constrained behavior in both types of nuclei. However, we found that the apparent confinement area for tagged loci in pavement cells is over 6 times larger than it is in guard cells. These findings suggest that chromatin is anchored to some component of the nucleus and that this might be responsible for the different dynamics of chromatin diffusion between diploid cells and endoreduplicated cells.

Arabidopsis↗

PubMed2006

The transcription factor B-Myb is essential for S-phase progression and genomic stability in diploid and polyploid megakaryocytes.

The cell-cycle-regulated Myb-family transcription factor B-Myb is crucial during S phase in many diploid cell types. We have examined the expression and function of B-Myb in megakaryocytic differentiation, during which cells progress from a diploid to a polyploid state. In contrast to terminal differentiation of most haematopoietic cells, during which B-myb is rapidly downregulated, differentiation of megakaryocytes is accompanied by continued B-myb RNA and protein expression. Overexpression of B-Myb in a megakaryoblastic cell line resulted in an increase in the number of cells entering S phase and, upon induction of differentiation, the fraction of cells actively endoreplicating increased. By contrast, reduction of B-Myb levels using short interfering (si)RNA resulted in a decline in S-phase progression during both normal and endoreplicative DNA synthesis. This effect correlated with aberrant localisation of initiation of DNA replication within the nucleus and an increased fraction of cells in mitosis. Chromosomal fragmentation and other aberrations, including shorter, thicker chromatids, end-to-end fusion, and loss of a chromatid, suggest that reduced B-Myb activity is also associated with structural chromosomal instability.

Cell Cycle Proteins↗

PubMed1995

'Chromosomal puffing' in diploid nuclei of Drosophila melanogaster.

In situ hybridization has become a powerful technique for dissecting nuclear structure. By localizing nucleic acids with high precision, it is possible to infer the native structure of chromosomes, replication factories and transcript processing complexes. To increase the value of this technique, we have established the limits of resolution of two-color in situ hybridization to chromosomal DNA in diploid chromosomes of Drosophila embryonic nuclei. Using high-resolution 3-dimensional optical microscopy and computational image analysis, we establish that we can distinguish the location of chromosomal sequences that lie 27-29 kb apart within a 40 kb transcription unit with an accuracy of about 100 nm. Contrary to observations made in mammalian tissue culture cells, we find that when the gene is expressed, it assumes an open configuration, and that the extent of decondensation is variable from chromosome to chromosome. Further experiments suggest that variation in gene structure results from asynchrony in transcriptional elongation. We suggest that the phenomenon we observe is the diploid analog to chromosomal puffing that occurs in the transcriptionally active regions of Drosophila polytene chromosomes.

Animals↗

PubMed1979

Production of serum proteins in normal diploid fibroblast-hepatoma cell hybrids and in A9-normal liver cell hybrids.

Two series of interspecific hybrids have been generated between liver cells (which actively secrete several serum proteins) and fibroblasts (which do not). In each series, one of the parental cells was a normal diploid cell: mouse hepatoma cells were fused with normal diploid rat fibroblasts, and normal rat liver cells were fused with mouse fibroblasts of the permanent line A9. The production of albumin, alpha-fetoprotein (AFP) transferrin and the third component of complement (C3) was analysed in these hybrids. Most hepatoma cell hybrids exhibit extinction of albumin, AFP and (to a lesser extent) transferrin; they retain the capacity to secrete C3. Normal liver cell hybrids are also characterized by the absence of albumin and transferrin production and by retention of C3 secretion. These results, when compared to previous results obtained with hybrids derived exclusively from different differentiated cells of permanent and transformed lines show that the phenotype of such hybrids is not determined by the abnormal character per se of the aneuploid parental cells. Amongst the rat fibroblast-mouse hepatoma cell hybrids, a few clones retain the capacity to actively secrete mouse albumin, AFP and transferrin, without the concomitant production of the rat serum proteins. These hybrids have lost more rat (fibroblast) chromosomes than the other clones and also have an increased number of mouse (hepatoma) chromosomes. Thus, their phenotype must result from either the complete loss of 'extinguisher' chromosomes, or gene dosage effects. The significance of the lack of rat serum protein production is also discussed, and it is suggested that retention, without concomitant activation, could be explained in terms of diffusible regulators and heritable differences in chromatin conformation.

Animals↗

PubMed1979

Necessary amino acids and vitamins for the growth of human diploid fibroblasts.

Only 2 amino acids and one vitamin were found to be essential for the growth of human embryonic diploid lung fibroblasts when 10% undialysed calf serum was used as a medium supplement. These amino acids were either glutamine + cysteine or serine + homocysteine. Replacing cysteine or homocysteine with cystine or homocystine, respectively, reduced growth. The growth rate in the glutamine + cysteine medium was slightly less than that in Eagle's medium, but the in vitro life time was similar. Folic acid was the only vitamin needed to support cell growth in vitro. The addition of other vitamins had no stimulatory effect with the possible exception of nicotinamide. When other amino acids were added to glutamine + cysteine none showed stimulatory effects but tryptophan was either toxic or inhibitory for the 3 human diploid strains examined. Serine was inhibitory for WI-38 but not for MRC5 cells. Subtle nutritional differences appear to exist between fibroblasts of the same type obtained from different embryos.

Amino Acids↗

PubMed1980

Human diploid fibroblast-like cells (CF-3) as a model system for the study of Golgi biogenesis.

Human diploid fibroblast-like cells were maintained in an arrested, essentially non-mitotic state for extended periods of time in culture by lowering the serum concentration in the medium from 10 to 0.5%. These arrested cells re-entered the proliferative state when subcultivated in medium containing 10% serum. The morphological distribution and enzyme activities associated with the Golgi complex were examined during growth, arrest, and recovery. Cells grown in medium containing 10% serum possessed a well-developed Golgi complex consisting of parallel arrays of membranes and associated vesicles. Galactosyl transferase activity was highest after 3 days growth (17.5 +/- 5.0 nmol galactose transferred/45 min/mg protein) and declined to 9.8 +/- 3.0 at day 7. When the serum concentration was reduced to 0.5%, Golgi complexes were rarely observed by electron microscopy and galactosyl transferase activity was further reduced into medium containing 10% serum recovered from the arrested state and ultrastructurally resembled cells continuously cultured at the higher serum level. Numerous Golgi complexes reappeared and galactosyl transferase activity increased to 13.0 +/- 3.34 days after subcultivation. These results indicate that the Golgi complex can be experimentally manipulated in human diploid fibroblast-like cells in a manner which may be useful for the study of the biogenesis of this organelle.

Cell Cycle↗

PubMed2006

Profiles of global gene expression in ionizing-radiation-damaged human diploid fibroblasts reveal synchronization behind the G1 checkpoint in a G0-like state of quiescence.

Cell cycle arrest and stereotypic transcriptional responses to DNA damage induced by ionizing radiation (IR) were quantified in telomerase-expressing human diploid fibroblasts. Analysis of cytotoxicity demonstrated that 1.5 Gy IR inactivated colony formation by 40-45% in three fibroblast lines; this dose was used in all subsequent analyses. Fibroblasts exhibited > 90% arrest of progression from G2 to M at 2 hr post-IR and a similarly severe arrest of progression from G1 to S at 6 and 12 hr post-IR. Normal rates of DNA synthesis and mitosis 6 and 12 hr post-IR caused the S and M compartments to empty by > 70% at 24 hr. Global gene expression was analyzed in IR-treated cells. A microarray analysis algorithm, EPIG, identified nine IR-responsive patterns of gene expression that were common to the three fibroblast lines, including a dominant p53-dependent G1 checkpoint response. Many p53 target genes, such as CDKN1A, GADD45, BTG2, and PLK3, were significantly up-regulated at 2 hr post-IR. Many genes whose expression is regulated by E2F family transcription factors, including CDK2, CCNE1, CDC6, CDC2, MCM2, were significantly down-regulated at 24 hr post-IR. Numerous genes that participate in DNA metabolism were also markedly repressed in arrested fibroblasts apparently as a result of cell synchronization behind the G1 checkpoint. However, cluster and principal component analyses of gene expression revealed a profile 24 hr post-IR with similarity to that of G0 growth quiescence. The results reveal a highly stereotypic pattern of response to IR in human diploid fibroblasts that reflects primarily synchronization behind the G1 checkpoint but with prominent induction of additional markers of G0 quiescence such as GAS1.

Cell Line↗

PubMed1988

Phosphofructokinase activity in normal diploid mice during development and in trisomy 16 fetal mice.

Although several of the genes mapped to human chromosome 21 have been assigned to mouse chromosome 16, it has not yet been possible to do this for the gene for the phosphofructokinase liver type subunit (PFKL). The goal of this study was to determine if there is a 1.5-fold increase of the PFK activity in fetal trisomy 16 mice, which, if present, would be indicative of a gene dosage effect. However, rather than an increase, an almost 100% decrease of the mean PFK activity was observed in fetal trisomy 16 liver at both 14 and 17 days of gestation when compared to littermate controls. This is the first biochemical abnormality detected in trisomy 16 fetal liver. In contrast, no significant differences in the mean PFK activity in homogenized whole fetus or brain trisomy 16 and diploid controls were observed. A developmental maturational effect of the PFK activity was observed in fetal liver from normal diploid mice, with a 3-fold increase of the activity from day 14 to day 18 of gestation and a further 2-fold increase to adulthood. The decreased PFK activity in fetal trisomy 16 liver may therefore be the result of delayed maturation of the liver in trisomic fetuses.

Animals↗

PubMed2004

Shared forces of sex chromosome evolution in haploid-mating and diploid-mating organisms: Microbotryum violaceum and other model organisms.

It is usually posited that the most important factors contributing to sex chromosome evolution in diploids are the suppression of meiotic recombination and the asymmetry that results from one chromosome (the Y) being permanently heterozygous and the other (the X) being homozygous in half of the individuals involved in mating. To distinguish between the roles of these two factors, it would be valuable to compare sex chromosomes in diploid-mating organisms and organisms where mating compatibility is determined in the haploid stage. In this latter group, no such asymmetry occurs because the sex chromosomes are equally heterozygous. Here we show in the fungus Microbotryum violaceum that the chromosomes carrying the mating-type locus, and thus determining haploid-mating compatibility, are rich in transposable elements, dimorphic in size, and carry unequal densities of functional genes. Through analysis of available complete genomes, we also show that M. violaceum is, remarkably, more similar to humans and mice than to yeast, nematodes, or fruit flies with regard to the differential accumulation of transposable elements in the chromosomes determining mating compatibility vs. the autosomes. We conclude that restricted recombination, rather than asymmetrical sheltering, hemizygosity, or dosage compensation, is sufficient to account for the common sex chromosome characteristics.

Base Sequence↗

PubMed2004

Nonadditive gene expression in diploid and triploid hybrids of maize.

The molecular basis of hybrid vigor (heterosis) has remained unknown despite the importance of this phenomenon in evolution and in practical breeding programs. To formulate a molecular basis of heterosis, an understanding of gene expression in inbred and hybrid states is needed. In this study, we examined the amount of various transcripts in hybrid and inbred individuals (B73 and Mo17) to determine whether the quantities of specific messenger RNAs were additive or nonadditive in the hybrids. Further, we examined the levels of the same transcripts in hybrid triploid individuals that had received unequal genomic contributions, one haploid genome from one parent and two from the other. If allelic expression were merely the additive value in hybrids from the two parents, the midparent values would be observed. Our study revealed that a substantial number of genes do not exhibit the midparent value of expression in hybrids. Instead, transcript levels in the diploid hybrids correlate negatively with the levels in diploid inbreds. Although transcript levels were clearly nonadditive, transcript levels in triploid hybrids were affected by genomic dosage.

Gene Expression Regulation, Plant↗

PubMed2005

Allele-specific, bidirectional silencing of an alcohol dehydrogenase gene in different organs of interspecific diploid cotton hybrids.

Interspecific hybridization is a common and important process that generates phenotypic novelty and can lead to hybrid speciation as well as to changes in gene expression. Using two different interspecific cotton (Gossypium) diploid hybrids, we show organ-specific, bidirectional allelic silencing at a heterozygous Adh locus, with alternate alleles being silenced in leaves and many floral organs, respectively. These results show that developmental regulation of gene expression is changed immediately upon hybridization between diploid species, possibly due to epigenetic factors or regulatory mismatch.

Alcohol Dehydrogenase↗

PubMed2004

Cremophor EL stimulates mitotic recombination in uvsH//uvsH diploid strain of Aspergillus nidulans.

Cremophor EL is a solubilizer and emulsifier agent used in the pharmaceutical and foodstuff industries. The solvent is the principal constituent of paclitaxel's clinical formulation vehicle. Since mitotic recombination plays a crucial role in multistep carcinogenesis, the study of the recombinagenic potential of chemical compounds is of the utmost importance. In our research genotoxicity of cremophor EL has been studied by using an uvsH//uvsH diploid strain of Aspergillus nidulans. Since it spends a great part of its cell cycle in the G2period, this fungus is a special screening system for the study of mitotic recombination induced by chemical substances. Homozygotization Indexes (HI) for paba and bi markers from heterozygous B211//A837 diploid strain were determined for the evaluation of the recombinagenic effect of cremophor EL. It has been shown that cremophor EL induces increase in mitotic crossing-over events at nontoxic concentrations (0.05 and 0.075% v/v).

Aspergillus nidulans↗

PubMed2005

Benznidazole-induced genotoxicity in diploid cells of Aspergillus nidulans.

Genotoxic effects of benznidazole were studied by the induction of homozygosis of genes previously present in heterozygous. UT448//A757 diploid strain was used in the benznidazole's recombinagenesis test. Although toxic effects on growth of colonies were not observed, 75 and 100 microM benznidazole induced an increasing of mitotic recombination events in diploid strain. Results were related to the induction of chromosomal breaks by the antiparasitic drug.

Aspergillus nidulans↗

PubMed2005

Loss of heterozygosity by mitotic recombination in diploid strain of Aspergillus nidulans in response to castor oil plant detergent.

Somatic recombination in heterozygous diploid cells may be a promotional agent of neoplasms by inducing homozygosity of defective genes. Tumor suppressor genes may in this way be completely suppressed in recombinant cells. In this work, the genotoxic effects of detergent derived from the castor oil plant (Ricinus communis) in heterozygous diploid cells of Aspergillus nidulans are evaluated. Previous studies have evaluated the application of this substance in endodontic treatments as an irrigating solution. The recombinogenic potential of the compound has been studied through the production of homozygous cells for nutritional markers riboA1, pabaA124, biA1, methA17, and pyroA4. Detergent was diluted to 1:10, 1:20, and 1:40, and morphologic alterations, delay in conidiophore development, and mitotic recombination occurrence were reported for the three dilutions. Although past studies have demonstrated the antimicrobial action of the detergent under analysis, our results revealed its cytotoxic effects and recombinogenic potential.

Aspergillus nidulans↗

PubMed2006

Metallothionein gene expression and protein levels in triploid and diploid oysters Crassostrea gigas after exposure to cadmium and zinc.

Quantitative real-time polymerase chain reaction (PCR) was used to compare for the first time the differential expression of metallothionein (MT) isoform genes, together with biosynthesis of the total MT proteins, in the gills of triploid and diploid juvenile Pacific oyster Crassostrea gigas in response to cadmium (Cd) and zinc (Zn) exposure. Oysters were exposed to Cd (0.133 microM), Zn (15.3 microM), and Cd+Zn for 14 d. Results showed similar response capacities to metal exposures in the two populations. No significant difference was revealed in terms of MT gene expression, MT protein synthesis, and Cd accumulation. However, triploid oysters bioaccumulated Zn 30% less efficiently than diploid oysters. Among the three MT isoform genes, CgMT2 appeared to be more expressed than CgMT1, whereas CgMT3 appeared to be anecdotal (10(6) times lower than CgMT2). CgMT2 and CgMT1 gene expression levels were increased sevenfold in the presence of Cd, whereas Zn appeared to have no effect. A twofold increase in MT protein levels occurred in response to Cd exposure. Discrepancies between mRNA and protein levels suggest that in C. gigas MT are regulated at the transcriptional level, as well as at the translational level.

Animals↗

PubMed2003

Androgenetic reproduction in a freshwater diploid clam Corbicula fluminea (Bivalvia: Corbiculidae).

Two shell color types of the exotic bivalve Corbicula fluminea were collected in Kyoto city, Japan. DNA microfluorometry revealed that both types were diploids with non-reductional spermatozoa. Maternal chromosomes were found to be extruded as two polar bodies at the first meiosis, and the second meiosis could not be observed. Only the male pronucleus was present in the egg cytoplasm and became metaphase chromosomes at the first mitosis. The present study indicates that the diploid C. fluminea in Japan has the same mode of androgenetic reproduction as the triploid C. leana.

Androgens↗