Pluripotency of a polyploid H1 (ES) cell system without leukaemia inhibitory factor.

OBJECTIVES: Tetraploid cells are strictly biologically inhibited from composition of embryos; by the same token, only diploid cells compose embryos. However, the distinction between diploid and tetraploid cells in development has not been well explained. To examine pluripotency of polyploid ES cells, a polyploid embryonic stem (ES)-cell system was prepared. MATERIALS AND METHODS: Diploid, tetraploid, pentaploid, hexaploid, octaploid and decaploid H1 (ES) cells (2H1, 4H1, 5H1, 6H1, 8H1 and 10H1 cells, respectively) were cultured for about 460 days in L15F10 medium without leukaemia inhibitory factor (LIF). The cells cultured under LIF-free conditions were denoted as 2H1(-), 4H1(-), 5H1(-), 6H1(-), 8H1(-) and 10H1(-) cells, respectively. Pluripotency and gene expression were examined. RESULTS: Ploidy alteration of H1(-) cells was similar to that of H1 cells. The polyploid H1(-) cells showed positive activity of alkaline phosphatase, suggesting that they maintained pluripotency in vitro without LIF. The polyploid H1(-) cells formed teratocarcinomas in mouse abdomen, suggesting they could differentiate in mouse abdomen in vivo. 2H1, 4H1 and polyploid H1(-) cells expressed nanog, oct3/4 and sox2 genes, suggesting that they fulfilled the criteria of ES cells. Nanog gene was significantly over-expressed in 4H1 and polyploid H1(-) cells, suggesting that overexpression of nanog gene was a characteristic of polyploid H1 cells. CONCLUSION: Polyploid H1 (ES) cells retained pluripotency in vitro, without LIF with nanog over-expression.

DNA-unstable decaploid mouse H1 (ES) cells established from DNA-stable pentaploid H1 (ES) cells polyploidized using demecolcine.

OBJECTIVES: DNA content of diploid H1 (ES) cells (2H1 cells) has been shown to be stable in long-term culture; however, tetraploid and octaploid H1 (ES) cells (4H1 and 8H1 cells, respectively) were DNA-unstable. Pentaploid H1 (ES) cells (5H1 cells) established recently have been found to be DNA-stable; how, then is cell DNA stability determined? To discuss ploidy stability, decaploid H1 (ES) cells (10H1 cells) were established from 5H1 cells and examined for DNA stability. MATERIALS AND METHODS: 5H1 cells were polyploidized using demecolcine (DC) and 10H1 cells were obtained by one-cell cloning. RESULTS: Number of chromosomes of 10H1 cells was 180 and durations of their G(1), S, and G(2)/M phases were 3, 7 and 6 h respectively. Volume of 10H1 cells was double that of 5H1 cells and morphology of 10H1 cells was flagstone-like in shape. 10H1 cells exhibited alkaline phosphatase activity and their DNA content decayed in 91 days of culture. 10H1 cells injected into mouse abdomen formed solid tumours that contained several kinds of differentiated cells with lower DNA content, suggesting that 10H1 cells were pluripotent and DNA-unstable. Loss of DNA stability was explained using a hypothesis concerning DNA structure of polyploid cells as DNA reconstructed through ploidy doubling was arranged in mirror symmetry in a new configuration. CONCLUSION: In the pentaploid-decaploid transition of H1 cells, cell cycle parameters and pluripotency were retained, but morphology and DNA stability were altered.

Establishment of a tetraploid cell line from mouse H-1 (ES) cells highly polyploidized with demecolcine.

OBJECTIVE: Establishment of tetraploid ES cells. MATERIALS AND METHODS: Mouse H-1 (ES) cells were polyploidized by demecolcine and released from the drug. RESULTS: A tetraploid cell line (4nH1 cells) was established from mouse H-1 (ES) cells (2nH1 cells) highly polyploidized by treatment with demecolcine. Cell cycle parameters of 4nH1 cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. Mode of chromosome number of 4nH1 cells was 76, about twice that of 2nH1 cells. Cell volume of 4nH1 cells was about twice of that of diploid cells, indicating that 4nH1 cells contained about twice as much total intracellular material as 2nH1 cells. Morphology of the 4nH1 cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that the transformation had occurred during the diploid-tetraploid transition. 4nH1 cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they would be pluripotent. CONCLUSION: A pluripotent tetraploid cell line (4nH1 cells) was established.

The reversion to diploid cells from established triploid V79 cells.

The triploid V79 cells are stable under usual culture conditions, and do not revert to being diploid. Here, the triploid-diploid transition of triploid V79 cells has been successfully induced in suspension culture in culture dishes with untreated surfaces. The diploid cells began to appear in a population of triploid V79 cells cultured under these conditions for 4 weeks. All of the triploid cells were transformed to diploid through subsequent monolayer culture for 5 weeks. It was confirmed that the revertant diploid cells had the same characteristics as original diploid V79 cells, with respect to DNA histograms, cell volume and chromosome number. Thus, it seems that suspension culturing is an important factor that induces the triploid-diploid transition.

Temperature dependence in proliferation of tetraploid Meth-A cells in comparison with the parent diploid cells.

The temperature dependency for the growth of tetraploid Meth-A cells established from diploid cells was examined in comparison with the parent diploid cells. Proliferation of the tetraploid cells was markedly suppressed below 35 degrees C. At above 40 degrees C, both the diploid and tetraploid Meth-A cells ceased growing. Flow cytometry (FCM) analysis showed that the hyperploid cell fraction increased in the tetraploid Meth-A cell population at low temperatures. The fluidity of cell membranes at different temperatures was measured by means of electron spin resonance (ESR), and it was almost the same between the diploid and tetraploid Meth-A cells. It was suggested that the decreased proliferation below 35 degrees C of the tetraploid Meth-A cells might be due to the increased volume of the cells.

[Alcohol-related high blood pressure, and erythrocyte Na+/K(+)-ATPase activity, sodium and potassium concentrations].

Erythrocyte Na+/K(+)-ATPase activity (Ery-ATPase) and intra-erythrocyte sodium and potassium concentrations (Ery-Na, Ery-K) were determined in 83 men aged between 36 and 60 years. Volumes of alcohol consumed during the preceding one week (Alc) correlated significantly with blood pressure (BP), but not correlated with Ery-ATPase and Ery-K. Ery-Na showed a week inverse correlation with Alc, but the partial correlation after adjusting Ery-ATPase was not significant. Therefore, elevations of BP found in alcohol consumers are not related to changes in the cell-membrane Na+/K(+)-ATPase activity and intracellular Na and K concentrations. Ery-ATPase showed a borderline significant positive correlation with diastolic BP (0.05 < or = p < 0.10), independently of age, body mass index and Alc. The significance of the weak association in the pathogenesis of hypertension remains unclear.

Frontal intermittent delta activity in schizophrenic patients receiving antipsychotic drugs.

FIRDA was found in 14 EEGs recorded from 338 schizophrenic patients receiving antipsychotic drugs, although they showed no FIRDA in the baseline EEG. The daily dose of antipsychotic drugs when FIRDA was found was larger than when FIRDA disappeared. FIRDA was assumed to be induced by relatively high doses of antipsychotic drugs. Thus, the effect of antipsychotic drugs should be added to the list of differential diagnoses associated with FIRDA. FIRDA did not correlate with the baseline psychopathology of schizophrenic patients. Patients without FIRDA tended to respond poorly to antipsychotic drugs in terms of negative symptoms. Improvement of positive symptoms in FIRDA patients was not as remarkable as in patients without FIRDA.

[Determination of human erythrocyte membrane Na+/K(+)-ATPase activity in small volume of blood sample].

Na+/K(+)-ATPase of the cell membrane is considered to be closely related to the pathology of various diseases including hypertension and heart failure. The activity of this enzyme in the erythrocyte membrane has been determined in earlier reports by the assay of inorganic phosphate generated from the substrate ATP or radioimmunoassay after binding 3H ouabain to the erythrocyte membrane, using a large volume of blood samples. However, as neither method was appropriate for wide routine use, we developed a method to assay this enzyme in a small volume (10 ml) of fresh human blood samples with re-evaluation of conditions for the inorganic phosphate assay. In this method, the coefficient value (CV) of membrane protein amount and the NA+/K(+)-ATPase activity were 2.2% and 2.5% respectively, indicating sufficient precision of the assay. Moreover, in 97 subjects without abnormalities in blood biochemical tests (77 males and 20 females) aged 35-59 years, the enzyme activity showed no differences according to age or sex, ranging from 0.217 to 0.071 mumols Pi/mg/hr with a mean of 0.130.

Choleretic actions of iridoid compounds.

The intravenous administration of 50 and 100 mg/kg of genipin (GP), gardenogenins (GAR-G), deacetylasperulosidic acid methylester genins (DAM-G), and scandoside methylester genin (SSM-G) exhibited the bile acid-independent choleretic actions. The action of DAM-G was stronger than the actions of other compounds tested. The choleretic action of SSM-G was milder, but longer lasting than those of GAR-G and DAM-G.

The Structural Transformation of Gardenoside and Its Related Iridoid Compounds by Acid and beta-Glucosidase.

In the course of studies on the metabolism of iridoid compounds, three new compounds derived from 6alpha-hydroxygeniposide and 6beta-hydroxygeniposide, obtained from gardenoside by the hydrochloric acid treatment, were isolated after the hydrolysis with beta-glucosidase. The aglycone of 6beta-hydroxygeniposide was elucidated as 6beta-hydroxygenipin. On the other hand, the aglycones of 6alpha-hydroxygeniposide were identified as the mixture of stereoisomers, 6alpha-hydroxygenipin and 6alpha-hydroxy-1- EPI-genipin.

Mutagenicity of 2,4-dinitrotoluene and its metabolites in Salmonella typhimurium.

2,4-Dinitrotoluene (2,4-DNT) and its metabolites (2A4NT, 4A2NT, 2,4-DNB, 2A4NB, 4A2NB, 2,4-DAT, 2N4AAT, 2A4AAT, 2A4AABA and 2,4-DNBA), and 2,4-dinitrobenzaldehyde (2,4-DNA1), putative metabolite of 2,4-DNT, were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100. 2,4-DAT, 2N4AAT, 2A4AAT, 2A4AABA and 2,4-DNBA were inactive for strains TA98 and TA100. 2,4-DNT itself was only a weak mutagen. Two aminonitrotoluenes (2A4NT and 4A2NT), two aminonitrobenzyl alcohols (2A4NB and 4A2NB) and 2,4-dinitrobenzyl alcohol (2,4-DNB) were increasingly mutagenic in that order, in both strains, however, they were only weak or weak mutagens at mM concentrations. In contrast with these compounds, 2,4-DNA1 was mutagenic even at microM concentrations in both strains. These results suggest that a high mutagenicity of 2,4-DNA1 may be correlated to the carcinogenicity of 2,4-DNT.