Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes

Rapidly proliferating, polyclonally stimulated mouse spleen lymphocytes were separated by density-gradient unit-gravity sedimentation. The following measurements were made on each fraction: the average intracellular water volume, the distribution of DNA content by flow microfluorometry, the rate of ³H-thymidine incorporation, and the intracellular pH. Fractions of cells with a small average intracellular volume were predominately in G0 or G1 phase of the cell cycle, while fractions of larger cells had higher proportions of cells in S or G2. Multiple regression analysis of the data for both T and B lymphocytes indicated that the intracellular pH of cells in G0, G1, or G2 is around pH 7.2, and that the intracellular pH of cells in S phase of the cell cycle is around pH 7.4.     

Control of cell cycle entry and progression in mitogen-stimulated human B lymphocytes

Tonsillar B lymphocytes were stimulated to proliferate by the mitogenic combination of phorbol dibutyrate and ionomycin. Progression through the cell cycle was monitored by measurements of cellular DNA and RNA content using flow cytometry. Changes in surface expression of class II MHC antigens and CD20 antigen were also monitored as early parameters of B lymphocyte activation and cell cycle progression. The results showed that about 60% of the population synchronously entered and progressed through the cell cycle. The transition from the resting state, signaled by increased RNA content, occurred about 12 to 24 hr after stimulation; S phase entry occurred at about 36 hr. Small, variable populations of cells appeared to be unresponsive to the stimuli, either because they were “preactivated” before in vitro stimulation or were already dying. The kinetics of appearance and accumulation of several cell cycle regulated/regulatory proteins were followed by immunoblotting. The proliferating cell nuclear antigen (PCNA) cyclin A and p33^cdk2 proteins were either absent or present in very low amounts in resting cells and first became detectable in increased amount beginning at about 24 hr after stimulation; increased p34^cdc2 protein was not detected until about 36 hr. Increased cellular content and phosphorylation of the p110^Rb protein was already obvious by 24 hr after stimulation. The effects of several immunosuppressive agents were examined using purified B cells. Both cyclosporin A and an FK506 analogue were shown to inhibit proliferation of B lymphocytes, at the low doses also inhibitory to T cells. © 1995 Wiley-Liss, Inc.     

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNA and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [³H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K⁺ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K⁺, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K⁺ transport, controlling mitosis at distinct phases of the cell cycle.     

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNa and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [3H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K+ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K+, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K+ transport, controlling mitosis at distinct phases of the cell cycle.     

Level of mIa expression on mitogen-stimulated murine B lymphocytes is dependent on position in cell cycle

Using correlated flow cytometric analysis of cell surface Ia antigen expression (immunofluorescence) and cell cycle phase (pulse-width of axial light extinction), we have quantitated changes in expression of mIa antigen on murine B cells during progression through cell cycle. Our results indicate that density of mIa expressed on mitogen-stimulated B cells increases fourfold to fivefold during the transition from G0 to G1. By early S phase, mIa density has decreased by fourfold to fivefold relative to peak expression. This decrease becomes more evident by late S, G2, and M phases, when an eightfold decrease in mIa antigen density is observed relative to peak levels. This decrease results in mIa antigen expression lower than that of resting, unstimulated B cells. Therefore, maximum mIa antigen expression occurs during G0 to G1 transition and in early G1, when a requirement for I region-restricted, antigen-driven T cell help for thymus-dependent, antigen-driven B cell activation has been demonstrated.     

The relationships between vitamin B12 and folic acid and the effect of methionine on folate metabolism

The relationship between vitamin B12 and folate and the effect of methionine on folate metabolism during B12 deficiency in rats is best explained by the prevention of the accumulation of 5-methyl-H4PteGlu by vitamin B12 and/or methionine. Although several points remain to be clarified, the 'methyl trap' hypothesis provides the most satisfactory explanation for the relation between vitamin B12, methionine and folic acid. This concept is extended by the hypothesis that H4PteGlu is the most active substrate for pteroylpolyglutamate synthetase, and thus accounts for the effect of methionine or vitamin B12 increasing liver folate levels.     

Assessment of cytogenetic response to folic acid deprivation in rat lymphocytes

Summary Rat lymphocyte cultures were initiated in minimal essential medium containing 0, 0.01, 1.0, or 10 mg/l of folic acid to investigate the influence of folic acid on cell kinetics, chromosome aberration, and sister chromatid exchange (SCE) frequencies. No significant difference was observed between cultures with and without folic acid in mitotic index or cell cycle kinetics as judged by the numbers of average lymphocyte divisions. However, a sequential reduction in the number of chromatid gaps occurred as the concentration of folic acid increased. On the other hand, addition of folic acid did not significantly affect the SCE frequency. Although folic acid does not seem to alter SCE formation, its significant influence in the reduction of chromatid gaps suggests that caution should be exercised in selecting a medium regarding folic acid content especially because gaps alone are produced by certain dose levels of some chemical clastogens.     

Sarcosine oxidase activity of rat liver tissue: effect of folic acid deficiency and induced hyperthyroidism

The sarcosine oxidase content of hepatic tissue from rats made deficient in folic acid was similar to that of normal rats. This is taken as evidence that sarcosine oxidase activity is not dependent upon the presence of folic acid vitamins.     

Dietary nickel and folic acid interact to affect folate and methionine metabolism in the rat

A previous experiment using rats indicated that dietary nickel (Ni), folic acid, and their interaction affected variables associated with one-carbon metabolism. That study used diets that produced only mild folate deficiency. Thus, an experiment was performed to determine the effect of a severe folate deficiency on nickel deprivation in rats. A 2×2 factorially arranged experiment used groups of six weanling Sprague-Dawley rats. Dietary variables were nickel, as NiCl₂·6H₂O, 0 or 1 μg/g and folic acid, 0 or 4 mg/kg. All diets contained 10 g succinylsulfathiazole/kg to suppress microbial folate synthesis. The basal diet contained <20 ng Ni/g. After 50 d, an interaction between nickel and folate affected the urinary excretion of formiminoglutamic acid (FIGLU) and the liver concentration of S-adenosylmethionine (SAM). Because of this, it is proposed that the physiological function of nickel is related to the common metabolism shared by SAM and FIGLU. Possibly the physiological function of nickel could be related to the tissue concentration of 5-methyltetrahydrofolate (MTHF) or tetrahydrofolate (THF).     

The effect of methionine on the uptake,distribution, and binding of the convulsant methionine sulfoximine in the rat

The effect of methionine on the uptake, distribution, and binding of the convulsant methionine sulfoximine (MSO) in 7 rat brain regions, the spinal cord, the liver, and the kidney was investigated. The administration of methionine decreased the uptake of MSO in all brain regions. The uptake of MSO by and its distribution in the nervous tissue was uniform and failed to result in any preferential accumulation of the drug. Methionine decreased the amount of MSO bound to cerebral structures and to the spinal cord. MSO bound to the spinal cord was less susceptible to release by Triton X-100 than was brain-bound MSO.     

Effects of folic acid deficiency and MTHFRC677T polymorphisms on cytotoxicity in human peripheral blood lymphocytes

Apoptosis (APO) and necrosis (NEC) are two different types of cell death occurring in response to cellular stress factors. Cells with DNA damage may undergo APO or NEC. Folate is an essential micronutrient associated with DNA synthesis, repair and methylation. Methylenetetrahydrofolate reductase (MTHFR) regulates intracellular folate metabolism. Folate deficiency and MTHFR C677T polymorphisms have been shown to be related to DNA damage. To verify the cytotoxic effects of folate deficiency on cells with different MTHFR C677T genotypes, 15 human peripheral lymphocyte cases with different MTHFR C677T genotypes were cultured in folic acid (FA)-deficient and -sufficient media for 9 days. Cytotoxicity was quantified using the frequencies of APO and NEC as endpoints, the nuclear division index (NDI), and the number of viable cells (NVC). These results showed that FA is an important factor in reducing cytotoxicity and increasing cell proliferation. Lymphocytes with the TT genotype proliferated easily under stress and exhibited different responses to FA deficiency than lymphocytes with the CC and CT genotypes. A TT individual may accumulate more cytotoxicity under cytotoxic stress, suggesting that the effects of FA deficiency on cytotoxicity are greater than the effects in individuals with the other MTHFR C677T variants.