Deficiency of calcium and magnesium induces apoptosis via scavenger receptor BI

AimsCells undergo apoptosis in stressed status such as in intracellular calcium overload or extracellular calcium/magnesium deficiency. The mechanisms of how deficiency of the divalent metal ions induces apoptosis remain to be defined. Scavenger receptor BI (SR-BI) is a high density lipoprotein (HDL) receptor. Recent studies demonstrated that SR-BI is a stress response molecule which induces apoptosis upon serum deprivation. In this study, we assessed our hypothesis that the deficiency of calcium/magnesium induces apoptosis via SR-BI apoptotic pathway.Main methodsWe employed CHO cell lines expressing vector and SR-BI to test the effect of SR-BI on apoptosis induced by deficiency of calcium, magnesium and zinc in culture medium. The regain of different metal ions in deficient medium was also performed, respectively. Cell death was detected by morphological changes and quantified by LDH cytotoxicity assay. Apoptosis was also assessed by DNA ladder assay and DNA condensation assay. The SR-BIC323G mutant cells which lack the apoptotic activity of SR-BI were employed to verify the SR-BI-dependent effect on calcium/magnesium induced apoptosis.Key findingsThe deficiency of calcium/magnesium induced cell apoptosis in CHO-SR-BI cells, but not in CHO-vector cells. Moreover, no apoptotic cell death was observed in SR-BIC323G mutant cells, indicating that the deficiency of divalent metal ions induces apoptosis in a SR-BI-dependent manner. Furthermore, the restoration of calcium or magnesium, but not zinc, protected CHO-SR-BI cells from apoptotic cell death, in a dose-dependent fashion.SignificanceThese findings extend our understanding about how calcium and magnesium deficiency induces apoptosis.     

Regulatory responses of hepatocytes,macrophages and vascular endothelial cells to magnesium deficiency

The liver is the organ that responds to nutritional disturbances including magnesium deficiency. The present study evaluated cellular responses to magnesium deficiency using model cells of the liver, namely, HepG2 cells as hepatocytes, RAW264.7 cells as Kupffer cells and human umbilical vein endothelial cells (HUVECs) as vascular endothelial cells; we examined effects of culture with magnesium deficient medium on cell responses in individual types of cells as well as interactive responses among cells. Metabolomic analyses indicated that magnesium deficiency differentially affected the cellular content of metabolites among HepG2 cells, RAW264.7 cells and HUVECs. The cellular content of the metabolites in HepG2 cells and HUVECs was also affected by the conditioned medium from RAW264.7 cells cultured with the magnesium-deficient media. The changes in HUVECs partly resembled those of the livers of magnesium-deficient rats previously described. RNA-seq analyses indicated that magnesium deficiency modulated the expression levels of molecules related to the ubiquitin-proteasome pathway and oxidative stress/antioxidant response in HepG2 cells and RAW264.7 cells, respectively. Furthermore, when HUVECs were co-cultured with RAW264.7 cells, lipopolysaccharide-induced expression of interleukin (IL)-1β and IL-6 was enhanced by magnesium deficiency, depending on the presence of RAW264.7 cells. The present study reveals that magnesium deficiency affects cellular metabolism in HepG2 liver cells, RAW264.7 macrophages and HUVECs, and that the modulation of cellular responses to extracellular magnesium deficiency in HUVECs depends on the presence of RAW264.7 cells. The complex responses in individual cells and through cell interactions partly explain the regulatory reaction to magnesium deficiency in the liver.     

Magnesium deficiency in the Japanese quail

Morphological effects of magnesium deficiency on liver cells and general aspects of its influence on the metabolism were investigated in young quails. Magnesium deficiency was characterized by a depressed growth, a high mortality rate, a decrease in hematocrit and magnesium and calcium plasma concentrations. Magnesium deficiency reduced the magnesium concentration in heart by 44%, but did not affect the concentration in liver. Ultrastructural aspect of liver parenchymal cells revealed that the number of mitochondria per cell section was decreased and the average area of a mitochondrion was greater in deficient quails than in control animals. The significance of these morphological changes was discussed in relation to disturbances in energy metabolism of these organelles. From these results, japanese quail appeared as an interesting experimental model for studies on metabolic disturbances in magnesium deficiency.     

Mutants of cultured chinese hamster cells deficient in adenine phosphoribosyl transferase

Spontaneous mutants of cultured Chinese hamster cells (line CHO) deficient in APRT have been isolated by selection in 8-azaadenine (AA). Loss of APRT activity occurs in two discrete steps. In the first step, about 65% of total activity is lost; in the second step, most or all of the remaining activity is lost. Cells totally deficient in APRT are highly resistant to AA and cannot utilize exogenous adenine as a source of purines for cell growth. Cells partially deficient in the enzyme exhibit resistance to AA intermediate to that of wild type and fully deficient cells. Growth of cells partially deficient in APRT is inhibited in medium containing drug by the presence of large numbers of wild type cells, but cells totally deficient in the enzyme are not inhibited by the presence of either partially deficient or wild type cells.Stepwise loss of APRT activity probably has a genetic origin. The mutants exhibit stable phenotypes, and the frequency of fully deficient cells in a partially deficient population is enhanced by treatment with a mutagen. The rate of spontaneous mutation from partial to total deficiency is 3 ± 0.8 × 10^?7 per cell generation, and reversion from full to partial deficiency can occur. Total lack of APRT activity is recessive to its presence, but the specific activity of the enzyme in hybrid cells depends quantitatively upon the specific activities in the two parents.     

Escherichia coli radC is deficient in the recA-dependent repair of X-ray-induced DNA strand breaks

Escherichia coli K-12 cells incubated in buffer can repair most of their X-ray-induced DNA single-strand breaks, but additional single-strand breaks are repaired when the cells are incubated in growth medium. While the radC102 mutant was proficient at repairing DNA single-strand breaks in buffer (polA-dependent repair), it was partially deficient in repairing the additional single-strand breaks (or alkali-labile lesions) that the wild-type strain can repair in growth medium (recA-dependent repair), and this repair deficiency correlated with the X-ray survival deficiency of the radC strain. In studies using neutral sucrose gradients, the radC strain consistently showed a small deficiency in rejoining X-ray-induced DNA double-strand breaks, and it was deficient in restoring the normal sedimentation characteristics of the repaired DNA.     

The effect of biotin on cellular functions in HeLa cells

HeLa cells cultured in a biotin-deficient medium show reduced rate of protein synthesis, DNA synthesis and growth. Addition of exogenous biotin to the cells cultured in biotin-deficient medium results in enhanced protein synthesis, DNA synthesis and cell growth. Continuous protein synthesis is required for the increase in DNA synthesis observed upon the addition of exogenous biotin to the cells cultured in biotin-deficient medium. These results suggest that cells cultured in biotin-deficient medium are arrested in the G1 stage of cell cycle and this block is removed upon the addition of biotin to the deficient medium.     

Starvation and Encystment of a Soil Amoeba Hartmannella castellanii*

SYNOPSIS. The behavior of the amoeba H. castellanii was investigated in various carbon and nitrogen deficient media with a view to developing a satisfactory replacement medium for the study of encystment and excystment. Media which had been devised for other soil amoebae did not cause H. castellanii to encyst. In these media there was an efflux of material from the cells which was independent of osmolarity but which was minimized by the addition of magnesium. Maximal encystment occurred in a medium containing magnesium chloride alone. The cysts produced in the magnesium chloride replacement medium are viable and readily excyst when resuspended in the growth medium. The cysts contain cellulose, which is not present in the vegetative amoebae, and differ from the amoebae in their greater resistance to induced lysis and mechanical injury.     

Arysulfatase A modulation with pH in multiple sulfatase deficiency disorder fibroblasts.

It has been observed that multiple sulfatase deficiency disorder (MSDD) fibroblasts contained from profoundly deficient to near normal amounts of arylsulfatase (ARS) A depending on the medium in which they were cultured. Our present findings show that the major factor determining the enzyme level is the pH of the medium during growth. In media which became acidic or was maintained at low pH (less than 7), the cells expressed the enzymopathy, while in high pH media (7.4), the cells produced enzyme. The high and low enzyme states were reversible. The ARS A deficiency in MSDD must, therefore, be a secondary manifestation of a mutation in another system.     

Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro.

We have compared several methods for reducing calcium and magnesium concentrations in tissue culture medium, with the objective of producing selective deficiency effects on the growth of mouse (L5178Y) and human (P1R) lymphoblasts. In experiments in which calcium- and magnesium- "free" McCoy's medium was supplemented with 15% horse or fetal calf serum, enough calcium and magnesium was provided by serum to support normal lymphoblast growth rate. Either dialysis or chelating-resin treatment of horse or fetal calf serum reduced calcium and magnesium contents approximately 100-fold. Use of dialyzed sera resulted in reduced growth rate, although in most cases the reduction in growth could be attributed to other effects of dialysis on serum, inasmuch as growth in those experiments was not restored to normal by the addition of calcium and magnesium to the medium. In contrast, the reduction of lymphoblast growth rate that occurred when resin-treated serum was used was always attributable to removal of calcium and magnesium, as normal growth always occurred in cultures to which calcium and magnesium were added. To demostrate a growth-inhibiting effect on either mouse or human lymphoblasts by severe reduction of either calcium or magnesium in the presence of normal amounts of the alternative cation, it was necessary to (a) expose McCoy's Ca-Mg-"free" medium to chelating-resin to reduce further the residual cation concentrations; (b) wash cells from stock cultures in a medium devoid of calcium and magnesium prior to inoculation into experimental cultures; (c) reduce the proportion of serum in the final medium from 15 to 5%; and (d) add 100 muM EGTA to cultures. Under these conditions, growth of both cell types was completely abolished in the presence of normal magnesium but in the absence of added calcium, and markedly reduced in the presence of normal calcium but in the absence of magnesium. These modifications did not compromise growth in cultures containing normal concentrations of both ions.     

外源NO对缺镁胁迫下玉米幼苗生长和离子平衡的影响

研究了在缺镁胁迫下,外源NO对缺镁玉米幼苗生长、根系活力和离子含量的影响。结果表明,缺镁胁迫使玉米幼苗株高、根长和干鲜重下降,根系活力降低,N元素在地上部和根部分配失调,新叶和老叶中Mg2＋、Cu2＋、Fe3＋、Mn2＋等离子含量下降,Ca2＋、K＋、Zn2＋等离子含量上升。根中Mg2＋离子含量下降,Ca2＋、K＋、Zn2＋、Cu2＋、Fe3＋、Mn2＋等离子含量上升。用100μmol·L-1一氧化氮供体硝普钠（SNP）处理后,玉米幼苗株高、根长、干重和鲜重均提高,根系活力增强,改善了N代谢,新叶中Ca2＋、K＋和Zn2＋等离子含量下降,Mg2＋、Cu2＋、Fe3＋和Mn2＋等离子含量提高,老叶中Mg2＋、Ca2＋、K＋和Zn2＋等离子含量下降,Cu2＋、Fe3＋和Mn2＋等离子含量提高,根中Mg2＋、Ca2＋、K＋、Cu2＋、Zn2＋、Fe3＋和Mn2＋离子含量均下降。实验结果表明,NO保护玉米幼苗免受缺镁胁迫的影响。     

The effect of magnesium deficiency on serum aldosterone in rats fed two levels of sodium.

Rats were fed 47 (deficient) and 606 ppm (adequate) magnesium with either 2,100 or 14,000 ppm sodium. Serum corticosterone and aldosterone levels were determined by randoimmunoassay in six rats from each treatment group killed on days 7, 14, and 28 of consumption of the experimental diets. Serum corticosterone levels were moderately, but not significantly, decreased in magnesium deficient animals. Serum aldosterone levels increased over time in the rats fed the lower sodium diet with adequate magnesium and were further elevated in magnesium deficient animals. In sodium loaded rats the increase in aldosterone levels in magnesium deficiency was less and occurred later. Retention and urinary excretion of sodium and potassium did not appear to be affected by magnesium status or the serum concentration of aldosterone. Possible mechanisms underlying the changes in aldosterone levels of magnesium depleted animals are discussed with reference to the known effects of magnesium deficiency on physiological functions.     

Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation

The rate of ethanol production per milligram of cell protein begins to decline in the early stage of batch fermentation before high concentrations of ethanol have accumulated. In yeast extract-peptone medium (20% glucose), this initial decline appears to be related to growth and to result in part from a nutrient deficiency. The addition of yeast extract, peptone, and ashed preparations of these restored the ability of glucose-reconstituted medium (in which cells had been previously grown) to support vigorous growth. Magnesium was identified as the active component. Supplementing fermentations with 0.5 mM magnesium prolonged exponential growth, resulting in increased yeast cell mass. The addition of magnesium also reduced the decline in fermentative activity (micromoles of CO2 evolved per hour per milligram of protein) during the completion of batch fermentations. These two effects reduced the time required for the conversion of 20% glucose into ethanol by 1/3 with no measurable loss in ethanol yield (98% of theoretical maximum yield). It is possible that some of the reported beneficial effects of complex nutrients (soy flour and yeast extract) for ethanol production also result from the correction of a simple inorganic ion deficiency, such as magnesium.     

Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation.

The rate of ethanol production per milligram of cell protein begins to decline in the early stage of batch fermentation before high concentrations of ethanol have accumulated. In yeast extract-peptone medium (20% glucose), this initial decline appears to be related to growth and to result in part from a nutrient deficiency. The addition of yeast extract, peptone, and ashed preparations of these restored the ability of glucose-reconstituted medium (in which cells had been previously grown) to support vigorous growth. Magnesium was identified as the active component. Supplementing fermentations with 0.5 mM magnesium prolonged exponential growth, resulting in increased yeast cell mass. The addition of magnesium also reduced the decline in fermentative activity (micromoles of CO2 evolved per hour per milligram of protein) during the completion of batch fermentations. These two effects reduced the time required for the conversion of 20% glucose into ethanol by 1/3 with no measurable loss in ethanol yield (98% of theoretical maximum yield). It is possible that some of the reported beneficial effects of complex nutrients (soy flour and yeast extract) for ethanol production also result from the correction of a simple inorganic ion deficiency, such as magnesium.     

Manganese-Resistant Mutants of Escherichia coli: Physiological and Genetic Studies

Manganese is growth inhibitory for Escherichia coli. The manganese concentration required for inhibition is dependent upon the magnesium concentration of the medium. Mutants have been isolated which are partially resistant to manganese inhibition in both liquid and solid media. From conjugation experiments, the genetic locus for manganese-resistance, mng, appears to be between 34 and 37 min on the E. coli genetic map. Experiments with radioactive (28)Mg lead to the tentative conclusion that the mng mutants are altered in the inhibition constant for manganese as a competitive inhibitor for the mangnesium accumulation system. Once high manganese enters the cells, it displaces internal magnesium and leads to a net cellular loss and hence growth inhibition. The mng mutants are somewhat less subject to manganese-induced magnesium loss under comparable conditions than are manganese-sensitive wild-type cells.     

Magnesium deprivation inhibits the expression of differentiation-related phenotypes in human promyelocytic leukemia HL-60 cells

The role of magnesium ions in the differentiation of human promyelocytic leukemia HL-60 cells was investigated. When HL-60 extracellular magnesium was deficient (less than 0.01 mM), the total intracellular magnesium content and [3H] leucine incorporation rates decreased to 61 and 28%, respectively, on day 3. When the cells were treated with various inducers (100 nM 1 alpha, 25 dihydroxyitamine D3 (1,25(OH)2D3), 100 nM beta-all-trans retinoic acid (RA), 20 nM 12-o-tetradecanoyl phorbol-13-acetate (TPA), 1.25% dimethylsulfoxide (DMSO) and 30 nM aclacinomycin (AcM] in magnesium-deficient medium, the expression of differentiation-related phenotypes (nitroblue tetrazolium (NBT) reducing ability, nonspecific esterase (NSE) activity and monoclonal antibody, OKM1 binding activity) was almost completely inhibited. After a 2-day treatment with 100 nM 1,25(OH)2D3 in magnesium-deficient medium, the expression of differentiation-related phenotypes was restored by further incubation in the absence of inducer in standard magnesium medium (0.4 mM). These results suggested that magnesium deprivation inhibited the expression of HL-60 differentiation-related phenotypes but not their commitment to differentiation. These phenotypes were expressed without inducer in standard magnesium medium after a 2-day simultaneous treatment with 1,25(OH)2D3 and cyclohexamide (protein synthesis inhibitor) in magnesium-deficient medium, but not after simultaneous pretreatment with 1,25(OH)2D3 and alpha-amanitin (RNA synthesis inhibitor). Thus, it was suggested that the magnesium-requiring step in HL-60 cell differentiation is in protein but not mRNA synthesis. This conclusion is supported by the findings that changes in c-myc and c-fms mRNA levels in HL-60 cells treated with 100 nM 1,25(OH)2D3 in magnesium-deficient medium and those in standard magnesium medium were the same. In addition, dibutyryl cyclic adenosine monophosphate (dbc AMP) could restore expression of differentiation-related phenotypes inhibited by magnesium deprivation but not those inhibited by cyclohexamide, even though magnesium deprivation inhibited protein synthesis as much as did cyclohexamide. This suggests that magnesium-requiring step in HL-60 cell differentiation is different from that inhibited by cyclohexamide.     

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

Dermal fibroblasts from Down''s syndrome patients share a cycloheximide-induced deficiency in collagen adhesion responses with normal aging cells

Human skin fibroblasts from three different Down's syndrome patients (trisomy 21) of very different ages have been tested for their adhesion responses on tissue culture substrata coated with type I collagen, fibronectin (FN), and their combination after or during treatment of cells with cycloheximide to evaluate limitations in specific responses. It was shown previously that in vitro-aged papillary and reticular dermal fibroblasts from normal individuals do not generate F-actin stress fibers when pretreated with cycloheximide on collagen substrata but do so on FN substrata, a deficiency linked to limiting amounts/function of collagen-specific receptors in aging cells. In these studies, all three Down's fibroblast populations demonstrated a similar deficiency in stress fiber formation, evaluated by rhodamine-phalloidin staining, upon cycloheximide treatment at all passage levels. They remained competent for stress fiber formation on FN substrata and for reorganization of microtubule and intermediate filament networks on all substrata, demonstrating the specificity for the collagen matrix and for the F-actin cytoskeleton in this deficiency. The cycloheximide-induced deficiency could be readily reversed in all three cell populations by further incubation of cells in drug-free medium and, in some cases, by prior growth of cells in ascorbate-supplemented medium to stimulate collagen and possibly collagen receptor production. However, several pieces of evidence indicate that reduced amounts of FN and collagen synthesized by fibroblasts do not contribute to the cycloheximide-induced deficiency, including the inability to reverse the effect by treatment of cells with TGF beta. Several conclusions are suggested from these studies: (a) Down's dermal fibroblasts become deficient in collagen-specific receptor(s) upon cycloheximide treatment, which leads to altered transmembrane signaling and inability to reorganize F-actin into stress fibers; (b) Down's dermal fibroblasts at all passage levels have matrix adhesive phenotypes similar to those of aging fibroblasts from normal individuals; and (c) these studies provide further support for cells from Down's patients as a genetic model of aging in normal populations.     

Effect of cultivation conditions on the age structure of a yeast population

The work was aimed at studying the effect exerted by mineral components of the medium and a carbon source limiting the growth of Candida boidinii and Saccharomyces cerevisiae as well as by the dilution rate in the course of chemostat cultivation and by the temperature of growth on the age structure of a population, i.e. on the proportion of cells at different phases of the cell cycle. Nitrogen, phosphorus and magnesium deficiency delayed the growth of cells in the G1 phase and, if the growth rate was low, at the end of budding. The rise of the growth rate increased the proportion of budding cells. A temperature drop below 23 degrees C delayed the separation of the daughter and mother cells.     

Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: Application to studies of lymphoblast proliferation in vitro

Summary We have compared several methods for reducing calcium and magnesium concentrations in tissue culture medium, with the objective of producing selective deficiency effects on the growth of mouse (L5178Y) and human (P1R) lymphoblasts. In experiments in which calcium- and magnesium-“free” McCoy’s medium was supplemented with 15% horse or fetal calf serum, enough calcium and magnesium was provided by serum to support normal lymphoblast growth rate. Either dialysis or chelating-resin treatment of horse or fetal calf serum reduced calcium and magnesium contents approximately 100-fold. Use of dialyzed sera resulted in reduced growth rate, although in most cases the reduction in growth could be attributed to other effects of dialysis on serum, inasmuch as growth in those experiments was not restored to normal by the addition of calcium and magnesium to the medium. In contrast, the reduction of lymphoblast growth rate that occurred when resin-treated serum was used was always attributable to removal of calcium and magnesium, as normal growth always occurred in cultures to which calcium and magnesium were added. To demonstrate a growth-inhibiting effect on either mouse or human lymphoblasts by severe reduction of either calcium or magnesium in the presence of normal amounts of the alternative cation, it was necessary to (a) expose McCoy’s Ca−Mg-“free” medium to chelating-resin to reduce further the residual cation concentrations; (b) wash cells from stock cultures in a medium devoid of calcium and magnesium prior to inoculation into experimental cultures; (c) reduce the proportion of serum in the final medium from 15 to 5%; and (d) add 100 μM EGTA to cultures. Under these conditions, growth of both cell types was completely abolished in the presence of normal magnesium but in the absence of added calcium, and markedly reduced in the presence of normal calcium but in the absence of magnesium. These modifications did not compromise growth in cultures containing normal concentrations of both ions. This work has been supported by a Research Grant from the U.S. Public Health Service (CA 12790), from the Monroe County Cancer and Leukemia Association, and by a contract from the Atomic Energy Project at the University of Rochester, and has been assigned publication no. UR-3490-624.     

Wall turnover deficiency of Bacillus subtilis Ni15 is due to a decrease in teichoic acid

Bacillus subtilis Ni15 is deficient in cell wall turnover. The deficiency is removed if the medium contains 0.2 M NaCl, which does not affect growth. The levels of amidase and glucosaminidase, the most likely enzymes involved in turnover, were, in stationary phase Ni15 cells, similar to those in late-exponential phase cells of a standard strain. The Ni15 enzymes were not salt sensitive. However, the Ni15 walls contained 4.7-fold less phosphorus than the walls of the standard strain. Since the phosphorus content of B. subtilis walls reflects the level of teichoic acid, it is proposed that the turnover deficiency of this strain is due to a decrease in wall teichoic acid.