Physiological Saline Solutions as a Useful Tool in Micronucleus and Metaphase Slide Preparations

The micronucleus test (Schmid 1975) is widely used as an indicator of cytogenetic damage induced in vivo by clastogens and spindle poisons. Yamamoto and Kikuchi (1980) have recently showed that by comparing the relative size of micronuclei it is possible to determine whether an agent acts as a clastogen or a spindle poison. This finding will undoubtedly increase the use of the technique in routine protocols for the testing of chemicals. Besides, the test is quite sensitive and much simpler and faster than chromosome analysis. One obstacle, however, hinders several laboratories: the increasing unavailability of fetal calf serum. Recently, Das and Kar (1980) have proposed sodium citrate as a substitute for fetal calf serum. However, 1% sodium citrate solution is hypotonic, which fact may pose difficulties for an experimenter having to sacrifice several animals within a short time, a common situation in routine tests. We were able to overcome the problem of hypotonia by replacing fetal calf serum with the isotonic solutions 0.9% NaCl and Ringer's saline for mammals (9.00 g NaCl, 0.42 g KCl, 0.24 g CaCl₂, 0.20 g NaHCO₃, 1000 ml distilled. H₂O) at room temperature.     

Sodium Citrate as a Substitute for Fetal Calf Serum in the Micronucleus Test

The micronucleus test developed recently by Schmid and coworkers (Boller and Schmid 1970, Ledebur and Schmid 1973, Schmid 1976) is a rapid, convenient, and sensitive procedure for the detection of induced chromosome aberrations in vivo. It is now widely used for evaluating the mutagenic potential of drugs and other chemicals. The test involves the demonstration of micronuclei which result from lagging of acentric chromosome fragments or even of whole chromosomes during mitosis due to spindle disruption in the anucleate young erythrocytes of bone marrow smears (for details see Schmid 1976). Success or failure of the technique largely depends on the quality of the smear. Cell clumping and cell damage render the smear valueless. Schmid (1976) recommends the use of fetal calf serum for preparing the best smears. However, as he also noted, fetal calf serum is very expensive. Moreover it is not readily available in certain countries, particularly developing ones. It is not easy to procure heat inactivated human AB serum either, which Schmid has suggested as a good substitute for fetal calf serum. Difficulty in obtaining these important elements of the procedure is overcome to a great extent by the brief use of 1% sodium citrate solution at 20-25 C as a substitute for fetal calf serum.     

Identification of sulphate-reducing ectosymbiotic bacteria from anaerobic ciliates using 16S rRNA binding oligonucleotide probes

The identity of ectosymbiotic bacteria of some marine, free-living anacrobic ciliates (Metopus contortus, Caenomorpha levanderi and Parablepharisma sp.) was studied using fluorescent-dye-conjugated oligonucleotides complementary to short sequence elements of 16S ribosomal RNA. The ectosymbiotic bacteria of all species hybridized with a eubacterial probe and those of the two former mentioned species hybridized with a general probe for sulphate-reducing bacteria, but not to a probe specific for Desulfobacter. The results support indirect evidence suggesting that ectosymbiotic bacteria of anaerobic ciliates are sulphate-reducers which depend on host metabolites for substrates.Abbreviations DMSO dimethyl sulfoxide - PBS phosphate-buffered saline, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH: 7.3 - TEAA triethylamonium acetate - 1 x SSC standard sodium citrate buffer, 150 mM NaCl, 15 mM Na₂ citrate, pH: 7.0 - 1 x Denh Denhardts solution, 0.02% ficoll, 0.02% bovine serum albumine, 0.02% polyvinol-pyralidone     

Ions and osmolality on post‐thaw sperm motility activation of the endangered Brycon insignis (Characiformes)

The aim of this study was to evaluate the effects of osmolality and the presence of ions on the activation of post‐thaw sperm motility of Brycon insignis. Sperm was frozen under a standardized methodology for this species. In experiment 1, 11 solutions were prepared with reverse osmosis (RO) water (～0 mOsm/kg) and glucose or NaCl adjusted to an osmolality of 50, 100, 150, 200 and 250 mOsm/kg. In experiment 2, six solutions were prepared with RO and adjusted to ~100 mOsm/kg with one of the following chemicals: NaHCO₃, sodium citrate (Na₃C₆H₅O₇), NaCl, KCl, CaCl₂ or glucose (as ion‐free control). Post‐thaw sperm of both experiments was evaluated for motility rate, velocities (curvilinear = VCL, among others) and beat‐cross frequency (BCF). In experiment 1, sperm motility rate and velocities were higher (p < 0.05) when triggered in solutions at osmolalities from 0 to 200 mOsm/kg (62–80% motility; 139–167 µm/s) than that at 250 mOsm/kg (36–44% motility; 94–99 µm/s VCL). BCF was not affected by osmolality and varied from 19 to 24 Hz in all samples. In experiment 2, samples activated in NaHCO₃, citrate, NaCl and KCl solutions yielded higher motility rates (76–85%) and BCF (24–25 Hz) compared to those activated in CaCl₂ (50%; 14 Hz). Samples activated in ion‐free control solution yielded higher motility rate (87%) than those activated in NaHCO₃ and in CaCl₂. Curvilinear velocity was higher in samples activated in NaHCO₃, citrate, KCl and control solutions (144–160 µm/s) than in those activated in CaCl₂ (104 µm/s); samples activated in NaCl yielded intermediate VCL values (127 µm/s). Post‐thaw sperm achieves maximum motility rate and velocities when activated in solutions composed of sodium citrate, NaCl, KCl or glucose. Thus, post‐thaw sperm motility of B. insignis can be triggered in ionic and non‐ionic solutions at osmolality between 0 and 200 mOsm/kg. The use of solutions containing calcium, however, should be avoided.     

The influence of dietary sodium on bone development in growing rats

The present study investigated the effects of dietary sodium on bone growth in young rats. Five-week-old rats were fed one of three different diets for 60 days: low sodium (NaCl, 0.32 g/kg diet), normal sodium (NaCl, 2.6 g/kg) and high sodium (NaCl, 20 g/kg). The proximal tibial metaphysis (PTM), the fifth lumbar vertebra (LV₅) and the middle part of the tibia shaft (TX) were analysed by bone histomorphometry. The expression of three osteogenesis genes, Runx2, osteopontin and osteocalcin, was determined by RT-PCR in bone samples from the skull. In both the PTM and LV₅, trabecular area and thickness were increased by the low-sodium diet, while the high-sodium diet decreased trabecular area in LV₅. Dynamic data revealed that sodium restriction increased bone formation parameters in the PTM and LV₅, but decreased bone resorption in LV₅. In TX, endosteal bone formation was enhanced by the low-sodium diet and depressed by the high-sodium diet compared to the normal sodium group. But there were no statistically changes in the cortical bone area of TX. Low-sodium intake significantly enhanced the expression of all three osteogenesis genes compared to the normal sodium group, while high-sodium intake suppressed osteogenic gene expression. Our results suggest that sodium restriction in growing rats promotes bone development by influencing both bone formation and resorption.     

Interactions of ganglioside GM1 with human and fetal calf sera. Formation of ganglioside-serum albumin complexes

The interactions of ganglioside G_M1 with human and fetal calf sera were studied, the following main results being obtained: (a) G_M1, upon incubation with both sera gave origin to two G_M1-protein complexes, which also occurred after interaction of G_M1 with the albumin fractions prepared from the same sera. Instead no complex formation occurred using the albumin-free fractions. Therefore G_M1 appeared to specifically bind serum albumin and to form G_M1-albumin complexes. (b) G_M1 binding to serum albumin started at ganglioside concentrations surely micellar (above 10^?6 M), was time and concentration dependent, and resulted in a relevant degree of G_M1 complexation (up to 80% of total G_M1 in human serum and up to 18% in fetal calf serum). (c) the binding kinetics appeared, in both serum and the correspondent albumin fraction, to be biphasic: in the first phase, occurring till about 2 · 10^?4 M G_M1, the ratio between bound and total G_M1 increased linearly with increasing G_M1 concentration; in the second phase, occurring above 2 · 10^?4 M, the ratio remained practically constant. After these findings it should be expected that G_M1, when present in serum containing systems, forms complexes with albumin. This should be appropriately considered when studying the effects of exogeneous G_M1 in in vivo and in vitro (tissue cultures) systems.     

Stimulation of heparan sulfate proteoglycan synthesis and secretion during G1 phase induced by growth factors and PMA

Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3′:5′-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([³H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G₁ phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G₁ phase. J. Cell. Biochem. 70:563–572, 1998. © 1998 Wiley-Liss, Inc.     

Antimicrobial activity of sodium citrate against Streptococcus pneumoniae and several oral bacteria

Aims: The aim of this study is to assess the antibacterial activity of sodium citrate against Streptococcus pneumoniae and several oral bacteria. Methods and Results: The antibacterial activity was determined by broth microdilution method. The results showed that although Enterocuccus faecium OB7084 and Klebsiella pneumoniae OB7088 had high tolerance to sodium citrate, several oral bacteria including Fusobacterium nucleatum JCM8532^T, Streptococcus mutans JCM5705^T and Strep. pneumoniae NBRC102642^T were susceptible. Furthermore, the bactericidal activity of sodium citrate against Strep. pneumoniae NBRC102642^T was not influenced by pH in the range of 5·0–8·0, whereas that of sodium lactate was weakened at neutral or weak alkaline pH. When Strep. pneumoniae NBRC102642^T was treated with sodium citrate for 2 h, many burst cells were observed. However, addition of MgCl₂ or CaCl₂ to an assay medium weakened the antimicrobial activity although ZnCl₂ or MnCl₂ did not influence. Conclusions: Independent of pH, sodium citrate inhibited the growth of oral bacteria, which suggests that the mechanism is different from that of sodium lactate. Significance and Impact of the Study: The results presented in this study would be available for understanding the antimicrobial property of sodium citrate.     

The regulation of cell growth: II. Some characteristics of a fetal calf serum factor (FF2) stimulating ornithine decarboxylase in mouse liver

A heat stable, non-dialysable fetal calf serum factor (FF₂), capable of stimulating ornithine decarboxylase in mouse liver, kidney and spleen, has been detected in fetal calf serum and commercial preparations of 81% pure fetuin.The factor has a molecular weight of approx. 17 500, contains sulfhydryl groups necessary for its activity, and is protease resistant.Stimulation of hepatic ornithine decarboxylase by the fetal calf serum factor is dose and time dependent and is blocked by both cycloheximide and by actinomycin D, if the latter is administered within 1 h of the factor. Theophylline enhances the effect of the fetal calf serum factor on ornithine decarboxylase in the liver and the factor stimulate ornithine decarboxylase in hypophysectomized and adrenalectomized rats.     

Dual Mechanisms of Tricarboxylate Transport and Catabolism by Acidaminococcus fermentans

Acidaminococcus fermentans utilized citrate or the citrate analog aconitate as an energy source for growth, and these tricarboxylates were used simultaneously. Citrate utilization and uptake showed biphasic kinetics. High-affinity citrate uptake had a K_t of 40 μM, but the V_max was only 25 nmol/mg of protein per min. Low-affinity citrate utilization had a 10-fold higher V_max, but the K_s was greater than 1.0 mM. Aconitate was a competitive inhibitor (K_i = 34μM) of high-affinity citrate uptake, but low-affinity aconitate utilization had a 10-fold-lower requirement for sodium than did low-affinity citrate utilization. On the basis of this large difference in sodium requirements, it appeared that A. fermentans probably has two systems of tricarboxylate uptake: (i) a citrate/aconitate carrier with a low affinity for sodium and (ii) an aconitate carrier with a high affinity for sodium. Citrate was catabolized by a pathway involving a biotin-requiring, avidin-sensitive, sodium-dependent, membrane-bound oxaloacetate decarboxylase. The cells also had aconitase, but this enzyme was unable to convert citrate to isocitrate. Since cell-free extracts converted either aconitate or glutamate to 2-oxoglutarate, it appeared that aconitate was being catabolized by the glutaconyl-CoA decarboxylase pathway. Exponentially growing cultures on citrate or citrate plus aconitate were inhibited by the sodium/proton antiporter, monensin. Because monensin had no effect on cultures growing with aconitate alone, it appeared that citrate metabolism was acting as an inducer of monensin sensitivity. A. fermentans cells always had a low proton motive force (<50 mV), and cells treated with the protonophore TCS (3,3′,4′,5-tetrachlorosalicylanide) grew even though the proton motive force was less than 20 mV. On the basis of these results, it appeared that A. fermentans was depending almost exclusively on a sodium motive force for its membrane energetics.     

A PERFUSING SOLUTION FOR THE LOBSTER (HOMARUS) HEART AND THE EFFECTS OF ITS CONSTITUENT IONS ON THE HEART

1. All inorganic perfusing solution for the heart of the lobster Homarus americanus, to allow prolonged normal beating (20 hours or more) must agree closely with the inorganic composition of the serum, which varies differentially with that of the environmental sea water. 2. All of the chief inorganic ions of the serum are necessary—Na, K, Ca, Mg, Cl, and SO₄; the critical numbers of the ions being 100, 3, 5, 2–3, 116, and 1–2 respectively. Absence of Mg and SO₄ will be tolerated for several hours. 3. The pH of the solution must agree with that of the serum within 0.2. 4. The osmotic pressure of the solution must agree with that of the serum within 15 per cent. 5. Beating of the heart will continue for several hours on improperly balanced solutions but changes in frequency, tone, or amplitude will occur. Hearts adapted to such solutions will show different responses to physical and chemical stimuli of the solution than those perfused on properly balanced solutions. 6. Arrest in systole is caused by isotonic NaCl, KCl, LiCl, and urea, and arrest in diastole by isotonic CaCl₂, MgCl₂, NaBr, NaI, MgSO₄, and glucose. 7. Lithium cannot replace sodium; neither can bromide or iodide replace chloride ions.     

The use of Box-Behnken design of experiments to study in vitro salt tolerance by Pisolithus tinctorius

The ectomycorrhizal fungus Pisolithus tinctorius was grown in vitro with different concentrations and combinations of three different sodium salts viz., sodium chloride, sodium sulphate and trisodium citrate (NaCl, Na₂SO4 and Na₃C₆H₅O₇) for 30 days. At the end of the experiment the mycelial biomass and protein content of the fungus was evaluated. Based on the salts tested the combination of NaCl and Na₂SO₄ in optimum concentrations actually promoted the growth of P. tinctorius. Box-Behnken design with three variables like NaCl, Na₂SO₄ and Na₃C₆H₅O₇ was used to identify a significant correlation between the effect of these variables on mycelial biomass production. The experimental values are in good agreement with predicted values, the correlation coefficient being 0.9880.     

Immunotherapy of EL4 lymphoma with reovirus

Summary EL4 lymphoma was grown as an ascitic tumor in the peritoneal cavity of C57Bl/6 mice. Animals with different tumor burdens (either 10⁷ or 10⁹ cells) were treated with a single intraperitoneal injection of BCNU using doses from 20–40 mg/kg. Response as measured by mean survival time and percent survival was dependent on tumor burden and dose of drug. The objective of chemotherapy was to increase the mean survival time, but not the percent survival, in order to evaluate the therapeutic effect of reovirus. Mice were given 10⁸, 10⁹, or 10¹⁰ Pfu of reovirus at various times with respect to chemotherapy. The number of mice cured after treatment with both BCNU and reovirus was significantly greater compared to mice treated with BCNU only. Mice cured with combination therapy developed tumor-specific immunity as measured by cytotoxic lymphocytes and serum, and resistance to a lethal tumor challenge. The Abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea; Saline: 0.9% NaCl solution; MEM: minimal essential medium; Pfu: plaque-forming units; FCS: fetal calf serum; BME: basal eagle's medium; SSC: sodium citrate-sodium chloride     

Muscular differentiation of chicken myotubes in a simple defined synthetic culture medium and in serum supplemented media: Expression of the molecular forms of acetylcholinesterase

We attempted to cultivate muscle cells from chick embryos in a serum-free, defined medium similar to that proposed by Bottenstein and Sato (1979) for the growth and differentiation of a murine neuronal cell-line. (1) We found that muscle cells from the legs of 11-day old chick embryos can be cultivated in a medium containing the different components indicated by Bottenstein and Sato, with 2 g/l bovine serum albumin, without serum or chick embryo extract. Myoblasts attached to the gelatin-coated dishes without any addition of attachment factors. They differentiated into myotubes in a similar manner as in classical serum supplemented media. (2) The level of cellular AchE activity was comparable in cultures grown in the presence of fetal calf serum (FCS), of horse serum (HS) and in the defined medium. The percentage of A₁₂ form was however higher in the defined medium (25–30%) than in FCS supplemented medium (about 5–6%). In HS supplemented medium the A₁₂ form was not detectable, partly because horse serum contains immunoglobulins which bind chicken AChE. The addition of defined medium components to FCS medium cultures did not lead to an increase of A₁₂. In contrast, the addition of a small amount (1%) of fetal calf serum to DM cultures reduced the level of A₁₂ in a drastic manner. FCS components therefore seem to repress the biosynthesis of A₁₂ AChE, or increase its degradation. (3) We estimated intracellular and extracellular compartments of AChE. The ratio of endocellular to ectocellular AChE decreased with the age of the cultures. The G₁ form was intracellular at all stages analyzed, but the other molecular forms were located in both cellular compartment, in different proportion: A₁₂ and G₄ seemed to be located preferentially in the external compartment, whereas G₂ was preferentially intracellular. (4) Muscle cultures grown in the defined medium and in the presence of serum secreted globular forms of AChE in a similar manner.     

Concanavalin A-mediated agglutinability of Balbc3T3 cells grown in media supplemented with different phosphatidylcholines

$\text{Balb}\text{c3T3}$ cells were grown near confluency in media supplemented with 10% fetal calf serum and than exposed for 24 hours to media containing different phosphatidylcholines bound to delipidated fetal calf serum. Compared to cells grown in regular media, 3T3 cells exposed to media containing dioleoyl-phosphatidylcholine dramatically increased their agglutinability by Concanavalin A. Exposure to several other phosphatidylcholines had no effect.     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B₂ (TXB₂) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 μg/g tissue) to both PGE₂ (10.7 μg/g tissue) and TXB₂ (6.2 μ/g tissue). Smaller amounts of PGF_2α (0.9 μ/g) and 6-oxoPGF_1α were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE₂, but formed very little TXB₂, PGF_2α or 6-oxoPGF_1α. Homogenates from rabbit lungs converted arachidonic acid (100 μg/g) mainly to PGE₂, both before and after birth. The amount of PGE₂ formed increased during gestation to a maximum of about 6 μg/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 μg/g) which was observed 8 days after birth, followed by an increase to about 4 μg/g in older rabbits.     

Chymotrypsin stimulated development of delayed implantation mouse blastocysts

Chymotrypsin-like enzyme activity increases transiently in the uterine lumen of ovariectomized mice upon administration of progesterone and estrogen (1). This is one of the few known macromolecular changes associated with conditions which result in activation of delayed implantation blastocysts $\text{in}$$\text{utero}$. $\text{In}$$\text{vitro}$, α-chymotrypsin (100 μg/ml) was found to shorten the time required for these embryos to attach to the glass culture dish and then form outgrowths in fetal calf serum-supplemented medium. Higher concentrations of the enzyme (250 μg/ml) prevented embryo attachment probably by digesting the fetuin present in fetal calf serum. Nevertheless, 250 μg/ml α-chymotrypsin could apparently replace fetal calf serum as a stimulator of development during the first 24 hours of culture. In contrast, bovine serum albumin (3.0 mg/ml) seemed to slow development of blastocysts $\text{in}$$\text{vitro}$. It is suggested that chymotrypsin-like enzyme activity may stimulate development of delayed implantation blastocysts $\text{in}$$\text{utero}$ (a) indirectly by removing inhibitory proteins such as albumin and (b) by directly affecting these embryos in a manner yet to be determined.     

Characterization of nuclear T3 receptors in human neuroblastoma cells SH-SY5Y: Effect of differentiation with sodium butyrate and nerve growth factor

Nuclear receptors for the thyroid hormone triiodothyronine (T₃) have been identified in vivo in brain tissues and in vitro in mouse and rat neuroblastoma and glioma cells. The present study characterizes nuclear T₃ receptors in human neuroblastoma SH-SY5Y cells and compares their levels before and after differentiation. Undifferentiated cells, grown in DME/HAM F-12 medium supplemented with 10% fetal calf serum, show an abundant single type of nuclear receptor, indicated by a straight Scatchard plot, with aK _d of 0.11 nmol/l. After treatment with sodium butyrate (0.5 mM for 4 days) or NGF (2 nM for 6 days), the cells showed neuronal-like patterns (extension of neurites, slowing of growth, increased tyrosine hydroxylase activity), with a decrease in the number of nuclear T₃ receptors. As sodium butyrate and NGF treatments differentiate neuroblastoma SH-SY5Y cells, these data suggest a down-regulation of T₃ receptors with cell maturation.     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum