Transport of cystine and cysteine and cell growth in cultured human diploid fibroblasts: effect of glutamate and homocysteate

Human diploid fibroblasts take up cystine in the culture medium and the cystine is immediately reduced to cysteine in the cells. It is found that cysteine thus formed is rapidly released from the cells into the medium and accumulates there. The system transporting cysteine is convincingly similar to the ASC system described by Christensen et al. (1967). Since cysteine in the medium is sensitive to autoxidation and readily changes back to cystine, the uptake of cystine seems crucial to the cells. Inhibitors of cystine uptake, such as glutamate and homocysteate, potently reduce the intracellular and extracellular levels of cysteine. These inhibitors modify the cell growth depending upon the cystine concentration is physiological. An excessive concentration of cystine is in itself inhibitory action is antagonized by glutamate or homocysteate.     

Regulation of glutathione levels in mouse spleen lymphocytes by transport of cysteine

Cysteine and cystine transport activities of resting and activated mouse spleen lymphocytes were characterized in order to examine the contributions of cysteine and cystine to intracellular glutathione contents. Following stimulation with lipopolysaccharide, the lymphocytes markedly increased their capacity to transport cysteine. The uptake of cysteine was mediated mainly by the ASC system (Na+-dependent neutral amino acid transport system especially reactive with alanine, serine, and cysteine). On the other hand, both the resting and the activated lymphocytes had extremely low cystine transport activities. Because of the instability of cysteine, the culture media usually contained cystine but not cysteine. Therefore, both the resting and the activated lymphocytes rapidly decreased their glutathione contents owing to their poor capacities to take up cystine. The effects of freshly added cysteine on the cellular glutathione contents were examined in the presence of bathocuproinedisulfonate, a nontoxic copper-specific chelator that inhibits autoxidation of cysteine. Cysteine added at 25-400 microM only partially prevented the rapid decrease of the glutathione contents in fresh resting lymphocytes. In the lipopolysaccharide-activated cells, however, cysteine enhanced the cellular glutathione contents in a dose-dependent manner. These results indicate that the enhanced activity of the ASC system increases the level of intracellular glutathione in the presence of cysteine.     

Cystine Uptake and Glutathione Level in Fetal Brain Cells in Primary Culture and in Suspension

Abstract— The glutathione level and the factors affecting this level were investigated in fetal rat brain cells in a primary culture. Early in the culture, the glutathione level of the brain cells decreased, but after 5 h it began to increase. This increase was not observed in a cystine-free medium and was prevented by excess glutamate. Cystine was taken up in freshly isolated brain cell suspensions, and its rate increased during the culture. The cystine uptake was mediated by a Na⁺-independent, glutamate-sensitive route previously found in various types of cells and designated as system x⁻_c. The uptake of cystine is a crucial factor in maintaining the glutathione level of the cells under culture, because it provides cysteine for the cells for glutathione synthesis. Cysteine was undetectable in the medium before the culture, but it appeared, though at a very low level, when the brain cells were cultured there. The source of this cysteine was the cystine in the medium. Presumably the decrease in the glutathione level of the cells in the early stage of the culture resulted from the fact that the medium did not contain cysteine. The enhancement of the cystine uptake during culture may constitute a protective mechanism against the oxidative stress to which the cultured cells are exposed. Regulation of the glutathione level in fetal brain cells in vivo by the transport of cystine and cysteine is discussed.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism

1. The ;initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the ;maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80-90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65-75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the ;low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the ;high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH. Studies with purified enzyme indicate that sulphur compounds do not interact directly with the enzyme, but that their effect is mediated by a number of other endogenous factors.     

Induction of cystine transport activity in human fibroblasts by oxygen

The transport activity for cystine in cultured human fibroblasts decreased after incubation of the cells under a low oxygen concentration. After the incubation for 48 h under 3% oxygen, the Vmax of the transport was decreased to less than one-third of that of the control cells, with little change in Km. The similar transport activity was observed in the cells cultured under 3% oxygen for 10-40 days with several times of passages. When these low oxygen-cultured cells were incubated under room air, the activity was enhanced with a lag of about 4 h and was almost completely restored within 24 h. This restoration required protein synthesis. The cystine transport activity increased by 50% after exposure of the cells to hyperoxia (40% oxygen). From these results it is concluded that the transport activity for cystine is induced by oxygen. In contrast, little change in the transport activities for alanine and leucine occurred in the cells exposed to the corresponding hypoxia or hyperoxia. Since the cystine transported into the cells is reduced to cysteine and the cysteine readily exits to the culture medium where it autoxidizes to cystine, a cystine-cysteine cycle across the plasma membrane has been postulated. Since the autoxidation of cysteine in the culture medium was markedly slowed down under the low oxygen concentration, the change in the cystine transport activity in response to the oxygen concentration was regarded as pertinent. Induction of the cystine transport activity may constitute a protective mechanism against the oxidative stress, to which the culture cells are exposed, by providing the cells with cysteine which is mainly incorporated into glutathione.     

Factors influencing the oxidation of cysteamine and other thiols: implications for hyperthermic sensitization and radiation protection

Some of the factors influencing the oxygen uptake and peroxide formation for cysteamine (MEA) and other thiols in serum-supplemented modified McCoy's 5A, a well-known medium used to cultivate a variety of cells in vitro, have been studied. The oxidation of MEA and cysteine in modified McCoy's 5A has been compared with that in Ham's F-12, MEM, and phosphate-buffered saline. All of the growth media were supplemented with 10% calf serum and 5% fetal calf serum. The rate of oxygen uptake for all of the studied thiols was greatest in McCoy's 5A. The data indicate that this medium may contain more copper than the other preparations. MEA and cysteine were found to be more effective at 0.4 mM at producing peroxide than dithiothreitol (DTT). N-acetylcysteine was the least reactive. The ability to produce peroxide is dependent upon the temperature, the concentration of thiol, the presence of copper ions, and pH of the medium. MEA and other thiol oxidation is inhibited by the copper chelator diethyldithiocarbamate. Catalase also reduces the oxygen uptake for all thiols. This inhibition involves the recycling of peroxide to oxygen. Superoxide dismutase (SOD) was found to stimulate the oxygen uptake in the case of MEA and cysteine, but had little or no effect with DTT and glutathione. The combined presence of SOD and catalase resulted in less inhibition of oxygen uptake than that obtained by catalase alone. Alkaline pH was found to enhance the oxidation of cysteine and MEA. An important observation was the inhibition of MEA oxidation at 0 degrees C and the stimulation at 42 degrees C. The results indicate that many problems may arise when thiols are added to various media. A major consideration is concerned with the production of peroxide, superoxide, and reduced trace metal intermediates. The presence of these intermediates may result in the production of hydroxyl radical intermediates as well as the eventual oxygen depletion from the medium. Oxygen depletion may alter the results of radiation sterilization and carcinogen activation. Radical production will cause cell damage that is temperature dependent. Therefore, careful consideration must be given to changes in oxygen tension when thiols are added to cells growing in complicated growth medium to protect against either chemical or radiation damage.     

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.     

Utilization of cystine by dermatophytes on glucose-peptone media

All the 16 strains of dermatophytes tested here metabolized cystine (3 mmol/L) in two glucose-peptone media with a different C: N ratio. Cystine was utilized as a sulfur source and, in addition, as a carbon and nitrogen source, in parallel with growth. Excess sulfur was excreted to the medium after its oxidation as inorganic sulfate and sulfite. In a physiologically alkaline medium the growth was fast and was accompanied by a pH increase and cystine was utilized intensively. Eleven species used up all cystine available. Sulfate was the main oxidation product, sulfite was produced at a low concentration, at the beginning of growth in particular. Only traces of thiol compounds (cysteine) were present in the medium. In a physiologically acid medium growth was soon limited by a decreased pH (below 5.0) but cystine continued to be utilized at an identical rate. All cystine was used up by 5 species. The tendency to produce sulfite in addition to sulfate further increased and sulfite was often the predominant product. Concentrations of thiol compounds were also substantially higher. Thus, dermatophytes can utilize cystine even under conditions that do not support good growth and increase the sulfite production.     

Cystine/cysteine metabolism in cultured Sf9 cells: influence of cell physiology on biosynthesis, amino acid uptake and growth

Spodoptera frugiperda (Sf9) insect cells proliferate in a cystine-free medium, with the same growth rate, reaching the same final cell density, as in a cystine-containing medium, provided that the inoculum is taken from a pre-culture sufficiently early, at 47–53 h. With an inoculum from a 103 h culture an extended lag phase accompanied by cell death was observed during the first 50 h of cystine-free culture, even though the culture had been adapted to cystine-free conditions for 10 passages. Cystine-free cultures seeded with a 103 h inoculum had lower growth rates and reached lower final cell densities than corresponding cystine-supplied cultures. Cysteine biosynthesis occurs from methionine via the β-cystathionine pathway. More methionine was consumed by the cells in cystine-free media, and cystathionine was secreted when methionine and cystine were supplied in excess. The data suggest that cysteine biosynthesis is up-regulated in proliferating cells but down-regulated when the cells enter the stationary phase. In cultures supplied with cystine (10–100 mg 1^-1), the specific uptake rate and total consumption of cystine, as well as the uptake of glutamate, glutamine and glucose increased with increasing cystine concentrations. These results are interpreted in view of system x _c ^– , a concentration dependent amino acid transporter. Similarly, the consumption of amino acids transported by system L (ile, leu, val, tyr) was enhanced in cystine-containing cultures, as compared to cystine-free cultures. Uptake of cystine, methionine and system L amino acids ceases abruptly in all cultures, even before growth ceased. The specific growth rate starts to decline early during the growth phase, but this growth behaviour could not be correlated to the depletion of nutrients. We therefore propose that the observed growth pattern is a result of (auto)regulatory events that control both proliferation and metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Redox imbalance in cystine/glutamate transporter-deficient mice

Cystine/glutamate transporter, designated as system x(-)(c), mediates cystine entry in exchange for intracellular glutamate in mammalian cells. This transporter consists of two protein components, xCT and 4F2 heavy chain, and the former is predicted to mediate the transport activity. This transporter plays a pivotal role for maintaining the intracellular GSH levels and extracellular cystine/cysteine redox balance in cultured cells. To clarify the physiological roles of this transporter in vivo, we generated and characterized mice lacking xCT. The xCT(-/-) mice were healthy in appearance and fertile. However, cystine concentration in plasma was significantly higher in these mice, compared with that in the littermate xCT(-/-) mice, while there was no significant difference in plasma cysteine concentration. Plasma GSH level in xCT(-/-) mice was lower than that in the xCT(-/-) mice. The embryonic fibroblasts derived from xCT(-/-) mice failed to survive in routine culture medium, and 2-mercaptoethanol was required for survival and growth. When 2-mercaptoethanol was removed from the culture medium, cysteine and GSH in these cells dramatically decreased, and cells started to die within 24 h. N-Acetyl cysteine also rescued xCT(-/-)-derived cells and permitted growth. These results demonstrate that system x(-)(c) contributes to maintaining the plasma redox balance in vivo but is dispensable in mammalian development, although it is vitally important to cells in vitro.     

Identification of cell culture conditions to control protein aggregation of IgG fusion proteins expressed in Chinese hamster ovary cells

The presence of aggregated forms of proteins can be problematic for therapeutics due to the potential for immunogenic and pharmacokinetic issues. Although downstream processing can remove the aggregated forms, inhibiting aggregate formation upstream during the cell culture stage could reduce the burden on downstream processing and potentially improve process yields. This study first examined the effects of environmental factors (temperature, pH, and dissolved oxygen) and medium components (bivalent copper ion, cysteine, and cystine) on the aggregation of two different recombinant fusion proteins expressed by Chinese hamster ovary (CHO) cells. Any strategy to reduce protein aggregation upstream during cell culture must also consider potential effects on critical upstream parameters such as cell growth, harvest titer, and protein sialylation levels. Manipulating the culture temperature shift and cystine concentration in the medium were both identified as effective and practical strategies for reducing protein aggregation in both CHO-cell expression systems. Furthermore, a combination of both strategies was more effective in reducing protein aggregation levels compared to either approach individually; and without any negative effects on harvest titer and protein sialylation. This study demonstrates a practical methodology for decreasing protein aggregation during upstream processing and emphasizes the importance of process understanding to ensure production of recombinant glycoprotein therapeutics with consistent product quality.     

Effect of amino acids containing sulfur on dithiolopyrrolone antibiotic productions by Saccharothrix algeriensis NRRL B-24137

AIMS: To study the effect of sulfur-containing amino acids (L-cysteine, L-cystine, L-methionine and DL-ethionine) on the production of dithiolopyrrolone antibiotics by Saccharothrix algeriensis NRRL B-24137. METHODS AND RESULTS: The production levels of dithiolopyrrolones were investigated by using high performance liquid chromatography in a chemically semi-synthetic medium. The production of the studied antibiotics depends upon the nature, concentration and the time of addition of these sources in the culture medium. Both cysteine and cystine favoured the specific productions of dithiolopyrrolones; iso-butyryl-pyrrothine (ISP) by cysteine, however butanoyl-pyrrothine, senecioyl-pyrrothine and tigloyl-pyrrothine by cystine, when added initially to the culture medium. The maximum specific productions of dithiolopyrrolones were observed in the presence of 5 mmol l(-1) cystine for thiolutin, 5 mmol l(-1) cysteine for ISP, and 10 mmol l(-1) cystine for others studied dithiolopyrrolones as shown in Fig. 3. The production of these antibiotics was decreased when the concentrations of cysteine and cystine were in excess. All dithiolopyrrolone specific productions were strongly inhibited by addition of methionine and ethionine, without inhibition of mycelial growth. CONCLUSIONS: Among all studied amino acids, cystine and cysteine can be used as supplements for improvement the production of dithiolopyrrolone antibiotics by S. algeriensis NRRL B-24137. SIGNIFICANCE AND IMPACT OF THE STUDY: Dithiolopyrrolone antibiotics have many important applications for employing them as medicaments, particularly in the treatment of human and animal cancers. In the present work, the influence of containing-sulfur amino acids on dithiolopyrrolone antibiotic productions was studied. The obtained results can be employed for the optimization of the culture medium for the dithiolopyrrolone productions in higher quantities.     

Full replacement of 2-mercaptoethanol by cysteine plus selenium compounds in augmenting DNA synthesis of mitogen-stimulated mouse spleen lymphocytes

Mouse spleen lymphocytes require 2-mercaptoethanol for maximal mitogenic activation in vitro. Previous studies indicate that the lymphocytes are defective in the cystine transport activity and that they require 2-mercaptoethanol to utilize cystine. 2-Mercaptoethanol catalytically carries cysteine moiety into the cells in a mixed disulfide form. Because cysteine is easily oxidized to cysteine in the culture medium, it has been not easy to precisely examine the effect of near-physiological concentrations of cysteine on the activation of lymphocytes. By controlling the cysteine content in the medium, we have reviewed the effect of cysteine to see if cysteine replaces 2-mercaptoethanol in enhancing the DNA synthesis of lipopolysaccharide-stimulated lymphocytes. It was found that cysteine was less effective than 2-mercaptoethanol, and that cysteine fully replaced 2-mercaptoethanol when a selenium compound was supplemented. The effects of cysteine and selenium compounds were apparently independent and additive. Among the selenium compounds examined, sodium selenite and L-selenocystine were much more effective in stimulating DNA synthesis than sodium selenate and L-selenomethionine.     

Mouse lymphoma L1210 cells acquire a new cystine transport activity upon adaptation in vitro

Summary Mouse lymphoma L1210 cells maintained in vitro at a high cell density for a certain time period adapted themselves to the in vitro environment and were able to grow indefinitely. From these adapted cells, more than 30 clones were isolated. They all had much higher activity to take up cystine than the original L1210 cells, supporting a previous view that the deficiency of the cystine uptake limits the survival and growth of L1210 cells in vitro. The cystine uptake of one cloned cell line was characterized. The enhanced uptake of cystine in these cells was mainly mediated by a Na⁺-independent, saturable system and was potently inhibited by glutamate and some other anionic amino acids, but less by aspartate. Such activity of cystine uptake was not observed in the original L1210 cells. The results suggest that, upon adaptation in vitro, L1210 cells acquire a new cystine transport activity necessary for survival and growth in vitro.     

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.     

NGF promotes copper accumulation required for optimum neurite outgrowth and protein methylation

The role of copper in biological phenomena that involve signal transduction is poorly understood. A well-defined cellular model of neuronal differentiation has been utilized to examine the requirement for copper during nerve growth factor (NGF) signal transduction that results in neurite outgrowth. Experiments demonstrate that NGF increases cellular copper content within 3 days of treatment. Copper chelators reduce the effects of NGF on neurite outgrowth and copper accumulation. The effects of tetraethylene pentamine (TEPA), a copper-specific chelator, are reversible by removal from the culture medium and/or by addition of equimolar copper chloride. Because previous work demonstrated that NGF increases protein methylation in PC12 cells, we examined whether TEPA also inhibits S-adenosylhomocysteine hydrolase (SAHH), an essential copper enzyme involved in all protein methylation reactions. In addition to direct in vitro inhibition of SAHH, we show that TEPA decreases protein arginine methyltransferase 1(PRMT1)-specific enzyme activity in PC12 cells and sympathetic neurons. These data comprise the first biochemical and cellular evidence to address the mechanism of copper involvement in neuronal differentiation.     

Cell death by pyruvate deficiency in proliferative cultured calvarial osteoblasts

Cell survival was significantly decreased in primary cultured rat calvarial osteoblasts in vitro at Day 0, 1, and 3 by replacement of the standard culture medium (alpha-modified minimum essential medium; alpha-MEM) with Dulbecco's modified eagle's medium (DMEM). Decreased cell survival was also observed following medium replacement in cultures of murine calvaria-derived osteoblastic cell line MC3T3-E1. Staining with Hoechst33342 revealed apoptotic cells with fragmented or condensed nuclei, while a fraction of the cell culture was stained with propidum iodide, indicating necrosis. Marked increases in DNA binding of both activator protein-1 and nuclear factor-kappaB were found in nuclear extracts of cells following medium replacement. The addition of either pyruvate or cysteine at each concentration found in alpha-MEM almost entirely prevented cell death associated with medium replacement at Day 3. These results suggest that pyruvate and cysteine may be essential factors for cell growth and survival in osteoblast cultures at the proliferative phase.     

A novel function of glutamine in cell culture: utilization of glutamine for the uptake of cystine in human fibroblasts

Transport and metabolism of glutamine has been investigated in human diploid fibroblasts, IMR-90. Glutamine was taken up via System ASC (Na+-dependent amino acid transport system especially reactive with short or polar side chain amino acids). In the routine culture medium the cells contained a large quantity of glutamate; its major source was shown to be glutamine in the medium. Previously we described a transport system that mediates the entrance of cystine in exchange for the exit of glutamate (Bannai, 1986). Since the cystine taken up is reduced to cysteine and the cysteine readily exits to the medium where it is oxidized to cystine, a cystine-cysteine cycle across the plasma membrane has been postulated. When the cells were cultured in glutamate/glutamine-free medium, intracellular glutamate decreased, depending on the amount of cystine in the medium; in the absence of cystine, glutamate decreased very slowly. When the cells were cultured in ordinary medium, glutamine in the medium decreased, and glutamate in the medium increased. Both changes were well correlated with cystine concentration in the medium. These results are consistent with the view that the intracellular glutamate, of which the source is glutamine in the medium, is released from the cells into the medium in order to take up cystine and thereby to rotate the cystine-cysteine cycle. In the routine culture one-third to one-half of the total consumption of glutamine seems to be used for the uptake of cystine.     

Use of 2-mercaptoethanol in cell culture]

Survival and growth in in vitro cultivation of lymphocytes, lymphoma cells and some other cells including human carcinomas are profoundly improved by 2-mercaptoethanol. These cells hardly take up cystine, an essential nutrient in the culture medium, but in the presence of 2-mercaptoethanol they can utilize cystine. Recently it has been found that 2-mercaptoethanol is effective in the in vitro cultivation of pathogenic trypanosomes and in the in vitro development of bovine embryos. The mechanisms by which 2-mercaptoethanol improves the survival and growth of these cells are described.     

Unique characteristics of rat spleen lymphocyte, L1210 lymphoma and HeLa cells in glutathione biosynthesis from sulfur-containing amino acids

Suspensions of rat spleen lymphocyte, murine L1210 lymphoma and HeLa cells were partially depleted of glutathione (GSH) with diethyl maleate and allowed to utilize either [35S]methionine, [35S]cystine or [35S]-cysteine for GSH synthesis. Lymphocytes preferentially utilized cysteine, compared to cystine, at a ratio of about 30 to 1, which was not related to differences in the extent of amino acid uptake. Only HeLa cells displayed a slight utilization of methionine via the cystathionine pathway for cysteine and GSH biosynthesis. HeLa and L1210 cells readily utilized either cystine or cysteine for GSH synthesis. The three cell types accumulated detectable levels of intracellular cysteine glutathione mixed disulfide when incubated in a medium containing a high concentration of cystine. Various enzyme activities were measured including gamma-glutamyl transpeptidase, GSH S-transferase and gamma-cystathionase. These results support the concept of a dynamic interorgan relationship of GSH to plasma cyst(e)ine that may have importance for growth of various cell types in vivo.