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PubMed2004

Effect of cystine on the methionine requirement of healthy Indian men determined by using the 24-h indicator amino acid balance approach.

BACKGROUND: The 1985 FAO/WHO/UNU requirement for methionine in healthy adults consuming a cystine-free diet is 13 mg.kg(-1).d(-1). It is unclear whether this daily requirement is influenced by dietary cystine. OBJECTIVE: We assessed the effect of 2 intakes of cystine (5 and 12 mg.kg(-1).d(-1)) on methionine requirements in well-nourished Indian men by using 7 test methionine intakes (3, 6, 9, 13, 18, 21 and 24 mg.kg(-1).d(-1)) and the 24-h indicator amino acid oxidation (24-h IAAO) and balance (24-h IAAB) methods. We combined these data with those from an experiment with zero cystine intake and in which the exact same method was used. DESIGN: Two studies were performed in which a diet containing either 5 or 12 mg cystine.kg(-1).d(-1) was fed to 21 well-nourished Indian men over three 7-d periods. The 24-h IAAO and 24-h IAAB values were measured on day 7 with the use of a 24-h intravenous [13C]leucine tracer infusion. The breakpoints in the relation between these values and methionine intake in each study were assessed by two-phase linear regression. RESULTS: Breakpoints in the response curve were obtained at methionine intakes of 20 (95% Fiellers CI: 17, 26) and 10 (95% Fiellers CI: 8, 16) mg.kg(-1).d(-1) with cystine intakes of 5 and 12 mg.kg(-1).d(-1) intakes, respectively, which suggested a sparing effect of cystine. Although the 5- and 12-mg cystine breakpoints differed from one another, they did not differ significantly from that estimated previously with 0 mg cystine. CONCLUSION: Cystine may spare the methionine requirement in healthy men, although the amount of sparing is difficult to quantify.

Adult↗
PubMed2002

Enhancement of L-cystine transport activity and its relation to xCT gene induction at the blood-brain barrier by diethyl maleate treatment.

The purpose of the present study was to elucidate the mechanism of enhancement of L-cystine uptake at the blood-brain barrier (BBB). The uptake of [(14)C]L-cystine and [(3)H]L-glutamic acid (L-Glu) was determined using a mouse brain endothelial cell line (MBEC4) as an in vitro BBB model. The mRNA levels of L-cystine/L-Glu exchanger, system x(c)(-), which consists of xCT and 4F2hc, were determined by quantitative real-time reverse transcription-polymerase chain reaction analysis. The [(14)C]L-cystine uptake by MBEC4 cells appeared to be mediated via an Na(+)-independent saturable process. The corresponding Michaelis-Menten constant (K(m)) was 63.7 microM. In the presence of L-Glu, there was competitive inhibition with an inhibition constant (K(i)) of 83.5 microM. [(3)H]L-Glu uptake in the absence of Na(+) was saturable with a K(m) of 48.1 microM, and it exhibited competitive inhibition with a K(i) of 24.9 microM in the presence of L-cystine. The mutual inhibition between L-cystine and L-Glu and the type of inhibition suggest that system x(c)(-) operates in MBEC4 cells. The xCT and 4F2hc mRNAs were expressed in MBEC4 cells and, following diethyl maleate (DEM) treatment, the xCT mRNA level and L-cystine uptake in MBEC4 cells were enhanced in parallel with an increase in DEM concentration (up to 500 microM). Concomitantly, the glutathione concentration in MBEC4 cells was increased. In conclusion, system x(c)(-)-mediated L-cystine uptake takes place in MBEC4 cells. L-Cystine transport via system x(c)(-) at the BBB is likely to be induced under oxidative stress conditions following DEM treatment due to enhanced transcription of the xCT gene.

Algorithms↗
PubMed1989

Cystine and lysine reabsorption in the isolated perfused rat kidney.

Renal tubular reabsorption of cystine and lysine were studied in the isolated perfused rat kidney to bridge the gap between in vivo clearance studies, and in vitro transport studies of tubule fragments, cells, and brush-border membranes. Lysine was reabsorped by a saturable transport system shared by the dibasics. Cystine was also reabsorbed by a saturable transport system, which was shared in part by the dibasics (maximum inhibition 30%). The lysine threshold (Fmin) was 0.9 mumol.min-1.g-1, with a tubular maximum (TM) of 2.4 mumol.min-1.g-1. The cystine Fmin was 0.06 mumol.min-1.g-1; the TM could not be estimated because it was above the limit of cystine solubility. There was no evidence of cystine "secretion." The gamma-glutamyltransferase inhibitor, AT-125, decreased cystine excretion, but only in the presence of glutathione, glycine, glutamate, and the diabasic amino acids. This suggests that cystine from glutathione degradation at the brush border may contribute to urinary cystine (an explanation of the phenomenon of cystine secretion), but only under certain conditions.

Amino Acids↗
PubMed1984

Pantethine and cystamine deplete cystine from cystinotic fibroblasts via efflux of cysteamine-cysteine mixed disulfide.

Children suffering from cystinosis, a genetic disease characterized by high levels of lysosomal cystine, are currently being treated with cysteamine to lower the cystine levels in their cells. In fibroblasts from these patients, cysteamine and its disulfide, cystamine, are equally effective in lowering cystine levels. We recently reported that pantethine, a dietary precursor of coenzyme A, depletes cystine from cultured, cystinotic fibroblasts as effectively as cystamine. To determine the mechanism of action of pantethine, and of cystamine, we have compared the fate of [35S]cystine-derived metabolites in the presence and absence of these agents. The results indicate that the ability of pantethine to deplete cystine resides in its being a metabolic precursor of cysteamine. Furthermore, both pantethine and cystamine act by generating the mixed disulfide of cysteamine and cysteine in the lysosomes, which is then rapidly excreted from the cells. The fall in intracellular [35S]cystine caused by these agents was not accompanied by a comparable increase in any intracellular metabolite; rather, it could be accounted for by the appearance of mixed disulfide in the medium. There was no accumulation of mixed disulfide in the cells. Radioactivity in cytoplasmic glutathione was, however, increased by cystamine or pantethine. Thus, cysteamine (formed intracellularly in these experiments) undergoes thiol-disulfide exchange with cystine in the lysosomes, producing cysteamine-cysteine mixed disulfide and free cysteine, which enter the cytoplasm. The free cysteine is available to several pathways, including oxidation to the disulfide or the mixed disulfide, and synthesis of glutathione. The mixed disulfide is excreted from the cell, which ultimately depletes the cell of its excess cystine.

Cells, Cultured↗
PubMed1976

Transport of L-cystine by cultivated skin fibroblasts of normal subjects and patients with cystinosis.

Uptake of L-cystine at the plasma membrane of fibroblasts derived from normal and cystinotic subjects was studied. L-Cystine accumulation after a 20-min period was increased in cystinotic fibroblasts incubated in 0.08 mM L-cystine. This effect appeared to be concentration-dependent since accumulation after 20 min at 0.004 mM concentration was decreased in cystinotic cells. Kinetic data suggested that at least two nondiffusional saturable processes with widely different substrate affinities mediate initial L-cystine uptake in skin fibroblasts. In addition, the transport process with high affinity for L-cystine may itself be a two-component system, as suggested by (1) additive inhibitory effect of other neutral amino acids, and (2) preincubation studies in which preincubation with cystathionine enhanced subsequent L-cystine uptake, whereas preincubation with other neutral amino acids depressed subsequent uptake. Affinity constants and maximal velocities of initial uptake did not appear to be altered in cells derived from patients with cystinosis. After 60-sec incubation with L-[35S] cystine, cystinotic cells retained more label as cystine than did normal cells at each concentration studied. These data indicate that initial L-cystine uptakein fibroblasts of patients with cystinosis proceeds at a normal rate by means of all transport systems currently shown to be present in normal cells.

Amino Acids↗
PubMed1979

Elevated cystine levels in cultured skin fibroblasts from patients with I-cell disease.

Cultured skin fibroblasts from patients with I-cell disease (mucolipidosis II) exhibit multiple deficiency of acid hydrolase activities associated with a defect in the mechanism of packaging of these enzymes into lysosomes. The authors have examined such cells to ascertain whether the impairment of lysosomal function is of so broad a nature as to result in the storage of the amino acid cystine in a manner similar to that seen in cells derived from patients with cystinosis, an unrelated lysosomal storage disease of unknown etiology. Of 10 I-cell lines examined by automated amino acid analysis, seven were found to possess abnormally high levels of total free cyst(e)ine (i.e., greater than 1 nmole 1/2 Cys/mg protein). The mean half-cystine content of those I-cell lines subjected to multiple analysis ranged from 3-10 nmole/mg protein. levels which are comparable to those seen in homozygous cystinotic cells. The cystine content of several of these lines appeared to increase with subculture. Cultured fibroblasts from two patients with the biochemically similar, but clinically less severe, mucolipidosis III (pseudo-Hurler polydystrophy) exhibited normal to marginally elevated levels of cystine, whereas cells from individuals with three different mucopolysaccharide storage disorders contained normal levels of the amino acid. It was concluded that cystine, and not cysteine, was the predominant form of this amino acid in these cells, because previous reaction of I-cell extracts with N-ethylmaleimide did not alter the observed cystine levels. The further identification of excess cystine in these cells was corroborated by analytical results obtained with a highly specific cystine-binding protein method as well as by high-voltage electrophoresis of extracts from cells pulsed with 35S-cystine. Comparative analysis of intracellular amino acids in normal and I-cell fibroblasts indicated that the elevation of csystine seen in the latter was unique to this amino acid and did not reflect a generalized increase in the total free amino acid content of these mutant cells.

Cell Line↗
PubMed1986

Developmental aspects of cystine transport in the dog.

Developmental changes in cystine transport by the canine kidney were examined both in vivo and in vitro. Renal clearance studies indicated that cystine was one of the more incompletely reabsorbed amino acids at birth, but its reabsorption approaches adult levels by 21 days. Concomitantly, cystine uptake by isolated renal cortical tubule fragments from immature dogs was slower than that by renal tubules from adult dogs. Both age groups rapidly metabolized the transported cystine. This metabolism was principally to cysteine, but also small amounts of reduced glutathione were formed from the transported cystine. Concentration dependence studies indicated two transport systems for cystine uptake in both the immature and the adult dog. Both transport systems in the 1-wk-old dog had a somewhat greater affinity for cystine than the corresponding system in the adult, but this was offset by the markedly lower maximal transport rates for these systems in the 1-wk-old dog. The high affinity system was inhibited by lysine in tubules from both age groups. In the dog, the rise in the tubular reabsorption of cystine with maturation could, in part, be explained by an increase in the number of transport sites for cystine.

Animals↗
PubMed1987

Cystine fluxes across the isolated jejunal epithelium in cystinuria: increased efflux permeability at the luminal membrane.

In cystinuria, renal clearance of cystine frequently exceeds creatinine clearance, suggesting net cystine secretion; and absorption of the (di)basic amino acid is impaired at the luminal membrane of the jejunal and probably also renal tubular epithelium. We studied cystine transport in vitro in jejunal biopsy specimens of eight subjects with homozygous cystinuria and in 12 controls. Cellular/medium cystine distribution ratio was reduced in cystinuria (1.36 +/- 0.36 versus 5.36 +/- 0.61, p less than 0.001). Cystine influx across the luminal membrane was normal (221 +/- 48 versus 261 +/- 79 pmol X h-1 cm-2). Measurement of transepithelial cystine fluxes showed net absorption in controls but secretion in cystinuria. Apparent permeability coefficients were close to normal in cystinuria except that the efflux permeability at the luminal membrane was significantly increased (0.839 +/- 0.22 versus 0.186 +/- 0.12 X h-1 cm-2), and, accordingly, at the luminal membrane, the influx over efflux permeability ratio was small (1.01 +/- 0.50 versus 4.95 +/- 0.80, p less than 0.001). The defect in cystine transport in cystinuria is apparently not caused by decreased influx but increased efflux of cystine (or cystine) from the cell to the lumen across the luminal membrane.

Adult↗
PubMed1980

Influence of dietary cystine on intestinal absorption and tissue distribution of methionine in the chick.

Studies were conducted on intestinal absorption and tissue distribution of methionine by chicks fed a crystalline amino acid diet containing .2% DL-methionine and .2 or .4% L-cystine or L-cysteine. Chicks fed the higher level of cystine or cysteine had previously been shown to exhibit depressed growth as opposed to chicks fed the lower level of these amino acids. In two experiments, 35S radioactivity of whole plasma and the protein and supernatant fractions were compared among chicks fed the different treatments at 30 or 60 min after oral intubation of L-[35S]-methionine into the crops. When the data were analyzed factorially, a significant level effect was noted for whole plasma and the protein and supernatant fractions, all of which were higher for chicks fed .2% L-cystine than those fed .4% L-cystine. Results with chicks fed the two levels of L-cysteine generally agreed with those of chicks fed L-cystine, although the differences were not as great. The labeled methionine was also added to diets containing .2 or .4% L-cystine of meal-fed chicks. Higher levels of radioactivity were observed in the liver but not in muscle or plasma of chicks fed the lower level of cystine at 7 hr after oral consumption of the isotope. No significant differences in liver, muscle, or plasma radioactivity were noted in a fourth experiment between chicks fed .2 or .4% L-cystine at 24 hr after intraperitoneal injection of L-[35S]-methionine. In vitro studies showed no differences in methionine accumulation by isolated intestinal segments incubated with three levels of cystine.

Animals↗
PubMed1990

Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake.

Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.

Animals↗
PubMed1980

Hydrolysis of some cystine amniopeptidase and aminopeptidase substrates by human placental enzymes after isoelectric focusing.

Several cystine aminopeptidase- and aminopeptidaselike activities of mature human placenta were demonstrated by chromatography on CM-Sephadex and subsequent preparative flat bed isoelectric focusing. Enzyme tests were carried out by using a micro modification of the Bratton-Marshall reaction with seven amino acid-rho-nitroanilides and one amino acid beta-naphthylamide as substrates. By this method three main activities were detected; each was nonhomogeneous and capable of hydrolyzing cystine aminopeptidase- as well as aminopeptidase substrates. The substrates benzyl-cystine-rho-nitroanilide, cystine di-rho-nitro-anilide and cystine di-beta-naphthylamide were split by different enzymes. By studying the effects of several effectors or of heating, no enzyme with the properties described in the literature for serum cystine aminopeptidase could be unequivocally demonstrated. The results report here suggest that serum cystine aminopeptidase--in contrast to placenta cystine aminopeptidase--is either altered severely in its structure or that it represents a multiplicity of enzymes, which attack "specific" substrates and are therefore jointly manifested as cystine aminopeptidase.

Aminopeptidases↗
PubMed1963

L-CYSTINE REQUIREMENT FOR PRODUCTION OF COXSACKIE B3 VIRUS IN CULTURED MONKEY HEART CELLS.

Tyndall, R. L. (The Pennsylvania State University, University Park) and E. H. Ludwig. l-Cystine requirement for production of Coxsackie B3 virus in cultured monkey heart cells. J. Bacteriol. 85:1339-1345. 1963.-The requirement for l-cystine in both the production of Coxsackie B3 virus in cultured monkey heart cells and in prolonging the survival of the cultured monkey heart cells is demonstrated. d-Cystine, dl-homocystine, dl-allo-cystathionine, dl-homocysteine thiolactone, 2-mercaptoethlyamine, allylglycine, 3,3'-dithiopropionic acid, and dl-ethionine could not replace l-cystine in either supporting Coxsackie B3 virus production or prolonging monkey heart cell culture survival time. By their ineffectiveness, these compounds suggest the specificity of the l-cystine requirement. Allylglycine, 3,3'-dithiopropionic acid, and dl-ethionine were likewise incapable of inhibiting the l-cystine effect in supporting both virus production and cell survival. Prolonged starvation of cell cultures prior to virus inoculation failed to reveal any additional marked nutritional requirements, but rather tended to accentuate the l-cystine requirement for virus production. Increased cell starvation did, however, lead to the establishment of a latent Coxsackie B3 virus infection of cultured monkey heart cells, the activation of which is l-cystine-dependent.

Amino Acids↗
PubMed2005

Prenatal diagnosis of cystinosis by quantitative measurement of cystine in chorionic villi and cultured cells.

OBJECTIVE: Prenatal diagnosis of cystinosis has been available for over 30 years by the incubation of cultured amniotic cells, intact chorionic villi and cultured chorionic cells with [35S]-cystine followed by thin layer chromatography and visual inspection of autoradiographs of the chromatograms for cystine. This method has proved highly reliable but because of the short half-life of [35S]-cystine, its cost and the length of the assay procedure, an alternative method of diagnosis was investigated. METHOD: Cystine was quantitatively measured in chorionic villi directly, in cultured chorionic villi and cultured amniotic cells using a cystine-binding protein from Escherichia coli. RESULTS: Twelve pregnancies at risk for cystinosis were monitored by both the [35S]-cystine uptake method and the new quantitative method in uncultured chorionic villi. There was no discrepancy between the results obtained with the two methods and subsequently 15 pregnancies have been monitored by the quantitative assay only--13 in chorionic villi directly, 1 in cultured chorionic villi cells and 1 in cultured amniotic cells. Grossly elevated levels of cystine were found in seven pregnancies. CONCLUSION: An unequivocal diagnosis of cystinosis can be made within 24 h of sampling by the quantitative measurement of cystine in uncultured chorionic villi.

Abortion, Eugenic↗
PubMed1995

Measurement of diurnal variations in urinary cystine saturation.

In an attempt to improve the diagnostic value of urine analysis in patients with homozygous cystinuria, we studied the diurnal variation in urine composition. A simplified estimate of the ion-activity product of cystine was used to increase the probability of identifying patients with a particular risk of stone formation. Eight 6-h urine samples were collected during two 24-h periods. The highest urinary excretion of cystine was recorded between 1200 and 1800 hours and the lowest between 0000 and 0600 hours, whereas the urinary cystine concentration was highest between 0000 and 0006 hours and lowest between 1200 and 1800 hours. The approximate ion-activity product of cystine had a maximal level between 0000 and 0600 hours but a minimal level between 0600 and 1200 hours. The differences between different periods were numerically more pronounced in terms of the ion-activity product of cystine than in terms of concentration. The peak concentrations of cystine in 6-h samples were about 90% higher than the corresponding concentrations in 24-h urine samples. It is concluded that the analysis of cystine in 6-h urine samples reveals transient episodes of cystine supersaturation that otherwise will remain undetected. Further studies are, however, needed to establish its usefulness in clinical practice.

Adult↗
PubMed1982

Utilization of L- and DL-cystine by the fungus Microsporum gypseum.

Growth of the fungus Microsporum gypseum and utilization of cystine during this growth was studied in a glucose-arginine medium containing either sodium sulphate, and L-cystine or DL-cystine. Replacement of sulphate with L-cystine brought about no significant changes in the growth of the microorganism. Utilization of L-cystine as a source of carbon and nitrogen was rapid and complete and excess sulphur was excreted into the medium in the form of sulphate. Similarly excreted were also minute amounts of sulphite which immediately reacted with the remaining cystine to form S. sulphocysteine. Growth of M. gypseum in a medium with DL-cystine was slow. Although this substance was not utilized as readily as L-cystine, its utilization was still complete and excess sulphur was similarly excreted in the form of sulphate and sulphite. The initial step in the utilization of the D-isomer is probably its extracellular deamination.

Culture Media↗
PubMed2005

BspA (CyuC) in Lactobacillus fermentum BR11 is a highly expressed high-affinity L-cystine-binding protein.

The BspA protein of Lactobacillus fermentum BR11 (BR11) is a cell envelope constituent that is similar to known solute-binding proteins and putative adhesins. BspA is required for L-cystine uptake and oxidative defense and is likely to be an L-cystine-binding protein. The aim of this study was to directly measure L-cystine-BspA binding and BspA expression. De-energized BR11 cells bound radiolabelled L-cystine with a Kd of 0.2 microM. A bspA mutant could not bind L-cystine. L-cystine-BR11 binding was unaffected by large excesses of L-glutamine, L-methionine, or collagen, indicating L-cystine specificity. BR11 and the bspA mutant were identical in their abilities to bind L-cysteine, indicating that L-cysteine is not a BspA ligand. BspA expression levels were deduced from radiolabelled L-cystine binding and it was found that there are 1-2 x 10(5) BspA molecules per cell, and that expression is slightly higher under oxidizing conditions. It is proposed that BspA be renamed CyuC.

Bacterial Proteins↗
PubMed1986

Further studies on the effect of chloroquine on the uptake, metabolism and intracellular translocation of [35S]cystine in cystinotic fibroblasts.

The present study uses the lysosomotropic drug chloroquine to investigate the mechanisms by which exogenous [35S]cystine is able to label the intracellular (intralysosomal) cystine pool(s) in cystinotic fibroblasts. When cystinotic fibroblasts were labelled for short periods of time (8 h or less), chloroquine (20 microM) inhibited the labelling of the intracellular cystine pool(s). However, when the cells were labelled for longer periods of time (24 h or more) chloroquine stimulated the labelling of the intracellular cystine pool(s). The short-term effect was selectively abolished when the cells were washed free of chloroquine, while the long-term effect was selectively abolished when the medium was depleted of cystine. Two routes of translocation of exogenous cystine to the lysosomes could be defined. One route was fast, had a low capacity, was inhibited by chloroquine and increased with increasing medium pH, while the other route was slow, had a high capacity, was stimulated by chloroquine and was more active at low pH. The former pathway probably consisted of plasma membrane transport of cystine into the cytosol followed by direct or indirect transport into the lysosomes. The latter route possibly consisted of pinocytosis with fusion of the cystine-containing pinosomes with lysosomes.

Biological Transport↗
PubMed1989

The effects of decreased growth temperature on the cystine content of cystinotic fibroblasts.

Cystinotic fibroblasts transferred from 37 degrees C to 28 degrees C accumulated additional cystine over the period from 4 to 7 days of incubation at 28 degrees C, after which the additional cystine was lost; warming (to 37 degrees C) of cells with elevated cystine stores led to rapid cystine loss. These results, taken together with previously published data showing cystine release from cystinotic fibroblasts incubated at above-normal temperature, are interpreted as indicating the presence in the cystinotic fibroblast lysosome membrane of a cystine-porter whose efficacy is increased by an increase in membrane fluidity. This porter may be the residual activity of the cystine porter that is known to be deficient in cystinosis, or it may be a second as yet unrecognized porter. It is further proposed that this porter is responsible for the presumed efflux of cystine from cystinotic lysosomes.

Cell Division↗