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PubMed1999

New method for determining cystine in leukocytes and fibroblasts.

BACKGROUND: Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required. METHODS: Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection. RESULTS: The assay was linear to 200 micromol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 micromol/L. Added cysteine, up to 200 micromol/L, was completely removed, and recovery of added cystine was 69-86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 degrees C CONCLUSIONS: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine.

Adult↗
PubMed1995

[Congenital metabolic disorder].

In children, tubulo-interstitial nephritis (TIN) is often associated with obstructive uropathy, metabolic disorders or hereditary diseases. The author reviewed congenital metabolic disorders (Fanconi syndrome, cystinosis, Lowe's syndrome, and hyperoxaluria) as causes of TIN. The Fanconi syndrome is caused by numerous disorders including cystinosis and Lowe's syndrome, and refers to a dysfunction of the proximal tubule leading to excessive urinary excretion of amino acids, glucose, phosphate, bicarbonate, etc. Prognosis of idiopathic Fanconi syndrome is not so bad if electrofluid balance is well maintained. On the other hand, prognosis of the infantile type of cystinosis, Lowe's syndrome, or hyperoxaluria "type 1" is poor. The pathophysiology of each disease should be fully understood for early diagnosis and treatment.

Child↗
PubMed1994

[Contribution of an ophthalmologic test to the diagnosis of familial nephropathies. Apropos of 10 cases].

The authors reported ten cases of familial nephropathy (two cystinosis, three Senior and Loken syndrome, one Alport's syndrome and four Oxalosis) associated to ocular manifestations. Aetiologic diagnosis was known from ocular symptoms in five cases (all cases of cystinosis and Senior and Loken syndrome). The authors undertook this study to analyse the value of ocular manifestations in determining the right aetiologic diagnosis in familial nephropathies. The results of this study showed that ocular manifestations are helpful for aetiologic diagnosis in the first diseases. Indeed, corneal injury is synonym of cystinosis and retinitis pigmentosa is usually associated with Senior and Loken syndrome. In Alport's syndrome, ocular manifestations: antcrior lenticonus cataractous and perimacular white points only have orientation value in the diagnosis of this disease. Oxalosis ocular manifestations which consist of retinal oxalate deposits appear late and are concomitant to familial renal insufficiency. They cannot help in the diagnostic search.

Adolescent↗
PubMed1995

Renal allograft survival according to primary diagnosis: a report of the North American Pediatric Renal Transplant Cooperative Study.

The data base of the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) was used to examine the effect of primary diagnosis on the outcome of renal transplantation in children. The relative risk of graft failure for eight diagnostic groups was determined, with patients with congenital and structural anomalies of the urinary tract serving as the reference group. Covariate analysis was used to control for the effects of age, race and transfusion history in recipients of living-related donor kidneys, and for age, donor age, antilymphocyte prophylaxis, prior transplantation, prior dialysis and cold ischemia time in recipients of cadaver kidneys. In recipients of living-related donor kidneys, the lowest graft failure rates were associated with the diagnoses of cystinosis, familial nephritis and hemolytic uremic syndrome (HUS), while the highest failure rates were observed in patients with a primary diagnosis of congenital nephrotic syndrome (CNS) or focal segmental glomerulosclerosis (FSGS). In cadaver allograft recipients, the lowest graft failure rates were associated with primary diagnoses of glomerulonephritis, congenital/structural disease and cystinosis, while patients with FSGS, HUS and CNS had the highest graft failure rates. This study suggests that patients with a primary diagnosis of cystinosis have superior outcomes, while the diagnoses of FSGS and CNS carry with them the highest risks of graft failure.

Canada↗
PubMed2004

Inhibition of pyruvate kinase activity by cystine in brain cortex of rats.

Cystinosis is a metabolic disturb associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending of the involved tissues. Accumulation of cystine in the brain may lead to severe neurological symptoms. However, the mechanisms by which cystine is neurotoxic are not fully understood. Considering that pyruvate kinase (PK) is a thiolic enzyme crucial for the glycolytic pathway, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on PK activity in the brain cortex of developing Wistar rats. We also performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol groups protector, and cysteamine (CysN), the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity by two different mechanisms, one through the competition with ADP and phosphoenolpyruvate (PEP), and the other non-competitively, probably through oxidation of the thiol groups of PK. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine. Considering that cysteamine is used in patients with cystinosis because it causes parenkimal organ cystine depletion, the present data provides a possible new beneficial effect for the use of this drug.

Animals↗
PubMed1977

Transformation of human cystinotic fibroblasts by SV40: characteristics of transformed cells with limited and unlimited growth potential.

Human skin fibroblasts derived from patients with nephropathic cystinosis were transformed with SV40 virions, cloned and permitted to enter the degenerative stage of growth termed "crisis," characteristic of SV40 transformed human cells. Nephropathic cystinosis is an autosomal recessively inherited metabolic disorder resulting in the intracellular accumulation of the amino acid cystine. A transformed cystinotic cell line which was recovered from the crisis stage was indistinguishable from its transformed precrisis parental cell strain in growth rate in media containing either 1% or 10% serum, cloning efficiency on plastic, in semisolid media, or upon confluent monolayers of normal skin fibroblasts, expression of SV40 T antigen, or production of virus. However, the modal DNA content of the recovered postcrisis transformed cystinotic cell line was different from that of the cloned parental precrisis transformed cell strain, suggesting that the postcrisis line was derived from a small subpopulation of the precrisis strain. The DNA content of the established cystinotic cell line continued to be unstable during subsequent subculturing and gave rise to subclones with both more and less DNA per cell. This line now has an apparently infinite growth potential and still has the hallmark of the cystinotic parental line, the storage of abnormally large amounts of intracellular nonprotein cystine.

Antigens, Viral↗
PubMed2002

Expression of CTNS alleles: subcellular localization and aminoglycoside correction in vitro.

Mutations in CTNS result in one of three forms of cystinosis: benign, intermediate, or nephropathic. Homozygosity for a nonsense mutation in CTNS (753G -->A), encoding a premature termination codon (PTC) at amino acid 138 (W138X), results in nephropathic cystinosis. Gentamicin is known to induce PTC readthrough and hence full-length protein production. We demonstrate that addition of gentamicin (300 microg/ml) to cystinotic fibroblasts leads to depletion of intracellular cystine in cell lines with a premature termination codon, but not in those with a large deletion or a deletion leading to a frameshift mutation. Plasmids were constructed with GFP as a C-terminal or N-terminal fusion to CTNS. The normal CTNS protein fused with either N- or C-terminal GFP colocalized with Lysotracker red, a fluorescent stain which selectively accumulates in lysosomes. PTC-GFP, a construct with GFP fused to the C-terminus of CTNS containing a PTC, allowed GFP to serve as a reporter of PTC readthrough. No significant fluorescence was observed in PTC-GFP-transfected cells in the absence of gentamicin but was seen and localized to lysosomes in its presence. A patient with a splice site mutation (IVS11 + 2T -->C) that eliminates the GYDQL lysosomal targeting sequence of cystinosin on one allele, and a PTC mutation (753G -->A) on the other, displays the intermediate phenotype. Transfection of the splice site mutant allele into CTNS null fibroblasts produced cystine depletion. Plasmids with GFP fused to the N-terminus of CTNS containing the splice site mutation (GFP-SS) were constructed. While the normal CTNS-GFP fusion protein was found to colocalize with Lysotracker red almost exclusively, the GFP-SS fusion product was found in the plasma membrane and cytoplasm, as well as lysosomes. A second lysosomal targeting motif in CTNS is present in this sequence, just proximal to the mutation, accounting for the partial lysosomal localization.

Alleles↗
PubMed1985

pH-profile of cystine and glutamate transport in normal and cystinotic human fibroblasts.

In the human recessive condition cystinosis, cystine transport has been reported to be normal in the plasma membrane but defective in the lysosome membrane. A possible explanation is that the transport systems at the two cellular sites are identical and that the defect in cystinosis affects the porter's ability to operate at the low pH of the lysosome. To test this hypothesis the uptake of 3H-labelled cystine and glutamate by normal and cystinotic human skin fibroblasts has been measured in vitro at pH 5.8, 6.5, 7.0, 7.4 and 8.0. Uptake of glutamate was more rapid than that of cystine. Uptake of cystine increased with increasing pH, but uptake of glutamate showed no marked pH-dependence. Transport in cystinotic cells was similar to that in normal cells, and similarly affected by pH. This finding is incompatible with the hypothesis proposed above. It is concluded that the cystine porters of the plasma membrane and the lysosome membrane are probably genetically distinct.

Biological Transport, Active↗
PubMed1985

A study of the low beta-galactosidase activity in cystinotic fibroblasts: effects of cysteamine.

beta-Galactosidase activity but not beta-glucuronidase, N-acetyl-beta-D-galactosaminidase or arylsulphatase A activity, is known to be significantly lower in cultured human skin fibroblasts from patients with cystinosis than in cells from control subjects. Incubation of cell homogenates with disulphide or thiol compounds did not affect beta-galactosidase activity, suggesting that decreased beta-galactosidase activity in affected cells was not caused by the presence of inhibiting substances or absence of activating substances. Incubating cells with 0.5 or 1.0 mmol/l cysteamine, a substance used in the clinical treatment of cystinosis because it depletes cells of excess cystine, greatly decreased beta-galactosidase activity in both cystinotic and normal cells. This effect is shown to result from enzyme instability in lysosomes with raised pH and increased thiol concentration. Thus, cysteamine, although effective in depleting cystinotic cells of excess cystine, may have the undesired side-effect of severely decreasing lysosomal beta-galactosidase.

Cells, Cultured↗
PubMed2006

Cystine inhibits creatine kinase activity in pig retina.

BACKGROUND: Cystinosis is an autosomal recessive disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Visual deficit is a possible consequence of cystine accumulation in cornea and retina. Fibroblasts from cystinotic patients present ATP deficit with intact mitochondrial energy-generating capacity by an unknown mechanism. Considering that creatine kinase is a thiol enzyme crucial for energy homeostasis in retina, and disulfides like cystine may alter thiol enzymes, the main objective of the present study was to investigate the effect of cystine and cysteamine, the drug used for treatment of cystinotic patients, on creatine kinase activity in cytosolic and mitochondrial fractions of the retina from adult pigs. METHODS: Retina was isolated from 6-month-old Landrace pigs, homogenized and mitochondrial and cytosolic fractions separated by centrifugation. Cytosolic and mitochondrial creatine kinase activities were determined in the presence of different concentrations of cystine and/or cysteamine. RESULTS: Cystine inhibited the enzyme activity in a dose- and time-dependent manner and cysteamine prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits creatine kinase activity by oxidation of the sulfhydryl groups of the enzyme. CONCLUSIONS: Considering that creatine kinase is a crucial enzyme for retina energy homeostasis, in case cystine leaves lysosome these results provide a possible mechanism for cystine toxicity and also another beneficial effect for the use of cysteamine in patients with cystinosis.

Animals↗
PubMed2005

Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex.

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.

Adenosine Diphosphate↗
PubMed2002

Retinal visualization in an eye with corneal crystals using indocyanine green videoangiography.

PURPOSE: To report a patient in whom clear imaging of the retina, impossible with conventional methods, was obtained using indocyanine green (ICG) videoangiography. DESIGN: Interventional case report. METHODS: A 31-year-old patient with nephropathic cystinosis and complaints of decreased vision in the left eye underwent a complete ophthalmologic evaluation, including conventional photography, fluorescein angiography, and ICG videoangiography. RESULTS: With conventional methods (direct and indirect ophthalmoscopy, photography, and fluorescein angiography), the view of the left retina was obscured by densely packed corneal cystine crystals. During ICG angiography, clear imaging of the retina was obtained with near-infrared illumination. CONCLUSION: Indocyanine green videoangiography can be a useful tool in cases with cystinosis and other corneal opacities, where visualization and imaging of the retina are important but impossible with conventional methods.

Adult↗
PubMed2001

The targeting of cystinosin to the lysosomal membrane requires a tyrosine-based signal and a novel sorting motif.

Cystinosis is a lysosomal transport disorder characterized by an accumulation of intra-lysosomal cystine. Biochemical studies showed that the lysosomal cystine transporter was distinct from the plasma membrane cystine transporters and that it exclusively transported cystine. The gene underlying cystinosis, CTNS, encodes a predicted seven-transmembrane domain protein called cystinosin, which is highly glycosylated at the N-terminal end and carries a GY-XX-Phi (where Phi is a hydrophobic residue) lysosomal-targeting motif in its carboxyl tail. We constructed cystinosin-green fluorescent protein fusion proteins to determine the subcellular localization of cystinosin in transfected cell lines and showed that cystinosin-green fluorescent protein colocalizes with lysosomal-associated membrane protein 2 (LAMP-2) to lysosomes. Deletion of the GY-XX-Phi motif resulted in a partial redirection to the plasma membrane as well as sorting to lysosomes, demonstrating that this motif is only partially responsible for the lysosomal targeting of cystinosin and suggesting the existence of a second sorting signal. A complete relocalization of cystinosin to the plasma membrane was obtained after deletion of half of the third cytoplasmic loop (amino acids 280-288) coupled with the deletion of the GY-DQ-L motif, demonstrating the presence of the second signal within this loop. Using site-directed mutagenesis studies we identified a novel conformational lysosomal-sorting motif, the core of which was delineated to YFPQA (amino acids 281-285).

Amino Acid Sequence↗
PubMed2003

Sialic acid storage disease and related disorders.

This paper gives an overview of the two sialic acid storage disorders, Salla disease and infantile sialic acid storage disease, and the related disorders cystinosis, sialuria, sialidosis, and galactosialidosis. Sialic acid storage disease and cystinosis are models for a deficient lysosomal transport of monosaccharides and amino acids, respectively. Several gene mutations leading to the production of the faulty membrane proteins sialin and cystinosin have been identified in recent years. Knowledge of the underlying pathophysiology is a prerequisite for future research projects, which will focus on the expression of the disease genes in living systems and the physical characterization of these proteins by X-ray crystallography and nuclear magnetic resonance spectroscopy.

Amino Acid Transport Systems, Neutral↗
PubMed2002

Lysosomal cystine storage augments apoptosis in cultured human fibroblasts and renal tubular epithelial cells.

Nephropathic cystinosis is a lethal disorder of lysosomal cystine storage due to defective lysosomal cystine transport. How lysosomal cystine causes this multisystemic disorder culminating in end-stage renal disease is not known, because the cystine is isolated from cellular metabolism by the lysosomal membrane. It is here reported that in both normal and nephropathic cystinotic fibroblasts and cultured renal proximal tubule epithelial cells, increased lysosomal cystine causes an increased rate of apoptosis. In nephropathic cystinotic fibroblasts, the rate of apoptosis is 14.8% after exposure to TNF-alpha versus 7.8% in control normal fibroblasts. Anti-Fas antibodies and UV exposure induced apoptosis in 18.1% and 17.4% of nephropathic cystinotic fibroblasts, respectively, versus 5.2% and 7.1% in normal fibroblasts when analyzed by CaspACE (P < 0.05). Similar results were found when the cells were analyzed by TdT-mediated dUTP nick end labeling (TUNEL). When the cystine content of normal fibroblasts is increased by exposure to cystine dimethylester (CDME), the apoptotic rate is increased to the rate seen in nephropathic cystinotic cells. Decreasing the cystinotic cells' cystine content by use of cysteamine results in normalization of the apoptotic rate. Renal proximal tubule epithelial (RPTE) cells are much more sensitive to CDME than fibroblasts, reaching 43.8% apoptosis 6 h after exposure to CDME alone, compared with 38.2% when exposed to TNF-alpha alone. Serum withdrawal causes an apoptotic rate of 8.7% in nephropathic cystinotic fibroblasts, compared with 6.1% in normal fibroblasts. That rate increases to 37.3% in normal fibroblasts after CDME exposure. Fibroblasts from two cystinotic variants, benign ocular and intermediate cystinosis, do not display increased apoptosis with increased lysosomal cystine. It is concluded that enhanced apoptosis resulting from lysosomal cystine storage may lead to inappropriate cell death and decreased cell numbers in many tissues and hence contribute to the nephropathic cystinotic phenotype. The variant forms may represent co-segregation or linkage of rare alleles that confer resistance to apoptosis, moderating the cell loss and causing the milder disease expression.

Apoptosis↗
PubMed1974

Cysteine activation in cultured cystinotic cells. The specific activity of cysteinyl-tRNA synthetase and tRNACys and the determination of the Michaelis-Menten constants for cysteinyl-tRNA synthetase.

This study explored the possibility whether an altered cysteinyl-tRNA synthetase might lead to the faulty regulation of cyst(e)ine levels in cystinotic cells. This hypotheses is attractive, since amino acid activation is important in the regulation of amino acid metabolism in microorganisms. By using cultured fibroblasts from patients with cystinosis, those cell components responsible for cysteine activation were examined: cyst(e)ine, the cysteinyl-tRNA levels, cysteinyl-tRNA synthetase activity, and the K(m) of cysteine, ATP, and tRNA(Cys) for cysteinyl-tRNA synthetase, Fibroblasts from two patients with the infantile form of cystinosis were labeled for three days with [(35)S]-cystine. In comparison with normal cells, these cells contained high levels of free cysteine and cystine. Labeled fibroblasts from a patient with the adolescent form of the disease contained elevated levels of cystine, although elevated cysteine levels were not detected. The ratio of acceptor activity of tRNA(Cys) to tRNA(Leu) in cystinotic cells was 0.46 in cystinotic cells and 0.54 in normal cells. The specific activity of cysteinyl-tRNA synthetase measured in fibroblasts of two infantile and one adolescent form was: 6.1, 2.2, and 2.1 pmol of [(14)C]aminoacyl-tRNA formed/mug protein/10 min, respectively. In addition, the cysteine K(m)'s for the same cells, respectively, were: 3.1 muM, 1.5 muM, and 1.2 muM. The corresponding data for specific activities of two normal cell lines were 2.0 and 5.1 pmol [(14)C]aminoacyl tRNA formed/mug protein/10 min, with K(m)'s of 3.0 muM and 1.7 muM. These data indicate that cystinotic cells contain levels of tRNA(Cys) and Cys-tRNA synthetase comparable to normal cells. In addition, within the cystinotic cells, the relative level of the Cys-tRNA synthetase and tRNA(Cys) to those of leucine and alanine are comparable to normal cells. Finally, the K(m) of Cys-tRNA synthetase for ATP and tRNA is similar in normal and cystinotic cells.

Amino Acids↗
PubMed1981

Pantetheinase activity and cysteamine content in cystinotic and normal fibroblasts and leukocytes.

Cysteamine is the most effective agent known for the reduction of the elevated cystine content of cells from patients with cystinosis. A defect in endogenous cysteamine generation could account for many of the metabolic features of this disorder. To test this hypothesis, we have developed improved methods for measuring pantetheinase (cysteamine-generating) activity and intracellular cysteamine levels and used these methods to measure such parameters in cystinotic and normal leukocytes and cultured skin fibroblasts. Pantetheinase activity as defined in the test was similar in extracts of cystinotic and normal cells [leucocytes, normal, 78 +/- 15 (S.E.), cystinotic, 56+/- 6.4; fibroblasts, normal, 9.4 +/- 1.5; cystinotic, 7.7 +/- 1.7]. Cysteamine levels were normal in leukocytes from cystinotics receiving no cysteamine or doses of oral cysteamine too low to reduce leukocyte cystine content. The results indicate that the cause of cystinosis is unlikely to be related to a failure to generate of sustain normal intracellular cysteamine levels.

Amidohydrolases↗
PubMed1988

[The initial presentation of renal tubular syndrome].

History, clinical presentation and first laboratory results were analyzed in three patients with cystinosis and in three patients with distal renal tubular acidosis. Growth failure, gastrointestinal tract disturbances, polydipsia, and polyuria were the most constant findings on history. Patients with distal renal tubular acidosis had a shorter period of clinical symptoms. All patients presented with dystrophy and dehydration. First laboratory investigation uniformly showed metabolic acidosis, high urinary-pH, and in spite of dehydration a low osmolarity of the first voided urine specimen. There were lower values of serum potassium and of urine osmolarity in cystinosis. In addition, metabolic acidosis was not so pronounced. Our study shows that history, clinical presentation and the results of a few simple laboratory investigations can indicate the presence of a renal tubular disorder even on admission of the patients.

Acidosis, Renal Tubular↗