Cystine Dimethyl Ester Research Articles

Synthesis of dehydroabietane-derived sulfonamides with fragments of L-amino acid esters and hydrazides

Ethyl ester of 12-chlorosulfodehydroabietic acid, when reacted with the methyl esters of glycine, methionine, leucine, glutamic acid, tyrosine, proline, histidine, for the first time yielded the corresponding sulfonamides, the ester group (-COOMe) of which was selectively transferred to the hydrazide (-COONHNH2) without affecting the ethyl group bounded with the dehydroabietic acid fragment. In the case of glutamic acid, the corresponding dihydrazides were obtained. The reaction of 12-chlorosulfodehydroabietic acid ethyl ester with cystine dimethyl ester led to the selective formation of bis-sulfonamide, while carrying out the same reaction in an acetone-contained solution was accompanied by the cleavage of the disulfide bond of the cystine fragment to give thioketal. The obtained bis-sulfonamide and thioketal were oxidized with chlorine dioxide to afford in both cases sulfochloride.

Effect of chain extender on microphase structure and performance of self‐healing polyurethane and poly(urethane‐urea)

AbstractIn this work, four aliphatic chain extenders, hexanediol (HDO), hexane diamine (HDA), cystamine (CY), and cystine dimethyl ester (CDE), were chosen to synthesize four kinds of polyurethane and poly(urethane‐urea)s (PUs), respectively. HDO extended polyurethanes, HDA extended poly(urethane‐urea), CY extended poly(urethane‐urea), and CDE extended poly(urethane‐urea) were denoted as OPU, APU, CPU, and SPU, respectively. The effect of chain extender type on microphase structure and performance of four PUs was investigated. Our research showed that mechanical strength increased in the following order: OPU < SPU < CPU < APU, and self‐healing performance increased in the opposite direction. This result is attributed to the increasing degree of microphase separation: OPU < SPU < CPU < APU. The optimal sample SPU has not only excellent mechanical properties (tensile strength of 27.1 MPa and elongation at break of 397.7%), but also exhibits superior self‐healing performance (self‐healing efficiencies of 95.3% and 93.5% based on tensile strength and elongation at break). The moderate degree of microphase separation between the soft segments and the hard segments, the introduction of disulfide bonds and low degree of hydrogen bonding are responsible for preparing a polyurethane or poly(urethane‐urea) system with high mechanical strength and excellent self‐healing performance simultaneously. This work provides useful information for us to develop self‐healing polyurethane or poly(urethane‐urea) materials in the future.

Cystine dimethyl ester cross-linked PEG-poly(urethane-urea)/nano-hydroxyapatite composited biomimetic scaffold for bone defect repair

Polyurethane (PU) and polyurea (PUA) materials have shown significant potential for application in tissue repair. Herein, we design a glycerol ethoxylate (PEG)-based poly(urethane-urea) for bone tissue repair. The polymer precursor was prepared from the reaction of PEG and isophorone diisocyanate (IPDI). The cystine dimethyl ester was used as a cross-linker for the preparation of poly(urethane-urea) elastomers. The material was further strengthened by physical blending of nano-hydroxyapatite (nHA). The physical and biological properties of final material were evaluated by mechanical testing, scanning electron microscopy characterization, degradation tests, cell proliferation and cell differentiation assays. The obtained scaffolds showed good mechanical strength, excellent biocompatibility and osteogenic capability. All the evidences demonstrated that this type of materials has good prospects for bone tissue repair application.

A Robust and High Self‐Healing Efficiency Poly(Urea‐Urethane) Based on Disulfide Bonds with Cost‐Effective Strategy

AbstractThe development of self‐healing poly(urea‐urethane)s with remarkable mechanical properties as well as self‐healing capability is an important challenge through a low‐cost approach. In this study, cystine dimethyl ester (CDE) is synthesized cost‐effectively using cystine from bio‐based sources as raw material, and a novel self‐healing poly(urea‐urethane) is successfully prepared using synthesized CDE as a disulfide‐containing chain extender through a two‐step approach. The as‐prepared disulfide‐containing poly(urea‐urethane) (SPU) presents an excellent self‐healing capability with self‐healing efficiency of 95.3% at a moderate healing temperature (≈60 °C). Meanwhile, the SPU exhibits robust mechanical properties possessing a maximum tensile strength of 25.80 MPa and a high elongation at break of over 460% simultaneously. The compromise between self‐healing capability and mechanical performance can be responsible for the exchange of dynamic disulfide bonds and the existence of quadruple hydrogen bonding. This strategy provides an available idea to develop robust self‐healing polymer materials cost‐effectively.

Dextran hydrogels by crosslinking with amino acid diamines and their viscoelastic properties

Amine functionalized polysaccharide hydrogels such as those based on chitosan are widely examined as biomaterials. Here we set out to develop a facile procedure for developing such hydrogels by crosslinking dextran with amino acid diamines. The dextran-amino acid gels were formed by the addition of the amino acid diamines to a dextran and epichlorohydrin solution once it became homogeneous. This was demonstrated with three amino acid diamines, lysine, lysine methyl ester, and cystine dimethyl ester. Hydrogel networks with albumin entrapped were also demonstrated. These hydrogels were characterized by FTIR, SEM, rotational rheometry, swelling studies and cell biocompatibility analysis. These hydrogels showed the unexpected pH-responsive behavior of greater swelling at more basic pH, similar to that of an anionic hydrogel. This is uncharacteristic for amine functionalized gels as they typically exhibit cationic hydrogel behavior. All hydrogels showed similar biocompatibility to that of dextran crosslinked without amino acids.

Formation of negative ions via resonant low-energy electron capture by cysteine and cystine methyl esters

The processes of resonance low-energy free electron attachment to methyl esters of some sulfur-containing amino acids were studied. The long-lived molecular negative ions of cystine dimethyl ester formed in the valence state via the Feshbach nuclear excited resonance mechanism were detected by mass spectrometry. The reactions of disulfide bond dissociation were identified in an electron energy range of 0—1 eV. They can be considered as model reactions regarding processes of peptide decomposition due to the resonance interaction with low-energy electrons. Predissociation of short-lived molecular ions of cysteine methyl ester formed by capture of electrons with energies of ~1.6 eV is accompanied by the intra-ionic transfer of negative charge from the carbonyl group to the sulfur atom leading to the elimination from the latter of hydrogen atom.

Cystine dimethyl ester induces apoptosis through regulation of PKC-δ and PKC-ε in prostate cancer cells.

Protein kinase C-δ (PKC-δ) and PKC-ε are reported to be effective in cancer prevention via S-thiolation-mediated mechanisms. This may be through stimulation of the pro-apoptotic, tumor-suppressive isozyme PKC-δ and/or inactivation of the growth stimulatory, oncogenic isozyme PKC-ε. We investigated oxidative regulatory responses of PKC-δ and PKC-ε to cystine dimethyl ester (CDME), a metabolic precursor of cystine, which, by inducing release of cellular cystine stimulates apoptosis in different prostate cancer cells, PC3 and LNCaP, compared to normal RWPE1 cells. Treatment of CDME in doses of 0.5mM and 5mM significantly induces apoptosis due to regulation of concentration-dependent PKC-δ stimulation and PKC-ε reduction in these prostate cancer cells. This apoptotic regulation was confirmed by immunoblot analyses and specific PKC enzyme assays in immunoprecipitated samples. Additionally, inhibition of PKC-δ by small interfering RNA (siRNA) proved that CDME-induced cell death was dependent on PKC-δ activity in prostate cancer cells. These data demonstrated that CDME induces apoptosis by cysteinylation of both PKC-δ and PKC-ε in tumorigenic prostate epithelial cells compared to control nontumorigenic cells. Cellular cystine may play a critical role in treatment and/or prevention of prostate cancer by regulating PKC activity.

PD32-06 ASSESSMENT OF PHARMACOLOGIC INTERVENTION ON CYSTINE STONE GROWTH USING IN VIVO IMAGING IN A MOUSE MODEL OF CYSTINURIA

You have accessJournal of UrologyStone Disease: Evaluation II1 Apr 2014PD32-06 ASSESSMENT OF PHARMACOLOGIC INTERVENTION ON CYSTINE STONE GROWTH USING IN VIVO IMAGING IN A MOUSE MODEL OF CYSTINURIA Jaspreet Parihar, Kathleen Capaccione, Min Yang, Derek Adler, Derek Gordon, Joseph Barone, David Goldfarb, Jay Tischfield, and Amrik Sahota Jaspreet PariharJaspreet Parihar More articles by this author , Kathleen CapaccioneKathleen Capaccione More articles by this author , Min YangMin Yang More articles by this author , Derek AdlerDerek Adler More articles by this author , Derek GordonDerek Gordon More articles by this author , Joseph BaroneJoseph Barone More articles by this author , David GoldfarbDavid Goldfarb More articles by this author , Jay TischfieldJay Tischfield More articles by this author , and Amrik SahotaAmrik Sahota More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2277AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Slc3a1 knockout mice (KO) are a model for human cystinuria and male mice beginning at age 3 months develop urinary tract stones. Computed tomography (CT) and magnetic resonance imaging (MRI) may allow us to follow stone growth and the effects of drug treatment in live animals. We evaluated the utility of these modalities for identifying cystine stone growth in Slc3a1 KO mice and for assessing the efficacy of cystine dimethyl ester (CDME), a cystine crystal growth inhibitor. METHODS Male mice were screened for bladder stones using the Albira CT scanner and the Aspect 2 MRI scanner. Initial bladder volume (an indicator of stone volume) was calculated using InviCRO VivoQuant software. Mice with stones were randomly assigned to treatment (200 μl of 10 mg/ml CDME, n=11) or control (200 μl water, n=7) groups, each given daily by gavage for 10 weeks. Volume measurements were repeated at 1-2 week intervals. Mice were then sacrificed and stone size and number analyzed using chi-square. RESULTS 60% of mice had bladder stones. Using CT, initial bladder volumes (mm3) in both groups were similar (12-96 for control and 11-86 for CDME). In both cases, there was a linear increase in stone volume that followed the equation y=mx+c. The equations for the CDME and the control groups were similar (range y=0.39x+11.0 to y=2.67x+73.3). Final MRI scans demonstrated a 15.5% and a 12.4% increase in kidney volume for the two groups, respectively, (p=.17). Thus, the two groups could not be distinguished by CT or MRI analysis. Upon histologic evaluation there was a significant difference in the percentage of stone sizes in the two groups (p=0.05). The treatment group had 9.4% more stones and the control group had 19.6% fewer stones in the 1.1-2.0 mm range. The treatment group had 26.1% fewer stones and the control group had 54.6% more stones in the 3.1-4.0 mm range. CONCLUSIONS CT can determine the rate of stone growth in vivo, but longitudinal measurement of stone volume is not suitable for evaluating CDME efficacy since total stone volumes in both groups were comparable. MRI was also unable to differentiate the two groups. These imaging modalities are useful for differentiating stone formers form non-stone formers, but cannot be used for the assessment of pharmacological intervention. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e838 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Jaspreet Parihar More articles by this author Kathleen Capaccione More articles by this author Min Yang More articles by this author Derek Adler More articles by this author Derek Gordon More articles by this author Joseph Barone More articles by this author David Goldfarb More articles by this author Jay Tischfield More articles by this author Amrik Sahota More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cystine dimethylester loading promotes oxidative stress and a reduction in ATP independent of lysosomal cystine accumulation in a human proximal tubular epithelial cell line

Using the cystine dimethylester (CDME) loading technique to achieve elevated lysosomal cystine levels, ATP depletion has previously been postulated to be responsible for the renal dysfunction in cystinosis, a genetic disorder characterized by an excessive accumulation of cystine in the lysosomes. However, this is unlikely to be the sole factor responsible for the complexity of cell stress associated with cystinosis. Moreover, CDME has been shown to induce a direct toxic effect on mitochondrial ATP generation. Using a human-derived proximal tubular epithelial cell line, we compared the effects of CDME loading with small interfering RNA-mediated cystinosin, lysosomal cystine transporter (CTNS) gene silencing on glutathione redox status, reactive oxygen species levels, oxidative stress index, antioxidant enzyme activities and ATP generating capacity. The CDME-loaded cells displayed increased total glutathione content, extensive superoxide depletion, augmented oxidative stress index, decreased catalase activity, normal superoxide dismutase activity and compromised ATP generation. In contrast, cells subjected to CTNS gene inhibition demonstrated decreased total glutathione content, increased superoxide levels, unaltered oxidative stress index, unaltered catalase activity, induction of superoxide dismutase activity and normal ATP generation. Our data indicate that many CDME-induced effects are independent of lysosomal cystine accumulation, which further underscores the limited value of CDME loading for studying the pathogenesis of cystinosis. CTNS gene inhibition, which results in intracellular cystine accumulation, is a more realistic approach for investigating biochemical alterations in cystinosis.

The synthesis and structural characterization of N-(ferrocenyl)2 and N-(ferrocenoyl)2 cystine dimethyl ester derivatives: Potential anion sensing agents

Standard peptide coupling reactions were use to prepare the N-(ferrocenyl)2 and N-(ferrocenoyl)2 cystine dimethyl ester derivatives 4–11. The ferrocene carboxylic acids 1 and 3 were treated with 1-hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), l-cystine methyl ester hydrochloride and triethylamine in dichloromethane at 0°C to furnish compounds 4–9. The preparation of compounds 10 and 11 employed the dipeptide derivatives (glycine)2-l-cystine dimethyl ester and (β-alanine)2-l-cystine dimethyl ester respectively. The N-(ferrocenyl)2 and N-(ferrocenoyl)2 cystine dimethyl ester derivatives 4–11, which are potential anion sensing agents, were spectroscopically characterized by a combination of 1H NMR, 13C NMR, IR, UV, DEPT-135 and 1H–13C COSY (HMQC) spectroscopy, mass spectrometry and cyclic voltammetry.The electrochemical detection of dihydrogen phosphate and adenosine nucleotide anions in aqueous electrolyte by monolayers of {N-(ferrocenoyl)-β-alanine}2-l-cystine dimethyl ester 11 immobilized on gold electrodes using cyclic voltammetry is described. Immobilization of this receptor on a gold electrode surface enabled the recognition process to be detected in aqueous media. The recognition process is as a result of electrostatic interactions between the ferricenium cation and the anion, and hydrogen bonding interactions between the peptide amide bonds and the anion. The complexation process was amperometrically sensed via a reduction in the peak current of the ferrocene/ferricenium redox couple. A linear relationship (R2=c. 0.99) was observed between anion concentration and change in peak current in both cases.

Synthesis of Disulfide-Derivatised Metallodeuteroporphyrins as Novel Catalysts to Imitate Cytochrome P450 in the Selective Oxidation of Cyclohexane

Metallodeuteroporphyrins with two side chains linked by a terminal cystine dimethyl ester [M(PDTEP), where M = ClFe(III), ClMn(III), Co(II)], have been synthesised. These metalloporpyirn complexes were found to act as efficient catalysts in the oxidation of cyclohexane to cyclohexanol and cyclohexanone using air as the oxygen source and in the absence of additives and solvents. The influence of the central metal, reaction temperature and pressure on the catalytic behavior of M(PDTEP) were evaluated. The high conversion and selectivity of M(PDTEP) was ascribed to the existence of intramolecular coordination by the M(PDTEP).

Cysteamine prevents inhibition of adenylate kinase caused by cystine in rat brain cortex

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in the lysosomes of almost all tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are still obscures. Adenylate kinase, along with creatine kinase, is responsible for the enzymatic phosphotransfer network, crucial for energy homeostasis. Taking into account that cystine is known to inhibit creatine kinase activity, the two enzymes have thiol groups, and the strong interaction between the two activities, our main objective was to investigate the effect of cystine on adenylate kinase activity in the brain cortex of Wistar rats. For the in vivo studies, the animals were injected twice a day with 1.6 micromol/g body weight of cystine dimethylester and/or 0.46 micromol/g body weight of cysteamine from the 25th to the 29th postpartum day and sacrificed after 12 h. Cystine inhibited the enzyme activity in vitro in a concentration dependent way, whereas cysteamine prevented the inhibition. Adenylate kinase activity was found diminished in the brain cortex of rats loaded with cystine dimethylester and co-administration of cysteamine prevented the diminution of the enzyme activity. Considering that adenylate kinase together with creatine kinase is crucial for energy homeostasis, the release of cystine from lysosomes with consequent enzymes inhibition could impair energy homeostasis, contributing to tissue damage in patients with cystinosis.

Cysteamine prevents inhibition of adenylate kinase caused by cystine in rat brain cortex

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in the lysosomes of almost all tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are still obscures. Adenylate kinase, along with creatine kinase, is responsible for the enzymatic phosphotransfer network, crucial for energy homeostasis. Taking into account that cystine is known to inhibit creatine kinase activity, the two enzymes have thiol groups, and the strong interaction between the two activities, our main objective was to investigate the effect of cystine on adenylate kinase activity in the brain cortex of Wistar rats. For the in vivo studies, the animals were injected twice a day with 1.6 micromol/g body weight of cystine dimethylester and/or 0.46 micromol/g body weight of cysteamine from the 25th to the 29th postpartum day and sacrificed after 12 h. Cystine inhibited the enzyme activity in vitro in a concentration dependent way, whereas cysteamine prevented the inhibition. Adenylate kinase activity was found diminished in the brain cortex of rats loaded with cystine dimethylester and co-administration of cysteamine prevented the diminution of the enzyme activity. Considering that adenylate kinase together with creatine kinase is crucial for energy homeostasis, the release of cystine from lysosomes with consequent enzymes inhibition could impair energy homeostasis, contributing to tissue damage in patients with cystinosis.

Cysteamine prevents inhibition of thiol-containing enzymes caused by cystine or cystine dimethylester loading in rat brain cortex

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in the lysosomes of all tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are still obscures. Considering that thiol-containing enzymes are critical for several metabolic pathways, our main objective was to investigate the effects of cystine or cystine dimethylester load on the thiol-containing enzymes creatine kinase and pyruvate kinase, in the brain cortex of young Wistar rats. The animals were injected twice a day with 1.6 micromol/g body weight of cystine dimethylester or 1 micromol/g body weight of cystine and/or 0.46 micromol/g body weight of cysteamine from the 16th to the 20th postpartum day and sacrificed after 12 h. Cystine or cystine dimethylester administration inhibited the two enzyme activities. Co-administration of cysteamine, the drug used to treat cystinotic patients, normalized the two enzyme activities. Lactate dehydrogenase activity, a nonthiol-containing enzyme was not affected by cystine dimethylester administration. Cystine inhibits creatine kinase and pyruvate activities possibly by oxidation of the sulfhydryl groups of the enzymes. Considering that creatine kinase and pyruvate kinase, like other thiol-containing enzymes, are crucial for energy homeostasis and antioxidant defenses, the enzymes inhibition caused by cystine released from lysosomes could be one of the mechanisms of tissue damage in patients with cystinosis.

Antioxidant Effect of Cysteamine in Brain Cortex of Young Rats

Cysteamine is a cystine-depleting drug used in the treatment of cystinosis, a metabolic disorder caused by deficiency of the lysosomal cystine carrier. As a result, cystine accumulates within lysosomes in many tissues and organs, including the nervous system. Studies with cystine dimethyl ester loaded cells suggest that cystine might induce apoptosis through oxidative stress. Our objective was to investigate the effects of co-administration of cysteamine with the oxidant cystine dimethyl ester on several parameters of oxidative stress in the brain cortex of rats. Animals were injected with 1.6 micromol/g cystine dimethyl ester and/or 0.26 micromol/g body weight cysteamine. Cystine dimethyl ester induced lipoperoxidation, protein carbonylation, and stimulated superoxide dismutase, glutathione peroxidase and catalase activities, probably through the formation of free radicals. Cysteamine prevented those effects, possibly increasing cellular thiol pool and acting as a scavenger of free radicals. These results suggest that the antioxidant effect of cysteamine may be important in the treatment of cystinosis.

Cystine Dimethylester Model of Cystinosis: Still Reliable?

The ability of cystine dimethylester (CDME) to load lysosomes with cystine has been used to establish the basic defect in cystinosis: defective cystine exodus from lysosomes. Using CDME loading, it has been postulated that cystine accumulation in cystinosis affects mitochondrial ATP production, resulting in defective renal tubular reabsorption. Recent studies in cystinotic fibroblasts, however, show normal adenosine triphosphate (ATP) generation capacity. To investigate the effect of CDME in more detail, mitochondrial ATP generation, reactive oxygen species production, and viability are compared in fibroblasts loaded with CDME with those of cystinotic cells with a defective cystine transporter. Intracellular cystine levels were comparable in fibroblasts loaded with CDME (1 mM, 30 min) and cystinotic fibroblasts. Intracellular ATP levels and mitochondrial ATP production were decreased in fibroblasts loaded with CDME, but normal in cystinotic fibroblasts. Superoxide production was increased with 300% after CDME loading, whereas no changes were observed in cystinotic fibroblasts. Exposure to CDME led to cell death in a time- and concentration-dependent manner. Our data demonstrate that CDME has a toxic effect on mitochondrial ATP production and cell viability. These effects are not observed in cystinotic cells, indicating that a more appropriate model is required for studying the pathogenesis of cystinosis.

Promotion of oxidative stress in kidney of rats loaded with cystine dimethyl ester

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in most tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are not fully understood. Studies performed in fibroblasts of cystinotic patients and in kidney cells loaded with cystine dimethyl ester (CDME) suggest that apoptosis is enhanced in this disease. Considering that oxidative stress is a known apoptosis inducer, our main objective was to investigate the effects of CDME loading on several parameters of oxidative stress in the kidney of young rats. Animals were injected twice a day with 1.6 micromol/g body weight CDME and/or 0.26 micromol/g body weight cysteamine (CSH) from the 16th to the 20th postpartum day and killed after 1 or 12 h. CDME induced lipoperoxidation and protein carbonylation and stimulated superoxide dismutase, glutathione peroxidase (GPx), and catalase activities, probably through the formation of superoxide anions, hydrogen peroxide, and hydroxyl free radicals. Coadministration of CSH, the drug used to treat cystinotic patients, prevented, at least in part, those effects, possibly acting as a scavenger of free radicals. These results suggest that the induction of oxidative stress might be one of the mechanisms leading to tissue damage in cystinotic patients.

Decreased Intracellular ATP Content and Intact Mitochondrial Energy Generating Capacity in Human Cystinotic Fibroblasts

Cystinosis is an autosomal recessive lysosomal storage disorder caused by a defect in the lysosomal cystine carrier cystinosin. Cystinosis is the most common cause of inherited Fanconi syndrome leading to renal failure, in which the pathogenesis is still enigmatic. Based on studies of proximal tubules loaded with cystine dimethyl ester (CDME), altered mitochondrial adenosine triphosphate (ATP) production was proposed to be an underlying pathologic mechanism. Thus far, however, experimental evidence supporting this hypothesis in humans is lacking. In this study, energy metabolism was extensively investigated in primary fibroblasts derived from eight healthy subjects and eight patients with cystinosis. Patient's fibroblasts accumulated marked amounts of cystine and displayed a significant decrease in intracellular ATP content. Remarkably, overall energy-generating capacity, activity of respiratory chain complexes, ouabain-dependent rubidium uptake reflecting Na,K-ATPase activity, and bradykinin-stimulated mitochondrial ATP production were all normal in these cells. In conclusion, the data presented demonstrate that mitochondrial energy-generating capacity and Na,K-ATPase activity are intact in cultured cystinotic fibroblasts, thus questioning the idea of altered mitochondrial ATP synthesis as a keystone for the pathogenesis of cystinosis.

Altered status of glutathione and its metabolites in cystinotic cells

Cystinosis is an autosomal recessive disorder, caused by mutations of the lysosomal cystine carrier cystinosin, encoded by the CTNS gene (17p13). The concomitant intralysosomal cystine accumulation leads to multi-organ damage, with kidneys being the first affected. Altered mitochondrial oxidative phosphorylation has been demonstrated in animal proximal tubules loaded with cystine dimethyl ester, mimicking cystine accumulation in cystinosis, but has not been confirmed in cells of patients with cystinosis. Furthermore, the link between cystine accumulation and mitochondrial damage is also missing. We hypothesized that cytosolic cysteine deficiency resulting in intracellular glutathione (GSH) shortage might be involved in cellular dysfunction in cystinosis. Components of the gamma-glutamyl cycle were measured in cultured skin fibroblasts (n = 9) and polymorphonuclear (PMN) leukocytes (n = 15) derived from patients with cystinosis and compared with the values in cultured fibroblasts (n = 9) and PMN cells (n = 18) of healthy controls. Cystine content in cystinotic fibroblasts and PMN cells was significantly elevated compared with the controls, consistent with the lysosomal cystine accumulation in these cells. Although no reduction of total intracellular GSH content was found in cystinotic cells, it inversely correlated with cystine levels. Furthermore, GSH disulfide (GSSG) was elevated in cystinotic cells, resulting in an increased GSSG/total GSH (%) ratio. No relationship between intracellular cystine and GSH was found in control fibroblasts and PMN cells. An elevated GSSG/total GSH (%) ratio might indicate increased oxidative stress present in cystinotic cells. Inverse correlation between cystine accumulation and intracellular GSH content indicates that under stress conditions such as intensive energy demand or increased oxidative insult, cystinotic cells may be more prone to GSH depletion.

Potential role of apoptosis in development of the cystinotic phenotype

Much still remains unclear about the proximal biochemical effects of mutations on development of the phenotype in inborn errors of metabolism. Cystinosis is an example of this phenomenon. We have recently shown that cystinotic cells undergo apoptosis at a two- to fourfold higher rate than controls. Cystinotic cells pre-treated with cysteamine, normalizing cystine content, display a four- to fivefold decrease in apoptosis, while normal cells pre-treated with cystine dimethylester, increasing lysosomal cystine, exhibit a fivefold increase in apoptosis. We speculate that cystine exits the lysosomal compartment during early apoptosis and affects apoptotic proteins in the cytosol, causing an inappropriate commitment to proceed to cell death. The resulting chronic hypocellularity could account for all the characteristics of the nephropathic cystinotic phenotype. The milder variants of cystinosis may result from modifying mutations within an apoptotic protein, ablating the proapoptotic effects of cystine. Failure of the mouse knockout for cystinosis to show renal involvement may be the result of differences in apoptotic processes between man and mouse. Apoptosis is a major final common pathway for many disease states. Therefore, a better understanding of the effect of lysosomal cystine on apoptosis may help to clarify development of other diseases.