Cystinosis is a rare disease characterized by abnormal lysosomal cystine accumulation of cystine due to impaired lysosomal transport. We previously reported the first case of cystinosis in Korea in a 12-year-old boy with short stature, general weakness, and photophobia. The diagnosis was confirmed based on ophthalmic findings and biochemical analyses (serum leukocyte cystine measurement). Major endocrine manifestations at diagnosis included hypothyroidism, growth retardation, and hypogonadism. Despite oral cysteamine administration and renal replacement therapy, multiple complications including both endocrine and nonendocrine disorders developed during and after adolescence. In this report, we review the presenting features and factors related to the long-term complications in a patient with cystinosis.
Adolescent* ; Child ; Cysteamine ; Cystine ; Cystinosis* ; Diagnosis ; Humans ; Hypogonadism ; Hypothyroidism ; Korea ; Leukocytes ; Lysosomal Storage Diseases ; Male ; Photophobia ; Rare Diseases ; Renal Replacement Therapy

PURPOSE: To investigate the effect of cysteamine on mixed peripheral blood mononuclear cells (PBMCs)-chemically injured keratocytes reaction (mixed lymphocyte-keratocyte reaction; MLKR). METHODS: PBMC stimulation assay was performed after keratocytes were chemically injured with 0.05 N NaOH for 60 seconds. MLKR was treated with various concentrations of cysteamine (0-10 mM). Intracellular reactive oxygen species (ROS) formation was measured using the oxidation-sensitive fluorescent probe, 2'7'-dichlorofluorescein diacetate (DCF-DA). Proliferation rate of PBMCs stimulated by NaOH-treated keratocytes and secretion profiles of matrix metalloprotease-9 (MMP-9), transforming growth factor-beta1 (TGF-beta1), interleukin-6 (IL-6), and macrophage migration inhibitory factor (MIF) were determined using the bromodeoxyuridine proliferation assay and enzyme-linked immunosorbent assay, respectively. RESULTS: Proliferation rate of PMBCs was suppressed by cysteamine in a dose-dependent manner (p = 0.019). Fluorescence of DCF-DA decreased depending on cysteamine concentration (p < 0.001). MMP-9, IL-6 and TGF-beta1 levels were suppressed by cysteamine in a dose-dependent manner (p < 0.05), whereas MIF levels increased with cysteamine concentration of 0.5-10 mM (p = 0.008). CONCLUSIONS: These study results indicate that cysteamine induced the ROS-mediated inhibition of inflammatory cytokine release and proliferation of PBMCs stimulated by chemically injured keratocytes. Thus, cysteamine can be used in the treatment of chemical corneal burns.
Bromodeoxyuridine ; Burns ; Cysteamine* ; Enzyme-Linked Immunosorbent Assay ; Fluorescence ; Humans* ; Interleukin-6 ; Macrophages ; Reactive Oxygen Species ; Transforming Growth Factor beta1

PURPOSE: Inhibition of the intimal hyperplasia after vascular surgery is an important issue. The purpose of this study is to define whether perivascular application of rapamycin, imatinib mesylate or cysteamine can reduce intimal hyperplasia in a carotid balloon injury model. METHODS: Each drug was mixed with 40% pluronic gel solution and was topically applied over the injured carotid artery evenly. Two or four weeks after injury, the arteries were harvested and morphometric analysis was done. RESULTS: The medial areas were not significantly different in each group and a thinning of the media as a toxic drug effect was not observed in any treatment group. The intimal area and intima-to-media (I/M) ratio were significantly reduced in rapamycin-treated group and imatinib-treated group (P < 0.05). But cysteamine-treated group showed a trend of decrease in I/M ratio in 2 weeks, but no difference in 4 weeks. CONCLUSION: Perivascular delivery of imatinib or rapamycin with pluronic gel attenuated the development of intimal hyperplasia. But cysteamine did not. Further studies are needed to refine the optimal drug dosages in large animal models.
Arteries ; Benzamides ; Carotid Arteries ; Carotid Artery Injuries ; Cysteamine ; Hyperplasia* ; Imatinib Mesylate ; Mesylates* ; Models, Animal ; Piperazines ; Pyrimidines ; Sirolimus*

OBJECTIVETo investigate the transduction efficiency of purified PEP-1-CAT fusion protein into rat heart and the protective effect of the fusion protein against myocardial ischemia-reperfusion injury.

METHODSPEP-1-CAT or CAT (500 microg) was injected in SD rats via the caudal vein, using normal saline as the control, and the hearts were harvested at 0.5, 1, 2, 4, 8, and 24 h after the injection. The transduction efficiency was evaluated by immunofluorescence technique, and the CAT activity was measured. Forty rats were randomized into 5 groups, namely the sham-operated group, ischemia-reperfusion group, and 3 PEP-1-CAT -treated groups (100, 300, and 500 microg). The left main coronary artery was occluded for 1 h followed by a 2-h reperfusion, and at the end of reperfusion, serum LDH and CK and MDA content in the myocardium were measured.

RESULTSNo green fluorescence was observed in saline group or CAT group. Bright green fluorescence was observed in PEP-1-CAT groups at different time points, most conspicuous at 8 h. No significant difference in CAT activity was found between CAT group and saline group (P>0.05); with the lapse of time, CAT activity in PEP-1-CAT group increased gradually, reaching the peak level at 8 h, which was 4.2 folds of that in the saline group. LDH ,CK and MDA were significantly lower in PEP-1-CAT- groups than in ischemia-reperfusion group (P<0.01).

CONCLUSIONPEP-1 can mediate the transduction of CAT in rat heart in a time-dependent manner, and PEP-1-CAT preconditioning provides a protective effect against ischemia- reperfusion injury in rats.

Animals ; Catalase ; metabolism ; pharmacology ; Cysteamine ; analogs & derivatives ; metabolism ; pharmacology ; Ischemic Preconditioning, Myocardial ; Male ; Myocardial Reperfusion Injury ; metabolism ; pathology ; prevention & control ; Peptides ; metabolism ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; pharmacology ; Transduction, Genetic

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor. Although it is well known to have various physiological roles in cancer, its inhibitory effect on inflammation remains poorly understood. In the present study, a human PTEN gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-PTEN fusion protein. The expressed and purified PEP-1-PTEN fusion protein were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose- dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-PTEN protein was stable for 24 h. Transduced PEP-1-PTEN fusion protein inhibited the LPS-induced cyclooxygenase 2 (COX-2) and iNOS expression levels in a dose-dependent manner. Furthermore, transduced PEP-1-PTEN fusion protein inhibited the activation of NF-kappa B induced by LPS. These results suggest that the PEP-1-PTEN fusion protein can be used in protein therapy for inflammatory disorders.
Animals ; Cell Line ; Cyclooxygenase 2/*metabolism ; Cysteamine/*analogs & derivatives ; Enzyme Activation ; Humans ; Lipopolysaccharides/*pharmacology ; Macrophages/*metabolism ; Mice ; NF-kappa B/metabolism ; Nitric Oxide/*biosynthesis ; Nitric Oxide Synthase Type II/metabolism ; PTEN Phosphohydrolase/*genetics ; Peptides/*genetics ; Recombinant Fusion Proteins/*biosynthesis/genetics ; Signal Transduction

OBJECTIVETo investigate the penetrating ability of fusion protein PEP-1-EGFP with human umbilical vein endothelial cells.

METHODSTwo prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E. coli BL21 (DE3) to express EGFP and fusion protein PEP-1-EGFP, respectively. The expressed EGFP and PEP-1-EGFP were purified with Ni(2+) -resin affinity chromatography, and their capabilities of transduction into human umbilical vein endothelial cells were evaluated. The time- and dose-dependent transduction of the fusion protein PEP-1-EGFP and its stability in the human umbilical vein endothelial cells were observed. The toxicity of the fusion protein PEP-1-EGFP was detected by MTT method.

RESULTSEGFP failed to be transduced into human umbilical vein endothelial cells, whereas PEP-1-EGFP fusion protein was transduced into cells shortly in 5 minutes. Its transduction was time- and dose-dependent and the fluorescence in the cells were detected even 27 hours later. No cytotoxicity of the fusion protein PEP-1-EGFP to human umbilical vein endothelial cells was detected even when the dose reached up to 200 micromol/L.

CONCLUSIONPEP-1-EGFP fusion protein can efficiently transduce the target protein into human umbilical vein endothelial cells, which provides a basis for future researches on the transduction of antioxidant enzymes mediated by the cell-penetrating peptide, PEP-1, in ischemia-reperfusion injury therapy.

Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Green Fluorescent Proteins ; metabolism ; Humans ; Peptides ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; metabolism ; toxicity ; Umbilical Veins ; cytology

This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or beta-mercaptoethanol (beta-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 micrometer CYS or 100 micrometer beta-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. beta-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos.
Animals ; Cysteamine ; Embryo Culture Techniques/*veterinary ; Embryo, Mammalian/*physiology ; Female ; Follicular Fluid ; Mercaptoethanol ; Nuclear Transfer Techniques/*veterinary ; Oocytes/*growth & development ; Sus scrofa/*physiology ; Time Factors

OBJECTIVETo construct prokaryotic expression vector of pET15b-PEP-1-SOD1 and investigate whether PEP-1-SOD1 fusion protein could be transduced into human umbilical vein endothelial cells (HUVECs) and the effects on hypoxia/reoxygenation injury.

METHODSThe recombinant plasmids pET15b-SOD1 and pET15b-PEP-1-SOD1 were constructed and transformed into E. coli BL21 (DE3) to express SOD1 and PEP-1-SOD1 with an N-terminal His-tag. The purified SOD1 and PEP-1-SOD1 were incubated with HUVECs and the viability (MTT assay) and the release of lactate dehydrogenase (LDH) in culture medium were determined in the hypoxia/reoxygenation injury model. The morphological changes were observed under an inverted phase contrast microscope. The content of malondialdehyde (MDA) in HUVECs was also determined with the method of thiobarbituric acid.

RESULTSPEP-1-SOD1 fusion protein could be transduced into cultured HUVECs in a time- and dose-dependent manner. The intracellular enzymatic activity of PEP-1-SOD1 after 30 min incubation with HUVECs was significantly higher than control group (60.88 U/ml +/- 6.73 U/ml vs. 41.06 U/ml +/- 4.19 U/ml, P < 0.01). The transduced PEP-1-SOD1 protein was enzymatically stable for 24 h within cells. After hypoxia/reoxygenation injury, control HUVECs shrunk, became round-shaped and intercellular space increased, while these morphological changes were not observed in PEP-1-SOD1 transduced HUVECs. PEP-1-SOD1 transduction also markedly increased the viability, decreased LDH leakage into culture media and reduced the content of MDA post hypoxia/reoxygenation.

CONCLUSIONSPEP-1-SOD1 fusion protein could be efficiently transduced into HUVECs in a natively active form, and the delivered enzymatically active PEP-1-SOD1 exhibits cellular protection against hypoxia/reoxygenation injury in HUVECs. The transduction of SOD1 mediated by cell-penetrating peptide, PEP-1, provides a basis for further research on the prevention of ischemia/reperfusion injury in vivo.

Cell Hypoxia ; Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Endothelial Cells ; cytology ; Humans ; Malondialdehyde ; metabolism ; Peptides ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reperfusion Injury ; prevention & control ; Superoxide Dismutase ; genetics ; metabolism ; Transduction, Genetic ; Umbilical Veins ; cytology

OBJECTIVETo investigate the transduction ability of PEP-1-CAT fusion protein into human umbilical vein endothelial cell (HUVECs) and the effects on hydrogen-peroxide (H2O2)-induced oxidative stress injury in these cells.

METHODSWith the use of TA-cloning program and isocaudamer technique, the pET15b-PEP-1-CAT of prokaryotic expression plasmid was successfully constructed. The recombinant plasmid was transformed into E.coli BL21 (DE3) and the protein expression was induced by IPTG. The recombinant protein has an N-terminal His-tag which could be used to purify the target protein by affinity chromatography on a Ni2+-NTA-resin column. The fusion protein PEP-1-CAT was prepared and confirmed by specific enzyme activity in vitro. The purified PEP-1-CAT fusion protein was added on cultured HUVECs in vitro. The transduction ability of PEP-1-CAT fusion protein into cells was analyzed by Western blot and specific enzyme activity. The cells were treated with H2O2 (0.5 mmol/L) alone and in combination with PEP-1-CAT fusion protein for 4 h. Then, the cell viability, lactate dehydrogenase (LDH) and malondialdehyde (MDA) contents were measured.

RESULTSThe PEP-1-CAT fusion protein could be transduced into the cultured HUVECs in a dose- and time-dependent manner and be stable for at least 48 h. After H2O2 administration, cell viability was significantly reduced compared with control group (37.23%+/-5.68% vs. 100%, P<0.05), while LDH leakage (849.3 U/L+/-95.1 U/L) and MDA (8.23 nmol/L+/-1.58 nmol/L) content were significantly higher than that in control group (540.6 U/L+/-65.7 U/L and 2.46 nmol/L+/-1.42 nmol/L, respectively, all P<0.05). Preincubation with PEP-1-CAT proteins at various concentrations (0.25-2 micromol/L) significantly attenuated H2O2-induced cell injury.

CONCLUSIONThe PEP-1-CAT fusion protein could efficiently penetrate HUVECs and the transduced protein could attenuate cellular oxidative stress injury induced by H2O2. The PEP-1-CAT fusion protein might be a new strategy for preventing and treating oxidative stress induced diseases.

Catalase ; metabolism ; Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Endothelial Cells ; metabolism ; Humans ; Hydrogen Peroxide ; Oxidative Stress ; physiology ; Peptides ; metabolism ; Umbilical Veins ; cytology

Ghrelin, a novel gastrointestinal peptide with 28 amino acids, is secreted from the A-like cells of the gastric fundus. This peptide hormone does not only promote the release of growth hormone, but also stimulates food intake, gastric motility and cardiac output. Increased plasma ghrelin level has been reported in patients with upper gastrointestinal (GI) disease or in their disease animal model, suggesting its important role in the pathogenesis of upper GI disease.
Appetite/*physiology ; Cysteamine/metabolism ; Dyspepsia/etiology ; *Eating ; Gastrointestinal Diseases/*etiology ; Ghrelin/*physiology ; Humans ; Peptic Ulcer/etiology