Effect of Advanced Glycation End Products on Oxidative Stress and Senescence of Trabecular Meshwork Cells

PURPOSE: To investigate the effect of advanced glycation end products (AGE) on oxidative stress and cellular senescence in cultured human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to 0, 10, 50, 100, 200 microg/mL of glycated bovine serum albumin (G-BSA) for 5 days. Also co-exposed were L-arginine, sepiapterin, and antioxidant N-acetylcysteine (NAC). Cellular survival and production of nitric oxide (NO), superoxide, and reactive oxygen species were assessed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay, Griess assay, cytochrome c assay, and dichlorofluorescin diacetate assay, respectively. Senescence-associated beta-galactosidase staining was performed to quantify the degree of cellular senescence. RESULTS: G-BSA decreased cellular survival, NO production, and increased superoxide production significantly in a dose-dependent manner. The effects of G-BSA were abolished with co-exposure of L-arginine, sepiapterin, and NAC. G-BSA enhanced cellular senescence accompanied by increased production of reactive oxygen species. G-BSA-induced cellular senescence was suppressed by application of L-arginine, sepiapterin, and NAC. CONCLUSIONS: AGE enhances cellular senescence of HTMC accompanied with increased oxidative stress. AGE-induced oxidative stress and cellular senescence could be delayed by application of anti-oxidants.

Mutations of the phenylalanine hydroxylase (PAH) gene in Filipino patients with phenylketonuria

INTRODUCTION: Phenylketonuria (PKU), an autosomal recessive metabolic disorder caused by phenylalanine hydroxylase (PAH) deficiency, leads to hyperphenylalaninemia and neurological damage if untreated. This is the first study in the Philippines to identify the disease-causing mutations in the PAH gene of clinically diagnosed Filipino PKU patients. METHODS: The study included four unrelated PKU patients detected by the Philippine Newborn Screening Program from 1996 to 2008. Plasma amino acid analyses for all patients showed increased phenylalanine and low to normal tyrosine levels consistent with the diagnosis of PKU. Mutations in the PAH gene were identified by genomic DNA extraction from dried blood spots of the patients, PAH exon amplification by polymerase chain reaction and subsequent bi-directional DNA sequence analysis. RESULTS: All patients presented with significantly elevated phenylalanine levels on bacterial inhibition assay and thin layer chromatography. Urinary pterins confirmed the diagnosis of Tetrahydrobiopterin deficiency in two patients while the other 2 patients had the Classical PKU phenotype. Four previously identified mutations in the PAH gene (p.I65T, p.R413P, p.EX6-96A>G, p.R243Q) were identified in those with Classical PKU. CONCLUSION: The present results confirm the heterogeneity of mutations at the PAH locus in Filipinos. Neonatal screening and the use of molecular diagnosis significantly aid in the medical management and genetic counseling of patients and their families.

Two Filipino patients with 6-pyruvoyltetrahydropterin synthase deficiency

Hyperphenylalaninemia can result from defects in either the phenylalanine hydroxylase (PAH) enzyme or in the synthesis or recycling of the active pterin, tetrahydrobiopterin (BH4), which is an obligate co-factor for the PAH enzyme, as well as tyrosine hydroxylase and tryptophan hydroxylase. One of the most common causes of BH4 deficiency is a defect in the synthesis of 6-pyruvoyltetrahydropterin synthase (PTPS) enzyme. Patients present with progressive neurological disease such as mental retardation, convulsions and disturbance of tone and posture despite strict adherence to diet and good metabolic control. The authors report the first two cases of PTPS deficiency in the Philippines. Both are females with initial phenylalanine levels of more than 1300 umol/L who continued to develop neurologic deterioration despite good metabolic control and strict adherence to diet. Further investigation showed that they both had PTPS deficiency. Treatment was started with BH4, L-dopa/carbidopa, and 5-hydroxytryptophan (5HT) with concomitant significant improvements in their neurologic and developmental outcomes.

Effect of High Glucose on the Oxidative Stress in Trabecular Meshwork Cells

PURPOSE: To investigate the effect of high glucose (HG) on the oxidative stress in cultured human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to low glucose (5 mM) and HG (25 mM) for 7 days. Additionally, 1 mM L-arginine, 5 mM DAHP, 10 microgram/ml insulin, 100 micrometer L-ascorbic acid, 10, and 100 micrometer sepiapterin were co-exposed. The cellular survival and nitric oxide (NO) production were assessed by MTT assay and Griess assay, respectively. Superoxide production was measured by modified cytochrome c assay. RESULTS: HG did not affect the survival of cultured HTMC significantly. HG decreased NO production. Co-exposed DAHP decreased but DAHP and insulin increased NO production. In addition, HG increased superoxide production, which was decreased by insulin, L-ascorbic acid, and sepiapterin. CONCLUSIONS: HG decreased NO production accompanied with increased superoxide production in HTMC. Thus HG induces oxidative stress in HTMC and may cause cellular dysfunction and damage of the trabecular meshwork.

Effect of Dexamethasone on the Production of Nitric Oxide in Trabecular Meshwork Cells

PURPOSE: To investigate the effects of dexamethasone (DEX) on the production of nitric oxide (NO) and its enzymatic synthetic pathway in cultured human trabecular meshwork (HTM) cells. METHODS: Primarily cultured HTM cells were exposed to 0, 10, 100, 1000 nM of DEX for 3 days. In addition, 100 micrometer sepiapterin, 100 micrometer ascorbic acid, and 10 micrometer methotrexate were co-exposed to DEX. The cellular survival and nitrite production rates were assessed by MTT assay and Griess assay, respectively. RESULTS: DEX did not significantly affect the survival of cultured HTM cells. DEX decreased the NO production in a dose-dependent manner. With co-exposure of DEX, ascorbic acid nullified the DEX-induced decrease of NO production. Sepiapterin and methotrexate did not affect DEX-induced decrease of NO production. CONCLUSIONS: DEX decreased NO production in HTM cells and the de novo pathway of tetrahydrobiopterin may be involved. This decrease may raise intraocular pressure by decreasing trabecular outflow.

Effect of Dexamethasone on the Production of Nitric Oxide in Trabecular Meshwork Cells

PURPOSE: To investigate the effects of dexamethasone (DEX) on the production of nitric oxide (NO) and its enzymatic synthetic pathway in cultured human trabecular meshwork (HTM) cells. METHODS: Primarily cultured HTM cells were exposed to 0, 10, 100, 1000 nM of DEX for 3 days. In addition, 100 micrometer sepiapterin, 100 micrometer ascorbic acid, and 10 micrometer methotrexate were co-exposed to DEX. The cellular survival and nitrite production rates were assessed by MTT assay and Griess assay, respectively. RESULTS: DEX did not significantly affect the survival of cultured HTM cells. DEX decreased the NO production in a dose-dependent manner. With co-exposure of DEX, ascorbic acid nullified the DEX-induced decrease of NO production. Sepiapterin and methotrexate did not affect DEX-induced decrease of NO production. CONCLUSIONS: DEX decreased NO production in HTM cells and the de novo pathway of tetrahydrobiopterin may be involved. This decrease may raise intraocular pressure by decreasing trabecular outflow.

Mutational analysis of patients with 6-pyruvoyltetrahydrobiopterin synthesis deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the gene mutation spectrum of patients with 6-pyruvoyltetrahydrobiopterin synthesis deficiency (PTPSD) in Mainland China.</p><p><b>METHODS</b>The 6-pyruvoyltetrahydrobiopterin synthesis gene lz(PTS)lz was analyzed in 55 PTPSD patients by using PCR-restriction fragment length polymorphism (PCR-RFLP) and direct DNA sequencing. The relationship between the genotype and phenotype was analyzed.</p><p><b>RESULTS</b>Eighteen mutations were identified and the detection rate of gene mutation was 95.28%. Four hot-spot mutations, namely P87S (40.57%), N52S(13.21%), D96N(12.26%) and IVS1nt-291A to G(10.38%) were found in this study, and the first three were associated with severe phenotype. The P87L was reported firstly in Chinese patients, and the Q13X, M80T, IVS4nt-2A to G, L93M and K131N were novel mutations.</p><p><b>CONCLUSION</b>The P87S, N52S, D96N and IVS1nt-291A to G mutations are the hot-spots mutations of the PTS gene in Chinese PTPSD patients. Using PCR-RFLP technique to screen the mutations in the PTS gene can increase the efficiency of gene diagnosis.</p>

6-pyruvoyltetrahydropterin synthase deficiency two-case report.

6-Pyruvoyltetrahydropterin Synthase (PTPS) deficiency is the most common cause of hyperphenylalaninemia due to tetrahydrobiopterin deficiency. The presenting symptoms of PTPS deficiency are mental retardation, convulsions, disturbance of tone and posture, drowsiness, irritability, abnormal movements, hypersalivation, and swallowing difficulties(1-3). The authors reported the first two cases of PTPS deficiency in Thailand. Both cases were male infants who showed phenylalanine levels of 25.23 mg/dl and 23.4 mg/dl respectively. The urinary pterins analysis showed low biopterin and high neopterin. The percentage of urinary biopterin was also found to be very low. The mutation analysis of the first case revealed a point mutation of exon 4, a homozygous C to T transition at nucleotide 200 in codon 67 (T67M), and the second case showed a compound heterozygous of exon 4, C to A transition at nucleotide 200, and exon 5, C to T transition at nucleotide 259 of the PTS gene confirming that they had PTPS deficiency. Treatment was started with neurotransmitters and a low phenylalanine diet. Family carriers were detected by means of urinary pterins determination and mutation analysis.