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Biomedical subjects

A S Jun

Publications and source records attributed to A S Jun.

12 recordsLinked to original sources
PubMed2003

Prospects for gene therapy in corneal disease.

Transfer of cDNA to corneal cells has been accomplished using viral and nonviral vectors. Studies examining the feasibility and optimal methods for vector-mediated gene transfer to the cornea have, as in other tissues, been performed using histochemical or fluorescent marker genes. These have used corneal cells or cell lines in vitro, and whole corneas maintained in ex vivo culture. Gene-based interventions have been examined in specific corneal disorders such as allograft rejection, postexcimer laser scarring, and herpes simplex keratitis using experimental models. As the feasibility of genetic modification of corneal cells has been successfully demonstrated, there is great potential for gene therapy vectors in the treatment of human corneal disease. Continued improvements in vectors for gene transfer will improve the efficacy and safety of gene therapy. In addition to use of cDNA transfer as an alternative to drug or protein treatments in acquired corneal disorders, our expanding knowledge of the genetic basis of inherited corneal disorders will ultimately lead to the development of specific and effective gene therapies in this category of diseases.

Corneal Diseases↗
PubMed2001

Microarray analysis of gene expression in human donor corneas.

OBJECTIVES: To use microarray analysis to identify genes expressed in human donor corneas and to create a preliminary, comprehensive database of human corneal gene expression. METHODS: A complementary DNA (cDNA) library was constructed from transplant-quality, human donor corneas. Biotin-labeled RNA was transcribed from the cDNA library and hybridized in duplicate to microarrays containing approximately 5600 human genes. Results were analyzed using a gene database of the National Institutes of Health, Bethesda, Md. Reverse transcriptase polymerase chain reaction analysis was performed to confirm corneal expression of genes identified by microarray analysis. RESULTS: Duplicate microarrays identified the expression of 1200 genes in human donor corneas. Chromosomal loci had been assigned to 1025 (85%) of these genes. A preliminary database of human corneal gene expression was compiled. A Web site containing these genes was created. Six collagen genes were identified that had not previously been localized within the cornea. Five apoptosis-related genes were identified, 4 of which had not previously been localized within the cornea. Three genes previously shown to cause corneal diseases were identified. Reverse transcriptase polymerase chain reaction analysis of genes identified by microarray analysis confirmed the corneal expression of 2 apoptosis-related genes and 1 collagen gene. CONCLUSIONS: Microarray analysis of healthy human donor corneas has produced a preliminary, comprehensive database of corneal gene expression. Large-scale analysis of gene expression has the potential to generate large amounts of data, which should be made readily accessible to the scientific community. The Internet offers many potential advantages as a medium for the maintenance of these large data sets. CLINICAL RELEVANCE: Identification of structural, apoptosis-related, and disease-causing genes within the cornea by microarrays may increase the understanding of normal and abnormal corneal function with likely relevance to corneal diseases and transplants.

Chromosomes, Human↗
PubMed2000

Functional analysis of lymphoblast and cybrid mitochondria containing the 3460, 11778, or 14484 Leber's hereditary optic neuropathy mitochondrial DNA mutation.

Leber's hereditary optic neuropathy (LHON) is a form of blindness caused by mitochondrial DNA (mtDNA) mutations in complex I genes. We report an extensive biochemical analysis of the mitochondrial defects in lymphoblasts and transmitochondrial cybrids harboring the three most common LHON mutations: 3460A, 11778A, and 14484C. Respiration studies revealed that the 3460A mutation reduced the maximal respiration rate 20-28%, the 11778A mutation 30-36%, and the 14484C mutation 10-15%. The respiration defects of the 3460A and 11778A mutations transferred in cybrid experiments linking these defects to the mtDNA. Complex I enzymatic assays revealed that the 3460A mutation resulted in a 79% reduction in specific activity and the 11778A mutation resulted in a 20% reduction, while the 14484C mutation did not affect the complex I activity. The enzyme defect of the 3460A mutation transferred with the mtDNA in cybrids. Overall, these data support the conclusion that the 3460A and 11778A mutants result in complex I defects and that the 14484C mutation causes a much milder biochemical defect. These studies represent the first direct comparison of oxidative phosphorylation defects among all of the primary LHON mtDNA mutations, thus permitting insight into the underlying pathophysiological mechanism of the disease.

Cell Line↗
PubMed1999

Mitochondrial disease in superoxide dismutase 2 mutant mice.

Oxidative stress has been implicated in many diseases. The chief source of reactive oxygen species within the cell is the mitochondrion. We have characterized a variety of the biochemical and metabolic effects of inactivation of the mouse gene for the mitochondrial superoxide dismutase (CD1-Sod2(tm1Cje)). The Sod2 mutant mice exhibit a tissue-specific inhibition of the respiratory chain enzymes NADH-dehydrogenase (complex I) and succinate dehydrogenase (complex II), inactivation of the tricarboxylic acid cycle enzyme aconitase, development of a urine organic aciduria in conjunction with a partial defect in 3-hydroxy-3-methylglutaryl-CoA lyase, and accumulation of oxidative DNA damage. These results indicate that the increase in mitochondrial reactive oxygen species can result in biochemical aberrations with features reminiscent of mitochondrial myopathy, Friedreich ataxia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

Aconitate Hydratase↗
PubMed1996

Mitochondrial DNA sequence analysis of four Alzheimer's and Parkinson's disease patients.

The mitochondrial DNA (mtDNA) sequence was determined on 3 patients with Alzheimer's disease (AD) exhibiting AD plus Parkinson's disease (PD) neuropathologic changes and one patient with PD. Patient mtDNA sequences were compared to the standard Cambridge sequence to identify base changes. In the first AD+PD patient, 2 of the 15 nucleotide substitutions may contribute to the neuropathology, a nucleotide pair (np) 4336 transition in the tRNA(Gln) gene found 7.4 times more frequently in patients than in controls, and a unique np 721 transition in the 12S rRNA gene which was not found in 70 other patients or 905 controls. In the second AD+PD patient, 27 nucleotide substitutions were detected, including an np 3397 transition in the ND1 gene which converts a conserved methionine to a valine. In the third AD+PD patient, 2 polymorphic base substitutions frequently found at increased frequency in Leber's hereditary optic neuropathy patients were observed, an np 4216 transition in ND1 and an np 13708 transition in the ND5 gene. For the PD patient, 2 novel variants were observed among 25 base substitutions, an np 1709 substitution in the 16S rRNA gene and an np 15851 missense mutation in the cytb gene. Further studies will be required to demonstrate a causal role for these base substitutions in neurodegenerative disease.

Alzheimer Disease↗
PubMed1996

Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia.

A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I.

Cell Line, Transformed↗
PubMed1995

Mitochondrial DNA mutations in human degenerative diseases and aging.

A wide variety of mitochondrial DNA (mtDNA) mutations have recently been identified in degenerative diseases of the brain, heart, skeletal muscle, kidney and endocrine system. Generally, individuals inheriting these mitochondrial diseases are relatively normal in early life, develop symptoms during childhood, mid-life, or old age depending on the severity of the maternally-inherited mtDNA mutation; and then undergo a progressive decline. These novel features of mtDNA disease are proposed to be the product of the high dependence of the target organs on mitochondrial bioenergetics, and the cumulative oxidative phosphorylation (OXPHOS) defect caused by the inherited mtDNA mutation together with the age-related accumulation mtDNA mutations in post-mitotic tissues.

Adult↗
PubMed1995

Leber's hereditary optic neuropathy plus dystonia is caused by a mitochondrial DNA point mutation.

A novel point mutation in the ND6 subunit of complex I at position 14,459 of the mitochondrial DNA (MTND6*LDY T14459A) was identified as a candidate mutation for the highly tissue-specific disease. Leber's hereditary optic neuropathy plus dystonia. Since the MTND6*LDYT14459A mutation was identified in a single family, other pedigrees with the mutation are needed to confirm its association with the disease. Clinical, biochemical, and genetic characterization is reported in two additional pedigrees. Leber's hereditary optic neuropathy developed in two family members in one pedigree. The daughter had clinically silent basal ganglia lesions. In a second pedigree, a single individual presented with childhood-onset generalized dystonia and bilateral basal ganglia lesions. Patient groups that included individuals with Leigh's disease, dystonia plus complex neurodegeneration, and Leber's hereditary optic neuropathy did not harbor the MTND6*LDYT14459A mutation, suggesting that this mutation displays a high degree of tissue specificity, thus producing a narrow phenotypic range. These results confirm the association of the MTND6*LDYT14459A mutation with Leber's hereditary optic neuropathy and/or dystonia. As the first genetic abnormality that has been identified to cause generalized dystonia, this mutation suggests that nuclear DNA or mitochondrial DNA mutations in oxidative phosphorylation genes are important considerations in the pathogenesis of dystonia.

Adolescent↗
PubMed1994

A mitochondrial DNA mutation at nucleotide pair 14459 of the NADH dehydrogenase subunit 6 gene associated with maternally inherited Leber hereditary optic neuropathy and dystonia.

A five-generation Hispanic family expressing maternally transmitted Leber hereditary optic neuropathy and/or early-onset dystonia associated with bilateral basal ganglia lesions was studied. Buffy coat mitochondrial DNA (mtDNA) from a severely affected child was amplified by the polymerase chain reaction and greater than 90% sequenced. The mtDNA proved to be a Native American haplogroup D genotype and differed from the standard "Cambridge" sequence at 40 nucleotide positions. One of these variants, a G-to-A transition at nucleotide pair (np) 14459, changed a moderately conserved alanine to a valine at NADH dehydrogenase subunit 6 (ND6) residue 72. The np 14459 variant was not found in any of 38 Native American haplogroup D mtDNAs, nor was it detected in 108 Asian, 103 Caucasian, or 99 African mtDNAs. Six maternal relatives in three generations were tested and were found to harbor the mutation, with one female affected with Leber hereditary optic neuropathy being heteroplasmic. Thus, the np 14459 G-to-A missense mutation is specific to this family, alters a moderately conserved amino acid in a complex I gene, is a unique mtDNA variant in Native American haplogroup D, and is heteroplasmic, suggesting that it is the disease-causing mutation.

Adenine↗
PubMedDate not supplied

High-performance liquid chromatography assay of amiodarone in rat plasma.

PURPOSE: A high-performance liquid chromatographic method is described for the determination of amiodarone in rat plasma. METHODS: After liquid-liquid extraction, the separation of amiodarone from internal standard and endogenous components was accomplished using reversed phase chromatography. The mobile phase, a combination of monobasic potassium phosphate, methanol and acetonitrile, was run isocratically through a C(8) analytical column. The UV detection was at 254 nm for ethopropazine, the internal standard, and subsequently changed to 242 nm for amiodarone detection. RESULTS: Analytical run time was less than 13 min. Mean recovery was 75% and 82% for lower (0.5 microg/ml) and higher concentrations (5 microg/ml), respectively. The assay exhibited excellent linear relationships between peak height ratios and plasma concentrations; quantitation limit was at least 0.035 microg/ml, based on 100 microl of rat plasma. Accuracy and precision were <17% over the concentration range of 0.035 to 5 microg/ml. CONCLUSION. The assay was applied successfully to the measurement of amiodarone plasma concentrations in rats given the drug orally.

Administration, Oral↗