Ornithine Delta-aminotransferase Research Articles

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

CRISPR correction of the Finnish ornithine delta-aminotransferase mutation restores metabolic homeostasis in iPSC from patients with gyrate atrophy

Hyperornithinemia with gyrate atrophy of the choroid and retina (HOGA) is a severe recessive inherited disease, causing muscular degeneration and retinochoroidal atrophy that progresses to blindness. HOGA arises from mutations in the ornithine aminotransferase (OAT) gene, and nearly one-third of the known patients worldwide are homozygous for the Finnish founder mutation OAT c.1205 T > C p.(Leu402Pro). We have corrected this loss-of-function OAT mutation in patient-derived induced pluripotent stem cells (iPSCs) using CRISPR/Cas9. The correction restored OAT expression in stem cells and normalized the elevated ornithine levels in cell lysates and cell media. These results show an efficient recovery of OAT function in iPSC, encouraging the possibility of autologous cell therapy for the HOGA disease.

Cloning and molecular characterization of Triticum aestivum ornithine amino transferase (TaOAT) encoding genes

BackgroundOrnithine aminotransferase (OAT, EC:2.6.1.13), alternatively known as ornithine delta aminotransferase (δOAT), is a pyridoxal phosphate (PLP)-dependent enzyme involved in the conversion of ornithine into glutamyl-5-semi-aldehyde (GSA) and vice versa. Up till now, there has been no study on OAT in wheat despite the success of its isolation from rice, maize, and sorghum. This study focuses on identification and molecular characterization of OAT in wheat.ResultsIn total, three homeologous OAT genes in wheat genome were found on chromosome group 5, named as TaOAT-5AL, TaOAT-5BL, and TaOAT-5DL. Sequence alignment between gDNA and its corresponding cDNA obtained a total of ten exons and nine introns. A phylogenetic tree was constructed and results indicated that OATs shared highly conserved domains between monocots and eudicots, which was further illustrated by using WebLogo to generate a sequence logo. Further subcellular localization analysis indicated that they functioned in mitochondria. Protein-protein interactions supported their role in proline biosynthesis through interactions with genes, such as delta 1-pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), involved in the proline metabolic pathway. Promoter analysis exposed the presence of several stress responsive elements, implying their involvement in stress regulation. Expression profiling illustrated that TaOAT was highly induced in the wheat plants exposed to drought or salt stress condition. Upregulated expression of TaOATs was observed in stamens and at the heading stage. A potential role of TaOAT genes during floret development was also revealed. Furthermore, the transgenic plants overexpressing TaOAT showed enhanced tolerance to drought stress by increasing proline accumulation. In addition, salt tolerance of the transgenic plants was also enhanced.ConclusionTaOATs genes were involved in proline synthesis and nitrogen remobilization because they interacted with genes related to proline biosynthesis enzymes and arginine catabolism. In addition, TaOAT genes had a role in abiotic stress tolerance and a potential role in floret development. The results of this study may propose future research in the improvement of wheat resistance to abiotic stresses.

Exogenous ethylene alleviates chilling injury of ‘Huangguan’ pear by enhancing the proline content and antioxidant activity

Peel browning spots (PBS) is a chilling injury (CI) symptom in cold-stored ‘Huangguan’ pear. In the present study, the effect of exogenous ethylene on CI and its correlation to proline and reactive oxygen species (ROS) metabolism was investigated. The results showed that ethylene treatment (100 μL L−1 for 24 h) could completely inhibit PBS appearance without significantly affecting fruit firmness, soluble solids content (SSC) and titratable acidity (TA), enhance ethylene production rate at the initiate stage of storage, and promote proline accumulation by increasing activities of pyrroline-5-carboxylate synthetase (P5CS), ornithine-delta-aminotransferase (OAT) and up-regulating expression of P5CS1 and OAT1 genes, decreasing proline dehydrogenase (PDH) activity and the expression of PDH1 gene in peel. Moreover, the ethylene treatment suppressed the accumulation of H2O2, O2- and malondialdehyde (MDA), maintained higher levels of ascorbic acid (AsA) and glutathione (GSH), and meanwhile enhanced activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as expression of the genes such as SOD1, CAT1, and APX1 which could encode these enzymes in peel. These results suggested that ethylene alleviated the CI of ‘Huangguan’ pear by promoting the proline accumulation and antioxidant activity of peel.

Characterization of Barley Leaf Tolerance to Drought Stress by Chlorophyll Fluorescence and Electron Paramagnetic Resonance Studies

AbstractTwo kinds of barley genotypes with various water‐stress tolerances, tolerant Cam/B1 and sensitive Maresi, were subjected to 10‐day soil‐drought stress in seedling and flag leaf developmental phases. After this time, both genotypes regardless of the growth stage showed a decrease in quantum yield of PSII photochemistry (ΦPSII) upon stress treatment; however, this effect was stronger in the sensitive plants than in the tolerant ones. The drought stress in the flag leaf stage was associated with an increase in superoxide dismutase (SOD) level in both genotypes, whereas in seedlings, this effect was observed only for Maresi. The activity of other enzymes (catalase and peroxidase) was changed only in small degree. An increase in proline levels and activities of Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS) and ornithine delta‐aminotransferase (OAT) were observed independently of genotype and the phase of plant development, whereas the activity pyruvate dehydrogenase (PDH) decreased in tolerant genotype. Moreover, changes in the concentration of monocarbohydrates (glucose and fructose) and dicarbohydrates (saccharose, raffinose and maltose) were found: in seedlings, the amount of all soluble sugars increased, while in flag leaves decreased. The drought treatment resulted in a drop in starch level in the tolerant genotype, but in the sensitive one, the content of this substance increased in both developmental stages. EPR studies allowed the determination of the amount and character of organic radicals present in leaves. In control conditions, the content of these radical species was higher in the sensitive genotype than in tolerant one and decreased upon water stress, with the exception of flag leaves of the sensitive plant. Simulation procedure revealed four types of signals in the EPR spectra. One of them was attributed to a chlorophyll a cation and decreased upon drought. The second, ascribed to semiquinone radicals, reflected the redox balance disturbed by water deficit. The two remaining signals were connected with carbon‐centred radicals situated in the carbohydrate matrix. Their number was correlated with starch concentration.

Ornithine‐delta‐aminotransferase and proline dehydrogenase genes play a role in non‐host disease resistance by regulating pyrroline‐5‐carboxylate metabolism‐induced hypersensitive response

Non-host disease resistance involves the production of hypersensitive response (HR), a programmed cell death (PCD) that occurs at the site of pathogen infection. Plant mitochondrial reactive oxygen species (ROS) production and red-ox changes play a major role in regulating such cell death. Proline catabolism reactions, especially pyrroline-5-carboxylate (P5C) accumulation, are known to produce ROS and contribute to cell death. Here we studied important genes related to proline synthesis and catabolism in the defence against host and non-host strains of Pseudomonas syringae in Nicotiana benthamiana and Arabidopsis. Our results show that ornithine delta-aminotransferase (δOAT) and proline dehydrogenases (ProDH1 and ProDH2) are involved in the defence against non-host pathogens. Silencing of these genes in N. benthamiana delayed occurrence of HR and favoured non-host pathogen growth. Arabidopsis mutants for these genes compromised non-host resistance and showed a decrease in non-host pathogen-induced ROS. Some of the genes involved in proline metabolism were also induced by a pathogen-carrying avirulence gene, indicating that proline metabolism is influenced during effector-triggered immunity (ETI). Our results demonstrate that δOAT and ProDH enzyme-mediated steps produce ROS in mitochondria and regulate non-host HR, thus contributing to non-host resistance in plants.

Analysis of transcriptional activity of the Arabidopsis thaliana ornithine-delta-aminotransferase gene promoter

Cloning of the Arabidopsis thaliana genomic DNA fragment presumably corresponding to the promoter region of the ornithine-delta-aminotransferase (OAT) gene is reported. The reporter-gene construct, containing the Escherichia coli beta-glucouronidase gene under control of the OAT gene promoter was generated. The Nicotian tabacum SR1 transformants carrying this construct were obtained. It was demonstrated that under normal conditions, expression of the reporter gene was associated with the meristems and the zones of intensive shoot growth. Possible role of the OAT gene in nitrogen metabolism and shoot development is discussed.

Cloning, expression and purification of plant ornithine delta-aminotransferase

Cloning, expression and purification of plant ornithine delta-aminotransferase

Secondary creatine deficiency in ornithine delta-aminotransferase deficiency

Aims Ornithine delta-aminotransferase (OAT) deficiency causes gyrate atrophy (GA) of the retina, as a consequence of high plasma ornithine concentrations. Because creatine synthesis requires the conversion of arginine and glycine into ornithine and guanidinoacetate, high ornithine concentration inhibits this reaction thus causing secondary creatine deficiency. The aim of this study was to evaluate the neuropsychological features and creatine metabolism in patients with GA. Methods The study involved 7 GA patients, aged from 11 to 27 years who underwent neuropsychological evaluation and cerebral proton magnetic resonance spectroscopy (MRS). Results Neurocognitive impairment was found in 5/7 patients, including mental retardation (3/7), school failure (1/7), major visuospatial dyspraxia (1/7), aggressive behavior (3/7) and epilepsy (2/7). Two patients had normal neuropsychological evaluation. Cerebral proton magnetic resonance spectroscopy revealed a profound creatine deficiency in all patients. MRS data were confirmed by decreased levels of creatine and/or guanidinoacetate in plasma and urine in all patients. Conclusions In our group of patients with GA, we found a high prevalence of neurological impairment, not reported so far, and possibly related to secondary creatine deficiency and hyperornithinemia. We propose to treat mentally retarded GA patients with high doses of creatine, as it may normalize brain creatine levels and help to reduce ornithine levels.

Functional genomics and SNP analysis of human genes encoding proline metabolic enzymes

Proline metabolism in mammals involves two other amino acids, glutamate and ornithine, and five enzymatic activities, Delta(1)-pyrroline-5-carboxylate (P5C) reductase (P5CR), proline oxidase, P5C dehydrogenase, P5C synthase and ornithine-delta-aminotransferase (OAT). With the exception of OAT, which catalyzes a reversible reaction, the other four enzymes are unidirectional, suggesting that proline metabolism is purpose-driven, tightly regulated, and compartmentalized. In addition, this tri-amino-acid system also links with three other pivotal metabolic systems, namely the TCA cycle, urea cycle, and pentose phosphate pathway. Abnormalities in proline metabolism are relevant in several diseases: six monogenic inborn errors involving metabolism and/or transport of proline and its immediate metabolites have been described. Recent advances in the Human Genome Project, in silico database mining techniques, and research in dissecting the molecular basis of proline metabolism prompted us to utilize functional genomic approaches to analyze human genes which encode proline metabolic enzymes in the context of gene structure, regulation of gene expression, mRNA variants, protein isoforms, and single nucleotide polymorphisms.

Ornithine Transport Via Cationic Amino Acid Transporter-1 Is Involved in Ornithine Cytotoxicity in Retinal Pigment Epithelial Cells

A prior report showed ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells in an in vitro model of gyrate atrophy of the choroid and retina. This study was intended to clarify the mechanism of ornithine cytotoxicity and to determine the responsible amino acid transporters. The mRNA expression of amino acid transporters in human telomerase reverse transcriptase (hTERT)-RPE cells was examined by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis. Carrier-mediated ornithine transport via the L-type amino acid transporter (LAT)1, LAT2, cationic amino acid transporter (CAT)-1, and y(+)LAT2 systems was evaluated by short interfering (si)RNA-mediated gene silencing. The cytoprotective effect of CAT-1-specific siRNA on ornithine cytotoxicity was measured using quantitative analysis of cellular adenosine triphosphate (ATP) at 24 hours after treatment with ornithine in OAT-deficient RPE cells. LAT1, LAT2, CAT-1, and y(+)LAT2 mRNA expression was detected by Northern blot analysis, whereas RT-PCR revealed that LAT1, LAT2, y(+)LAT1, y(+)LAT2, CAT-1, and b(0,+)AT mRNAs were expressed together with the heterodimeric glycoproteins 4F2hc and rBAT in hTERT-RPE cells. l-[(14)C]ornithine uptake in hTERT-RPE cells was decreased by 46.6% and 22.0% by CAT-1 and y(+)LAT2 siRNA, respectively, whereas LAT1 and LAT2 siRNA had no significant effect. Further, CAT-1 silencing by siRNA reduced ornithine cytotoxicity in OAT-deficient RPE cells. The results suggest that ornithine transport via CAT-1 may play a crucial role in ornithine cytotoxicity in hTERT-RPE cells. Reduction of the ornithine transport via CAT-1 may be a new target for treatment of gyrate atrophy.

Transgenic Medicago truncatula plants that accumulate proline display nitrogen‐fixing activity with enhanced tolerance to osmotic stress

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.

Osteoporosis Associated with Gyrate Atrophy: a Case Report

Gyrate atrophy (GA) is a rare degenerative, hereditary disease characterized by markedly high serum ornithine levels resulting from the deficiency of ornithine-delta-amino transferase (OAT), a mitochondrial matrix enzyme. Hyperornithinaemia is accompanied by lysinuria and reduced lysine plasma levels in GA. Type I collagen is known to play a role in osteoporosis pathogenesis. Lysine has a role in cross ligament formation of type I collagen, a bone matrix element, and thus, in bone strength. Although the most common complaint in GA is visual problems, the disease may include muscle involvement, as well. Reduced physical activity resulting from muscle involvement and hypolysinemia in GA may lead to osteoporosis. However, there is no data in the literature concerning the relation between GA and osteoporosis. In this report a GA case with early osteoporosis, besides visual deterioration and muscular signs, is reported, and the relation between GA and osteoporosis is emphasized for the first time.

Dietary compliance in ornithine aminotransferase deficiency

Ornithine delta-aminotransferase (OAT) deficiency (McKusick 258870) is associated with hyperornithinaemia, thought to be the cause of the progressive retinal degeneration that occurs in this disorder. For the large majority of cases unresponsive to the co-factor pyridoxine, treatment is based on reducing ornithine plasma levels below 400 micromol/L with an arginine-restricted diet. This has been shown to slow the progression of retinal disease. (Santinelli et al 2004). In Table 1 we present our experience in the dietary management of 12 patients (7 female) from 8 families. Compliance was defined as good, intermediate or poor according to plasma ornithine levels. Only one patient could be categorized as a good complier, 5 were intermediate, and 6 were poor. The age at start of treatment was the most important factor as regards ability to comply with diet. Our study emphasizes the difficulty with dietary treatment and need for early diagnosis. For the older patients, alternative treatments such as the use of oral lysine to increase renal losses of ornithine need to be investigated further (Peltola et al 2000).

B6‐responsive disorders: A model of vitamin dependency

Pyridoxal phosphate is the cofactor for over 100 enzyme-catalysed reactions in the body, including many involved in the synthesis or catabolism of neurotransmitters. Inadequate levels of pyridoxal phosphate in the brain cause neurological dysfunction, particularly epilepsy. There are several different mechanisms that lead to an increased requirement for pyridoxine and/or pyridoxal phosphate. These include: (i) inborn errors affecting the pathways of B(6) vitamer metabolism; (ii) inborn errors that lead to accumulation of small molecules that react with pyridoxal phosphate and inactivate it; (iii) drugs that react with pyridoxal phosphate; (iv) coeliac disease, which is thought to lead to malabsorption of B(6) vitamers; (v) renal dialysis, which leads to increased losses of B(6) vitamers from the circulation; (vi) drugs that affect the metabolism of B(6) vitamers; and (vii) inborn errors affecting specific pyridoxal phosphate-dependent enzymes. The last show a very variable degree of pyridoxine responsiveness, from 90% in X-linked sideroblastic anaemia (delta-aminolevulinate synthase deficiency) through 50% in homocystinuria (cystathionine beta-synthase deficiency) to 5% in ornithinaemia with gyrate atrophy (ornithine delta-aminotransferase deficiency). The possible role of pyridoxal phosphate as a chaperone during folding of nascent enzymes is discussed. High-dose pyridoxine or pyridoxal phosphate may have deleterious side-effects (particularly peripheral neuropathy with pyridoxine) and this must be considered in treatment regimes. None the less, in some patients, particularly infants with intractable epilepsy, treatment with pyridoxine or pyridoxal phosphate can be life-saving, and in other infants with inborn errors of metabolism B(6) treatment can be extremely beneficial.

Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features.

The model legume plant Medicago truncatula accumulates free proline in response to hyperosmotic stress as do many other organisms. In order to analyse the transcriptional regulation of proline biosynthesis in M. truncatula, three cDNAs encoding Delta(1)-pyrroline-5-carboxylate synthetase (P5CS1, P5CS2; EC not assigned) and ornithine delta-aminotransferase (OAT; EC 2.6.1.13) were isolated. The cDNAs shared high homologies with the other plant sequences and genomic organization analysis indicated the presence of two P5CS and two putative OAT genes. The two P5CS genes showed differing transcript level regulation according to organs and in response to osmotic stress. MtP5CS1 steady-state transcript levels in the different plant organs were correlated with proline levels but transcript abundance was unaffected by osmotic stresses. MtP5CS2 transcripts were poorly detected in all organs but were strongly accumulated in shoots of salt-stressed plants. We suggest a specific of MtP5CS1 and MtP5CS2 as a housekeeping product and as a stress specific isoform, respectively. MtOAT transcripts were predominantly detected in roots and shoots of unstressed plants. Salt-stress treatment induced the accumulation of MtOAT transcripts in the whole plant whatever the developmental stage. In salt-stressed roots, a positive correlation was found between proline and MtOAT transcript accumulation. These results suggest that both ornithine and glutamate biosynthesis pathways contribute to the osmotic stress-induced proline accumulation in M. truncatula.

Prevention of ornithine cytotoxicity by nonpolar side chain amino acids in retinal pigment epithelial cells.

To investigate the effect of amino acids on ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy (GA) of the choroid and retina. RPE cells were treated with 0.5 mM 5-fluoromethylornithine (5-FMOrn), a specific and irreversible OAT inhibitor. OAT-deficient RPE cells were incubated with 10 mM ornithine in the presence of 20 mM of 1 of 18 amino acids or 10 mM 2-amino-2-norbornane-carboxylic acid (BCH), a conventional inhibitor of the amino acid transporter system L. Ornithine cytotoxicity and cytoprotective effects of each amino acid was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay 72 hours after treatment with ornithine in OAT-deficient RPE cells. Ornithine incorporation into RPE cells was evaluated using DL-[14C]ornithine. An MTT colorimetric assay revealed that small and large zwitterionic amino acids, but not acidic or basic amino acids, decreased ornithine cytotoxicity in OAT-deficient RPE cells. Incorporation of DL-[14C]ornithine by RPE cells decreased to 79% of the control level after incubation for 48 hours with 20 mM leucine, the most effective cytoprotective amino acid. Further, BCH prevented ornithine cytotoxicity in a dose-dependent manner. Both light and heavy chains of L-type amino acid transporter (LAT)-1, LAT2, y+LAT1, and 4F2hc were expressed in RPE cells. The present results demonstrate that L-type amino acid transporter(s) may be involved in protection against ornithine cytotoxicity in human RPE cells. Thus, amino acid transportation in RPE cells may be a good target for a new therapy for GA as well as other kinds of chorioretinal degeneration.

Ornithine aminotransferase, a potential target for the treatment of hyperammonemias.

Ornithine-delta-aminotransferase (OAT) (EC 2.6.1.13) is a pyridoxal-5' phosphate dependent mitochondrial matrix enzyme. It controls the L-ornithine (Orn) level in tissues by catalysing the transfer of the delta-amino group of Orn to 2-oxoglutarate. The products of this reaction are L-glutamate-gamma-semialdehyde and L-glutamate. Among the compounds known to inhibit (or inactivate) OAT, only L-canaline and (SS)-5-(fluoromethyl)ornithine [(SS)-5FMOrn] are selective for OAT. Treatment of laboratory animals with 5FMOrn causes a dramatic accumulation of Orn in most tissues and organs, and the enhanced formation of urea due to saturation of ornithine:carbamoyltransferase with its substrate. The enhancement of urea formation by increased endogenous levels of Orn is comparable with that produced by large doses of Orn and arginine, a treatment known to enhance the detoxification of ammonia. However, protection to lethal doses of ammonium salts by exogenous Orn is rapidly fading. In contrast, inactivation of OAT by a small dose of 5FMOrn renders a long-lasting protective effect against various forms of hyperammonemic states. Among these the reduction of ammonia concentrations in blood and tissues, and the reduction of the pathologic excretion of orotic acid to normal levels in mice with hereditary defects of the urea cycle, were most impressive. In human hereditary OAT deficiency the elevated intraocular concentrations of Orn are considered to be a cause of gyrate atrophy. This is presumably the reason, why OAT has not been considered as a therapeutically useful target. Chronic inactivation of OAT by repeated administration of 5FMOrn, caused elevated intraocular Orn concentrations, but this treatment had no effect on the function and histology of the visual system, or the behaviour of adult mice. The confirmation of this and related observations in higher species will show, whether OAT inactivation has potentials in the treatment of hyperammonemic states.

Correction of ornithine accumulation prevents retinal degeneration in a mouse model of gyrate atrophy of the choroid and retina.

Deficiency of ornithine-delta-aminotransferase (OAT) in humans results in gyrate atrophy of the choroid and retina (GA), an autosomal recessive disorder characterized by ornithine accumulation and a progressive chorioretinal degeneration of unknown pathogenesis. To determine whether chronic, systemic reduction of ornithine can prevent this form of retinal degeneration, we used an arginine-restricted diet to maintain long term reduction of ornithine in a mouse model of OAT-deficiency (Oat(-/-)) produced by gene targeting. We evaluated the mice over a 12-month period by measurement of plasma amino acids, electroretinograms, and retinal histologic and ultrastructural studies. We found that an arginine-restricted diet substantially reduces plasma ornithine levels and completely prevents retinal degeneration in Oat(-/-). This result indicates that ornithine accumulation is a necessary factor in the pathophysiology of the retinal degeneration in GA and that restoration of OAT activity in retina is not required for effective treatment of GA.

Ornithine-delta-aminotransferase expression and ornithine metabolism in cultured epidermal keratinocytes: toward metabolic sink therapy for gyrate atrophy.

There is now strong evidence that the chorioretinal degeneration associated with ornithine-delta-aminotransferase (OAT) deficiency is a consequence of hyperornithinemia. Therefore development of a metabolic system for clearing ornithine from the circulation is being pursued as a potential treatment. The skin is considered an attractive location for such a metabolic system because autologous cells can be safely and easily utilized. This study was undertaken to determine the ornithine metabolizing capacity of epidermal keratinocytes expressing normal and superphysiologic amounts of OAT. The data show that overexpression of OAT in keratinocytes cultured from a gyrate atrophy patient restores ornithine metabolism and results in a rate of ornithine disappearance from the medium that is significantly higher than the rate of disappearance from the medium bathing normal keratinocytes. In addition, OAT activity determined in soluble protein prepared from sonicates suggests that the capacity to maintain plasma ornithine within the normal range is contained within an accomplishable graft of keratinocytes overexpressing OAT. However, the actual rate of ornithine disappearance from the media was significantly less than predicted from enzyme activity assays. Following ornithine metabolite production by intact cells suggests that ornithine metabolism is limited primarily by clearance of downstream metabolites, as opposed to substrate delivery.