RESUMO
BackgroundPyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5'-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available.MethodsWe combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening.ResultsWe identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system.ConclusionThis study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients.FundingSociety for Inborn Errors of Metabolism for Netherlands and Belgium (ESN), United for Metabolic Diseases (UMD), Stofwisselkracht, Radboud University, Canadian Institutes of Health Research, Dutch Research Council (NWO), and the European Research Council (ERC).
Assuntos
Epilepsia/metabolismo , Metabolômica , Ácidos Pipecólicos/metabolismo , Aldeído Desidrogenase/deficiência , Aldeído Desidrogenase/metabolismo , Animais , Biomarcadores/metabolismo , Criança , Epilepsia/genética , Feminino , Humanos , Camundongos , Camundongos Knockout , Espectrofotometria Infravermelho , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Plant-parasitic nematodes are devastating pathogens of many important agricultural crops. They have been successful in large part due to their ability to modify host plant metabolomes to their benefit. Both root-knot and cyst nematodes are endoparasites that have co-evolved to modify host plants to create sophisticated feeding cells and suppress plant defenses. In contrast, the ability of migratory ectoparasitic nematodes to modify host plants is unknown. Based on global metabolomic profiling of sting nematodes in African bermudagrass, ectoparasites can modify the global metabolome of host plants. Specifically, sting nematodes suppress amino acids in susceptible cultivars. Upregulation of compounds linked to plant defense have negative impacts on sting nematode population densities. Pipecolic acid, linked to systemic acquired resistance induction, seems to play a large role in protecting tolerant cultivars from sting nematode feeding and could be targeted in breeding programs.
Assuntos
Cynodon/parasitologia , Metaboloma/imunologia , Ácidos Pipecólicos/metabolismo , Doenças das Plantas/imunologia , Tylenchoidea/patogenicidade , Animais , Cynodon/imunologia , Cynodon/metabolismo , Resistência à Doença , Interações Hospedeiro-Parasita , Metabolômica , Ácidos Pipecólicos/imunologia , Melhoramento Vegetal , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Tylenchoidea/imunologia , Tylenchoidea/metabolismoRESUMO
L-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like L-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.
Assuntos
Aminoácidos Neutros/biossíntese , Lisina/biossíntese , Aminoácidos/química , Ácido Aminocaproico/química , Materiais Biocompatíveis/química , Cadaverina/metabolismo , Caprolactama/química , Química Farmacêutica , Corynebacterium glutamicum/metabolismo , Escherichia coli/metabolismo , Fermentação , Química Verde , Microbiologia Industrial , Lactamas/química , Ácidos Pipecólicos/metabolismo , Piperidonas/química , Polímeros/químicaRESUMO
The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/µ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction.
Assuntos
Arabidopsis/imunologia , Ácidos Pipecólicos/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal , Pseudomonas syringae/imunologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Cetoácidos/imunologia , Cetoácidos/metabolismo , Leucina/imunologia , Leucina/metabolismo , Lisina/imunologia , Lisina/metabolismo , Metionina/imunologia , Metionina/metabolismo , Ácidos Pipecólicos/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Pseudomonas syringae/fisiologia , Transaminases/genética , Transaminases/imunologia , Transaminases/metabolismoRESUMO
Regulation of taste is important for improving meat quality and glutamate (Glu) is one of the important taste-active components in meat. Here, the effects of dietary lysine (Lys) content on taste-active components in meat, especially free Glu, were investigated. Fourteen-day-old broiler chicks (Gallus gallus) were fed on diets containing 100% or 150% of the recommended Lys content for 10 days. Concentrations of free amino acids in plasma, muscle and liver were measured. The levels of messenger RNAs (mRNAs) for enzymes related to Glu metabolism were determined in muscle and liver. The concentration of muscle metabolites was also determined. The free Glu content in muscle of chicks fed the Lys150% diet was increased by 44.0% compared with that in chicks fed the Lys100% diet (P < 0.01). The mRNA level of lysine α-ketoglutarate reductase, which is involved in Lys degradation and Glu production, was significantly increased (P < 0.05) in the Lys150% group. Metabolome analysis showed that the Lys degradation products, muscular saccharopine, pipecolic acid and α-aminoadipic acid, were increased in the Lys150% group. Our results suggest that free Glu content in muscle is regulated by Lys degradation. These results suggest that a short-term feeding of high-Lys diet could improve the taste of meat.
Assuntos
Ração Animal , Galinhas/metabolismo , Qualidade dos Alimentos , Ácido Glutâmico/análise , Lisina/administração & dosagem , Carne/análise , Ácido 2-Aminoadípico/metabolismo , Animais , Suplementos Nutricionais , Feminino , Ácido Glutâmico/metabolismo , Fígado/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Músculos/metabolismo , Ácidos Pipecólicos/metabolismo , RNA Mensageiro/metabolismo , Sacaropina Desidrogenases/genética , Sacaropina Desidrogenases/metabolismoRESUMO
Here, we describe the development and validation of a quantitative analytical method for rapid evaluation of protein-compound interactions. The method uses size-exclusion chromatography in a 96-well format with liquid chromatography/mass spectrometry (qSEC-LC/MS) by which the amount of a compound that was originally in complex with a target protein is determined as an indicator of the binding affinity. Proof of concept of this new analytical approach was performed using a thrombin-inhibitor model. The results showed that the qSEC-LC/MS could be developed into an effective affinity-based analytical technique, despite a few limitations such as difficulty in determining the K(d) value accurately.
Assuntos
Cromatografia em Gel/métodos , Cromatografia Líquida/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Espectrometria de Massas/métodos , Proteínas/metabolismo , Animais , Antitrombinas/metabolismo , Antitrombinas/farmacologia , Arginina/análogos & derivados , Bovinos , Concentração Inibidora 50 , Ácidos Pipecólicos/metabolismo , Ácidos Pipecólicos/farmacologia , Proteínas/análise , Reprodutibilidade dos Testes , Sulfonamidas , Trombina/análise , Trombina/metabolismoRESUMO
The mammalian degradation of lysine is believed to proceed via two distinct routes, the saccharopine and the pipecolic acid routes, that ultimately converge at the level of alpha-aminoadipic semialdehyde (alpha-AASA). alpha-AASA dehydrogenase-deficient fibroblasts were grown in cell culture medium supplemented with either L-[alpha-(15)N]lysine or L-[epsilon-(15)N]lysine to explore the exact route of lysine degradation. L-[alpha-(15)N]lysine was catabolised into [(15)N]saccharopine, [(15)N]alpha-AASA, [(15)N]Delta(1)-piperideine-6-carboxylate, and surprisingly in [(15)N]pipecolic acid, whereas L-[epsilon-(15)N]lysine resulted only in the formation of [(15)N]saccharopine. These results imply that lysine is exclusively degraded in fibroblasts via the saccharopine branch, and pipecolic acid originates from an alternative precursor. We hypothesize that pipecolic acid derives from Delta(1)-piperideine-6-carboxylate by the action of Delta(1)-pyrroline-5-carboxylic acid reductase, an enzyme involved in proline metabolism.
Assuntos
Aldeído Desidrogenase/deficiência , Fibroblastos/enzimologia , Lisina/metabolismo , Proteínas de Neoplasias/deficiência , Ácidos Pipecólicos/metabolismo , Linhagem Celular , Humanos , L-Aminoadipato-Semialdeído Desidrogenase , Ácidos Picolínicos/metabolismo , Pirróis/metabolismoRESUMO
Unbalanced diets generate oxidative stress commonly associated with the development of diabetes, atherosclerosis, obesity and cancer. Dietary flavonoids have antioxidant properties and may limit this stress and reduce the risk of these diseases. We used a metabolomic approach to study the influence of catechin, a common flavonoid naturally occurring in various fruits, wine or chocolate, on the metabolic changes induced by hyperlipidemic diets. Male Wistar rats ( n = 8/group) were fed during 6 weeks normolipidemic (5% w/w) or hyperlipidemic (15 and 25%) diets with or without catechin supplementation (0.2% w/w). Urines were collected at days 17 and 38 and analyzed by reverse-phase liquid chromatography-mass spectrometry (LC-QTOF). Hyperlipidic diets led to a significant increase of oxidative stress in liver and aorta, upon which catechin had no effect. Multivariate analyses (PCA and PLS-DA) of the urine fingerprints allowed discrimination of the different diets. Variables were then classified according to their dependence on lipid and catechin intake (ANOVA). Nine variables were identified as catechin metabolites of tissular or microbial origin. Around 1000 variables were significantly affected by the lipid content of the diet, and 76 were fully reversed by catechin supplementation. Four variables showing an increase in urinary excretion in rats fed the high-fat diets were identified as deoxycytidine, nicotinic acid, dihydroxyquinoline and pipecolinic acid. After catechin supplementation, the excretion of nicotinic acid was fully restored to the level found in the rats fed the low-fat diet. The physiological significance of these metabolic changes is discussed.
Assuntos
Aorta/metabolismo , Catequina/farmacologia , Gorduras na Dieta/farmacologia , Fígado/metabolismo , Espectrometria de Massas/métodos , Animais , Antioxidantes/metabolismo , Aorta/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Catequina/metabolismo , Catequina/urina , Colesterol/sangue , Cromatografia Líquida de Alta Pressão/métodos , Desoxicitidina/metabolismo , Desoxicitidina/urina , Ingestão de Alimentos/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Malondialdeído/metabolismo , Malondialdeído/urina , Análise Multivariada , Niacina/metabolismo , Niacina/urina , Ácidos Pipecólicos/metabolismo , Ácidos Pipecólicos/urina , Quinolinas/metabolismo , Quinolinas/urina , Ratos , Ratos Wistar , Triglicerídeos/sangueRESUMO
Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active alpha-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1-) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3'-end region. P. chrysogenum SR1- lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1- was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1- was grown with L-lysine as the sole nitrogen source and supplemented with DL-alpha-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1- with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from alpha-aminoadipic acid and not from L-lysine catabolism.
Assuntos
Ácido 2-Aminoadípico/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Penicillium chrysogenum/enzimologia , Ácidos Pipecólicos/metabolismo , Sacaropina Desidrogenases/genética , Ácidos Carboxílicos/metabolismo , Meios de Cultura , Proteínas Fúngicas/metabolismo , Lisina/metabolismo , Penicillium chrysogenum/genética , Sacaropina Desidrogenases/metabolismo , Transformação GenéticaRESUMO
Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.
Assuntos
Lisina/biossíntese , Penicillium chrysogenum/genética , Ácidos Pipecólicos/metabolismo , Sacaropina Desidrogenases/genética , Sequência de Aminoácidos , Clonagem Molecular , Genes Fúngicos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Penicillium chrysogenum/metabolismo , Plasmídeos , Sacaropina Desidrogenases/metabolismo , Homologia de Sequência de Aminoácidos , Transformação BacterianaRESUMO
The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 microg ml(-1). Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 2-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition.
Assuntos
Fungos Mitospóricos/metabolismo , Swainsonina/metabolismo , Ácido 2-Aminoadípico/metabolismo , Cromatografia Líquida de Alta Pressão , Lisina/análogos & derivados , Lisina/metabolismo , Fungos Mitospóricos/genética , Fungos Mitospóricos/crescimento & desenvolvimento , Ácidos Pipecólicos/metabolismo , Swainsonina/análiseRESUMO
Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain. An additional H2O2-producing sarcosine oxidase has now been purified from rabbit kidney. A corresponding cDNA was cloned from rabbit liver and the gene designated sox. This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme. Sequence analysis revealed an N-terminal ADP-betaalphabeta-binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1. Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases. Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver. Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney. Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway.
Assuntos
Microcorpos/enzimologia , Oxirredutases N-Desmetilantes/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/enzimologia , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/genética , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Rim/enzimologia , Cinética , Dados de Sequência Molecular , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Ácidos Pipecólicos/metabolismo , Prolina/metabolismo , Coelhos , Sarcosina/metabolismo , Sarcosina Oxidase , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Frações Subcelulares/químicaRESUMO
Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.
Assuntos
Ácido 2-Aminoadípico/metabolismo , Lisina/metabolismo , Penicilinas/biossíntese , Penicillium chrysogenum/metabolismo , Lisina/análogos & derivados , Lisina/farmacologia , Penicillium chrysogenum/enzimologia , Ácidos Pipecólicos/metabolismoRESUMO
Phencyclidine (PCP) inhibits the uptake of the neurotransmitter dopamine (DA), and blocks N-methyl-D-aspartate (NMDA) receptor-regulated ion channels. PCP also binds to sigma receptors in vivo and in vitro in rat brain. Prolonged exposure to PCP in adults has been observed to reduce the number of PCP binding sites in brain. We designed these experiments to evaluate whether prolonged prenatal exposure to PCP produces alterations in the development of DA and NMDA systems in brain. To do so, we characterized the normal course of development of basal and stimulated DA release in striatal slices, the ontogeny of striatal DA concentrations, and the development of NMDA receptor channels and associated glutamate binding sites in frontal cortex. We compared these developmental profiles to those in rats exposed to prenatal PCP, in an attempt to characterize the effect of prenatal PCP exposure on the pattern of brain development. Pregnant CD rats were injected s.c. with either 0, 10 or 20 mg/kg PCP daily on gestational days 8 through 20. On postnatal days (PND) 8, 21, 45, or 100, rats were sacrificed and brain tissues isolated for in vitro assessment. In vitro [3H]DA release from striatal slices evoked by either 40 microM glutamate or 15 mM K+ increased over 250% from PND 8 to PND 45, and glutamate-stimulated release was still significantly below adult levels at PND 45. In contrast, D-methamphetamine (D-METH)-evoked [3H]DA release, frontal cortical glutamate binding sites and NMDA channels developed early, reaching adult levels on or before PND 21.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Lobo Frontal/metabolismo , N-Metilaspartato/metabolismo , Fenciclidina/farmacologia , Efeitos Tardios da Exposição Pré-Natal , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/crescimento & desenvolvimento , Feminino , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/crescimento & desenvolvimento , Glutamatos/farmacologia , Ácido Glutâmico , Ácido Hidroxi-Indolacético/metabolismo , Masculino , Metanfetamina/farmacologia , N-Metilaspartato/antagonistas & inibidores , Ácidos Pipecólicos/metabolismo , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
The role of pipecolic acid in the biosynthesis of lysine was investigated in Rhodotorula glutinis, an aerobic red yeast. Supplementation of pipecolic acid in the minimal medium supported the growth of mutants lys2, lys3, and lys5; alpha-aminoadipic acid supported the growth of lys5; but neither alpha-aminoadipic acid nor pipecolic acid supported the growth of mutants MNNG42 and MNNG37. During the growth of the appropriate mutants, pipecolic acid was removed from the growth medium and the intracellular pool. In tracer experiments, radioactivity from [(14)C]pipecolic acid was selectively incorporated into the cellular lysine of lys5 and the wild-type strain. l-Pipecolic acid-dependent enzyme activity did not require any cofactor and was inhibited by mercuric chloride and potassium cyanide. This activity was present in the wild-type strain and all of the mutants tested and was repressed in mutant lys5 when grown in the presence of higher concentration of lysine. The reaction product of pipecolic acid was converted to saccharopine by lys5 enzyme in the presence of glutamate and reduced nicotin-amide adenine dinucleotide phosphate. Mutant MNNG37 lacked the saccharopine dehydrogenase activity, indicating that this step is involved in the conversion of alpha-aminoadipic acid and pipecolic acid to lysine. Mutants MNNG37 and MNNG42 accumulated a p-dimethylaminobenzaldehyde-reacting product in the culture supernatant and in the intracellular pool. Chromatographic properties of the p-dimethylaminobenzaldehyde adduct and that of the pipecolic acid-dependent reaction product were similar. The reaction product and the accumulation product were characterized on the basis of mass and absorption spectra as alpha-aminoadipic-semialdehyde, which in solution remains in equilibrium with Delta(1)-piperideine-6-carboxylic acid. Since alpha-aminoadipic-semialdehyde is a known intermediate of the alpha-aminoadipic acid pathway for the biosynthesis of lysine, it is concluded that pipecolic acid is converted to lysine in R. glutinis via alpha-aminoadipic-semialdehyde and saccharopine.