RESUMEN
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.
Asunto(s)
Arabidopsis/inmunología , Ácidos Pipecólicos/inmunología , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta , Pseudomonas syringae/inmunología , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Interacciones Huésped-Patógeno/inmunología , Cetoácidos/inmunología , Cetoácidos/metabolismo , Leucina/inmunología , Leucina/metabolismo , Lisina/inmunología , Lisina/metabolismo , Metionina/inmunología , Metionina/metabolismo , Ácidos Pipecólicos/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Pseudomonas syringae/fisiología , Transaminasas/genética , Transaminasas/inmunología , Transaminasas/metabolismoRESUMEN
The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free alpha-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN alpha-ketoglutarate, pyruvate PMN superoxide anion (O2-) generation, hydrogen peroxide (H2O2) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in alpha-ketoglutarate, pyruvate, MPO release and H2O2 generation. Formation of O2- on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and alpha-ketobutyrate levels, decreased O2- and H2O2 formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-alpha-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.
Asunto(s)
Arginina/farmacología , Dipéptidos/farmacología , Cetoácidos/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Taurina/farmacología , Adulto , Activación Enzimática/efectos de los fármacos , Humanos , Líquido Intracelular/metabolismo , Cetoácidos/química , Masculino , Neutrófilos/química , Oxidación-Reducción , Peroxidasa/efectos de los fármacos , Factores de TiempoRESUMEN
This study determined effects of leucine and its catabolites on in vitro, mitogen-stimulated DNA synthesis by bovine lymphocytes. Cultures grown in leucine-free or leucine-replete (0.4 mmol/L leucine) medium were supplemented with 0-10.0 mmol/L leucine or individual catabolites. Leucine at greater than or equal to 0.08 mmol/L was necessary for normal DNA synthesis by mitogen-stimulated bovine lymphocytes. beta-Hydroxy-beta-methylbutyrate (HMB) and beta-hydroxy-beta-methylglutarate (HMG) had minimal effect on unresponsiveness of mitogen-stimulated bovine lymphocytes in leucine-free medium; however, alpha-ketoisocaproate (KIC) at 0.4 and 2.0 mmol/L partially or completely restored DNA synthesis. In leucine-replete medium, 0.016-0.4 mmol/L KIC and 0.016-2.0 mmol/L HMB and HMG did not affect DNA synthesis. At 2.0 and 10.0 mmol/L, KIC inhibited (P less than 0.01) DNA synthesis, whereas HMB and HMG at 10.0 mmol/L enhanced (P less than 0.01) DNA synthesis. Overall, these results suggest that leucine is necessary for mitogen-induced DNA synthesis by bovine lymphocytes, and that this requirement for leucine can be partially met by KIC. When leucine was not limiting, KIC, HMB and HMG at concentrations that might occur in vivo did not alter lymphocyte DNA synthesis in vitro.
Asunto(s)
ADN/biosíntesis , Leucina/farmacología , Linfocitos/efectos de los fármacos , Mitógenos/metabolismo , Adulto , Animales , Bovinos , Células Cultivadas , Femenino , Humanos , Cetoácidos/inmunología , Cetoácidos/farmacología , Leucina/inmunología , Linfocitos/metabolismo , MasculinoRESUMEN
Haptens with bridge at the 2-position have not yet been explored. Radioimmunoassays with antibodies directed against 2 alpha-alkyl bridged steroid haptens are expected to be highly specific due to greater topographical exposure and similarity in conformation to the native steroid. The 2 alpha-alkyl bridged haptens were synthesized by first adding a cyclopropane ring to 2-methylene-4-en-3-one. Selective opening of the three-membered ring with trimethyl silyl iodide and transformation of the iodo group gave a carbocyclic acid, the desired analog for conjugation with protein.