RESUMEN
PURPOSE: In humans, deficiency of ornithine-δ-aminotransferase (OAT) results in progressive degeneration of the neural retina (gyrate atrophy) with blindness in the fourth decade. In this study, we used the Xenopus embryonic developmental model to study functions of the OAT gene on embryonic development. METHODS: We cloned and sequenced full-length OAT cDNA from Xenopus oocytes (X-OAT) and determined X-OAT expression in various developmental stages of Xenopus embryos and in a variety of adult tissues. The phenotype, gene expression of neural developmental markers, and enzymatic activity were detected by gain-of-function and loss-of-function manipulations. RESULTS: We showed that X-OAT is essential for Xenopus embryonic development, and overexpression of X-OAT produces a ventralized phenotype characterized by a small head, lack of axial structure, and defective expression of neural developmental markers. Using X-OAT mutants based on mutations identified in humans, we found that substitution of both Arg 180 and Leu 402 abrogated both X-OAT enzymatic activity and ability to modulate the developmental phenotype. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development. CONCLUSIONS: Neurogenesis is inhibited by X-OAT during Xenopus embryonic development, but it is essential for Xenopus embryonic development. The Arg 180 and Leu 402 are crucial for these effects of the OAT molecule in development.
Asunto(s)
Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Neurogénesis/genética , Ornitina-Oxo-Ácido Transaminasa/genética , ARN/genética , Xenopus laevis/embriología , Animales , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
There is functional zonation of metabolism across the liver acinus, with glutamine synthetase restricted to a narrow band of cells around the terminal hepatic venules. Portacaval anastomosis, where there is a major rerouting of portal blood flow from the portal vein directly to the vena cava bypassing the liver, has been reported to result in a marked decrease in the activity of glutamine synthetase. It is not known whether this represents a loss of perivenous hepatocytes or whether there is a specific loss of glutamine synthetase. To answer this question, we have determined the activity of glutamine synthetase and another enzyme from the perivenous compartment, ornithine aminotransferase, as well as the immunochemical localization of both glutamine synthetase and ornithine aminotransferase in rats with a portacaval shunt. The portacaval shunt caused a marked decrease in glutamine synthetase activity and an increase in ornithine aminotransferase activity. Immunohistochemical analysis showed that the glutamine synthetase and ornithine aminotransferase proteins maintained their location in the perivenous cells. These results indicate that there is no generalized loss of perivenous hepatocytes, but rather, there is a significant alteration in the expression of these proteins and hence metabolism in this cell population.
Asunto(s)
Glutamato-Amoníaco Ligasa/biosíntesis , Hepatocitos/enzimología , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Derivación Portocava Quirúrgica/métodos , Vena Porta/enzimología , Vena Porta/cirugía , Animales , Glutamato-Amoníaco Ligasa/metabolismo , Hepatocitos/metabolismo , Masculino , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Vena Porta/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The Medicago truncatula ornithine aminotransferase cDNA was cloned under the potent constitutive 35S RNA promoter of the cauliflower mosaic virus and transferred into the genome of tobacco Nicotiana tabacum SR1 plants. Transformed tobacco plants grew better in salinity stress, but did not differ in proline content under normal or stress conditions from control plants. It was assumed that the role of ornithine aminotransferase in the molecular mechanisms of stress resistance is not associated with additional proline synthesis.
Asunto(s)
Expresión Génica , Medicago truncatula , Nicotiana , Ornitina-Oxo-Ácido Transaminasa , Proteínas de Plantas , Plantas Modificadas Genéticamente , Caulimovirus/genética , ADN Complementario/genética , Medicago truncatula/enzimología , Medicago truncatula/genética , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Ornitina-Oxo-Ácido Transaminasa/genética , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas/fisiología , Nicotiana/enzimología , Nicotiana/genéticaRESUMEN
Previous reports have shown elevated arginase activity in prostate cancer patients. This study was designed to compare expression levels of arginase II (AII) in various human prostate cancer cell lines and tissues. Expression levels of AII and other enzymes involved in arginine metabolism were examined in androgen-dependent (LNCaP, LAPC-4) and androgen-independent (PC3, DU145, CL-1, CL-2) prostate cancer cell lines by real-time RT-PCR and Western blot analysis. Further expression analysis of AII was accomplished by immunohistochemical staining of a tissue microarray comprised of 246 primary prostatectomy specimens. In addition, polyamine levels were measured within the prostate cancer cell lines by HPLC. Total polyamines were significantly lower in the androgen-dependent cell lines compared to the androgen-independent cell lines. AII expression was found to be most prominent in the androgen-dependent cell lines and least prominent in the androgen-independent cell lines. Additionally, we found expression of ornithine aminotransferase (OAT), an enzyme also responsible for ornithine production, to be inversely correlated with AII expression. The tissue microarray data revealed that the highest AII expression was seen in BPH, followed by PIN and normal samples, with the lowest expression levels observed in prostate cancer tissues. Moreover, we observed an expression gradient across Gleason grades revealing stronger AII expression in low-grade tumors. The polyamine data, combined with the expression analysis studies, support a possible correlation between AII, OAT, and polyamine synthesis. Based on these results, arginase II expression may play a role in prostate cancer progression. More specifically, the elevated AII expression seen in androgen-dependent and in more differentiated prostate cancers suggests that AII could be a potentially useful marker of early stage prostate adenocarcinoma.
Asunto(s)
Arginasa/biosíntesis , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/enzimología , Andrógenos/metabolismo , Arginina/metabolismo , Biomarcadores de Tumor , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Progresión de la Enfermedad , Humanos , Masculino , Neoplasias Hormono-Dependientes/metabolismo , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Poliaminas/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismoRESUMEN
We describe the gene expression profile of third leaves of rice (cv. Nipponbare) seedlings subjected to salt stress (130 mM NaCl). Transcripts of Mn-SOD, Cu/Zn-SOD,cytosolic and stromal APX, GR and CatB were regulated, whereas expression of thylakoid-bound APX and CatA were down-regulated. The levels of the compatible solute proline and of transcripts of its biosynthetic gene, Delta1-pyrroline-5-carboxylate synthetase (P5CS), were strongly increased by salt stress. Interestingly, a potential compatible solute, gamma-aminobutyric acid (GABA), was also found to be strongly induced by salt stress along with marked up-regulation of transcripts of GABA-transaminase. A dye-swap rice DNA microarray analysis identified a large number of genes whose expression in third leaves was altered by salt stress. Among 149 genes whose expression was altered at all the times assayed (3, 4 and 6 days) during salt stress, there were 47 annotated novel genes and 76 unknown genes. These results provide new insight into the effect of salt stress on the expression of genes related to antioxidant enzymes, proline and GABA as well as of genes in several functional categories.
Asunto(s)
Oryza/efectos de los fármacos , Oryza/genética , Hojas de la Planta/metabolismo , Cloruro de Sodio/farmacología , 4-Aminobutirato Transaminasa/biosíntesis , Ascorbato Peroxidasas , Catalasa/biosíntesis , Citosol/enzimología , Regulación hacia Abajo , Perfilación de la Expresión Génica , Glutatión Reductasa/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Peroxidasas/biosíntesis , Prolina/metabolismo , Superóxido Dismutasa/biosíntesis , Tilacoides/enzimología , Transcripción Genética , Regulación hacia Arriba , Ácido gamma-Aminobutírico/metabolismoRESUMEN
BACKGROUND: The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression. RESULTS: Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells--D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1)--most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. CONCLUSION: AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general, response is stronger in C4-2B compared to LNCaP cells. Some of the genes near AR-occupied regions appear to be regulated by the AR in vivo as evidenced by their expression levels in prostate cancer tumors of various stages. Several AR target genes discovered in the present study, for example PRKCD and PYCR1, may open avenues in PCa research and aid the development of new approaches for disease management.
Asunto(s)
Adenocarcinoma/genética , Andrógenos , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , Receptores Androgénicos/fisiología , Adenocarcinoma/metabolismo , Sitios de Unión , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Cromosomas Humanos/efectos de los fármacos , Cromosomas Humanos/metabolismo , Dihidrotestosterona/farmacología , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Mucina 6 , Mucinas/biosíntesis , Mucinas/genética , Proteínas de Neoplasias/biosíntesis , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Ornitina-Oxo-Ácido Transaminasa/genética , Neoplasias de la Próstata/metabolismo , Proteína Quinasa C-delta/biosíntesis , Proteína Quinasa C-delta/genética , Pirrolina Carboxilato Reductasas/biosíntesis , Pirrolina Carboxilato Reductasas/genética , Receptores Androgénicos/genética , Canales Catiónicos TRPV/biosíntesis , Canales Catiónicos TRPV/genética , Transcripción Genética , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
The arginine catabolism gene otaA encoding ornithine transaminase (OTAse) is specifically induced by arginine and is under the control of the broad-domain carbon and nitrogen repression systems. Arginine induction is mediated by a product of arcA gene coding for Zn(2)C(6) activator. We have identified a region responsible for arginine induction in the otaA promoter (AnUAS(arg)). Deletions within this region result in non-inducibility of OTAse by arginine, whether in an arcA(+) strain or in the presence of the arcA(d)47 gain of function allele. AnUAS(arg) is very similar to the Saccharomyces cerevisiae UAS(arg), a sequence bound by the Zn(2)C(6) activator (ArgRIIp), acting in a complex with two MADS-box proteins (McmIp and ArgRIp). We demonstrate here that two CREA in vitro binding sites in the otaA promoter are functional in vivo. CREA is directly involved in carbon repression of the otaA gene and it also reduces its basal level of expression. Although AREA binds to the otaA promoter in vitro, it probably does not participate in nitrogen metabolite repression of the gene in vivo. We show here that another putative negatively acting GATA factor AREB participates directly or indirectly in otaA nitrogen repression. We also demonstrate that the high levels of OTAse activity are an important factor in the suppression of proline auxotrophic mutations. This suppression can be achieved neither by growing of the proline auxotroph under carbon/nitrogen derepressing conditions nor by introducing of a creA(d) mutation.
Asunto(s)
Arginina/metabolismo , Aspergillus nidulans/genética , Ornitina-Oxo-Ácido Transaminasa/genética , Regiones Promotoras Genéticas , Aspergillus nidulans/metabolismo , Sitios de Unión , Carbono/metabolismo , Inducción Enzimática , Proteínas Fúngicas/metabolismo , Factores de Transcripción GATA , Regulación de la Expresión Génica , Glucosa/metabolismo , Nitrógeno/metabolismo , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Prolina , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Ureohidrolasas/metabolismoRESUMEN
We have cloned and analysed the arcA gene which encodes a transcriptional activator necessary for the high-level expression of two genes for enzymes of the arginine catabolic pathway in Aspergillus nidulans: agaA (for arginase) and otaA (for ornithine transaminase, OTAse). Here we present complete genomic and cDNA sequences for, and describe the pattern of expression of, the arcA gene. This gene contains one intron and encodes a polypeptide of 600 amino acids. The deduced protein belongs to the family of Zn(2)Cys(6) fungal regulatory proteins. ARCA is the first known protein of this family that has glycine instead of the conserved proline at the fifth position in the second, six-residue, loop of the Zn cluster domain. We have established that transcription of the arcA gene is not self-regulated and does not depend on arginine. Two mutations in arcA, one gain-of-function and one loss-of-function, have been sequenced and the effects of these mutations on the expression of the agaA gene at the transcriptional level are reported.
Asunto(s)
Arginina/metabolismo , Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Reguladores , Transactivadores/genética , Secuencia de Aminoácidos , Arginasa/biosíntesis , Arginasa/genética , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Mutación , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Ornitina-Oxo-Ácido Transaminasa/genética , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Dedos de ZincRESUMEN
Citrulline synthesis from glutamine is enhanced remarkably in enterocytes of weanling pigs, but the molecular mechanism(s) involved are not known. The objective of this study was to determine whether a cortisol surge mediates the enhanced expression of intestinal citrulline-synthetic enzymes during weaning. Jejunal enterocytes were prepared from 29-d-old weanling pigs treated with or without metyrapone (an inhibitor of cortisol synthesis), or from age-matched unweaned pigs. The mRNA levels and activities of phosphate-dependent glutaminase (PDG), pyrroline-5-carboxylate synthase (P5CS), ornithine aminotransferase (OAT), carbamoyl-phosphate synthase I (CPS-I) and ornithine carbamoyltransferase (OCT) were determined. The mRNA levels for PDG, P5CS, OAT and OCT were 139, 157, 102 and 55% higher, respectively, in weanling pigs compared with suckling pigs. The activities of PDG and P5CS were 38 and 692% higher, respectively, in weanling pigs compared with unweaned pigs, but the activities of OAT, CPS-I and OCT did not differ between these two groups of pigs. The effects of metyrapone administration to weanling pigs were as follows: 1) prevention of a cortisol surge, 2) abolition of the increases in both mRNA levels and activity of P5CS, 3) no alteration in the mRNA levels and activities of PDG and CPS-I, 4) increases in the mRNA levels for OAT (216%) and OCT (39%) and in OAT activity (30%), and 5) prevention of the increase in intestinal synthesis of citrulline from glutamine. These results suggest that increased P5CS activity reflects in large part the increased levels of P5CS mRNA and is responsible for the increased synthesis of citrulline from glutamine in enterocytes of weanling pigs; these increases may be mediated by a cortisol surge during weaning that can be blocked by metyrapone administration.
Asunto(s)
Hidrocortisona/sangre , Intestinos/enzimología , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Destete , Animales , Citrulina/biosíntesis , Femenino , Glutamina/metabolismo , Masculino , Ornitina-Oxo-Ácido Transaminasa/genética , Flujo Pulsátil , ARN Mensajero/metabolismo , PorcinosRESUMEN
Expression of inducible nitric oxide (NO) synthase (iNOS) and related enzymes of arginine metabolism in the mouse lung exposed to filamentous fungus Fusarium kyushuense was studied by RNA blot, immunoblot, and histological analyses. When mice were exposed intranasally to the fungi only once, no induction of iNOS mRNA was observed. However, when the animals were infected again 6 days after the first exposure, iNOS mRNA was induced, reached a maximum 12-24 h after the exposure, and decreased to an undetectable level at 48 h. mRNAs for cationic amino acid transporter-2 (CAT2) and argininosuccinate synthetase were induced gradually, reached a maximum at 24 h, and decreased at 48 h. Arginase II mRNA increased at 24 h and decreased markedly at 48 h. On the other hand, arginase I mRNA started to increase at 24 h and reached to a much higher level at 48 h. Ornithine decarboxylase and ornithine aminotransferase mRNAs were also induced. Immunoblot analysis showed that iNOS, argininosuccinate synthetase, and arginase I and II proteins were induced with similar kinetics as those of their respective mRNAs. In histological examination, fungal elements were observed in the bronchoalveolar lumen at 3-6 h, decreased at 12 h, and almost disappeared at 48 h. Small granuloma appeared 3 h after the infection and their size increased with time. These results suggest that NO is produced in the mouse lung in response to F. kyushuense exposure and that the NO production is regulated by CAT2, the citrulline-NO cycle, and arginase isoforms. Enhanced synthesis of polyamines and proline (and thus collagen) is also suggested.
Asunto(s)
Arginasa/biosíntesis , Arginina/metabolismo , Argininosuccinato Sintasa/biosíntesis , Proteínas Portadoras/biosíntesis , Fusarium , Isoenzimas/biosíntesis , Enfermedades Pulmonares Fúngicas/enzimología , Proteínas de la Membrana/biosíntesis , Óxido Nítrico Sintasa/biosíntesis , Ornitina Descarboxilasa/biosíntesis , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Sistemas de Transporte de Aminoácidos Básicos , Animales , Arginasa/genética , Argininosuccinato Sintasa/genética , Proteínas Portadoras/genética , Citrulina/metabolismo , Inducción Enzimática , Femenino , Isoenzimas/genética , Cinética , Enfermedades Pulmonares Fúngicas/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Ornitina Descarboxilasa/genética , Ornitina-Oxo-Ácido Transaminasa/genética , ARN Mensajero/biosíntesisRESUMEN
The liver plays a central role in nitrogen metabolism. Nitrogen enters the liver as free ammonia and as amino acids of which glutamine and alanine are the most important precursors. Detoxification of ammonia to urea involves deamination and transamination. By applying quantitative in situ hybridization, we found that mRNA levels of the enzymes involved are mainly expressed in periportal zones of liver lobules. Free ammonia, that is not converted periportally, is efficiently detoxified in the small rim of hepatocytes around the central veins by glutamine synthetase preventing it from entering the systemic circulation. Detoxification of ammonia by glutamine synthetase may be limited due to a shortage of glutamate when the nitrogen load is high. Adaptations in metabolism that prevent release of toxic ammonia from the liver were studied in rats that were fed diets with different amounts of protein, thereby varying the nitrogen load of the liver. We observed that mRNA levels of periportal deaminating and transaminating enzymes increased with the protein content in the diet. Similarly, mRNA levels of pericentral glutamate dehydrogenase and ornithine aminotransferase, the main producers of glutamate in this zone, and pericentral glutamine synthetase all increased with increasing protein levels in the diet. On the basis of these changes in mRNA levels, we conclude that: (a) glutamate is produced pericentrally in sufficient amounts to allow ammonia detoxification by glutamine synthetase and (b) in addition to the catalytic role of ornithine in the periportally localized ornithine cycle, pericentral ornithine degradation provides glutamate for ammonia detoxification.
Asunto(s)
Amoníaco/metabolismo , Proteínas en la Dieta/metabolismo , Glutamato Deshidrogenasa/biosíntesis , Glutamatos/metabolismo , Hígado/metabolismo , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Animales , Glutamato Deshidrogenasa/genética , Masculino , Ornitina-Oxo-Ácido Transaminasa/genética , Ratas , Ratas WistarRESUMEN
Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of alpha-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes.
Asunto(s)
Proteínas Portadoras/biosíntesis , Transformación Celular Neoplásica/genética , Fibroblastos/metabolismo , Genes jun , Genes ras , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas Nucleares/biosíntesis , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/genética , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas Portadoras/genética , Línea Celular Transformada , Dioxigenasas , Fibroblastos/efectos de los fármacos , Flavonoides/farmacología , Ribonucleoproteínas Nucleares Heterogéneas , MAP Quinasa Quinasa 1 , Espectrometría de Masas , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Nucleares/genética , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Ornitina-Oxo-Ácido Transaminasa/genética , Factor 2 de Elongación Peptídica , Factores de Elongación de Péptidos/biosíntesis , Factores de Elongación de Péptidos/genética , Fosfoglicerato Quinasa/biosíntesis , Fosfoglicerato Quinasa/genética , Fosfopiruvato Hidratasa/biosíntesis , Fosfopiruvato Hidratasa/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/fisiología , Ratas , Ribonucleoproteínas/biosíntesis , Ribonucleoproteínas/genética , TransfecciónRESUMEN
To evaluate the relative importance of ornithine (Orn) as a precursor in proline (Pro) synthesis, we isolated and sequenced a cDNA encoding the Orn-delta-aminotransferase (delta-OAT) from Arabidopsis thaliana. The deduced amino acid sequence showed high homology with bacterial, yeast, mammalian, and plant sequences, and the N-terminal residues exhibited several common features with a mitochondrial transit peptide. Our results show that under both salt stress and normal conditions, delta-OAT activity and mRNA in young plantlets are slightly higher than in older plants. This appears to be related to the necessity to dispose of an easy recycling product, glutamate. Analysis of the expression of the gene revealed a close association with salt stress and Pro production. In young plantlets, free Pro content, Delta1-pyrroline-5-carboxylate synthase mRNA, delta-OAT activity, and delta-OAT mRNA were all increased by salt-stress treatment. These results suggest that for A. thaliana, the Orn pathway, together with the glutamate pathway, plays an important role in Pro accumulation during osmotic stress. Conversely, in 4-week-old A. thaliana plants, although free Pro level also increased under salt-stress conditions, the delta-OAT activity appeared to be unchanged and delta-OAT mRNA was not detectable. Delta1-pyrroline-5-carboxylate synthase mRNA was still induced at a similar level. Therefore, for the adult plants the free Pro increase seemed to be due to the activity of the enzymes of the glutamate pathway.
Asunto(s)
Arabidopsis/enzimología , Arabidopsis/genética , ADN Complementario/aislamiento & purificación , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Ornitina-Oxo-Ácido Transaminasa/genética , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN Complementario/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Datos de Secuencia Molecular , Ornitina-Oxo-Ácido Transaminasa/efectos de los fármacos , Ornitina-Oxo-Ácido Transaminasa/aislamiento & purificación , Concentración Osmolar , Prolina/metabolismo , Cloruro de Sodio/farmacologíaRESUMEN
Altered nitrogen metabolism is a feature of chronic renal failure (CRF). The present study examined changes in renal expression of mRNA for enzymes involved in ornithine and polyamine metabolism, i.e. ornithine aminotransferase (OAT), ornithine decarboxylase (ODC), and S-adenosylmethionine synthetase (S-ADMase), during the early phase of renal insufficiency in rats after 5/6 nephrectomy (Nx). Involvement of androgens, the most potent stimulators of renal ODC, in these changes, was also evaluated inasmuch as testoseronemia is known to be significantly decreased in male uremic subjects. The abundance of mRNA was evaluated by quantitative Northern analysis of total RNA extracted from the remnant kidney of male or female Nx rats. The level mRNA for ODC was depressed by 76, 83, and 79%, that for OAT by 60, 76 and 63%, and that for S-ADMase by 37, 58 and 30%, at, respectively, 2, 7 and 35 days after Nx, in both male and female rats. ODC but not OAT enzyme activity was decreased. The expression of glyceraldehyde-3-phosphate dehydrogenase was only slightly lowered and that of c-myc was unaltered. Renal polyamine content of the remnant kidney was unchanged. It is concluded that in CRF: (1) intrarenal ornithine metabolism and polyamine biosynthesis are greatly impaired; (2) decreased androgens are not involved in these changes; (3) increased ODC is not a prerequisite for kidney hypertrophy; (4) extrarenal polyamines accumulation into the remnant likely compensates for defective renal biosynthesis.
Asunto(s)
Lesión Renal Aguda/enzimología , Fallo Renal Crónico/enzimología , Riñón/enzimología , Metionina Adenosiltransferasa/biosíntesis , Nefrectomía , Ornitina Descarboxilasa/biosíntesis , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Transcripción Genética , Lesión Renal Aguda/fisiopatología , Análisis de Varianza , Animales , Femenino , Riñón/fisiopatología , Fallo Renal Crónico/fisiopatología , Masculino , Metionina Adenosiltransferasa/metabolismo , Tamaño de los Órganos , Ornitina/metabolismo , Ornitina Descarboxilasa/metabolismo , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Poliaminas/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Caracteres SexualesRESUMEN
The purposes of this study were to determine whether expression of the gene encoding ornithine aminotransferase (OAT) in the rat liver and kidney is regulated by retinoic acid (RA) and to characterize further the role of thyroid hormone in regulating the expression of this gene. The level of OAT messenger RNA (mRNA) was reduced 70% in the liver of animals fed a vitamin A-deficient diet relative to that in animals fed a vitamin A-sufficient diet. RA, administered at a dose of 20 micrograms/rat to A-deficient rats for 1 or 3 days, restored OAT mRNA to near the level observed in animals fed the A-sufficient diet. Retinol was also effective in this regard. T3, when injected alone at a dose of 10 micrograms/100 g BW, had no effect on the level of OAT mRNA in the liver. However, when injected concurrently with RA, T3 blocked the ability of RA to induce OAT mRNA in the liver of rats fed the vitamin A-deficient diet. Animals made both vitamin A deficient and hypothyroid responded to RA in a manner similar to vitamin A-deficient animals. The vitamin A-deficient, hypothyroid rats responded somewhat differently to T3, however. T3 was unable to block the induction of OAT mRNA in the liver of vitamin A-deficient, hypothyroid rats when injected concurrently with RA for 1 day, but did block the induction of OAT mRNA by RA when these two hormones were injected concurrently for 3 days. These data indicate that RA and T3 exert opposing effects on the level of OAT mRNA in the liver. The effects of RA and T3 on OAT mRNA were markedly different in the kidney. Neither vitamin A deficiency nor RA had any apparent affect on the level of OAT mRNA in the kidney. T3, however, increased the level of OAT mRNA in the kidney of vitamin A-deficient rats. In the kidney of vitamin A-deficient, hypothyroid rats, T3 was unable to increase OAT mRNA when injected for 1 day, but did increase this mRNA when injected for 3 days. Together, these data indicate cell-type specific effects of both RA and T3 on the OAT gene.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Riñón/enzimología , Hígado/enzimología , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Propiltiouracilo/farmacología , Tretinoina/farmacología , Triyodotironina/farmacología , Animales , Animales Recién Nacidos , Femenino , Hipotiroidismo/enzimología , Riñón/efectos de los fármacos , Lactancia , Hígado/efectos de los fármacos , Masculino , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas Lew , Tiroxina/metabolismo , Vitamina A/metabolismo , Deficiencia de Vitamina A/enzimologíaRESUMEN
Human liver ornithine aminotransferase was expressed in Escherichia coli and purified by ammonium sulfate fractionation and anion exchange column chromatography. The purified recombinant enzyme is fully active and crystallized readily over a wide range of polyethylene glycol concentrations. The crystals belong to the trigonal space group P3(1)21 (or its enantiomorph P3(2)21) with unit cell parameters a = b = 116.3 A, and c = 190.0 A, alpha = beta = 90 degrees, gamma = 120 degrees. There are three monomers per asymmetric unit. Self-rotation function studies revealed both 2-fold and 3-fold non-crystallographic symmetry, with the local 3-fold axis being tilted 15 degrees from the c axis and perpendicular to a crystallographic dyad. A complete native data set to 2.3 A resolution was collected using synchrotron radiation.
Asunto(s)
Ornitina-Oxo-Ácido Transaminasa/química , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli , Humanos , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
Ornithine delta aminotransferase (OAT) is a nuclear-encoded mitochondrial matrix enzyme that catalyzes the reversible transamination of ornithine to glutamate semialdehyde. In humans, genetic deficiency of OAT results in gyrate atrophy of the choroid and retina, a blinding chorioretinal degeneration usually beginning in late childhood. This disorder has been shown to be autosomal recessive, and is often caused by missense, nonsense, and/or frameshift mutations in the OAT gene. With the view of applying gene therapy, a Moloney murine leukemia virus (MoMLV)-based recombinant retrovirus vector has been constructed. The human OAT cDNA was placed under the control of the enhancer-promoter regulatory elements derived from the MoMLV long terminal repeat (LTR). The construct was transfected into the retroviral packaging cell lines GP + E - 86 and psi CRIP to produce virus particles. Supernatant from these OAT retrovirus producer cell lines were used to transduce mouse C57B1/6 embryonal fibroblasts. We showed that the recombinant retrovirus transfers the OAT gene to the recipient cells, which produce an OAT RNA transcript when analyzed by Northern blot. Western blot analysis and enzymatic assays confirmed the presence of an OAT polypeptide that has a high enzymatic activity in the transduced cell lines, even after a long period of time in vitro.
Asunto(s)
ADN Recombinante/genética , Embrión de Mamíferos/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Virus de la Leucemia Murina de Moloney/genética , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Animales , Secuencia de Bases , Línea Celular , Fibroblastos/enzimología , Genoma Viral , Humanos , Kanamicina Quinasa , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transducción GenéticaRESUMEN
Ornithine-delta-aminotransferase catalyzes the conversion of ornithine to glutamate-gamma-semialdehyde. In humans, deficiency of this mitochondrial matrix enzyme results in the progressive blinding disorder, gyrate atrophy of the choroid and retina. To explore yeast as an expression system, we introduced a cDNA encoding human ornithine-delta-aminotransferase into an ornithine aminotransferase-deficient strain of Saccharomyces cerevisiae. The human enzyme was expressed at high levels, with activity 20-fold greater than that of wild-type yeast and 10-fold higher than in human fibroblasts. Although the normal location of ornithine-delta-aminotransferase in S. cerevisiae is cytosolic, human ornithine-delta-aminotransferase expressed in S. cerevisiae was localized to the mitochondrial matrix with correct proteolytic processing of its mitochondrial leader sequence. Despite this anomalous location in yeast, human ornithine-delta-aminotransferase complemented the phenotype of the mutant strain, restoring its ability to utilize ornithine as a sole nitrogen source. We also expressed a vitamin B6-responsive missense allele of ornithine-delta-aminotransferase (V332M) and showed that the biochemical phenotype of this allele is easily demonstrated confirming the usefulness of this system for examining mutations causing gyrate atrophy.
Asunto(s)
Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Ornitina-Oxo-Ácido Transaminasa/genética , Secuencia de Bases , Clonación Molecular/métodos , Cartilla de ADN , Expresión Génica , Humanos , Datos de Secuencia Molecular , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Plásmidos , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Ornithine-delta-aminotransferase (OAT) catalyzes the reversible transamination of ornithine to glutamate semialdehyde. OAT is abundant in liver, kidney and retina; hereditary deficiency of the enzyme leads to chorioretinal degeneration. Studies of OAT regulation in retinoblastomas have revealed an alternatively spliced OAT mRNA, which contains an additional exon (exon 2) in the 5' untranslated region. Estrogen and thyroid hormone were previously shown to increase OAT mRNA levels approximately 3-fold and 5-fold, respectively, in these cells. To determine the mechanism of hormonal action in retinoblastomas, we performed nuclear transcription assays and analyzed the distribution of OAT mRNAs in individual fractions of a polysome gradient. Thyroid hormone increased the rate of transcription of the OAT mRNA in these cells. Estrogen did not stimulate transcription; it was associated with increased translation, since it resulted in a shift of the major (spliced) OAT mRNA species into denser fractions of the polysome gradient. Cycloheximide treatment suggested that the latter effect was due to increased initiation of translation. The unspliced OAT mRNA, which is inefficiently compared to the spliced mRNA, was insensitive to estrogen in these experiments.