RESUMEN
The catalytic properties of bulky water-soluble Co-, Cu-, Fe- and Mn-salen complexes in the oxidation of phenolic lignin model compounds have been studied in aqueous water--dioxane solutions (pH 3--10). Mn catalysts were found to oxidize coniferyl alcohol in a same reaction time as horseradish peroxidase (HRP) enzyme and Mn and Co catalysts showed different regioselectivity suggesting a different substrate to catalyst interaction in the oxidative coupling. When the oxidation of material more relevant to plant polyphenolics was studied, the results indicated that the complexes catalyze one- and two-electron oxidations depending on the bulk of the substrate.
Asunto(s)
Bioquímica/métodos , Etilenodiaminas/química , Compuestos Organometálicos/química , Fenoles/metabolismo , Cobalto/química , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Concentración de Iones de Hidrógeno , Imidazoles/química , Lignina/química , Lignina/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , Oxidación-Reducción , Fenoles/químicaRESUMEN
We used differential display analysis to identify mRNAs responsive to changes in polyamine synthesis. As an overproducing model we used the kidneys of transgenic hybrid mice overexpressing ornithine decarboxylase and S-adenosylmethionine decarboxylase, two key enzymes in polyamine biosynthesis. To identify mRNAs that respond to polyamine starvation, we treated Rat-2 cells with alpha-difluoromethylornithine, a specific inhibitor of polyamine biosynthesis. We isolated 41 partial cDNA clones, representing 37 differentially expressed mRNAs. Of these, 15 have similarity with known genes, five appear to be similar to reported expressed sequence tags and seventeen clones were novel sequences. Of the 35 mRNAs expressed differentially after alpha-difluoromethylornithine treatment, 26 were up-regulated. The expression of only three mRNAs was altered in the transgenic animals and all three were down-regulated. Determination of mRNA half-life of three of the mRNAs up-regulated in response to polyamine depletion revealed that the accumulation results from stabilization of the messages. Because most of the transcripts identified from Rat-2 cells suffering polyamine starvation were accumulated, we conclude that polyamine depletion, while blocking cell growth, is stabilizing mRNAs. This may be due to the lack of spermidine for post-translational modification of the eukaryotic initiation factor 5A, which plays a major role in mRNA turnover. The coupling of mRNA stabilization with cell-growth arrest in response to polyamine starvation provides cells with an economical way to resume growth after recovery from polyamine deficiency.
Asunto(s)
Regulación de la Expresión Génica/genética , Poliaminas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Adenosilmetionina Descarboxilasa/genética , Adenosilmetionina Descarboxilasa/metabolismo , Animales , División Celular , Línea Celular , Clonación Molecular , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Eflornitina/farmacología , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica/efectos de los fármacos , Semivida , Riñón/enzimología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Poliaminas/antagonistas & inhibidores , Putrescina/farmacología , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , Ratas , Espermidina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Recent work has shown that transgenic mice overexpressing human ornithine decarboxylase display no marked changes in the tissue concentrations of spermidine or spermine in spite of a dramatic increase in putrescine levels. In the tissues of transgenic mice carrying the human spermidine synthase gene and in those of hybrid mice overexpressing both ornithine decarboxylase and spermidine synthase, spermidine and spermine levels remain within normal limits. To test whether the amount of the propylamine group donor, decarboxylated S-adenosylmethionine, limits the conversion of putrescine into the higher polyamines, we have produced transgenic mouse lines harbouring the rat S-adenosylmethionine decarboxylase gene in their genome. However, neither these mice nor the hybrid mice overexpressing both ornithine decarboxylase and S-adenosylmethionine decarboxylase displayed significant changes in their spermidine and spermine tissue levels. To study the mechanism by which cells maintain the constancy of the polyamine concentrations, we have determined the metabolic flux of polyamines in transgenic primary fibroblasts using pulse labelling. The results indicate that the polyamine flow is faster in transgenic primary fibroblasts than in non-transgenic fibroblasts and that the intracellular homoeostasis of higher polyamines is maintained at least partly by the acetylation of spermidine and spermine and their secretion into the medium.
Asunto(s)
Adenosilmetionina Descarboxilasa/metabolismo , Ornitina Descarboxilasa/metabolismo , Poliaminas/metabolismo , Animales , Northern Blotting , Encéfalo/metabolismo , Homeostasis , Humanos , Hígado/enzimología , Ratones , Ratones Transgénicos , ARN/metabolismo , RatasRESUMEN
We have investigated expression of the S-adenosylmethionine decarboxylase (AdoMetDC) gene in H4-II-E rat hepatoma cells treated with growth factors (epidermal growth factor and transforming growth factor beta 1) and inducers (cAMP and insulin). Treatment with insulin caused a marked increase in both RNA level and enzyme activity. The stability of AdoMetDC mRNA was not altered by insulin treatment: the accumulation of mRNA in hepatoma cells therefore seems to be due to an increase in the transcription rate. Cycloheximide was found to be a strong inducer of AdoMetDC mRNA transcription and the effects of insulin and cycloheximide were additive, suggesting that they increase expression by separate mechanisms. Chloramphenicol acetyltransferase assays in rat hepatoma cells using 5' flanking regions of different lengths revealed that the promoter region extending 337 bp upstream from the transcription start site contains elements involved in insulin response.
Asunto(s)
Adenosilmetionina Descarboxilasa/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Neoplasias Hepáticas Experimentales/enzimología , Animales , Cloranfenicol O-Acetiltransferasa/biosíntesis , AMP Cíclico/farmacología , Cicloheximida/farmacología , Inducción Enzimática , Factor de Crecimiento Epidérmico/farmacología , Cinética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes/biosíntesis , Transcripción Genética/efectos de los fármacos , Transfección , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
S-Adenosylmethionine decarboxylase (AdoMetDC) is a ubiquitous enzyme in eukaryotic cells and responds to a wide variety of stimuli affecting growth. To provide a framework for understanding the molecular basis of the mechanisms responsible for regulating the expression of this enzyme activity, we recently cloned and sequenced the rat gene for AdoMetDC. Now we have isolated another, slightly different AdoMetDC gene from a rat genomic library. Comparison of the two genes shows a high degree of conservation of sequence and structural organization. The two genes share the following characteristics: (1) both are approximately 16 kb in size, have identical exon/intron boundaries, and exhibit similar intron/exon structural organization; and (2) the nucleotide sequences are highly conserved in the coding regions and in many introns. Analysis of mouse-rat somatic cell hybrids has localized both rat genes to chromosome 20. The most interesting feature of these genes is that their 5' flanking regions are totally different. The promoter activities of the 5' regulatory regions were assessed by transient gene expression assays in Rat-2 cells after fusion to the chloramphenicol acetyltransferase gene. Transient transfections with the chimeric DNAs demonstrated that these fragments were able to function as efficient promoters, indicating that the diverging 5' regions of two AdoMetDC genes contain functional, but different, regulatory transcription elements.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Genes , Ratas/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Exones , Biblioteca de Genes , Células Híbridas , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Seudogenes , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Homología de Secuencia de Ácido NucleicoRESUMEN
L-Ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis and thus a unique marker of the proliferation and maturation processes, has been localized by means of immunohistochemistry in the human central nervous system. The enzyme protein has not only been found in the developing nervous system but also in the adult brain. The first immunopositive neuroblasts occurred in the 15th week of gravidity, whereas an increasing number of structures in the medulla oblongata and cerebellum showed ODC immunoreactivity in weeks 18-24 of gravidity. ODC immunoreactivity was revealed in multiple neurons and in the ependymal lining of ventricles. In the adult human brain ODC immunoreactivity was found in the hippocampus and in the spinal cord. In no case were immunoreactive glial cells observed.
Asunto(s)
Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Ornitina Descarboxilasa/metabolismo , Adolescente , Adulto , Anciano , Encéfalo/embriología , Niño , Femenino , Edad Gestacional , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Neuroglía/enzimología , Neuronas/enzimología , Ornitina Descarboxilasa/inmunología , Embarazo , Médula Espinal/embriología , Médula Espinal/enzimología , Médula Espinal/crecimiento & desarrolloRESUMEN
The nucleotide sequence of a cDNA encoding the proenzyme of mouse S-adenosylmethionine decarboxylase (AdoMetDC) including 257 nucleotides of the 5' untranslated region has been determined. Comparison of the nucleotide sequence of the mouse 5' untranslated region with those of other mammals shows it to be highly conserved. The 52 nucleotides upstream from the translation initiation codon are identical in human, rat, bovine and mouse. The polyamines, spermidine and spermine, have been shown to inhibit AdoMetDC mRNA translation. An RNA gel retardation assay demonstrated that a cytoplasmic extract from mouse brain forms an RNA-protein complex with the completely conserved 5' untranslated sequence and that the complex formation is highly dependent on the presence of spermine. Crosslinking by UV irradiation shows that the complex contains a 39-kDa protein interacting with the 5' untranslated sequence. These data demonstrate spermine-dependent specific protein binding to a highly conserved 5' untranslated region of an mRNA translationally regulated by polyamines.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Encéfalo/enzimología , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismo , Adenosilmetionina Descarboxilasa/metabolismo , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Citosol/metabolismo , ADN/genética , ADN/aislamiento & purificación , Precursores Enzimáticos/genética , Biblioteca de Genes , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/aislamiento & purificación , Poliaminas/farmacología , Unión Proteica , ARN Mensajero/genética , Ratas , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
The activities of the two key enzymes in mammalian polyamine synthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) in mouse brain show distinct, but inverse, changes during ontogeny. The level of ODC activity is about 70 fold higher at the time of birth than in the adult mouse, whereas AdoMetDC activity is very low after birth and increases as the brain matures. The correlation between the changes in enzyme activities and in the levels of the corresponding mRNAs diminishes dramatically during development. The increase in AdoMetDC mRNA level exceeds the increase in enzyme activity by 100%. Whereas ODC mRNA level falls initially, in concert with decreasing enzyme activity, but then shows an abrupt rise to a very high level during the late period of brain maturation while the enzyme activity continues to decrease to an almost undetectable level. These data suggest the development-dependent appearance of post-transcriptional regulation mechanisms.
Asunto(s)
Adenosilmetionina Descarboxilasa/metabolismo , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Ornitina Descarboxilasa/metabolismo , Adenosilmetionina Descarboxilasa/genética , Envejecimiento , Animales , Sondas de ADN , Regulación Enzimológica de la Expresión Génica , Cinética , Ratones , Ratones Endogámicos , Ornitina Descarboxilasa/genética , Putrescina/metabolismo , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/genética , Espermidina/metabolismo , Espermina/metabolismoAsunto(s)
Encéfalo/enzimología , Ornitina Descarboxilasa/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Trasplante de Tejido Encefálico/fisiología , Dexametasona/farmacología , Epilepsia/inducido químicamente , Epilepsia/metabolismo , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Hipocampo/trasplante , Inmunohistoquímica , Masculino , Ratas , Ratas EndogámicasRESUMEN
The gene for S-adenosylmethionine decarboxylase (AdoMetDC) was isolated from a rat genomic library using AdoMetDC cDNA as a probe. Nucleotide sequence analysis shows that the rat AdoMetDC gene consists of 8 exons which encode a protein identical to that inferred by a rat AdoMetDC cDNA sequence. The exons range in length from 43 to 1964 base pairs spanning 15672 bases of chromosomal DNA. All of the exon/intron junctions were found to conform to the consensus splice donor and acceptor sequences. Exon 8 corresponds to the 3' noncoding region of the 2 species of AdoMetDC mRNA which are formed by alternative utilization of 2 polyadenylation signals separated from each other by 1272 nucleotides. The transcription initiation site was located by S1 nuclease protection and by primer extension analysis, -325 nucleotides upstream of the translation initiation codon. The promoter region of the rat AdoMetDC gene contains a TATA box at -29 base pairs. No typical GC or CAAT boxes are located in the promoter, but six GC boxes and several putative binding sites for both tissue-specific and non-specific transcription factors are found in the proximal part of intron 1. Southern blot analyses reveal a complex hybridization pattern suggesting that there are multiple copies of the AdoMetDC gene in the rat genome.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Genes , Adenosilmetionina Descarboxilasa/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/química , Intrones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
In the present study efforts are made to localize ornithine decarboxylase enzyme protein--the key enzyme of polyamine biosynthesis--in the adenohypophysis of different vertebrates by means of immunocytochemistry. The antigenic expression of ornithine decarboxylase was revealed in the pituitary of the clawed frog (Xenopus laevis D.), but not in rat and human adenohypophysis. The immunocytochemical results are compared with the staining pattern of the periodic acid-Schiff-reaction. No correlation between these results and the immunocytochemically obtained data has been found. Conclusions are drawn from the location of the enzyme and possible phylogenetic and humoral regulation mechanism.
Asunto(s)
Ornitina Descarboxilasa/análisis , Adenohipófisis/enzimología , Hipófisis/enzimología , Animales , Citoplasma/enzimología , Humanos , Técnicas para Inmunoenzimas , Ratas , Ratas Endogámicas , Xenopus laevisRESUMEN
Anesthetized Mongolian gerbils were subjected to 5-min ischemia and 8 h of recirculation. Vibratom sections were taken for studying changes in ornithine decarboxylase (ODC) immunoreactivity using an antiserum to ODC, and tissue samples were taken for measuring ODC activity. After 5-min ischemia and 8-h recirculation ODC activity increased 11.5-, 5.9-, and 7.9-fold in the cerebral cortex, striatum and hippocampus, respectively (P less than or equal to 0.05 to 0.01). In the cortex, striatum and hippocampus of control animals immunoreactivity was low but clearly above the detection limit. The reaction was confined to neurons. After 5-min ischemia and 8-h recirculation a sharp increase in immunoreactivity was observed confined to neurons, indicating that the postischemic activation of polyamine metabolism is a neuronal response to ischemia. The immunoreactivity was markedly increased in the perinuclear cytoplasm and the dendrites. In the striatum the density of neurons exhibiting a sharp increase in immunoreactivity was more pronounced in the lateral than in the ventral part. In the hippocampus a strong reaction was present in all subfields but the CA1 subfield was particularly affected. The present study demonstrates for the first time that biosynthesis of a protein is markedly activated during the first 24 h of recirculation after 5-min cerebral ischemia of gerbils even in the vulnerable CA1 subfield, in which the overall protein synthesis is sharply reduced at the same time. Studying polyamine metabolism after ischemia may, thus, provide new information about the basic molecular mechanisms responsible for the altered gene expression after metabolic stress.
Asunto(s)
Isquemia Encefálica/enzimología , Ornitina Descarboxilasa/metabolismo , Animales , Isquemia Encefálica/patología , Arterias Carótidas/fisiología , Corteza Cerebral/patología , Cuerpo Estriado/patología , Femenino , Gerbillinae , Hipocampo/patología , Técnicas para Inmunoenzimas , Inmunohistoquímica , Coloración y EtiquetadoRESUMEN
Due to two different polyadenylation signals, two forms of S-adenosylmethionine decarboxylase (AdoMetDC) mRNA (2.1 and 3.4 kb) are present in human and rodent tissues. The nucleotide sequences of rat and human cDNAs corresponding to the shorter mRNA were published previously by us [Pajunen et al., J. Biol. Chem. 263 (1988) 17040-17049]. These sequences covered the coding regions but were incomplete at their 5' ends. Here we report the sequence of rat cDNA spanning the entire longer mRNA with a substantially extended leader region, and compare the sequence with that of a rat psi AdoMetDC pseudogene isolated from a rat genomic library. Relative to the mRNA, the pseudogene has multiple base changes as well as insertions, and deletions. Furthermore, it lacks introns, and is flanked by a short direct repeat. These are typical characteristics of a processed retrogene.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Carboxiliasas/genética , Genes , Intrones , Seudogenes , Animales , Secuencia de Bases , Clonación Molecular/métodos , ADN/genética , ADN/aislamiento & purificación , Biblioteca de Genes , Masculino , Datos de Secuencia Molecular , Próstata/enzimología , ARN Mensajero/genética , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido NucleicoRESUMEN
The cDNA coding for rat S-adenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50) has been cloned into a plasmid expression vector, pKK-223-3, and expressed in E. coli. The authenticity of the expressed protein has been demonstrated by reactivity with antibodies specific for rat AdoMetDC, by size analysis on SDS gels visualized with immunotransblots, and, finally, by catalytic activity. The expression of the enzyme results in a decrease in the activity of the bacterial enzyme suggesting the replacement of the bacterial enzyme by the rat AdoMetDC. Similarly, the addition of exogenous spermidine to the growth medium reduces bacterial enzyme activity affecting only marginally the expression of the recombinant protein.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Carboxiliasas/genética , Escherichia coli/enzimología , Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Adenosilmetionina Descarboxilasa/biosíntesis , Animales , Western Blotting , Catálisis , Clonación Molecular , ADN/metabolismo , Escherichia coli/metabolismo , Plásmidos , Ratas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Espermidina/farmacologíaRESUMEN
In order to understand the structure and regulation of S-adenosylmethionine decarboxylase, cDNA clones encoding this enzyme have been isolated from rat prostate and human fibroblast cDNA libraries. The authenticity of the cDNAs was verified by: (a) transfecting the Chinese hamster ovary cells with the human cDNA in the pcD vector which resulted in a transient 10-20-fold increase in S-adenosylmethionine decarboxylase activity in recipient cells; and (b) translating the mRNA formed by transcription of the cDNA insert in a reticulocyte lysate and recording an increase in S-adenosylmethionine decarboxylase activity. The amino acid sequences deduced from the cDNAs indicate that the human proenzyme for this protein contains 334 amino acids and has a molecular weight of 38,331 whereas the rat proenzyme contains 333 amino acid residues. The human and rat enzymes are very similar having only 11 amino acid differences and the cDNAs are also closely related showing over 90% homology in the 1617-nucleotide overlap which was sequenced. A further indication of the highly conserved nature of mammalian S-adenosylmethionine decarboxylases is that the amino acid sequences deduced from the human and the rat cDNAs contained peptide sequences identical to those previously reported for the purified bovine enzyme. In vitro transcription/translation experiments showed that the proenzyme is converted to two polypeptides of molecular weights about 32,000 and 6,000 in a processing reaction which generates the prosthetic pyruvate group and that the final enzyme contains both polypeptides. Two forms of S-adenosylmethionine decarboxylase mRNA (2.1 and about 3.4-3.6 kilobases) are present in human and rodent tissues and may originate from the utilization of two different polyadenylation signals. Southern blots of rat genomic DNA indicated that the S-adenosylmethionine decarboxylase gene belongs to a multigene family. Depletion of cellular polyamines by inhibitors or ornithine decarboxylase or the aminopropyltransferases led to an increase in the content of S-adenosylmethionine decarboxylase protein and mRNA, but the elevation in the mRNA was not sufficient to account for all of the change in the enzyme level, particularly in cells in which spermine was depleted.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Carboxiliasas/genética , ADN/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Eflornitina/farmacología , Fibroblastos/análisis , Humanos , Sustancias Macromoleculares , Masculino , Datos de Secuencia Molecular , Poli A/análisis , ARN/análisis , ARN Mensajero/metabolismo , Ratas , Espermidina/farmacologíaRESUMEN
L-Ornithine decarboxylase, the rate limiting enzyme of polyamine synthesis and a possible marker enzyme for tissue proliferation and maturation, has been found in the developing guinea pig cochlea using the unlabelled horseradish-peroxidase-antiperoxidase technique. Ornithine decarboxylase-like immunoreactive material was detected in the neurons of the Ganglion spirale and in their axonal and/or dendritic fibers. The location of the enzyme and the possible functional role of ornithine decarboxylase plays in the development and maturation of the auditory organ and of the hearing process are discussed.
Asunto(s)
Oído Interno/enzimología , Ornitina Descarboxilasa/metabolismo , Envejecimiento , Animales , Nervio Coclear/enzimología , Nervio Coclear/crecimiento & desarrollo , Oído Interno/crecimiento & desarrollo , Femenino , Cobayas , Histocitoquímica , Masculino , Neuronas/enzimologíaRESUMEN
Previous work in which the synthesis of S-adenosylmethionine decarboxylase was studied by translation of its mRNA indicated that it was formed as a proenzyme having a M.W. of about 37,000 that was cleaved to form the enzyme sub-unit of M.W. 32,000 in a putrescine-stimulated reaction. The extent to which the proenzyme accumulates in vivo and is affected by the putrescine concentration was studied by subjecting prostate extracts to Western immunoblotting procedures. The proenzyme form was readily detectable in control prostates (about 4% of the total) and this proportion was increased to 25% when the rats were pretreated for 3 days with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. Conversely, it was decreased to almost undetectable levels after treatment with methylglyoxal bis(guanylhydrazone). These results indicate that the processing of the proenzyme form of S-adenosylmethionine decarboxylase is regulated by the cellular putrescine concentration. This conversion provides another step at which polyamine biosynthesis may be controlled.