RESUMO
c-Myc is an essential component of the regulatory mechanisms controlling cell growth. Max is the obligatory partner of c-Myc for all its biological functions analysed to date. Recently two Max interacting proteins, Mad and Mxi1, have been identified. It has been suggested that these two proteins modulate c-Myc function, in the simplest model by competing with c-Myc for the interaction with Max. We have analysed different aspects of Mad function in comparison to Max. Native Mad/Max heterodimers bound specifically to a c-Myc/Max consensus DNA binding site. Furthermore Mad inhibited efficiently c-Myc, mutant p53, adenovirus E1a, or human papilloma virus type 16 transformation of rat embryo cells in cooperation with activated Ha-Ras. Myc transformed clones showed an increased cell cycle time and a reduced immortalization frequency after cotransfection with either mad or max. In contrast to Mad, Max did not inhibit E1a/Ha-Ras cotransformation but repressed c-Myc/Ha-Ras transformation efficiently. Mad delta N, an N-terminal deletion mutant of Mad, was as efficient in repressing c-Myc/Ha-Ras cotransformation as full length Mad but showed little inhibitory activity when assayed on E1a/Ha-Ras. Unlike wt Mad, Mad delta N had little effect on cell growth. Our data suggest that Mad affects cell growth at least in part by a c-Myc independent mechanism.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/fisiologia , Proteínas Oncogênicas Virais/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Proteínas Repressoras , Fatores de Transcrição , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Ciclo Celular , Linhagem Celular , Chlorocebus aethiops , Expressão Gênica , Genes myc , Genes ras , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genética , Relação Estrutura-Atividade , TransfecçãoRESUMO
Uptake and distribution of transferrin and iron in perfused rat liver are dependent on perfusion temperature, time and uptake affinity. Transferrin passes at least two different compartments on its receptor-mediated recycling pathway, which are separable by centrifugation in a shallow Nycodenz gradient. Perfusion at lowered temperature (16 degrees C) is sufficient for internalization of transferrin and iron. Passage of radiolabelled iron to other than endosomal compartments as well as recycling of labelled transferrin are largely suppressed at this perfusion temperature, as much less is released by further perfusion with unlabelled transferrin than at 4 degrees C where the ligand is largely washed off the surface, or 37 degrees C, where the recycling pathway is operating. But also at lowered temperature only a part of the iron in endosomal fractions can be assigned to transferrin. A considerable part of the total uptake of transferrin and iron can be attributed to low-affinity mechanisms even at very low transferrin concentrations. Transferrin receptors are concentrated in endosomal fractions in comparison to fractions representing different plasma membrane domains of the liver. Endosomal fractions specifically display detergent-activated NADH-acceptor oxidoreductase which may be part of the iron uptake system.
Assuntos
Assialoglicoproteínas , Endocitose , Ferro/metabolismo , Fígado/metabolismo , Transferrina/metabolismo , Animais , Técnicas In Vitro , Fígado/citologia , Fígado/enzimologia , NADH Desidrogenase/metabolismo , NADH NADPH Oxirredutases/metabolismo , Orosomucoide/análogos & derivados , Orosomucoide/metabolismo , Oxirredução , Ratos , TemperaturaRESUMO
Treatment of L-tyrosine in a peroxidase/H2O2 system results in the formation of dityrosine. However, the phosphoester derivative of tyrosine, O-phospho-L-tyrosine, was unable to form dityrosine even in mixtures with free L-tyrosine. Dephosphorylation of O-phospho-L-tyrosine by alkaline phosphatase followed by horseradish peroxidase/H2O2 treatment resulted in the formation of dityrosine. Our in vitro results indicate that phosphorylation/dephosphorylation of L-tyrosine may regulate dityrosine formation, and is supposed to play an important role in protein-protein interactions, i.e. cross-linking.
Assuntos
Tirosina , Fosfatase Alcalina/metabolismo , Fenômenos Químicos , Química , Peroxidase do Rábano Silvestre/metabolismo , Cinética , Fosforilação , FosfotirosinaRESUMO
It is known that a tyrosine free radical is produced during the hormonogenic iodination reaction of tyrosine residues on thyroglobulin. In the hormonogenic region of thyroglobulin, phosphorylated tyrosine residues have been detected. Using an vitro tyrosine iodinating system we report that the hormonogenic reaction cannot go off if tyrosine becomes phosphorylated. Enzymatic dephosphorylation of the modified amino acid restored the ability of the molecule to become iodinated. Considering the mechanism of the tyrosine free radical formation, these observations are due to the inability of the phosphorylated amino acid to form a free radical. Our data may suggest a putative regulatory mechanism in thyroid hormone synthesis by phosphorylation of hormonogenic tyrosine residues on thyroglobulin.
Assuntos
Iodo/metabolismo , Tireoglobulina/metabolismo , Tirosina/metabolismo , Dimerização , Radicais Livres , Fosforilação , Fosfotirosina/metabolismoRESUMO
Simultaneously produced superoxide/nitric oxide radicals (O2*-/NO*) could form peroxynitrite (OONO-) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO- has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O2*-/NO* by scavenging the OONO reactive decomposition products. When LDL was exposed to simultaneously produced O2*-/NO* using the sydnonimine SIN-1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN-1 pre-oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN-1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN-1. 2,3- and 2,5-DHBA were even more effective to protect LDL from oxidation by O2*-/NO*. Because O2*-/NO* can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O2*-/NO* initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.
Assuntos
Sequestradores de Radicais Livres/metabolismo , Lipoproteínas LDL/metabolismo , Óxido Nítrico/metabolismo , Ácido Salicílico/metabolismo , Superóxidos/metabolismo , Humanos , Molsidomina/análogos & derivados , OxirreduçãoRESUMO
The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. Tyrosyl radicals generated by myeloperoxidase (MPO) can act as prooxidants of LDL oxidation. Taking into consideration, that monophenolic compounds are able to form phenoxyl radicals in presence of peroxidases, we have tested salicylate, in its ability to act as a prooxidant in the MPO system. Measurement of conjugated dienes and lipid hydroperoxides were taken as indicators of lipid oxidation. Exposure of LDL preparations to MPO in presence of salicylate revealed that the drug could act as a catalyst of lipid oxidation in LDL. The radical scavenger ascorbic acid as well as heme poisons (cyanide, azide) and catalase were inhibitory. The main metabolite of salicylic acid, gentisic acid, showed inhibitory action in the MPO system. Even when lipid oxidation was maximally stimulated by salicylate the LDL oxidation was efficaciously counteracted in presence of gentisic acid at salicylate/gentisic acid ratios that could be reached in plasma of patients receiving aspirin medication. Gentisic acid was also able to impair the tyrosyl radical catalyzed LDL peroxidation. The results suggest that salicylate could act like tyrosine via a phenoxyl radical as a catalyst of LDL oxidative modification by MPO. But the prooxidant activity of this radical species is effectively counteracted by the salicylate metabolite gentisic acid.
Assuntos
Gentisatos , Hidroxibenzoatos/farmacologia , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Peroxidase/antagonistas & inibidores , Peroxidase/metabolismo , Ácido Salicílico/farmacologia , Arteriosclerose/etiologia , Aspirina/metabolismo , Aspirina/farmacologia , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/metabolismo , Humanos , Hidroxibenzoatos/metabolismo , Técnicas In Vitro , Peroxidação de Lipídeos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oxidantes/química , Oxidantes/metabolismo , Oxirredução , Fenóis/metabolismo , Ácido Salicílico/metabolismo , Tirosina/metabolismoRESUMO
Oxidation of low density lipoprotein (LDL) by glucose-derived radicals may play a role in the aetiology of atherosclerosis in diabetes. Salicylate was shown to scavenge certain radicals. In the present study, aspirin, salicylate and its metabolites 2,5- and 2, 3-dihydroxybenzoic acid (DHBA) were tested for their ability to impair LDL oxidation by glucose. Only the DHBA derivatives, when present during LDL modification, inhibited LDL oxidation and the increase in endothelial tissue factor synthesis induced by glucose oxidised LDL. The LDL glycation reaction was not affected by DHBA. The antioxidative action of DHBA may be attributed to free radical scavenging and/or chelation of transition metal ions catalysing glucose autoxidation.
Assuntos
Arteriosclerose/prevenção & controle , Aspirina/farmacologia , Angiopatias Diabéticas/prevenção & controle , Gentisatos , Glucose/metabolismo , Hidroxibenzoatos/farmacologia , Lipoproteínas LDL/metabolismo , Aspirina/metabolismo , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Hidroxibenzoatos/metabolismo , Quelantes de Ferro/metabolismo , Quelantes de Ferro/farmacologia , Masculino , Oxirredução , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia , Tromboplastina/biossínteseRESUMO
A sucrose-rich diet stimulates hepatic lipogenesis and induces net production of very low density lipoproteins in the liver. To study changes of hepatic apolipoprotein gene expression in response to such a diet, we measured the mRNA abundance of apolipoproteins A-I, C-III and A-IV in livers of rats fed a sucrose-rich diet or a control diet for 3 weeks. In livers of sucrose-fed rats, the abundance of cellular and nuclear apo A-IV mRNA increased to 185% +/- 21% and 142% +/- 22% of control values (P less than 0.01), respectively. In sucrose-fed rats, the transcriptional activity of the apo A-IV gene, measured in a cell-free transcription system using isolated liver nuclei, increased to 144% +/- 23% of control (P less than 0.05). In contrast, this diet neither affected the abundance of cellular and nuclear apo A-I and apo C-III mRNA nor the transcriptional activity of these genes in liver. These results are consistent with specialization of the regulatory elements of the genes coding for apolipoproteins A-I, C-III and A-IV. Alternatively, enhanced transcription of the apo A-IV gene may preclude increased synthesis of apo A-I and/or apo C-III mRNA due to the close linkage of the three genes in the rat genome.
Assuntos
Apolipoproteína A-I/genética , Apolipoproteínas A/genética , Apolipoproteínas C/genética , Expressão Gênica/efeitos dos fármacos , Fígado/fisiologia , Sacarose/administração & dosagem , Animais , Apolipoproteína C-III , Sequência de Bases , Dieta , Fígado/metabolismo , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sacarose/farmacologia , Transcrição GênicaRESUMO
This review focuses on the influence of tyrosine phosphorylation on the biological reactions of tyrosine. Reactions that are modulated by this amino acid modification include dityrosine formation, thyroid hormone synthesis, and DOPA formation. In addition, we show that the reactivity of tyrosine in the common Lowry method of determination of protein concentrations is lost upon phosphorylation of the amino acid.
Assuntos
Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Fosfotirosina/fisiologia , Tirosina/fisiologia , Animais , Di-Hidroxifenilalanina/metabolismo , Di-Iodotirosina/metabolismo , Radicais Livres , Humanos , Monoiodotirosina/metabolismo , Fosforilação , Tirosina/análogos & derivados , Tirosina/metabolismoAssuntos
Divisão Celular/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Repressoras , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação/genética , Ciclo Celular/fisiologia , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fatores de Ligação de DNA Eritroide Específicos , Humanos , Modelos Biológicos , Fatores de Transcrição/fisiologia , Fator de Transcrição YY1RESUMO
Pretreatment of calf intestine alkaline phosphatase with phosphatidylserine resulted in an inhibition of the phosphatase activity towards low - (p-nitrophenylphosphate) and high (phosphohistone) molecular weight substrate. Phosphatidylcholine, irrespectively of the substrate used did not cause enzyme modulation. 12-O-tetradecanoylphorbol-13-acetate, 1,2-diolein as well certain retinoids, known to effect phosphatidylserine-sensitive enzyme systems (Castagna, M. et al. 1982, J. Biol. Chem. 257, 7847-7851; Gmeiner, B. 1986, Biochim. Biophys. Acta 856, 392-394) had no influence on the modulated phosphatase. The lipid interacting drug trifluoperazine inhibited the enzyme activity towards phosphohistone, but not towards p-nitrophenylphosphate as a substrate. The results indicate that acidic phospholipid may play a role in activity modulation of calf intestine membranous alkaline phosphatase activity.
Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Intestinos/enzimologia , Fosfatidilserinas/farmacologia , Animais , Bovinos , Concentração de Íons de Hidrogênio , Peso Molecular , Fosfatidilcolinas/farmacologia , Fosfolipídeos/farmacologia , Fosfolipídeos/fisiologia , Trifluoperazina/farmacologiaRESUMO
Alteration of the nuclear matrix protein composition during active cell death was investigated by high resolution 2-dimensional gel electrophoresis and computer-assisted image analysis. Nuclear matrices were isolated from purified nuclei of a rat embryo cell line showing an immediate apoptotic response to serum reduction. While cell shrinkage and cytoplasmic compaction, characteristic features of apoptosis, were induced, the nuclear matrix protein pattern was not altered 1 h after induction of apoptosis. However, two sets of novel nuclear matrix protein spots appeared with differing kinetics within the following 5 h of apoptosis. They consisted of five and six protein spots, respectively. In addition, the intensity of five nuclear matrix protein spots that had already been present in the uninduced cells increased continuously within an observation period of 12 h. These coincidences point to a potential involvement of the described nuclear matrix proteins in the apoptotic process.
Assuntos
Apoptose/fisiologia , Proteínas Nucleares/análise , Animais , Antígenos Nucleares , Linhagem Celular Transformada , Núcleo Celular/química , Eletroforese em Gel Bidimensional , Embrião de Mamíferos , Processamento de Imagem Assistida por Computador , RatosRESUMO
An assay for the estimation of phosphotyrosine phosphatase using the Folin-Ciocalteu phenol reaction to monitor enzyme activity is presented. The method is based on the property of the substrate phosphotyrosine not to react as a phenol until it is dephosphorylated. The method is sensitive, there is no interference from the use of detergents and it does not rely on special laboratory equipment to distinguish tyrosine from phosphotyrosine.
Assuntos
Indicadores e Reagentes , Molibdênio , Proteínas Tirosina Fosfatases/metabolismo , Compostos de Tungstênio , Sequência de Aminoácidos , Animais , Carcinoma , Neoplasias do Colo , Fígado/enzimologia , Dados de Sequência Molecular , Ratos , Sensibilidade e Especificidade , Células Tumorais Cultivadas , Tirosina/análiseRESUMO
Incubation of bovine aortic as well as human umbilical vein endothelial cells with either oxidized or native low-density-lipoprotein in the presence of trace amounts of copper induced morphological changes of the cells and chromatin fragmentation characteristic for programmed cell death. Shrinkage of cells was evident after 6 to 8 hours of incubation and clearly preceded release of lactate dehydrogenase as a marker of cell permeability. Condensation of nuclear chromatin and internucleosomal cleavage was demonstrated by Hoechst staining and gel electrophoresis, respectively. Thus, by inducing active death of endothelial cells oxidized low-density-lipoprotein might negatively influence tissue homeostasis of the endothelium and thereby promote the development of atherosclerotic plaques.
Assuntos
Apoptose/efeitos dos fármacos , Endotélio Vascular/patologia , Lipoproteínas LDL/farmacologia , Animais , Bovinos , Células Cultivadas , Endotélio Vascular/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismoRESUMO
Fibronectin has been found to bind metal ions. Using metal chelate chromatography and limited proteolysis to generate the distinct functional domains of fibronectin we set out to address the metal binding sites to well-defined regions of fibronectin. The results show that the affinity binding of fibronectin to Co2+ is mediated exclusively via the collagen binding domain of the molecule, whereas fibronectin will bind to Zn2+, Ni2+, and Cu2+ by metal binding sites located in two, three, and four well-defined regions of fibronectin, respectively. Fe2+ and Mn2+ chelates did not bind any of the isolated fibronectin domains. Combined metal chelate affinity chromatography opens a possibility to isolate particular fibronectin domains.
Assuntos
Quelantes , Fibronectinas/química , Metais , Sítios de Ligação , Cátions , Cromatografia de Afinidade , Fibronectinas/sangue , Fibronectinas/isolamento & purificação , Humanos , Cinética , Metais/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificaçãoRESUMO
Previous studies have shown that the elongation phase of apoA-I gene transcription is regulated and contributes to hormone-induced changes in the expression of this gene in rat liver. We have now identified, by in vitro transcription studies with HeLa nuclear extracts, two transcriptional arrest sites within exon 3 and intron 3, respectively. Two truncated transcripts of 510 and approximately 1100 nucleotides in length, termed attenuator 1 RNA and attenuator 2 RNA, respectively, were observed when a rat apoA-I genomic fragment extending from -309 to +1842 relative to the transcription start site was transcribed in vitro in the presence of KCl or Sarkosyl. The attenuation events were promoter-independent as transcription of the apoA-I gene driven by the cytomegalovirus promoter resulted in transcriptional arrest at both sites. Transcription studies using deletion constructs as templates identified nucleotides +976 to +1158 as a region that contained the signal for transcriptional arrest at attenuator site 2. Computational analysis predicted a stem;-loop structure in the nascent RNA immediately upstream of the arrest site. Deletion of attenuator 2 signal or deletion of sequences +147 to +216 located far upstream of the actual elongation block site 1 abrogated arrest at site 1. Thus, complex long-range interactions may be involved in the transcriptional arrest at site 1. These elongation blocks identified in vitro are consistent with earlier in vivo data based on nuclear run-on assays and represent, to our knowledge, the first example describing transcriptional attenuation as a mechanism controlling the expression of a member of the apolipoprotein gene family.
Assuntos
Apolipoproteína A-I/genética , Mapeamento Cromossômico , Transcrição Gênica , Animais , Sequência de Bases , Sistema Livre de Células , DNA/química , Pegada de DNA , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/química , RatosRESUMO
The oxidative modification of LDL may play a significant role in atherogenesis. Myeloperoxidase (MPO) expressed in human atherosclerotic plaques has been suggested to be operative in vivo, making LDL atherogenic. Tyrosyl radicals generated by MPO have been shown to act as physiological pro-oxidants of lipid peroxidation in LDL. Assuming that a variety of phenolic compounds are able to form phenoxyl radicals when exposed to peroxidases, we tested the ability of paracetamol, a known analgesic drug with a tyrosine-like monophenolic structure, to act as a pro-oxidant of lipid peroxidation in LDL. Spectroscopic analyses indicated that paracetamol, similar to tyrosine, could undergo peroxidase-induced phenoxyl radical formation, which was inhibited by the radical scavenger ascorbic acid as well as by heme poisons and catalase. Measurement of conjugated dienes and lipid hydroperoxides in LDL preparations exposed to MPO/H2O2 in the absence or presence of paracetamol revealed that the drug could act as a catalyst of lipid oxidation in LDL. Similar results were found when LDL oxidation was performed with activated human neutrophils, which use MPO to promote lipid peroxidation. In conclusion, the results suggest that paracetamol could act, via a phenoxyl radical, as a catalyst of LDL oxidative modification by MPO.
Assuntos
Acetaminofen/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Oxidantes/farmacologia , Peroxidase/metabolismo , Acetaminofen/química , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Azidas/farmacologia , Catalase/farmacologia , Catálise , Cianetos/farmacologia , Dimerização , Sequestradores de Radicais Livres/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Neutrófilos/enzimologia , Oxidantes/química , Oxirredução , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Tirosina/químicaRESUMO
There is now growing evidence that the oxidative modification of LDL plays a potential role in atherosclerosis. In this study, genistein, a compound derived from a soy diet with a flavonoid chemical structure (4',5,7-trihydroxyisoflavone), which was found to inhibit angiogenesis, has been evaluated for its ability to act as an LDL antioxidant and a vascular cell protective agent against oxidized LDL. The results showed that genistein was able to inhibit the oxidation of LDL in the presence of copper ions or superoxide/nitric oxide radicals as measured by thiobarbituric acid-reactive substance formation, alteration in electrophoretic mobility, and lipid hydroperoxides. Bovine aortic endothelial cell- and human endothelial cell-mediated LDL oxidation was also inhibited in the presence of genistein. The 7-O-glucoside of genistein, genistin, was much less effective in inhibiting LDL oxidation in the cell-free and cell-mediated lipoprotein-oxidating systems. Incubating human endothelial cells in the absence or presence of genistein and challenging the cells with already oxidized lipoprotein revealed that in addition to its antioxidative potential during LDL oxidating processes, genistein effectively protected the vascular cells from damage by oxidized lipoproteins. The tyrosine kinase inhibitor genistein was found to block upregulation of two tyrosine-phosphorylated proteins of 132 and 69 kDa in endothelial cells induced by oxidized LDL. Parallel experiments with the inactive analogue daidzein, however, showed that the cytoprotective effect of the isoflavones seems not to be dependent on tyrosine phosphorylation. Our findings will support the suggested and documented beneficial action of a soy diet in preventing chronic vascular diseases and early atherogenic events.
Assuntos
Antioxidantes/farmacologia , Endotélio Vascular/efeitos dos fármacos , Genisteína/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Aorta/citologia , Bovinos , Sistema Livre de Células , Células Cultivadas , Cobre/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Isoflavonas/farmacologia , Lipoproteínas LDL/toxicidade , Óxido Nítrico/metabolismo , Oxidantes/farmacologia , Fosfoproteínas/biossíntese , Fosfoproteínas/fisiologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Superóxidos/metabolismo , Veias Umbilicais/citologiaRESUMO
Chronic administration of thyroid hormone (T3) increases apolipoprotein (apo) A-I gene expression in rat liver. That transcriptional activity of the apoA-I gene is reduced to 50% of control, whereas abundance levels of nuclear and total cellular apoA-I mRNA are increased 3-fold, implies more effective apoA-I mRNA maturation. To study hormonal effects on apoA-I RNA processing, we quantified mRNA precursors in control and T3-treated rats (50 micrograms/100 g body weight for 7 days). Northern blotting, amplification of reverse-transcribed RNA, and ribonuclease protection assays showed that the splicing pathway is branched, in that either intron 1 or intron 2 is removed first from the primary transcript, whereas intron 3 is removed last. In T3-treated rats, abundance levels of the primary transcript, the intron 1-containing precursor devoid of intron 2, the intron 2-containing precursor devoid of intron 1, the intron 3-containing precursor lacking both introns 1 and 2, and nuclear mRNA were 65, 183, 78, 195, and 268% of controls. Compared with control rats, the half-life of the intron 1-containing precursor, measured after injection of actinomycin D, was increased 2-fold in T3-treated rats. In contrast, half-lives of the primary transcript and the intron 2-containing precursor were similar in control and T3-treated rats. Ribonuclease protection assays revealed an RNA species extending from the transcription start site close to the 3' end of intron 1. The abundance of this RNA fragment, probably representing a degradation product, was 2.5-fold higher in control than in T3-treated animals (p < 0.001). Sequences of apoA-I mRNA precursors were identical in control and T3-treated rats which excluded hormonal effects on splice-site selection or post-transcriptional editing of apoA-I transcripts. Compartmental modeling of apoA-I mRNA processing suggested that chronic thyroid hormone administration enhances apoA-I mRNA maturation more than 7-fold by protecting the intron 1-containing precursor devoid of intron 2 from degradation and by facilitating the splicing of intron 1 from this precursor.
Assuntos
Apolipoproteína A-I/genética , Fígado/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Tri-Iodotironina/fisiologia , Animais , Sequência de Bases , DNA Complementar , Íntrons , Masculino , Dados de Sequência Molecular , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Homologia de Sequência do Ácido NucleicoRESUMO
BACKGROUND/AIM: Survival after orthotopic liver transplantation for hepatocellular carcinoma is limited by a high rate of tumor recurrence. A polymerase chain reaction assay based on the detection of albumin mRNA expression in peripheral blood for detection of hematogenous micrometastasis of hepatocellular carcinoma has been described, which may help to select candidates for orthotopic liver transplantation. METHODS: The prognostic value of a highly sensitive nested reverse transcription-polymerase chain reaction assay was evaluated in comparison with the TNM-classification of the International Union against Cancer in a population of liver transplant candidates. RESULTS: Eighty patients with liver disease and 42 control patients were evaluated. Six of 21 patients with hepatocellular carcinoma and 11 of 59 patients with other diseases of the liver were positive for albumin reverse transcription-polymerase chain reaction, making this assay an indicator of ongoing liver damage without absolute specificity for hepatocellular carcinoma. Twelve patients with hepatoma were followed after liver transplantation and seven of those patients had a tumor recurrence within 12 months. Six of these patients with recurrence had International Union against Cancer stage IV A tumors preoperatively, while only one of them was positive for albumin reverse transcription-polymerase chain reaction before transplantation. Only one patient with a stage I to III tumor had a recurrence within 12 months. CONCLUSIONS: Detection of albumin mRNA in peripheral blood by reverse transcription-polymerase chain reaction seems to be an unreliable marker for assessing hematogenous spread of hepatocellular carcinoma. With International Union against Cancer stage IV A being a much better predictor of tumor recurrence, the practical value of albumin mRNA reverse transcription-polymerase chain reaction for patient selection in liver transplant candidates seems to be very limited.