RESUMEN
OBJECTIVES: The toll-like receptor (TLR) family is thought to be expressed in many cell types in the skin and play a role in various diseases. The expression pattern and role of TLRs in systemic sclerosis (SSc) is to be clarified. We investigated the expression profiles of TLR-related genes in SSc fibroblasts, and tried to clarify their roles in the pathogenesis of this disease. METHODS: The expression profile of TLR-related genes was assessed by gene array. Real-time PCR was used to confirm the array result. The protein expression of TLRs and type I collagen was determined by immunoblotting and immunohistochemistry. RESULTS: PCR array revealed that several genes were up- or down-regulated in SSc fibroblasts compared to normal cells. Among them, both mRNA and protein levels of TLR5 and TLR10 were up-regulated in SSc fibroblasts. The transfection of Smad3 siRNA into SSc fibroblasts resulted in the down-regulation of TLR proteins. There was no significant difference in mRNA half-lives of TLR5 and TLR10 between normal and SSc fibroblasts. Immunohistochemical staining revealed that TLRs expression was strongly detected in SSc fibroblasts in vivo. The stimulation of TLR5 signal with flagellin reduced the expression of type I collagen in SSc fibroblasts, but not in normal fibroblasts. CONCLUSIONS: TLR5 and TLR10 expression is increased in SSc fibroblasts in vitro and in vivo, probably at transcript level via the TGF-ß/Smad3 activation. Furthermore, TLR5 itself may have suppressive effects on collagen expression, and its overexpression in SSc fibroblasts may be the negative feedback against tissue fibrosis.
Asunto(s)
Colágeno Tipo I/metabolismo , Citocinas/genética , Fibroblastos/metabolismo , ARN Mensajero/metabolismo , Esclerodermia Sistémica/genética , Receptores Toll-Like/genética , Western Blotting , Células Cultivadas , Citocinas/metabolismo , Dermis/citología , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerodermia Sistémica/metabolismo , Receptores Toll-Like/metabolismo , TransfecciónRESUMEN
BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by intense proliferation and abnormal differentiation of keratinocytes, although the pathogenesis is still not completely clarified. OBJECTIVES: We investigated the mechanism of keratinocyte proliferation seen in psoriasis, focusing on microRNA (miRNA). MATERIALS AND METHODS: miRNAs were extracted from tissues and sera of psoriasis, atopic dermatitis and healthy control. To determine pathogenic miRNAs, we performed miRNA polymerase chain reaction (PCR) array analysis. The results were confirmed with quantitative real-time PCR, in situ hybridization, immunohistochemistry, transient transfection of siRNA and inhibitor in cultured keratinocytes and Western blotting. RESULTS: PCR array analysis using tissue miRNA demonstrated miR-424 level was markedly decreased in psoriasis skin in vivo. Protein expression of mitogen-activated protein kinase kinase 1 (MEK1) or cyclin E1, predicted target genes of miR-424, was increased in psoriatic skin, although their mRNA levels were not. The transfection of specific inhibitor of miR-424 in normal human keratinocytes led to upregulation of MEK1 or cyclin E1 protein, and resulted in increased cell proliferation. On the other hand, cell number was significantly decreased when cells were transfected with siRNA for MEK1 or cyclin E1. Furthermore, we first investigated serum miRNA levels in psoriasis. Although not significant, serum miR-424 concentration tended to be decreased in patients with psoriasis compared with healthy controls. CONCLUSIONS: Decreased miR-424 expression and subsequently increased MEK1 or cyclin E1 may play a key role in the pathogenesis of psoriasis. Investigation of the regulatory mechanisms of keratinocyte proliferation by miRNA may lead to new treatments and a disease activity marker.
Asunto(s)
Proliferación Celular , Queratinocitos/patología , MicroARNs/metabolismo , Psoriasis/patología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Células Cultivadas , Ciclina E/metabolismo , Femenino , Humanos , MAP Quinasa Quinasa 1/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Oncogénicas/metabolismoRESUMEN
Interferon (IFN) gamma induces replacements of the proteasomal subunits X and Y by LMP7 and LMP2, respectively, resulting in an alteration of the proteolytic specificity. We found a third pair of proteasome subunits expressed reciprocally in response to IFN-gamma. Molecular cloning of a cDNA encoding one subunit designated as Z, downregulated by IFN-gamma, showed that it is a novel proteasomal subunit with high homology to MECL1, which is markedly induced by IFN-gamma. Thus, IFN-gamma induces subunit replacements of not only X and Y by LMP7 and LMP2, respectively, but also of Z by MECL1, producing proteasomes responsible for immunological processing of endogenous antigens. When processed from their precursors, three pairs of the 10 homologous, but distinct, beta-type subunits of eukaryotic proteasomes, that is, X/LMP7, Y/LMP2, and Z/MECL1, have an NH2-terminal threonine residue, assumed to be part of a catalytic center. These findings suggest that the altered molecular organization of the proteasome induced by IFN-gamma may be responsible for acquisition of its functional change.
Asunto(s)
Cisteína Endopeptidasas/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Interferón gamma/farmacología , Complejos Multienzimáticos/efectos de los fármacos , Secuencia de Aminoácidos , Presentación de Antígeno , Secuencia de Bases , Bandeo Cromosómico , Mapeo Cromosómico , Clonación Molecular , Cisteína Endopeptidasas/biosíntesis , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Regulación hacia Abajo , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejo de la Endopetidasa Proteasomal , Biosíntesis de Proteínas , Conformación Proteica , Procesamiento Proteico-Postraduccional , Homología de Secuencia de AminoácidoAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Quimiocina CCL22/metabolismo , Fármacos Dermatológicos/uso terapéutico , Psoriasis/tratamiento farmacológico , Artritis Psoriásica/tratamiento farmacológico , Femenino , Humanos , Infliximab , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Proteasomes are the proteolytic complex responsible for major histocompatibility complex (MHC) class I-restricted antigen presentation. Interferon gamma treatment increases expression MHC-encoded LMP2 and LMP7 subunits of the proteasome and decreases expression of two proteasome subunits, named X and Y, which alters the proteolytic specificity of proteasomes. Molecular cloning of complementary DNAs encoding X and Y showed that their proteins are proteasomal subunits with high amino acid similarity to LMP7 and LMP2, respectively. Thus, interferon gamma may induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing proteasomes perhaps more appropriate for the immunological processing of endogenous antigens.
Asunto(s)
Cisteína Endopeptidasas , Regulación hacia Abajo , Interferón gamma/farmacología , Complejos Multienzimáticos , Complejo de la Endopetidasa Proteasomal , Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Endopeptidasas/química , Endopeptidasas/genética , Humanos , Complejo Mayor de Histocompatibilidad , Datos de Secuencia Molecular , Proteínas/química , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
The present study was performed to compare the long-term effects of 24-h ambulatory blood pressure (BP) control with amlodipine versus valsartan on vascular damage in untreated hypertensive patients. Amlodipine and valsartan have benefits on cardiovascular mortality and morbidity in hypertensive patients. Although ambulatory BP is associated with severity of target-organ damage in hypertensive patients, beneficial effects of ambulatory BP control with amlodipine versus valsartan on vascular damage have not been compared. Pulse wave velocity (PWV), intima-media thickness (IMT) of the carotid arteries, urinary albumin excretion (UAE) and 24-h ambulatory BP were determined in 100 untreated hypertensive patients before and 12 months after the start of antihypertensive therapy with amlodipine or valsartan. Amlodipine and valsartan decreased ambulatory BP similarly, but the variability of 24-h and daytime ambulatory systolic BP was significantly reduced by amlodipine but not by valsartan. The reduced variability of ambulatory systolic BP caused by amlodipine significantly contributed to the improvement of PWV, although both drugs decreased PWV similarly. Carotid IMT was unaffected by treatment with either drug. Valsartan significantly decreased UAE independently of its depressor effect, but amlodipine had no effect on UAE. These results suggest that the 24-h control of ambulatory BP with amlodipine had functionally improved the stiffened arteries of hypertensive patients by the end of 12 months of treatment, in part through reducing BP variability, whereas ambulatory BP control with valsartan decreased the arterial stiffness to the same degree as amlodipine without affecting BP variability maybe through some pleiotropic effects.
Asunto(s)
Amlodipino/administración & dosificación , Antihipertensivos/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Arterias Carótidas/fisiopatología , Hipertensión/tratamiento farmacológico , Tetrazoles/administración & dosificación , Valina/análogos & derivados , Monitoreo Ambulatorio de la Presión Arterial , Endotelio Vascular/lesiones , Endotelio Vascular/fisiopatología , Femenino , Humanos , Hipertensión/mortalidad , Hipertensión/fisiopatología , Hipertensión/orina , Masculino , Persona de Mediana Edad , Pulso Arterial , Valina/administración & dosificación , Valsartán , Resistencia Vascular/efectos de los fármacosRESUMEN
Blood pressure (BP) variability is estimated as the standard deviation of 24-h ambulatory BP. The present study was performed to determine the effect of the mean 24-h ambulatory BP values and standard deviations on arterial wall stiffness assessed by brachial-ankle pulse wave velocity (baPWV). Brachial-ankle pulse wave velocity, carotid intima-media thickness (IMT), urinary albumin excretion (UAE) and 24-h ambulatory BP were measured before the start of antihypertensive therapy in 203 newly diagnosed hypertensive patients (53.3+/-0.7 years old; clinic systolic/diastolic BP: 154+/-1/98+/-1 mm Hg), and univariate and multivariate regression analyses of these clinical and biological parameters were performed. Univariate regression analyses revealed a significant association between mean baPWV values and the standard deviations of ambulatory systolic/diastolic BP. Mean ambulatory systolic/diastolic BP values were also associated with UAE, and the standard deviations of ambulatory systolic BP were associated with maximum carotid IMT. Quintile analyses showed that patients with a mean 24-h ambulatory mean BP value and standard deviation below 110 and 20 mm Hg, respectively, had the lowest baPWV. Moreover, the multivariate regression analyses confirmed a significant correlation between baPWV and the standard deviation of 24-h ambulatory systolic BP. In conclusion, untreated hypertensive patients with a higher 24-h ambulatory systolic BP variability had stiffer arterial walls. Ambulatory systolic BP variability may be involved in stiffening of the arteries of hypertensive patients.
Asunto(s)
Tobillo/irrigación sanguínea , Monitoreo Ambulatorio de la Presión Arterial/métodos , Presión Sanguínea/fisiología , Arteria Braquial/fisiopatología , Hipertensión/fisiopatología , Anciano , Anciano de 80 o más Años , Arterias/patología , Arterias/fisiopatología , Velocidad del Flujo Sanguíneo/fisiología , Fenómenos Fisiológicos Cardiovasculares , Femenino , Humanos , Hipertensión/patología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Análisis de Regresión , Resistencia Vascular/fisiologíaRESUMEN
Proteasomes and ubiquitin (Ub) are essential components of the energy-dependent, nonlysosomal proteolytic pathway. To clarify the physiological role of this proteasome/Ub-dependent pathway, we meaured the levels of expressions of proteasomes and Ub in human renal cancers by Northern blot and immunochemical analyses. The mRNAs for two of the multiple subunits of proteasomes, C2 and C9, were expressed at abnormally high levels in most neoplastic lesions of patients with various primary renal cell carcinomas and in all renal cancer cell lines examined. However, no significant difference was found by enzyme immunoassay in the proteasomal contents of cancerous and normal parts of the kidney. The levels of mRNAs for the subunits of proteasomes were high in rapidly proliferating renal cells and appeared to be correlated with the activities of these cells for proteasome synthesis, but the cellular contents of proteasomes in these cells were normal, suggesting rapid turnover of proteasomes in rapidly proliferating cancer cells. Consistent with the increased expressions of proteasomal mRNAs, the expressions of three Ub genes, mono-UbA80, mono-UbA52, and poly-UbC, were found to be greatly increased in these renal cancer cells. Immunohistochemical staining of normal kidney showed that the levels of both proteasomes and Ub were high in cells of renal tubules and collecting ducts, but low in the glomerulus. The levels of both proteins appeared to be considerably increased in the nuclei of granular and clear carcinoma cells of the kidney. Moreover, the profiles of cellular proteins conjugated with Ub in normal kidney tissues were different from those in cancerous parts of the kidney and in established renal cancer cells. These results suggest that the proteasome- and ubiquitin-mediated system is functionally involved in the cancerous state in human kidney.
Asunto(s)
Cisteína Endopeptidasas/genética , Neoplasias Renales/genética , Riñón/fisiología , Complejos Multienzimáticos/genética , Ubiquitinas/genética , Adulto , Anciano , Northern Blotting , División Celular , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Riñón/citología , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal , ARN Mensajero/genética , ARN Neoplásico/genética , Células Tumorales Cultivadas , Ubiquitinas/inmunologíaRESUMEN
Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferse (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rat limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate. For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1- 14C]tyrosine. Then their respiratory 14CO2 and the incorporation of 14C into total lipids of liver were measured. The amounts of radioactivity in CO2 and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U- 14C]acetate the radioactivities in CO2 and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studied gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.
Asunto(s)
Cuerpos Cetónicos/biosíntesis , Lípidos/biosíntesis , Hígado/enzimología , Tirosina/metabolismo , 4-Hidroxifenilpiruvato Dioxigenasa/metabolismo , Animales , Ácido Ascórbico/farmacología , Proteínas en la Dieta , Inducción Enzimática/efectos de los fármacos , Hidrocortisona/farmacología , Hígado/efectos de los fármacos , Masculino , Ratas , Tirosina Transaminasa/metabolismoRESUMEN
L-Lysine-alpha-ketoglutarate reductase (N5-(1,3-dicarboxypropyl)-L-lysine: NADP+ oxidoreductase (L-lysine-forming, EC 1.5.1.8) was purified from rat liver mitochondria to a homogeneous state judged by SDS polyacrylamide gel electrophoresis, and its molecular weight was estimated as 52000. On Sepharose 4B filtration it has a molecular weight of 230 000 and it is suggested that the active enzyme is a tetramer of subunits of similar size. The purified enzyme was clearly separated from saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine:NAD+ oxidoreductase (L-glutamate-forming, EC 1.5.1.9). The reactions of purified L-lysine-alpha-ketoglutarate reductase favored the forward reaction (saccharopine formation) and the rate of the reverse reaction (lysine formation) was only 3--5% that of the forward reaction. The forward reaction was specific for L-lysine, alpha-ketoglutarate and NADPH and followed Michaelis-Menten kinetics, whereas the dose vs. response curve of the reverse reaction was sigmoidal with saccharopine. Among the amino acids examined, ornithine, leucine and tryptophan inhibited the forward reaction competitively. These results are different from earlier reports on human and yeast enzymes. The fact that rats fed on lysine-deficient diet do not lose weight much is discussed in relation to the properties of this enzyme.
Asunto(s)
Mitocondrias Hepáticas/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Sacaropina Deshidrogenasas/metabolismo , Animales , Cinética , Masculino , Peso Molecular , Ratas , Sacaropina Deshidrogenasas/aislamiento & purificación , Especificidad por SustratoRESUMEN
The developmental change in gene expression of tryptophan 2,3-dioxygenase (EC 1.13.11.11) in rat liver was studied by dot-blot hybridization with cDNA of the enzyme as a probe. The mRNA of tryptophan oxygenase is not expressed in fetal liver, but is expressed very slightly 1 day after birth. Its expression increases first gradually until 12 days after birth and then rapidly, and reaches the adult level about 22 days after birth. On the other hand, mRNA of albumin in the liver, measured with its cDNA, increases rapidly in the late fetal period and reaches almost the adult level at the time of birth. Studies on in vitro transcription by the nuclear run-off technique showed that the developmental increases in the mRNAs of tryptophan oxygenase and albumin are caused by an increase in the rates of transcription of their genes. Treatment of rats with cortisol significantly increased the amount of tryptophan oxygenase mRNA in the liver from soon after birth. This treatment did not increase mRNA of albumin. It is suggested from these findings that the gene of tryptophan oxygenase is switched on as early as the first day after birth in the few differentiated hepatocytes present in the liver and that the number of these differentiated cells gradually increases during early postnatal development. Although injected glucocorticoid stimulated transcription of the gene of tryptophan oxygenase precociously during this period, presumably in vivo the activity of tryptophan oxygenase normally increases about 2 weeks after birth, because this is when the plasma concentrations of glucocorticoid and glucagon increase sufficiently to be effective.
Asunto(s)
Hígado/enzimología , Triptófano Oxigenasa/metabolismo , Triptófano/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa , Hígado/crecimiento & desarrollo , ARN Mensajero/genética , Ratas , Albúmina Sérica/genética , Transcripción Genética/efectos de los fármacos , Triptófano Oxigenasa/genéticaRESUMEN
An aminotransferase (transaminase) which is active for leucine and methionine, but not for valine or isoleucine, was purified from rat liver mitochondria. The purified preparation appeared homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was shown to be 55 000 by gel filtration. It differed from enzyme II (leucine aminotransferase, EC 2.6.1.6) in the supernatant fraction, another transaminase which is also specific for leucine and methionine, in molecular weight, Km values for substrates, electrophoretic mobility, chromatographic behavior and heat stability. From comparison with related transaminases it was concluded to be a new enzyme and named mitochondrial leucine (methionine) transaminase.
Asunto(s)
Mitocondrias Hepáticas/enzimología , Transaminasas , Animales , Estabilidad de Medicamentos , Electroforesis Discontinua , Leucina , Masculino , Metionina , Peso Molecular , Ratas , Relación Estructura-Actividad , Transaminasas/aislamiento & purificación , Transaminasas/metabolismoRESUMEN
L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP) in the liver of adult rats increased 4-5 times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, in controlled by both glucagon and glucocorticoid.
Asunto(s)
Dexametasona/farmacología , Glucagón/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/biosíntesis , Sacaropina Deshidrogenasas/biosíntesis , Glándulas Suprarrenales/fisiología , Factores de Edad , Animales , Células Cultivadas , Dactinomicina/farmacología , Diabetes Mellitus Experimental/enzimología , Inducción Enzimática/efectos de los fármacos , Feto/enzimología , Insulina/farmacología , Hígado/enzimología , Masculino , RatasRESUMEN
Hormonal inductions of lipogenic enzyme activities (fatty acid synthetase, malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) and ATP-citrate lyase) were studied in primary cultured rat hepatocytes. Insulin, triiodothyronine and dexamethasone markedly stimulated the inductions of the enzymes (particularly G6PD and ME) in the presence of pyruvate. Lactate also induced their activities. The activities of these enzymes in the presence of appropriate hormone combinations and a substrate amount of pyruvate were as high as, or higher than those in the liver of rats on high-carbohydrate, low-fat diet. The aldolase and glucokinase activities induced by these hormones were not enhanced by the addition of pyruvate. The induction by pyruvate was inhibited by actinomycin D or cycloheximide. The ATP content of rat hepatocytes was maintained without increase during culture with pyruvate for 6 days. These results indicate that the additions of pyruvate, or its metabolites to cultures of isolated hepatocytes have specific effects on the inductions of certain hepatic enzymes, possibly acting at the level of transcription. Their effects are similar to those of feeding a high-carbohydrate, low-fat diet to intact animals.
Asunto(s)
Ácido Graso Sintasas/biosíntesis , Hormonas/farmacología , Hígado/enzimología , Malato Deshidrogenasa/biosíntesis , Piruvatos/farmacología , ATP Citrato (pro-S)-Liasa/biosíntesis , Adenosina Trifosfato/análisis , Animales , Células Cultivadas , Cicloheximida/farmacología , Dactinomicina/farmacología , Inducción Enzimática , Glucosafosfato Deshidrogenasa/biosíntesis , Lactatos/farmacología , Hígado/efectos de los fármacos , Ratas , Ratas Wistar , Regulación hacia ArribaRESUMEN
The quantity of translatable mRNA of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) in primary cultures of adult rat hepatocytes subjected to different hormonal conditions was determined with a reticulocyte-lysate, cell-free system. The level of glucose-6-phosphate dehydrogenase mRNA was about 5-fold higher in the presence of insulin than in its absence. This increase of glucose-6-phosphate dehydrogenase mRNA reached a maximum 12 h after the addition of insulin. The maximum level of induction of glucose-6-phosphate dehydrogenase mRNA required 10(-8) M insulin. Glucagon and triiodothyronine had no effect on the glucose-6-phosphate dehydrogenase mRNA level. The increase of glucose-6-phosphate dehydrogenase activity correlated with the increase in level of mRNA of this enzyme. This suggests that the changes in glucose-6-phosphate dehydrogenase activity in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.
Asunto(s)
Glucagón/farmacología , Glucosafosfato Deshidrogenasa/genética , Insulina/farmacología , Hígado/enzimología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , Animales , Sistema Libre de Células , Células Cultivadas , Cinética , Hígado/efectos de los fármacos , Conejos , Ratas , Reticulocitos/metabolismoRESUMEN
Developmental increase of tryptophan oxygenase (L-tryptophan: oxygen 2, 3-oxidoreductase (decyclizing), EC 1.13.11.11) was studied using hepatocytes of neonatal rats in primary culture. Hepatocytes from rats of 2-30-days-old were isolated and cultured for 2 days. In cultured hepatocytes of 2-day-old rats, tryptophan (2.5 mM), dexamethasone (1 x 10(-5) M) and glucagon (1 x 10(-7) M) did not cause the appearance of tryptophan oxygenase. But the enzyme activity became detectable, when hepatocytes from 5-day-old rats were incubated with tryptophan, the oxygenase could be induced precociously by dexamethasone, but by glucagon. The effect of glucagon was first seen 2 weeks after birth. However, in hepatocytes of 9-day-old rats glucagon stimulated formation of cyclic AMP and protein kinase activity (EC 2.7.1.37) and also induced tyrosine aminotransferase (EC 2.6.1.5). When hepatocytes of 9-day-old rats were cultured for 4 days, their tryptophan oxygenase became inducible by glucagon. Insulin almost completely inhibited precocious appearance of the enzyme activity evoked by tryptophan plus dexamethasone in hepatocytes of 9-day-old rats. These studies suggest that the appearance of tryptophan oxygenase in rat liver during development is due to first the onset of gene coding for tryptophan oxygenase and then stimulation by the sequential actions of glucocorticoid and glucagon.
Asunto(s)
Dexametasona/farmacología , Glucagón/farmacología , Hígado/enzimología , Triptófano Oxigenasa/biosíntesis , Triptófano/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , Inducción Enzimática/efectos de los fármacos , Indolamina-Pirrol 2,3,-Dioxigenasa , Hígado/citología , Hígado/crecimiento & desarrollo , RatasRESUMEN
Proteasomes play a major role in non-lysosomal pathways of protein turnover mediated by distinct multiple proteolytic activities. Identification of their active-site residues is important for elucidating their catalytic mechanisms. Here we report the nucleotide sequences of three human proteasomal subunits, HsN3, HsC7-I and HsC10-II, coding for proteins with 264, 201 and 205 amino acid residues with calculated molecular weights of 29,192, 22,836 and 22,931, respectively. Sequence comparison showed that all three proteins belong to the beta-type superfamily and that they are the human counterparts of subunits reported to participate in the peptidyl-glutamyl-peptide hydrolyzing, chymotrypsin-like and trypsin-like activity of this complex. Alignments of the putative catalytically active subunits of various species revealed several family-specifically conserved serinyl residues within highly conserved amino acid stretches. Based on localization and hydrophobicity, the roles of these amino acid residues as active site and substrate binding site candidates are discussed.
Asunto(s)
Cisteína Endopeptidasas/química , Complejos Multienzimáticos/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Secuencia de Consenso , ADN Complementario/genética , Datos de Secuencia Molecular , Peso Molecular , Familia de Multigenes , Complejo de la Endopetidasa Proteasomal , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , SolubilidadRESUMEN
To determine why the rat serine dehydratase gene becomes transcriptionally activated just after birth, we examined the interactions of DNA binding proteins of fetal and adult rat livers with the serine dehydratase gene promoter by DNase I protection analysis and gel mobility shift assay. Several binding regions of nuclear proteins were found to be common to fetal and adult livers and interaction of factors with the characteristics of Sp1 or NF-Y was suggested. Two additional regions, named regions B and I, were specific to fetal liver. These regions contain GATA-like sequences and competition experiments by gel mobility shift assay suggested that the fetal liver-enriched factor binds to the GATA-like sequences. The function of the regions B and I in transcription regulation was investigated in fetal and adult hepatocytes by transient DNA transfer experiments with serine dehydratase-chloramphenicol acetyltransferase fusions. These experiments showed that these regions functioned as negative cis-acting elements in fetal hepatocytes, but not in adult hepatocytes.
Asunto(s)
L-Serina Deshidratasa/genética , Hígado/metabolismo , Proteínas Represoras/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Regulación de la Expresión Génica , Hígado/embriología , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Ratas , Ratas Wistar/embriologíaRESUMEN
Mature adult rat hepatocytes were cultured as monolayers in serum-free Williams medium E containing 10(-7) M each of insulin (Ins), dexamethasone (Dex) and triiodothyronine (T3) and 30 mM pyruvate. The hepatocytes remained morphologically intact for at least 14 days, during which period they maintained normal liver functions such as the expressions of cytochrome P-450 mRNA and glucokinase and secretion of albumin. They also retained the ability to resume proliferation. Cells cultured with pyruvate had a much higher ATP level than those without pyruvate, suggesting that pyruvate can sustain functional hepatocytes for a long period in culture in the presence of Ins, Dex and T3, probably by producing enough energy for their maintenance.
Asunto(s)
Medios de Cultivo , Hormonas/farmacología , Hígado/citología , Piruvatos/farmacología , Adenosina Trifosfato/metabolismo , Albúminas/metabolismo , Animales , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , ADN/biosíntesis , Dexametasona/farmacología , Glucoquinasa/metabolismo , Insulina/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Microscopía Electrónica , Ácido Pirúvico , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Triyodotironina/farmacologíaRESUMEN
In cell-free extracts of Alternaria solani, an enzymatic activity converting prosolanapyrone II to solanapyrones A and D via oxidation and subsequent Diels-Alder reaction has been found. Chromatography with DEAE-Sepharose provided two active fractions, pools 1 and 2. The former fraction converted prosolanapyrone II to solanapyrones A and D in a ratio of 2.2:1 with optical purities of 99% and 45% ee, respectively. The latter fraction did so in a ratio of 7.6:1 with 99% and nearly 0% ee, respectively. The enzyme partially purified from pool 2 native molecular weight of 40-62 kD and a pl of 4.25. The high reactivity of prosolanapyrone III in aqueous solution and the chromatographic behavior of the enzyme in pool 2 suggest that a single enzyme catalyzes both the oxidation and Diels-Alder reaction.