RESUMEN
Trisomy karyotype occurs in 5%-10% of AML. Its mutational landscape and prognostic significance are not well defined. A cohort of 156 trisomy AML patients was analysed, with reference to 615 cytogenetically normal (CN) AML patients. Trisomy AML showed distinct mutational landscape with more prevalent SMC1A, N/KRAS, ASXL1 and BCOR but fewer CEBPAbZIP and NPM1 mutations in patients ≤60, and fewer NPM1 mutations in those >60. NRAS mutations were associated with poor outcome in trisomy AML, whereas DNMT3A and FLT3-ITD mutations had neutral effect. Trisomy AML appeared biologically distinct from CN-AML.
Asunto(s)
Leucemia Mieloide Aguda , Proteínas Nucleares , Humanos , Proteínas Nucleares/genética , Nucleofosmina , Leucemia Mieloide Aguda/genética , Trisomía , Mutación , Cariotipo , Pronóstico , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
BACKGROUND: Papaya is widely grown in Malaysia and normally only the fruits are consumed. Other parts of the plant such as leaves, roots, bark, peel, seeds and pulp are also known to have medicinal properties and have been used to treat various diseases. Papaya leaves also contain flavonoids, alkaloids phenolic compounds and cynogenetic compounds, and are also reported to be able to treat dengue fever. RESULTS: Studies were carried out on drying of papaya leaves using hot air (60, 70 and 80 °C), shade and freeze drying. Effective diffusivities were estimated ranging from 2.09 × 10-12 to 2.18 × 10-12 m2 s-1 from hot air drying, which are within the order of magnitudes reported for most agricultural and food products. The activation energy to initiate drying showed a relatively low value (2.11 kJ mol-1 ) as a result of the thin leave layer that eased moisture diffusion. In terms of total polyphenols content and antioxidant activities, freeze-dried sample showed a significantly higher (P < 0.05) total polyphenols content [2158 mg gallic acid equivalent 100 g dry weight-1 ] and antioxidant activities [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) = 571 mg TE 100 g DW-1 and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) = 215 µg mg-1 ] compared to hot air and shade dried samples. CONCLUSION: Freeze dried sample retained the most total polyphenols content and showed the highest antioxidant activities in both ABTS and DPPH antioxidant assays. Hot air and shade drying are not conducive with repect to preserving the antioxidants as a result of possible thermal degradation at elevated temperatures and oxidations under prolonged drying condition. © 2020 Society of Chemical Industry.
Asunto(s)
Antioxidantes/análisis , Carica/química , Desecación/métodos , Liofilización/métodos , Polifenoles/análisis , Calor , Hojas de la Planta/químicaRESUMEN
The present study aimed to define a subtype of complex/monosomal karyotype (CK/MK) acute myeloid leukemia (AML) by its distinct clinical features, p53 signaling and responses to p53 targeting agents. Ninety-eight young adults (range: 21-60 years; median: 49 years) with CK/MK AML were studied. They received standard induction, consolidation and allogeneic hematopoietic stem cell transplantation from siblings or matched unrelated donors if available. Chromosomal abnormalities most commonly affected chromosome 5 (30%), 7 (22%) and 17 (21%). Next generation sequencing of a 54-myeloid gene panel were available in 76 patients. Tumor protein 53 (TP53) mutations were most common (49%) and associated with the presence of -5/5q- (P < .001) and -17/17p- (P < .001), but not -7/7q- (P = .370). This "typical" CK/MK AML subtype was associated with significantly lower presenting white cell counts, higher number of karyotypic abnormalities, and inferior leukemia-free and overall survivals, compared with CK/MK AML without the typical features. Blood or bone marrow samples from typical CK/MK AML patients showed defective p53 signaling upon induction by etoposide. In vitro drug sensitivity analysis showed that they were sensitive to APR-246 that targeted mutant p53, but resistant to MDM2 antagonist MI-77301. Novel therapeutic strategies targeting TP53 mutations in CK/MK AML should be developed and tested in clinical trials.
Asunto(s)
Cariotipo Anormal , Antineoplásicos/administración & dosificación , Cromosomas Humanos , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Monosomía , Proteína p53 Supresora de Tumor , Adolescente , Adulto , Cromosomas Humanos/genética , Cromosomas Humanos/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Leucemia Mieloide Aguda , Adolescente , Adulto , Anciano , Supervivencia sin Enfermedad , Femenino , Humanos , Idarrubicina/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Mitoxantrona/administración & dosificación , Tasa de Supervivencia , Adulto JovenRESUMEN
A competitive protein-binding (CPB) assay, suitable for measuring corticosterone levels in 20 mul of mouse plasma or 100 mg of brain, is described. The postnatal development of adrenocortical function was determined in C57BL/10 and DBA/1 mice by CPB assay of basal and stressinduced levels of plasma corticosterone and resting levels of brain corticosterone. Marked increases in both basal and stressed levels of plasma corticosterone were found beginning at day 12 after birth: mean basal levels rose from about 1 mug/u99 ml on day 12 to peak values of about 10-15 mug/100 ml on days 18-20, and then declined by day 30 to the 13-day level of 2.6 mug/100 ml. This pattern differs significantly from results obtained with standard fluorometric assays for corticosterone; it was determined that a major part of this discrepancy is due to the lack of specificity of the fluorometric assay. The developmental change in brain corticosterone was similar to the pattern found in plasma. Only the stress-induced levels of plasma corticosterone showed significant genetic variation, and this did not appear until about one week after the end of the relative stress-nonresponsive period. These findings should be useful in evaluating hypotheses concerning the developmental regulation of adrenocortical function and the action of glucocorticoids in regulating the biochemical differentiation of other tissues.
Asunto(s)
Química Encefálica , Corticosterona/análisis , Factores de Edad , Animales , Unión Competitiva , Corticosterona/sangre , Estimulación Eléctrica , Fluorometría , Masculino , Métodos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Unión ProteicaRESUMEN
Administration of corticosterone (10 mg/kg, ip, twice daily for 3 days) to mice during the second week of postnatal development led to an increase of tyrosine hydroxylase (TH) activity in the locus coeruleus, but not in the substantia nigra. The corticosterone effect was observed only transiently during this developmental period. Tritiated corticosterone can bind to a cytosol fraction prepared from mouse locus coeruleus, with a specific binding capacity of 110 fmol/mg protein. There is a correlation between the ability of various steroids to increase TH activity and their binding to the cytosol glucocorticoid receptor. Cortexolone and progesterone, two antiglucocorticoids that can bind to the cytosol receptor, were found to abolish the effect of corticosterone in increasing TH activity. It appears that the noradrenergic neurons in the locus coeruleus may be target cells for glucocorticoids, and that the glucocorticoid effect on TH may be a receptor-mediated mechanism.
Asunto(s)
Corticosterona/farmacología , Locus Coeruleus/crecimiento & desarrollo , Tirosina 3-Monooxigenasa/metabolismo , Animales , Unión Competitiva , Corticosterona/metabolismo , Cortodoxona/farmacología , Locus Coeruleus/efectos de los fármacos , Locus Coeruleus/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Progesterona/farmacología , Receptores de Glucocorticoides/metabolismoRESUMEN
A versatile expression vector system for construction of gene and protein fusions, specific radiolabeling of gene products and high-level protein production is described. Expression cassettes were constructed containing structural genes encoding native and analog forms of connective tissue-activating peptide-III (CTAP-III), beta-thromboglobulin, neutrophil-activating protein and modified regulatory sequences derived from the colicin E1 operon. Gene expression was enhanced by changes in the colicin promoter that increased the transcription initiation rate both in vivo and in vitro, and by deletion of a sequence affecting catabolite repression. High-level expression, producing recombinant protein up to 30% of the total cellular protein, was induced rapidly after stimulation of the SOS response by using either mitomycin C or nalidixic acid, by temperature shift using temperature-sensitive mutations in the LexA or RecA proteins, or by UV light. The presence of radiolabeled amino acids during induction resulted in greater than 95% preferential labeling of recombinant proteins. CTAP-III remained stable for more than 6 h following decay of the inducing signal. The use of CTAP-III in protein fusions improved stability of several therapeutically useful proteins including the thrombin-specific inhibitor, hirudin and a cell receptor-binding domain of laminin, when they were produced in Escherichia coli.
Asunto(s)
Factores de Coagulación Sanguínea/genética , Colicinas , Genes Sintéticos , Vectores Genéticos , Péptidos , Proteínas Recombinantes de Fusión/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Recombinante , Escherichia coli , Humanos , Datos de Secuencia Molecular , Operón , PlásmidosRESUMEN
The high affinity uptake of [3H]choline by the superior cervical ganglion, isolated from the rat, was found to be increased by dexamethasone. Maximal increase (60-65% above control values) occurred at the steroid concentration of 5 X 10(-5) M. Other glucocorticoids (triamcinolone, corticosterone and hydrocortisone) were without an effect on the [3H]choline uptake. Following administration of dexamethasone (25 mg/kg, i.p.), there was a marked increase in the level of choline in the ganglion. The increase was 3-fold at 1 hr and 10-fold at 6 hr, and by 24 hr the choline levels still remained higher in the steroid-treated animals than in the controls. Levels of acetylcholine in the ganglion were also increased, beginning at 1 hr after the injection of steroid. The increase was 85% by 3 hr and 60% by 6 hr. Triamcinolone, a glucocorticoid that was without an effect on [3H]choline uptake in vitro, was also ineffective in altering the levels of choline and acetylcholine in vivo. It seems probable that the increase of choline uptake in the ganglion induced by dexamethasone may, at least in part, occur in the preganglionic cholinergic terminals, leading to increased synthesis of acetylcholine. Such an effect of dexamethasone provides another case of a selective steroid acting directly on nerve terminals by altering a transport mechanism.
Asunto(s)
Colina/metabolismo , Dexametasona/farmacología , Ganglios Simpáticos/metabolismo , Acetilcolina/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Ganglios Simpáticos/efectos de los fármacos , Glucocorticoides/farmacología , Cinética , Masculino , Ratas , Ratas EndogámicasRESUMEN
Mammalian brain sodium channels consist of an alpha subunit and two smaller beta subunits. The role of the beta 1 subunit in modulating ligand interactions at these channels was examined using a cell line stably expressing human beta1 and rat brain IIA alpha subunits. Coexpression of the beta 1 subunit had no effect on the potencies of sodium channel blockers in inhibiting whole cell [3H]batrachotoxinin A benzoate ([3H]BTX) binding or veratridine-stimulated [14C]guanidinium influx. Coexpression of the beta 1 subunit also had no effect on the potencies of alpha scorpion toxin, brevetoxin, or RU 39568 in stimulating [14C]guanidinium influx. By contrast, coexpression of the beta 1 subunit had dramatic effects on ligand interactions in isolated membranes. In isolated membranes of cells expressing only the alpha subunit, the neurotoxins had no stimulatory effect on [3H]BTX binding and the potencies of local anesthetic-like channel inhibitors were 10-100-fold lower than those at native sodium channels. Whereas in membranes of cells coexpressing the beta 1 subunit, the neurotoxins increased [3H]BTX binding 30-fold and the potencies of the sodium channel inhibitors closely matched those found at native sodium channels. These findings indicate that the beta 1 subunit is not required for the binding of sodium channel activators or inhibitors but rather, that the beta 1 subunit may stabilize the alpha subunit in a functional conformation thereby allowing detection of these interactions in disrupted membranes.
Asunto(s)
Anestésicos Locales/farmacología , Encéfalo/efectos de los fármacos , Interacciones Farmacológicas , Canales Iónicos/efectos de los fármacos , Neurotoxinas/farmacología , Canales de Sodio/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Ratas , Ratas Sprague-Dawley , Veratridina/farmacologíaRESUMEN
Expression from the promoter for the large subunit (ICP6) of the ribonucleotide reductase encoded by herpes simplex virus type 1 (HSV-1) has been examined. Using the lacZ reporter gene fused in-frame with ICP6 regulatory sequences to assay expression quantitatively, we showed that the ICP6 promoter responded very weakly to the alpha-transinducing factor (TIF) in the absence of all other viral gene products, but much more strongly to immediate early proteins. Similar patterns of regulation were observed when the reporter gene construct was located at two different positions within the the viral genome or in a stably transfected Vero cell line. Infection of the stably transfected cells with various HSV-1 mutants identified ICP0 as the major transactivator of the ICP6 promoter.
Asunto(s)
Simplexvirus/genética , Proteínas Virales/genética , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Viral/genética , Regulación Viral de la Expresión Génica , Genes Virales , Operón Lac , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Células VeroRESUMEN
This study was undertaken to investigate the biochemical events underlying the inhibitory action of ethanol on dihydropyridine-sensitive voltage-dependent Ca2+ channels in brain synaptosomes. The binding of radiolabeled dihydropyridine was used to determine functional Ca2+ channels in synaptosomes following exposure to ethanol. No effect on [3H]PN 200-110 binding was found when disrupted synaptosomal membranes were incubated with ethanol concentrations as high as 300 mM, suggesting that ethanol did not interact directly with sites on or near the Ca2+ channels. However, when intact synaptosomes were first incubated with ethanol (100 mM) at 37 degrees and then disrupted, a significant reduction in membrane binding of [3H]PN 200-110 was found. Ethanol incubation of synaptosomes at 0 degree was ineffective. It appears that metabolic processes involving intracellular factors were required in the ethanol action. In examining this possibility, [3H]PN 200-110 binding was activated by incubation of disrupted membranes with MgATP and Ca(2+)-calmodulin, and ethanol was found to inhibit the activation in a concentration-dependent manner (50-200 mM). [3H]PN 200-110 binding to membranes was also activated by incubation with MgATP and cyclic AMP-dependent protein kinase, but this activation was not inhibited by ethanol. These findings are consistent with the interpretation that ethanol acts on Ca2+ channels by inhibiting calmodulin-dependent activation of the channels.
Asunto(s)
Encéfalo/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Dihidropiridinas/metabolismo , Etanol/farmacología , Sinaptosomas/efectos de los fármacos , Adenosina Trifosfato/farmacología , Alcoholismo/metabolismo , Animales , Sitios de Unión , Encéfalo/metabolismo , Canales de Calcio/metabolismo , AMP Cíclico/farmacología , Dihidropiridinas/farmacología , Isradipino/metabolismo , Magnesio/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Sinaptosomas/metabolismo , TritioRESUMEN
The present study was designed to demonstrate that endogenous calmodulin (CaM) content in synaptic plasma membranes (SPM) is altered by acute and chronic administration of ethanol and is a sequel to the kinetic characterization of ethanol inhibition of [125I]CaM binding to SPM reported in our previous study. In rats, an acute ethanol injection (2 g/kg, i.p.) rapidly reduced CaM content in SPM from cerebral cortex, whereas chronic ethanol treatment [6% (w/v) in a liquid diet for 3 weeks] led to an up-regulation of the CaM content. In both cases, the alteration of CaM content in SPM occurred in the EGTA-dissociable pool of CaM (77% of total membrane CaM); the EGTA-nondissociable pool (23% of total CaM) was not affected. In animals receiving chronic ethanol treatment, CaM content in SPM was not altered significantly by the acute ethanol dose that produced rapid reduction of CaM content in control animals, indicating that resistance to ethanol develops. This resistance to ethanol can be attributed to alterations of membrane properties. In control SPM, ethanol at 50 mM markedly accelerated the temperature-dependent dissociation of endogenous CaM, whereas in SPM from animals chronically treated with ethanol, significant acceleration of CaM dissociation required ethanol concentrations as high as 150-200 mM. These findings on SPM in vitro were consistent with the data on CaM content obtained in vivo. Since CaM mediates a variety of biochemical processes in synaptic membranes, we hypothesize that the effects of ethanol in altering the content of membrane-bound CaM may lead to a cascade of consequences in synaptic membrane function.
Asunto(s)
Encéfalo/efectos de los fármacos , Calmodulina/metabolismo , Etanol/farmacología , Membranas Sinápticas/efectos de los fármacos , Animales , Encéfalo/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Membranas Sinápticas/metabolismoRESUMEN
Depolarization-induced Ca2+ influx in brain synaptosomes is known to be inhibited by ethanol and stimulated by glucocorticoids. The present study was undertaken to characterize the interactions of corticosterone (CORT) with ethanol effects on 45Ca2+ uptake in synaptosomes depolarized by high K+ (70 mM). CORT was shown to antagonize the inhibitory effects of ethanol on the fast-phase component of the K(+)-induced 45Ca2+ uptake (the initial 3 s following depolarization). Glucocorticoid antagonism of ethanol inhibition of the 45Ca2+ uptake exhibited a high degree of steroid specificity; steroids with glucocorticoid activity including cortisol, dexamethasone and triamcinolone were effective, whereas gonadal steroids and excitatory neuroactive steroid metabolites were ineffective. From the shift of concentration-response relationships when CORT and ethanol were present in combination, the interaction between steroid stimulation and ethanol inhibition of 45Ca2+ uptake occurred in an additive manner over the range of their effective concentrations. Parallel to 45Ca2+ uptake, the binding sites for [3H]PN 200-110 were reduced by ethanol and increased by CORT. These opposite effects on [3H]dihydropyridine labeled sites were found also to antagonize each other, and the antagonism again occurred in an additive relationship. These results demonstrate that glucocorticoids antagonized ethanol inhibition of voltage-dependent Ca2+ channel activity in brain synaptosomes, and support the notion that these steroids may be among the endogenous factors that modulate neuronal sensitivity to ethanol.
Asunto(s)
Encéfalo/efectos de los fármacos , Calcio/metabolismo , Etanol/farmacología , Glucocorticoides/farmacología , Sinaptosomas/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Masculino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Ethanol inhibits [125I]calmodulin binding to synaptic plasma membranes from rat brain, and this inhibition is correlated in a concentration-dependent manner with the increase of membrane fluidity, as determined by diphenylhexatriene fluorescence polarization. Moreover, several short-chain alcohols that increase membrane fluidity are also effective inhibitors of [125I]calmodulin binding. These data support the notion that ethanol inhibits calmodulin binding by increasing lipid fluidity of the synaptic membranes.
Asunto(s)
Calmodulina/efectos de los fármacos , Etanol/farmacología , Metabolismo de los Lípidos , Membranas Sinápticas/efectos de los fármacos , Animales , Sitios de Unión , Masculino , Fluidez de la Membrana , Ratas , Ratas Sprague-DawleyRESUMEN
C57BL/6Bg mice had silver bead electrodes chronically implanted on the surface of the cortex and had their cortical EEG recorded during audiogenic seizures following ethanol withdrawal. For 7 days, the experimental groups were fed a liquid diet containing 6% v/v ethanol ad lib as the only source of food and water. The control group was fed a similar diet containing an isocaloric amount of sucrose. The cortical EEG's of experimental and control groups before, during, and after treatment were virtually identical. Only the experimental group was susceptible to audiogenic seizures. During audiogenic seizures, the cortical EEG showed no sign of spike waves or paroxysmal activity. This is in contrast to picrotoxin convulsions with these same mice as well as to spontaneous convulsions in animals following ethanol withdrawal. Similar EEG observations have been reported on audiogenic seizures from genetic and acoustically primed susceptibilities. Consequently, we suggest that all audiogenic seizure responses, including those during ethanol withdrawal, are a type of subcortical epilepsy.
Asunto(s)
Alcoholismo , Convulsiones/fisiopatología , Síndrome de Abstinencia a Sustancias/fisiopatología , Estimulación Acústica , Animales , Electroencefalografía , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Male C57 mice were divided into two experimental groups: "Isolated" and "Grouped". Isolated mice were housed individually for 30 or 45 days; Grouped mice were housed five per cage. Midbrain tryptophan hydroxylase activity was determined at the end of the isolation period. Isolated mice showed 35% less tryptophan hydroxylase activity than grouped mice (P less than 0.001). Prolongation of the isolation (45 vs 30 days) did not further reduce the enzyme activity. The changes in tryptophan hydroxylase activity may be related to behavioral changes induced by isolation.
Asunto(s)
Mesencéfalo/enzimología , Aislamiento Social , Triptófano Hidroxilasa/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Serotonina/metabolismoRESUMEN
We have previously shown that glucocorticoids accelerate depolarization-induced 45Ca2+ influx in synaptosomes isolated from rat cerebral cortex, indicating that the steroids may modulate voltage-dependent Ca2+ channels. The present study was undertaken to characterize the biochemical action of glucocorticoids on dihydropyridine-sensitive voltage-dependent Ca2+ channels known to be present in brain synaptosomes. The [3H]dihydropyridine labeled site was used as a marker to determine the levels of functional Ca2+ channels. No effect on equilibrium binding of [3H]PN 200-110 was found when membranes from disrupted synaptosomes were incubated with corticosterone as high as 1 microM. However, when intact synaptosomes were first incubated with corticosterone at 37 degrees C and then disrupted, a significant increase in [3H]PN 200-110 binding was found. Steroid incubation of synaptosomes at 0 degree C was ineffective. It appears that metabolic processes requiring intracellular factors were involved in the steroid action. In examining this possibility, [3H]PN 200-110 binding was activated in disrupted membranes by MgATP and Ca(2+)-calmodulin, and corticosterone was found to enhance the activation in a concentration-dependent manner. [3H]PN 200-110 binding to membranes was also activated by incubation with MgATP and cAMP-dependent protein kinase, but this activation was not enhanced by the steroid. These findings are consistent with the interpretation that the steroid promotes Ca2+ channel activity by enhancing calmodulin-dependent activation of the channels. The action on voltage-dependent Ca2+ channels in synaptic terminals may well be a mechanism whereby glucocorticoids modulate neuronal activity.
Asunto(s)
Canales de Calcio/fisiología , Corteza Cerebral/fisiología , Glucocorticoides/farmacología , Sinapsis/fisiología , Sinaptosomas/fisiología , Adenosina Trifosfato/farmacología , Animales , Unión Competitiva , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Canales de Calcio Tipo L , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , AMP Cíclico/farmacología , Dihidropiridinas/metabolismo , Isradipino/metabolismo , Cinética , Masculino , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiología , Ratas , Ratas Sprague-Dawley , Esteroides/farmacología , Sinapsis/efectos de los fármacos , Sinaptosomas/efectos de los fármacosRESUMEN
The effects of corticosterone on Ca(2+)-dependent binding of [125I]calmodulin to purified synaptic plasma membranes (SPM) from rats brain were characterized. The enhancement of [125I]calmodulin binding was a sigmoidal function of steroid concentration, with the maximal increase (> 55% above control) occurring at a steroid concentration of 1 x 10(-6) M and EC50 estimated at 1-2 x 10(-7) M. Other glucocorticoids including hydrocortisone, dexamethasone and triamcinolone produced similar effects, whereas steroids without glucocorticoid activity such as 11-deoxycortisol, 11-deoxycorticosterone and cholesterol were ineffective. The steroid-induced increase of binding was correlated with an increase of membrane affinity for [125I]calmodulin as shown by Scatchard analysis, and a decrease of the rate of dissociation of [125I]calmodulin from the membranes as shown by kinetic analysis. Arrhenius analysis indicates that [125I]calmodulin binding was influenced by lipid transition of the membranes and that corticosterone resulted in a shift of membrane transition toward a higher temperature. Since a variety of biochemical processes associated with synaptic membranes are dependent upon calmodulin for their regulation, we hypothesize that the effects of glucocorticoids in promoting membrane binding of calmodulin may lead to a cascade of alterations in synaptic function.
Asunto(s)
Calmodulina/metabolismo , Glucocorticoides/farmacología , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/metabolismo , Animales , Calcio/farmacología , Corticosterona/farmacología , Dexametasona/farmacología , Hidrocortisona/farmacología , Radioisótopos de Yodo , Cinética , Masculino , Ratas , Ratas Sprague-Dawley , Termodinámica , Triamcinolona/farmacologíaRESUMEN
The binding of ATP to brain l-glutamate decarboxylase (GAD) was studied by means of ATP-agarose chromatography, utilizing partially purified GAD from mouse brain after DEAE-cellulose chromatography and ammonium sulfate fractional precipitation. GAD was found to bind with a high affinity to the ATP-agarose with the ATP molecule linked to the beaded agarose through the N(6)-amino group. Agarose with ATP attached through the ribosyl hydroxyls was totally ineffective to bind the enzyme. GAD bound to the immobilized ATP could be dissociated by free ATP (10-50 mM), but not by ADP at a concentration as high as 100 mM. Mg(2+) was not a required factor for the binding. The enzyme binding to the ATP-agarose occurred under a saturating concentration (50 ?M) of pyridoxal 5?-phosphate (PLP). Moreover, GAD bound to the ATP-agarose was not dissociated by PLP even at 1.0 mM, indicating no competition of PLP with ATP for the same binding site on the enzyme. Kinetic characterization showed that binding of ATP raised the K(m) of the enzyme for PLP. Our approach provides direct evidence that there is a specific binding site on GAD for ATP, which is distinct from the binding site for PLP.
RESUMEN
[(125)I]LSD (labeled at the 2 position) has been introduced as the first (125)I-labeled ligand for serotonin 5-HT(2) (S2) receptors. In the present study we examined the binding of [(125)I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [(125)I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75-80% and Scatchard plots of the binding data reveal K(d) = 1.1 nM, B(max) = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0 degrees C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC(50) values for a variety of compounds show a clear 5-HT(2) (S2) serotonergic pattern at this [(125)I]LSD site. Blockage of this primary 5-HT(2) (S2) caudate binding site by 0.3 ?M mianserin reveals the presence of a weaker [(125)I]LSD binding site with a K(d) = 9.1 nM, B(max) = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [(125)I]LSD is a sensitive and selective label for 5-HT(2) (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [(125)I]LSD binding site with mianserin allows the high sensitivity of [(125)I]LSD to be applied to D2 dopamine receptor studies as well.