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191.
192.
Antonio Miralles Catalina Ribas Gabriel Olmos Jesús A. García-Sevilla 《Journal of neurochemistry》1993,61(5):1602-1610
Abstract— The alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a peptide-coupling agent that is being used to inactivate irreversibly α2-adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain l2-imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10?8-10?5M) markedly decreased (20–90%) the specific binding of the selective antagonist [3H]R821002 to α2-adrenoceptors without affecting that of [3H]idazoxan (in the presence of adrenaline) to l2-imidazoline sites. In EEDQ-pretreated membranes (10?5M, 30 min at 25°c), the density of l2-imidazoline sites (Bmax= 80 ± 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of 10?6M (-)-adrenaline (Bmax= 83 ± 4 fmol/mg of protein), and both densities were lower (24%, p < 0.05) than the total native density of [3H]idazoxan binding sites (Bmax= 107 ± 6 fmol/mg of protein) (l2-imidazoline sites plus a2-adrenoceptors). Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 ± 1%) the specific binding of [3H]-R821002 to α2-adrenoceptors, but also the binding of [3H]idazoxan to l2-imidazoline sites (by 44 ± 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ-induced α2-adrenoceptor inactivation. In contrast, pretreatment with cirazoline (1 mg/kg, i.p.), did not protect against EEDQ-induced inactivation of l2-imidazoline sites. Treatment with EEDQ (1.6 mg/kg, i.p., for 6 h) did not alter the density of brain monoamine oxidase-A sites labeled by [3H]Ro 41–1049 or that of monoamine oxidase-B sites labeled by [3H]Ro 19–6327 (lazabemide), two relevant mitochondrial markers. Competition experiments with cirazoline against the specific binding of [3H]idazoxan to l2-imidazoline sites demonstrated the presence of the expected two affinity states for the drug in EEDQ-pretreated membranes as well as in rats treated with EEDQ. The results indicate that EEDQ in vitro is a useful tool for quantitating l2-imidazoline sites when using [3H]-imidazoline ligands that also recognize α2-adrenoceptors. In vivo, however, EEDQ is also able to inactivate partially brain l2-imidazoline sites probably by an indirect mechanism. Key Words: Brain l2-imidazoline sites—[3H]-Idazoxan—α2-Adrenoceptors—[3H] R821002—N -Ethoxycarbonyl-2-ethoxy-li2-dihydroquinoline—Monoamine oxidase-A—[3H]Ro 41–1049—Monoamine oxidase-B—[3H]Ro 19–6327. 相似文献
193.
194.
V Felipo V Miralles E Knecht J Hernandez-Yago S Grisolia 《European journal of biochemistry》1983,133(3):641-644
The cytosolic precursor for the mitochondrial glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using messenger RNA from rat liver. To check whether this precursor had enzymatic activity, a highly sensitive fluorimetric method, which can measure picogram quantities of enzyme, was used together with competitive dissociation of the precursor from an immunoprecipitate with inactive glutamate dehydrogenase. Glutamate dehydrogenase activity, corresponding to that estimated from incorporation of [35S]-methionine, was detected in the precursor. The significance of this finding is discussed. 相似文献
195.
We have analyzed the developmental properties of kainate receptors in cerebellar membranes prepared from chick and rat, two vertebrate species with contrasting patterns of functional maturation. Single populations of binding sites have been characterized in the avian and rodent membranes with apparent dissociation constants (Kd) in the 210–280 nM and 40–55 nM ranges, respectively; the number of binding sites (Bmax) increases with age in both species, reaching a maximum of 187 pmol/mg in the case of 10-day chicks vs. 1.28 pmol/mg in 75-day rats. The ontogenetic profiles of kainate receptors in chick and rat cerebella are in consonance with the patent differences in motor development at birth. 相似文献