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991.
Ave Patrick Colucci-Guyon Emma Babinet Charles Huerre Michel-Rene 《Transgenic research》1997,6(1):37-40
The Escherichia coli -galactosidase gene is frequently used as a reporter gene in transgenic studies because its activity can be easily detected at the cellular level. Here we report a procedure for monitoring -galactosidase activity directly in tissue sections, which involves the use of a mixture of ethanol and poly-ethylene-glycol as a fixative (Kryofix) and a special paraffin characterized by a lower fusion point of 42 °C. After embedding and cutting, the sections are stained by the chromogenic substrate 5-bromo-4-chloro-3-indoyl--d galactopyranoside (X-Gal). This procedure allows both the retention of a high level of -galactosidase activity and the preservation of good tissue morphology. Furthermore, it can be combined with immunohistochemical methods to detect other cellular components without compromising reporter gene detection 相似文献
992.
Spectroscopic techniques were used to investigate the interaction between vanadate and human erythrocyte ghosts. Direct evidence from 51V nuclear magnetic resonance (NMR) studies suggested that the monomeric and polymeric vanadate species may bind to the anion binding sites of band 3 protein of the erythrocyte membrane. The results of 51V NMR studies and the quenching effect of vanadate on the intrinsic fluorescence of the membrane proteins indicated that in the low concentration range of vanadate (<0.6 mm), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mm. The experiments of sulfhydryl content titration by the method of Ellman and residue sulfhydryl-labeled fluorescence spectroscopies clearly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (c. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of intact ghost membrane were reduced by the reaction with vanadate, as shown by the ESR studies employing the protein- and lipid-specific spin labels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate may oxidize the residue sulfhydryl groups of membrane proteins, and thus decrease the fluidity of erythrocyte membrane. 相似文献
993.
用Blue Sepharose CL-6B快速纯化天花粉蛋白 总被引:8,自引:0,他引:8
差光谱显示染料cibacron blue F3GA与天花粉蛋白(TCS)有特异性结合,复合物在可见光部分的最大吸收波长在690 nm,摩尔消光系数ε=2.6×10-3(mol/L)-1·cm-1,解离常数Kd=1.8 μmol/L,0.5 mol/L NaCl可使复合物解离.根据这一特点,用Blue-Sepharose CL-6B凝胶从栝篓块茎中亲和纯化了TCS.此法快速、简便、高效,易于大量制备. 相似文献
994.
Kenji Hara Henneke Pangkey Kiyoshi Osatomi Keiko Yatsuda Atsushi Hagiwara Katsuyasu Tachibana Tadashi Ishihara 《Hydrobiologia》1997,358(1-3):89-94
We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl2 and MnCl_2. 相似文献
995.
We have investigated the phylogenetic relationships of monotremes and marsupials using nucleotide sequence data from the
neurotrophins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The study included
species representing monotremes, Australasian marsupials and placentals, as well as species representing birds, reptiles,
and fish. PCR was used to amplify fragments encoding parts of the neurotrophin genes from echidna, platypus, and eight marsupials
from four different orders. Phylogenetic trees were generated using parsimony analysis, and support for the different tree
structures was evaluated by bootstrapping. The analysis was performed with NGF, BDNF, or NT-3 sequence data used individually
as well as with the three neurotrophins in a combined matrix, thereby simultaneously considering phylogenetic information
from three separate genes. The results showed that the monotreme neurotrophin sequences associate to either therian or bird
neurotrophin sequences and suggests that the monotremes are not necessarily related closer to therians than to birds. Furthermore,
the results confirmed the present classification of four Australasian marsupial orders based on morphological characters,
and suggested a phylogenetic relationship where Dasyuromorphia is related closest to Peramelemorphia followed by Notoryctemorphia
and Diprotodontia. These studies show that sequence data from neurotrophins are well suited for phylogenetic analysis of mammals
and that neurotrophins can resolve basal relationships in the evolutionary tree.
Received: 27 January 1997 / Accepted: 20 March 1997 相似文献
996.
Benoit Cousineau Fabrice Leclerc Robert Cedergren 《Journal of molecular evolution》1997,45(6):661-670
Sequence similarity has given rise to the proposal that IF-2, EF-G, and EF-Tu are related through a common ancestor. We evaluate
this proposition and whether the relationship can be extended to other factors of protein synthesis. Analysis of amino acid
sequence similarity gives statistical support for an evolutionary affiliation among IF-1, IF-2, IF-3, EF-Tu, EF-Ts, and EF-G
and suggests further that this association is a result of gene duplication/fusion events. In support of this mechanism, the
three-dimensional structures of IF-3, EF-Tu, and EF-G display a predictable domain structure and overall conformational similarity.
The model that we propose consists of three consecutives duplication/fusion events which would have taken place before the
divergence of the three superkingdoms: eubacteria, archaea, and eukaryotes. The root of this protein superfamily tree would
be an ancestor of the modern IF-1 gene sequence. The repeated fundamental motif of this protein superfamily is a small RNA
binding domain composed of two α-helices packed along side of an antiparallel β-sheet.
Received: 17 October 1996 / Accepted: 10 June 1997 相似文献
997.
The effect of ethanol on maxi Ca2+-activated K+ channels (BK channels) in GH3 pituitary tumor cells was investigated using single-channel recordings and focusing on intracellular
signal transduction. In outside-out patches, ethanol caused a transient concentration-dependent increase of BK-channel activity.
30 mm (1.4‰) ethanol significantly increased mean channel open time and channel open probability by 26.3 ± 9% and 78.8 ± 10%, respectively;
single-channel current amplitude was not affected by ethanol. The augmenting effect of ethanol was blocked in the presence
of protein kinase C (PKC) inhibitors staurosporine, bisindolylmaleimide, and PKC (19–31) pseudosubstrate inhibitor as well
as by AMP-PNP (5′-adenylylimidodiphosphate), a nonhydrolyzable ATP-analogue, but not by the phospholipase C blocker U-73122.
Phosphatase inhibitors microcystin-LR and okadaic acid promoted the ethanol effect. The blocking effect was released at higher
concentrations of ethanol (100 mm) suggesting a second site of action or a competition between blockers and ethanol. Our results suggest that the effect of
ethanol on BK-channels is mediated by PKC stimulation and phosphorylation of the channels which increases channel activity
and hence may influence action potentials duration and hormone secretion.
Received: 24 July 1996/Revised: 27 December 1996 相似文献
998.
Prostaglandins are important in signaling pathways involved in modulating the rates of Na+ transport in a diverse group of tissues possessing apical membrane epithelial channels. PGE2 is known to cause either stimulation, inhibition or transient stimulatory changes of Na+ transport. We have continued our studies of frog skins that are known to respond to forskolin and PGE2 with large steady-state increases of transport and have used noninvasive methods of blocker-induced noise analysis of Na+ channels to determine their channel densities (N
T
) and open probabilities (P
o
). In the absence of exogenous hormones, baseline rates of Na+ transport are especially high in scraped skins (R. pipiens pipiens) studied in the fall of the year. Na+ transport was inhibited by indomethacin and by removal of the unstirred layers of the corium (isolated epithelia) alone suggesting
that PGE2 is responsible for the sustained and elevated rates of transport in scraped skins. Changes of transport caused by indomethacin,
forskolin or PGE2 were unquestionably mediated by considerably larger changes of N
T
than compensatory changes of P
o
. Since cAMP caused no change of P
o
in tissues pretreated with indomethacin, PGE2 appears in this tissue to serve a dual role, increasing the steady state N
T
by way of cAMP and decreasing P
o
by unknown mechanisms. Despite appreciable PGE2-related decreases of P
o
, the net stimulation of transport occurs by a considerably greater cAMP-mediated increase of N
T
.
Received: 28 February 1996/Revised: 22 August 1996 相似文献
999.
Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum 总被引:2,自引:1,他引:1
Yasushi Ikarashi Akira Takahashi Hirohisa Ishimaru Tadashi Arai Yuji Maruyama 《Journal of neurochemistry》1997,69(3):1246-1251
Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D2 antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D2 agonist (±)-2-( N -phenylethyl- N -propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µ M hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake. 相似文献
1000.
Abstract: Oxygen radicals have been implicated in the neurodegenerative and other neurobiological effects evoked by methamphetamine (MA) in the brain. It has been reported that shortly after a single large subcutaneous dose of MA to the rat, the serotonergic neurotoxin 5,6-dihydroxytryptamine (5,6-DHT) is formed in the cortex and hippocampus. This somewhat controversial finding suggests that MA potentiates formation of the hydroxyl radical (HO?) that oxidizes 5-hydroxytryptamine (5-HT) to 5,6-DHT, which, in turn, mediates the degeneration of serotonergic terminals. A major and more stable product of the in vitro HO?-mediated oxidation of 5-HT is 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). In this investigation, a method based on HPLC with electrochemical detection (HPLC-EC) has been developed that permits measurement of very low levels of 5-HEO in rat brain tissue in the presence of biogenic amine neurotransmitters/metabolites. After intracerebroventricular administration into rat brain, 5-HEO is transformed into a single major, but unknown, metabolite that can be detected by HPLC-EC. One hour after administration of MA (100 mg/kg s.c.) to the rat, massive decrements of 5-HT were observed in all regions of the brain examined (cortex, hippocampus, medulla and pons, midbrain, and striatum). However, 5-HEO, its unidentified metabolite, or 5,6-DHT were not detected as in vivo metabolites of 5-HT. MA administration, in particular to rats pretreated with pargyline, resulted in the formation of low levels of N-acetyl-5-hydroxytryptamine (NAc-5-HT) in all brain regions examined. These results suggest that MA does not potentiate the HO?-mediated oxidation of 5-HT. Furthermore, the rapid MA-induced decrease of 5-HT might not only be related to oxidative deactivation of tryptophan hydroxylase, as demonstrated by other investigators, but also to the inhibition of tetrahydrobiopterin biosynthesis by NAc-5-HT. The massive decrements of 5-HT evoked by MA are accompanied by small or no corresponding increases in 5-hydroxyindole-3-acetic acid (5-HIAA) levels. This is due, in part, to the relatively rapid clearance of 5-HIAA from the brain and monoamine oxidase (MAO) inhibition by MA. However, the loss of 5-HT without corresponding increases in its metabolites point to other mechanisms that might deplete the neurotransmitter, such as oxidation by superoxide radical anion (O2??), a reaction that in vitro does not generate 5-HEO or 5,6-DHT but rather another putative neurotoxin, tryptamine-4,5-dione. One hour after administration, MA evokes large depletions of norepinephrine (NE) throughout the brain but somewhat smaller decrements of dopamine (DA) that are restricted to the nigrostriatal pathway. Furthermore, MA evokes a major shift in the metabolism of both NE and DA from the pathway mediated by MAO to that mediated by catechol-O-methyltransferase. The profound and widespread effects of MA on the noradrenergic system, but more anatomically localized influence on the dopaminergic system, suggests that NE in addition to DA, or unusual metabolites of these neurotransmitters, might play roles in the neurodegenerative effects evoked by this drug. 相似文献