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31.
K Han 《Nucleic acids research》1996,24(21):4362-4363
I have developed an efficient method for transfecting Drosophila S2 cells using DDAB, a cationic liposome reagent. The optimized DDAB method resulted in a 10 times or greater increase in transfection efficiency compared with the conventional calcium phosphate method which has been essentially the only way for transfecting S2 cells. 相似文献
32.
重组水蛭素HV2的稳定性 总被引:3,自引:0,他引:3
重组水蛭素HV2是凝血酶的特异性抑制剂,是一种非常稳定的蛋白质。温度的升高(100℃水浴)和pH(1─13)的改变不影响其活力,在某些变性剂(8mol/L尿素、1%SDS和6mol/L盐酸胍)存在的条件下也非常稳定,0.1mol/L的DTT在70℃时使其部分失活,只有pH和温度同时升高其活力才开始下降,pH13、80℃处理15min即完全失活,氨基酸组成和活性分析发现失活样品的Cys和Lys被破坏。重组水蛭素HV2含有一个结构紧密的N端核心区和一个无序的C端尾部。其N端的3个Lys-Xaa键均不被胰蛋白酶水解;胃蛋白酶及糜蛋白酶消化后,分离所得片段,氨基酸组成分析发现N端核心区依然保持很高的抗凝血酶活性,继续消化24h,核心区不被进一步降解。 相似文献
33.
The entire mitochondrial genome of Schizosaccharomyces pombe ura4-294h
-was analyzed by the 2D pulsed field gel electrophoresis technique developed by Brewer and Fangman. The genome consists of multimers with an average size of 100 kb and analysis of the overlapping restriction fragments of the complete mitochondrial DNA (mtDNA) genome resulted in simply Y 2D gel patterns. Large single-stranded DNA molecules or double-stranded DNA molecules containing large or numerous single-stranded regions were found in the S. pombe mtDNA preparation. The replication of mtDNA monomers was found to occur in either direction. On the basis of these results, a replication mechanism for S. pombe mtDNA that is most consistent with a rolling circle model is suggested. 相似文献
34.
Factors affecting viability of IVF-derived bovine blastocysts after freezing and thawing were investigated. A total of 1,101 ova matured and fertilized in vitro were cultured under 2 different conditions, 1) in TCM-199 on granulosa cell monolayers at 5% CO(2) in air and 2) in synthetic oviduct fluid (SOF) medium without somatic cell support at 5% CO(2), 5% O(2), 90% N(2). All blastocysts that developed from the 2 different culture systems were individually classified into 4 grades of embryo quality and were then frozen by conventional slow freezing. Developmental rates of the IVF-derived ova to blastocysts and the survival rates of the frozen-thawed blastocysts were not different between the SOF medium (16 and 49%) and the co-culture system (13 and 61%, respectively). Survival of frozen-thawed blastocysts was affected by embryo quality in both the SOF and co-culture systems (P<0.001). Blastocysts produced in vitro were also individually classified into 3 developmental stages and were then cultured for 3 d in the co-culture system with granulosa cells after freezing and thawing. There was a difference in the survival rate of frozen-thawed embryos between blastocyst developmental stages (early vs mid, P<0.05; mid vs expanded, P<0.01; early vs expanded, P<0.001). The post-thawing survival rate of blastocysts frozen at Day 7 (62%) of culture was higher compared with that of Day 8 (45%), but there was no difference in survival rate between Day 7 and 8 of culture. The results indicate that the quality and developmental stage of blastocysts are important factors influencing their survival after freezing and thawing. 相似文献
35.
Further Evidence for Multiple Forms of an N-Methyl-d-Aspartate Recognition Domain in Rat Brain Using Membrane Binding Techniques 总被引:1,自引:1,他引:0
Pingping Zuo Kiyokazu Ogita Takeo Suzuki Daiken Han Yukio Yoneda 《Journal of neurochemistry》1993,61(5):1865-1873
Abstract— Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsul-fonic acid, invariably resulted in marked inhibition of the binding of dl -(E)-2-amino-4-[3H]propyl-5-phosphono-3-pentenoic acid ([3H]CGP 39653), a competitive antagonist at an N-methyl-d -aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X-100, but did not significantly affect the binding of L-[3H]-glutamic acid ([3H]Glu), an endogenous agonist. The pre-treatment was effective in reducing the binding of [3H]-CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl-reactive agents also enhanced the potencies of NMDA agonists to displace [3H]-CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [3H]Glu binding. The binding of both [3H]CGP 39653 and [3H]Glu was similarly sensitive to pretreatment with four different proteases in Tritontreated membranes, whereas pretreatment with phospho-lipase A2 or C markedly inhibited [3H]CGP 39653 binding without altering [3H]Glu binding. Moreover, both phospho-lipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [3H]CGP 39653 and [3H]Glu, but also caused diminution of those of NMDA antagonists. These results suggest that both sulfhydryl-reactive agents and phospholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [3H]CGP 39653, with concomitant facilitation of that in an agonist-preferring form by [3H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data. 相似文献
36.
Abstract: Pretreatment with Triton X-100 more than doubled the binding of radiolabeled 5,7-dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N-methyl-d -aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.87 pmol/mg of protein). The binding of both [3H]DCKA and [3H]Gly was similarly displaced by numerous putative agonists and antagonists at the Gly domain in a concentration-dependent manner at a concentration range of 100 nM to 0.1 mM. Among the 24 putative ligands tested, DCKA was the second most potent displacer of the binding of both radioligands with no intrinsic affinity for the binding of [3H]kainic acid and α-amino-3-hydroxy-5-[3H]methylisoxazole-4-propionic acid (AMPA) to the non-NMDA receptors. In contrast, the other proposed potent Gly antagonist, 5,7-dinitroquinoxaline-2,3-dione, was active in displacing the binding of [3H]glutamic ([3H]Glu) and D,L-(E)-2-amino-4-[3H]propyl-5-phosphono-3-pentenoic acids to the NMDA recognition domain with a relatively high affinity for the non-NMDA receptors. In addition, the proposed antagonist at the AMPA-sensitive receptor, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline, not only displaced weakly the binding of both [3H]- Gly and [3H]DCKA, but also inhibited the binding of (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) to an ion channel associated with the NMDA-sensitive receptor in the presence of added Glu alone in a manner sensitive to antagonism by further added Gly. Clear correlations were seen between potencies of the displacers to displace [3H]DCKA binding and [3H]Gly binding, in addition to between the potencies to displace [3H]-DCKA or [3H]Gly binding and to potentiate or inhibit [3H]MK-801 binding. All quinoxalines tested were invariably more potent displacers of [3H]DCKA binding than [3H]Gly binding, whereas kynurenines were similarly effective in displacing the binding of both [3H]Gly and [3H]-DCKA. These results undoubtedly give support to the proposal that [3H]DCKA is one useful radioligand available in terms of its high selectivity and affinity for the Gly domain in the brain. Possible multiplicity of the Gly domain is suggested by the differential pharmacological profiles between the binding of [3H]Gly and [3H]DCKA. 相似文献
37.
38.
Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site. 总被引:18,自引:7,他引:11 下载免费PDF全文
Site-specific recombination of bacteriophage lambda starts with the formation of higher-order protein--DNA complexes, called 'intasomes', and is followed by a series of steps, including the initial DNA cleavage, top-strand exchange, branch migration and bottom-strand exchange, to produce recombinant products. One of the intasomes formed during excisive recombination (the attL complex) is composed of the phage-encoded integrase (Int), integration host factor (IHF) and one of the recombination substrates, attL DNA. Int is the catalytic recombinase and has two different DNA binding domains. When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and the core-type sites (B and C') where the reciprocal strand exchange takes place. The Tyr342 residue of Int serves as a nucleophile during strand cleavage and covalently attaches to the DNA through a phosphotyrosyl bond. In vitro complementation assays have been performed for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases. We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cleavage at the B site. It is likely that the active site is formed by two Int monomers. 相似文献
39.
从崖椒(Zanthoxglum schinifolutm Sieb.et Zucc.)茎的石油醚、二氯甲烷提取物中分离得到8个化合物。经物理常数测定及光谱(UV,IR,MS,NMR)分析鉴定其为(1)白鲜碱(dictamning),(2)茵芋碱(skimmianine),(3)滨蒿内酯(scoparone),(4)崖椒内酯(schinifolin),(5)莨菪亭(scopoletin),(6)7-羟基-8-甲氧基香豆素(7-hydroxy-8-methoxycoumarin),(7)N-甲基弗林辛(N-methylflindersine),(8)β-谷甾醇(β-sitosterol),其中化合物(5)、(6)和(7)为首次从该植物中分离。 相似文献
40.
Xu Yuping Wang Lu Tang Quehui Naselli-Flores Luigi Jeppesen Erik Han Bo-Ping 《Ecosystems》2023,26(4):752-767
Ecosystems - The relationship between species diversity and ecosystem functioning is one of central topics in modern ecology, but variable and controversial patterns have been found depending on... 相似文献