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141.
Introduction – Artemisia annua is a rich source of biologically active substances such as terpenoids, coumarins and polyacetylenes. These chemicals have been reported to show beneficial pharmacological properties such as antitumor and antibacterial activities. In genetically transformed root cultures of A. annua, three bioactive metabolites, namely, ponticaepoxide (an insecticidal polyacetylene, 1 ), drimartol A (an anticancer sesquiterpene coumarin, 2 ) and (Z)‐7‐acetoxy‐methyl‐11‐methyl‐3‐methylene‐dodeca‐1,6,10‐triene (a new anticancer sesquiterpene, 3 ) were isolated and identified in our recent work. However, no quantitative analysis methods for any of them are yet available, nor for their simultaneous analysis. Objective – To develop an HPLC‐PAD method for simultaneous determination of 1 , 2 and 3 in hairy root cultures of A. annua. Methodology – HPLC operating conditions were optimised and the chromatographic separation was performed on a C18 column with a gradient acetonitrile : water as mobile phase. Results – Linear relationships within the range of investigated concentrations were observed for the three metabolites with their correlation coefficients greater than 0.997. The method was validated for repeatability (RSD <3.59%) and intra‐ and inter‐day precision (RSD <3.1%) with recovery between 94.8 and 107.6% and the RSD less than 3.40%. The method was successfully applied to the time‐course of accumulation of the bioactive compounds in genetically transformed root cultures of A. annua. Conclusion – The HPLC‐PAD method developed for the simultaneous determination of three bioactive metabolites 1 , 2 and 3 was simple, reproducible and sensitive. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
142.
Christine E. Brotherton-Pleiss Michael P. Dillon Anthony P.D.W. Ford Joel R. Gever David S. Carter Shelley K. Gleason Clara J. Lin Amy G. Moore Anthony W. Thompson Marzia Villa Yansheng Zhai 《Bioorganic & medicinal chemistry letters》2010,20(3):1031-1036
Despite the extensive literature describing the role of the ATP-gated P2X3 receptors in a variety of physiological processes the potential of antagonists as therapeutic agents has been limited by the lack of drug-like selective molecules. In this paper we report the discovery and optimization of RO-85, a novel drug-like, potent and selective P2X3 antagonist. High-throughput screening of the Roche compound collection identified a small hit series of heterocyclic amides from a large parallel synthesis library. Rapid optimization, facilitated by high-throughput synthesis, focusing on increasing potency and improving drug-likeness resulted in the discovery of RO-85. 相似文献
143.
Cashman JR MacDonald M Ghirmai S Okolotowicz KJ Sergienko E Brown B Garcia X Zhai D Dahl R Reed JC 《Bioorganic & medicinal chemistry letters》2010,20(22):6560-6564
High-throughput screening of 66,000 compounds using competitive binding of peptides comprising the BH3 domain to anti-apoptotic Bfl-1 led to the identification of 14 validated 'hits' as inhibitors of Bfl-1. N-Aryl maleimide 1 was among the validated 'hits'. A chemical library encompassing over 280 analogs of 1 was prepared following a two-step synthesis. Structure-activity studies for inhibition of Bfl-1 by analogs of N-aryl maleimide 1 revealed a preference for electron-withdrawing substituents in the N-aryl ring and hydrophilic amines appended to the maleimide core. Inhibitors of Bfl-1 are potential development candidates for anti-cancer therapeutics. 相似文献
144.
145.
Chih-Wen Shu Marcin Drag Miklos Bekes Dayong Zhai Guy S Salvesen John C Reed 《Autophagy》2010,6(7):936-947
Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A2 (LC3B-PLA2), which upon cleavage releases active PLA2 for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA2 substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases and the LC3B-PLA2 substrate afforded substantially higher catalytic rates (kcat/Km 5.26 × 105 M−1/sec−1) than Ac-GTFG-AFC peptide (0.92 M−1/sec−1), consistent with substrate-induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24 and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominant-negative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.Key words: autophagin, fluorogenic assay, tetrapeptide, phospholipase A2, LC3 相似文献
146.
? Premise of the study: The development of compound microsatellite markers was conducted in Neolitsea sericea to investigate genetic diversity and population genetic structure of this endangered insular species. ? Methods and Results: Using the compound microsatellite marker technique, 10 compound microsatellite markers that were successfully amplified showed polymorphism when assessed in 55 individuals from two populations in East China and Japan. Overall, the number of alleles ranged from 3 to 17, with an average of 7.9 alleles per locus. In addition, these primers could be easily amplified in Neolitsea aurata var. paraciculata and N. aurata var. chekiangensis. ? Conclusions: The highly polymorphic markers developed and characterized in this study will be useful for population genetic studies of N. sericea. 相似文献
147.
148.
Bruey JM Bruey-Sedano N Luciano F Zhai D Balpai R Xu C Kress CL Bailly-Maitre B Li X Osterman A Matsuzawa S Terskikh AV Faustin B Reed JC 《Cell》2007,129(1):45-56
Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. We have identified a mammalian analog of this caspase-regulatory system in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-X(L) bind and suppress NALP1, reducing caspase-1 activation and interleukin-1beta (IL-1beta) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1beta production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1beta production. The findings reveal an interaction of host defense and apoptosis machinery. 相似文献
149.
Mutations in the Type II protein arginine methyltransferase AtPRMT5 result in pleiotropic developmental defects in Arabidopsis 总被引:2,自引:1,他引:1 下载免费PDF全文
Human PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) encodes a type II protein arginine (Arg) methyltransferase and its homologs in animals and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) are known to regulate RNA processing, signal transduction, and gene expression. However, PRMT5 homologs in higher plants have not yet been reported and the biological roles of these proteins in plant development remain elusive. Here, using conventional biochemical approaches, we purified a plant histone Arg methyltransferase from cauliflower (Brassica oleracea) that was nearly identical to AtPRMT5, an Arabidopsis (Arabidopsis thaliana) homolog of human PRMT5. AtPRMT5 methylated histone H4, H2A, and myelin basic protein in vitro. Western blot using symmetric dimethyl histone H4 Arg 3-specific antibody and thin-layer chromatography analysis demonstrated that AtPRMT5 is a type II enzyme. Mutations in AtPRMT5 caused pleiotropic developmental defects, including growth retardation, dark green and curled leaves, and FlOWERING LOCUS C (FLC)-dependent delayed flowering. Therefore, the type II protein Arg methyltransferase AtPRMT5 is involved in promotion of vegetative growth and FLC-dependent flowering time regulation in Arabidopsis. 相似文献
150.
Fine mapping of S32(t), a new gene causing hybrid embryo sac sterility in a Chinese landrace rice (Oryza sativa L.) 总被引:2,自引:0,他引:2
Li D Chen L Jiang L Zhu S Zhao Z Liu S Su N Zhai H Ikehashi H Wan J 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2007,114(3):515-524
Ketan Nangka, the donor of wide compatibility genes, showed sterility when crossed to Tuanguzao, a landrace rice from Yunnan
province, China. Genetic and cytological analyses revealed that the semi-sterility was primarily caused by partial abortion
of the embryo sac. Genome-wide analysis of the linkage map constructed from the backcross population of Tuanguzao/Ketan Nangka//Ketan
Nangka identified two independent loci responsible for the hybrid sterility located on chromosomes 2 and 5, which explained
18.6 and 20.1% of phenotypic variance, respectively. The gene on chromosome 5 mapped to the previously reported sterility
gene S31(t), while the gene on chromosome 2, a new hybrid sterility gene, was tentatively designated as S32(t). The BC1F2 was developed for further confirmation and fine mapping of S32(t). The gene S32(t) was precisely mapped to the same region as that detected in the BC1F1 but its position was narrowed down to an interval of about 1.9 cM between markers RM236 and RM12475. By assaying the recombinant
events in the BC1F2, S32(t) was further narrowed down to a 64 kb region on the same PAC clone. Sequence analysis of this fragment revealed seven predicted
open reading frames, four of which encoded known proteins and three encoded putative proteins. Further analyses showed that
wide-compatibility variety Dular had neutral alleles at loci S31(t) and S32(t) that can overcome the sterilities caused by these two genes. These results are useful for map-based cloning of S32(t) and for marker-assisted transferring of the neutral allele in hybrid rice breeding. 相似文献