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911.
Brodin P de Jonge MI Majlessi L Leclerc C Nilges M Cole ST Brosch R 《The Journal of biological chemistry》2005,280(40):33953-33959
Proteins of the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 of Mycobacterium tuberculosis are not only strongly involved in the anti-mycobacterial Th1-host immune response but are also key players for virulence. In this study, protein engineering together with bioinformatic, immunological, and virulence analyses allowed us to pinpoint regions of the ESAT-6 molecule that are critical for its biological activity in M. tuberculosis. Mutation of the Trp-Xaa-Gly motif, conserved in a wide variety of ESAT-6-like proteins, abolished complex formation with the partner protein CFP-10, induction of specific T-cell responses, and virulence. Replacement of conserved Leu residues interfered with secretion, coiled-coil formation, and virulence, whereas certain mutations at the extreme C terminus did not affect secretion but caused attenuation, possibly because of altered ESAT-6 targeting or trafficking. In contrast, the mutation of several residues on the outer surface of the four-helical bundle structure of the ESAT-6.CFP-10 complex showed much less effect. Construction of recombinant BCG expressing ESAT-6 with a C-terminal hexahistidine tag allowed us to co-purify ESAT-6 and CFP-10, experimentally confirming their strong interaction both in and outside of the mycobacterial cell. The strain induced potent, antigen-specific T-cell responses and intermediate in vivo growth in mice, suggesting that it remained immunogenic and biologically active despite the tag. Together with previous NMR data, the results of this study have allowed a biologically relevant model of the ESAT-6.CFP-10 complex to be constructed that is critical for understanding the structure-function relationship in tuberculosis pathogenesis. 相似文献
912.
Bornet C Chollet R Malléa M Chevalier J Davin-Regli A Pagès JM Bollet C 《Biochemical and biophysical research communications》2003,301(4):985-990
Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants. 相似文献
913.
Gruber Véronique Berna Patrick P. Arnaud Thierry Bournat Philippe Clément Christèle Mison Dominique Olagnier Béatrice Philippe Laurence Theisen Manfred Baudino Sylvie Bénicourt Claude Cudrey Claire Bloës Carole Duchateau Nathalie Dufour Sylvie Gueguen Catherine Jacquet Séverine Ollivo Catherine Poncetta Christine Zorn Nathalie Ludevid Dolores Van Dorsselaer Alain Verger Robert Doherty Annette Mérot Bertrand Danzin Charles 《Molecular breeding : new strategies in plant improvement》2001,7(4):329-340
A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants. 相似文献
914.
The chiral β-nitroacrylate 2 derived from the (R)- or (S)-4-(3-hydroxy-4,4-dimethyl-2-oxopyrrolidin-1-yl) benzoic acid 1 acts as a reactive dienophile in a diastereoselective Diels-Alder reaction with 1,3-cyclohexadiene. The major cycloadducts have been isolated and transformed into enantiopure trans(2S,3S)- or (2R,3R)-N-Boc-3-aminobicyclic[2,2,2]octane-2-carboxylic acids 5. The trans-(2S,3S)- or (2R,3R)-N-Boc 3-(hydoxymethyl)-2-aminobicyclic[2,2,2]octane 6 derivatives were also obtained. 相似文献
915.
谭翠燕 徐春和 沈均人 SAKUMA Shinsuke YAMAMOTO Yasushi BALNY Claude 阮康成 《Acta biochimica et biophysica Sinica》2003,(7)
利用荧光光谱学等方法结合高压力技术研究了光合作用系统II中的一个外周蛋白——— 2 3kD(以P2 3k表示 )蛋白的去折叠。热力学研究表明 ,在 2 0℃、180MPa(1MPa =10 .0大气压 )可使该蛋白质完全去折叠 ,而在3℃ ,16 0MPa即可使该蛋白质完全去折叠 ,这是迄今为止有关研究中最易被高压力去折叠的一个蛋白质。在2 0℃ ,该蛋白质在常压下去折叠反应的标准自由能与标准体积变化分别为 2 3.4 5kJ mol和 - 15 0 .3ml mol;动力学研究揭示该蛋白质的折叠反应的活化体积ΔV f 为正值 (84 .1ml mol) ,而去折叠反应的活化体积ΔV u 为负值(- 6 6 .2ml mol)。在常压下 ,折叠和去折叠反应的速度常数 (K0f,K0u)分别为 1.87s- 1 和 1.3× 10 - 4s- 1 ,这些结果为解释该蛋白质易被压力去折叠提供了线索 相似文献
916.
Georgios A. Dalkas Damien Marchand Jean‐Claude Galleyrand Jean Martinez Georgios A. Spyroulias Paul Cordopatis Florine Cavelier 《Journal of peptide science》2010,16(2):91-97
Human ACE is a central component of the renin–angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
917.
918.
Abstract. The effects of disturbance and microtopography on the organization and dynamics of plant communities were studied in a European salt marsh located in the Bay of Mont St. Michel, France. The existence of seed trapping mechanisms was also tested. The study took place in the lower and middle marsh plant communities dominated by the perennials Puccinellia maritima and Halimione portulacoides, respectively and associated with the annual Suaeda maritima. Three treatments were used in series of plots placed in each community: (1) vegetation removal and root destruction to a depth of 10 cm and refilling, (2) non‐remnant herbicide treatment without vegetation removal and (3) creation of depressions (20 cm deep). These treatments were compared with adjacent control plots. The first year of the experiment showed that the perennials facilitated the establishment of Suaeda by trapping its seeds. Estimation of cover, density and biomass over 5 yr following the disturbances showed that in the first 2 yr Suaeda dominated the disturbed plots. Thereafter Suaeda was gradually eliminated by competitive exclusion after ca. 3 yr in the zone originally dominated by Puccinellia maritima and after 4 yr in the zone occupied by Halimione portulacoides. Depressions constituted refuge habitats for Suaeda by limiting competition with the perennials but also led to a high risk of mortality with temporal fluctuations in density. Despite a period of investigation limited to 5 yr, our study demonstrated that natural disturbances of various types occurred and influenced the dynamics of Suaeda, Halimione and Puccinellia. We deduced that natural disturbances and microtopography are responsible for the maintenance of the habitat in a state of non‐equilibrium by favouring the establishment of both spatial and temporal environmental heterogeneity. These conditions appear to be particularly favourable for the maintenance of annual species such as Suaeda maritima. 相似文献
919.
920.
Hanwen Zhang Keelara Abiraj Daniel L. J. Thorek Beatrice Waser Peter M. Smith-Jones Michael Honer Jean Claude Reubi Helmut R. Maecke 《PloS one》2012,7(9)
Bombesin receptors are under intense investigation as molecular targets since they are overexpressed in several prevalent solid tumors. We rationally designed and synthesized a series of modified bombesin (BN) peptide analogs to study the influence of charge and spacers at the N-terminus, as well as amino acid substitutions, on both receptor binding affinity and pharmacokinetics. This enabled development of a novel 64/67Cu-labeled BN peptide for PET imaging and targeted radiotherapy of BN receptor-positive tumors. Our results show that N-terminally positively charged peptide ligands had significantly higher affinity to human gastrin releasing peptide receptor (GRPr) than negatively charged or uncharged ligands (IC50: 3.2±0.5 vs 26.3±3.5 vs 41.5±2.5 nM). The replacement of Nle14 by Met, and deletion of D-Tyr6, further resulted in 8-fold higher affinity. Contrary to significant changes to human GRPr binding, modifications at the N-terminal and at the 6th, 11th, and 14th position of BN induced only slight influences on affinity to mouse GRPr. [CuII]-CPTA-[βAla11] BN(7–14) ([CuII]-BZH7) showed the highest internalization rate into PC-3 cells with relatively slow efflux because of its subnanomolar affinity to GRPr. Interestingly, [64/67Cu]-BZH7 also displayed similar affinities to the other 2 human BN receptor subtypes. In vivo studies showed that [64/67Cu]-BZH7 had a high accumulation in PC-3 xenografts and allowed for clear-cut visualization of the tumor in PET imaging. In addition, a CPTA-glycine derivative, forming a hippurane-type spacer, enhanced kidney clearance of the radiotracer. These data indicate that the species variation of BN receptor plays an important role in screening radiolabeled BN. As well, the positive charge from the metallated complex at the N-terminal significantly increases affinity to human GRPr. Application of these observations enabled the novel ligand [64/67Cu]-BZH7 to clearly visualize PC-3 tumors in vivo. This study provides a strong starting point for optimizing radiopeptides for targeting carcinomas that express any of the BN receptor subtypes. 相似文献