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91.
Neale BM Rivas MA Voight BF Altshuler D Devlin B Orho-Melander M Kathiresan S Purcell SM Roeder K Daly MJ 《PLoS genetics》2011,7(3):e1001322
Technological advances make it possible to use high-throughput sequencing as a primary discovery tool of medical genetics, specifically for assaying rare variation. Still this approach faces the analytic challenge that the influence of very rare variants can only be evaluated effectively as a group. A further complication is that any given rare variant could have no effect, could increase risk, or could be protective. We propose here the C-alpha test statistic as a novel approach for testing for the presence of this mixture of effects across a set of rare variants. Unlike existing burden tests, C-alpha, by testing the variance rather than the mean, maintains consistent power when the target set contains both risk and protective variants. Through simulations and analysis of case/control data, we demonstrate good power relative to existing methods that assess the burden of rare variants in individuals. 相似文献
92.
A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the milligram scale. The newly designed vectors combines two different promoters (lpp(p)-5 and T7 RNA polymerase ?10), two different endoprotease recognition sites for the His?-tag removal (enterokinase and tobacco etch virus), different antibiotic selectable markers (ampicillin and erythromycin resistance), and different placements of the His?-tag (N- and C-terminus). A single gene can be cloned and further expressed in the eight pURI vectors by using six nucleotide primers, avoiding the restriction enzyme and ligation steps. A unique NotI site was introduced to facilitate the selection of the recombinant plasmid. As a case study, the new vectors have been used to clone the gene coding for the phenolic acid decarboxylase from Lactobacillus plantarum. Interestingly, the obtained results revealed markedly different production levels of the target protein, emphasizing the relevance of the cloning strategy on soluble protein production yield. Efficient purification and tag removal steps showed that the affinity tag and the protease cleavage sites functioned properly. The novel family of pURI vectors designed for parallel cloning is a useful and versatile tool for the production and purification of a protein of interest. 相似文献
93.
Volpato G Filice M de las Rivas B Rodrigues RC Heck JX Fernandez-Lafuente R Guisan JM Mateo C Ayub MA 《Biotechnology progress》2011,27(3):717-723
Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated. 相似文献
94.
Balsera M Arellano JB Revuelta JL de las Rivas J Hermoso JA 《Journal of molecular biology》2005,350(5):1051-1060
We report the high-resolution structure of the spinach PsbQ protein, one of the main extrinsic proteins of higher plant photosystem II (PSII). The crystal structure shows that there are two well-defined regions in PsbQ, the C-terminal region (residues 46-149) folded as a four helix up-down bundle and the N-terminal region (residues 1-45) that is loosely packed. This structure provides, for the first time, insights into the crucial N-terminal region. First, two parallel beta-strands cross spatially, joining the beginning and the end of the N-terminal region of PsbQ. Secondly, the residues Pro9-Pro10-Pro11-Pro12 form a left-handed helix (or a polyproline type II (PPII) structure), which is stabilized by hydrogen bonds between the Pro peptide carbonyl groups and solvent water molecules. Thirdly, residues 14-33 are not visible in the electron density map, suggesting that this loop might be very flexible and presumably extended when PsbQ is free in solution. On the basis of the essential role of the N-terminal region of PsbQ in binding to PSII, we propose that both the PPII structure and the missing loop are key secondary structure elements in the recognition of specific protein-protein interactions between PsbQ and other oxygen-evolving complex extrinsic and/or intrinsic proteins of PSII. In addition, the PsbQ crystal coordinates two zinc ions, one of them is proposed to have a physiological role in higher plants, on the basis of the full conservation of the ligand protein residues in the sequence subfamily. 相似文献
95.
A role for the P-body component GW182 in microRNA function 总被引:2,自引:0,他引:2
In animals, the majority of microRNAs regulate gene expression through the RNA interference (RNAi) machinery without inducing small-interfering RNA (siRNA)-directed mRNA cleavage. Thus, the mechanisms by which microRNAs repress their targets have remained elusive. Recently, Argonaute proteins, which are key RNAi effector components, and their target mRNAs were shown to localize to cytoplasmic foci known as P-bodies or GW-bodies. Here, we show that the Argonaute proteins physically interact with a key P-/GW-body subunit, GW182. Silencing of GW182 delocalizes resident P-/GW-body proteins and impairs the silencing of microRNA reporters. Moreover, mutations that prevent Argonaute proteins from localizing in P-/GW-bodies prevent translational repression of mRNAs even when Argonaute is tethered to its target in a siRNA-independent fashion. Thus, our results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA. 相似文献
96.
Background
To compare the characteristics and prognostic features of ischemic stroke in patients with diabetes and without diabetes, and to determine the independent predictors of in-hospital mortality in people with diabetes and ischemic stroke. 相似文献97.
98.
Domínguez MG Vásquez AI Troyo R Ortiz-Aranda M Padilla JR Hernández-Zaragoza G Rivas F Rivera H 《Genetic counseling (Geneva, Switzerland)》2006,17(4):413-419
We characterized two Y-ring microchromosomes (MC) found in an azoospermic patient with Turner stigmata (case A) and a male infant with hypospadias (case B). The karyotypes, as assessed by banding, FISH, and STRs/STSs analyses, were 46,X,r(Y).ish r(Y)(p11.3q11.222)(SRY+,DYZ3+) and 46,X,+r(Y)/45,X.ish r(Y)(p 11.2q11.2)(Xp/Yp-,SRY+,DYZ3+) respectively. In both cases, we evaluated the association of each MC with the centromere of the nearest and second nearest chromosomes in G-banded metaphases by means of measuring the intervening distance according to two criteria: < or =1 time or < or =3 times the size of the MC in each metaphase. The case A's MC was associated 84 times in 98 cells according to the latter or less strict criterion and two times in 98 cells according to the strict criterion; the corresponding values for case B were 84 and two in 95 cells respectively. The centromeric association appears to be related to centromeric attraction mediated by heterochromatin or centromere-specific proteins, the replication time, and the Rabl orientation. 相似文献
99.
Mar��a Lucas Blanca Gonz��lez-P��rez Matilde Cabezas Gabriel Moncalian Germ��n Rivas Fernando de la Cruz 《The Journal of biological chemistry》2010,285(12):8918-8926
TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR2). Mutation analysis in the nic region indicated that recognition of the IR2 proximal arm and the nucleotides located between IR2 and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR2 cruciform and recognition of the distal IR2 arm and loop were not necessary for these reactions to take place. On the other hand, IR2 was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR2-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place. 相似文献
100.