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971.
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975.
Habermann FA André S Kaltner H Kübler D Sinowatz F Gabius HJ 《Histochemistry and cell biology》2011,135(6):539-552
Gene divergence has given rise to the galectin family of mammalian lectins. Since selective binding to distinct β-galactosides
underlies the known bioactivities of galectins, they could find application in cyto- and histochemistry. The pertinent question
on the characteristics of their individual reactivity profiles therefore needs to be answered. Toward this end, comparative
studies of a panel of galectins in defined systems are required. We here characterise the staining profiles of seven human
lectins as well as five natural derivatives originating from proteolytic truncation and serine phosphorylation and one engineered
variant. As test system, bovine germinal vesicle oocytes with their glycoprotein envelope (zona pellucida), which presents
bi- to tetraantennary complex-type N-glycans with N-acetyllactosamine repeats and core fucosylation, were processed. Technically, confocal laser scanning microscopy was used,
first with plant lectins to map the sialylation status. Hereby, α2,3/6-sialylation was detected in the superficial filamentous
meshwork of the zona pellucida, while sialic acid-free glycan chains were found to characterise the main inner part of the
compact layer of the zona pellucida. Galectin staining was specific and non-uniform. Significant differences in reactivity
were detected for the superficial filamentous meshwork and the compact layer of the zona pellucida between galectins-1 to
-4 versus galectins-8 and -9. The typical staining profiles intimate a spatially organised display of N-glycans in the different layers of the zona pellucida, underscoring the potential of galectins as cyto- and histochemical
tools. Our results encourage further comparative analysis and research to trace the underlying structural and/or topological
properties. 相似文献
976.
André T. Fernandes Manuela M. Pereira Catarina S. Silva Peter F. Lindley Isabel Bento Eduardo Pinho Melo Lígia O. Martins 《Journal of biological inorganic chemistry》2011,16(4):641-651
The contribution of the disulfide bridge in CotA-laccase from Bacillus subtilis is assessed with respect to the enzyme’s functional and structural properties. The removal of the disulfide bond by site-directed
mutagenesis, creating the C322A mutant, does not affect the spectroscopic or catalytic properties and, surprisingly, neither
the long-term nor the thermodynamic stability parameters of the enzyme. Furthermore, the crystal structure of the C322A mutant
indicates that the overall structure is essentially the same as that of the wild type, with only slight alterations evident
in the immediate proximity of the mutation. In the mutant enzyme, the loop containing the C322 residue becomes less ordered,
suggesting perturbations to the substrate binding pocket. Despite the wild type and the C322A mutant showing similar thermodynamic
stability in equilibrium, the holo or apo forms of the mutant unfold at faster rates than the wild-type enzyme. The picosecond
to nanosecond time range dynamics of the mutant enzyme was not affected as shown by acrylamide collisional fluorescence quenching
analysis. Interestingly, copper uptake or copper release as measured by the stopped-flow technique also occurs more rapidly
in the C322A mutant than in the wild-type enzyme. Overall the structural and kinetic data presented here suggest that the
disulfide bridge in CotA-laccase contributes to the conformational dynamics of the protein on the microsecond to millisecond
timescale, with implications for the rates of copper incorporation into and release from the catalytic centres. 相似文献
977.
Tosi T Nickerson NN Mollica L Jensen MR Blackledge M Baron B England P Pugsley AP Dessen A 《Molecular microbiology》2011,82(6):1422-1432
A crucial aspect of the functionality of bacterial type II secretion systems is the targeting and assembly of the outer membrane secretin. In the Klebsiella oxytoca type II secretion system, the lipoprotein PulS, a pilotin, targets secretin PulD monomers through the periplasm to the outer membrane. We present the crystal structure of PulS, an all-helical bundle that is structurally distinct from proteins with similar functions. Replacement of valine at position 42 in a charged groove of PulS abolished complex formation between a non-lipidated variant of PulS and a peptide corresponding to the unfolded region of PulD to which PulS binds (the S-domain), in vitro, as well as PulS function in vivo. Substitutions of other residues in the groove also diminished the interaction with the S-domain in vitro but exerted less marked effects in vivo. We propose that the interaction between PulS and the S-domain is maintained through a structural adaptation of the two proteins that could be influenced by cis factors such as the fatty acyl groups on PulS, as well as periplasmic trans-acting factors, which represents a possible paradigm for chaperone-target protein interactions. 相似文献
978.
Marc‐André Dubois Alain Grandbois Shawn K. Collins Andreea R. Schmitzer 《Journal of molecular recognition : JMR》2011,24(2):288-294
Dimerization of a hydroxycarbazole produces an axially chiral biaryl, BICOL ( 2 ). One enantiomer (R)‐ 2 , is capable of enantioselective binding to different polymorphs of DNA. The biaryl (R)‐ 2 was shown by fluorescence and circular dichroism to induce a shift of Z‐DNA to B‐DNA. The opposite enantiomer (S)‐ 2 shows no specific binding. The significant difference in behaviour between the two enantiomers (S)‐ 2 and (R)‐ 2 is in line with molecular modelling studies which show two very different binding geometries between the enantiomers with each polymorph of DNA. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
979.
The monoclonal antibody B13-DE1 binds fluorescein, several fluorescein derivatives, and three peptide mimotopes. Our results revealed that this antibody also catalyzed the redox reaction of resazurin to resorufin, which are both structurally related to fluorescein. By using sodium sulfite as a reducing agent, the antibody B13-DE1 lowered the activation energy of this reaction. The Michaelis-Menten constant and turnover number of the catalyzed reaction were determined as 4.2 μmol/l and 0.0056 s(-1) , respectively. Because the results showed that fluorescein inhibited the catalytic activity of the antibody, we assume that the antigen-binding site and the catalytic active site are identical. 相似文献
980.
Rahman LN Bamm VV Voyer JA Smith GS Chen L Yaish MW Moffatt BA Dutcher JR Harauz G 《Amino acids》2011,40(5):1485-1502
Dehydrins are intrinsically unstructured proteins that are expressed in plants experiencing extreme environmental conditions
such as drought or low temperature. Although their role is not completely understood, it has been suggested that they stabilize
proteins and membrane structures during environmental stress and also sequester metals such as zinc. Here, we investigate
two dehydrins (denoted as TsDHN-1 and TsDHN-2) from Thellungiella salsuginea. This plant is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and
experiences periods of both extreme cold and drought. We show using circular dichroism and attenuated total reflection-Fourier
transform infrared spectroscopy that ordered secondary structure is induced and stabilized in these proteins, both in free
and vesicle-bound form, by association with zinc. In membrane-associated form, both proteins have an increased proportion
of β-strand conformation induced by the cation, in addition to the amphipathic α-helices formed by their constituent K-segments.
These results support the hypothesis that dehydrins stabilize plant plasma and organellar membranes in conditions of stress,
and further that zinc may be an important co-factor in stabilization. Whereas dehydrins in the cytosol of a plant cell undergoing
dehydration or temperature stress form bulk hydrogels and remain primarily disordered, dehydrins with specific membrane- or
protein-associations will have induced ordered secondary structures. 相似文献