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91.
92.
Jonathan G. Messer Robin G. Hopkins Deborah E. Kipp 《Journal of cellular biochemistry》2015,116(9):1857-1866
93.
94.
Balamurali MM Sharma D Chang A Khor D Chu R Li H 《Protein science : a publication of the Protein Society》2008,17(10):1815-1826
Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins. 相似文献
95.
B. L. Phillips J. Llewelyn A. Hatcher S. Macdonald C. Moritz 《Journal of evolutionary biology》2014,27(12):2687-2694
The two foremost hypotheses on the evolutionary constraints on an organism's thermal sensitivity – the hotter‐is‐better expectation, and the specialist–generalist trade‐off – have received mixed support from empirical studies testing for their existence. Could these conflicting results reflect confusion regarding the organizational level (i.e. species > population > individual) at which these constraints should manifest? We propose that these evolutionary constraints should manifest at different organizational levels because of differences in their underlying causes and requirements. The hotter‐is‐better expectation should only manifest across separate evolutionary units (e.g. species, populations), and not within populations. The specialist–generalist trade‐off, by contrast, should manifest within as well as between separate evolutionary units. We measured the thermal sensitivity of sprint performance for 440 rainforest sun skinks (Lampropholis coggeri) representing 10 populations, and used the resulting performance curves to test for evidence for the hypothesized constraints at two organizational levels: (i) across populations and (ii) within populations. As predicted, the hotter‐is‐better expectation was evident only at the across‐population level, whereas the specialist–generalist trade‐off was evident within, as well as across, populations. Our results suggest that, depending on the processes that drive them, evolutionary constraints can manifest at different organizational levels. Consideration of these underlying processes, and the organizational level at which a constraint should manifest, may help resolve conflicting empirical results. 相似文献
96.
DSC thermal analysis has been carried out for human hair samples with various moisture contents to investigate the melting temperature depression behavior of alpha-form crystallites in human hair. This is achieved by adopting a novel technique using silicon oil as the thermal medium, which permits hair samples to retain a range of moisture contents in between completely dry and fully saturated. The results show that the melting temperature of the alpha-form crystallites in human hair varies with moisture content from 205 degrees C for dry hair to 155 degrees C for the hair sample with moisture content of 23%. These experimental results are particularly useful for clarification of the conceptual ambiguities associated with the molecular properties of alpha-helices and alpha-form crystallites. Furthermore, the Flory-Huggins theory was employed to determine the water-helix interaction parameter (chi = 4.5) and the alpha-form crystallinity of human hair (22%), a figure consistent with that obtained by the XRD method (21%). 相似文献
97.
Shepard JA Stevans AC Holland S Wang CE Shikanov A Shea LD 《Biotechnology and bioengineering》2012,109(3):830-839
Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration. 相似文献
98.
The development and application of a flexible process controller in fed‐batch yeast fermentations using pO2 cascade control was performed. A new algorithm for fed‐batch fermentations using pO2 cascade control was developed, the concept of which could be used as a realizable solution in fermentation systems equipped according to the basic configuration. The algorithm is based on the combined influence of pO2 and pH on the substrate feeding intensity. To test and develop this algorithm, Saccharomyces cerevisiae DY 7221 and Candida tropicalis CK‐4 fermentations were carried out. As a result of the use of the combined algorithm, the specific growth rate and productivity grew in both fermentations. In this case, the effect of the use of the algorithm was most pronounced in the C. tropicalis fermentation. 相似文献
99.
To develop a cost effective process for bioinsecticides production by Photorhabdus temperata, dissolved oxygen (DO) requirements were investigated in both the complex and the optimized media using diluted seawater as a source of micronutrients. By varying DO concentrations, tolerance to hydrogen peroxide was shown to be medium dependant. Indeed, P. temperata cells grown in the complex medium, exhibited higher tolerance than cells grown in the optimized medium (OM). Tolerance to H2O2 was shown to be related to intracellular reactive oxygen species (ROS) accumulation during soya bean meal or glucose assimilation, as shown by flow cytometry analysis. To avoid oxidative stress damages in P. temperata cells cultured in the OM, DO concentration should be constant 50% saturation throughout the fermentation. However, a DO‐shift control strategy was demonstrated to be beneficial for P. temperata bioinsecticide production in the complex medium. By using such a strategy biomass, culturability, and oral toxicity reached 16.5 × 108, 1.15 × 108 cells/mL and 64.2%, respectively, thus was 16.19, 26.37, and 12.2% more than in the cultures carried out at a constant 50% saturation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012 相似文献
100.
Taking advantage of the phenomenon that animal cells adhering to a culture substrate are temporarily immobilized by light irradiation, we established a technique to manipulate the cells adhering to a culture substrate under microscopic observation. Using this technique, we demonstrated a separation of cells adhering to a culture substrate and fabrication of an elaborately patterned co-culture system. 相似文献