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31.
Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes. 相似文献
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Lack of demographic equilibrium indicates natural,large‐scale forest dynamics,not a problematic forest conservation policy – a reply to Brzeziecki et al. 下载免费PDF全文
Brzeziecki et al. 2016 (Journal of Vegetation Science 27: 460–467.) describe a decrease in population densities and proportion of younger individuals for several tree species in permanent research plots in the core zone of the Bia?owie?a National Park. They attribute insufficient tree recruitment inter alia to the strict protection. Although the authors performed a thorough analysis of tree population dynamics, the scales of the study mean that their far‐reaching conclusions on the causes and consequences of the lack of demographic equilibrium cannot be supported by the data. The inadequate spatial and temporal scales of the study did not allow for the observation of representative population dynamics. Furthermore, they did not compare the results obtained in the strictly protected area with known demographic dynamics of trees in surrounding managed forests or under other forms of nature conservation. Looking from the wider ecosystem perspective, it is clear that strict protection is not a cause for concern and, instead, that such a near‐natural forest manifests population dynamics at rather larger scales. 相似文献
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Maximizing microbial degradation of perchlorate using a genetic algorithm: Media optimization 总被引:1,自引:0,他引:1
Kucharzyk KH Crawford RL Paszczynski AJ Soule T Hess TF 《Journal of biotechnology》2012,157(1):189-197
Microbial communities are under constant influence of physical and chemical components in ecosystems. Shifts in conditions such as pH, temperature or carbon source concentration can translate into shifts in overall ecosystem functioning. These conditions can be manipulated in a laboratory setup using evolutionary computation methods such as genetic algorithms (GAs). In work described here, a GA methodology was successfully applied to define sets of environmental conditions for microbial enrichments and pure cultures to achieve maximum rates of perchlorate degradation. Over the course of 11 generations of optimization using a GA, we saw a statistically significant 16.45 and 16.76-fold increases in average perchlorate degradation rates by Dechlorosoma sp. strain KJ and Dechloromonas sp. strain Miss R, respectively. For two bacterial consortia, Pl6 and Cw3, 5.79 and 5.75-fold increases in average perchlorate degradation were noted. Comparison of zero-order kinetic rate constants for environmental conditions in GA-determined first and last generations of all bacterial cultures additionally showed marked increases. 相似文献
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Improvement in stability of an immobilized fungal laccase 总被引:4,自引:0,他引:4
Andrzej Leonowicz Jawed M. Sarkar Jean-Marc Bollag 《Applied microbiology and biotechnology》1988,29(2-3):129-135
Summary A laccase of the basidiomyceteTrametes versicolor was immobilized on porous glass beads that were activated with 3-aminopropyltriethoxysilane and glutaraldehyde. The support immobilized 100% of the enzyme, whereupon 90% of the original activity was retained. After immobilization, the enzyme was active in a wider pH and temperature range, and its heat stability and reuse were greatly improved compared to those of the free laccase. The immobilized enzyme was found reusable in treating different substrates, either recycled alone or in a sequential order. 相似文献
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When cells are briefly exposed to cis-diamminedichloroplatinum (II) before lysis in high sodium dodecyl sulfate-urea solutions, the high molecular-weight nucleic acids pelleted by ultracentrifugation contain an increased level of bound proteins when compared to a similar fraction from untreated cells. Subsequent shearing of the pelleted DNA followed by treatment with DNase permits electrophoretic and immunoblot analysis of the crosslinked proteins. In the present study such experiments were carried out with reference to nuclear envelope pore complex proteins in the differentiating L8 rat skeletal muscle cells. The results show that (i) whereas the major lamin proteins crosslinked to DNA in both myoblast and myotubes, lamin B is crosslinked to a greater extent to DNA in myotubes; (ii) a 62-kDa lectin-binding glycoprotein is apparently situated differently with respect to DNA in myotube nuclei; and (iii) the crosslinking pattern of the nuclear matrix proteins to DNA is qualitatively similar in myoblast and myotubes. In addition, lamin C′, a modified form of lamin C, not observed in intact nonmuscle cells previously [[31.] J. Biol. Chem. 260, 1895–1900], exists as a native component of the nuclear lamina in rat skeletal myotubes but not in myoblasts. These results point to significant structural alterations in the proteins of the nuclear lamina-pore complex during myogenesis. 相似文献