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31.
32.
Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3–8.3-fold and 19–22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications. 相似文献
33.
Jerome J. Howard 《Journal of Insect Behavior》1993,6(4):441-453
Patterns of resource fidelity, switching, and variation in individual diet were examined in the polyphagous grasshopper Taeniopoda equesforaging in experimental cages over an 11-day period. Ten novel dicots were provided in the cages, but grasshoppers fed primarily on kale (Brassica oleraceavar. acephala),lobelia (Lobelia erinus),pansy (Viola x wittrockiana),and dry bermuda grass (Cynodon dactylon).Grasshoppers showed significant fidelity to a single plant within a meal but tended to eat less of the same plant in the next meal and, in the case of pansy, on the next day. Grasshoppers switched nonrandomly, suggesting that compensatory feeding on complementary resources may have occurred. There was no evidence that behavioral interactions among individuals increased switching rate or lowered fidelity. Overall patterns of resource use varied significantly among grasshoppers during the study, despite frequent switching among resources and a significant tendency to aggregate while feeding. All individuals were polyphagous but differed in relative consumption of available plants. The results suggest that individual grasshoppers express different feeding patterns that are consistent over time and that variation in diet among individual observed in the field may be more than simple sampling error. 相似文献
34.
35.
We describe the optimized effective potential method of density functional theory and the semi-analytical approximation due to Krieger, Li and Iafrate. Results for atomic and molecular systems including correlation contributions are presented and compared with conventional Kohn–Sham methods. The combination of the exact exchange energy functional with the correlation energy functional of Colle and Salvetti works extremely well for atomic systems, while further improvement is required for molecular systems. 相似文献
36.
Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a
process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into
the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which
a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly,
solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport
with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by
fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient,
are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation
product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into
the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume
cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different
types of secondary metabolic energy generation will be discussed. 相似文献
37.
Hints on the evolutionary design of a dimeric RNase with special bioactions. 总被引:2,自引:2,他引:0 下载免费PDF全文
A. Di Donato V. Cafaro I. Romeo G. D'Alessio 《Protein science : a publication of the Protein Society》1995,4(8):1470-1477
Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases. 相似文献
38.
The aim of this work was to investigate the mechanism of formation of triose phosphates and 3-phosphoglycerate during photosynthetic induction in leaves of Zea mays. Simultaneous measurements of gas exchange, chlorophyll a fluorescence and metabolite contents of maize leaves were made. Leaves illuminated in the absence of CO2 showed a build-up of triose phosphates during the first 2 min of illumination which was comparable to the build-up observed in the presence of CO2. Isolated mesophyll protoplasts, which lack the Calvin cycle, also showed a build-up of triose phosphates upon illumination. Leaves contained amounts of phosphoglycerate mutase and enolase adequate to account for the formation of triose phosphates and 3-phosphoglycerate from intermediates of the C4 cycle and their precursors. 相似文献
39.
The effect of caffeine on Chinese hamster V79 cells after treatment with the highly mutagenic (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-7,8,9,10-tetrahydrobenzo[a]pyrene, and the weaker mutagen (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, B[a]P-deiol-epoxide II, was studied at both the biological and molecular levels. Caffeine, at nontoxic dose levels, caused a synergistic reduction in cell survival induced by both isomers and also inhibited DNA elongation as measured by alkaline sucrose-gradient analysis of nascent DNA. However, caffeine did not affect the induction of either ouabain-resistant mutants or sister-chromatid exchanges by either isomer. These results suggest that enhanced cell killing by caffeine in benzo[a]pyrene-diol-epoxide treated V79 cells may be related to caffeine's inhibitory effect on DNA elongation. However, inhibition of DNA elongation by caffeine did not influence the resulting induced levels of mutagenesis or sister-chromatid exchanges. 相似文献
40.
Synopsis
Raja erinacea and R. ocellata are sibling species which are positively correlated with each other by occurrence and numerical abundance. In sympatry the species undergo interactive segregation; R. erinacea feeds on a higher percentage of epifauna and R. ocellata feeds on a higher percentage of infauna.An isolated allopatric population of R. ocellata occurs in the Gulf of St. Lawrence which is phenotypically intermediate between the sympatric populations of R. erinacea and R. ocellata in characters related to feeding e.g. size, number of tooth rows in the upper jaw, and shape of the upper jaw. It appears probable that the allopatric population represents the morphological state of R. ocellata before it became sympatric with R. erinacea; divergence in size, number of tooth rows, and shape of the upper jaw between the two species developed after establishment of sympatry. These divergences in character traits, related to feeding, reduced competition between the two sympatric species and permitted the present wide overlap in their ranges.Character displacement is evidently rare in demersal fishes inhabiting the flat and soft bottoms of the northwestern Atlantic because the three other pairs of sibling species that occur there are parapatrically distributed and thus would not compete for resources. Raja erinacea and R. ocellata may have been restrained from establishing parapatry by another species pair of skates (R. senta and R. radiata) which have a complementary distribution and similar feeding habits of R. erinacea and R. ocellata but which occur in deeper water. 相似文献