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1.
Tomohiro Hamasaki Takahiro Matsumoto Naoya Sakamoto Akiko Shimahara Shiori Kato Ayumi Yoshitake Ayumi Utsunomiya Hisayoshi Yurimoto Esteban C. Gabazza Tadaaki Ohgi 《Nucleic acids research》2013,41(12):e126
Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of 18O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging 18O atom into the phosphate group during the oxidation step of the synthetic cycle by using 18O water as the oxygen donor. The 18O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The 18O/16O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of 18O-labeled RNA, and this technique was used to determine the blood concentration of 18O-labeled RNA after administration to mice. 18O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies. 相似文献
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Yusuke Nakamura Michio Ogawa Takahiro Nishide Mitsuru Emi Goro Kosaki Seiichi Himeno Kenichi Matsubara 《Gene》1984,28(2):263-270
The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase. 相似文献
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H Shibata F W Robinson T R Soderling T Kono 《The Journal of biological chemistry》1991,266(27):17948-17953
Okadaic acid, a potent inhibitor of Type 1 and Type 2A protein phosphatases, was used to investigate the mechanism of insulin action on membrane-bound low Km cAMP phosphodiesterase in rat adipocytes. Upon incubation of cells with 1 microM okadaic acid for 20 min, phosphodiesterase was stimulated 3.7- to 3.9-fold. This stimulation was larger than that elicited by insulin (2.5- to 3.0-fold). Although okadaic acid enhanced the effect of insulin, the maximum effects of the two agents were not additive. When cells were pretreated with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), the level of phosphodiesterase stimulation by okadaic acid was rendered smaller, similar to that attained by insulin. In cells that had been treated with 2 mM KCN, okadaic acid (like insulin) failed to stimulate phosphodiesterase, suggesting that ATP was essential. Also, as reported previously, the effect of insulin on phosphodiesterase was reversed upon exposure of hormone-treated cells to KCN. This deactivation of previously-stimulated phosphodiesterase was blocked by okadaic acid, but not by insulin. The above KCN experiments were carried out with cells in which A-kinase activity was minimized by pretreatment with H-7. Okadaic acid mildly stimulated basal glucose transport and, at the same time, strongly inhibited the action of insulin thereon. It is suggested that insulin may stimulate phosphodiesterase by promoting its phosphorylation and that the hormonal effect may be reversed by a protein phosphatase which is sensitive to okadaic acid. The hypothetical protein kinase thought to be involved in the insulin-dependent stimulation of phosphodiesterase appears to be more H-7-resistant than A-kinase. 相似文献
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Takahiro Ishii Tatsufumi Okino Yosuke Mino Hiroaki Tamiya Fuyuhiko Matsuda 《Plant Growth Regulation》2007,52(2):131-139
Starfish waste has been shown to be an effective compost material not only in the promotion of plant growth but also in terms
of having insecticidal activity. In the present study, plant growth regulation by chemicals from starfish was examined. The
aqueous fraction from a hot water extract of the starfish Asterias amurensis Lütken showed plant-growth activity, while the aqueous fraction from a methanol extract inhibited growth of Brassica campestris. The lipophilic fraction from the methanol extract also exhibited a plant growth-promoting effect. The active components
from each extract were identified. Asterubine from the hot water extract promoted plant growth. A ceramide from the lipophilic
fraction showed root growth promoting effect, and three glucocerebrosides had promotive effects on the entire plant. Asterosaponins
were identified as the main growth inhibitors in the aqueous fraction of the methanol extract. These active compounds from
starfish waste could be analyzed as potential plant growth regulators in agricultural applications in the future. 相似文献
8.
K Fukuzawa W Ikebata A Shibata I Kumadaki T Sakanaka S Urano 《Chemistry and physics of lipids》1992,63(1-2):69-75
Studies were made on the position and dynamics of the OH-group of alpha-tocopherol in phospholipid membranes. There was no difference in the spin-lattice (T1) relaxation times at the 5a-position of alpha-tocopherol labeled with 13C- or C19F3-determined from the nuclear magnetic resonance (NMR) spectra of liposomes positively charged with stearylamine (SA) and negatively charged with dicetylphosphate (DCP). The zeta-potentials of egg yolk phosphatidylcholine (EYPC) liposomes with and without SA or DCP were not affected by incorporation of 20 mol% alpha-tocopherol, though incorporation of 10 mol% ascorbyl-palmitate decreased the zeta-potentials of EYPC and EYPC-SA liposomes. The P==O stretching band (1235 cm-1) of the phosphate group and C==O stretching band (1734 cm-1) of the acyl ester linkage in dimyristoylphosphatidylcholine (DMPC) liposomes, measured by Fourier transform-infrared (FT-IR) spectroscopy, were not changed by incorporation of alpha-tocopherol. These results suggest that no specific interaction occurred between the OH-group of alpha-tocopherol and the polar interfacial region of the bilayer. The dynamic quenching effects of n-(N-oxy-4,4'-dimethyloxazolidine-2-yl)stearic acids (n-NSs) on the intrinsic fluorescence of alpha-tocopherol were in the order 5-NS > 7-NS = 12-NS > 16-NS. Acrylamide, a water-soluble fluorescence quencher with a very low capacity to penetrate through phospholipid bilayers, had very low quenching efficiency. These results indicate that the bulk of the chromanol moiety of alpha-tocopherol is located in a position close to that occupied by the nitroxide group of 5-NS in the membranes and is poorly exposed at the membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain were treated with an Arthrobacter sp. beta-1,3-glucanase, Zymolyase-60,000, in the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride. Fractionation of the solubilized materials with Cetavlon (cetyltrimethylammonium bromide) yielded a purified mannan-protein complex, which had a molecular weight of ca. 150,000, approximately three times higher than that of the mannan isolated from the same cells by the hot-water extraction method at 135 C. The amino acid composition of the mannan-protein complex was found to be very similar to that of the mannan-protein complexes of S. cerevisiae X2180-1A wild and S. cerevisiae X2180-1A-5 mutant strains, indicating the presence of large amounts of serine and threonine. It was unexpected that the antibody-precipitating activity of this complex against the homologous anti-whole cell serum was about twice as great as that of the mannan isolated by hot-water extraction. Treatment of this complex with 100 mM NaOH, hot water at 135 C, and pronase, respectively, gave degradation products having the same molecular weight and antibody-precipitating activity as those of the hot-water extracted mannan, allowing the assumption that the protein moiety participated in a large part of this activity. 相似文献