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991.
992.
The present study demonstrates that H(2)O(2) and OH(.-) cause fibril aggregation and catalytic inactivation of porcine fumarase. In the aggregated (oxidized) enzyme, modifications in both secondary and tertiary protein structure occur and the enzyme aggregation obeys to fractal geometry. We then collected information on the fractal dimension and on the size and shape of fumarase aggregates by using Synchrotron Radiation (SR) Small Angle X-ray Scattering (SAXS) analysis. The geometrical self-similarity assessment of aggregates has been revealed by both AFM and SEM measurements at different scale of magnification. Micrographs collected remarkably demonstrate that the oxidized enzyme shows dendritic fractal structure over a large range of sizes. 相似文献
993.
Koch M Bhattacharya S Kehl T Gimona M Vasák M Chazin W Heizmann CW Kroneck PM Fritz G 《Biochimica et biophysica acta》2007,1773(3):457-470
Human S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca2+ S100A2 binds Zn2+ with a high affinity. Studies have been carried out to investigate whether Zn2+ acts as a regulatory ion for S100A2, as in the case of Ca2+. Using the method of competition with the Zn2+ chelator 4-(2-pyridylazo)-resorcinol, an apparent Kd of 25 nM has been determined for Zn2+ binding to S100A2. The affinity lies close to the range of intracellular free Zn2+ concentrations, suggesting that S100A2 is able to bind Zn2+ in the nucleus. Two Zn2+-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd2+ and Co2+ as probes. In site 1 Zn2+ is bound by Cys21 and most likely by His 17. The binding of Zn2+ in site 2 induces the formation of a tetramer, whereby the Zn(2+) is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn2+ to site 2 substantially weakens the affinity of S100A2 for Ca2+. Analysis of the individual Ca2+-binding constants revealed that the Ca2+ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn2+ and Ca2+, and suggest that Zn2+ might deactivate S100A2 by inhibiting response to intracellular Ca2+ signals. 相似文献
994.
Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine 总被引:1,自引:0,他引:1
Donadelli M Costanzo C Beghelli S Scupoli MT Dandrea M Bonora A Piacentini P Budillon A Caraglia M Scarpa A Palmieri M 《Biochimica et biophysica acta》2007,1773(7):1095-1106
We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5'-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5'-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy. 相似文献
995.
Horjales S Cota G Señorale-Pose M Rovira C Román E Artagaveytia N Ehrlich R Marín M 《Archives of biochemistry and biophysics》2007,467(2):139-143
As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34 kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers. 相似文献
996.
Ludivina Avila Marc Wirtz Richard A. Bunce Mario Rivera 《Journal of biological inorganic chemistry》1999,4(5):664-674
The gene coding for putidaredoxin has been synthesized using a combination of chemical and enzymatic methods and subsequently
expressed in Escherichia coli. The recombinant protein characterized by electronic spectroscopy, mass spectrometry, and electrochemistry was found to be
identical to putidaredoxin obtained from Pseudomonas putida. Polylysine was found to promote the fast and reversible electrochemistry of putidaredoxin at negatively charged electrodes
such as indium-doped tin oxide or gold surfaces modified with mercaptoalkanoate groups. The value of the heterogeneous electron
transfer rate constant obtained from solutions containing a mixture of putidaredoxin and polylysine (k
s
=1.3×10–3 cm/s) is one order of magnitude larger than the values reported previously at gold electrodes modified with mercaptoethylamine
or at antimony-doped tin oxide semiconductor electrodes. It was observed that when the reduction potential of putidaredoxin
is measured by cyclic voltammetry, the resultant value is consistently more positive (64 mV) than the reduction potential
measured with potentiometric titrations. A comparison between the electrochemical responses of putidaredoxin and spinach ferredoxin,
combined with the examination of their corresponding three-dimensional structures, indicates that the positive shift in the
reduction potential of putidaredoxin originates from the formation of a transient complex between putidaredoxin and polylysine
at the electrode surface. The formation of this transient complex modulates the reduction potential of putidaredoxin by lowering
the value of the dielectric constant around its iron-sulfur cluster microenvironment, specifically by neutralizing negative
charges surrounding the active site and by excluding water from the solvent exposed iron sulfur cluster. The observed positive
shift in E°′, which is induced by complexation with polylysine at the electrode-surface, suggests that similar factors are
likely to contribute to the anodic shift in the E°′ of cytochrome P450cam-bound putidaredoxin (+44 mV) with respect to the E°′ measured for free putidaredoxin.
Received: 14 June 1999 / Accepted: 6 August 1999 相似文献
997.
Ectopic mesodermal expression promoted by the eight-cell stage Bufo arenarum subequatorial cytoplasm
Mesodermal determinants were investigated by cytoplasmic transfer and blastomere isolation in the eight-cell stage of Bufo arenarum. Their existence was confirmed by assaying the subequatorial cytoplasm’s ability to respecify the developmental potency of
animal quartets. The gray subequatorial cytoplasm, but not animal cytoplasm, is able to divert the ectodermal fate of animal
quartets to several mesodermal components. The source of the transplanted cytoplasm was important in determining the category
of the resulting structures. Ventral subequatorial cytoplasm from ventrovegetal blastomeres generated ventral derivatives,
namely erythrocytes and mesenchyma. Dorsal subequatorial cytoplasm from dorsovegetal blastomeres produced dorsolateral derivatives,
such as notochord, muscle, nephric tubules, and coelomic epithelium, including mesenchyma. On the other hand, transfer of
vegetal pole cytoplasm to animal quartets resulted in the formation of groups of endoderm-like cells dispersed among epidermal
cells. However, the presence of such cells did not cause any mesodermal induction. The present findings suggest the existence
of cytoplasmic information responsible for mesodermal specification. The alternative hypothesis that animal blastomeres become
mesoderm due to vegetal induction is questioned.
Received: 9 October 1998 / Accepted: 10 March 1999 相似文献
998.
Axis development: the mouse becomes a dachshund. 总被引:1,自引:0,他引:1
Targeted deletion of the gene for GDF11, a novel member of the TGFbeta family, has been found to cause an increase in the number of thoracic and lumbar vertebrae in the mouse. This is the first hint that a secreted factor may influence the specification of segment identity. 相似文献
999.
Safety Evaluation of Transgenic Tilapia with Accelerated Growth 总被引:2,自引:0,他引:2
Isabel Guillén Jorge Berlanga Carmen M. Valenzuela Antonio Morales José Toledo Mario P. Estrada Pedro Puentes Orlando Hayes José de la Fuente 《Marine biotechnology (New York, N.Y.)》1999,1(1):2-14
Recent advances in modern marine biotechnology have permitted the generation of new strains of economically important fish
species through the transfer of growth hormone genes. These transgenic fish strains show improved growth performance and therefore
constitute a better alternative for aquaculture programs. Recently, we have obtained a transgenic tilapia line with accelerated
growth. However, before introducing this line into Cuban aquaculture, environmental and food safety assessment was required
by national authorities. Experiments were performed to evaluate the behavior of transgenic tilapia in comparison to wild tilapia
as a way to assess the environmental impact of introducing transgenic tilapia into Cuban aquaculture. Studies were also conducted
to evaluate, according to the principle of substantial equivalence, the safety of consuming transgenic tilapia as food. Behavior
studies showed that transgenic tilapia had a lower feeding motivation and dominance status than controls. Food safety assessment
indicated that tilapia growth hormone has no biological activity when administered to nonhuman primates. Furthermore, no effects
were detected in human healthy volunteers after the consumption of transgenic tilapia. These results showed, at least under
the conditions found in Cuba, no environmental implications for the introduction of this transgenic tilapia line and the safety
in the consumption of tiGH-transgenic tilapia as an alternative feeding source for humans. These results support the culture
and consumption of these transgenic tilapia.
Received: March 9, 1998; accepted June 25, 1998. 相似文献
1000.