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971.
972.
Optical Single Transporter Recording (OSTR) is a technique for analyzing membrane transport kinetics at high sensitivity, selectivity, and spatial resolution. Cellular membranes are firmly attached to microarrays of small test compartments (TCs) with diameters between approximately 0.1 and 100 microm and depths between approximately 10 and 100 microm. This permits to generate either "small" membrane patches containing few transporters or "large" patches containing many transporters. Transport of substrates across membrane patches is recorded by confocal microscopy. The present article reviews recent applications of OSTR to the nuclear pore complex (NPC). The results show that the transport functions of the NPC, previously studied almost exclusively in intact and permeabilized cells, are conserved in isolated nuclei and can be fully reconstituted in purified nuclear envelopes by addition of recombinant transport factors. This opens new avenues to the analysis of nuclear transport including the export of nucleic-acid-protein and ribosomal particles. 相似文献
973.
We have developed a screening assay for erythrocyte stability, which is rapid, easy, inexpensive, robust, and suitable for handling a large number of samples in parallel. Erythrocytes are incubated overnight in 96-well microtiter plates in absence or presence of various oxidants, intact cells are pelleted by centrifugation, and lysis is determined by release of intracellular constituents into the supernatant as either activity of lactate dehydrogenase (LDH) or absorbance of hemoglobin at 406 nm. There is good correlation between the methods. A number of advantages by the present method are that only small amounts of blood is needed, washing is optional, erythrocytes may be stored for at least one day before assay, and large numbers of samples can be handled in parallel. Using this set-up, we have compared erythrocyte stability from several different animal species. We find that erythrocyte susceptibility towards lysis induced by H2O2 and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is highly species dependent. The different susceptibility between species is due to cellular components, since swapping of plasma between species has little or no effect. As a novel observation, we find that erythrocytes from chicken are the most sensitive of the species tested towards lysis by H2O2 and are almost four orders of magnitude more sensitive than erythrocytes from man. This is due to a much lower content of catalase in erythrocytes from chicken. A more narrow range is observed for susceptibility towards AAPH and the ranking between the species is different. Thus, chicken erythrocytes are more resistant towards AAPH than some mammals by up to two orders of magnitude. This differential stability towards different oxidative stressors is likely due to evolution/selection of different defense mechanisms. 相似文献
974.
Demello DE Mahmoud S Ryerse J Hoffmann JW 《In vitro cellular & developmental biology. Animal》2002,38(3):154-164
The Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2Kb-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2Kb. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression. 相似文献
975.
Prediction and overview of the RpoN-regulon in closely related species of the Rhizobiales 总被引:3,自引:1,他引:2
Dombrecht B Marchal K Vanderleyden J Michiels J 《Genome biology》2002,3(12):research0076.1-research007611
976.
Various aspects of excitation energy conversion in anoxygenic photosynthetic bacteria are surveyed. This minireview discusses
different models that have been proposed during the past 60 years to describe excitation energy transfer from an antenna molecule
to the reaction center. First, a simple one-dimensional model was suggested, but over time the models became more detailed
when structural and dynamic information was included. This review focuses mainly on the picture of purple bacteria and green
sulfur bacteria developed during the past decades.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
977.
Depreitere J Durinx C Wang Z Coen E Lambeir AM Scharpé S De Potter W Nouwen EJ 《Regulatory peptides》2002,106(1-3):71-79
Using the pig splenic nerve as a model, we investigated the proteolytic processing of porcine chromogranin B (CgB) during its axonal transport. An ELISA was developed for SR-17 (CgB(586-602)), a novel CgB-derived peptide, originally found in the adrenal medulla. The results demonstrate that CgB is processed in an early stage during its axonal transport. Immunohistochemical data, based on a rabbit anti-SR-17 antiserum, show that the spleen CgB/SR-17 is exclusively present in the nerve endings. No SR-17 immunoreactivity (IR) was found in splenocytes. We also provide evidence that SR-17 is co-released with noradrenaline (NA) upon electrical stimulation of the splenic nerve. Its release is frequency-dependent and strongly enhanced in the presence of the alpha-blocking agent phentolamine. In addition, we show that the new CgB-peptide can serve as a substrate for the lymphocyte surface glycoprotein CD26, also known as dipeptidyl peptidase IV (DPP IV), generating a new peptide ER-15 (CgB(588-602)). 相似文献
978.
The influence of exogenous gibberellic acid (GA3) andpaclobutrazol, an inhibitor of gibberellin biosynthesis, on growth of callusandsomatic embryogenesis in petiole-derived tissue cultures of Medicagosativa L. has been investigated. GA3 (0.5–500M) or paclobutrazol(5–100 M) were added to either an induction (with 2,4 Dand kinetin) or a differentiation medium (without plant growth regulators).Gibberellin A3, applied during the induction as well as thedifferentiation stage, reduced the weight of callus and increased the number ofsomatic embryos in Medicago sativa L. tissue cultures.Somatic embryo production was increased more by the presence of exogenousGA3 in the differentiation than induction medium. The inclusion ofpaclobutrazol in the induction or differentiation medium caused the inhibitionof callus growth and embryo production. Callus growth was much less affectedthan embryogenesis. These results indicate that gibberellins are beneficial forboth embryoinduction and formation. The level of endogenous gibberellins is presumablysufficient for callus induction and growth. However, it seems not optimal forthe induction and particularly for the differentiation of embryos. 相似文献
979.
Elżbieta Galoch Joanna Czaplewska Elżbieta Burkacka-Łaukajtys Jan Kopcewicz 《Plant Growth Regulation》2002,37(3):199-205
The influence of BA, GA3 and IAA applied successively onflower bud formation in shoot apices of Pharbitis nil hasbeen investigated. The shoot apices were isolated from seedlings cultivatedunder non-inductive continuous light and from seedlings exposed to asubinductive (12 h) dark period. BA and GA3 introducedsuccessively into culture medium replaced the inductive night, causing theflowering of plantlets in completely non-inductive continuous light (optimalconcentration of BA – 10–7–10–6mol dm–3, GA3 –10–7–10–6 moldm–3) and stimulated this process under thesubthresholdinduction. These hormones applied in reverse sequence (in the first placeGA3, then BA) did not affect flowering of explants. IAA nullifiedthestimulating effect of BA and GA3. The influence of phytohormones onflowering may result from the change of growth correlations within the shootapical meristem. 相似文献
980.
Verhaert RM Beekwilder J Olsthoorn R van Duin J Quax WJ 《Journal of biotechnology》2002,96(1):103-118
Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display we selected alpha-amylase variants, which have the ability to bind starch substrate at industrially preferred low pH conditions. First we displayed active alpha-amylase on the surface of phage fd. Secondly we developed a selection system that is based on the ability of alpha-amylase displaying phages to bind to cross-linked starch. This system was used to probe the involvement of specific beta-strands in substrate interaction. Finally, a saturated library of alpha-amylase mutants with one or more amino acid residues changed in their Cbeta4 starch-binding domain was subjected to phage display selection. Mutant molecules with good starch-binding and hydrolytic capacity could be isolated from the phage library by repeated binding and elution of phage particles at lowered pH value. Apart from the wild type alpha-amylase a specific subset of variants, with only changes in three out of the seven possible positions, was selected. All selected variants could hydrolyse starch and heptamaltose at low pH. Interestingly, variants were found with a starch hydrolysis ratio at pH 4.5/7.5 that is improved relative to the wild type alpha-amylase. These data demonstrate that useful alpha-amylase mutants can be selected via surface display on the basis of their binding properties to starch at lowered pH values. 相似文献