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A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K+-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K+-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)−1 · min−1 were obtained, the value decreasing with age and K+-deficiency. Complete inhibition of the K+-dependent phosphatase was obtained with 10−3 M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K+-dependent phosphatase activity and (Na+ + K+)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min−1 was estimated from simultaneous determination of K+-dependent phosphatase activity and [3H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [3H]ouabain binding sites measured in intact muscles or biopsies hereof. 相似文献
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14.
Fluorescent antibody staining indicated differences in surface antigenicity in Anabaena azollae cells fresh from the leaf cavities of the fern, Azolla caroliniana, and algae which were isolated and subcultured from this fern. Such results suggest that either changes in antigenicity occur in this phycobiont during culturing or that isolation selects for an antigenically different mutant strain capable of in vitro growth.Non-Standard Abbreviations FA
fluorescent antibody staining
- PBS
phosphate buffered saline
- W
microwatt
- Anti-F
antiserum prepared against fresh cells
- Anti-N
antiserum prepared against Newton's culture
- FTTC
fluorescein isothiocyanate
To whom offprint requests should be sent 相似文献
15.
Bile and serum samples were collected from calves with an implanted cannula throughout a 20-week period of infection with Fasciola hepatica. Using indirect fluorescent antibody labelling and plastic-embedded sections of juvenile and adult flukes as antigens, estimates were made of the relative concentrations of IgG and IgA specific for fluke tegumental and gut antigens in the samples of serum and bile. In serum, antibodies against juvenile (t1) tegument and gut antigens reached peak concentrations 4–6 weeks postinfection and declined slowly thereafter as flukes became established in the bile ducts. IgG against adult tegument (t2) antigens appeared in the serum 6 weeks after infection, but no IgA against t2 was detected. In the bile, both IgG and IgA titres against t1 and gut antigens rose to peak values at 4–6 weeks after infection, but there was no activity against t2 antigen. The Ig levels in bile were considerably lower than in serum. Much more IgA relative to IgG occurred in bile as compared to serum (IgG/IgA ratio in serum was 16–32, in bile 1–2) suggesting a role for IgA in defence at mucosal surfaces. Comparison of the antibody profiles in bile and serum suggested that IgG in the bile was derived from circulating IgG whereas IgA may have been preferentially concentrated in the bile. 相似文献
16.
Shimaa Mohammad Yousof Horeya Erfan Marwa Mohamed Hosny Shaimaa A. Shehata Karima El-Sayed 《Saudi Journal of Biological Sciences》2022,29(5):3890
Products containing Silver nanoparticles (Ag NPs) are becoming vastly used in our daily life. The widespread increased introduction of Ag NPs in many aspects of life has raised researchers'' concerns regarding their safety and toxicity for biological and environmental life in the past few years. The current study aimed to explore the subsequent effects of Ag NPs withdrawal, following short-term oral administration. Eighteen rats were assigned randomly into three groups (control group "1" and AG NPs treated groups "2" and "3"; 6 animals each). The control group received normal food and tap water while groups 2 & 3 received 0.5 ml of a solution containing 25 ppm Ag NPs for 14 days. Group 2 rats were sacrificed on day 14 whereas group 3 was left for another 14 days of particle cessation followed by euthanasia on day 28. Functional assessment was done by liver enzyme assays, hydrogen peroxide activity, hepatic Bdnf expression, and P53 immunoreactivity. Hepatic tissue structural assessment was done via hematoxylin and eosin, periodic acid-Schiff as well as Masson''s trichrome stains. The results revealed a significant elevation of Hydrogen peroxide in group 2 only compared to the control group. Hepatic Bdnf and liver enzymes were both insignificantly affected. Structural abnormalities and enhanced apoptosis in hepatic tissue were found 14 days after ceasing the nanoparticles. In conclusion: Structural and functional insults following Ag NPs oral administration continues after particle withdrawal, and interestingly they do not necessitate apparent reflection on liver enzyme assays. 相似文献
17.
E. Iwarsson Monalill Lundqvist José Inzunza Lars Ährlund-Richter Peter Sjöblom Örjan Lundkvist Niklas Simberg Magnus Nordenskjöld Elisabeth Blennow 《Human genetics》1999,104(5):376-382
We have studied the chromosomal content in 68 normally fertilised freeze-thawed human embryos of good morphology from 34
patients with an average maternal age of 32,6 years. Forty embryos showed post-thaw cellular division and twenty-eight post-thaw
cleavage arrest. After spreading of the embryos on microscope slides, analysis of chromosomes X, Y, 15, 16, 17 and 18 was
performed using two rounds of fluorescent in situ hybridisation (FISH). According to the results, the embryos were divided into four groups: (I) normal, all nuclei uniformly
diploid, (II) diploid mosaics, normal diploid blastomeres in combination with abnormal blastomeres, (III) abnormal, all nuclei
abnormal, (IV) chaotic, the chromosome constitution varies randomly from cell to cell. Approximately 25% of the embryos had
normal number of the chromosomes tested, while the majority of the embryos were abnormal. Most of the abnormal embryos were
diploid mosaics (57%). This was true for the embryos showing cleavage division as well as the embryos showing cleavage arrest.
Our data show a slightly higher incidence of abnormal embryos compared to those obtained with FISH in non-cryopreserved embryos
and confirm that the majority of preimplantation embryos fertilised in vitro contain abnormal blastomeres. The results, mechanisms, significance and implications are discussed.
Received: 19 November 1998 / Accepted: 4 March 1999 相似文献
18.
Emilie Blanc Patrick Wagner Fabrice Plaisier Martine Schmitt Thierry Durroux Jean-Jacques Bourguignon Michel Partiseti Elodie Dupuis Frederic Bihel 《Analytical biochemistry》2015
Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries. 相似文献
19.
Confocal fluorescence microscopy of plant cells 总被引:14,自引:0,他引:14
Summary The confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the z axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM. 相似文献
20.
We designed cassettes allowing the systematic fusion of fluorescent or luminescent proteins preceded by the calmodulin binding peptide tag to the C–terminus of Escherichia coli proteins. The chromosomal insertion, and thus physiological expression level of these fusions, permits the study of protein localization by fluorescent microscopy and protein quantification, in vivo and dynamically in diverse conditions. Furthermore, the calmodulin binding peptide tag allows standard detection, affinity purification, and co–purification experiments. These cassettes are therefore very valuable for the versatility of experiments they make available for a given strain, from biochemistry to dynamic and in vivo studies. 相似文献