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Achim von Bomhard Alexander Els?sser Lucas Maximilian Ritschl Silke Schwarz Nicole Rotter 《PloS one》2016,11(2)
Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco''s modified Eagle medium Ham’s F-12 (DMEM)and K+-modified TiProtec (K+TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm2 cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K+TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1.6%). The most viable cells in proliferation after three days of culture were obtained in cells vitrificated by using GLY with K+TiP (308 ±34%) and PG with DMEM in slow freezing (280 ±27%). 相似文献
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Natsumi Doi Yuuki Kunimatsu Kouhei Fujiura Hiro Togari Kenji Minagi Koichi Nakaoji Kazuhiko Hamada Achim Temme Masaaki Tatsuka 《Journal of cellular physiology》2019,234(9):15134-15146
The molecular signals that regulate mitotic spindle orientation to determine the proper division axis play a critical role in the development and maintenance of tissue homeostasis. However, deregulation of signaling events can result in spindle misorientation, which in turn can trigger developmental defects and cancer progression. Little is known about the cellular signaling pathway involved in the misorientation of proliferating cells that evade apoptosis after DNA damage. In this study, we found that perturbations to spindle orientation were induced in ultraviolet C (UVC)-irradiated surviving cells. N-terminal truncated Rho GDP-dissociation inhibitor β (RhoGDIβ), which is produced by UVC irradiation, distorted the spindle orientation of HeLa cells cultured on Matrigel. The short hairpin RNA-mediated knockdown of RhoGDIβ significantly attenuated UVC-induced misorientation. Subsequent expression of wild-type RhoGDIβ, but not a noncleavable mutant, RhoGDIβ (D19A), again led to a relative increase in spindle misorientation in response to UVC. Our findings revealed that RhoGDIβ impacts spindle orientation in response to DNA damage. 相似文献
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Achim Brockmeier Manuel Skopnik Brigitte Koch Christian Herrmann Stefan Welti Klaus Scheffzek 《Biochemical and biophysical research communications》2009,388(4):630-636
Eubacteria can import and simultaneously phosphorylate a range of different carbohydrates by means of sugar specific phosphoenolpyruvate (PEP) dependent sugar phosphotransferase systems (PTSs). Here, we report the biochemical characterization of the gluconate specific PTS component EIIAgnt from Enterococcus faecalis and its unexpectedly strong complex with EIIBgnt. We analyze the activity of the complex regarding phosphoryl transfer using kinetic measurements and demonstrate by mutagenesis that His-9 of EIIAgnt is essential for this process and represents most likely the phosphoryl group carrier of EIIAgnt. With a combination of isothermal titration calorimetry (ITC), analytical ultracentrifugation (AUC), native gel electrophoresis and chemical crosslinking experiments we show that EIIAgnt and EIIBgnt form a strong 2:2 heterotetrameric complex, which seems to be destabilized upon phosphorylation of EIIBgnt. 相似文献
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Kaiser A Hammels I Gottwald A Nassar M Zaghloul MS Motaal BA Hauber J Hoerauf A 《Bioorganic & medicinal chemistry》2007,15(18):6200-6207
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Since the late 1990s, the Pacific oyster (Crassostrea gigas) has spread into the East Frisian Wadden Sea (Germany). This invasion provided an opportunity to study the population dynamics
and the patterns of spread during the initial bioinvasion process. With its source area in The Netherlands, the bioinvasion
continues in an eastward direction, as documented by a gradient of high abundances in the west and low abundances in the east
during the first study year. One year later, abundances of the Pacific oyster were more heterogenic and differed between adjacent
tidal basins. The increase in population sizes at all study sites was very high, reaching levels similar to native occurrence
populations. The growth constant (K) varied between 0.300 and 0.990 year−1. The mussel bed with the highest densities had a mean abundance of >300 ind. m−2, and a maximum of 1,460 ind. m−2. Furthermore, the bioinvasion was facilitated by a low mortality (Z) found for populations between 0.5 and 1.5 years old (Z = 0.03–0.13 year−1). At present, Pacific oysters are well established at several locations in the East Frisian Wadden Sea and may become with
these reproductive potential self-sustaining populations. 相似文献
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Cell focal adhesions are micrometer-sized aggregates of proteins that anchor the cell to the extracellular matrix. Within the cell, these adhesions are connected to the contractile, actin cytoskeleton; this allows the adhesions to transmit forces to the surrounding matrix and makes the adhesion assembly sensitive to the rigidity of their environment. In this article, we predict the dynamics of focal adhesions as a function of the rigidity of the substrate. We generalize previous theories and include the fact that the dynamics of proteins that adsorb to adhesions are also driven by their coupling to cell contractility and the deformation of the matrix. We predict that adhesions reach a finite size that is proportional to the elastic compliance of the substrate, on a timescale that also scales with the compliance: focal adhesions quickly reach a relatively small, steady-state size on soft materials. However, their apparent sliding is not sensitive to the rigidity of the substrate. We also suggest some experimental probes of these ideas and discuss the nature of information that can be extracted from cell force microscopy on deformable substrates. 相似文献