全文获取类型
收费全文 | 469篇 |
免费 | 42篇 |
国内免费 | 1篇 |
专业分类
512篇 |
出版年
2022年 | 3篇 |
2020年 | 3篇 |
2019年 | 6篇 |
2018年 | 7篇 |
2017年 | 10篇 |
2016年 | 9篇 |
2015年 | 13篇 |
2014年 | 19篇 |
2013年 | 20篇 |
2012年 | 14篇 |
2011年 | 24篇 |
2010年 | 8篇 |
2009年 | 18篇 |
2008年 | 24篇 |
2007年 | 20篇 |
2006年 | 10篇 |
2005年 | 24篇 |
2004年 | 10篇 |
2003年 | 12篇 |
2002年 | 12篇 |
2001年 | 16篇 |
2000年 | 11篇 |
1999年 | 12篇 |
1998年 | 8篇 |
1997年 | 3篇 |
1996年 | 4篇 |
1993年 | 4篇 |
1992年 | 10篇 |
1991年 | 7篇 |
1990年 | 11篇 |
1989年 | 15篇 |
1988年 | 7篇 |
1987年 | 7篇 |
1986年 | 6篇 |
1985年 | 6篇 |
1984年 | 5篇 |
1983年 | 8篇 |
1982年 | 14篇 |
1981年 | 5篇 |
1980年 | 3篇 |
1979年 | 8篇 |
1978年 | 6篇 |
1977年 | 9篇 |
1976年 | 5篇 |
1974年 | 5篇 |
1972年 | 7篇 |
1971年 | 5篇 |
1970年 | 4篇 |
1968年 | 8篇 |
1967年 | 3篇 |
排序方式: 共有512条查询结果,搜索用时 0 毫秒
11.
R S Power H E David M H A Z Mutwakil K Fletcher C Daniells M A Nowell J L Dennis A Martinelli R Wiseman E Wharf D I de Pomerai 《Journal of biosciences》1998,23(4):513-526
This paper reviews the current status of nematodes with stress-inducible transgenes as biosensors responsive to a range of
external stressors, e.g., soil or water pollution, microwave radiation or immunological attack. TransgenicCaenorhabditis elegans carrying reporter genes under heat shock promoter control express reporter products only under stressful conditions. Although
relatively insensitive to single metal ions, these worms respond to complex mixtures present in metal-contaminated watercourses
and to laboratory mixtures containing similar constituents, but not to any of their components singly at comparable concentrations.
Responses to metal mixtures are enhanced by a non-ionic surfactant, Pluronic F-127. Metals taken up by food bacteria and insoluble
metal carbonates can also evoke stress responses, both in soil and aqueous media. However, high concentrations of added metals
are needed to induce clear-cut responses in soil, owing to metal sorption onto clays and organic matter. Transgenic worms
are also stressed by exposure to microwave radiation; pulsed signals generate responses that diminish markedly with distance
from the source. Finally, stress responses are inducible by anti-epicuticle antisera and complement, suggesting that immune
attack can also activite the heat shock system. The development of rapid microplate toxicity assays based on transgenic nematodes
is discussed. 相似文献
12.
We present higher-order moment analysis of fluorescence intensity fluctuations from individual laser scanning microscopy images applied to study monomer-oligomer distributions. We demonstrate that the number densities and brightness ratios of a mixed population of monomers and oligomers can be determined by analyzing higher-order moments of the fluorescence intensity fluctuations from individual images for specific ranges of densities and particle brightness ratios. Computer simulations and experiments with fluorescent microspheres and cells were performed to illustrate the detection limits and accuracy of this statistical approach. The simulation results show that the concentration of the dimer or oligomer population should be less than or equal to the monomeric concentration for the method to provide accurate results, and that the upper density detection limit of the population of monomers is one order-of-magnitude higher than the concentration of the oligomers. We implemented this technique to resolve two populations of fluorescent microspheres with different brightness ratios and we also applied the moment-analysis method to examine the distribution of aggregation states of PDGF-beta receptors in human fibroblast cells. The method was able to resolve a tetrameric population of the PDGF-beta receptors relative to the background distribution of nonspecifically bound fluorophore. 相似文献
13.
Jeffrey J. Brault Natalie M. Pizzimenti John N. Dentel Robert W. Wiseman 《Journal of cellular biochemistry》2013,114(6):1445-1455
Muscle contractions strongly activate p38 MAP kinases, but the precise contraction‐associated sarcoplasmic event(s) (e.g., force production, energetic demands, and/or calcium cycling) that activate these kinases are still unclear. We tested the hypothesis that during contraction the phosphorylation of p38 isoforms is sensitive to the increase in ATP demand relative to ATP supply. Energetic demands were inhibited using N‐benzyl‐p‐toluene sulphonamide (BTS, type II actomyosin) and cyclopiazonic acid (CPA, SERCA). Extensor digitorum longus muscles from Swiss Webster mice were incubated in Ringer's solution (37°C) with or without inhibitors and then stimulated at 10 Hz for 15 min. Muscles were immediately freeze‐clamped for metabolite and Western blot analysis. BTS and BTS + CPA treatment decreased force production by 85%, as measured by the tension time integral, while CPA alone potentiated force by 310%. In control muscles, contractions resulted in a 73% loss of ATP content and a concomitant sevenfold increase in IMP content, a measure of sustained energetic imbalance. BTS or CPA treatment lessened the loss of ATP, but BTS + CPA treatment completely eliminated the energetic imbalance since ATP and IMP levels were nearly equal to those of non‐stimulated muscles. The independent inhibition of cytosolic ATPase activities had no effect on contraction‐induced p38 MAPK phosphorylation, but combined treatment prevented the increase in phosphorylation of the γ isoform while the α/β isoforms unaffected. These observations suggest that an energetic signal may trigger phosphorylation of the p38γ isoform and also may explain how contractions differentially activate signaling pathways. J. Cell. Biochem. 114: 1445–1455, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
14.
Michael D. Woodrow Stuart P. Ballantine Michael D. Barker Beth J. Clarke John Dawson Tony W. Dean Christopher J. Delves Brian Evans Sharon L. Gough Steven B. Guntrip Stuart Holman Duncan S. Holmes Michael Kranz Mika K. Lindvaal Fiona S. Lucas Margarete Neu Lisa E. Ranshaw Yemisi E. Solanke Don O. Somers Peter Ward Joanne O. Wiseman 《Bioorganic & medicinal chemistry letters》2009,19(17):5261-5265
Crystallography driven optimisation of a lead derived from similarity searching of the GSK compound collection resulted in the discovery of quinoline-3-carboxamides as highly potent and selective inhibitors of phosphodiesterase 4B. This series has been optimized to GSK256066, a potent PDE4B inhibitor which also inhibits LPS induced production of TNF-α from isolated human peripheral blood mononuclear cells with a pIC50 of 11.1. GSK256066 also has a suitable profile for inhaled dosing. 相似文献
15.
Mode of action of the alpha toxin of Staphylococcus aureus 总被引:3,自引:0,他引:3
16.
van het Hof KH Wiseman SA Yang CS Tijburg LB 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1999,220(4):203-209
Epidemiological studies suggest that antioxidant flavonoids in tea may reduce the risk of cardiovascular disease, possibly via protection of low-density lipoproteins (LDL) against oxidation. However, the extent of absorption of tea flavonoids and their accumulation in LDL during regular consumption of tea is not clear. Therefore we investigated plasma and lipoprotein levels of catechins during tea consumption and the impact on LDL oxidizability ex vivo. Eighteen healthy adults consumed, in an incomplete balanced cross-over design, green tea, black tea, black tea with milk or water, one cup every 2 hr (eight cups/day) for three days. Blood samples were obtained in the mornings and evenings of each day. Plasma total catechin concentration was determined in all blood samples, and the distribution of catechins among lipoproteins was determined at the end of the third day (t = 60 hr). The resistance of LDL to copper-induced oxidation ex vivo was assessed before tea consumption and at t = 60 hr. Repeated tea consumption during the day rapidly increased plasma total catechin levels whereas they declined overnight when no tea was consumed. There was a gradual increase in plasma levels in the mornings (respectively, 0.08 microM vs. 0.20 microM on first and last day of black tea consumption) and evenings (respectively, 0.29 microM vs. 0.34 microM on first and last day of black tea consumption). Green tea catechins were mainly found in the protein-rich fraction of plasma (60%) and in high-density lipoproteins (23%). Although present in LDL, the concentration of catechins in LDL was not sufficient to enhance the resistance of LDL to oxidation ex vivo. Addition of milk to black tea did not affect any of the parameters measured. In conclusion, the present study shows that catechin levels in blood rapidly increase upon repeated tea consumption. The accumulation of catechins in LDL particles is not sufficient to improve the intrinsic resistance of LDL to oxidation ex vivo. 相似文献
17.
18.
Martin MJ Yin D Adams C Houtz J Shen J Chong AS Sharma A Byrne GW Wiseman BS Logan JS 《Cloning and stem cells》2003,5(2):117-121
Nuclear transfer technology allows for the reprogramming of somatic cells, and the production of embryonic stem cells and animals that are genetically identical in terms of nuclear DNA to the parental somatic cell. It is assumed that these products of nuclear transfer technology will be immunologically compatible to each other in spite of the fact that there are data that show differences in the expression patterns and phenotypes between animals produced by nuclear transfer. We have produced a series of cloned pigs from embryonic fibroblasts. Microsatellite analysis was used to confirm that the clones were genetically identical. Skin transplants were performed to assess immunological reactivity. Skin transplants between genetically identical cloned pigs were accepted, whereas third party grafts were rejected. Histological analysis of the grafts showed edema and mononuclear cell infiltrates in the recipient's skin in rejected grafts and not in grafts that were accepted. Our data supports the notion that genetically identical cloned pigs are immunologically compatible. 相似文献
19.
Nicholas Mikolajewicz Simon Sehayek Paul W. Wiseman Svetlana V. Komarova 《Biophysical journal》2019,116(10):2009-2022
The skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically relevant mechanical forces lead to small repairable membrane injuries in bone-forming osteoblasts, resulting in release of ATP and stimulation of purinergic (P2) calcium responses in neighboring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, their extracellular diffusion and degradation, and purinergic responses in neighboring cells. After validation using experimental data for intracellular free calcium elevations, ATP, and vesicular release after mechanical stimulation of a single osteoblast, the model was scaled to a tissue-level injury to investigate how purinergic signaling communicates information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration, and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed spatial factors, such as the maximal distance from the injury at which purinergic responses were stimulated. The peak ATP concentration reflected the severity of an individual cell injury, allowing to discriminate between minor and severe injuries that released similar amounts of ATP because of differences in injury repair, and determined temporal aspects of the response, such as signal propagation velocity. ADP-mediated signaling became relevant only in larger tissue-level injuries, conveying information about the distance to the injury site and its geometry. Thus, we identified specific features of extracellular ATP and ADP spatiotemporal signals that depend on tissue mechanoresilience and encode the severity, scope, and proximity of the mechanical stimulus. 相似文献
20.
George?W. Ashdown Andrew Cope Paul?W. Wiseman Dylan?M. Owen 《Biophysical journal》2014,107(9):L21-L23
We combine total internal reflection fluorescence structured illumination microscopy with spatiotemporal image correlation spectroscopy to quantify the flow velocities and directionality of filamentous-actin at the T cell immunological synapse. These techniques demonstrate it is possible to image retrograde flow of filamentous-actin at superresolution and provide flow quantification in the form of velocity histograms and flow vector maps. The flow was found to be retrograde and radially directed throughout the periphery of T-cells during synapse formation.Many biological processes are now being visualized with the use of superresolution fluorescence microscopy techniques. However, localization-based techniques primarily rely on fixed or slow moving samples to permit the collection of structural information. The 10-fold gains in resolution afforded by these superresolution techniques are usually possible through sacrificing the factors that originally made microscopy such a powerful tool: the ability to image live cells. In the case of stimulated emission depletion imaging, the scanning approach associated with this technique may fail to detect faster molecular events when imaging whole cellular regions.Structured illumination microscopy (SIM) is an alternative to these methods (1). It increases the resolution of conventional fluorescence microscopy twofold; it has the advantage of using a wide-field system, providing fast acquisition speeds of whole cells with relatively low laser powers; and it is compatible with standard fluorophores. By using a physical grating to produce interference patterns from a laser, periodic illumination is created. This patterned illumination causes information from higher spatial frequencies to be downmodulated (i.e., shifted) into the optical transfer function (support region) of the lens, resulting in higher-resolution spatial information being captured than is ordinarily obtainable.To quantify the directional motion of intracellular molecules, spatiotemporal image correlation spectroscopy (STICS (2)) was applied. Using spatial image correlation in time, STICS measures the similarity of pixels with those surrounding in lagging frames via a correlation function. The correlation function provides information on both flow velocities and directionality, while discounting static structures through the immobile object filter, achieved by subtracting a moving average of pixel intensities.The formation of an immunological synapse between T cells and antigen-presenting cells is a process requiring many dynamic (3) and subdiffraction-limited clustering events (4–6) to take place. The polymerization of actin is important for the spreading of cells over their target antigen-presenting cells (7), as well as cell mobility and migration (8). Retrograde flow of densely meshed cortical actin is observed at the basal membrane of synapse-forming T cells, where it may have a role in the corralling and clustering of signaling molecules at the plasma membrane (9), as well as at the leading edge of migrating cells (10). Filamentous actin is an extremely dynamic (7), densely packed, and thin (7-nm) structure (11,12).Here, we perform STICS on SIM data acquired on a total internal reflection fluorescence (TIRF) microscope system, which generated an evanescent field of 75-nm depth for excitation. To our knowledge, this is the first demonstration of an image correlation approach to quantify molecular dynamics on subresolution length scales using wide-field microscopy. To demonstrate the technique, we analyze two-dimensional actin flows in CD4+ T cells during immunological synapse formation, performed after cross-linking of antigen T cell receptors on a coverslip coated with specific antibodies.Fig. 1
a shows a schematic of the TIRF SIM setup. Excitation light (488 nm) passes through a polarizing module and then a phase-grating block, producing diffracted beams. These are then passed through a diffraction filter module to isolate the −1 and +1 order laser beams. These first-order laser beams are angled through the objective to produce total internal reflection conditions at the glass-water interface. The two evanescent waves interfere at the sample, producing structured illumination. The setup then produces lateral and rotational shifts through three orientations, producing nine raw images containing higher spatial frequencies than can normally be acquired by an objective using standard light microscopy. Fig. 1
b demonstrates the increased resolution obtained from TIRF SIM. Shown are the collected Fourier frequencies compared to those of a conventional microscope (dotted red line). Resolution was also measured using sparse 100-nm diameter fluorescent beads. Fig. 1
c shows a magnified image of these beads from which a line profile was obtained (yellow arrow). The full width at half-maximum of this profile (Fig. 1
d) gives a lateral resolution for the system of 120 nm.Open in a separate windowFigure 1(a) Schematic of the TIRF SIM setup. (b) Demonstration of the doubling of spatial resolution of collected frequencies through a Fourier transform (superimposed red circle demonstrating regular spatial frequency limits). (c) SIM reconstructed image of 100-nm bead (scale bar 0.5 μm). (d) (Plotted line) Bead showing full width at half-maximum of 120 nm.We then applied STICS analysis to quantify actin flow in T cell synapses acquired using TIRF SIM (Fig. 2). Fig. 2
a shows a schematic of the STICS analysis. From the raw data, immobile objects are first filtered by subtracting a moving average of the pixel values. Vector maps were obtained from correlation analysis of the time-series as previously published in Hebert et al. (2) and Brown et al. (13). Fig. 2
b shows a reconstructed TIRF SIM image of a mature T cell immunological synapse, representative of a time-point derived from the time series acquired at 1.28 fps (see Movie S1 in the Supporting Material). From this reconstructed image, two representative regions have been selected. In these regions, pseudo-colored actin flow vectors are overlaid onto the fluorescence intensity image. These range in magnitude from 0.01 μm/min (blue) to 5.61 μm/min (red). It can be observed that all flow vectors are directed radially toward the synapse center. A histogram of this flow is shown in Fig. 2
c. The histogram shows a peak retrograde flow velocity of 1.91 ± 1.27 μm/min. These data are representative of n = 7 T-cell synapses imaged by TIRF SIM.Open in a separate windowFigure 2(a) STICS analysis, performed by isolating mobile from immobile structures through a moving average filter (i) and binning a subset of pixels into blocks of superpixels (ii); the STICS software correlates spatial fluorescence fluctuations through time (iii). The code then outputs vector maps showing directionality and flow velocities. (b) TIRF SIM image of actin flow in a T cell 5 min after contact with a stimulatory coverslip. (Zoomed regions) Retrograde actin flow at the synapse periphery. (c) Histograms showing flow speed statistics of vectors from T-cell synapses (n = 7). 相似文献