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251.
Luminescent quantum dots (QDs)--semiconductor nanocrystals--are a promising alternative to organic dyes for fluorescence-based applications. We have developed procedures for using QDs to label live cells and have demonstrated their use for long-term multicolor imaging of live cells. The two approaches presented are (i) endocytic uptake of QDs and (ii) selective labeling of cell surface proteins with QDs conjugated to antibodies. Live cells labeled using these approaches were used for long-term multicolor imaging. The cells remained stably labeled for over a week as they grew and developed. These approaches should permit the simultaneous study of multiple cells over long periods of time as they proceed through growth and development. 相似文献
252.
The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains 总被引:9,自引:0,他引:9
Woodside DG Obergfell A Talapatra A Calderwood DA Shattil SJ Ginsberg MH 《The Journal of biological chemistry》2002,277(42):39401-39408
Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities (beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains. 相似文献
253.
Attachment to plant surface waxes by an insect predator 总被引:4,自引:0,他引:4
Insects foraging on plant surfaces must attach to the layerof lipophilic materials known as epicuticular waxes (EW) thatcover these surfaces. In this paper, we briefly review the evidencethat variation in EW morphology can influence the ecology ofherbivorous insects directly, by affecting their attachmentto plant surfaces, and indirectly by affecting attachment byactively foraging predatory insects to plant surfaces. We thenpresent new data examining how EW micromorphology and chemicalcomposition of Brassica oleracea influence attachment by thepredatory beetle, Hippodamia convergens (Coccinellidae). Bioassayswith genotypes of B. oleracea differing in wax characteristics,and with EW extracts from these plants applied to glass, showthat wax crystals disrupt attachment. In addition, bioassaysshow that attachment by H. convergens differs among EW extractsprepared to have smooth surfaces without crystals. The differencesin attachment under these conditions are evidently due to thechemical composition of the waxes. Bioassays with two pure waxconstituents show that wax composition can significantly affectattachment by H. convergens. The study opens the way for usinga similar approach to understand attachment by insects to waxyplant surfaces. 相似文献
254.
Complementary Roles for Receptor Clustering and Conformational Change in the Adhesive and Signaling Functions of Integrin αIIbβ3
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Integrin αIIbβ3 mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to αIIbβ3 function, αIIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified αIIb subunits were expressed with β3 in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when αIIbβ3 affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of αIIbβ3 also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72Syk and fibrinogen-dependent phosphorylation of pp125FAK, even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in αIIbβ3 function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of αIIbβ3 in platelets. 相似文献
255.
256.
Biolistic transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles 总被引:2,自引:0,他引:2
Jeanette L. Rasmussen Julie R. Kikkert Mihir K. Roy John C. Sanford 《Plant cell reports》1994,13(3-4):212-217
Summary We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.Abbreviations BMS
Black Mexican Sweet
- RPM
revolutions per minute
-
uidA
-glucuronidase gene
- GUS
-glucuronidase protein
- LB
Luria-Bertani broth
- OD600
optical density at 600 nm
- psi
pounds per square inch
- Apr
ampicillin resistance
- Knr
kanamycinresistance 相似文献
257.
258.
Chou-Chik Ting Dr. Katherine K. Sanford Floyd M. Price 《In vitro cellular & developmental biology. Plant》1978,14(2):207-211
Summary Expression of fetal antigens in early and late passages of tissue culture cells derived from C3H/HeN and C57BL/KaLw mouse
fetal cells and from lung tissue of young C57BL/6N mice was investigated by the isotopic antiglobulin technique. The late
passage lines of fetal cells had undergone “spontaneous” neoplastic transformation in culture. The antisera were produced
by syngeneic immunization with 5000 R x-irradiated tissues from C3H/HeN and C57BL/6N fetuses of 1 to 2 weeks gestation. Fetal
antigens were found to be retained even after 5 years in cell lines derived from fetal tissues In these lines no consistent
change in fetal antigen expression could be correlated with neoplastic transformation. In contrast, the early passages of
adult cells did not have detectable amounts of fetal antigens. However, fetal antigen(s) was demonstrated in cells of the
late passages, and cells of both lines grew as sarcomas when next assayed 55 days later. In addition, fetal antigens were
also present in established tumor lines in culture. 相似文献
259.
Effect of the composition of sodium dodecyl sulfate preparations on the renaturation of enzymes after polyacrylamide gel electrophoresis 总被引:6,自引:0,他引:6
Sanford A. Lacks Sylvia S. Springhorn Allan L. Rosenthal 《Analytical biochemistry》1979,100(2):357-363
The extent of renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate depended on the source of the detergent. Analysis of commercial preparations of sodium dodecyl sulfate revealed appreciable amounts of tetradecyl and hexadecyl sulfates in some preparations. Inhibition of renaturation was correlated with the amount of hexadecyl sulfate and, to a much lesser extent, of tetradecyl sulfate present. The higher alkyl sulfates appeared to bind more tenaciously to proteins in the gel. More extensive washing was required to remove them than to remove dodecyl sulfate, and they were inhibitory to enzyme activity at lower detergent concentrations. A system is described for gas chromatographic analysis of alkyl sulfates containing chains of 10 to 16 carbon atoms in length. 相似文献
260.
Masahiro Tetsuo Sanford P. Markey Robert W. Colburn Irwin J. Kopin 《Analytical biochemistry》1981,110(1):208-215
A method for assay of urinary 6-hydroxymelatonin, the major metabolite of the pineal hormone melatonin is described. After addition of an internal standard of deuterated 6-hydroxymelatonin sulfate, human urine was hydrolyzed enzymatically and free 6-hydroxymelatonin extracted, reacted to form a stable t-butyldimethylsilylpentafluoropropionyl derivative which was separated on silica gel column chromatography, and quantified using electron capture negative-ion chemical ionization mass spectrometry. Intrassay variability over an 18-h period was 5.4% [53.8 ng/3 ml urine ± 2.94 (SD)] and interassay variability over a 2-week period was 2.1% [51.8 ng/3 ml urine ± 1.08 (SD)]. 相似文献