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71.
Dinh M Grunberger D Ho H Tsing SY Shaw D Lee S Barnett J Hill RJ Swinney DC Bradshaw JM 《The Journal of biological chemistry》2007,282(12):8768-8776
Bruton's tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr(551) within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr(551) causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 +/- 20 microM and KmS1 = 37 +/- 8 microM. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase. 相似文献
72.
73.
Alex Whittle Robert L. Barnett Dan J. Charman Angela V. Gallego-Sala 《Ecology letters》2022,25(1):17-25
The salinisation of many coastal ecosystems is underway and is expected to continue into the future because of sea-level rise and storm intensification brought about by the changing climate. However, the response of soil microbes to increasing salinity conditions within coastal environments is poorly understood, despite their importance for nutrient cascading, carbon sequestration and wider ecosystem functioning. Here, we demonstrate deterioration in the productivity of a top-tier microbial group (testate amoebae) with increasing coastal salinity, which we show to be consistent across phylogenetic groups, salinity gradients, environment types and latitude. Our results show that microbial changes occur in the very early stages of marine inundation, presaging more radical changes in soil and ecosystem function and providing an early warning of coastal salinisation that could be used to improve coastal planning and adaptation. 相似文献
74.
Jason E. Chung Hannah R. Joo Jiang Lan Fan Daniel F. Liu Alex H. Barnett Supin Chen Charlotte Geaghan-Breiner Mattias P. Karlsson Magnus Karlsson Kye Y. Lee Hexin Liang Jeremy F. Magland Jeanine A. Pebbles Angela C. Tooker Leslie F. Greengard Vanessa M. Tolosa Loren M. Frank 《Neuron》2019,101(1):21-31.e5
75.
76.
Klein AT Barnett P Bottger G Konings D Tabak HF Distel B 《The Journal of biological chemistry》2001,276(18):15034-15041
We have studied how Pex5p recognizes peroxisomal targeting signal type 1 (PTS1)-containing proteins. A randomly mutagenized pex5 library was screened in a two-hybrid setup for mutations that disrupted the interaction with the PTS1 protein Mdh3p or for suppressor mutations that could restore the interaction with Mdh3p containing a mutation in its PTS1. All mutations localized in the tetratricopeptide repeat (TPR) domain of Pex5p. The Pex5p TPR domain was modeled based on the crystal structure of a related TPR protein. Mapping of the mutations on this structural model revealed that some of the loss-of-interaction mutations consisted of substitutions in alpha-helices of TPRs with bulky amino acids, probably resulting in local misfolding and thereby indirectly preventing binding of PTS1 proteins. The other loss-of-interaction mutations and most suppressor mutations localized in short, exposed, intra-repeat loops of TPR2, TPR3, and TPR6, which are predicted to mediate direct interaction with PTS1 amino acids. Additional site-directed mutants at conserved positions in intra-repeat loops underscored the importance of the loops of TPR2 and TPR3 for PTS1 interaction. Based on the mutational analysis and the structural model, we put forward a model as to how PTS1 proteins are selected by Pex5p. 相似文献
77.
Apical abortion in calabrese (Brassica oleracea var.
italica), a highly destructive disorder which occurs
in overwintered transplants, has been investigated using a model system in
which blindness (abortion of the apical meristem) can be reproducibly and
predictably induced. An initial experiment examined the susceptibility of
12 cultivars to apical abortion when grown throughout a winter period under
commercial conditions. Three of those varieties showed very high levels of
blindness (100%). Subsequently, plants of the susceptible cultivar PETO
7204 were subjected to an inductive period of low light intensity (30
mol m-2
s-1) and low temperature (4 C). Apical meristematic cells of all plants ceased
mitotic activity within 3 d of being transferred to a regime comprising
higher light intensity (100 mol
m-2 s-1) and temperature (15
C). Using this system the structures of
normal apices were compared with those which became blind. Blindness was
characterized by a cessation of leaf primordium production by the
vegetative apex, the last formed primordium growing on in some cases to
form a mature normal leaf, or in others, a deformed structure known as a
whip-tail. The inactive apical bud became embedded in the tissues of this
last-formed structure. The cells of the inactivated apical bud remained
alive, but lost their meristematic capability, becoming enlarged, highly
vacuolated parenchyma cells with amyloplasts.Keywords:
Apical abortion, apical meristem, blindness, calabrese.
相似文献
78.
79.
Cross-subtype neutralizing antibodies induced in baboons by a subtype E gp120 immunogen based on an R5 primary human immunodeficiency virus type 1 envelope 下载免费PDF全文
VanCott TC Mascola JR Loomis-Price LD Sinangil F Zitomersky N McNeil J Robb ML Birx DL Barnett S 《Journal of virology》1999,73(6):4640-4650
Global human immunodeficiency virus type 1 (HIV-1) diversity may require engineering vaccines to express antigens representing strains prevalent in the target population of vaccine testing. The majority (90%) of incident infections in Thailand are genetic subtype E, with a small percentage of subtype B infections in the intravenous drug user populations. We have evaluated and compared the binding and HIV-1 neutralizing properties of serum antibodies induced in baboons by CHO cell-expressed monomeric gp120 derived from a CCR5-using (R5) subtype E primary HIV-1CM235 or a CXCR4-using (X4) subtype B T-cell line-adapted (TCLA) HIV-1SF2 isolate. In contrast to the subtype-specific HIV-1 neutralizing antibodies induced with recombinant HIV-1SF2 gp120 (rgp120SF2), rgp120CM235 immunization induced antibodies capable of neutralizing both subtype E and subtype B TCLA HIV-1 isolates. However, neither immunogen induced antibodies capable of neutralizing primary HIV-1 isolates. Antibody induced by rgp120CM235 preferentially bound natively folded gp120 and retained strong cross-reactivity against multiple gp120 strains within subtype E as well as subtype B. In contrast, antibody responses to rgp120SF2 were directed predominantly to linear epitopes poorly exposed on native gp120 and had more limited cross-recognition of divergent gp120. Fine epitope mapping revealed differences in antibody specificities. While both rgp120CM235 and rgp120SF2 induced antibodies to regions within C1, V1/V2, V3, and C5, unique responses were induced by rgp120CM235 to multiple epitopes within C2 and by rgp120SF2 to multiple epitopes within C3, V4, and C4. These data demonstrate that strain and/or phenotypic differences of HIV-1 subunit gp120 immunogens can substantially alter antibody binding specificities and subsequent HIV-1 neutralizing capacity. 相似文献
80.
Comparison of immunity generated by nucleic acid-, MF59-, and ISCOM-formulated human immunodeficiency virus type 1 vaccines in Rhesus macaques: evidence for viral clearance 下载免费PDF全文
Verschoor EJ Mooij P Oostermeijer H van der Kolk M ten Haaft P Verstrepen B Sun Y Morein B Akerblom L Fuller DH Barnett SW Heeney JL 《Journal of virology》1999,73(4):3292-3300
The kinetics of T-helper immune responses generated in 16 mature outbred rhesus monkeys (Macaca mulatta) within a 10-month period by three different human immunodeficiency virus type 1 (HIV-1) vaccine strategies were compared. Immune responses to monomeric recombinant gp120SF2 (rgp120) when the protein was expressed in vivo by DNA immunization or when it was delivered as a subunit protein vaccine formulated either with the MF59 adjuvant or by incorporation into immune-stimulating complexes (ISCOMs) were compared. Virus-neutralizing antibodies (NA) against HIV-1SF2 reached similar titers in the two rgp120SF2 protein-immunized groups, but the responses showed different kinetics, while NA were delayed and their levels were low in the DNA-immunized animals. Antigen-specific gamma interferon (IFN-gamma) T-helper (type 1-like) responses were detected in the DNA-immunized group, but only after the fourth immunization, and the rgp120/MF59 group generated both IFN-gamma and interleukin-4 (IL-4) (type 2-like) responses that appeared after the third immunization. In contrast, rgp120/ISCOM-immunized animals rapidly developed marked IL-2, IFN-gamma (type 1-like), and IL-4 responses that peaked after the second immunization. To determine which type of immune responses correlated with protection from infection, all animals were challenged intravenously with 50 50% infective doses of a rhesus cell-propagated, in vivo-titrated stock of a chimeric simian immunodeficiency virus-HIVSF13 construct. Protection was observed in the two groups receiving the rgp120 subunit vaccines. Half of the animals in the ISCOM group were completely protected from infection. In other subunit vaccinees there was evidence by multiple assays that virus detected at 2 weeks postchallenge was effectively cleared. Early induction of potent type 1- as well as type 2-like T-helper responses induced the most-effective immunity. 相似文献