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981.
Molecular Mechanism of Insulin-Induced Degradation of Insulin Receptor Substrate 1 总被引:14,自引:0,他引:14 下载免费PDF全文
Rachel Zhande John J. Mitchell Jiong Wu Xiao Jian Sun 《Molecular and cellular biology》2002,22(4):1016-1026
Insulin receptor substrate 1 (IRS-1) plays an important role in the insulin signaling cascade. In vitro and in vivo studies from many investigators have suggested that lowering of IRS-1 cellular levels may be a mechanism of disordered insulin action (so-called insulin resistance). We previously reported that the protein levels of IRS-1 were selectively regulated by a proteasome degradation pathway in CHO/IR/IRS-1 cells and 3T3-L1 adipocytes during prolonged insulin exposure, whereas IRS-2 was unaffected. We have now studied the signaling events that are involved in activation of the IRS-1 proteasome degradation pathway. Additionally, we have addressed structural elements in IRS-1 versus IRS-2 that are required for its specific proteasome degradation. Using ts20 cells, which express a temperature-sensitive mutant of ubiquitin-activating enzyme E1, ubiquitination of IRS-1 was shown to be a prerequisite for insulin-induced IRS-1 proteasome degradation. Using IRS-1/IRS-2 chimeric proteins, the N-terminal region of IRS-1 including the PH and PTB domains was identified as essential for targeting IRS-1 to the ubiquitin-proteasome degradation pathway. Activation of phosphatidylinositol 3-kinase is necessary but not sufficient for activating and sustaining the IRS-1 ubiquitin-proteasome degradation pathway. In contrast, activation of mTOR is not required for IRS-1 degradation in CHO/IR cells. Thus, our data provide insight into the molecular mechanism of insulin-induced activation of the IRS-1 ubiquitin-proteasome degradation pathway. 相似文献
982.
Rachel B Kapust Karen M Routzahn David S Waugh 《Protein expression and purification》2002,24(1):61-70
Due to its high degree of sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV protease) is a useful reagent for cleaving genetically engineered fusion proteins. However, the overproduction of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility. Here we demonstrate that the low yield can be attributed to the presence of arginine codons in the TEV protease coding sequence that are rarely used in E. coli and specifically to a tandem pair of AGA codons. The yield of protease can be improved by replacing these rare arginine codons with synonymous ones or by increasing the supply of cognate tRNA that is available to the cell. Furthermore, we show that when ribosomes become stalled at rare arginine codons in the TEV protease mRNA, the nascent polypeptides are targeted for proteolytic degradation in BL21(DE3) cells by a mechanism that does not involve tmRNA-mediated peptide tagging. 相似文献
983.
Expression of a synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize 总被引:2,自引:0,他引:2
Chikwamba Rachel McMurray Jennifer Shou Huixia Frame Bronwyn Pegg Sue Ellen Scott Paul Mason Hugh Wang Kan 《Molecular breeding : new strategies in plant improvement》2002,10(4):253-265
We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R1 kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R1 kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R3 seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R3 seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens. 相似文献
984.
Andrew B. Kingston Jon Marsden‐Smedley Don A. Driscoll Sib Corbett Janet Fenton Rachel Anderson Cathie Plowman Frances Mowling Margie Jenkin Kiyoshi Matsui Kevin J. Bonham Mick Ilowski Peter B. Mcquillan Belinda Yaxley Tim Reid David Storey Lionel Poole Stephen A. Mallick Nicholas Fitzgerald Jamie B. Kirkpatrick Justin Febey Andrew G. Harwood Karyl F. Michaels Mick J. Russell Paul G. Black Louise Emmerson Mic Ah Visoiu John Morgan Shane Breen Simon Gates Mark N. Bantich Jolyon M. Desmarchelier 《Austral ecology》2002,27(2):162-172
Abstract Observations of the large earth bumblebee, Bombus terrestris (L.), in native vegetation were collated to determine the extent to which this exotic species has invaded Tasmanian native vegetation during the first 9 years after its introduction. The range of B. terrestris now encompasses all of Tasmania's major vegetation types, altitudes from sea level to 1260m a.s.L, and the entire breadth of annual precipitation in the state from more than 3200 mm to less than 600 mm. Observations of workers carrying pollen, together with the presence of large numbers of bumblebees at many localities across this range indicate that colonies are frequently established in native vegetation. Evidence that colonies are often successful was obtained from repeated observations of the species during more than 1 year at particular sites. Unequivocal evidence of colonies was obtained from six National Parks, including four of the five in the Tasmanian Wilderness World Heritage Area (WHA). Indeed, the species has been present in the WHA for at least as long as it has in the city of Hobart, where it was first recorded. In southwestern Tasmania, evidence of colonies was obtained up to 40km from gardens, 61 km from small towns and 93 km from large towns. Hence, contrary to previous suggestions, the species is established in the most remote parts of Tasmania and is not dependent on introduced garden plants. Given their strong record of invasion, it is likely that B. terrestris will form feral populations on the mainland of Australia and in many other parts of the world if introduced. Because of their likely negative impacts on native animals and plants, and potential to enhance seed production in weeds, the spread of bumblebees should be avoided. 相似文献
985.
986.
987.
Influence of plant development and environment on transgene expression in potato and consequences for insect resistance 总被引:8,自引:0,他引:8
Down Rachel E. Ford Louise Bedford Simon J. Gatehouse Laurence N. Newell Christine Gatehouse John A. Gatehouse Angharad M.R. 《Transgenic research》2001,10(3):223-236
Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance. 相似文献
988.
Kurt H. Schütte Ellen A. Maas Vasileios Exadaktylos Daniel Berckmans Rachel E. Venter Benedicte Vanwanseele 《PloS one》2015,10(10)
Small wireless trunk accelerometers have become a popular approach to unobtrusively quantify human locomotion and provide insights into both gait rehabilitation and sports performance. However, limited evidence exists as to which trunk accelerometry measures are suitable for the purpose of detecting movement compensations while running, and specifically in response to fatigue. The aim of this study was therefore to detect deviations in the dynamic center of mass (CoM) motion due to running-induced fatigue using tri-axial trunk accelerometry. Twenty runners aged 18–25 years completed an indoor treadmill running protocol to volitional exhaustion at speeds equivalent to their 3.2 km time trial performance. The following dependent measures were extracted from tri-axial trunk accelerations of 20 running steps before and after the treadmill fatigue protocol: the tri-axial ratio of acceleration root mean square (RMS) to the resultant vector RMS, step and stride regularity (autocorrelation procedure), and sample entropy. Running-induced fatigue increased mediolateral and anteroposterior ratios of acceleration RMS (p < .05), decreased the anteroposterior step regularity (p < .05), and increased the anteroposterior sample entropy (p < .05) of trunk accelerometry patterns. Our findings indicate that treadmill running-induced fatigue might reveal itself in a greater contribution of variability in horizontal plane trunk accelerations, with anteroposterior trunk accelerations that are less regular from step-to-step and are less predictable. It appears that trunk accelerometry parameters can be used to detect deviations in dynamic CoM motion induced by treadmill running fatigue, yet it is unknown how robust or generalizable these parameters are to outdoor running environments. 相似文献
989.
Biological processes that govern bacterial proliferation and survival in the host-environment(s) are likely to be vastly different from those that are required for viability in nutrient-rich laboratory media. Consequently, growth-based antimicrobial screens performed in conditions modeling aspects of bacterial disease states have the potential to identify new classes of antimicrobials that would be missed by screens performed in conventional laboratory media. Accordingly, we performed screens of the Selleck library of 853 FDA approved drugs for agents that exhibit antimicrobial activity toward the Gram-negative bacterial pathogen Acinetobacter baumannii during growth in human serum, lung surfactant, and/or the organism in the biofilm state and compared those results to that of conventional laboratory medium. Results revealed that a total of 90 compounds representing 73 antibiotics and 17 agents that were developed for alternative therapeutic indications displayed antimicrobial properties toward the test strain in at least one screening condition. Of the active library antibiotics only four agents, rifampin, rifaximin, ciprofloxacin and tetracycline, exhibited antimicrobial activity toward the organism during all screening conditions, whereas the remainder were inactive in ≥ 1 condition; 56 antibiotics were inactive during serum growth, 25 and 38 were inactive toward lung surfactant grown and biofilm-associated cells, respectively, suggesting that subsets of antibiotics may outperform others in differing infection settings. Moreover, 9 antibiotics that are predominantly used for the treatment Gram-positive pathogens and 10 non-antibiotics lacked detectable antimicrobial activity toward A. baumannii grown in conventional medium but were active during ≥ 1 alternative growth condition(s). Such agents may represent promising anti-Acinetobacter agents that would have likely been overlooked by antimicrobial whole cell screening assays performed in traditional laboratory screening media. 相似文献
990.
Robert M Bennett Ronald Friend Kim D Jones Rachel Ward Bobby K Han Rebecca L Ross 《Arthritis research & therapy》2009,11(4):R120-14