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A high‐throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation 下载免费PDF全文
Lam Raga Anggara Markely Lila Cheung Young Jun Choi Thomas Ryll Scott Estes Shashi Prajapati Iva Turyan Ruth Frenkel Zoran Sosic James Lambropoulos Lia Tescione Thomas Ryll Melissa Berman 《Biotechnology progress》2016,32(1):235-241
The serum half‐life, biological activity, and solubility of many recombinant glycoproteins depend on their sialylation. Monitoring glycoprotein sialylation during cell culture manufacturing is, therefore, critical to ensure product efficacy and safety. Here a high‐throughput method for semi‐quantitative fingerprinting of glycoprotein sialylation using capillary isoelectric focusing immunoassay on NanoPro (Protein Simple) platform was developed. The method was specific, sensitive, precise, and robust. It could analyze 2 μL of crude cell culture samples without protein purification, and could automatically analyze from 8 samples in 4 h to 96 samples in 14 h without analyst supervision. Furthermore, its capability to detect various changes in sialylation fingerprints during cell culture manufacturing process was indispensable to ensure process robustness and consistency. Moreover, the changes in the sialylation fingerprints analyzed by this method showed strong correlations with intact mass analysis using liquid chromatography and mass spectrometry. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:235–241, 2016 相似文献
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Hammond Hammond Stacy Denise Viehmannova Iva Zamecnik Jiri Panis Bart Hlasna Cepkova Petra 《Plant Cell, Tissue and Organ Culture》2019,138(3):559-570
Plant Cell, Tissue and Organ Culture (PCTOC) - Slow-growth is a biotechnological tool for medium-term conservation of plant germplasm under in vitro conditions. In the present study, we assessed... 相似文献
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Dakic Tamara Jevdjovic Tanja Lakic Iva Djurasevic Sinisa F. Djordjevic Jelena Vujovic Predrag 《Neurochemical research》2019,44(2):388-399
Neurochemical Research - Our group previously reported that 6-h fasting increased both insulin II mRNA expression and insulin level in rat hypothalamus. Given that insulin effects on central... 相似文献
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Cell prestress. II. Contribution of microtubules 总被引:7,自引:0,他引:7
Stamenović D Mijailovich SM Tolić-Nørrelykke IM Chen J Wang N 《American journal of physiology. Cell physiology》2002,282(3):C617-C624
The tensegritymodel hypothesizes that cytoskeleton-based microtubules (MTs) carrycompression as they balance a portion of cell contractile stress. Totest this hypothesis, we used traction force microscopy to measuretraction at the interface of adhering human airway smooth muscle cellsand a flexible polyacrylamide gel substrate. The prediction is that ifMTs balance a portion of contractile stress, then, upon theirdisruption, the portion of stress balanced by MTs would shift to thesubstrate, thereby causing an increase in traction. Measurements weredone first in maximally activated cells (10 µM histamine) and thenagain after MTs had been disrupted (1 µM colchicine). We found that after disruption of MTs, traction increased on average by ~13%. Because in activated cells colchicine induced neither an increase inintracellular Ca2+ nor an increase in myosin light chainphosphorylation as shown previously, we concluded that the observedincrease in traction was a result of load shift from MTs to thesubstrate. In addition, energy stored in the flexible substrate wascalculated as work done by traction on the deformation of thesubstrate. This result was then utilized in an energetic analysis. Weassumed that cytoskeleton-based MTs are slender elastic rods supportedlaterally by intermediate filaments and that MTs buckle as the cellcontracts. Using the post-buckling equilibrium theory of Euler struts,we found that energy stored during buckling of MTs was quantitativelyconsistent with the measured increase in substrate energy afterdisruption of MTs. This is further evidence supporting the idea thatMTs are intracellular compression-bearing elements. 相似文献
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Marko Samardzija Martina Karadjole Iva Getz Zdenko Makek Marijan Cergolj Tomislav Dobranic 《Reproductive biology and endocrinology : RB&E》2006,4(1):58
The aim of our research was to examine the ability of density gradient preparation BoviPure? and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental
bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity
and acrosomal status were evaluated and compared before and after sperm processing using BoviPure? and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were
significant differences (P < 0.05) between the sperm characteristics before and after BoviPure?, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods.
A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and
the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly
higher using BoviPure? method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of
Day 7 morulas and blastocysts showed that BoviPure? treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure? method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So,
we concluded that BoviPure? could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for
IVP of bovine embryos. 相似文献
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Francesco V. Rao Alexander W. Schüttelkopf Helge C. Dorfmueller Andrew T. Ferenbach Iva Navratilova Daan M. F. van Aalten 《Open biology》2013,3(10)
The dynamic modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification present in higher eukaryotes. Removal of O-GlcNAc is catalysed by O-GlcNAcase, a multi-domain enzyme that has been reported to be bifunctional, possessing both glycoside hydrolase and histone acetyltransferase (AT) activity. Insights into the mechanism, protein substrate recognition and inhibition of the hydrolase domain of human OGA (hOGA) have been obtained via the use of the structures of bacterial homologues. However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood. Here, we describe the crystal structure of a putative acetyltransferase (OgpAT) that we identified in the genome of the marine bacterium Oceanicola granulosus, showing homology to the hOGA C-terminal AT domain (hOGA-AT). The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel. The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA. Thus, the C-terminal domain of hOGA is a catalytically incompetent ‘pseudo’-AT. 相似文献