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991.
SubbaRao V. Madhunapantula Arati Sharma Raghavendra Gowda Gavin P. Robertson 《Pigment cell & melanoma research》2013,26(6):886-899
Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA‐mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis‐inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR‐A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma. 相似文献
992.
B. Nussberger M. P. Greminger C. Grossen L. F. Keller P. Wandeler 《Molecular ecology resources》2013,13(3):447-460
Introgression can be an important evolutionary force but it can also lead to species extinction and as such is a crucial issue for species conservation. However, introgression is difficult to detect, morphologically as well as genetically. Hybridization with domestic cats (Felis silvestris catus) is a major concern for the conservation of European wildcats (Felis s. silvestris). The available morphologic and genetic markers for the two Felis subspecies are not sufficient to reliably detect hybrids beyond first generation. Here we present a single nucleotide polymorphism (SNP) based approach that allows the identification of introgressed individuals. Using high‐throughput sequencing of reduced representation libraries we developed a diagnostic marker set containing 48 SNPs (Fst > 0.8) which allows the identification of wildcats, domestic cats, their hybrids and backcrosses. This allows assessing introgression rate in natural wildcat populations and is key for a better understanding of hybridization processes. 相似文献
993.
Historical collections held in ancient archives and libraries constitute a cultural and artistic heritage of inestimable value. These collections contain a series of organic materials which may alter and perish in time, especially if they are conserved in inadequate environmental conditions and without any specific precautionary devices to protect them from damage caused by chemical, biological and physical factors. It is essential to prevent ‘at risk’ situations to conserve library and archive heritage. Early detection of fungal spores and moulds in conservation environments will permit us to intervene at the infection site at the right moment and also to detect the micro‐environments where climatic parameters are not suitable for conservation purposes. The aim of this study is to examine potentially biodeteriogen fungal micro‐flora in the environment of the “Doctorate Library”, one of the most important buildings of the University of Perugia, which contains frescos and library collection of great value. The study was carried out through quantitative and qualitative analysis of airborne fungal species and through the evaluation of viable aerobiological particles as potential agents of biodeterioration. The airspore monitoring was made using the active method (by Personal Volumetric Air Sampler Spore Trap, Burkard Company Ltd.) and the passive one (by sedimentation technique on Petri dishes). The results obtained by both methods allowed us to detect numerous potentially biodeteriogen species, to determine their quantity and influence and to evaluate the levels of their variability in time. 相似文献
994.
Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening α-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the l-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either l- or d-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 d-amino acid aminotransferase displaying a decrease in kcat/KM of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate l-leucine than l-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering. 相似文献
995.
Chunxia Qiao Ming Lv Xinying Li Jing Geng Yan Li Jiyan Zhang 《Journal of biomolecular structure & dynamics》2013,31(5):511-521
Increased affinities mainly equal to improved biological efficacy in many cases. By now, display methods including phage library are widely exploited to obtain higher affinity antibodies. Traditional library methods mainly focus on complementary determining region mutagenesis, in which extensive screening of variant combinations as well as large library capacity is required to find higher affinity clones. In this study, based on the modeling 3D complex structure of antigen (HER2)–antibody (MIL5) complex, the key residues of contact surface were predicted and identified to guide the synthetic phage library design. Then, epitope-specific site-directed mutagenesis phage library comprised of MIL5_scFv mutants was constructed, from which a higher affinity single chain antibody (M5scFv_ph) was screened out. Following experimental results showed that the novel antibody M5scFv_ph retained superimposed epitope to the parent antibody MIL5_scFv, and possessed similar tumor growth inhibitory activity in vivo on ovarian carcinoma xenografts. 相似文献
996.
M. Rejwan Ali Mostafa Sadoqi Allal Boutajangout Mihaly Mezei 《Journal of biomolecular structure & dynamics》2013,31(17):4494-4506
AbstractMolecular dynamics (MD) simulation using the AMBER force field has been performed on the neurotensin (NT) receptor, a class A type G-protein-coupled receptor in its activated conformation co-crystallized with the non-peptide agonists. For structure-based hit molecule identification via natural chemical compound library, orthosteric sites on NT receptor have been mapped by docking using AutoDock4.0 and Vina with the known agonists and antagonists SR48692, SR142948, ML301 and ML314 of the receptor. Furthermore, clustering analysis on the MD trajectories by SIMULAID has been performed to filter receptor conformations for the allosteric binders from the Otava natural compound library. Comparative mappings of contrasting binding region patterns have been done between the crystal structure orthosteric sites as well as the binding regions in the SIMULAID-based cluster center conformations from MD trajectories with the FTmap server using the small organic molecule fragments as the probes. The distinct binding region in the cluster-based conformations in the extracellular region of the receptor has been identified for targeted docking by Otava natural chemical compound library using AutoDock4.0 and Vina docking suites to obtain putative allosteric binders. A group of compounds from the Otava library has been identified as showing high free energy in both AutoDock4.0 and Vina docking suites. Biophysical assessments on the natural compound computational hit molecules will be done to identify lead structures from the hit molecules.Communicated by Ramaswamy H. Sarma 相似文献
997.
Kunio Nakagawa Ikuo Yoshimura Noriyoshi Sueda Hideaki Fukawa 《Bioscience, biotechnology, and biochemistry》2013,77(8):1431-1433
Pyridoxal-5′-phosphate was synthesized in excellent yield by phosphorylation of 1-secondaryammo-l,3-dihydro-7-hydroxy-6-methyl-furo(3,4-c)pyridine which was readily obtained by a condensation reaction between pyridoxal and a secondary amine. 相似文献
998.
《Bioscience, biotechnology, and biochemistry》2013,77(10):2739-2749
The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. Here we describe an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes large-scale ligand screening with two libraries, proteins and chemicals, the use of cell lysates that express proteins of interest fused with red fluorescent protein, and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. Using our systematic platform, we detected novel inhibitors of carbonic anhydrase II. It is suggested that our systematic platform is a rapid and robust approach to screen novel ligands for human proteins of interest. 相似文献
999.
Jonathan W. Day Chan Hyuk Kim Vaughn V. Smider Peter G. Schultz 《Bioorganic & medicinal chemistry letters》2013,23(9):2598-2600
The bidentate metal binding amino acid bipyridylalanine (BpyAla) was incorporated into a disulfide linked cyclic peptide phage displayed library to identify metal ion binding peptides. Selection against Ni2+–nitrilotriacetic acid (NTA) enriched for sequences containing histidine and BpyAla. BpyAla predominated when selections were carried out at lower pH, consistent with the differential pKa’s of histidine and BpyAla. Two peptides containing BpyAla were synthesized and found to bind Ni2+ with low micromolar dissociation constants. Incorporation of BpyAla and other metal binding amino acids into peptide and protein libraries should enable the evolution of novel binding and catalytic activities. 相似文献
1000.