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101.
Askoxylakis V Garcia-Boy R Rana S Krämer S Hebling U Mier W Altmann A Markert A Debus J Haberkorn U 《PloS one》2010,5(12):e15962
Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.
Methods
Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.Results
In vitro binding experiments of 125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.Conclusions
These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX. 相似文献102.
Osiak A Radecke F Guhl E Radecke S Dannemann N Lütge F Glage S Rudolph C Cantz T Schwarz K Heilbronn R Cathomen T 《PloS one》2011,6(12):e28911
Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. 相似文献
103.
Industrial enzyme applications 总被引:12,自引:0,他引:12
The effective catalytic properties of enzymes have already promoted their introduction into several industrial products and processes. Recent developments in biotechnology, particularly in areas such as protein engineering and directed evolution, have provided important tools for the efficient development of new enzymes. This has resulted in the development of enzymes with improved properties for established technical applications and in the production of new enzymes tailor-made for entirely new areas of application where enzymes have not previously been used. 相似文献
104.
Eric Guenzi Anne-Marie Gasc Michel A. Sicard Regine Hakenbeck 《Molecular microbiology》1994,12(3):505-515
Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for β-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this β-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, claR and claH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase ClaH into two putative domains: an N-terminal extracelluiar sensor part, and an intracelluiar C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of ClaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated claH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation. 相似文献
105.
Outcrossing and hybridization in wild and cultivated foxtail millets: consequences for the release of transgenic crops 总被引:3,自引:0,他引:3
I. Till-Bottraud X. Reboud P. Brabant M. Lefranc B. Rherissi F. Vedel H. Darmency 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1992,83(8):940-946
Summary Outcrossing rates within the wild green foxtail, Setaria viridis, and the cultivated foxtail millet, S. italica, are very low. However, spontaneous interspecific hybridizations in the experimental garden occurred in both directions at rates ranging from 0.002% to 0.6% according to plant density and distance between parents. Offtypes found in farmers' fields where foxtail millet is cultivated were shown to have originated from such interspecific crosses. Differences in the EcoR1 patterns of chloroplast DNA between cultivated and wild plants indicated that reciprocal crosses do occur in the field. These findings indicate that even a largely selfing cultivated species may exchange genetic information with wild relatives at rates that may cause problems if transgenic cultivars are released. 相似文献
106.
Ohne Zusammenfassung 相似文献
107.
108.
The explosive pollination mechanism of the prayer plants (Marantaceae) is unique among plants. After a tactile stimulus by a pollinator, the style curls up rapidly and mediates pollen exchange. It is still under discussion whether this explosive movement is released electrophysiologically, i.e. by a change in the membrane potential (as in Venus flytrap), or purely mechanically. In the present study, electrophysiological experiments are conducted to clarify the mechanism. Artificial release experiments (chemical and electrical) and electrophysiological measurements were conducted with two phylogenetically distant species, Goeppertia bachemiana (E. Morren) Borchs. & S. Suárez and Donax canniformis (G. Forst.) K. Schum. Electric responses recorded after style release by extracellular measurements are characterised as variation potentials due to their long repolarization phase and lack of self-perpetuation. In both species, chemical and electric stimulations do not release the style movement. It is concluded that the style movement in Marantaceae is released mechanically by relieving the tissue pressure. Accordingly, the variation potential is an effect of the movement and not its cause. The study exemplarily shows that fast movements in plants are not necessarily initiated by electric changes of the membrane as known from the Venus flytrap. 相似文献
109.
H. Baatout M. Marrakchi C. Mathieu F. Vedel 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1985,70(6):577-584
Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian
populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation
was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the
two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes. 相似文献
110.
Marie-Christine Montel Regine Talon Monique Cantonnet Jeanne Fournaud 《Letters in applied microbiology》1992,15(2):73-77
Fifty-one strains of staphylococci isolated from French dry sausages were mainly identified with Staphylococcus carnosus, S. xylosus, S. warneri and S. saprophyticus. The API Staphylococcus identification system proved to be reliable for S. xylosus and S. carnosus. The identification of S. warneri and S. saprophyticus was performed by DNA-DNA hybridization. These species are better identified by taking into account not only the API Staphylococcus system but also the following characters: novobiocin and lysozyme susceptibilities, production of D-lactate. hydrolysis of tri-olein. 相似文献