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51.
Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE) 总被引:5,自引:0,他引:5
Kügler Marion Jänsch Lothar Kruft Volker Schmitz Udo K. Braun Hans-Peter 《Photosynthesis research》1997,53(1):35-44
Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b
6
f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed. 相似文献
52.
53.
54.
Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight 总被引:7,自引:0,他引:7
Bohmert K Balbo I Kopka J Mittendorf V Nawrath C Poirier Y Tischendorf G Trethewey RN Willmitzer L 《Planta》2000,211(6):841-845
Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described.
These plants accumulated more than 4% of their fresh weight (≈40% of their dry weight) in the form of PHB in leaf chloroplasts.
These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of
a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis
in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating
the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe
effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing
lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that
the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount
of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and
sugar alcohols.
Received: 2 February 2000 / Accepted: 31 March 2000 相似文献
55.
56.
Zhongli Gao William J. Hurst Etienne Guillot Werngard Czechtizky Ulrike Lukasczyk Raisa Nagorny Marie-Pierre Pruniaux Lothar Schwink Juan Antonio Sanchez Siegfried Stengelin Lei Tang Irvin Winkler James A. Hendrix Pascal G. George 《Bioorganic & medicinal chemistry letters》2013,23(11):3416-3420
A series of structurally novel aryl ureas was derived from optimization of the HTS lead as selective histamine H3 receptor (H3R) antagonists. The SAR was explored and the data obtained set up the starting point and foundation for further optimization. The most potent tool compounds, as exemplified by compounds 2l, 5b, 5d, and 5e, displayed antagonism potencies in the subnanomolar range in in vitro human-H3R FLIPR assays and rhesus monkey H3R binding assays. 相似文献
57.
Anabaenopsis spp. are heterocytous cyanobacteria commonly found in tropical, subtropical, and temperate water bodies. So far, the knowledge
about the phylogeny of this genus is poor. Therefore, we have isolated 15 Anabaenopsis spp. strains from Kenyan and Mexican alkaline and saline water bodies and from a Ugandan freshwater body and studied the
morphology and phylogeny in a polyphasic approach. Morphologically, the investigated strains could be discriminated in two
groups. One group was containing six Anabaenopsis abijatae and A. cf. abijatae strains with up to more than 500 vegetative cells in one filament, mostly single intercalary heterocyte formation, and the
ability to branch out. The other group comprised nine strains of Anabaenopsis elenkinii with short filaments with up to 38 vegetative cells, intercalary heterocytes in pairs, and no ability to branch out. The
morphological differences were reflected in the two distinct clusters, which were found in the phylogenetic trees of 16S rDNA
and PC-IGS. While the high 16S rDNA similarity values >97.5% found between all investigated A. abijatae and A. elenkinii strains support the assignment of these two species to one single genus, the morphological differences and the low similarity
values (<87.3) found in PC-IGS sequences between the two clusters indicate two separate genera. A close morphological and
phylogenetic relationship was found for A. abijatae and Anabaenopsis (Cyanospira) rippkae. 相似文献
58.
Beutin L Miko A Krause G Pries K Haby S Steege K Albrecht N 《Applied and environmental microbiology》2007,73(15):4769-4775
We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans. 相似文献
59.
Scanlan CN Ritchie GE Baruah K Crispin M Harvey DJ Singer BB Lucka L Wormald MR Wentworth P Zitzmann N Rudd PM Burton DR Dwek RA 《Journal of molecular biology》2007,372(1):16-22
The HIV envelope has evolved a dense array of immunologically "self" carbohydrates that efficiently protect the virus from antibody recognition. Nonetheless, one broadly neutralising antibody, IgG1 2G12, has been shown to recognise a cluster of oligomannose glycans on the HIV-1 surface antigen gp120. Thus the self carbohydrates of HIV are now regarded as potential targets for viral neutralisation and vaccine design. Here, we show that chemical inhibition of mammalian glycoprotein synthesis, with the plant alkaloid kifunensine, creates multiple HIV (2G12) epitopes on the surface of previously non-antigenic self proteins and cells, including HIV gp120. This formally demonstrates the structural basis for self/non-self discrimination between viral and host glycans, by a neutralising antibody. Moreover, this study provides an alternative protein engineering approach to the design of a carbohydrate vaccine for HIV-1 by chemical synthesis. 相似文献
60.
Zoltán Jászberényi Loïck Moriggi Philipp Schmidt Claudia Weidensteiner Rainer Kneuer André E. Merbach Lothar Helm Éva Tóth 《Journal of biological inorganic chemistry》2007,12(3):406-420
Generation 4 polyamidoamine (PAMAM) and, for the first time, hyperbranched poly(ethylene imine) or polyglycerol dendrimers
have been loaded with Gd3+ chelates, and the macromolecular adducts have been studied in vitro and in vivo with regard to MRI contrast agent applications.
The Gd3+ chelator was either a tetraazatetracarboxylate DOTA-pBn4− or a tetraazatricarboxylate monoamide DO3A-MA3− unit. The water exchange rate was determined from a 17O NMR and 1H Nuclear Magnetic Relaxation Dispersion study for the corresponding monomer analogues [Gd(DO3A-AEM)(H2O)] and [Gd(DOTA-pBn-NH2)(H2O)]− (k
ex298 = 3.4 and 6.6 × 106 s−1, respectively), where H3DO3A-AEM is {4-[(2-acetylaminoethylcarbamoyl)methyl]-7,10-bis(carboxymethyl-1,4,7,10-tetraazacyclododec-1-yl)}-acetic acid
and H4DOTA-pBn-NH2 is 2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. For the macromolecular complexes, variable-field
proton relaxivities have been measured and analyzed in terms of local and global motional dynamics by using the Lipari–Szabo
approach. At frequencies below 100 MHz, the proton relaxivities are twice as high for the dendrimers loaded with the negatively
charged Gd(DOTA-pBn)− in comparison with the analogous molecule bearing the neutral Gd(DO3A-MA). We explained this difference by the different
rotational dynamics: the much slower motion of Gd(DOTA-pBn)−-loaded dendrimers is likely related to the negative charge of the chelate which creates more rigidity and increases the overall
size of the macromolecule compared with dendrimers loaded with the neutral Gd(DO3A-MA). Attachment of poly(ethylene glycol)
chains to the dendrimers does not influence relaxivity. Both hyperbranched structures were found to be as good scaffolds as
regular PAMAM dendrimers in terms of the proton relaxivity of the Gd3+ complexes. The in vivo MRI studies on tumor-bearing mice at 4.7 T proved that all dendrimeric complexes are suitable for
angiography and for the study of vasculature parameters like blood volume and permeability of tumor vessels.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献