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41.
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Giovanna Cacciapuoti Maria Angela Moretti Sabrina Forte Assunta Brio Laura Camardella Vincenzo Zappia Marina Porcelli 《European journal of biochemistry》2004,271(23-24):4834-4844
The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins. 相似文献
43.
Alexander Christmann Jacqueline Christmann Petra Schiller Burkhard Frenzel 《Trees - Structure and Function》1996,10(5):331-338
Levels of indole-3-acetic acid (IAA) were determined in needles from silver fir (Abies alba Mill.) trees in the northern Black Forest. IAA was quantified by gas chromatography (GC) as 1-heptafluorobutyryl-IAA-methylester
(HFB-IAA-ME) using electron capture detection. Prior to GC analysis, extensive purification of needle extracts was performed
employing two HPLC steps. Peak identity of HFB-IAA-ME was confirmed by combined gas chromatography-mass spectrometry in selected
samples. Levels of IAA in needles belonging to different needle age-classes exhibited a cyclic seasonal pattern with highest
concentrations in winter and lowest levels in spring when bud-break occurred. Such a cyclic seasonal pattern of IAA levels
was also observed in needles from declining fir trees or fir trees suffering from a strong sulfur impact (S-impact) in the
field due to a local SO2 source. Levels of IAA increased with increasing needle age. This age dependency of IAA concentrations was most pronounced
in late autumn when IAA levels were high and nearly disappeared in spring when IAA levels reached their minimum. In needles
from declining fir trees or fir trees suffering from a strong S-impact in the field, IAA levels hardly increased with increasing
needle age. It is suggested that in healthy trees high levels of IAA protect older needles from abscission and that the considerable
losses of older needles of declining fir trees or of fir trees under S-impact are a consequence of the low levels of IAA found
in older needles of such trees.
Received: 4 May 1995 / Accepted: 29 August 1995 相似文献
44.
Petra R. Moog Tom A. W. van der Kooij Wolfgang Brüggemann John W. Schiefelbein Pieter J. C. Kuiper 《Planta》1995,195(4):505-513
Arabidopsis thaliana (L.) Heynh. Columbia wild type and a root hair-less mutant RM57 were grown on iron-containing and iron-deficient nutrient solutions. In both genotypes, ferric chelate reductase (FCR) of intact roots was induced upon iron deficiency and followed a Michaelis-Menten kinetic with a K
m of 45 and 54 M FeIII-EDTA and a V
max of 42 and 33 nmol Fe2+·(g FW)–1·min–1 for the wild type and the mutant, respectively. The pH optimum for the reaction was around pH 5.5. The approximately four fold stimulation of FCR activity was independent of formation of root hairs and/or transfer cells induced by iron deficiency. Iron-deficiency-induced chlorosis and the development of a rigid root habit disappeared when ferric chelate was applied to the leaves, while FCR activity remained unchanged. The time course of the responses to iron deficiency showed that morphological and physiological responses were controlled separately.Abbreviations FCR
ferric chelate reductase
- FW
fresh weight
Thanks are due to Klaas Sjollema (Department of Electronmicroscopy, University of Groningen, The Netherlands) for help with the electron microscopy sample preparation and especially to Dr. Uwe Santore (Heinrich-Heine-University for electron microscopy. This work was supported by the SCIENCE programm of the European community; P.R.M.) and a Personal Research Grant by the Ministerium für Wissenschaft und Forschung of Nordrhein-Westfalen (P.R.M.) and last, not least by the productive discussions in ECOTRANS B.V. 相似文献
45.
Yasmeen Hashim Ioannis Ragoussis Lyndal Kearney Sabrina Tosi Alex K. So 《Immunogenetics》1995,41(6):337-342
Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human T-cell chain gene complex (TCRB). Variable region genes (BV) for the 25 known subfamilies were used as probes to screen the ICRF AM4x YAC library. Of the five positive YACs identified, one YAC designated B3, 820 kilobase pairs (kbp) in size, scored positive for all 25 TCRBV subfamilies plus the constant region genes (BC) when analyzed by pulse field gel electrophoresis. Restriction enzyme mapping of B3 located TCRBV and TCRBC gene regions to 4 Sfi I fragments of 280 110, 90, and 125 kbp and was in accordance with published data. In addition comparison of hybridization results of Sfi I-restricted B3 and genomic DNA from the parental cell line GM1416B revealed identical banding patterns. The data thus showed YAC B3 encoded a complete and unrearranged TCRB gene locus of some 600–620 kbp. The map was further resolved by locating restriction sites for Sal I and Bss HII on B3, giving more precise localization of the individual TCRBV gene families. Flourescent in situ hybridization of B3 to spreads of human metaphase chromosomes localized B3 to 7q35. However, two additional signals were obtained; one attributable to the TCRBV orphon cluster on 9p21, the second to the long arm of chromosome 2. Polymerase chain reaction amplification of a chromosome 2 somatic cell hybrid, using primers for all 25 TCRBV gene families, revealed that the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCRB locus flanked by chromosome 2 sequences. 相似文献
46.
Petra Düx Brian Whitehead Rolf Boelens Robert Kaptein Geerten W. Vuister 《Journal of biomolecular NMR》1997,10(3):301-306
A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 1173J(NH) coupling constants. In addition, 293J(NH(i–1)) coupling constantscould be measured, providing information about the backbone angle .These data, in conjunction with the magnitudes of the3J(HNH) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theH resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate H resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the H resonances for these residues. In addition,reliable and precise , dihedral constraints were also derived forthese residues from the J-coupling data. 相似文献
47.
48.
49.
Eight compounds exuded from young roots of black locust (Robinia pseudoacacia) were separated by two-dimensional HPTLC, by HPLC and GC, and were identified by spectroscopic methods (ultraviolet/visible
spectroscopy and mass spectrometry) as 4′,7-dihydroxyflavone, apigenin, naringenin, chrysoeriol and isoliquiritigenin. Structural
assignments were confirmed by comparison with authentic standards. The capacity to induce β-galactosidase activity in Rhizobium sp. NGR234 containing a nod box::lacZ fusion on plasmid pA27 identified these flavonoids and the chalcone as nod gene inducers. This indicates the important role of these compounds in nodulation of this legume tree.
Received: 26 July 1996 / Accepted: 9 September 1996 相似文献
50.