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11.
By application of immunocytochemical techniques at the electron microscope level, glucoamylase was localized to the cell periphery in Clostridium thermosaccharolyticum during and following growth on starch, sucrose or glucose. Levels of immunolabelling were found to be relatively independent of growth substrate and of phase of growth, whereas previous studies had demonstrated strong dependence of glucoamylase activity on growth conditions; previously high levels of glucoamylase activity had been detected after growth on starch (i.e. during the stationary phase after growth) and only very low activities detected during exponential growth and following growth on glucose. The results presented demonstrate that levels of the glucoamylase protein are independent of measurable enzyme activity, and imply that the protein is constitutive. This indicates that the protein can exist in active and inactive states in the cell. By analogy with similar systems, we consider it likely that maturation or activation of newly synthesized glucoamylase occurs during (or following) transport through the cytoplasmic membrane. Electron microscopy of individual protein molecules which had been subjected to negative staining revealed that the enzyme consists of two domains of approximately equal size which are linked by a hinge region. 相似文献
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Tanja Albrecht Sophie Haebel Anke Koch Ulrike Krause Nora Eckermann Martin Steup 《European journal of biochemistry》2004,271(20):3978-3989
Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis. 相似文献
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16.
The Penicillium frequentans strain Bi 7/2, using phenol as a sole source of carbon and energy,transformed the fluorinated
phenols 2,3-, 2,4-, 2,5-and 3,4-difluorophenol rapidly. After growth on phenol, resting mycelia of the fungus converted the
difluorophenols completely at an initial concentration of 0.5 mM within 6 hours. The corresponding difluorinated catechols
were found to be intermediates of all difluorophenols investigated. A relatively unspecific phenol hydroxylase catalyzed this
hydroxylation step and showed activities towards all difluorophenols tested. One difluorocatechol was formed from each difluorophenol
substituted with fluorine in the ortho-position, whereas two catechols were formed from 3,4-difluorophenol, due to its two
vacant ortho-positions. A partial defluorination (50-77%) was observed in all cases.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
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18.
Characteristics of single chemoreceptive units sensitive to amino acids and related substances in the crayfish leg 总被引:2,自引:0,他引:2
Ulrike Bauer J. Dudel H. Hatt 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1981,142(1):67-74
Summary Impulses in single afferent fibres from amino acid receptors were recorded extracellularly. Doseresponse relations were determined for different superfused amino acids; the relations all had a slope of 1, a common saturation level, and the action of different amino acids was characterized by a specific half saturation concentration,K
M. The most effective amino acids were always L-serine, L-alanine and L-histidine, having aK
M of 10–5, 2·10–5 and 1.5·10–4 mol/l, respectively. The sequence of effective amino acids was the same for all units tested. Structural requirements for optimal stimulatory action of the amino acid molecules were concluded.Abbreviation
vH
van Harreveld solution
This work was supported by the Deutsche ForschungsgemeinschaftWe gratefully acknowledge assistance in electronics from Mr. W. Zeitz, and in mechanics from Mr. D. Beyer and Mr. L. Müller. Technical help was provided by Mrs. E. Köster, secretarial help by Mrs. L. Bauer. 相似文献
19.
In-vitro binding of labeled auxins to sedimentable particles was tested in subcellular fractions from homogenates of maize (Zea mays L.) coleoptiles. The material was fractionated by differential centrifugation or on sucrose density gradients. It was confirmed that the major saturable binding activity (site I) for 1-naphthyl[1-14C]acetic acid is associated with vesicles derived from the endoplasmatic reticulum. A second type of specific auxin binding (site II) could be distinguished by several criteria, e.g. by the low affinity towards phenylacetic acid. The particles carrying site II could be clearly separated from markers of the endoplasmatic reticulum, the plasmalemma, the mitochondria and the nuclei, while their density as well as sedimentation velocity correlated with particle-bound acid phosphatase, indicating a localization at the tonoplast. In contrast to site I, binding at site II was hardly affected by a supernatant factor and by sulfhydryl groups. However, the specificity pattern of site II towards auxins and auxin analogs was very similar to that of site I tested in the presence of supernatant factor. The existence of a third auxin receptor localized in plasma membrane-rich gradient fractions was indicated by a preferential in-vitro binding of 2,4-dichlorophenoxyacetic acid.Abbreviations 1-NAA
1-naphthyl acetic acid
- 2-NAA
2-naphthyl acetic acid
- IAA
3-indolyl acetic acid
- PAA
phenyl acetic acid
- 2,4-D
2,4-D-dichlorophenoxy acetic acid
- D-2,4-DP
dichlorophenoxy isopropionic acid
- NPA
1-N-naphthyl phthalamic acid
- ER
endoplasmatic reticulum
- SF
supernatant factor 相似文献
20.
Ulrike Nolte 《Hydrobiologia》1991,222(3):197-211
Chironomid communities of mosses in a small upland stream in central Germany were highly dynamic across the year with respect to their abundance, biomass and dominant taxa. During 1988 semi-submersed mosses near a main spring and those occurring some 700 m downstream were compared with permanently submersed mosses in immediate vicinity of the downstream site. All the chironomids sampled were conspicuously small, with nearly 98% being less than 5 mm in length. A total of 65 chironomid species from 26 genera were found, with a higher diversity occurring near the source and a change in dominant taxa along the upper stream section. The mean abundance in permanently submersed mosses (250 larvae/10 cm2, n = 125) was about five times higher than in semi-submersed mosses. The maximum value of 830 larvae/10 cm2 (n = 1) is the highest chironomid density ever reported, which is explained by the sampling method used. The mean standing crop was also highest in permanently submersed mosses (1.5 mg AFDW/10 cm2 (n = 125)), even though the highest individual value was recorded in semi-submersed mosses near the spring (10.4 mg AFDW/10 cm2). The evidence suggested that the dominance of chironomid taxa depended mainly on the location of the moss along the stream, whereas abundance and biomass were determined mainly by constancy in the ambient discharge as well as the factors influenced by this (e.g. temperature, detritus deposition). A trend was seen towards a seasonal succession among the chironomid taxa colonizing lotic mosses. 相似文献