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51.
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Tuo Li Annika J. E. Borg Leo Krammer Rolf Breinbauer Bernd Nidetzky 《Biotechnology and bioengineering》2023,120(6):1506-1520
Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4–0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates. 相似文献
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Mechanisms of the reactions of representative dipeptides (Gly2, Gly-Ala), oligopeptides (Gly3, Gly4) and the polypeptide (poly-Gly)n) in solution and clay suspensions at 85 degrees C were investigated. The reaction products and their yields were analysed and determined by means of HPLC. Interestingly, hydrolysis, where water molecules act as the reactant, was not the main reaction, even for oligopeptides. Formation of cyclic dipeptides prevailed in the reactions of dimers as well as oligopeptides. The breakdown of oligopeptide molecules proceeded via an intramolecular cyclization reaction. For example, the reaction of Gly3 led to the formation of equal amounts of cyclic dipeptide, c(Gly)2 and Gly. The presence of clay (montmorillonite) significantly increased yields in the reactions of dipeptides but it did not have much effect on the reactions of oligopeptides. However, an opposite effect of clay, protection of poly(Gly)n against decomposition, was proven. 相似文献
56.
Richard Griessler Alexandra Schwarz Jan Mucha Bernd Nidetzky 《European journal of biochemistry》2003,270(10):2126-2136
Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate. 相似文献
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Lone Baunsgaard Kees Venema Kristian B. Axelsen José Manuel Villalba Annikki Welling Bernd Wollenweber Michael G. Palmgren 《The Plant journal : for cell and molecular biology》1996,10(3):451-458
Transport across the plasma membrane is driven by an electrochemical gradient of H+ ions generated by the plasma membrane proton pump (H+ -ATPase). Random mutants of Arabidopsis H+ -ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H+ -ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H+ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H+ extrusion and in their ability to sustain an electrochemical H+ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H+ -ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H+ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters. 相似文献
59.
Isolated lungs from male Wistar rats (250–350 g) were perfused at a constant flow rate (10 ml/min, non -recirculating) with Krebs-Ringerbicarbonate buffer containing 4.5 % bovine serum albumin, and were ventilated at a positive pressure (60 breaths/min). Pulmonary arterial pressure and lung weight (as a measure of edema formation) were recorded continuously. After an equilibration period of 20 minutes the various test compounds were added to the perfusion fluid and experimental recording was continued for another 60 minutes.The effects of the stable PGI2-mimetic, iloprost, of PGE1, and of the biologically active PGE1-metabolite, 13,14-dihydro-PGE,, were evaluated in this model (n=6). Iloprost showed slight, but not significant vasodilation; however, lung weight remained unchanged. PGE1 and 13,14-dihydro-PGE1 also caused slight vasodilation, but in contrast to iloprost these compounds induced distinct pulmonary edema. The lung weight gain was discernible at concentrations of 2.8 × 10-6 mol/1 (significant at 2.8 × 10-5 mol/l; p 0.05) and was accompanied by increases in the wet-weight to dry-weight ratios. These findings were duplicated in a second set of experiments (n = 6) from which the same results were obtained.The results indicate that at high concentrations PGE, (and 13,14-dihydro-PGE1), but not iloprost, can induce pulmonary edema in rats probably by increasing the permeability of the pulmonary vasculature. 相似文献
60.
The lateral leaflets of Desmodium motorium exhibit rhythmic upward and downward movements with a period in the minute range. Apoplasmic K+ and H+ activities were monitored in situ in the abaxial part of the pulvini with ion-selective microelectrodes. An extracellular electric potential was recorded simultaneously. The apoplasmic H+ activity of all pulvini exhibiting a regular rhythm of the extracellular electric potential oscillated with the same period between about 10 and 20 mM. The apoplasmic K+ activity was high when the membrane potential of the motor cells was depolarized (about 36 mV) and the cells were shrunken. In contrast, the apoplasmic K+ activity was low in the swollen state of the motor cells, when the membrane potential was hyperpolarized (about -136 mV). The volatile anesthetic enflurane suppressed reversibly the movement of the leaflets. The same treatment also arrested spontaneous oscillations in the apoplasmic K+ activity in the pulvinus. The apoplasmic K+ activity oscillated roughly in phase with the K+ activity between pH 6.6 and 6.0. Application of white light disturbed the rhythm and increased the extracellular pH. Our results indicate that the physiological mechanism that drives the lateral leaflet movements of Desmodium motorium is closely related to the osmotic motors mediating the leaf movements of Mimosa, Samanea and Phaseolus.Abbreviations Em
membrane potential
- Eex
extracellular electric potential
- Hex
extracellular H+ activity
- Kex
extracellular K+ activity
- Rex
extracellular electrical resistance
B. Antkowiak was supported by the Stiftung Volkswagenwerk. 相似文献