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101.
Preparation and structural characterization of large heparin-derived oligosaccharides 总被引:4,自引:2,他引:2
Pervin Azra; Gallo Cindy; Jandik Kenneth A; Han Xue-Jun; Linhardt Robert J. 《Glycobiology》1995,5(1):83-95
Porcine mucosal heparin was partially depolymerized with heparinlyase I and then fractionated into low-molecularweight (<5000)and high-molecular-weight (>5000) oligosaccharides by pressurefiltration. The high-molecular-weight oligosaccharide mixture({small tilde}50 wt% of the starting heparin) also containedintact heparin. This intact polymer complicates oligosacsharidepurification. Thus, the low-molecular-weight fraction was usedto prepare homogeneous oligosaccharides for structural characterization.The low-molecular-weight oligosaccharide mixture was first fractionatedby low pressure gel permeation chromatography into size-uniformmixtures of disaccharides, tetrasaccharides, hexasaccharides,octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharidesand higher oligosaccharides. Each size-fractionated mixturewas then purified on the basis of charge by repetitive semi-preparativestrong-anion-exchange high-performance liquid chromatography.This approach has led to the isolation of 14 homogeneous oligosaccharidesfrom disaccharide to tetradecasaccharide. The purity of theseheparin-derived oligosaccharides was determined by gradientpolyacrylamide gel electrophoresis, analytical strong-anion-exchangehigh-performance liquid chromatography, capillary electrophoresisand one-dimensional nuclear resonance spectroscopy. The structureof these oligosaccharides was established using 600 MHz two-dimensionalnuclear resonance spectroscopy . The spectral methods used includedhomonuclear correlation spectroscopy, nuclear Overhauser effectspectroscopy and heteronuclear multiple quantum coherence spech-clscopy.The 1H/1H connectivities of the protons of each sugar residuein an oligosaccharide were established by two-dimensional homonuclearcorrelation spectroscopy, while 1H/13C assignments were madeusing 1H inverse detection. One- and two-dimensional nuclearresonance spectroscopic analysis of these heparin oligosaccharidesshowed two closely related groups of heparin-oligosaccharidesare afforded by enzymatic depolymerization of heparin. One groupis fully sulphated, having the structures 相似文献
102.
A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters 总被引:20,自引:0,他引:20
Scrapie hamster brains contain at least 5–10 g of scrapie-associated fibrils (SAF) per brain as estimated by the amount of its major constituent, a protein of about 26 000 daltons (SAF-protein). It can be extracted efficiently by a 10% solution of sarkosyl and can be enriched by differentia] centrifugation and buffer extraction. Scrapie infectivity, SAF, and SAF-protein copurify. 相似文献
103.
Norbert Madry Rainer Zocher Karola Grodzki Horst Kleinkauf 《Applied microbiology and biotechnology》1984,20(2):83-86
Summary The multifunctional enzyme enniatin synthetase was immobilized by adsorption to propyl agarose. The immobilized multienzyme retained 45% of the activity of the free enzyme; an operational half-life of about 15 h was estimated. Selective synthesis of several different enniatin homologues was achieved with propyl agarose-bound enniatin synthetase. In addition to enniatin A, B, and C formation, a selective synthesis of non-naturally occurring depsipeptides, containing norvaline, norleucine, or -aminobutyric acid as sole amino acid moieties, was observed. 相似文献
104.
105.
Cindy Lloyd John R. Kennedy Joseph Mendicino 《In vitro cellular & developmental biology. Plant》1984,20(5):416-432
Summary Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability
of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the
active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin
glycoprotein was about 0.035 mg per cm2 per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with
radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal
incorporation was observed at 200 μM glucosamine. A higher concentration of35SO4, 1000 μM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 μg/ml increased the rate of secretion twofold,
whereas 0.1 to 100 μg/ml of hydrocortisone and 0.1 to 100 μg/ml of epinephrine significantly decreased the rate of secretion.
Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10−5
M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10−9
M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats.
Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified.
The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified
by chromatography on Sepharose CL-6B columns under dissociating conditions in 2M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually
indistingushable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified
glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More
than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same
molar ratio ofN-acetylgalactosamine,N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations.
This investigation was supported by U.S. Public Health Service Grants HL 20868, HL 24688, and HL 24718 from the National Heart,
Lung and Blood Institute, Bethesda, MD, and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and
Kidney Diseases, Bethesda, MD. 相似文献
106.
Ronald S. Oremland Cindy Umberger Charles W. Culbertson Richard L. Smith 《Applied microbiology》1984,47(5):1106-1112
The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO3− + NO2− concentrations (≤26 μM) were below the apparent Km (50 μM) for nitrate. During the rainy season, when ambient NO3− + NO2− concentrations were higher (37 to 89 μM), an accurate estimate of the Km could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N2O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N2O in the presence of C2H2 was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N2O loss was caused by incomplete blockage of N2O reductase by C2H2 at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 μmol of N2 m−2 h−1 (for undisturbed sediments) to 17 to 280 μmol of N2 m−2 h−1 (for shaken sediment slurries). 相似文献
107.
G E Lester R L Horst J L Napoli 《Biochemical and biophysical research communications》1984,120(3):919-925
The results of normal mode calculations on the beta 4.4, beta 6.3, beta 5.6, and beta 7.2 structures of gramicidin A are compared with infrared and Raman spectra of crystalline native, crystalline Cs+-bound, and vesicle-bound gramicidin A. The observed frequencies and frequency splittings are in good agreement with an assignment of beta 5.6, beta 7.2, and beta 6.3 structures, respectively, to the gramicidin A molecules in the above three systems. 相似文献
108.
The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
109.
Horst Blüthmann 《Molecular biology reports》1978,4(2):97-100
Stimulation of bovine lymphocytes with phytohemagglutinin results in quantitative as well as qualitative changes in the nonhistone chromosomal proteins. Analysis of these proteins by hydroxyapatite chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis shows not only a selective increase in the amount of some nonhistone proteins but also a decrease of other nonhistone protein bands. This observation is compatible with the view that nonhistone proteins have an inhibitory as well as an activating function at the genome level. 相似文献
110.
Summary Amino acid transport and incorporation have been studied in vitro in rat pancreatic lobules after maximal and supramaximal hormonal stimulation with caerulein. Incorporation into proteins was increased already after 30 and 120 min of maximal stimulation, but was decreased after the infusion of a supramaximal dose. Uptake of neutral amino acids was monitored using labeled leucine and -aminoisobutyric acid (AIB). In the case of leucine the free pool was consistently reduced after maximal stimulation, while supramaximal doses led to an increase which could be potentiated by the addition of 2mM tetracaine. Using AIB, a significant increase in the intracellular pool was observed after maximal stimulation, conversely a decrease after supramaximal stimulation. Release of labeled leucine and AIB from preloaded lobules during incubation in the cold was significantly reduced after maximal secretory stimulation, but was found enhanced by 200 to 300 percent after supramaximal stimulation. No fine structural alterations at junctional complexes or at both the lateral and luminal plasma membranes were observed after maximal stimulation except an increased number of exocytotic figures at the luminal face. However, supramaximal stimulation led to progressive rarefaction of the tight junctional network and disintegration of the gap junctions. Concomitantly, an equal distribution of membrane particles on both faces of the plasma membrane together with a random occurrence of exocytotic figures were observed.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (SFB 122, project C 5). Dedicated to Professor Dr. Gerhard Petry, Marburg, on the occasion of his 65th birthday 相似文献