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31.
Stefan Highsmith Mark Kubinec Devendra K. Jaiswal Hiromi Morimoto Philip G. Williams David E. Wemmer 《Journal of biomolecular NMR》1993,3(3):325-334
Summary The synthesis of [2-3H]ATP with specific activity high enough to use for 3H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single 3H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg2+, S1 converts [2-3H]ATP to [2-3H]ADP and the complex S1.Mg[2-3H]ADP has ADP bound in the active site. At 0°C, 1D 3H NMR spectra of S1.Mg[2-3H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-3H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-3H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (Vi) to produce S1.Mg[2-3H]ADP.Vi shifts both peaks slightly more upfield without chaning their widths or relative areas. 相似文献
32.
Toshiro Matsui Miho Imamura Hiromi Oka Katsuhiro Osajima Ko-Ichi Kimoto Terukazu Kawasaki Kiyoshi Matsumoto 《Journal of peptide science》2004,10(9):535-545
The distribution of an antihypertensive dipeptide, Val-Tyr (VY), in the tissues of spontaneously hypertensive rats (SHR) was investigated in this study. A single oral administration of VY (10 mg/kg) to 18-week-old SHR resulted in a prolonged reduction of systolic blood pressure (SBP) up to 9 h (SBP0h 198.0+/-3.6 mmHg; SBP9h 154.6+/-3.5 mmHg). As a result of VY determination, a roughly 10-fold higher increment of plasma VY level was observed at 1 h than that at 0 h, whereas thereafter the level declined rapidly. In tissues, VY was widely accumulated in the kidney, lung, heart, mesenteric artery and abdominal aorta with the area under the curve over 9 h of more than 40 pmol h/g tissue; of these a higher VY level was observed in the kidney and lung. In addition, a mean resident time (MRT) for each tissue (>5 h except for liver) revealed that VY preferably accumulated in the tissues rather than in the plasma (MRT 3.8 h). Significant reductions of tissue angiotensin I-converting enzyme activity and angiotensin II level were found in the abdominal aorta as well as in the kidney, suggesting that these organs could be a target site associated with the antihypertensive action of VY. 相似文献
33.
The localization of the antigen for monoclonal antibody 9F11-B-E4 was clarified by immuno-electron microscopy. The antigens were localized on the mitochondria and Golgi bodies in the male germ cells and on the secretory granules of various glands cells in the penis bulb and subepidermal parenchymal tissue of Phagocata vivida. The results of the interspecific cross-reaction tests with seven other freshwater triclads showed that these secretory granules are species-specific. A positive interspecific reaction was showed with Dugesia (family Dugesiidae), but not with Polycelis within the same family Planariidae which suggests the position of Phagocata within the Planariidae needs to be reassesed. 相似文献
34.
The universal genetic code is determined by the aminoacylation of tRNAs. In spite of the universality of the code, there are barriers to aminoacylation across taxonomic domains. These barriers are thought to correlate with the co-segregation of sequences of synthetases and tRNAs into distinct taxonomic domains. By contrast, we show here examples of eukaryote-like synthetases that are found in certain prokaryotes. The associated tRNAs have retained their prokaryote-like character in each instance. Thus, co-segregation of domain-specific synthetases and tRNAs does not always occur. Instead, synthetases make adaptations of tRNA-protein contacts to cross taxonomic domains. 相似文献
35.
Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica. 相似文献
36.
The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor 总被引:1,自引:0,他引:1
The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule. 相似文献
37.
The cellular site and characteristics of the phosphorylation of a nucleolus-specific phosphoprotein (molecular weight, 120 000) in mouse ascites tumor cells were studied. The phosphoprotein was strongly labeled with 32P when the isolated nucleoli were incubated with [γ-32P]ATP in vitro. This phosphoprotein, and protein kinase for the protein phosphorylation were both purified from 0.3 M KCl soluble protein fraction of the nucleoli by hydroxylapatite and phosphocellulose column chromatographies. It was found that phosphorylation of the nucleolus-specific phosphoprotein was catalyzed selectively by a guanosine 3:5-monophosphate-dependent protein kinase in the nucleoli and the reaction product was the same phosphoprotein as the substrate used. 相似文献
38.
An anticomplementary agent, K-76 monocarboxylic acid: its site and mechanism of inhibition of the complement activation cascade. 总被引:11,自引:0,他引:11
K Hong T Kinoshita W Miyazaki T Izawa K Inoue 《Journal of immunology (Baltimore, Md. : 1950)》1979,122(6):2418-2423
A monocarboxylic acid derivative (K-76 COOH) of K-76, purified from the culture filtrate of Stachybotrys complement I nov. sp. K-76, inhibits complement (C) activity. Its inhibitory action is mainly on C5 step. It strongly inhibits the generation of EAC1,4b,2a,3b,5b from C5 and EAC1,4b,2a,3b, and accelerates the decay of EAC1,4b,2a,3b,5b. It also causes some inhibition of the reactions of the reactions of C2,C3,C6,C7 and C9 with their respective preceding intermediate cells. It has no effect on the generation of EAC1,4b from C4 and EAC1, or of EAC-8 from C8 and EAC-7, and apparently increases the generation of EAC1,4b from C1 and EAC4b probably by inhibiting transfer or turnover of C1. It does not affect the rate of decay of EAC1,4b,2a or the T max of generation of EAC1,4b,2a, and it inhibits immune adherence only at high concentration. K-76 COOH also strongly inhibits hemolysis through the alternative pathway of C activation by cobra venom factor, but it does not seem to inhibit the early steps of the alternative pathway, because it has little affect on the consumption of C3 or the conversion of beta 1C to beta 1A on treatment of C serum with zymosan. K-76 COOH probably combines with C5 molecules, forming the inactive complexes, or it causes the structural alteration of C5. 相似文献
39.
The characteristics of root respiration of melon were examinedwith an oxygen electrode. The Hofstee plot of root respirationbreaks into two straight lines. The results of cyanide inhibitionexperiments and curve-fitting analysis suggest that one cyanide-insensitiveand two cyanide-sensitive oxidases operate in melon roots. (Received December 24, 1976; ) 相似文献
40.
It was found that an increase in fluorescence intensity at 340 nm is observed on the binding of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' in the pH range 6--10. The dissociation constant, Ki, of the enzyme-inhibitor complex was determined as a function of pH and temperature by direct fluorometric titration utilizing the single photon counting technique in the protein concentration range of 10(-9) M. Ki values as low as 10(-10) M could be obtained with reasonable accuracy by this high-sensitivity detection method. From the temperature dependence of Ki, it was found that the binding is endothermic, and is entirely "entropy-driven" in nature. The effect of pH on Ki suggested the participation of an ionizable group with pKapp = 8.5 in the binding. 相似文献