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141.
Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase. 总被引:3,自引:1,他引:2 下载免费PDF全文
G. C. Ferreira P. J. Neame H. A. Dailey 《Protein science : a publication of the Protein Society》1993,2(11):1959-1965
5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer. The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5'-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein. 相似文献
142.
R. Ashton Lavoie Jeffrey T. Zugates Andrew T. Cheeseman Matt A. Teten Srivatsan Ramesh Julia M. Freeman Summer Swango Jeremy Fitzpatrick Amod Joshi Bradley Hollers Zufan Debebe Tyler K. Lindgren Amber N. Kozak Vinay K. Kondeti Mary K. Bright Eric J. Yearley Alexander Tracy Jacob A. Irwin Michael Guerrero 《Biotechnology and bioengineering》2023,120(10):2953-2968
Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%. 相似文献
143.
Bruno Mhul Michle Aubery Hans-Georg Mannherz Patrice Codogno 《Journal of cellular biochemistry》1993,52(3):266-274
The myoblast cell surface activity of ecto-5′-nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto-5′-nucleotidase. This increase was related to a higher expression of ecto-5′-nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E′1 and E8 modified the AMPase activity of the ecto-5′-nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E8, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto-5′-nucleotidase. By contrast, the large fragment derived from the short arms, designated E′1, inhibited the AMPase activity. Furthermore, the monoclonal anti-ecto-5′-nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E8 on the AMPase activity of ecto-5′-nucleotidase but did not reverse the inhibitory effect of fragment E′1. In conclusion, laminin stimulates the AMPase activity of ecto-5′-nucleotidase by two mechanisms: inducing the expression of ecto-5′-nucleotidase to the cell surface and direct modulation of the enzymatic activity. 相似文献
144.
To study the roles of m5C in the differentiation of rice calli derived from protoplasts (protoclones), the m5C level of the total DNA was analyzed using the32P post-labeling method. The level of m5C in regenerable and nonregenerable protoclones was similar, as was in calli and leaves of plants regenerated from the same
protoclones. Treatment with 0.5 mM 5-azacytidine caused significant reduction of the level of m5C and of the regeneration frequency of callus. Significantly increased m5C levels were observed during prolonged culture. 相似文献
145.
新生大鼠脊髓切片运动神经元的电生理参数测定 总被引:1,自引:0,他引:1
汪萌芽 《中国应用生理学杂志》1993,9(2):164-167
用微电极技术对新生大鼠脊髓横切薄片运动神经元(MN)进行细胞内记录,测得静息电位为-62±4mV(n=26),膜电阻为67±31MΩ,时间常数3.8±1.6ms,动作电位幅度68±7mV(n=26),阈电位-50±8mV,超射值6±4mV。灌流谷氨酸(1~30mmol/L)诱导伴膜电阻降低的缓慢去极化反应,5-羟色胺(50μmol/L)介导伴膜电导降低的电压依赖性内向电流。结果表明新生大鼠脊髓切片MN的细胞内生物电记录是一种稳定可靠的电生理学和药理学研究方法。 相似文献
146.
Kojic acid (KA), produced mainly by Aspergillus species, is a product of fungal secondary metabolism and has great potential in biotechnological applications. The use of KA has steadily increased, chiefly in the pharmaceutical industry, where KA is used for skin lightning. The market for KA has grown considerably in recent years and is expected to reach $39 million by 2026. In this review, we summarise the relevant information regarding the application of KA, describe the optimal cultivation conditions for Aspergillus species used in the production of KA, and assess the prospects for the KA market. Based on our findings, we established that the highest yields of KA can be achieved using submerged fermentation with glucose and yeast extract as the primary sources of carbon and nitrogen, respectively. Furthermore, according to literature, the main species/strains reported as the best producers of KA are Aspergillus flavus (44-L), Aspergillus oryzae (AR-47 and NRRL 484), and Aspergillus terreus (C5-10 mutant of the strain PTCC 5283). Given the commercial importance of KA and the growing demand for this natural product, further studies are needed to identify novel strains of Aspergillus as potential high producers of this acid. Similarly, it will be desirable to identify novel sources of substrate for the low-cost production of KA, thereby promoting its production for use in pharmaceutical, healthcare, and other potential industrial applications. In addition, given the current limited knowledge regarding the biosynthetic pathway of KA, further studies are required to elucidate that biosynthetic pathway. 相似文献
147.
Piotr Orlewski Vassilios Tsikaris Maria Sakarellos-Daitsiotis Constantinos Sakarellos Ketty P. Soteriadou Michel Marraud Manh Thong Cung 《Letters in Peptide Science》1996,3(5):317-326
Summary The IASRYDQL synthetic octapeptide (250–257) of the Leishmania major surface glycoprotein gp63 efficiently inhibits parasite attachment to the macrophage receptors in in vitro experiments, and the SRYD-containing tetrapeptide mimics antigenically and functionally the RGDS sequence of fibronectin. The conformational properties of the octapeptide were investigated in dimethylsulfoxide (DMSO) with the combined use of NMR data (vicinal coupling constants, nuclear Overhauser effects (NOEs) and temperature coefficient values), molecular modeling by energy minimization and molecular dynamics. The structure is characterized by the high occurrence, exceeding 95%, of the Arg-Asp side-chain-side-chain ionic interaction, which plays a key role in the backbone folding through a distorted type-I -turn involving the Gln256-NH to Arg253-CO hydrogen bond. 相似文献
148.
Tiecheng Qiao Robert Witkowski Robin Henderson G. McLendon 《Journal of biological inorganic chemistry》1996,1(5):432-438
The kinetics of methemoglobin reduction by cytochrome b
5 has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k
f = 2.44×104 M–1 s–1 and a reverse rate constant k
b = 540 M–1s–1 have been observed at 10 mm, pH 6.20, 25 °C. The ratio k
f/k
b = k
eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b
5 and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b
5 to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such
collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin.
Received: 20 February 1996 / Accepted: 4 June 1996 相似文献
149.
Summary The monoclonal antibody (MAb) JIM5, marking acidic pectins, was used to localize ultrastructurally pectin molecules in the pollen tube wall ofNicotiana tabacum. Longitudinal sections of LR-White embedded pollen tubes were exposed to antibody treatment; accumulations of pectins were identified by counting the density of the gold particles representing the pectin epitopes along the pollen tube wall. Significant accumulations of gold grains were marked and the distances between them were measured. In many pollen tubes a more or less regular distribution of the accumulations was observed along the tube indicating a periodical deposition of pectin. The distances between the accumulations were 4–6 m. Most of the label was found in the inner part of the outer layer of the bilayered cell wall. These findings correspond to and confirm the earlier observation by our group reporting ring-shaped periodical deposits in pollen tubes after immunofluorescence labelling with the MAb JIM5 under the confocal laser scanning microscope.Abbreviations Ab
antibody
- MAb
monoclonal antibody 相似文献
150.
Xavier Khawaja Non Evans Yvonne Reilly Christine Ennis Michael C. W. Minchin 《Journal of neurochemistry》1995,64(6):2716-2726
Abstract: The specific binding of [3H]WAY-100635 {N-[2-[4-(2-[O-methyl-3H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [3H]WAY-100635 association (k+1 = 0.069 ± 0.015 nM?1 min?1) and dissociation (k?1 = 0.023 ± 0.001 min?1) followed monoexponential kinetics. Saturation binding isotherms of [3H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (Bmax) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [3H]WAY-100635 was ~36% higher compared with that of 8-hydroxy-2-(di-n-[3H]-propylamino)tetralin ([3H]8-OH-DPAT). The binding affinity of [3H]WAY-100635 was significantly lowered by the divalent cations CaCl2 (2.5-fold; p < 0.02) and MnCl2 (3.6-fold; p < 0.05), with no effect on Bmax. Guanyl nucleotides failed to influence the KD and Bmax parameters of [3H]WAY-100635 binding to 5-HT1A receptors. The pharmacological binding profile of [3H]WAY-100635 was closely correlated with that of [3H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine1A (5-HT1A) sites in rat hippocampus. [3H]WAY-100635 competition curves with 5-HT1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites. 相似文献