全文获取类型
收费全文 | 1381篇 |
免费 | 89篇 |
国内免费 | 1篇 |
出版年
2023年 | 7篇 |
2022年 | 6篇 |
2021年 | 22篇 |
2020年 | 24篇 |
2019年 | 11篇 |
2018年 | 20篇 |
2017年 | 19篇 |
2016年 | 33篇 |
2015年 | 42篇 |
2014年 | 78篇 |
2013年 | 73篇 |
2012年 | 112篇 |
2011年 | 102篇 |
2010年 | 57篇 |
2009年 | 57篇 |
2008年 | 87篇 |
2007年 | 112篇 |
2006年 | 107篇 |
2005年 | 87篇 |
2004年 | 82篇 |
2003年 | 93篇 |
2002年 | 83篇 |
2001年 | 10篇 |
2000年 | 21篇 |
1999年 | 18篇 |
1998年 | 12篇 |
1997年 | 11篇 |
1996年 | 7篇 |
1995年 | 4篇 |
1994年 | 2篇 |
1993年 | 4篇 |
1991年 | 2篇 |
1990年 | 3篇 |
1989年 | 4篇 |
1988年 | 4篇 |
1987年 | 2篇 |
1986年 | 3篇 |
1985年 | 6篇 |
1983年 | 3篇 |
1982年 | 2篇 |
1981年 | 3篇 |
1980年 | 6篇 |
1978年 | 3篇 |
1976年 | 5篇 |
1975年 | 2篇 |
1974年 | 2篇 |
1973年 | 5篇 |
1972年 | 5篇 |
1971年 | 2篇 |
1970年 | 2篇 |
排序方式: 共有1471条查询结果,搜索用时 31 毫秒
21.
Alexei V. Vorobiev Noah K. Scarr Yevgeniy Belousov Eugeny A. Lukhtanov 《Nucleosides, nucleotides & nucleic acids》2013,32(8):421-438
Several 7-(hydroxy, amino, methylureido, and guanidino)alkynyl-substituted 8-aza-7-deaza- hypoxanthine analogues were investigated as potential universal nucleobases. 7-Aminobutynyl-8-aza-7-deazahypoxanthine was found to be the most promising quasi-universal nucleobase with improved hybridization and polymerase chain reaction (PCR) enhancing properties as compared to commonly used hypoxanthine (the nucleobase of inosine). It demonstrated improved ambiguity for pairing with A, T, and C bases and its base pairing properties can be summarized as follows: X:C~X:A~X:T > X:G. The improvement in PCR performance directly correlated with primer's Tm. Primers containing multiple 7-aminobutynyl-8-aza-7-deazahypoxanthines were successfully used without noticeable inhibition of Taq polymerase activity provided the modifications are positioned more than two bases away from the 3′ end. 相似文献
22.
23.
Silvia C. Locatelli-Hoops Inna Gorshkova Klaus Gawrisch Alexei A. Yeliseev 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(10):2045-2056
Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB2 and immobilization at solid interfaces. Expression of CB2 as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB2 was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB2 from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB2 and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB2. 相似文献
24.
25.
Alexei Kharitonenkov John M. Beals Radmila Micanovic Beth A. Strifler Radhakrishnan Rathnachalam Victor J. Wroblewski Shun Li Anja Koester Amy M. Ford Tamer Coskun James D. Dunbar Christine C. Cheng Christopher C. Frye Thomas F. Bumol David E. Moller 《PloS one》2013,8(3)
Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans. 相似文献
26.
Hongyan Zheng Alina Beliavsky Anatoli Tchigvintsev Joseph S. Brunzelle Greg Brown Robert Flick Elena Evdokimova Zdzislaw Wawrzak Radhakrishnan Mahadevan Wayne F. Anderson Alexei Savchenko Alexander F. Yakunin 《Proteins》2013,81(6):1031-1041
Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD+ and NADP+ as cofactors, although affinity to NAD+ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD+ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD+ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD+. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc. 相似文献
27.
Alexei N. Nekrasov Alexei A. Zinchenko 《Journal of biomolecular structure & dynamics》2013,31(5):553-561
Abstract The novel method allowing identification of protein structure elements responsible for catalytic activity manifestation is proposed. Structural organization of various hydrolases was studied using the ANIS (ANalysis of Informational Structure) method. ANIS allows to reveal a hierarchy of the ELements of Information Structure (ELIS) using protein amino acid sequence. The ELIS corresponds to the variable length sites with an increased density of structural information. The amino acid residues forming the enzyme catalytic site were shown to belong to the different top-ranking ELIS located in the contact area of the corresponding spatial structure clusters. In the protein spatial structure catalytic sites are located in the area of contact between fragments of polypeptide chain (structural blocs) allocation to the different top-ranking ELIS. According to our results we concluded that structural blocks corresponding to top-ranking ELIS are crucial for protein functioning. Such regions are structurally independent, and their determinate mobility relative to each other is vital for an efficient enzymatic reaction to occur. 相似文献
28.
Nikolai A. Pavlov Dmitry I. Cherny Thomas M. Jovin Alexei I. Slesarev 《Journal of biomolecular structure & dynamics》2013,31(2):207-214
Abstract The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (Rw) of ≈3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw ≤ 3 revealed formation of isometric loops encompassing 71 +/- 7 bp of DNA duplex. At high values of Rw (≥9) thickened compact nucleoprotein structures were observed; no individual loops were seen within the complexes. Gel filtration chromatography and chemical fixation indicated that in the absence of DNA the dominant form of the MkaH in solution, unlike other archaeal histones, is a stable dimer (pseudo-tetramer of the histone-fold domain) apparently resembling the eukaryal (H3-H4)2 tetramer. Similarly, dimers are the dominant form of the protein interacting with DNA. The properties of MkaH supporting the assignment of its intermediate position between other archaeal and eukaryal histones are discussed. 相似文献
29.
Alex Burdyga Alan Conant Lee Haynes Jin Zhang Kees Jalink Robert Sutton John Neoptolemos Eithne Costello Alexei Tepikin 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2013,1833(12):2664-2672
We demonstrated that increasing intracellular cAMP concentrations result in the inhibition of migration of PANC-1 and other pancreatic ductal adenocarcinoma (PDAC) cell types. The rise of cAMP was accompanied by rapid and reversible cessation of ruffling, by inhibition of focal adhesion turnover and by prominent loss of paxillin from focal adhesions. All these phenomena develop rapidly suggesting that cAMP effectors have a direct influence on the cellular migratory apparatus. The role of two primary cAMP effectors, exchange protein activated by cAMP (EPAC) and protein kinase A (PKA), in cAMP-mediated inhibition of PDAC cell migration and migration-associated processes was investigated. Experiments with selective activators of EPAC and PKA demonstrated that the inhibitory effect of cAMP on migration, ruffling, focal adhesion dynamics and paxillin localisation is mediated by PKA, whilst EPAC potentiates migration. 相似文献
30.
Sébastien L'Hoste Alexei Diakov Olga Andrini Mathieu Genete Laurent Pinelli Teddy Grand Mathilde Keck Marc Paulais Laurent Beck Christoph Korbmacher Jacques Teulon Stéphane Lourdel 《生物化学与生物物理学报:生物膜》2013
Several Cl− channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl− absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl− > Br− > NO3− > I−. Single-channel recordings revealed a unit conductance of ~ 40 pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~ − 65 mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~ 20 pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~ + 25 mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253–260). 相似文献