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排序方式: 共有513条查询结果,搜索用时 265 毫秒
51.
John Fuesler Yasunori NagahamaJoseph Szulewski Joshua MundorffStephanie Bireley Jonathon S. Coren 《Gene》2012
The various iterations of the HapMap Project and many genome-wide association studies (GWAS) have identified hundreds of potential genes involved in monogenic and multifactorial traits. We constructed an arrayed 115,000-member human genomic library in the PAC shuttle vector pJCPAC-Mam2 that can be propagated in both bacterial and human cells. The library appears to represent a two-fold coverage of the human genome. Transient transfection of a p53-containing PAC clone into p53-null Saos-2 human osteosarcoma cells demonstrated that both p53 mRNA and protein were produced. Additionally, expression of the p53 protein triggered apoptosis in a subset of the Saos-2 cells. This library should serve as a valuable resource to validate potential disease genes identified by GWAS in human cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders. 相似文献
52.
Sarah E. Stewart Michael E. D'Angelo Phillip I. Bird 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(1):59-67
Cytotoxic lymphocytes (CLs) are responsible for the clearance of virally infected or neoplastic cells. CLs possess specialised lysosome-related organelles called granules which contain the granzyme family of serine proteases and perforin. Granzymes may induce apoptosis in the target cell when delivered by the pore forming protein, perforin. Here we follow the perforin-granzyme pathway from synthesis and storage in the granule, to exocytosis and finally delivery into the target cell. This review focuses on the controversial subject of perforin-mediated translocation of granzymes into the target cell cytoplasm. It remains unclear whether this occurs at the cell surface with granzymes moving through a perforin pore in the plasma membrane, or if it involves internalisation of perforin and granzymes and subsequent release from an endocytic compartment. The latter mechanism would represent an example of cross talk between the endo-lysosomal pathways of individual cells. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. 相似文献
53.
Blanchard B Nurisso A Hollville E Tétaud C Wiels J Pokorná M Wimmerová M Varrot A Imberty A 《Journal of molecular biology》2008,383(4):837-853
The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL. 相似文献
54.
Ahmad MF Singh D Taiyab A Ramakrishna T Raman B Rao ChM 《Journal of molecular biology》2008,382(3):812-824
Oxidative stress and Cu2+ have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu2+, is also known to induce the expression of the small heat shock proteins α-crystallins. However, the role of α-crystallins in oxidative stress and in Cu2+-mediated processes is not clearly understood. We demonstrate using fluorescence and isothermal titration calorimetry that α-crystallins (αA- and αB-crystallin and its phosphorylation mimic, 3DαB-crystallin) bind Cu2+ with close to picomolar range affinity. The presence of other tested divalent cations such as Zn2+, Mg2+, and Ca2+ does not affect Cu2+ binding, indicating selectivity of the Cu2+-binding site(s) in α-crystallins. Cu2+ binding induces structural changes and increase in the hydrodynamic radii of α-crystallins. Cu2+ binding increases the stability of α-crystallins towards guanidinium chloride-induced unfolding. Chaperone activity of αA-crystallin increases significantly upon Cu2+ binding. α-Crystallins rescue amyloid beta peptide, Aβ1-40, from Cu2+-induced aggregation in vitro. α-Crystallins inhibit Cu2+-induced oxidation of ascorbate and, hence, prevent the generation of reactive oxygen species. Interestingly, α-synuclein, a Cu2+-binding protein, does not inhibit this oxidation process significantly. We find that the Cu2+-sequestering (or redox-silencing) property of α-crystallins confers cytoprotection. To the best of our knowledge, this is the first study to reveal high affinity (close to picomolar) for Cu2+ binding and redox silencing of Cu2+ by any heat shock protein. Thus, our study ascribes a novel functional role to α-crystallins in Cu2+ homeostasis and helps in understanding their protective role in neurodegenerative diseases and cataract. 相似文献
55.
Shibata T Nakahara H Kita N Matsubara Y Han C Morimitsu Y Iwamoto N Kumagai Y Nishida M Kurose H Aoki N Ojika M Uchida K 《Journal of neurochemistry》2008,107(5):1248-1260
Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF-dependent neuritogenesis enhancer potential of a food-derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor-initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic-promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6-HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low-concentration of NGF that alone was insufficient to induce neuronal differentiation. 6-HITC also facilitated the sustained-phosphorylation of the extracellular signal-regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr-490 of TrkA and was inactivated by 6-HITC in a redox-dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates. 相似文献
56.
Louzao MC Espiña B Vieytes MR Vega FV Rubiolo JA Baba O Terashima T Botana LM 《Glycoconjugate journal》2008,25(6):503-510
There are presently many methods of detecting complex carbohydrates, and particularly glycogen. However most of them require radioisotopes or destruction of the tissue and hydrolysis of glycogen to glucose. Here we present a new method based on the incorporation of 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1, 3-diazol 4-yl] amino}-2-deoxyglucose), a D-glucose fluorescent derivative, into glycogen. Two kinds of approaches were carried out by using Clone 9 rat hepatocytes as a cellular model; (1) Incubation of cell lysates with 2-NBDG, carbohydrate precipitation in filters and measurement of fluorescence in a microplate reader (2) Incubation of living hepatocytes with 2-NBDG and recording of fluorescence images by confocal microscopy. 2-NBDG labeled glycogen in both approaches. We confirmed this fact by comparison to the labeling obtained with a specific monoclonal anti-glycogen antibody. Also drugs that trigger glycogen synthesis or degradation induced an increase or decrease of fluorescence, respectively. This is a simple but efficient method of detecting glycogen with 2-NBDG. It could be used to record changes in glycogen stores in living cells and cell-free systems and opens the prospect of understanding the role of this important energy reserve under various physiological and pathophysiological conditions. 相似文献
57.
As a novel approach to the mode of medicinal action of garlic, its constituents were comparatively studied with respect to
their interactions with membrane lipids to modify the membrane fluidity. Allyl derivatives rigidified tumor cell and platelet
model membranes consisting of unsaturated phospholipids and cholesterol at 20–500 μM with the potency being diallyl trisulfide
(DATS) > diallyl disulfide (DADS) by preferentially acting on the hydrocarbon cores of lipid bilayers. They were also effective
in rigidifying candida cell model membranes prepared with ergosterol and phospholipids at 100–500 μM with the potency being
DADS > DATS > diallyl sulfide (DAS), but not bacteria cell model membranes without ergosterol. Alliin, a precursor of these
DASs, was not active on any membranes at 500 μM. Both relative intensity and selectivity in membrane effects correlated with
those in antiproliferative, antiplatelet and antimicrobial effects. In cell culture experiments, membrane-active DASs inhibited
the growth of tumor cells cultured for 24 and 48 h at 20–500 μM to show the potency being DATS > DADS, together with rigidifying
cell membranes by acting on their deeper regions more intensively. However, membrane-inactive allyl derivatives were not growth-inhibitory
on tumor cells. The membrane lipid interactions of DASs appear to be one of possible mechanisms underlying different effects
of garlic. 相似文献
58.
Radical graft functional modification of cellulose with allyl monomers: Chemistry and structure characterization 总被引:1,自引:0,他引:1
Cotton cellulose was successfully functionalized via a free radical graft polymerization process. Potassium persulfate served as an effective water soluble radical initiator to generate cellulosic radicals. The polymeric radicals could react with allyl monomers such as allyl-dimethylhydantion (ADMH) to form surface grafted cellulose. The reaction sites generated by potassium persulfate were probably at carbon 3 and 4 in glucose ring via oxidative hydrogen abstraction. The cellulosic radicals can initiate grafting polymerization of ADMH with a maximum polymerization degree of about 12 based on LC–MS results. The radical graft polymerization mechanisms were proposed based on LC–ESI/MS analysis. The ideal covalent bonding between cellulose and poly (allyl-dimethylhydantion) (PADMH) ensured permanent graft of the monomers on cotton and durability of the expected functions on the treated cotton. 相似文献
59.
Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death 总被引:1,自引:1,他引:0
Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death. 相似文献
60.