全文获取类型
收费全文 | 12359篇 |
免费 | 684篇 |
国内免费 | 404篇 |
出版年
2023年 | 134篇 |
2022年 | 140篇 |
2021年 | 289篇 |
2020年 | 264篇 |
2019年 | 277篇 |
2018年 | 358篇 |
2017年 | 255篇 |
2016年 | 234篇 |
2015年 | 331篇 |
2014年 | 520篇 |
2013年 | 748篇 |
2012年 | 401篇 |
2011年 | 450篇 |
2010年 | 403篇 |
2009年 | 519篇 |
2008年 | 647篇 |
2007年 | 604篇 |
2006年 | 643篇 |
2005年 | 549篇 |
2004年 | 517篇 |
2003年 | 471篇 |
2002年 | 452篇 |
2001年 | 314篇 |
2000年 | 292篇 |
1999年 | 299篇 |
1998年 | 291篇 |
1997年 | 255篇 |
1996年 | 245篇 |
1995年 | 252篇 |
1994年 | 226篇 |
1993年 | 243篇 |
1992年 | 198篇 |
1991年 | 187篇 |
1990年 | 187篇 |
1989年 | 150篇 |
1988年 | 138篇 |
1987年 | 118篇 |
1986年 | 94篇 |
1985年 | 98篇 |
1984年 | 140篇 |
1983年 | 82篇 |
1982年 | 85篇 |
1981年 | 84篇 |
1980年 | 70篇 |
1979年 | 54篇 |
1978年 | 44篇 |
1977年 | 27篇 |
1976年 | 24篇 |
1975年 | 13篇 |
1973年 | 12篇 |
排序方式: 共有10000条查询结果,搜索用时 140 毫秒
991.
992.
Frahnert C Rao ML Grasmäder K 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,794(1):35-47
Therapeutic drug monitoring necessitates efficient, fast and reliable analytical methods validated by external quality control. We therefore devised an isocratic reversed-phase HPLC method with ultraviolet detection and optimised this to quantify mirtazapine, reboxetine, moclobemide, venlafaxine, O-desmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OH-risperidone in human serum. After solid-phase extraction of the drugs and metabolites, the chromatographic separation was achieved on a Nucleosil 100-Protect 1 column with acetonitrile-potassium dihydrogenphosphate buffer as mobile phase. The method was validated for therapeutic and toxic serum ranges. A linear relationship (r>0.998) was obtained between the concentration and the detector signal. Recoveries were between 75 and 99% for the drugs and metabolites. The accuracy of the quality control samples, expressed as percent recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard deviations were 0.9-10.2% and 0.9-9.7%, respectively. Additional external quality control is carried out since 3 years. This method is applicable to rapidly and effectively analyze serum or plasma samples for therapeutic drug monitoring of about 30 antidepressants and atypical antipsychotics. 相似文献
993.
Regulation of apoptosis by Bcl-2 family proteins 总被引:9,自引:0,他引:9
Burlacu A 《Journal of cellular and molecular medicine》2003,7(3):249-257
For multicellular organisms, the rigorous control of programmed cell death is as important as that of cell proliferation. The mechanisms involved in the regulation of cell death are not yet understood, but a key component is the family of caspases which are activated in a cascade and are responsible for the apoptotic-specific changes and disassembly of the cell. Although the caspases represent a central point in apoptosis, their activation is regulated by a variety of other factors. Among these, Bcl-2 family plays a pivotal role in caspases activation, by this deciding whether a cell will live or die. Bcl-2 family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, but their functions are not yet well defined. This review summarizes the recent data regarding the Bcl-2 proteins and the ways they regulate the apoptosis. 相似文献
994.
Multiple stress proteins are recruited in response to stress in living cells. There are limited reports in the literature analyzing multiple stress protein shifts and their functional consequences on stress response. Using two-dimensional electrophoresis we have analyzed shifts in stress protein profiles in response to energy deprivation as a model of ischemic injury to kidneys. A group of chaperones and stress-induced mitogen activated protein (MAP) kinases were analyzed. In addition to examining stress protein induction and phosphorylation we have also examined the mechanism of cytoprotection by heat shock protein 70 (Hsp70). Our results show that, of the different stress proteins examined, only binding protein (BiP) and Hsp70 were significantly induced upon energy deprivation. Other stress proteins, including Hsp27, calnexin, Hsp90 and ERp57 showed alterations in their phosphorylation profiles. Three different MAP kinases, namely p38, extracellular signal regulated kisase and c-jun N-terminal kinase (JNK) were activated in response to energy deprivation. While JNK activation was linked to apoptosis, activated-p38 was involved in phosphorylation of Hsp27. Study of inhibitors of Hsp70 induction or pre-induction of Hsp70 indicated that induced Hsp70 was involved in the suppression of JNK activation thereby inhibiting apoptotic cell death. Our results provide important insights into the flux in stress protein profiles in response to simulated ischemia and highlight the antiapoptotic, cytoprotective mechanism of Hsp70 action. 相似文献
995.
Duranti M Barbiroli A Scarafoni A Tedeschi G Morazzoni P 《Protein expression and purification》2003,30(2):167-170
A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations. 相似文献
996.
Hu SH Gee CL Latham CF Rowlinson SW Rova U Jones A Halliday JA Bryant NJ James DE Martin JL 《Protein expression and purification》2003,31(2):305-310
Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies. 相似文献
997.
Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers 总被引:1,自引:0,他引:1
The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface
of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually
brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves
recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers
followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed
that the most frequent force values (of the force) were in the range of 0.4–0.6 nN. The observed rupture force most likely
represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent
crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor
pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip
was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface
and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological
research for the application of AFM. 相似文献
998.
Apoptosis,necrosis and cellular senescence: chaperone occupancy as a potential switch 总被引:7,自引:0,他引:7
Chaperone function plays a key role in repairing proteotoxic damage and in the maintenance of cell survival. Here we compare the regulatory role of molecular chaperones (heat shock proteins, stress proteins) in cellular senescence, apoptosis and necrosis. We also review the current data on chaperone level and function in aging cells, and list some possible therapeutic interventions. Finally, we postulate a hypothesis, that increasing chaperone occupancy might be an important event which forces cells out of the normal cell cycle towards senescence. In the case of severe stress, this may lead to apoptosis or, following lethal stress, to cell necrosis. 相似文献
999.
The toxicity and effects on protein synthesis of the phthalate esters diethyl phthalate (DEP) and di(2-ethylhexyl) phthalate (DEHP) was studied in radish seedlings (Raphanus sativus cv. Kööpenhaminan tori). Phthalate esters are a class of commercially important compounds used mainly as plasticizers in high molecular-weight polymers such as many plastics. They can enter soil through various routes and can affect plant growth and development. First the effect of DEP and DEHP on the growth of radish seedlings was determined in an aqueous medium. It was found that DEP, but not DEHP, caused retardation of growth in radish. A further investigation on protein synthesis during DEP-stress was executed by in vivo protein labeling combined with two-dimensional gel electrophoresis (2D-PAGE). For comparisons with known stress-induced proteins a similar experiment was done with heat shock, and the induced heat shock proteins (HSPs) were compared with those of DEP-stress. The results showed that certain HSPs can be used as an indicator of DEP-stress, although the synthesis of most HSPs was not affected by DEP. DEP also elicited the synthesis of numerous proteins found only in DEP-treated roots. The toxic effect of phthalate esters and the roles of the induced proteins are discussed. 相似文献
1000.
AIMS: The aim of this study was to investigate the links between survival of Escherichia coli in sea water microcosms in the laboratory and the presence of porins in the outer membrane. The E. coli strains studied were a wild-type strain and a series of outer membrane protein (omp) mutants. METHODS AND RESULTS: Bacteria were suspended in natural or filtered-autoclaved sea water microcosms and numbers determined over an incubation period by plate count and by count of cells capable of respiration. CONCLUSIONS: The type of omp mutation has a significant impact in bacterial survival. The double OmpC-OmpF mutant and the OmpR mutant (which was incapable of synthesizing OmpC and OmpF) survived poorly compared with single omp mutants and the wild-type strain. This suggests that these proteins are important in determining the entry of E. coli into the survival mode. The EnvZ mutant, which lacks the protein by which the cell senses some changes in the environment, survived as well as the wild-type strain when compared by plate counts and by respiring cell count. The loss of the EnvZ protein has no effect on survival but it could prevent the organism sensing the changes in the environment through which entry into the survival state is triggered. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is another piece in the puzzle as to how bacteria survive stress conditions. 相似文献