全文获取类型
收费全文 | 5817篇 |
免费 | 332篇 |
国内免费 | 511篇 |
出版年
2023年 | 81篇 |
2022年 | 103篇 |
2021年 | 159篇 |
2020年 | 153篇 |
2019年 | 205篇 |
2018年 | 196篇 |
2017年 | 155篇 |
2016年 | 205篇 |
2015年 | 184篇 |
2014年 | 309篇 |
2013年 | 557篇 |
2012年 | 190篇 |
2011年 | 253篇 |
2010年 | 238篇 |
2009年 | 256篇 |
2008年 | 242篇 |
2007年 | 264篇 |
2006年 | 233篇 |
2005年 | 213篇 |
2004年 | 199篇 |
2003年 | 164篇 |
2002年 | 170篇 |
2001年 | 120篇 |
2000年 | 126篇 |
1999年 | 144篇 |
1998年 | 123篇 |
1997年 | 123篇 |
1996年 | 120篇 |
1995年 | 142篇 |
1994年 | 130篇 |
1993年 | 140篇 |
1992年 | 121篇 |
1991年 | 96篇 |
1990年 | 66篇 |
1989年 | 55篇 |
1988年 | 67篇 |
1987年 | 56篇 |
1986年 | 41篇 |
1985年 | 47篇 |
1984年 | 50篇 |
1983年 | 26篇 |
1982年 | 19篇 |
1981年 | 23篇 |
1980年 | 16篇 |
1979年 | 9篇 |
1978年 | 17篇 |
1976年 | 13篇 |
1975年 | 7篇 |
1974年 | 11篇 |
1972年 | 7篇 |
排序方式: 共有6660条查询结果,搜索用时 31 毫秒
91.
Zhao Rongrui Wang Wenze Wu Bowei Hoebeke Johan Hjalmarson Åke Fu Michael L. X. 《Molecular and cellular biochemistry》1996,163(1):185-193
The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K+ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated Ek of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward Ica and outward Ik simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated ICa but not the isoprenaline-stimulated Ik. The antibody- or carbachol-induced outward K+ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated Ica were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events. 相似文献
92.
Alexander Schouten Jan Roosien Fred A. van Engelen G. A. M. de Jong A. W. M. Borst-Vrenssen Jacoline F. Zilverentant Dirk Bosch Willem J. Stiekema Fred J. Gommers Arjen Schots Jaap Bakker 《Plant molecular biology》1996,30(4):781-793
The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed. 相似文献
93.
Margaret Smallwood Edwin A. Yates William G. T. Willats Helen Martin J. Paul Knox 《Planta》1996,198(3):452-459
Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP
arabinogalactan-protein
- GlcY
-glucosyl Yariv reagent
- ELISA
enzyme-linked immunosorbent assay
We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society. 相似文献
94.
Summary The ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 m long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, -D-mannose and -D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (Mr 133,000 and 146,000). Reductions in the Mr of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.Abbreviations ECM
extracellular matrix
- BPA
Bauhinia purpurea agglutinin
- BSA
bovine serum albumin
- DIC
differential interference contrast
- FITC
fluorescein isothiocyanate
- GNL
Galanthus nivalis lectin
- GSI-B4
Griffonia simplicifolia isolectin B4
- HEPES
(N-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid)
- IIF
indirect immunofluorescence
- IPC
isopycnic centrifugation
- MAb
monoclonal antibody
- PEG
polyethylene glycol
- PBS
phosphate buffered saline
- PNGase
peptideN-glycosidase
- SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis
- TCS
tissue culture supernatant
- TEM
transmission electron microscopy
- TFMS
trifluoromethane sulphonic acid 相似文献
95.
Ignacio García-De La Torre Mario Salazar-Páramo Gerardo Salmón-De La Torre 《Molecular biology reports》1996,23(3-4):153-157
Mixed connective tissue disease (MCTD) was described as a distinct clinical syndrome in 1972. Since then many cases have been reported in the literature worldwide. In this study we present our experience with a group of 17 Mexican patients with this syndrome, and we analyze their clinical and serological features, as well as the causes of death in these patients. The patients are Mexican mestizos living in Guadalajara and most of them have been followed-up at Hospital General de Occidente for a period of 1–10 years. The female/male ratio was 16:1, and their age ranged from 14–55 years with a mean of 29 years. The disease duration has ranged from 1–17 years, with a mean of 6 years. Among the clinical manifestations we have found a high frequency of lymphadenopathy when compared with published series (13/17 or 76%), and the laboratory findings in our patients included a very high polyclonal increase of gammaglobulins (93%), lymphopenia (76%), direct immunofluorescence speckled nuclear epidermal deposits in skin biopsies (75%) and positive rheumatoid factor (65%). Other clinical and serological features were similar to those reported in other series of patients with MCTD. Six of the 17 patients have died (35%), and in 3 of them (17.5%) the cause of death was due to an infectious disease that suddenly presented, and apparently was not related to a concomitant high dose of steroids or malnutrition in the patients. It seems that in addition to the already well known autoimmune abnormalities that occur in MCTD, there are other features like the presence of lymphadenopathy, the high polyclonal increase of gammaglobulins, and the lymphopenia, that reflect the profound disturbance of the immune system in this syndrome, possibly contributing to the sudden appearance of a severe infectious disease in some of our patients.Abbreviations ANA
antinuclear antibody
- CIE
counterimmunoelectrophoresis
- MCTD
mixed connective tissue disease
- PHA
passive hemagglutination
- PM
polymyositis
- RF
rheumatoid factor
- SLE
systemic lupus erythematosus
- SS
systemic sclerosis (SS) 相似文献
96.
Formaldehyde treated cherry mottle leaf virus (ChMLV) and the isolated coat protein were used successfully for the production of polyclonal and monoclonal antibodies. The monoclonal antibodies had a titre of 1:51 200 and consisted of IgG1 and IgG2. The antibodies reacted with all 11 isolates of ChMLV, from five locations in Canada and the USA, included in this study. Several serological procedures were assessed to compare their sensitivity for detecting ChMLV. Plate-trapped antigen ELISA (PTA-ELISA) and dot-blot immunobinding assay (DBIA), using virus specific MAbs, were the most sensitive tests in this study. Triple antibody sandwich ELISA (TAS-ELISA) and Western blot were found to be less sensitive. Dilution of the samples appeared to increase the sensitivity of both PTA-ELISA and Western blot detection. Young leaves and flowers of Prunus avium were the best tissue for detecting the virus which could also be detected in the fruit and leaves of P. tomentosa. April and May were optimal for detection of the virus in the field, whereas both April to May and August to September were optimal for screenhouse-grown plants. 相似文献
97.
Separate polyclonal antibodies have previously been developed against the domoic-acid-producingPseudonitzschia multiseries (=Pseudo-nitzschia pungens f.multiseries) and the non-toxicP. pungens (=P. pungens f.pungens). These antibodies bind to the surface of the diatoms as shown by immunofluorescence studies. Here we examine the molecular nature of the antigens by Western blotting (electro-immunoblotting) analysis. The major antigens for both polyclonal antibodies migrated as high molecular-weight diffuse bands, mostly remaining in the stacking gel, using an SDS-PAGE system. The antibodies prepared againstP. multiseries strongly labelled the high molecular-weight antigens of allP. multiseries strains tested and showed little reactivity towardsP. pungens extracts on Western blots.P. pungens antibodies strongly labelled high molecular-weightP. pungens antigens and faintly labelled a fewP. multiseries antigens. The selectivity of the antibodies for their respective species correlates with the results of the immunofluorescence experiments, suggesting that the antigens examined in this study are responsible for the selective labelling in immunofluorescence studies. The electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disruption of carbohydrates in the pellets by treatment with periodic acid resulted in loss of the antigen. These data suggest that the major antigens of toxicP. multiseries and non-toxicP. pungens are high molecular-weight (°>100kDa) polysaccharides located on the surface of these diatoms.Author for correspondence 相似文献
98.
99.
用人工合成的丁型肝炎病毒抗原(HDV-Ag)肽建立了检测抗HDV-IgM抗体的ELISA方法,本法操作简便、快速,重复性好,特异性强,与抗HAV-IgM、抗Hk-IgM、抗HBs-IgM、抗HCV-IgI、抗CMV-IgM、抗RV-IgM、类风湿因子(RF)及抗核抗体(ANA)阳性血清均不起反应,且可被2-巯基乙醇阻断而不起反应。经初步临床应用,31例正常人血清抗HDV-IgM全部阴性,28例慢活肝患者检出率为32.1%(9/28),17例慢迁肝患者血清阳性率为11.8%(2/17)18例肝癌和肝硬化病人血清阳性率为22.2%(4/18)这三组病人与正常对照者相比较均有显著性差异(P<0.001)。此外,抗HDV-IgM阳性血清的ALT值均明显高于正常参考范围,提示在HDV感染过程中,患者肝细胞进一步受损。实验结果证明,抗HDV-IgM是诊断HDV感染的重要指标,对HDV感染早期诊断具有重要价值。 相似文献
100.