全文获取类型
收费全文 | 3163篇 |
免费 | 80篇 |
国内免费 | 157篇 |
出版年
2023年 | 33篇 |
2022年 | 42篇 |
2021年 | 49篇 |
2020年 | 43篇 |
2019年 | 69篇 |
2018年 | 72篇 |
2017年 | 36篇 |
2016年 | 49篇 |
2015年 | 45篇 |
2014年 | 188篇 |
2013年 | 200篇 |
2012年 | 153篇 |
2011年 | 215篇 |
2010年 | 143篇 |
2009年 | 143篇 |
2008年 | 159篇 |
2007年 | 193篇 |
2006年 | 127篇 |
2005年 | 120篇 |
2004年 | 72篇 |
2003年 | 73篇 |
2002年 | 59篇 |
2001年 | 44篇 |
2000年 | 43篇 |
1999年 | 65篇 |
1998年 | 39篇 |
1997年 | 42篇 |
1996年 | 34篇 |
1995年 | 40篇 |
1994年 | 38篇 |
1993年 | 41篇 |
1992年 | 52篇 |
1991年 | 54篇 |
1990年 | 39篇 |
1989年 | 33篇 |
1988年 | 27篇 |
1987年 | 43篇 |
1986年 | 17篇 |
1985年 | 54篇 |
1984年 | 53篇 |
1983年 | 47篇 |
1982年 | 35篇 |
1981年 | 37篇 |
1980年 | 45篇 |
1979年 | 31篇 |
1978年 | 35篇 |
1977年 | 24篇 |
1976年 | 29篇 |
1975年 | 25篇 |
1973年 | 21篇 |
排序方式: 共有3400条查询结果,搜索用时 31 毫秒
151.
Devappa S. Lamani K. R. Venugopala Reddy H. S. Bhojya Naik K. S. R. Pai Ravishankar Kumar Fasiulla 《Nucleosides, nucleotides & nucleic acids》2013,32(8):591-605
This article deals with the synthesis of 4-(2-hydroxyquinolin-3-yl)-6-phenyl-5,6-dihydropyrimidin derivatives (2a–f), on condensation with various aromatic aldehydes and ketones in aqueous ethanolic NaOH solution yielding the corresponding chalcones (3). These chalcones were further reacted with thiourea/urea in the presence of a base, which led to the formation of the titled derivatives (2a–f). The newly synthesized heterocyles were characterized by elemental analysis, FTIR, 1HNMR, and electronic and mass spectral data. The compounds (2a and 2b) were evulated for in vitro cyctotoxicity against human breast adenocarcinoma cell (MCF-7). In MTT cytotoxicity studies, both quinolinde derivatives were found most effective. The binding interaction behavior of the compound (2a) and (2d) with calf thymus-DNA (CT-DNA) was studied by electronic spectra, viscosity measurements, and thermal denaturation studies. On binding to CT-DNA, the absorption spectrum underwent bathochromic and hypochromic shifts. The binding constant (Kb) observed 4.3 × 105 M?1 for (2a), and 3.8 × 105 M?1 for (2d) suggested that compound (2a) binds more strongly with base pairs than (2d). 相似文献
152.
153.
Kohjiro Nagao Minami Maeda Noralyn B. Mañucat Kazumitsu Ueda 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2013,1831(2):398-406
ATP-binding cassette protein A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL). However, the detailed mechanism of HDL formation remains unclear; in order to reveal it, chemicals that specifically block each step of HDL formation would be useful. Cyclosporine A inhibits ABCA1-mediated cholesterol efflux, but it is not clear whether this is mediated via inhibition of calcineurin. We analyzed the effects of cyclosporine A and related compounds on ABCA1 function in BHK/ABCA1 cells. Cyclosporine A, FK506, and pimecrolimus inhibited ABCA1-mediated cholesterol efflux in a concentration-dependent manner, with IC50 of 7.6, 13.6, and 7.0 μM, respectively. An mTOR inhibitor, rapamycin also inhibited ABCA1, with IC50 of 18.8 μM. The primary targets for these drugs were inhibited at much lower concentrations in BHK/ABCA1 cells, suggesting that they were not involved. Binding of [3H] cyclosporine A to purified ABCA1 could be clearly detected. Furthermore, a non-immunosuppressive cyclosporine, PSC833, inhibited ABCA1-mediated cholesterol efflux with IC50 of 1.9 μM, and efficiently competed with [3H] cyclosporine A binding to ABCA1. These results indicate that cyclosporine A and PSC833 inhibit ABCA1 via direct binding, and that the ABCA1 inhibitor PSC833 is an excellent candidate for further investigations of the detailed mechanisms underlying formation of HDL. 相似文献
154.
Yolande Rouiller Arnaud Périlleux Natacha Collet Martin Jordan Matthieu Stettler Hervé Broly 《MABS-AUSTIN》2013,5(3):501-511
An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame. 相似文献
155.
156.
Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis
David W. Greening Hong Ji Eugene A. Kapp Richard J. Simpson 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(11):2293-2307
Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1 mM sulindac over 16 h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433–451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1 mM sulindac treatment for 8 h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30 K, 3 K and 1 K). Proteins isolated in the > 30 K and 3–30 K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1–3 K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS–MS. Collectively, our data show that LIM1215 cells treated with 1 mM sulindac for 8 h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5 AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8 h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1 mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell–cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome. 相似文献
157.
David W. Greening Eugene A. Kapp Hong Ji Terry P. Speed Richard J. Simpson 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(11):2396-2407
The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-Mr (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome. 相似文献
158.
Immacolata Del Giudice Danila Limauro Emilia Pedone Simonetta Bartolucci Gabriella Fiorentino 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(10):2071-2079
Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a kcat/KM value of 1.2 × 104 M− 1 s− 1. It also exhibited weak phosphatase activity with a kcat/KM value of 2.7 × 10− 4 M− 1 s− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase. 相似文献
159.
Many attempts on optimization of sorghum [Sorghum bicolor (L.)
Moench] tissue culture induction media have been made, but the culture system
remains with some bottlenecks compared to that of other crops. This study aimed
at assessing the suitability of various induction media to produce embryogenic
callus (yellow and friable) with high induction rates and reduced phenolic
exudation. The six culture medium modifications: 3 based on Murashige and
Skoog (MS) medium and one each based on Chu N6, Gamborg B5 and 190-2
media respectively were applied in the culture of mature embryos from 10
sorghum genotypes. Although there was a genotype influence on the attainment
of a yellow callus, friability of the callus was determined to be dependent on the
culture medium and not the genotype. Half strength MS medium with 0.2 mg/l
2,4-D with 2.8 g/l Gelrite® as the gelling agent modified with 1.0 g/l KH2PO4,
1.0 g/l L-proline, 1.0 g/l L-asparagine and 0.16 mg/l CuSO4·5H2O (type E) was
found to be the most effective resulting in about 60% yellow coloured callus
induction with 25% friability. Addition of CuSO4·5H2O, KH2PO4, L-proline and
L-asparagine significantly reduced the phenolic production. Half strength MS
medium was observed to contribute to quality callus production when compared
to full strength MS media modified with the compounds. The half strength MS
medium was also observed to suppress phenolic production. Medium 190-2
produced the highest regeneration frequency (40%) among the 3-regeneration
media tested. The results provide information on a suitable sorghum callus
induction medium necessary for embryogenesis. 相似文献
160.
Jan F. Kamler Khamtai Thatdokkham Susana Rostro-García Anita Bousa Anthony Caragiulo Rachel Crouthers Visattha In Chen Pay Chanratana Pin Sovanna Prum Chantavy Vongkhamheng Arlyne Johnson David W. Macdonald 《The Journal of wildlife management》2020,84(7):1396-1405
Endangered dholes (Cuon alpinus) are restricted to small and declining populations in Southeast Asia, and little is known about how their ecology differs within the region. We used DNA-confirmed scats and prey surveys to determine the seasonal diet and prey selection of dholes in 2 different landscapes that dominate Southeast Asia: closed evergreen forests in hilly terrain in northern Laos, and open deciduous forests in relatively flat terrain in eastern Cambodia. On both sites, muntjac (Muntiacus spp.; 20–28 kg) was the dominant prey item and was selectively consumed over other ungulates in all seasons. Our findings differ from previous conclusions, based largely on studies from India, that the preferred prey weight range of dholes was either 40–60 kg or 130–190 kg. Other important prey were sambar (Rusa unicolor) in Laos, and wild pig (Sus scrofa) and banteng (Bos javanicus) in Cambodia. Seasonal differences in overall diet occurred in Laos, but not Cambodia, primarily because of an increase in livestock consumption. The mean number of dhole scats in group defecation sites was higher in Cambodia (5.9 ± 0.5 [SE]) than Laos (2.4 ± 0.2), suggesting pack sizes were larger in Cambodia. Our results suggest that regardless of land cover type, prey diversity, or pack size, the management of muntjac will be important for conserving dhole populations in Southeast Asia. In Laos, we recommend that local villagers remove livestock from the protected area during the hot-dry season to reduce livestock predation by dholes. © 2020 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society. 相似文献