全文获取类型
收费全文 | 14612篇 |
免费 | 1661篇 |
国内免费 | 4071篇 |
出版年
2024年 | 77篇 |
2023年 | 316篇 |
2022年 | 642篇 |
2021年 | 928篇 |
2020年 | 758篇 |
2019年 | 832篇 |
2018年 | 725篇 |
2017年 | 605篇 |
2016年 | 736篇 |
2015年 | 1078篇 |
2014年 | 1207篇 |
2013年 | 1250篇 |
2012年 | 1565篇 |
2011年 | 1412篇 |
2010年 | 886篇 |
2009年 | 865篇 |
2008年 | 917篇 |
2007年 | 887篇 |
2006年 | 886篇 |
2005年 | 701篇 |
2004年 | 526篇 |
2003年 | 417篇 |
2002年 | 391篇 |
2001年 | 261篇 |
2000年 | 246篇 |
1999年 | 201篇 |
1998年 | 125篇 |
1997年 | 97篇 |
1996年 | 94篇 |
1995年 | 81篇 |
1994年 | 67篇 |
1993年 | 51篇 |
1992年 | 70篇 |
1991年 | 67篇 |
1990年 | 54篇 |
1989年 | 35篇 |
1988年 | 35篇 |
1987年 | 38篇 |
1986年 | 30篇 |
1985年 | 37篇 |
1984年 | 20篇 |
1983年 | 19篇 |
1982年 | 22篇 |
1981年 | 9篇 |
1980年 | 6篇 |
1978年 | 8篇 |
1973年 | 6篇 |
1971年 | 6篇 |
1970年 | 6篇 |
1950年 | 5篇 |
排序方式: 共有10000条查询结果,搜索用时 46 毫秒
991.
Tao Pan Zhilong Wang Jian-He Xu Zhenqiang Wu Hanshi Qi 《Applied microbiology and biotechnology》2010,85(6):1789-1796
Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with
a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase
(coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus,
a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation
process. This is the first report about extractive fermentation of proteins in cloud point system. 相似文献
992.
Yi Zhang Tao Zheng Yue Wang Yingjun Guo Feixiang Ding Mingyu Fei Huixian Cui Shuhan Sun 《Applied microbiology and biotechnology》2010,85(3):605-614
Annexin B1 is a novel Ca2+-dependent phospholipid-binding protein from metacestodes of Taenia solium and has been shown to have many potential biomedical applications. Although annexin B1 has been produced successfully in
Escherichia coli, the purified protein has poor stability at room temperature, which has hindered our attempts to further study its structure–function
relationship. To increase the stability of the protein, the construction and purification procedures were examined and changed
to hopefully increase its effectiveness. In this study, we describe a new recombinant annexin B1 expressed with a hexahistidine
tag fused to its N-terminal end, which was purified to homogeneity in two steps using immobilized metal affinity followed
by size exclusion chromatography. The final yield was approximately 23 mg/L of bacterial culture. Isoelectric focusing and
mass spectrometry analysis showed that the protein purified by this method was quite stable at room temperature, even greater
than 3 days later. A series of functional tests indicated that the recombinant protein had high anticoagulant activity, and
fluorescence-labeled annexin B1 could bind to the outer membranes of apoptotic mammalian cells and efficiently detect them
in the early stages of apoptosis. 相似文献
993.
简要介绍了应用于中华鲟(Acipenser sinensis)的pop-up标志固定方法,包括预埋体的制作、植入及pop-up标志悬挂等技术,并对其应用效果进行了评价。 相似文献
994.
Megan Cully Alice Genevet Patricia Warne Caroline Treins Tao Liu Julie Bastien Buzz Baum Nic Tapon Sally J. Leevers Julian Downward 《Molecular and cellular biology》2010,30(2):481-495
The target of rapamycin (TOR) complex 1 (TORC1) signaling pathway is a critical regulator of translation and cell growth. To identify novel components of this pathway, we performed a kinome-wide RNA interference (RNAi) screen in Drosophila melanogaster S2 cells. RNAi targeting components of the p38 stress-activated kinase cascade prevented the cell size increase elicited by depletion of the TOR negative regulator TSC2. In mammalian and Drosophila tissue culture, as well as in Drosophila ovaries ex vivo, p38-activating stresses, such as H2O2 and anisomycin, were able to activate TORC1. This stress-induced TORC1 activation could be blocked by RNAi against mitogen-activated protein kinase kinase 3 and 6 (MKK3/6) or by the overexpression of dominant negative Rags. Interestingly, p38 was also required for the activation of TORC1 in response to amino acids and growth factors. Genetic ablation either of p38b or licorne, its upstream kinase, resulted in small flies consisting of small cells. Mutants with mutations in licorne or p38b are nutrition sensitive; low-nutrient food accentuates the small-organism phenotypes, as well as the partial lethality of the p38b null allele. These data suggest that p38 is an important positive regulator of TORC1 in both mammalian and Drosophila systems in response to certain stresses and growth factors.The target of rapamycin, TOR, is a highly conserved serine/threonine kinase that is a critical regulator of cell growth. It is a core component of two signaling complexes, TORC1 and TORC2 (60, 74). TORC1 is defined by the presence of Raptor in the complex, while TORC2 contains Rictor. Rictor and Raptor are mutually exclusive. Activation of the TORC1 pathway leads to increased protein translation, increased cell size, and increased proliferation, making this pathway an important target for emerging cancer therapies. Rapamycin is an inhibitor of TORC1 that is commonly used as an immunosuppressant following kidney transplantation (51). At least three analogs of rapamycin are currently being tested in solid and hematological tumors and have shown some promising results (21).The TORC1 pathway responds to numerous inputs, sensing both the desirability of and the capacity for growth. Many of these pathways control TORC1 signaling through phosphorylation of the tuberous sclerosis protein TSC2. TSC2 associates with TSC1 to form a heterodimeric GTPase-activating protein complex (GAP) that inactivates the small GTPase Rheb (24, 29, 67). While the exact molecular mechanism remains a topic of debate, activation of Rheb promotes the kinase activity of TORC1 (24, 29, 67). Rheb is required for the activation of TORC1 in response to both amino acids and growth factors (55, 62). In Drosophila melanogaster, mutation of either TOR or Rheb inhibits growth, leading to reduced body size and reduced cell size in mutant clones (42, 64). Mutation of either TSC1 or TSC2 has the predicted opposite effect, as tissue deficient for either of these proteins overgrows and contains large cells (49, 66).TORC1 is activated via the phosphatidylinositol 3′ kinase (PI3′K) pathway by growth-promoting mitogens, such as insulin and growth factors. Drosophila mutants with mutations of PI3′K pathway components have size phenotypes similar to those of the TOR and Rheb mutants (71). In mammalian cells, the PI3′K-mediated activation of TORC1 occurs at least in part through the phosphorylation of TSC2 by the PI3′K target AKT (30, 50). Interestingly, mutation of these residues in Drosophila has no impact on TSC2 function in vivo, suggesting that there may be other mechanisms through which PI3′K can activate Drosophila TOR (20). Recent work has suggested that the proline-rich AKT substrate PRAS40 may provide part of this link (23, 59, 69, 70). In addition, signaling through RAS activates extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK), which can phosphorylate TSC2 and Raptor to activate TORC1 (13, 40, 56). There are also likely to be additional mechanisms through which growth factors activate Drosophila TOR that have not yet been identified.TORC1 activity is also controlled by the intracellular building blocks necessary to support cellular growth. The energy-sensing AMP-activated protein kinase (AMPK) pathway relays information about the energy status of the cell to TORC1 by phosphorylating TSC2. Unlike the inactivating phosphorylation of TSC2 by Akt, phosphorylation of TSC2 by AMPK promotes the GAP activity of the TSC complex (31). AMPK also phosphorylates Raptor, leading to decreased TORC1 activity (28). Thus, when energy levels are low, active AMPK inhibits TORC1.Amino acids also activate the TORC1 pathway, through a mechanism that requires Rheb, as well as the type III PI3′K VPS34 and the serine/threonine kinase mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) (11, 22, 43). TORC1 thereby integrates information about the availability of amino acids and the amount of energy available for growth with growth factor signaling. Given its ancient function in adapting growth rates to environmental conditions, it is likely that TOR responds to a variety of stimuli, suggesting that many TOR control mechanisms remain to be uncovered. The Rag family of Ras-related small GTPases has recently been identified as a key component of the amino acid-sensing pathway, acting in parallel to Rheb (34, 58). Rag GTPases form heterodimers; RagA or RagB interacts with RagC or RagD. RagA and RagB are active when GTP bound, while RagC and RagD are active when bound to GDP (34, 58). Activation of the Rags by amino acids results in TOR relocalization to Rab7-containing vesicles (58). While the function of these vesicles in TORC1 signaling remains unclear, this relocalization is associated with increased TORC1 activity.TORC1 controls cell growth and translation through the phosphorylation and activation of components of the translational machinery, such as S6 kinase (S6K) and 4EBP1, an inhibitor of eukaryotic translation initiation factor 4E (eIF4E) activity (reviewed in reference 27). S6K phosphorylates the S6 ribosomal subunit, thereby increasing translation. Mice deficient for S6K1 are small and have small pancreatic beta cells and a correspondingly low level of circulating insulin (45). Mutation of the phosphorylation sites on S6 results in a similar phenotype, with small beta cells and fibroblasts (57). In Drosophila, mutation of S6K again reduces both cell and organism size (42), as does the overexpression of 4EBP (41).Interestingly, while mutation of the TORC1 pathway in mammalian cells reduces cell size by 10 to 15%, ablation of core TORC1 pathway components in Drosophila cells can affect cell size by up to 40% (73). In an attempt to identify novel components of the TORC1 pathway, we undertook an RNA interference (RNAi)-based screen of Drosophila S2 cells. We reasoned that the extreme size phenotypes observed in Drosophila cells upon TORC1 manipulations would facilitate the identification of modulators. In order to increase the likelihood of isolating novel regulators of TOR, we uncoupled TOR activity from many of its known nutritional controls by depleting TSC2 and screened for double-stranded RNAs (dsRNAs) that could reverse the cell size increase elicited by loss of TSC2. Depletion of multiple components of the p38 pathway was found to revert the TSC2 RNAi-induced cell size increase. Furthermore, activation of p38 is necessary and sufficient for the activation of TOR. Strikingly, mutation of components of the stress-activated p38 pathway in Drosophila has a similar phenotype to mutations in the TOR and insulin signaling pathway: a cell-autonomous cell size decrease, reduced body size, and a sensitization to the effects of nutritional stress. 相似文献
995.
Ping Huang Yuejuan Zhang Tao Jiang Wenjiao Zeng Nong Zhang 《Molecular biology reports》2010,37(7):3097-3103
Glomerulosclerosis is considered to be the final pathway leading to the progressive loss of renal function in several kidney
diseases, transforming growth factor β1 (TGF-β1) plays a critical role in glomerulosclerosis. However, the mechanisms of TGF-β1
stimulating glomerulosclerosis remain poorly understood. Here we report that TGF-β1-induced expression of fibronectin (FN)
depends on the activity of aldose reductase (AR) in human mesangial cells (HMCs).The results show that TGF-β1 increased the
expression of FN, which attenuated by pharmacological inhibition of AR or knockdown of the enzyme by small interfering RNA
(siRNA). MAPKs (ERK, JNK and p38) signalling pathways were activated in HMCs after stimulated by TGF-β1, inhibition of AR
blunted the activation ERK, p38 and JNK signalling pathways. These changes were associated with decreased TGF-β1-induced expression
of FN. These results indicate that AR is a potent regulator of TGF-β1 induced expression of FN in human mesangial cells: it
suggests that inhibition of this enzyme may be useful to prevented extracellular matrix (ECM) deposition in glomerulosclerosis. 相似文献
996.
Liping Yang Ning Liu Xiang Hu Wenfeng Zhang Tao Wang Hong Li Bo Zhang Shuanglin Xiang Jianlin Zhou Jian Zhang 《Molecular biology reports》2010,37(6):2967-2973
TNFAIP1 is a protein which can be induced by tumor necrosis factorα (TNFα) and interleukin-6 (IL-6), it may play roles in
DNA synthesis, DNA repair, cell apoptosis and human diseases. However, very little has been known about how TNFAIP1 acts in
these physiological processes. In this paper, CK2β was identified as a partner of TNFAIP1 by screening the HeLa cDNA library
in yeast two-hybrid system with TNFAIP1 as a bait. Furthermore, it was demonstrated that CK2 could phosphorylate TNFAIP1 in vitro
and in vivo, which facilitated the distribution of TNFAIP1 in nucleus and enhanced its interaction with PCNA. It is suggested
that the phosphorylation of TNFAIP1 may be required for its functions. 相似文献
997.
Yi Liang Jia Cheng Lin Kun Wang Yi Jie Chen Hong Hong Liu Rong Luan Shuai Jiang Tao Che Yong Zhao De Feng Li Da Cheng Wang Lin Guo Hui Sun 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
We have previously reported a novel fungal galectin Agrocybe aegerita lectin (AAL) with apoptosis-induced activity and nuclear migration activity. The importance of nuclear localization for AAL's apoptosis-induced activity has been established by mutant study. However, the mechanism remains unclear.Methods
We further investigated the mechanism using a previously reported carbohydrate recognition domain (CRD) mutant protein H59Q, which retained its nuclear localization activity but lost most of its apoptotic activity. The cell membrane-binding ability of recombinant AAL (rAAL) and H59Q was analyzed by FACS, and their cellular partners were identified by affinity chromatography and mass spectroscopy. Furthermore, the interaction of AAL and ligand was proved by mammalian two-hybrid and pull down assays. A knockdown assay was used to confirm the role of the ligand.Results
The apoptotic activity of AAL could be blocked by lactose. Mutant H59Q retained comparable cell membrane-binding ability to rAAL. Four cellular binding partners of AAL in HeLa cells were identified: glucose-regulated protein 78 (GRP78); mortality factor 4-like protein 1 (MRG15); elongation factor 2 (EEF2); and heat shock protein 70 (Hsp70). CRD region of AAL was required for the interaction between AAL/mutant AAL and MRG15. MRG15 knockdown increased the cells' resistance to AAL treatment.Conclusion
MRG15 was a nuclear ligand for AAL in HeLa cells. These data implied the existence of a novel nuclear pathway for the antitumor activity of fungal galectin AAL.General significance
These findings provide a novel explanation of AAL bioactivity and contribute to the understanding of mushroom lectins' antitumor activity. 相似文献998.
Gomez-Godinez V Wu T Sherman AJ Lee CS Liaw LH Zhongsheng Y Yokomori K Berns MW 《Nucleic acids research》2010,38(22):e202
In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1-2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ~ 34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories. 相似文献
999.
1000.