全文获取类型
收费全文 | 5632篇 |
免费 | 519篇 |
国内免费 | 746篇 |
专业分类
6897篇 |
出版年
2024年 | 26篇 |
2023年 | 95篇 |
2022年 | 222篇 |
2021年 | 315篇 |
2020年 | 233篇 |
2019年 | 264篇 |
2018年 | 206篇 |
2017年 | 156篇 |
2016年 | 201篇 |
2015年 | 362篇 |
2014年 | 405篇 |
2013年 | 380篇 |
2012年 | 515篇 |
2011年 | 470篇 |
2010年 | 309篇 |
2009年 | 281篇 |
2008年 | 278篇 |
2007年 | 309篇 |
2006年 | 272篇 |
2005年 | 218篇 |
2004年 | 182篇 |
2003年 | 173篇 |
2002年 | 173篇 |
2001年 | 101篇 |
2000年 | 81篇 |
1999年 | 60篇 |
1998年 | 50篇 |
1997年 | 57篇 |
1996年 | 54篇 |
1995年 | 49篇 |
1994年 | 45篇 |
1993年 | 40篇 |
1992年 | 41篇 |
1991年 | 23篇 |
1990年 | 27篇 |
1989年 | 37篇 |
1988年 | 24篇 |
1987年 | 15篇 |
1986年 | 24篇 |
1985年 | 15篇 |
1984年 | 9篇 |
1983年 | 13篇 |
1982年 | 8篇 |
1981年 | 9篇 |
1980年 | 13篇 |
1979年 | 12篇 |
1977年 | 6篇 |
1976年 | 5篇 |
1975年 | 5篇 |
1971年 | 6篇 |
排序方式: 共有6897条查询结果,搜索用时 0 毫秒
21.
PerR-Regulated Manganese Ion Uptake Contributes to Oxidative Stress Defense in an Oral Streptococcus
Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H2O2, whereas Dpr (an Fe2+-chelating protein)-dependent H2O2 protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H2O2-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl2, S. oligofermentans increased survival after H2O2 stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn2+-mediated H2O2 resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ∼0.4 μM Mn2+) unless it was supplemented with ≥2.5 μM MnCl2 and caused 82 to 95% reduction of the cellular Mn2+ level, while mntABC overexpression increased cellular Mn2+ 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn2+ transporter in S. oligofermentans. mntA mutation reduced the survival of H2O2-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H2O2-challenged survival 12-fold, indicating that MntABC-mediated Mn2+ uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H2O2 pulsing upregulated mntABC, while H2O2-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H2O2 can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H2O2 stress defenses and may be distributed across all oral streptococci lacking catalase. 相似文献
22.
Jia Liu Feng-Yu Liu Zhi-Qian Tong Zhi-Hua Li Wen Chen Wen-Hong Luo Hui Li Hong-Jun Luo Yan Tang Jun-Min Tang Jie Cai Fei-Fei Liao You Wan 《PloS one》2013,8(3)
Background
Bone cancer pain seriously affects the quality of life of cancer patients. Our previous study found that endogenous formaldehyde was produced by cancer cells metastasized into bone marrows and played an important role in bone cancer pain. However, the mechanism of production of this endogenous formaldehyde by metastatic cancer cells was unknown in bone cancer pain rats. Lysine-specific demethylase 1 (LSD1) is one of the major enzymes catalyzing the production of formaldehyde. The expression of LSD1 and the concentration of formaldehyde were up-regulated in many high-risk tumors.Objective
This study aimed to investigate whether LSD1 in metastasized MRMT-1 breast cancer cells in bone marrows participated in the production of endogenous formaldehyde in bone cancer pain rats.Methodology/Principal Findings
Concentration of the endogenous formaldehyde was measured by high performance liquid chromatography (HPLC). Endogenous formaldehyde dramatically increased in cultured MRMT-1 breast cancer cells in vitro, in bone marrows and sera of bone cancer pain rats, in tumor tissues and sera of MRMT-1 subcutaneous vaccination model rats in vivo. Formaldehyde at a concentration as low as the above measured (3 mM) induced pain behaviors in normal rats. The expression of LSD1 which mainly located in nuclei of cancer cells significantly increased in bone marrows of bone cancer pain rats from 14 d to 21 d after inoculation. Furthermore, inhibition of LSD1 decreased the production of formaldehyde in MRMT-1 cells in vitro. Intraperitoneal injection of LSD1 inhibitor pargyline from 3 d to 14 d after inoculation of MRMT-1 cancer cells reduced bone cancer pain behaviors.Conclusion
Our data in the present study, combing our previous report, suggested that in the endogenous formaldehyde-induced pain in bone cancer pain rats, LSD1 in metastasized cancer cells contributed to the production of the endogenous formaldehyde. 相似文献23.
Tong L Lin Q Wong WK Ali A Lim D Sung WL Hew CL Yang DS 《Protein expression and purification》2000,18(2):175-181
HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) from the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium. Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG induction, a biologically active rAFP was expressed. The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm. After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC. Mass spectrometric and amino acid composition analyses confirmed the identity of the purified product. The rAFP, which lacked amidation at the C-terminal, was about 70% active when compared to the amidated wild-type protein, thus confirming the importance of C-terminal cap structure in protein stability and function. 相似文献
24.
汉族马凡综合征(MFS)患者FBN1基因两种新发突变分析 总被引:1,自引:0,他引:1
为调查马凡综合征(Marfan syndrome, MFS)患者的原纤维蛋白-1(Fibrillin-1, FBN1)基因突变情况, 应用聚合酶链反应(PCR)和变性高效液相色谱法(Denaturing high-performance liquid chromatography, DHPLC)对MFS患者的FBN1基因进行突变筛查, 对DHPLC初筛异常的DNA片段进行测序分析。结果在两个MFS家系中发现FBN1基因两种新的突变: 一种为复合突变包含第55号外显子的缺失突变c.6862_6871delGGCTGTGTAG (p.Gly2288MetfsX109)、同义突变c.6861A>G和内含子的突变c.[6871+1_6871+11delGTAAGAGGATC; 6871+34dupCATCAGAAGTGACAGTGGACA]; 另一种为第20号外显子的错义突变c.2462G>A(p.Cys821Tyr)。研究表明, FBN1基因的缺失突变c.[6862_6871delGGCTGTGTAG; 6871+1_6871+11delGTAAGAGGATC] (p.Gly2288MetfsX109)和错义突变c.2462G>A(p.Cys821Tyr)可能分别是这两个家系患者的致病原因。 相似文献
25.
Ping Xu Yangxi Zheng Jiujiang Liao Mingyu Hu Yike Yang Baozhen Zhang Mark D. Kilby Huijia Fu Yamin Liu Fumei Zhang Liling Xiong Xiyao Liu Huili Jin Yue Wu Jiayu Huang Tingli Han Li Wen Rufei Gao Yong Fu Xiujun Fan Hongbo Qi Philip N. Baker Chao Tong 《Cell proliferation》2023,56(2)
Pre‐eclampsia (PE) is deemed an ischemia‐induced metabolic disorder of the placenta due to defective invasion of trophoblasts during placentation; thus, the driving role of metabolism in PE pathogenesis is largely ignored. Since trophoblasts undergo substantial glycolysis, this study aimed to investigate its function and regulatory mechanism by AMPK in PE development. Metabolomics analysis of PE placentas was performed by gas chromatography–mass spectrometry (GC–MS). Trophoblast‐specific AMPKα1‐deficient mouse placentas were generated to assess morphology. A mouse PE model was established by Reduced Uterine Perfusion Pressure, and placental AMPK was modulated by nanoparticle‐delivered A769662. Trophoblast glucose uptake was measured by 2‐NBDG and 2‐deoxy‐d‐[3H] glucose uptake assays. Cellular metabolism was investigated by the Seahorse assay and GC–MS.PE complicated trophoblasts are associated with AMPK hyperactivation due not to energy deficiency. Thereafter, AMPK activation during placentation exacerbated PE manifestations but alleviated cell death in the placenta. AMPK activation in trophoblasts contributed to GLUT3 translocation and subsequent glucose metabolism, which were redirected into gluconeogenesis, resulting in deposition of glycogen and accumulation of phosphoenolpyruvate; the latter enhanced viability but compromised trophoblast invasion. However, ablation of AMPK in the mouse placenta resulted in decreased glycogen deposition and structural malformation. These data reveal a novel homeostasis between invasiveness and viability in trophoblasts, which is mechanistically relevant for switching between the ‘go’ and ‘grow’ cellular programs.Pre‐eclampsia (PE) is associated with trophoblast AMPK hyperactivation, presumably due to LKB1 phosphorylation, and glucose uptake is consequently increased via trafficking of GLUT3 from the cytosol to the plasma membrane. Such translocation enhances glycolytic flux and redirects glucose metabolic intermediates into gluconeogenesis, resulting in PEP accumulation, which not only benefits cell survival but also suppresses invasion by repressing MMPs, and thus in turn modulates switching between the ‘go’ and ‘grow’ cellular programs. 相似文献
26.
27.
28.
Construction of a recombinant BHV-1 expressing the VP1 gene of foot and mouth disease virus and its immunogenicity in a rabbit model 总被引:1,自引:0,他引:1
Xian-Gang Ren Fei Xue Yuan-Mao Zhu Guang-Zhi Tong Yan-Hui Wang Jun-Ke Feng Hong-Fei Shi Yu-Ran Gao 《Biotechnology letters》2009,31(8):1159-1165
Foot-and-mouth disease (FMD) and infectious bovine rhinotracheitis (IBR) are two important infectious diseases of cattle.
Using bovine herpesvirus type 1 (BHV-1) as a gene delivery vector for development of live-viral vaccines has gained widespread
interest. In this study, a recombinant BHV-1 was constructed by inserting the synthetic FMDV (O/China/99) VP1 gene in the
the gE locus of BHV-1 genome under the control of immediately early gene promoter of human cytomegalovirus (phIE CMV) and
bovine growth hormone polyadenylation (BGH polyA) signal. After homologous recombination and plaque purification, a recombinant
virus named BHV-1/gE−/VP1 was acquired and identified. The immunogenicity was confirmed in a rabbit model by virus neutralization test and enzyme-linked
immunosorbent assay (ELISA). The result indicated that the BHV-1/gE−/VP1 has the potential for being developed as a bivalent vaccine for FMD and IBR. 相似文献
29.
Religious priming has been found to have both positive and negative consequences, and recent research suggests that the activation of God-related and community-related religious cognitions may cause outgroup prosociality and outgroup derogation respectively. The present research sought to examine whether reminders of God and religion have different effects on attitudes towards ingroup and outgroup members. Over two studies, little evidence was found for different effects of these two types of religious primes. In study 1, individuals primed with the words “religion”, “God” and a neutral control word evaluated both ingroup and outgroup members similarly, although a marginal tendency towards more negative evaluations of outgroup members by females exposed to religion primes was observed. In study 2, no significant differences in attitudes towards an outgroup member were observed between the God, religion, and neutral priming conditions. Furthermore, the gender effect observed in study 1 did not replicate in this second study. Possible explanations for these null effects are discussed. 相似文献
30.
低氧对胚胎干细胞增殖的影响 总被引:3,自引:1,他引:3
目的:观察间歇性低氧和持续性低氧对体外培养的胚胎干细胞(ES细胞)增殖的影响.方法:利用细胞记数法和BrdU (5-溴脱氧尿苷)掺入的流式细胞分析检测细胞增殖,并用RT -PCR的方法检测低氧诱导因子(HIF-1a)的表达变化.结果:①将ES细胞分别放在低氧(3%~10% O2)和常氧(20% O2)的环境中培养24 h后,在低氧环境中培养的ES细胞数较常氧组明显减少;②将ES细胞分别给予间歇性低氧刺激(3%~10% O2),每天10 min,连续4 d后,发现3%低氧组较常氧对照组的细胞增殖明显升高.③用RT-PCR方法观察HIF-1a的表达与细胞增殖的关系,发现在常氧环境中培养的ES即有HIF-1a的表达,ES细胞在持续低氧24 h或间歇性低氧(3%~10% O2)刺激4 d后对HIF-1a的表达均无明显影响.结论:间歇性低氧(3% O2)可明显促进体外培养的ES细胞增殖,而持续性低氧抑制ES细胞增殖,间歇性低氧(3% O2)刺激促进ES细胞增殖的机制尚有待于进一步的研究. 相似文献