全文获取类型
收费全文 | 5326篇 |
免费 | 491篇 |
国内免费 | 628篇 |
出版年
2024年 | 17篇 |
2023年 | 77篇 |
2022年 | 186篇 |
2021年 | 295篇 |
2020年 | 209篇 |
2019年 | 245篇 |
2018年 | 196篇 |
2017年 | 143篇 |
2016年 | 195篇 |
2015年 | 338篇 |
2014年 | 371篇 |
2013年 | 339篇 |
2012年 | 473篇 |
2011年 | 429篇 |
2010年 | 286篇 |
2009年 | 258篇 |
2008年 | 253篇 |
2007年 | 295篇 |
2006年 | 250篇 |
2005年 | 205篇 |
2004年 | 181篇 |
2003年 | 176篇 |
2002年 | 171篇 |
2001年 | 100篇 |
2000年 | 80篇 |
1999年 | 61篇 |
1998年 | 51篇 |
1997年 | 58篇 |
1996年 | 56篇 |
1995年 | 50篇 |
1994年 | 47篇 |
1993年 | 40篇 |
1992年 | 42篇 |
1991年 | 23篇 |
1990年 | 28篇 |
1989年 | 37篇 |
1988年 | 24篇 |
1987年 | 15篇 |
1986年 | 24篇 |
1985年 | 14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6445条查询结果,搜索用时 31 毫秒
911.
Shizhen Wang Yewande Alimi Ailing Tong Colin G. Nichols Decha Enkvetchakul 《The Journal of biological chemistry》2009,284(5):2854-2860
Potassium channels are tetrameric proteins that mediate
K+-selective transmembrane diffusion. For KcsA, tetramer stability
depends on interactions between permeant ions and the channel pore. We have
examined the role of pore blockers on the tetramer stability of KirBac1.1. In
150 mm KCl, purified KirBac1.1 protein migrates as a monomer
(∼40 kDa) on SDS-PAGE. Addition of Ba2+
(K1/2 ∼ 50 μm) prior to loading results
in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue
located near the expected Ba2+-binding site, decreased tetramer
stabilization by Ba2+ (K1/2 ∼ 300
μm), whereas I131C, located nearby, stabilized tetramers in the
absence of Ba2+. Neither mutation affected Ba2+ block of
channel activity (using 86Rb+ flux assay). In contrast
to Ba2+, Mg2+ had no effect on tetramer stability (even
though Mg2+ was a potent blocker). Many studies have shown
Cd2+ block of K+ channels as a result of cysteine
substitution of cavity-lining M2 (S6) residues, with the implicit
interpretation that coordination of a single ion by cysteine side chains along
the central axis effectively blocks the pore. We examined blocking and
tetramer-stabilizing effects of Cd2+ on KirBac1.1 with cysteine
substitutions in M2. Cd2+ block potency followed an α-helical
pattern consistent with the crystal structure. Significantly, Cd2+
strongly stabilized tetramers of I138C, located in the center of the inner
cavity. This stabilization was additive with the effect of Ba2+,
consistent with both ions simultaneously occupying the channel:
Ba2+ at the selectivity filter entrance and Cd2+
coordinated by I138C side chains in the inner cavity.Potassium channels are expressed in many cell types and are key players in
a wide range of physiological processes. One subset of potassium channels, the
inward-rectifying potassium (Kir) channels, are functionally blocked by
cytosolic cations such as Mg2+ and polyamines and contribute to the
regulation of membrane excitability, cardiac rhythm, vascular tone, insulin
release, and salt flow across epithelia
(1–3).
There are seven subfamilies of eukaryotic Kir channel genes. Among them, Kir1
encodes weak rectifiers, whereas Kir2 and Kir5 encode strong rectifiers; Kir3
encodes G-protein-regulated channels; and Kir6 encodes ATP-sensitive channels
(4). Recently, a related
bacterial family of genes (KirBac) has been identified
(5,
6), and in 2003, the first
member (KirBac1.1) was crystallized
(7), providing a structural
model for eukaryotic channels.The crystal structure of KirBac1.1 revealed a tetrameric pore structure
similar to that seen in KcsA and a novel cytoplasmic domain
(7,
8). The selectivity filter of
both KirBac1.1 and KcsA consists of an extremely conserved pore loop followed
by a central cavity, forming a transmembrane ion-selective permeation pore
(7,
8). The linear arrangement of
five oxygen rings (four from carbonyl oxygens and one from a Thr side chain)
in the selectivity filter coordinates with ions, compensating for the energy
barrier caused by K+ dehydration, thereby facilitating the rapid
diffusion of K+ across the membrane
(8–12).
Two-thirds of the KirBac1.1 amino acid residues constitute the cytosolic
domain that is highly conserved among the Kir subfamilies and form the
cytosolic vestibule
(13–16),
which, together with the transmembrane pore, generates an 88-Å-long ion
conduction pore (7).The prototypic potassium channel KcsA exists very stably as a tetramer,
even in the harsh conditions of SDS-PAGE
(17). In addition to
protein-protein interaction between monomers, protein-lipid and protein-ion
interactions play important roles in stabilizing the KcsA tetramer
(17–20).
The selectivity filter of KcsA, coordinated with K+ ions, can serve
as a bridge between the four monomers to maintain the structure of the
selectivity filter and the tetrameric architecture of the channel as a whole
(11,
21). Blocking ions, such as
Ba2+, also act as strong stabilizers
(17). In the crystal structure
of KcsA, Ba2+ occupies a site equivalent to the S4
K+-binding site within the selectivity filter
(22). Other permeant ions
(Rb+, Cs+, Tl+, and
NH+4) and strong blockers (Sr2+) can also
contribute to the thermostability of the KcsA tetramer in SDS-PAGE
(17). In contrast, impermeant
ions such as Na+ and Li+ or weak blockers such as
Mg2+ tend to destabilize the KcsA tetramer
(17,
19).Like KcsA, KirBac1.1 purified using decylmaltoside or tridecylmaltoside is
active and presumably stable as a tetramer in mild detergent solutions.
However, in SDS-PAGE, KirBac1.1 migrates exclusively as a monomer
(23). Because KcsA and
KirBac1.1 are structurally similar in the transmembrane region of the pore, we
hypothesized that permeant and blocking ions would also affect KirBac1.1
tetramer stability in SDS-PAGE. In the present work, the effects of blocking
ions such as Ba2+ and Mg2+ on KirBac1.1 tetramer
stability were examined to provide insight to the physical nature of their
interaction with KirBac1.1, particularly in the selectivity filter and TM2
cavity. The data reveal important differences in the nature of the interaction
of Mg2+ and Ba2+ with the channel as well as provide
previously unavailable evidence for the nature of Cd2+ coordination
within the channel. 相似文献
912.
本文研究了天山北麓干旱、湿润2种不同水分条件下拟南芥(Arabidopsis thaliana)光合生理、形态及发育特征的自然适应特点。结果表明:分枝数、株高、相对适合度(角果质量)、莲座叶面积等特征在不同水分下差异极显著;根质量与相对适合度、莲座叶面积、水分利用效率和光合速率之间表现了正相关,胞间二氧化碳浓度与光合速率、水分利用效率之间表现了负相关;选择梯度分析得出,干湿水分条件下分枝数、株高、莲座叶面积均表现定向选择;主成分分析发现,光合生理特征整合在处理间有一定的规律性,表现出拟南芥物种特征整体协调一致性较强,对环境响应具有较大的趋同性;通径分析发现,水分利用效率、枝质量、株高、角果数对相对适合度直接产生较大影响,因而在通径模型中设置为直接路径特征,将莲座叶面积通过分枝数、花期、角果数,花期通过角果数到达相对适合度的过程设置为间接路径,这些特征通过间接路径对相对适合度间接产生较大影响。 相似文献
913.
Chung-Hin Chui Raymond Siu-Ming Wong Roberto Gambari Gregory Yin-Ming Cheng Marcus Chun-Wah Yuen Kit-Wah Chan See-Wai Tong Fung-Yi Lau Paul Bo-San Lai Kim-Hung Lam Cheuk-Lam Ho Chi-Wai Kan Kelvin Sze-Yin Leung Wai-Yeung Wong 《Bioorganic & medicinal chemistry》2009,17(23):7872-7877
A list of diethynylfluorenes and their gold(I) derivatives have been studied for their antitumor activity as a function of their structure–activity relationships. End-capping the fluoren-9-one unit with gold(I) moieties could significantly strengthen the cytotoxic activity in vitro on three human cancer cell lines with induction of reactive oxygen species generation on Hep3B hepatocellular carcinoma cells and exhibit attractive antitumor activity from in vivo nude mice Hep3B xenograft model with limited adverse effects on vital organs including liver and kidney. 相似文献
914.
Hucho taimen are listed as endangered in China. The population size has declined recently, prompting an increase in the level of listing from grade three in 2002 to grade five in 2006. We analyzed the genetic diversity of wild populations using 17 microsatellite markers to establish a scientific basis for conservation of this species. We collected tissue samples from four populations in the Heilongjiang River basin: Huma River (HM), Hutou (HT), Haiqing (HQ), and Zhuaji (ZJ). A total of 21 loci were amplified, 18 of which were polymorphic. The number of alleles per locus ranged from 2 to 9 (mean: 4.1905). There were 13 highly polymorphic loci and 5 moderately polymorphic loci. Analysis of five genetic diversity parameters (Na, Ne, Ho, He, and PIC) suggested moderate levels of diversity within the populations. The populations were ranked HT > HQ > ZJ > HM, but the differences in diversity were not statistically significant (P > 0.05). A comparison of variation among all four populations suggested Hardy–Weinberg disequilibrium at 20% of the loci. Genetic differentiation (Fst) was 0.0644 and the gene flow among populations was estimated at 3.36 individuals per generation. The majority of diversity (93.88%) occurred among individuals within a population. In contrast, relatively little (6.12%) of the genetic diversity was distributed between the populations. An analysis of genetic differentiation and genetic distance between pairs of populations revealed that both parameters were higher in comparisons of the HM population to the HT, HQ, and ZJ populations than among the three latter populations. This suggests that the HM population has a distinct genetic structure. We hypothesize that habitat degradation and excessive fishing, not low genetic diversity, has caused the decline in H. taimen populations. However, this species should be protected from further declines in genetic diversity. 相似文献
915.
Ivan F.N. Hung Pierre Chan Sally Leung Fion S.Y. Chan Axel Hsu David But Wai Kay Seto Siu Yin Wong Chi Kuen Chan Qing Gu Teresa S.M. Tong Ting Kin Cheung Kent Man Chu Benjamin C.Y. Wong 《Helicobacter》2009,14(6):505-511
Background: Recent studies have suggested the eradication rate for Helicobacter pylori infection with standard amoxycillin–clarithromycin‐containing triple therapy as first‐line treatment have fallen below 80%. Levofloxacin‐containing triple therapy was proposed as an alternative. The aim of this study is to compare the efficacy and tolerability of the standard 7‐day clarithromycin‐containing triple therapy against the 7‐day levofloxacin‐containing triple therapy, and to assess whether the classical triple therapy is still valid as empirical first‐line treatment for H. pylori infection in Hong Kong. Methods: Three hundred consecutive H. pylori‐positive patients were randomized to receive either 1 week of EAL (esomeprazole 20 mg b.d., amoxycillin 1 g b.d., and levofloxacin 500 mg daily) or EAC (esomeprazole 20 mg b.d., amoxycillin 1 g b.d., and clarithromycin 500 mg b.d.). H. pylori status was rechecked by 13C‐urea breath test 6 weeks after treatment. Patients who failed either of the first‐line eradication therapy were invited to undergo H. pylori susceptibility testing. Results: H. pylori eradication was achieved in 128 of 150 (85.3%) patients in EAL and 139 of 150 (92.7%) patients in EAC groups, respectively (p = .043), for both intention‐to‐treat and per‐protocol analysis. More patients in the clarithromycin‐ than the levofloxacin‐containing therapy group developed side effects from the medication (21.3% vs 13.3%, p = .060). Nine patients (six from the EAL group and three from the EAC group) who failed their corresponding eradication therapy returned for susceptibility testing. All nine isolates were highly resistant to levofloxacin (minimum inhibitory concentration or MIC > 32 μg/mL), whereas only two of the six isolates from the EAL group were resistant to clarithromycin (MIC > 0.5 μg/mL). Conclusions: The standard 7‐day clarithromycin‐containing triple therapy is still valid as the most effective empirical first‐line eradication therapy for H. pylori infection in Hong Kong, as prevalence of primary resistance of H. pylori to amoxycillin and clarithromycin remains low. Patients who failed their empirical first‐line eradication therapy should undergo H. pylori susceptibility testing to guide further treatment. 相似文献
916.
表达O型口蹄疫病毒VPl基因的重组病毒BHV-1的构建与鉴定 总被引:1,自引:0,他引:1
[目的]为了构建表达口蹄疫病毒(O/china/99)VP1基因的牛疱疹病毒1型,将人工合成的口蹄疫病毒VP1基因插入到巨细胞病毒(CMV)启动子之下构建gE基因缺失转移载体.[方法]利用磷酸钙介导转染法将该转移载体与亲本病毒BHV-1/gE-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒.通过筛选白色病毒蚀斑,得到重组病毒BHV-1/gE-/VP1.[结果]PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中,间接免疫荧光试验和Westem blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达.[结论]本研究成功地构建了表达口蹄疫病毒VP1基因的重组病毒BHV-1/gE-/VP1,为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础. 相似文献
917.
918.
Yan Liang Qiang Xu An Kang Yuan Xie Tong Xie Li Liu Haiping Hao Lin Xie Guang-ji Wang 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2009,877(18-19):1765-1770
We herein describe the development of an LC–MS method for simultaneous determination of astilbin and 3′-O-methylastilbin in rat plasma. A simple liquid–liquid extraction procedure was followed by injection of the extracts on to a Shim-pack C18 column (150 mm × 2.0 mm I.D., 5 μm) with gradient elution and detection in negative ionization mode. Initially, the method was validated regarding linearity, accuracy and precision. The correlation coefficients of all the calibration curves showed good linearity (r > 0.999) within test ranges, and the relative deviation was less than 10% for intra- and inter-day assays. Besides, this method was also validated for its stability, extraction efficiency, matrix effect and so on. Finally, this proposed method was successfully applied to rat pharmacokinetic study and yielded the most comprehensive data on systemic exposure of them to date. 相似文献
919.
Shuyang Tu Yan Ren Wei Tong Shirong Zheng Ningzhi Xu Aruni Bhatnagar Siqi Liu 《Proteomics》2009,9(22):5090-5100
The aldo–keto reductase (AKR) proteins catalyze reduction of diverse aldehydes and play detoxification roles in many organisms. Since many substrates are shared among AKR, it is generally accepted that these enzymes can functionally compensate each other in response to oxidative stress. Their overall abundances are the important factor that partially reflects the capacity of antioxidant and detoxification in tissues. In this study, the strategy was proposed for generation of Pan‐AKR antibodies to recognize most AKR proteins in mouse tissues. Derived from bioinformatic analysis, several consensus peptides with different potential antigenicities were synthesized, conjugated to hemocyanin from keyhole limpets and further delivered to rabbits to generate polyclonal antibodies. Three Pan‐AKR antibodies exhibited the immune specificities and immune sensitivities, Pan‐AKR‐P1 for AKR1B and AKR1C, Pan‐AKR‐P3 for AKR1C and Pan‐AKR‐P4 for all the AKR proteins. Pan‐AKR‐P4 antibody was employed to 2‐DE Western blot to examine the AKR abundances in mouse liver and kidney, resulting in seven immune‐reactive spots from each tissue. Protein identification with MS revealed that most immune‐positive spots were the members of AKR superfamily. Furthermore, Pan‐AKR‐P4 antibody was implemented to compare the different abundances of the AKR proteins in liver and kidney between normal and diabetic mice, suggesting that diabetes did cause some abnormal changes in the AKR protein abundances. 相似文献
920.
The roles of weather variability and sunspots in the occurrence of cyanobacteria blooms, were investigated using cyanobacteria
cell data collected from the Fred Haigh Dam, Queensland, Australia. Time series generalized linear model and classification
and regression tree (CART) model were used in the analysis. Data on notified cell numbers of cyanobacteria and weather variables
over the periods 2001 and 2005 were provided by the Australian Department of Natural Resources and Water, and Australian Bureau
of Meteorology, respectively. The results indicate that monthly minimum temperature (relative risk [RR]: 1.13, 95% confidence
interval [CI]: 1.02–1.25) and rainfall (RR: 1.11; 95% CI: 1.03–1.20) had a positive association, but relative humidity (RR:
0.94; 95% CI: 0.91–0.98) and wind speed (RR: 0.90; 95% CI: 0.82–0.98) were negatively associated with the cyanobacterial numbers,
after adjustment for seasonality and auto-correlation. The CART model showed that the cyanobacteria numbers were best described
by an interaction between minimum temperature, relative humidity, and sunspot numbers. When minimum temperature exceeded 18°C
and relative humidity was under 66%, the number of cyanobacterial cells rose by 2.15-fold. We conclude that weather variability
and sunspot activity may affect cyanobacteria blooms in dams. 相似文献