全文获取类型
收费全文 | 5858篇 |
免费 | 407篇 |
国内免费 | 9篇 |
专业分类
6274篇 |
出版年
2023年 | 19篇 |
2022年 | 53篇 |
2021年 | 115篇 |
2020年 | 59篇 |
2019年 | 98篇 |
2018年 | 136篇 |
2017年 | 113篇 |
2016年 | 134篇 |
2015年 | 257篇 |
2014年 | 251篇 |
2013年 | 375篇 |
2012年 | 459篇 |
2011年 | 502篇 |
2010年 | 288篇 |
2009年 | 261篇 |
2008年 | 408篇 |
2007年 | 394篇 |
2006年 | 403篇 |
2005年 | 349篇 |
2004年 | 330篇 |
2003年 | 304篇 |
2002年 | 300篇 |
2001年 | 44篇 |
2000年 | 38篇 |
1999年 | 61篇 |
1998年 | 96篇 |
1997年 | 30篇 |
1996年 | 49篇 |
1995年 | 35篇 |
1994年 | 31篇 |
1993年 | 30篇 |
1992年 | 30篇 |
1991年 | 19篇 |
1990年 | 19篇 |
1989年 | 13篇 |
1988年 | 10篇 |
1987年 | 5篇 |
1986年 | 8篇 |
1985年 | 9篇 |
1984年 | 12篇 |
1983年 | 7篇 |
1982年 | 22篇 |
1981年 | 12篇 |
1980年 | 10篇 |
1979年 | 6篇 |
1978年 | 6篇 |
1977年 | 9篇 |
1976年 | 7篇 |
1975年 | 6篇 |
1974年 | 7篇 |
排序方式: 共有6274条查询结果,搜索用时 15 毫秒
91.
92.
Kvasnica M Budesinsky M Swaczynova J Pouzar V Kohout L 《Bioorganic & medicinal chemistry》2008,16(7):3704-3713
Twelve steroidal platinum(II) complexes were synthesized by reaction of potassium tetrachloroplatinate with steroidal esters of L-methionine and L-histidine. The steroidal esters coordinated as bidentate ligands via S and N donor atoms of L-methionine and via two N donor atoms of L-histidine. Cholesterol, cholestanol, diosgenine, pregnenolone, dehydroepiandrosterone, testosterone, estrone, and estradiol were used as the steroidal compounds. The esters and complexes prepared were characterized by infrared, mass, and (1)H NMR spectroscopy and elemental analysis. Platinum complexes were tested for in vitro cytotoxicity against several cancer cell lines: T-lymphoblastic leukemia CEM, breast carcinoma MCF-7, lung carcinoma A-549, multiple myeloma RPMI 8226, and one normal cell line human fibroblast BJ. 相似文献
93.
Ilyinskii PO Gambaryan AS Meriin AB Gabai V Kartashov A Thoidis G Shneider AM 《PloS one》2008,3(1):e1417
The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed. 相似文献
94.
Energetics of vesicle fusion intermediates: comparison of calculations with observed effects of osmotic and curvature stresses 下载免费PDF全文
We reported previously the effects of both osmotic and curvature stress on fusion between poly(ethylene glycol)-aggregated vesicles. In this article, we analyze the energetics of fusion of vesicles of different curvature, paying particular attention to the effects of osmotic stress on small, highly curved vesicles of 26 nm diameter, composed of lipids with negative intrinsic curvature. Our calculations show that high positive curvature of the outer monolayer "charges" these vesicles with excess bending energy, which then releases during stalk expansion (increase of the stalk radius, r(s)) and thus "drives" fusion. Calculations based on the known mechanical properties of lipid assemblies suggest that the free energy of "void" formation as well as membrane-bending free energy dominate the evolution of a stalk to an extended transmembrane contact. The free-energy profile of stalk expansion (free energy versus r(s)) clearly shows the presence of two metastable intermediates (intermediate 1 at r(s) approximately 0 - 1.0 nm and intermediate 2 at r(s) approximately 2.5 - 3.0 nm). Applying osmotic gradients of +/-5 atm, when assuming a fixed trans-bilayer lipid mass distribution, did not significantly change the free-energy profile. However, inclusion in the model of an additional degree of freedom, the ability of lipids to move into and out of the "void", made the free-energy profile strongly dependent on the osmotic gradient. Vesicle expansion increased the energy barrier between intermediates by approximately 4 kT and the absolute value of the barrier by approximately 7 kT, whereas compression decreased it by nearly the same extent. Since these calculations, which are based on the stalk hypothesis, correctly predict the effects of both membrane curvature and osmotic stress, they support the stalk hypothesis for the mechanism of membrane fusion and suggest that both forms of stress alter the final stages, rather than the initial step, of the fusion process, as previously suggested. 相似文献
95.
Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5′-end and a non-fluorescent quencher at the 3′-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2–4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB–quencher interaction and concealment of the MGB moiety inside the minor groove. 相似文献
96.
Magalhães ML Czekster CM Guan R Malashkevich VN Almo SC Levy M 《Protein science : a publication of the Protein Society》2011,20(7):1145-1154
We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ~10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions. 相似文献
97.
Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) (Biochemistry 35, 8742-8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast kon constants at 4.5 °C: 6.6 × 108 M− 1 s− 1 for nNOS726-749, 2.9 × 108 M− 1 s− 1 for eNOS492-511 and 6.1 × 108 M− 1 s− 1 for iNOS507-531, 3-4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca2+ chelation with ETDA: 3.7 s− 1 for nNOS726-749, 4.5 s− 1 for eNOS492-511, and 0.063 s− 1 for iNOS507-531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS507-531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM. 相似文献
98.
Pospichalova V Tureckova J Fafilek B Vojtechova M Krausova M Lukas J Sloncova E Takacova S Divoky V Leprince D Plachy J Korinek V 《Genesis (New York, N.Y. : 2000)》2011,49(3):142-151
HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located on chromosome 17p13.3, a region frequently hypermethylated or deleted in human neoplasias. In mouse, Hic1 is essential for embryonic development and exerts an antitumor role in adult animals. Since Hic1-deficient mice die perinatally, we generated a conditional Hic1 null allele by flanking the Hic1-coding region by loxP sites. When crossed to animals expressing Cre recombinase in a cell-specific manner, the Hic1 conditional mice will provide new insights into the function of Hic1 in developing and mature tissues. Additionally, we used gene targeting to replace sequence-encoding amino acids 186-893 of Hic1 by citrine fluorescent protein cDNA. We demonstrate that the distribution of Hic1-citrine fusion polypeptide corresponds to the expression pattern of wild-type Hic1. Consequently, Hic1-citrine "reporter" mice can be used to monitor the activity of the Hic1 locus using citrine fluorescence. 相似文献
99.
Muñoz A Sojo F Arenas DR Kouznetsov VV Arvelo F 《Chemico-biological interactions》2011,189(3):215-221
Two tetrahydroquinoline compounds, called DM8 and DM12, from a new series of the cis-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines, were selected for cytotoxic effects studies on cellular lines of human breast cancer. The synergistic, additive and antagonistic effects in combination of these compounds with anticancer drugs, such as paclitaxel and gemcitabine, were studied. The isobolograms and their analysis demonstrated models of synergism, additivity and antagonism of these tetrahydroquinolines in the presence of paclitaxel and gemcitabine. Results showed that compounds DM8 and DM12 individually induced growth inhibition on breast cancer cell lines MCF-7 and SKBR3, and the addition of paclitaxel and gemcitabine intensified their cytotoxic activity on both cell lines at conc. below 1 μg/mL. During these studies the compound DM12 was identified as new, perspective and safe agent for adjuvant therapy. 相似文献
100.
Organ printing, or the layer by layer additive robotic biofabrication of functional three-dimensional tissue and organ constructs using self-assembling tissue spheroid building blocks, is a rapidly emerging technology that promises to transform tissue engineering into a commercially successful biomedical industry. It is increasingly obvious that similar well-established industries implement automated robotic systems on the path to commercial translation and economic success. The use of robotic bioprinters alone however is not sufficient for the development of large industrial scale organ biofabrication. The design and development of a fully integrated organ biofabrication line is imperative for the commercial translation of organ printing technology. This paper presents recent progress and challenges in the development of the essential components of an organ biofabrication line. 相似文献