全文获取类型
收费全文 | 768篇 |
免费 | 65篇 |
出版年
2022年 | 5篇 |
2021年 | 5篇 |
2019年 | 5篇 |
2018年 | 9篇 |
2017年 | 3篇 |
2016年 | 14篇 |
2015年 | 31篇 |
2014年 | 35篇 |
2013年 | 45篇 |
2012年 | 52篇 |
2011年 | 44篇 |
2010年 | 30篇 |
2009年 | 26篇 |
2008年 | 49篇 |
2007年 | 45篇 |
2006年 | 41篇 |
2005年 | 49篇 |
2004年 | 35篇 |
2003年 | 41篇 |
2002年 | 47篇 |
2001年 | 7篇 |
2000年 | 3篇 |
1999年 | 8篇 |
1998年 | 6篇 |
1997年 | 5篇 |
1996年 | 7篇 |
1995年 | 5篇 |
1994年 | 6篇 |
1991年 | 3篇 |
1988年 | 3篇 |
1984年 | 7篇 |
1983年 | 6篇 |
1981年 | 6篇 |
1978年 | 4篇 |
1977年 | 11篇 |
1974年 | 4篇 |
1973年 | 3篇 |
1970年 | 3篇 |
1956年 | 3篇 |
1949年 | 5篇 |
1948年 | 5篇 |
1946年 | 3篇 |
1942年 | 3篇 |
1935年 | 4篇 |
1934年 | 3篇 |
1932年 | 5篇 |
1929年 | 3篇 |
1911年 | 2篇 |
1910年 | 3篇 |
1908年 | 2篇 |
排序方式: 共有833条查询结果,搜索用时 398 毫秒
31.
Schizophyllan is a beta(1-->3)-D-glucan polysaccharide with beta(1-->6)-branched lateral glucose residues that presents a very stiff triple-helical structure under most experimental conditions. Despite the remarkable stability of this structure (which persists up to 120 degrees C in aqueous solution), schizophyllan undergoes a major change of state around 7 degrees C in water that has been hypothesized to result from an order-disorder transition in the lateral residues. This hypothesis is only supported by indirect experimental evidence and detailed knowledge (at the atomic level) concerning hydrogen-bonding networks, interactions with the solvent molecules, orientational freedom of the lateral residues, and orientational correlations among them is still lacking. In this study explicit-solvent molecular dynamics simulations of a schizophyllan fragment (complemented by simulations of its tetrasaccharide monomer) are performed at three different temperatures (273 K, 350 K, and 450 K) and with two different types of boundary conditions (finite nonperiodic or infinite periodic fragment) as an attempt to provide detailed structural and dynamical information about the triple-helical conformation in solution and the mechanism of the low-temperature transition. These simulations suggest that three important driving forces for the high stability of the triple helix are i), the limited conformational work involved in its formation; ii), the formation of a dense hydrogen-bonding network at its center; and iii), the formation of interchain hydrogen bonds between main-chain and lateral glucose residues. However, these simulations evidence a moderate and continuous variation of the simulated observables upon increasing the temperature, rather than a sharp transition between the two lowest temperatures (that could be associated with the state transition). Although water-mediated hydrogen-bonded association of neighboring lateral residues is observed, this interaction is not strong enough to promote the formation of an ordered state (correlated motions of the lateral residues), even at the lowest temperature considered. 相似文献
32.
This protocol presents a new method to purify plasmid DNA using temperature-triggered precipitation. The principle is based on the specific DNA-binding affinity of a bacterial metalloregulatory (MerR) protein to its cognate DNA sequence and the temperature responsiveness of elastin-like protein (ELP). A bifunctional ELP-MerR fusion protein is created to enable the precipitation of plasmid DNA, designed to contain the MerR recognition sequence, by a simple temperature trigger. The protocol covers all stages of the process from the design of ELP-MerR fusion proteins and MerR-binding plasmids, to the isolation of plasmid DNA from Escherichia coli cultures after boiling lysis, the subsequent temperature-triggered precipitation of plasmid DNA-fusion protein complexes and final elution of plasmid DNA by mild heating. This protocol is well suited to laboratory research-scale applications, producing plasmid DNA of better purity and similar yield as one of the most commonly used laboratory methods, standard alkaline lysis (known as the midiprep procedure). The protocol takes approximately 30 min to obtain pure plasmid DNA from cell cultures using the temperature-triggered precipitation method. 相似文献
33.
Blodgett JA Thomas PM Li G Velasquez JE van der Donk WA Kelleher NL Metcalf WW 《Nature chemical biology》2007,3(8):480-485
Phosphinothricin tripeptide (PTT, phosphinothricylalanylalanine) is a natural-product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 21705 (ref. 1) and Streptomyces viridochromogenes DSM 40736 (ref. 2). PTT has attracted widespread interest because of its commercial applications and unique phosphinic acid functional group. Despite intensive study since its discovery in 1972 (see ref. 3 for a comprehensive review), a number of steps early in the PTT biosynthetic pathway remain uncharacterized. Here we report a series of interdisciplinary experiments involving the construction of defined S. viridochromogenes mutants, chemical characterization of accumulated intermediates, and in vitro assay of selected enzymes to examine these critical steps in PTT biosynthesis. Our results indicate that early PTT biosynthesis involves a series of catalytic steps that to our knowledge has not been described so far, including a highly unusual reaction for carbon bond cleavage. In sum, we define a pathway for early PTT biosynthesis that is more complex than previously appreciated. 相似文献
34.
To investigate the cause of accumulation of oxidised proteins in the livers of rats with carbon tetrachloride (CCl4) induced liver cirrhosis, the activity of alkaline protease (a high molecular weight, multisubunit cysteine proteinase) was determined in the cirrhotic livers. A significant decrease (P < 0.05) in the activity of hepatic alkaline protease was observed in the cirrhotic rats. Decreased activity of alkaline protease in the liver of cirrhotic rats may contribute to the accumulation of the oxidised proteins in the liver. 相似文献
35.
36.
Kostal J Yang R Wu CH Mulchandani A Chen W 《Applied and environmental microbiology》2004,70(8):4582-4587
The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd(2+) or Zn(2+) accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water. 相似文献
37.
Yadav R Yoshimura Y Boesteanu A Christianson GJ Ajayi WU Shashidharamurthy R Stanic AK Roopenian DC Joyce S 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(10):5133-5142
Minor histocompatibility (H) Ag disparities result in graft-vs-host disease and chronic solid allograft rejection in MHC-identical donor-recipient combinations. Minor H Ags are self protein-derived peptides presented by MHC class I molecules. Most arise as a consequence of allelic variation in the bound peptide (p) that results in TCR recognizing the p/MHC as foreign. We used a combinational peptide screening approach to identify the immune dominant H2K(b)-restricted epitope defining the mouse H4(b) minor H Ag. H4(b) is a consequence of a P3 threonine to isoleucine change in the MHC-bound peptide derived from epithelial membrane protein-3. This allelic variation also leads to phosphorylation of the H4(b) but not the H4(a) epitope. Further, ex vivo CD8(+) T lymphocytes bind phosphorylated Ag tetramers with high efficiency. Although we document the above process in the minor H Ag system, posttranslational modifications made possible by subtle amino acid changes could also contribute to immunogenicity and immune dominance in tumor immunotherapeutic settings. 相似文献
38.
Optimizing chemotherapy by measuring reversal of P-glycoprotein activity in plasma membrane vesicles
The appearance of multidrug resistance (MDR) of cancer cells is a major obstacle to successful chemotherapy. Several proteins have been identified that pump chemotherapeutic drugs out of cells, thus bringing about MDR. One representative pump is the P-glycoprotein, whose function can be inhibited by blockers (also known as reversers, modulators or chemosensitizers). In clinical application, many of these blockers are often not effective because they become bound to the plasma of the patients. The extent of plasma binding of the blocker varies in different persons and we have developed a 96-well kit to assay such inter-person differences. The assay uses membrane vesicles isolated from a human lymphoblastoid cell line (CEM Col1000). Uptake of rhodamine into the vesicles was measured with different concentrations of the blockers verapamil and XR9576 in presence of human plasma. The reverser XR9576 is nearly 30 times more effective than the classical blocker verapamil, the relevant K(m) values ranging from 2.66 to 45 nM for XR 9576 and 0.7 to 5.5 microM for verapamil. An even greater difference between these two drugs, nearly 1,000-fold, could be shown also in intact cells by calcein AM uptake experiments. 相似文献
39.
Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents 总被引:7,自引:0,他引:7
Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman. 相似文献
40.
Photoactive yellow protein (PYP) is a prototype of the PAS domain superfamily of signaling proteins. The signaling process
is coupled to a three-state photocycle. After the photoinduced trans-cis isomerization of the chromophore, 4-hydroxycinnamic acid (pCA), an early intermediate (pR) is formed, which proceeds to a
second intermediate state (pB) on a sub-millisecond time scale. The signaling process is thought to be connected to the conformational
changes upon the formation of pB and its recovery to the ground state (pG), but the exact signaling mechanism is not known.
Experimental studies of PYP by solution NMR and X-ray crystallography suggest a very flexible protein backbone in the ground
as well as in the signaling state. The relaxation from the pR to the pB state is accompanied by the protonation of the chromophore's
phenoxyl group. This was found to be of crucial importance for the relaxation process. With the goal of gaining a better understanding
of these experimental observations on an atomistic level, we performed five MD simulations on the three different states of
PYP: a 1 ns simulation of PYP in its ground state [pG(MD)], a 1 ns simulation of the pR state [pR(MD)], a 2 ns simulation
of the pR state with the chromophore protonated (pRprot), a 2 ns simulation of the pR state with Glu46 exchanged by Gln (pRGln)
and a 2 ns simulation of PYP in its signaling state [pB(MD)]. Comparison of the pG simulation results with X-ray and NMR data,
and with the results obtained for the pB simulation, confirmed the experimental observations of a rather flexible protein
backbone and conformational changes during the recovery of the pG from the pB state. The conformational changes in the region
around the chromophore pocket in the pR state were found to be crucially dependent on the strength of the Glu46-pCA hydrogen
bond, which restricts the mobility of the chromophore in its unprotonated form considerably. Both the mutation of Glu46 with
Gln and the protonation of the chromophore weaken this hydrogen bond, leading to an increased mobility of pCA and large structural
changes in its surroundings. These changes, however, differ considerably during the pRGln and pRprot simulations, providing
an atomistic explanation for the enhancement of the rate constant in the Gln46 mutant.
Electronic supplementary material to this article is available at and is accessible for athorized users.
Electronic Publication 相似文献