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141.
Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins fromE. coli
Jeffrey G. Thomas Amanda Ayling François Baneyx 《Applied biochemistry and biotechnology》1997,66(3):197-238
The high-level expression of recombinant gene products in the gramnegative bacteriumEscherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular proteases or its deposition
into biologically inactive aggregates known as inclusion bodies. It has recently become clear that in vivo protein folding
is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE
and GroEL-GroES systems, suppress off-pathway aggregation reactions and facilitate proper folding through ATP-coordinated
cycles of binding and release of folding intermediates. On the other hand, folding catalysts (foldases) accelerate rate-limiting
steps along the protein folding pathway such as thecis/trans isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges. Manipulating the cytoplasmic
folding environment by increasing the intracellular concentration of all or specific folding modulators, or by inactivating
genes encoding these proteins, holds great promise in facilitating the production and purification of heterologous proteins.
Purified folding modulators and artificial systems that mimic their mode of action have also proven useful in improving the
in vitro refolding yields of chemically denatured polypeptides. This review examines the usefulness and limitations of molecular
chaperones and folding catalysts in both in vivo and in vitro folding processes. 相似文献
142.
Bunai K Ariga M Inoue T Nozaki M Ogane S Kakeshita H Nemoto T Nakanishi H Yamane K 《Electrophoresis》2004,25(1):141-155
We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium). 相似文献
143.
For lattices with two kinds of points (black and white), distributed according to a translation-invariant joint probability distribution, we study statistical properties of the sequence of consecutive colors encountered by a random walker moving through the lattice. The probability distribution for the single steps of the walk is considered to be independent of the colors of the points. Several exact results are presented which are valid in any number of dimensions and for arbitrary probability distributions for the coloring of the points and the steps of the walk. They are used to derive a few general properties of random walks on lattices containing traps.Presented at the Symposium on Random Walks, Gaithersburg, MD, June 1982. 相似文献
144.
Jin Chen Lin-xi Zhang A-gen Xia 《高分子科学》2006,(1):13-19
It is important to know the rate of intra-molecular contact formation in proteins in order to understand how proteins fold clearly. Here we investigate the rate of intra-molecular contact formation in short two-dimensional compact polymer chains by calculating the probability distribution p(r) of end-to-end distance r using the enumeration calculation method and HP model on two-dimensional square lattice. The probability distribution of end-to-end distance p(r) of short two-dimensional compact polymers chains may consist of two parts, i.e. p(r) = p1(r) p2(r), where p1(r) and p2(r) are different for small r. The rate of contact formation decreases monotonically with the number of bonds N, and the rate approximately conforms to the scaling relation of k(N) ∝ N-α. Here the value of α increases with the contact radius a and it also depends on the percentage of H (hydrophobic) residues in the sequences of compact chains and the energy parameters of εHH, εHP and εPP . Some comparisons of theoretical predictions with experimental results are also made. This investigation may help us to understand the protein folding. 相似文献
145.
Assessment of various carbon sources and nutrient feeding strategies for Panax ginseng cell culture 总被引:1,自引:0,他引:1
Ginseng (root of Panax ginseng C. A. Meyer) cells were cultivated on medium supplemented with various carbohydrates including sucrose, glucose, and fructose,
at initial concentrations ranging from 10 to 110 g/L. Sucrose was shown to be the superior carbon source to the monosaccharides
for ginseng cell growth and the optimal concentration was between 30 and 50 g/L. An increase in the initial concentration
within this range increased the maximum cell density and growth index significantly, whereas much higher concentrations inhibited
cell growth. Feeding of sucrose and some other medium components during the growth (fed-batch mode) was more effective in
enhancing the cell growth and biomass productivity, increasing the growth index by more than 60–70% and biomass productivity
by more than 50%. 相似文献
146.
van de Waterbeemd H Smith DA Jones BC 《Journal of computer-aided molecular design》2001,15(3):273-286
Lipophilicity, often expressed as distribution coefficients (log D) in octanol/water, is an important physicochemical parameter influencing processes such as oral absorption, brain uptake and various pharmacokinetic (PK) properties. Increasing log D values increases oral absorption, plasma protein binding and volume of distribution. However, more lipophilic compounds also become more vulnerable to P450 metabolism, leading to higher clearance. Molecular size and hydrogen bonding capacity are two other properties often considered as important for membrane permeation and pharmacokinetics. Interrelationships among these physicochemical properties are discussed. Increasing size (molecular weight) often gives higher potency, but inevitably also leads to either higher lipophilicity, and hence poorer dissolution/solubility, or to more hydrogen bonding capacity, which limits oral absorption. Differences in optimal properties between gastrointestinal absorption and uptake into the brain are addressed. Special attention is given to the desired lipophilicity of CNS drugs. In examples using -blockers, Ca channel antagonists and peptidic renin inhibitors we will demonstrate how potency and pharmacokinetic properties need to be balanced. 相似文献
147.
Present proteomics and biomedicine require sensitive analytical methods for all proteins. Recent progress in electrochemical analysis of peptides and proteins based on their intrinsic electroactivity is reviewed. Tyrosine and/or tryptophan‐containing proteins are oxidizable at carbon electrodes. At mercury electrodes all peptides and proteins (about 13 peptides and >25 proteins were tested) produce chronopotentiometric peak H at nanomolar concentrations. This peak is sensitive to changes in protein structure. Microliter sample volumes are sufficient for the analysis. Electrochemical methods can be used in studies of nucleic acid‐protein interactions and can be applied in biomedicine. Examples of such applications in neurogenerative diseases and cancer are presented. 相似文献
148.
Alessandra Sussulini Jerusa S. Garcia Márcia F. Mesko Diogo P. Moraes Érico M. M. Flores Carlos A. Pérez Marco A. Z. Arruda 《Mikrochimica acta》2007,158(1-2):173-180
Two methods of protein extraction for soybean seeds were evaluated in terms of preservation of the metal ions bound to proteins
after the extraction and separation procedures. The proteins were firstly separated according to their molar masses by polyacrylamide
gel electrophoresis. Then, the protein bands were mapped by synchrotron radiation X-ray fluorescence in order to establish
which metal ions were present in each one. Finally, some mapped protein bands were decomposed by microwave-assisted combustion
and Ca, Cu, K, Mg, Mn, and Zn were quantified by inductively coupled plasma mass spectrometry or inductively coupled plasma
optical emission spectrometry. The extraction methods studied were Method A (based on the treatment of ground soybean seeds with hexane and their extraction with Tris–HCl and β-mercaptoethanol) and
Method B (based on the treatment of ground soybean seeds with petroleum ether and their extraction with Tris–HCl, dithiothreitol,
phenylmethanesulfonyl fluoride, sodium dodecyl sulfate and potassium chloride). The best method was Method B, in which a 78% higher extraction efficiency was obtained when compared to Method A. Additionally, the metal-protein interactions were more appropriately preserved when Method B was applied, where the most affected ions were those that are bound weakly to proteins, such as Ca, K, and Mg. 相似文献
149.
Volkhard Helms 《Chemphyschem》2007,8(1):23-33
Proteins are key components of biological cells. For example, enzymes catalyze biochemical reactions, membrane transporters are responsible for uptake and release of critical and superfluous components from the cell environment, and structural proteins are responsible for the stability of the cell wall and cytoskeleton. Many of the diverse protein functions involve dynamic transitions ranging from small local atomic displacements up to large allosteric conformational changes. In any conformation, proteins are in contact with the universal solvent medium of cells, water. Water not only surrounds proteins but is often an integral part of proteins and also is involved in key mechanistic steps. This Minireview discusses recent experimental and theoretical results on the role of water for protein dynamics and function. 相似文献
150.
Thazha P PrakashAndrew M Kawasaki Elena A LesnikNamir Sioufi Muthiah Manoharan 《Tetrahedron》2003,59(37):7413-7422
Synthesis of a series of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified 5-methyluridine nucleoside phosphoramidites and solid supports are described. Using these monomers, modified oligonucleotides containing phosphodiester linkages were synthesized in high yields. These modified oligonucleotides showed enhanced binding affinity to the complementary RNA (and not to DNA) and excellent nuclease stability with t1/2>24 h. The human serum albumin binding properties of modified oligonucleotides have been evaluated to assess their transport and toxicity properties. 相似文献