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161.
Jacques Ricard Jacques Vergne Jean-Luc Decout Marie-Christine Maurell 《Journal of molecular evolution》1996,43(4):315-325
A polyallylamine carrying long hydrophobic dodecyl groups and adenine residues as side chains (PALAD C12) may be able to catalyze the hydrolysis ofN-carbobenzoxy-l-alaninep-nitrophenyl ester (N-Cbz-Ala) as well asp-nitrophenyl acetate (pNPA). The progress curve of hydrolysis of the former displays a long lag and apparently no steady state.
After this transient the rate falls off due to the accumulation of the products. Conversely, the hydrolysis ofp-nitrophenyl acetate displays classical burst kinetics followed by a slow decline of the reaction rate.
Theoretical considerations show that a steady state may be expected to occur only if the concentration of the free catalyst
is very small during the reaction. This condition is sufficient to allow the rate of disappearance of the substrate to be
equal to the rate of appearance of the products, which is precisely a condition for the existence of a steady state. If the
catalyst is poorly active and has a loose affinity for its substrate and product, the measurement of a significant reaction
rate will require a much larger concentration of the catalyst. Therefore, under these conditions, one cannot expect a steady
state to occur. The mathematical expression of the error made in the steady-state assumption has been derived. This error
increases with the catalyst concentration and decreases if the affinity of the substrate for the catalyst is high. Therefore
the lack of steady state is associated with the affinity (or the dissociation) of the substrate and the product for the catalyst.
When this affinity is low, the free concentration of the catalyst during the reaction is high and one cannot expect a steady
state to occur. This is precisely what takes place with N-Cbz-Ala.
A mathematical expression of the rate of hydrolysis of N-Cbz-Ala and of any reactant that displays this type of kinetics may
be derived at the end of the transient when the rate is close to its maximum value. Under these conditions the rate cannot
follow classical Michaelis-Menten kinetics and displays positive cooperativity.
It may therefore be speculated that primordial template-like catalysts that were displaying a poor affinity for their substrates
and products were already exhibiting apparent positive cooperativity in the kinetic reactions they were able to catalyze.
Correspondence to: J. Ricard 相似文献
162.
James H. Fitzpatrick Jr. Douglas Kintner †Mark Anderson †William M. Westler ‡Sherrie E. Emoto David D. Gilboe 《Journal of neurochemistry》1996,66(6):2612-2620
Abstract: The inorganic phosphate (Pi) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of Pi. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated Pi spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of Pi buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the Pi peaks arising from the three compartments with different pH values if each peak made up 10–35% of total Pi area. In vivo, we identified the plasma compartment by intraarterial infusion of Pi. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that Pi compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pHi. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger Pi peaks represented intracellular spaces. 相似文献
163.
Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85S6K . These data indicate that p85S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago. 相似文献
164.
Abstract: C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na+-dependent l -[3H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated l -[3H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC50 value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in Vmax with no change in Km. PMA also increased the transport of the nonmetabolizable analogue, d -[3H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Na+-dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4α-phorbol 12,13-didecanoate, did not stimulate l -[3H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca2+ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na+/H+ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na+-dependent l -[3H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS. 相似文献
165.
166.
An in vitro procedure for large scale multiplication of Sterculia urens Roxb. (Gum Kadaya Tree) has been developed using cotyledonary node segments. An average of 4.0 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 2.0 mgl–1 6-benzyl amino-purine (BAP) within 21 days of initial culture. Upon subsequent subculture 16 shoots/node could be harvested every three weeks and upto three times. Sixty per cent of the shoots were successfully rooted. Rooted plantlets were transferred to plastic pots containing soil under mist house conditions before they were finally exposed to an external environment. Fifty seven per cent of the plantlets survived in nursery sheds. 相似文献
167.
Marianne Jegouic Valerij Ya. Grinberg André Guingant Thomas Haertlé 《Journal of Protein Chemistry》1996,15(6):501-509
Denaturation and aggregation of-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding of-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation of-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield of-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers of-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecular-sheets. 相似文献
168.
169.
Summary Polyclonal antibodies directed against polypeptide epitopes of conglutin , a glycosylated lectin in lupin seed, have been used to identify and quantify this protein in root extracts of germinating lupins. The highest conglutin content was found in protein extracts of root elongation zones after 5 to 7 days germination. Root conglutin showed the same subunit composition, glycosylation pattern, isoelectric point, and lectin activity as the cotyledonary one. Immunolocalization experiments on root thin sections demonstrated that conglutin is chiefly present in the intercellular spaces of the cortical parenchyma, where it forms large aggregates. Labelling of the Golgi complexes and the area between the plasmalemma and cell wall revealed the conglutin pathway from post-synthetic processing to excretion via the secretory system.Abbreviations EDTA
ethylene diamino tetraacetic acid
- IEF
isoelectric focusing
- LRW
London resin white
- NC
nitrocellulose
- PAGE
polyacrylamide gel electrophoresis
- pI
isoelectric point
- Pi
inorganic phosphate
- SDS
sodium dodecylsulphate
- TEM
transmission electron microscopy 相似文献
170.
Robert N. Fontaine Ruanna E. Gossett Friedhelm Schroeder Barbara A. O'Toole Thomas Doetschman Ann B. Kier 《Molecular and cellular biochemistry》1996,159(2):149-153
The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP
liver fatty acid binding protein
- I-FABP
intestinal fatty acid binding protein
- TGF1
transforming growth factor beta-1
- TNF-
tumor necrosis factor-
- MIP-
macrophage inflammatory protein-
- PMSF
phenylmethyl sulfonyl fluoride
- PBS
phosphate buffered saline 相似文献