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1.
2.
The applicability of 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride (MUGB) as active-site titrant for tissue-type plasminogen activator (t-PA) was studied in comparison to urokinase. Although t-PA was capable of cleaving MUGB, active-site titration of t-PA (one-chain form as well as two-chain form) with MUGB was not possible, whereas MUGB titration of urokinase could be performed. We therefore studied the kinetics of the interaction of these two plasminogen activators with MUGB. The equilibrium dissociation constant, KS, for the interaction between MUGB and urokinase was 2.9 X 10(-6) M, and for the interaction with t-PA 3.13 X 10(-5) M. However, one main requirement for active-site titration, namely a stable acyl enzyme intermediate (ES'), was only fulfilled for MUGB urokinase but not for MUGB t-PA. Whereas for the reaction of MUGB and urokinase the first-order acylation rate constant k2 was found to be about 10(6)-times higher than the first-order deacylation rate constant k3 (k2 = 3.76 X 10(-1) s-1, k3 = 3.7 X 10(-7) s-1), the k2/k3 ratio for the reaction of MUGB and t-PA (one- and two-chain form) was 0.77 to 3.85. Therefore, urokinase and t-PA differ in their reaction with this fluorogenic substrate and MUGB cannot be used for active-site titration of tPA. 相似文献
3.
Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Ca2+ ions are necessary for re-activation. 下载免费PDF全文
The soluble form of the homogeneous quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus is reversibly inactivated at temperatures above 35 degrees C. An equilibrium is established between active and denatured enzyme, this depending on the protein concentration and the inactivation temperature used. Upon thermal inactivation the enzyme dissociates into the prosthetic group pyrroloquinoline quinone and the apo form of glucose dehydrogenase. After inactivation at 50 degrees C active enzyme is re-formed again at 25 degrees C. Ca2+ ions are necessary for the re-activation process. The velocity of re-activation depends on the protein concentration, the concentration of the prosthetic group pyrroloquinoline quinone and the Ca2+ concentration. The apo form of glucose dehydrogenase can be isolated, and in the presence of pyrroloquinoline quinone and Ca2+ active holoenzyme is formed. Even though native glucose dehydrogenase is not inactivated in the presence of EDTA or trans-1,2-diaminocyclohexane-NNN'NH-tetra-acetic acid, Ca2+ stabilizes the enzyme against thermal inactivation. Two Ca2+ ions are found per subunit of glucose dehydrogenase. The data suggest that pyrroloquinoline quinone is bound at the active site via a Ca2+ bridge. Mn2+ and Cd2+ can replace Ca2+ in the re-activation mixture. 相似文献
4.
Seeds of the root parasitesStriga (several spp.) andBuchnera americana were examined by means of SEM. The surface patterns of the seeds in both genera resemble each other closely, especially those ofS. angustifolia andB. americana. SomeStriga spp. can be clearly distinguished by their surface characteristics, while this is quite difficult in others. The taxonomic value of the seed surface features ofStriga andBuchnera is discussed. 相似文献
5.
Janusz Janiszewski Dietmar Otto 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1988,162(6):739-746
1. | Intracellular recordings of suboesophageal neurons were performed in the cricketGryllus bimaculatus during applied changes of head temperature in the range 8 to 32.5 °C. The temperature was controlled by perfusing the head with Ringer solution of appropriate temperature. Subsequent staining with Lucifer Yellow revealed descending, ascending or T-shaped cells with ventrally located somata (Fig. 1). |
2. | In 6 out of 7 neurons recorded (Fig. 1, neurons A, B, C, D, E, G) the firing rate was correlated with abdominal ventilatory pumping (Fig. 2a, b). These neurons also received input from cereal sensory hairs (Fig. 2c). Furthermore, one of them (Fig. 1, neuron A) showed responses to auditory (Fig. 2d) and another (Fig. 1, neuron E) to visual input (Fig. 2e). |
3. | Activity of every tested neuron was correlated with the temperature of the perfusing Ringer solution: the amplitude and duration of spikes and excitatory postsynaptic potentials increased with cooling (Fig. 3). Two types of temperature-dependent changes in firing rate were identified. In type I the spiking rate was higher at higher temperature (Figs. 4a, b; 5). In type II spiking rate was related to the direction of temperature change (Fig. 4c, d). |
4. | The possible involvement of one of the recorded cells (Fig. 1, neuron F) in thermoreception processes is discussed. Activity of this neuron was not related to the rhythm of abdominal ventilatory pumping, nor did the cell receive cereal, visual or auditory input. Its activity was related mainly to the direction of temperature changes i.e. with an increase in firing rate during cooling, independent of the temperature at which the cooling started and with a transient decrease in firing rate during warming from starting point of 10 °C. |
6.
Derivatives of D-luciferin, D-luciferin methyl ester, D-luciferin O-sulfate, D-luciferin O-phosphate, D-luciferyl-L-N alpha-arginine and D-luciferyl-L-phenylalanine were used as highly sensitive substrates for carboxylic esterase, arylsulfatase, alkaline phosphatase and carboxypeptidases A, B and N. Enzymatic cleavage of the compounds by enzymes leading to the release of D-luciferin was demonstrated. Kinetic constants have been determined for D-luciferin methyl ester and carboxylic esterase, for D-luciferin O-sulfate and arylsulfatase, for D-luciferin O-phosphate and alkaline phosphatase, for D-luciferyl-L-phenylalanine and carboxypeptidase A, and for carboxypeptidases B and N and D-luciferyl-L-N alpha-arginine. All compounds proved to be highly sensitive substrates for the respective enzymes, permitting a limit of detection for enzymes between 10 and 500 fg per assay. 相似文献
7.
Spyridon Vamvakas Wolfgang Dekant Dietmar Schiffmann Dietrich Henschler 《Cell biology and toxicology》1988,4(4):393-403
S-(chloroethyl)-cysteine (CEC) and S-(1,2-dichlorovinyl)cysteine (DCVO) have been proposed as intermediates in the metabolic transformation of the carcinogens 1,2-dichloroethane and 1,1,2-trichloroethylene. We have tested the ability of CEC and DCVC to induce DNA repair and genotoxic effects at the chromosomal level by comparative assessment of unscheduled DNA synthesis induction and micronucleus formation in Syrian hamster embryo fibroblasts. CEC induced a potent and dose-dependent response in both assays, whereas DCVC treatment resulted in a comparatively weak induction of DNA repair and failed to raise micronucleus formation above control rates. Inhibition of cysteine conjugate \gB-lyase diminished the effect of DCVC, but had no influence on the genotoxicity of CEC either in the unscheduled DNA synthesis or micronucleus assay.Abbreviations AOAA
aminooxyacetic acid
- CEC
S-(chloroethyl)-cysteine; \gB-lyase, cysteine conjugate -lyase
- DCE
1,2-dichloroethane
- DCVC
S(1,2-dichlorovinyl)-cysteine
- GSH
glutathione
- HU
hydroxyurea
- IBR
IBR-modified Dulbecco's Eagle's reinforced medium
- MN2
micronuclei/2,000 cells
- 4-NQO
4-nitroquinoline-1-oxide
- SHE
Syrian hamster embryo fibroblasts; 3H-Thd, 3H-thymidine
- TCE
1,1,2-trichloroethylene
- UDS
unscheduled DNA synthesis 相似文献
8.
Heiko Traupe Dietmar Müller David Atherton D. Chester Kalter Frans P. M. Cremers Bernard A. van Oost Hans-Hilger Ropers 《Human genetics》1992,89(6):659-665
Summary Homology with the mouse bare patches mutant suggests that the gene for the X-linked dominant chondrodysplasia punctata / ichthyosis / cataract / short stature syndrome (Happle syndrome) is located in the human Xq28 region. To test this hypothesis, we performed a linkage study in three families comprising a total of 12 informative meioses. Multiple recombinations appear to exclude the Xq28 region as the site of the gene. Surprisingly, multiple crossovers were also found with 26 other markers spread along the rest of the X chromosome. Two-point linkage analysis and analysis of recombination chromosomes seem to exclude the gene from the entire X chromosome. Three different mechanisms are discussed that could explain the apparent exclusion of an X-linked gene from the X chromosome by linkage analysis: (a) different mutations on the X chromosome disturbing X inactivation, (b) metabolic interference, i.e. allele incompatibility of an X-linked gene, and (c) an unstable pre-mutation that can become silent in males. We favour the last explanation, as it would account for the unexpected sex ratio (MF) of 1.21 among surviving siblings, and for the striking clinical variability of the phenotype, including stepwise increases in disease expression in successive generations. 相似文献
9.
P J Geiger 《Biochemical medicine and metabolic biology》1992,48(1):74-80
This paper discusses the application of the data handling-graphics-statistics program Stata (Computing Resources Center, Santa Monica, CA) to radioimmunoassay. We have found that this program is more powerful and easier to use than a spreadsheet for analyzing various kinds of laboratory data generated from chromatography, radiolabeling experiments, enzyme-linked immunosorbent assays, and radioimmunoassays, to name several examples. Data from a radioimmunoassay procedure, originally analyzed using a spreadsheet, Lotus 1-2-3, have been processed with Stata. Simple programs (batch files) have been devised for computations and graphics. The original data and a comparison of results are presented. 相似文献
10.
G Helmlinger R V Geiger S Schreck R M Nerem 《Journal of biomechanical engineering》1991,113(2):123-131
Endothelial cells (EC) appear to adapt their morphology and function to the in vivo hemodynamic environment in which they reside. In vitro experiments indicate that similar alterations occur for cultured EC exposed to a laminar steady-state flow-induced shear stress. However, in vivo EC are exposed to a pulsatile flow environment; thus, in this investigation, the influence of pulsatile flow on cell shape and orientation and on actin microfilament localization in confluent bovine aortic endothelial cell (BAEC) monolayers was studied using a 1-Hz nonreversing sinusoidal shear stress of 40 +/- 20 dynes/cm2 (type I), 1-Hz reversing sinusoidal shear stresses of 20 +/- 40 and 10 +/- 15 dynes/cm2 (type II), and 1-Hz oscillatory shear stresses of 0 +/- 20 and 0 +/- 40 dynes/cm2 (type III). The results show that in a type I nonreversing flow, cell shape changed less rapidly, but cells took on a more elongated shape than their steady flow controls long-term. For low-amplitude type II reversing flow, BAECs changed less rapidly in shape and were always less elongated than their steady controls; however, for high amplitude reversal, BAECs did not stay attached for more than 24 hours. For type III oscillatory flows, BAEC cell shape remained polygonal as in static culture and did not exhibit actin stress fibers, such as occurred in all other flows. These results demonstrate that EC can discriminate between different types of pulsatile flow environments.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献