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1.
Four kinds of active sites of bacterial fatty acid synthetase were mapped on distinct regions within a subunit. Active sites were specifically labeled with radioactive substrates and active-site-directed inhibitors. Labeled enzymes were cleaved with proteases, and the fragments thus produced were identified with respect to specific labels by SDS-polyacrylamide gel electrophoresis and a fluorographic technique. The linear alignment of such fragments in the original subunit was established and when the results were combined with those of our previous work, five active sites were located in three regions as follows. Starting from the N-terminal of the subunit, we located acetyl, malonyl and palmitoyl transferases in the first region, the acyl carrier site in the second region (Morishima & Ikai (1985) Biochim. Biophys. Acta 832, 297-307), and beta-ketoacyl synthetase in the third region. The observed order of active sites of bacterial fatty acid synthetase can be correlated with that of the yeast enzyme, which has two kinds of subunits. 相似文献
2.
H Murofushi A Ikai K Okuhara S Kotani H Aizawa K Kumakura H Sakai 《The Journal of biological chemistry》1988,263(25):12744-12750
Kinesin was purified from bovine adrenal medulla. The sedimentation coefficient was 8.8 S. Sedimentation equilibrium ultracentrifugation studies showed the molecular weight of kinesin to be 300,000. The calculated axial ratio was 1:16. The Stokes radius was estimated to be 8.9 nm by gel filtration. Circular dichroism showed the alpha-helix content to be about 50%. Purified kinesin preparation contained a major polypeptide with a molecular weight of 120,000 and minor ones with molecular weights of 71,000, 68,000, and 65,000. Bovine adrenal kinesin had an ATPase activity which was stimulated severalfold by microtubules to a specific activity of about 0.1 mumol/min.mg. Kinesin molecules adsorbed to a glass slide promoted the movement of microtubules on the glass surface at a rate of about 0.5 micron/s. Immunostaining of EBTr (bovine embryonic trachea fibroblast) cells and bovine adrenal chromaffin cells in interphase with an affinity-purified antibody against the major polypeptide of kinesin showed that some kinesin was located on microtubules and the rest distributed throughout the cytoplasm in a diffuse manner. EBTr cells in mitotic phase gave a staining pattern showing that kinesin was present throughout the cytoplasm with higher concentration in the region of mitotic apparatus. 相似文献
3.
Atsushi Ikai Masaaki Nishigai Toshiya Osada Hideo Arakawa Masako Kikuchi 《The protein journal》1987,6(1):81-93
The plasma α2-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f 0 of human α2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f 0 and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as α2-macroglobulin. 相似文献
4.
David O. Hall Sergei A. Markov Yoshitomo Watanabe K. Krishna Rao 《Photosynthesis research》1995,46(1-2):159-167
Natural photosynthesis may be adapted to advantage in the development of clean energy technologies. Efficient biocatalysts that can be used in solar energy conversion technologies are the cyanobacteria. Photobioreactors incorporating cyanobacteria have been used to demonstrate (a) the production of hydrogen gas, (b) the assimilation of CO2 with the production of algal biomass, (c) the excretion of ammonium, and (d) the removal of nitrate and phosphate from contaminated waters.Abbreviations Chl
chlorophyll
- DW
dry weight
- MSX
L-methionine-D-L-sulphoximine
- PAR
photosynthetically active radiation
- PU
polyurethane
- PV
polyvinyl
- PVC
polyvinylchloride 相似文献
5.
Electron microscopy of pig intestinal proline-β-naphthyltamidase revealed that the enzyme is composed of 3 subunits, which are assembled in a trifoliolate shape, At pH 4.5 and 4°C, the enzyme dissociates reversibly into active subunits in 4h. Dissociation also occurs at higher pHs when the enyzme concentration is very low. The activity per mg protein of the native, trimeric enzyme is about 2.5-fold higher than that of the dissociated enzyme. 相似文献
6.
Mast cells contain spleen-type prostaglandin D synthetase 总被引:2,自引:0,他引:2
Y Urade M Ujihara Y Horiguchi M Igarashi A Nagata K Ikai O Hayaishi 《The Journal of biological chemistry》1990,265(1):371-375
Prostaglandin D synthetase activity in the cytosol (100,000 x g, 1-h supernatant) fraction of peritoneal mast cells of adult rats (105.0 nmol/min/mg protein) was the highest among such activities in various rat tissues and cells. As judged by the absolute requirement for glutathione for the reaction (Km = 300 microM), the Km value for prostaglandin H2 (200 microM), and insensitivity of the activity to 1 mM 1-chloro-2,4-dinitrobenzene, the enzyme in mast cells was similar to rat spleen prostaglandin D synthetase and differed from rat brain prostaglandin D synthetase or glutathione S-transferase, all of which catalyze the isomerase reaction from prostaglandin H2 to prostaglandin D2. In immunotitration analyses, the activity in mast cells showed a titration curve exactly identical with that of the purified spleen-type enzyme and almost completely absorbed by an excess amount of antibody against this enzyme, but it remained unchanged after incubation with antibodies against the brain-type enzyme and glutathione S-transferase isozymes thus far purified. In Western blot after two-dimensional electrophoresis of crude extracts of mast cells, a single immunoreactive spot was observed with antibody against the spleen-type enzyme at the same position as that of the purified enzyme (Mr = 26,000, pI = 5.2). Furthermore, the immunoreactive protein obtained from mast cells showed the same peptide fingerprints as those of the purified spleen-type enzyme, after partial digestion with Staphylococcus aureus V8 protease or trypsin. In immunoperoxidase staining, the immunoreactivity of the spleen-type enzyme was found in the cytosol of tissue mast cells in various organs such as thymus, intestine, stomach, and skin of adult rats. These findings indicate that prostaglandin D2 is produced by the spleen-type synthetase in mast cells of various tissues. 相似文献
7.
A Ikai 《Biochimica et biophysica acta》1976,445(1):182-193
The denaturation of subtilisin BPN' (EC 3.4.21.14) in guanidine hydrochloride was studied in order to find possible reasons for the exceptional stability of this enzyme against the action of denaturing agents including guanidine hydrochloride. Chemically modified subtilisins, i.e., phenylmethanesulfonylsubtilisin and thio-subtilisin, were completely denatured in 2 M guanidine hydrochloride at pH 7 without autolysis but they were stable in 0.5 M guanidine hydrochloride for at least 60 h. On the other hand, once completely denatured, the subtilisins remained inactive and in highly unfolded conformations for 60 h or longer after transfer into 0.5 M guanidine solution at pH 7 or 9. No enzymatic activity was regained when the guanidine concentration was lowered to almost zero. We concluded from these and other results described in this paper that this enzyme was thermodynamically unstable in 2 M guanidine hydrochloride at 20 degrees C and at pH 7. We wish to point out the possibility that the denaturation of this enzyme could indeed be irreversible. 相似文献
8.
9.
Qiang Li Kazuhiro Hori Yoshitomo Minagi Takahiro Ono Yong-jin Chen Jyugo Kondo Shigehiro Fujiwara Kenichi Tamine Hirokazu Hayashi Makoto Inoue Yoshinobu Maeda 《PloS one》2013,8(8)
Background
Swallowing dysfunction (also known as dysphagia), which results in a deterioration of nutritional intake, slows rehabilitation and causes aspiration pneumonia, is very common following neurological impairments. Although videofluorographic (VF) examination is widely used for detecting aspiration, an objective and non-invasive method for assessing swallowing function has yet to be established because of a lack of adequate devices and protocols. In this paper, a bend sensor whose resistance is altered by bending was introduced to monitor swallowing-related laryngeal movement.Methods
Six healthy male volunteers were recruited in the present study. Specific time points on the signal waveform produced by the bend sensor were defined to describe laryngeal movement by differential analysis. Additionally, the physiological significance of the obtained waveform was confirmed by analyzing the sequential correlations between the signal waveform from the bend sensor and hyoid bone kinetics simultaneously recorded by VF.Results
Seven time points were successfully defined on the signal waveform to reference laryngeal movement. Each time point was well correlated with certain VF events, with evidence of no significant time lags, and there were positive correlations between waveform time points and matched VF events. Furthermore, obvious similarities were noticed between the duration of each phase on the signal waveform and the duration of the matched hyoid bone activity.Conclusions
The present monitoring system using a bend sensor might be useful for observing the temporal aspects of laryngeal movement during swallowing, and it was well coordinated with hyoid bone movement. 相似文献10.
W.?Mark Abbott Melanie Snow Sonia Eckersley Jonathan Renshaw Gareth Davies Richard?A. Norman Peter Ceuppens Jerry Slootstra Joris J. Benschop Yoshitomo Hamuro Jessica?E. Lee Peter Newham 《Bioscience reports》2013,33(4)
TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the Ki in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays. 相似文献