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181.
Kiichiro Teruya Yoshihito Daimon Xiao-Yan Dong Yoshinori Katakura Takumi Miura Akira Ichikawa Tsukasa Fujiki Makiko Yamashita Tetsuya Mori Hideya Ohashi Sanetaka Shirahata 《Cytotechnology》2005,47(1-3):29-36
The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase
hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 μg 10−6 cells day−1, one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line
A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number
and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein
accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level
in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-κB in D29 cells. These
results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact
hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit
hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the
effect of pyrrolidinedithiocarbamate, an inhibitor of NF-κB activation, was examined. Suppression of NF-κB activation in D29
cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the
release of negative feedback signals could increase the total amount of recombinant protein secretion. 相似文献
182.
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184.
Mayumi Shiohara Masatomo Kawakubo Takehisa Matsumoto Toshiko Kumagai Kazuyoshi Yamauchi Kozue Oana Hiroyoshi Ota Yoshiyuki Kawakami 《Microbiology and immunology》2009,53(5):251-258
An attempt was made to assess the hitherto undescribed optimal gaseous conditions for growth of zoonotic Helicobacter felis, focusing on the ratio of spiral-forms amongst the whole cells examined. The largest mean colony diameter was obtained under the gaseous condition of O2 12% and CO2 10%. In analyzing the five day old colonies, the highest percentage of spiral forms (85.5%) was observed under the condition of O2 18% and CO2 5%. In contrast, the lowest percentage of spiral forms (2.3%) was demonstrated under the condition of O2 1% and CO2 10%. The condition of O2 12% and CO2 10% was concluded to be optimal for obtaining cells with the largest colony sizes, although colonies proliferated under such conditions definitely contain many more coccoid cells than spiral forms. In culturing H. felis strains, optimal gaseous conditions should be employed according to the purposes or preferences of study designs. 相似文献
185.
186.
Satoshi Okada Kazuhisa Ota Takashi Ito 《Protein science : a publication of the Protein Society》2009,18(12):2518-2527
Quantitative measurement of small molecules with high spatiotemporal resolution provides a solid basis for correct understanding and accurate modeling of metabolic regulation. A promising approach toward this goal is the FLIP (fluorescent indicator protein) nanosensor based on bacterial periplasmic binding proteins (PBPs) and fluorescence resonance energy transfer (FRET) between the yellow and cyan variants of green fluorescent protein (GFP). Each FLIP has a PBP module that specifically binds its ligand to induce a conformation change, leading to a change in FRET between the two GFP variant modules attached to the N‐ and C‐termini of the PBP. The larger is the dynamic range the more reliable is the measurement. Thus, we attempted to expand the dynamic range of FLIP by introducing a circular permutation with a hinge loop deletion to the PBP module. All the six circularly permutated PBPs tested, including structurally distinct Type I and Type II PBPs, showed larger dynamic ranges than their respective native forms when used for FLIP. Notably, the circular permutation made three PBPs, which totally failed to show FRET change when used as their native forms, fully capable of functioning as a ligand binding module of FLIP. These FLIPs were successfully used for the determination of amino acid concentration in complex solutions as well as real‐time measurement of amino acid influx in living yeast cells. Thus, the circular permutation strategy would not only improve the performance of each nanosensor but also expand the repertoire of metabolites that can be measured by the FLIP nanosensor technology. 相似文献
187.
Ryotaro Koike Akinori Kidera Motonori Ota 《Protein science : a publication of the Protein Society》2009,18(10):2060-2066
Transferases and hydrolases catalyze different chemical reactions and express different dynamic responses upon ligand binding. To insulate the ligand molecule from the surrounding water, transferases bury it inside the protein by closing the cleft, while hydrolases undergo a small conformational change and leave the ligand molecule exposed to the solvent. Despite these distinct ligand‐binding modes, some transferases and hydrolases are homologous. To clarify how such different catalytic modes are possible with the same scaffold, we examined the solvent accessibility of ligand molecules for 15 SCOP superfamilies, each containing both transferase and hydrolase catalytic domains. In contrast to hydrolases, we found that nine superfamilies of transferases use two major strategies, oligomerization and domain fusion, to insulate the ligand molecules. The subunits and domains that were recruited by the transferases often act as a cover for the ligand molecule. The other strategies adopted by transferases to insulate the ligand molecule are the relocation of catalytic sites, the rearrangement of secondary structure elements, and the insertion of peripheral regions. These findings provide insights into how proteins have evolved and acquired distinct functions with a limited number of scaffolds. 相似文献
188.
Sixty-two isolates from basidiocarps of Armillaria spp. were obtained from Hokkaido Island, northern Japan. Six species (Armillaria cepistipes, A. gallica, A. nabsnona, A. ostoyae, A. sinapina, and an undescribed species, “Nag. E”) were identified by pairing tests with known tester strains and one subspecies (Armillaria mellea subsp. nipponica, a non-heterothallic form of A. mellea) was identified by its macro- and micro-morphological characters of the basidiocarps. This is the first case of “Nag. E”
being reported from Hokkaido Island. 相似文献
189.
Sawako Tokuda Tsutomu Hattori Yu-Cheng Dai Yuko Ota Peter K. Buchanan 《Mycoscience》2009,50(3):190-202
190.
Shin-Ichi Morinaga Yuko Kumano Ayako Ota Ryohei Yamaoka Satoki Sakai 《Population Ecology》2009,51(1):187-195
We examined the contribution of diurnal and nocturnal pollination to male and female reproductive success in Lilium auratum. Plants were bagged for either 12 h during the day or at night to allow either only nocturnal or only diurnal visitors to
forage throughout the flowering period. We found that there was no significant difference in the seed:ovule ratio among diurnally
pollinated, nocturnally pollinated, or control flowers. However, in terms of male reproductive success, it was more advantageous
for the plants to be pollinated both diurnally and nocturnally: the numbers of pollen grains remaining in diurnally pollinated
or nocturnally pollinated flowers were significantly greater than those in control flowers. The total amount of floral volatiles
of L. auratum was significantly higher at night than during the day. The constituents of floral scent of all time series examined were
mostly monoterpenoids, many of which serve as attractants for nocturnal hawkmoths. Such nocturnally biased floral scent emission
of L. auratum might achieve male reproductive success by attracting nocturnal visitors, which may suggest that the relative contribution
of floral scent in this species is biased towards male reproductive success. 相似文献