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91.
In the present study we have studied how [Ca2+]
i
is influenced by H2O2 in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric
system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H2O2 caused a slow sustained [Ca2+]
i
increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H2O2 was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application
of 1 mm H2O2 to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration
of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial
uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H2O2-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and
FCCP in the presence of H2O2 did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable
calcium pools, but was followed by a sustained increase of [Ca2+]
i
. In addition, H2O2 was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca2+]
i
induced by H2O2 was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H2O2 releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H2O2 is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases.
Received: 15 May 2000/Revised: 4 October 2000 相似文献
92.
Vollmayer P Koch M Braun N Heine P Servos J Israr E Kegel B Zimmermann H 《Journal of neurochemistry》2001,78(5):1019-1028
The physiological action of extracellular ATP and other nucleotides in the nervous system is controlled by surface-located enzymes (ecto-nucleotidases) of which several families with partially overlapping substrate specificities exist. In order to identify ecto-nucleotidases potentially associated with neural cells, we chose PC12 cells for analysis. PC12 cells revealed surface-located ATPase and ADPase activity with apparent K(m)-values of 283 microM and 243 microM, respectively. Using PCR we identified the mRNA of all members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated (NTPDase1 to NTPDase3, NTPDase5/6), of ecto-nucleotide pyrophosphatase/phosphodiesterase3 (NPP3), tissue-non-specific alkaline phosphatase and ecto-5'-nucleotidase. The surface-located catalytic activity differed greatly between the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto-nucleoside triphosphate diphosphohydrolase family. Activity of ecto-5'-nucleotidase and alkaline phosphatase was very low and activity of NPP3 was absent. For a detailed analysis of the cellular distribution of ecto-nucleotidases single and double transfections of PC12 cells were performed, followed by fluorescence analysis. Ecto-nucleotidases were distributed over the entire cell surface and accumulated intracellularly in varicosities and neurite tips. PC12 cell ecto-nucleotidases are likely to play an important role in terminating autocrine functions of released nucleotides and in producing extracellular nucleosides supporting the survival and neuritic differentiation of PC12 cells. 相似文献
93.
Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography 总被引:3,自引:0,他引:3
Varley DL Hitchcock AG Weiss AM Horler WA Cowell R Peddie L Sharpe GS Thatcher DR Hanak JA 《Bioseparation》1999,8(1-5):209-217
We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feedstock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography.The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration. 相似文献
94.
Yutaka Tokiwa Masaru Kitagawa Hong Fan Tetsuji Yokochi Takao Raku Yoichi Hiraguri Shigeo Shibatani Yoshihiko Maekawa Naoki Kashimura Ryuichiro Kurane 《Biotechnology Techniques》1999,13(8):563-566
The transesterification of divinyladipate with adenosine in DMF containing 20% (v/v) DMSO was catalyzed by Streptomyces sp. alkaline protease and esterification occurred exclusively at the 3-position of hydroxyl group of ribofuranose in adenosine to give 3-O-vinyladipoyl adenosine without other products. 相似文献
95.
Simulated acid rain (SAR) combined with higher concentration of aluminium (SAR+Al) influenced the ecophysiology of three arbuscular
mycorrhizal fungi (AMF) in both the germination and symbiotic phases of their life cycle. Acaulospora tuberculata, an isolate
from the soil with low pH, exhibited a higher tolerance to environmental stress as compared to Glomus mosseae and G. fistulosum.
This higher tolerance may be related to the edaphic conditions of soil of the isolate origin. The histochemical staining of
the alkaline phosphatase and NADH-diaphorase activities in the extraradical mycelium (ERM) of the AMF proved to be more sensitive
indication of negative effects of the SAR or SAR+Al stress compared to commonly measured parameters of the AMF such as mycorrhizal
colonisation or growth of the ERM.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
96.
Two experiments were conducted using completely randomized designs to study the bioavailability of Si from three sources to
growing rats and turkeys fed semipurified diets. The basal diets were dextrose-egg albumin for rats and dextrose-casein for
turkeys. The Si sources were tetraethylorthosilicate (TES), sodium silicate (NaSil), and sodium zeolite A (NaZA). Rats and
turkeys were supplemented at 500 and 270 ppm Si, respectively, from each source. A control group of unsupplemented rats and
turkeys was included in each experiment. In general, irrespective of Si source, Si supplementation slowed (p < 0.05 orp < 0.01) growth rates in both rats and turkeys. Although dietary Si supplementation reduced (p < 0.05) plasma Mg levels and liver Zn concentrations in rats, it increased (p < 0.05) plasma P and reduced (p < 0.05) plasma Cu levels in turkeys. Rats on TES had significantly slower (p < 0.05 orp < 0.01) growth rates (5–10%) than those on NaSil or NaZA. In rats, NaZA and TES reduced (p < 0.05) hemoglobin concentrations and plasma Zn, respectively. However, plasma Mg levels were higher (p < 0.05) in TES than
NaSil-or NaZA-fed rats. The source of the dietary Si did not affect (p < 0.05) the organ weights of rats and their mineral concentrations. Turkeys on TES diets grew at a significantly faster (p < 0.05) rate (15%) than those on NaSil or NaZA diets during the first 2 wk of experimentation. However, after 4 wk, there were
no significant (p > 0.05) differences in growth between the Si sources. In turkeys, NaZA increased (p < 0.05) hematocrit levels and plasma Mg levels. Turkeys on NaZA diets had larger (p < 0.05) hearts and livers than those on
NaSil but not TES. Liver Mn content was higher (p < 0.05) in turkeys on NaSil than TES or NaZA. Heart Zn was lower (p < 0.05) in turkeys on NaSil than TES, but not NaZA. 相似文献
97.
AIMS: Different indicator enzymes and fluorogenic or chromogenic substrates were compared as detector systems in a novel polymyxin-based enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157 lipopolysaccharide (LPS) antigens. METHODS AND RESULTS: An ELISA system was developed using polymyxin immobilized in the wells of a microtitre plate as a high-affinity adsorbent for E. coli O157 LPS antigens, which were immunoenzymatically detected using anti-E. coli O157 antibody-enzyme conjugates. With peroxidase as the indicator enzyme the fluorogenic substrates Amplex Red and QuantaBlu produced only slight improvement in the performance characteristics of the polymyxin-ELISA compared with the use of the chromogenic substrate tetramethylbenzidine (TMB). On the other hand, with alkaline phosphatase as the indicator enzyme a pronounced improvement in assay performance was noted using the fluorogenic substrate Attophos compared with the chromogenic substrate p-nitrophenylphosphate. CONCLUSIONS: The detection system exhibiting the best characteristics with respect to cost, ease of use and overall performance in the detection of E. coli O157 in enrichment cultures from a variety of solid foods was based on the use of peroxidase as the indicator enzyme with the chromogenic substrate TMB. SIGNIFICANCE AND IMPACT OF THE STUDY: The polymyxin-ELISA provides a rapid, simple and inexpensive assay system for the detection of E. coli O157 in foods. 相似文献
98.
The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816. 相似文献
99.
Renata?A.?ZvyagilskayaEmail author Olga?A.?Parkhomenko Anna?V.?Gordeeva Yuliya?I.?Deryabina Bengt?L.?Persson 《Bioscience reports》2004,24(2):117-125
Energy status of the novel alkalitolerant Yarrowia lipolytica yeast strain grown at alkaline conditions (pH 9.7) was examined. Cells grown under such severe conditions were found to preserve high respiratory activity. The oxidative phosphorylation system dominated in the energy budget of the cell. A procedure was specially design to isolate tightly coupled mitochondria from yeast cells grown at alkaline conditions. The isolated mitochondrial preparations met known criteria of physiological intactness, as inferred from their ability to maintain distinctive state 4–3 respiration transition upon addition of ADP, high respiratory rates, good respiratory control values, and ADP/O ratios close to the theoretically expected maxima for the substrates used. 相似文献
100.
We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves. Here we characterized the
structure and function of an SAG-Osh69 encoding alkaline α-galactosidase that belongs to a novel family of glycosyl hydrolases. Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8. The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid,
salicylic acid), and stresses (H2O2 and wounding). The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal α-galactosidase activity at pH 8.0. The enzyme showed good hydrolytic activities towards α-1,6-galactosyl
oligosaccharides and galactolipid digalactosyl diacylglycerol. Immunoelectron microscopic analysis demonstrates that Osh69
is specifically localized in the chloroplasts of senescing leaves. These findings strongly suggest an important role for Osh69
in the degradation of chloroplast galactolipids during leaf senescence. The nucleotide sequence data reported will appear
in the GenBank Nucleotide Sequence Database under the accession number AF251068. 相似文献