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81.
Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different
refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column
refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl
S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA
inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 m guanidine hydrochloride (Gdm·HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm·HCl with a G25
column and simultaneously dissolved in 8 m urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing
urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was
increased from 3.6 to 13.8% in comparison with the usual dilution refolding.
Revisions requested 31 October 2005; Revisions received 20 December 2005 相似文献
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Human mesenchymal stem cells (hMSCs) hold great promise in cell therapy and regenerative medicine. Various preclinical and clinical trials have been carried out to illustrate the therapeutic potential of these cells. However, one major challenge for manufacturing clinical grade hMSCs is the requisition of current good manufacturing practice (cGMP) grade practices in cell isolation, processing, storage, and distribution. Development of non-toxic and animal serum-free preservation medium is critical for storage and distribution of mesenchymal stem cells (MSCs). In this study, we developed a solution formula that could preserve MSCs at 4°C for up to 3 weeks. In the solution, trehalose is a key ingredient for maintaining survival of MSCs. Among the concentrations investigated, 40 mM trehalose showed the best outcome with the viability maintained more than 92.7 ± 1.5% for 7 days. Cells preserved in the solution formula for 3 weeks still remained about 70% viability, and produced results similar to those of freshly harvested hMSCs in terms of growth kinetics, expression profile of cell surface antigens, and differentiation potential. In summary, storage of MSCs in the medium makes it far easier for transporting the cells from processing units to clinical sites. 相似文献
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Tyrosyl O-sulfation is a common posttranslational derivatization of proteins that may also modify regulatory peptides. Among these are members of the cholecystokinin (CCK)/gastrin family. While sulfation of gastrin peptides is without effect on the bioactivity, O-sulfation is crucial for the cholecystokinetic activity (i.e. gallbladder emptying) of CCK peptides. Accordingly, the purification of CCK as a sulfated peptide was originally monitored by its gallbladder emptying effect. Since then, the dogma has prevailed that CCK peptides are always sulfated. The dogma is correct in a semantic context since the gallbladder expresses only the CCK-A receptor that requires sulfation of the ligand. CCK peptides, however, are also ligands for the CCK-B receptors that do not require ligand sulfation. Consequently, unsulfated CCK peptides may act via CCK-B receptors. Since in vivo occurrence of unsulfated products of proCCK with an intact α-amidated C-terminal tetrapeptide sequence (-Trp-Met-Asp-PheNH(2)) has been reported, it is likely that unsulfated CCK peptides constitute a separate hormone system that acts via CCK-B receptors. This review discusses the occurrence, molecular forms, and possible physiological as well as pathophysiological significance of unsulfated CCK peptides. 相似文献
87.
An ultrasensitive electrochemical DNA biosensor was constructed by assembling probe labeled gold nanoparticles (ssDNA-AuNP) on electrochemically reduced graphene oxide (ERGO) modified electrode with thiol group tagged (GT) DNA strand (d(GT)(29)SH) and coupling with horseradish peroxidase (HRP) functionalized carbon sphere (CNS) as tracer. The heteronanostructure formed on the biosensor surface appeared relatively good conductor for accelerating the electron transfer, while the HRP tagged CNS provided dual signal amplification for electrochemical biosensing. The triplex signal amplification strategy produced an ultrasensitive electrochemical detection of DNA down to attomolar level (5 aM) with a linear range of 5 orders of magnitude (from 1 × 10(-17)M to 1 × 10(-13)M), and appeared high selectivity to differentiate single-base mismatched and three-base mismatched sequences of DNA. The proposed approach provided a simple and reliable method for DNA detection with high sensitivity and specificity, indicating promising application in bioanalysis and biomedicine. 相似文献
88.
Ketones are a class of important organic compounds. As the simplest ketone, acetone is widely used as solvents or precursors for industrial chemicals. Presently, million tonnes of acetone is produced worldwide annually, from petrochemical processes. Here we report a biotechnological process that can produce acetone from CO(2), by designing and creating a modularized synthetic pathway in engineered cyanobacterium Synechocystis sp. PCC 6803. The engineered Synechocystis cells are able to produce acetone (36.0 mgl(-1) culture medium) using CO(2) as the sole carbon source, thus opens the gateway for biosynthesis of ketones from CO(2). 相似文献
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BackgroundSome diet factors are risk factors for dental erosion.MethodsWe performed computer searches of PubMed, Cochrane Library, EBSCO, CALIS, et al., to search for studies investigating risk factors for dental erosion. For risk factors investigated in a comparative way, we computed pooled odds ratios (ORs) using the Mantel and Haenszel method.ResultsA total of 9 studies met the inclusion criteria, and 6 risk factors were considered, including soft drinks, sports drinks, juice, vitamin C, milk, and yoghourt. The following associations were found for soft drinks (OR = 2.41, 95%CI = 2.03–2.85) and vitamin C (OR = 1.16, 95%CI = 1.10–1.22). While juice (OR = 0.90, 95%CI = 0.25–3.24), sports drinks (OR = 1.58, 95%CI = 0.88–2.85), milk (OR = 0.67, 95%CI = 0.11–4.01), and yoghourt products (OR = 1.05, 95%CI = 0.28–3.96) were not associated with dental erosion.ConclusionsThis meta-analysis provides comprehensive evidence-based assessment of diet-related factors for dental erosion. Preventive strategies should be taken to reduce dental erosion. 相似文献
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