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Sophie Seigneurin-Venin Elaine Parrish Isabelle Marty François Rieger Georges Romey Michel Villaz Luis Garcia 《Experimental cell research》1996,223(2):301
The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation–contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca2+associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca2+is mobilized via a machinery similar to that involved in excitation–contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 μM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca2+but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (α1 subunit of the L-type Ca2+channel) and the internal stores of Ca2+. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation–contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca2+is triggered by the type of mechanism already described in skeletal muscle. 相似文献
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Isabelle Chevalot Athanase Visvikis Pierre Nabet Jean-Marc Engasser Annie Marc 《Cytotechnology》1994,16(2):121-129
Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h–1, the highest cell density (near 1.3×106 cells ml–1), and the highest enzyme activity around 300 mU ml–1, which corresponded to a specific cellular level of 20 mU 10–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems. 相似文献
40.
Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells 总被引:6,自引:0,他引:6
Alain Garnier Johanne Côté Isabelle Nadeau Amine Kamen Bernard Massie 《Cytotechnology》1994,15(1-3):145-155
Human 293S cells, a cell line adapted to suspension culture, were grown to 5×106 cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×105 cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×106 cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM
calcium-free DMEM
- CS
bovine calf serum
- hpi
hours post-infection
- J+
enriched Joklik medium
- MLP
major late promoter
- MOI
multiplicity of infection (# of infectious viral particle/cell)
- q
specific consumption rate (mole/cell.h)
- pfu
plaque forming unit (# of infectious viral particle)
- Y
yield (g/E6 cells or mole/cell) 相似文献