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991.
992.
The chromosomes of ciliates are fragmented at reproducible sites during the development of the polyploid somatic macronucleus, but the mechanisms involved appear to be quite diverse in different species. In Paramecium aurelia, the process is imprecise and results in de novo telomere addition at locally heterogeneous positions. To search for possible determinants of chromosome fragmentation, we have studied an ~21-kb fragmentation region from the germ line genome of P. primaurelia. The mapping and sequencing of alternative macronuclear versions of the region show that two distinct multicopy elements, a minisatellite and a degenerate transposon copy, are eliminated by an imprecise mechanism leading either to chromosome fragmentation and the formation of new telomeres or to the rejoining of flanking sequences. Heterogeneous internal deletions occur between short direct repeats containing TA dinucleotides. The complex rearrangement patterns produced vary slightly among genetically identical cell lines, show non-Mendelian inheritance during sexual reproduction, and can be experimentally modified by transformation of the maternal macronucleus with homologous sequences. These results suggest that chromosome fragmentation in Paramecium is the consequence of imprecise DNA elimination events that are distinct from the precise excision of single-copy internal eliminated sequences and that target multicopy germ line sequences by homology-dependent epigenetic mechanisms. 相似文献
993.
Malham SK Lacoste A Gélébart F Cueff A Poulet SA 《Journal of experimental zoology. Part A, Comparative experimental biology》2003,295(2):136-144
Stress is thought to cause increased disease outbreaks and mortality in a number of invertebrates but currently very little information is available on mechanisms linking physiological states of stress and reduced disease resistance in these organisms. In the present study, we examined the possibility that stress alters immune functions, the principal line of defense against pathogens, in a molluscan model, the abalone Haliotis turbeculata. Immune parameters were investigated in abalones subjected to a 15 min mechanical disturbance which, as indicated by noradrenaline and dopamine hemolymphatic levels, resulted in a transient state of physiological stress. During the application of the stressor, immune parameters such as the number of circulating hemocytes, the migratory activity, the phagocytic capacity and the respiratory burst responses of hemocytes, decreased significantly. All parameters returned to initial values within 15-30 min after the end of the disturbance and a transient period of immunostimulation occurred between 100 and 480 min after the stress for all immune parameters except intracellular superoxide anion production. These results indicate that in the abalone H. tuberculata, as in vertebrates, a link exists between stress and the immune system. This may begin to answer why stress and disease outbreaks are linked in shellfish. 相似文献
994.
Altering the rate of translation initiation of a specific gene can tightly regulate the synthesis of the corresponding polypeptide and is an important mechanism in the control of gene expression. For some time it has been known that many genes involved in cell proliferation, cell growth and apoptosis have atypical 5' untranslated regions (UTRs) containing a high degree of RNA secondary structure, upstream open reading frames and internal ribosome entry segments. These features play a key role in the regulation of protein synthesis. In this review we discuss how the rate of translation initiation of proto-oncogenes and tumour suppressor genes is affected by elements in their 5' and 3' UTRs and we focus on how changes in the controlof gene expression at this level can contribute towards tumorigenesis. 相似文献
995.
Asad Y Cropp G Adams A O'Donnell A Raynaud F Judson I Workman P 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,785(1):175-186
The validation of an analytical method to quantify the antiangiogenic, (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid (SU006668) for pharmacokinetic determination in a phase I clinical trial, is described. HPLC, with a gradient mobile phase and UV detection at 440 nm, was used. SU006668 was extracted from plasma by precipitation of proteins with acetonitrile. The assay was linear from 25 to 2000 ng/ml (r(2)=0.997); sensitive (limit of quantification 25 ng/ml), accurate (RE 2.6-11.9%) and reproducible (inter-batch precision C.V. 3.2%). Pharmacokinetic data for six patients are presented. They show linear pharmacokinetics with a low volume of distribution and induction at doses of 50, 100 and 200 mg/m(2). 相似文献
996.
Jönsson BA Malmberg B Amilon A Helene Garde A Orbaek P 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,784(1):63-68
The aim of this work was to develop a method for determination of cortisol in saliva by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Saliva was sampled on Salivette tubes. These were centrifuged, deuterium-labeled cortisol was added as internal standard and the proteins precipitated by acetonitrile. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LC-MS-MS. The with-in run precision, tested by pooling saliva samples from volunteers and then analyzing these in a single run, was found to be 7% at 0.7 microgram l(-1). The between-run precision was tested by analysis of the same samples at different days and found to be 11% at 2.5 microgram l(-1). The limit of quantification was 0.5 microgram l(-1). The method was applied for analysis of saliva samples from three volunteers during their last week before vacation and the first and second week on vacation. In addition, the method was compared to analysis by an immunological method. The values from the immunological method were 2.7 times higher than the LC-MS-MS results. 相似文献
997.
998.
Stöcker M Tur MK Sasse S Krüssmann A Barth S Engert A 《Protein expression and purification》2003,28(2):211-219
Immunotoxins consist of a target-cell-specific binding moiety, chemically or recombinantly linked to a cytotoxic component. A number of different immunotoxins (IT) have increasingly been evaluated for immunotherapy. Since these foreign proteins are highly immunogenic in human, we have developed recombinant IT using the human ribonuclease angiogenin. Due to their potential toxic effects on eucaryotic cells, these IT are usually expressed in bacteria. Depending on the structure, size, and sequence of the desired IT, bacterial expression can be limited and the yield after purification be unsatisfactory. Therefore, the expression of IT in eucaryotic cells could provide a promising alternative. For this purpose we genetically fused the anti-CD30 single-chain variable fragment (scFv) Ki4 to the N- and C-termini of recombinant angiogenin. Both IT possess leader sequences, which mediate their secretion into the cell culture supernatant. Using a bicistronic mRNA the IT were simultaneously expressed together with enhanced green fluorescent protein (EGFP). This allows direct monitoring of transfected cells. An additional plasmid encoded Zeocin resistance enhances the cultivation of transfected cells under selection pressure. Three days after transfection of 293T-cells, unpurified IT were analyzed by flow cytometry and competitive cell proliferation assays. This is the first report on the use of eucaryotic cells for the secretion of functionally active IT with a human effector domain. 相似文献
999.
Thuesen MH Nørgaard A Hansen AM Caspersen MB Christensen HE 《Protein expression and purification》2003,27(1):175-181
The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein was purified to apparent homogeneity and analyzed by electronic absorption spectroscopy, nanoflow electrospray ionization time-of-flight mass spectrometry, and electrochemistry. Cyclic voltammograms and UV-vis electronic absorption spectra were indistinguishable from the equivalent data of native P. stutzeri cytochrome c(4). Furthermore, the calculated and experimentally determined molecular masses of recombinant cytochrome c(4) were identical. Biochemical characterization of both wild-type and mutant derivatives of the protein will be greatly enhanced and facilitated by the described high-yield fermentation and rapid isolation procedure. 相似文献
1000.