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91.
Roberto E. Izquierdo Kimberly Breese Shalini Jain Daniel Carestio Lawrence Jung James Figge 《In vitro cellular & developmental biology. Animal》1995,31(1):71-76
Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH),
that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered
C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard,
a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture.
A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were
positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression
from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components
of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation
of transferred genes in animals. 相似文献
92.
乌奴龙胆中五个新的环烯醚萜甙 总被引:4,自引:0,他引:4
从藏药乌奴龙胆(GentianaurnulaSmith)(龙胆科)的全草中分离到5个新的环烯醚萜甙,命名为乌奴龙胆甙(gentioumoside)A-E;它们的结构主要通过光谱分析得以确定。其中,乌奴龙胆A-C是二聚环烯醚萜甙,而乌奴龙胆甙D和E为马钱素型的环烯醚萜甙,所有这些化合物的分子中都具有一个2,3-二羟基苯甲酰基或其衍生物的取代基。 相似文献
93.
Richard Wales Hazel C. Gorham Khalid Hussain Lynne M. Roberts J. Michael Lord 《Glycoconjugate journal》1994,11(4):274-281
Deleted forms of ricin B chain (RTB) containing only one of the two galactose binding sites were produced inE. coli and targeted to the periplasm by fusion to theompA orompF signal sequences. The proteins were then isolated from the periplasm and their sugar binding properties assessed. Previous studies investigating the properties of such proteins produced inXenopus laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugars, unlike the full-length unglycosylated proteins. When produced inE. coli however we found that only one, EB733, of a number of deleted forms of RTB closely related to those previously produced inXenopus laevis oocytes, bound to simple sugars. All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which has been previously utilized to measure binding of lectins to the terminal galactose residues of glycoprotein oligosaccharides. However, in contrast to glycosylated RTB, binding of the deletion mutants could be competed to only a small degree or not at all with galactose. The only deletion mutant observed to bind to free galactose when produced inE. coli corresponded closely to the complete domain 2 of RTB. It is assumed that this mutant forms a stable structure similar to that of the C-terminal domain in the full-length protein. The structural integrity of EB733 was not only suggested by its sugar binding properties and solubility but also by its consistently higher level of expression and the absence of any apparent susceptibility toE. coli proteases.Abbreviations RTA
ricin toxin A chain
- RTB
ricin toxin B chain
- ER
endoplasmic reticulum
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
- IPTG
isopropyl -d-thiogalactopyranoside 相似文献
94.
Gabriel Gutiérrez Josep Casadesús Jose L. Oliver Antonio Marine 《Journal of molecular evolution》1994,39(4):340-346
E. coli genes that contain a high frequency of the tetranucleotide CTAG are also rich in the tetramers CTTG, CCTA, CCAA, TTGG, TAGG, and CAAG (group-I tetramers). Conversely, E. coli genes lacking CTAG are rich in the tetranucleotides CCTG, CCAG, CTGG, and CAGG (group-II tetramers). These two gene samples differ also in codon usage, amino acid composition, frequency of Dcm sites, and contrast vocabularies. Group-I tetramers have in common that they are depleted by very-short-patch repair (VSP), while group-II tetramers are favored by VSP activity. The VSP system repairs G:T mismatches to G:C, thereby increasing the overall G+C content of the genome; for this reason the CTAG-rich sample has a lower G+C content than the CTAG-poor sample. This compositional heterogeneity can be tentatively explained by a low level of VSP activity on the CTAG-rich sample. A negative correlation is found between the frequency of group-I tetramers and the level of gene expression, as measured by the Codon Adaptation Index (CAI). A possible link between the rate of VSP activity and the level of gene expression is considered.Correspondence to: A. Marine 相似文献
95.
Abstract: The effects of prostaglandin E2 (PGE2) on 86Rb efflux from rat brain synaptosomes were studied to explore its role in nerve ending potassium (K+) channel modulation. A selective dose-dependent inhibition of the calcium-activated charybdotoxin-sensitive component of efflux was found upon application of PGE2. No significant effect was seen on basal and voltage-dependent components over the concentration range of 10–8 to 10–5M. The protein kinase C (PKC) inhibitors H-7 (10 μM) and staurosporine (100 nM), as well as prolonged preincubation (90 min) with 40-phorbol 12, 13-dibutyrate, which has been reported to down-regulate PKC, abolished the PGE2-in- duced inhibition, whereas HA1004 (10 μM) and Rp-3′,5’cyclic phosphorothioate (100 nM), which are relatively more selective for protein kinase A than PKC, did not. 4β-Phorbol 12, 13-dibutyrate (100 nM), an activator of PKC, produced a similar inhibition of the Ca2+-dependent component of 86Rb efflux but also had no effect on the basal and voltage-dependent components. These data suggest that PGE2 can inhibit rat brain nerve ending calcium-activated 86Rb efflux, and this inhibition may involve PKC activation. 相似文献
96.
Abstract: A synthetic peptide corresponding to residues 226–240 (E9 peptide) of human τ, which contains an Lys-Ser-Pro motif, was used to raise a polyclonal antibody. The antibody, E9, was 10-fold less reactive with phospho-E9 peptide than with native E9 peptide. E9 antibody was used to study the extent of phosphorylation in a modified form of τ (PHF-τ) that is found in Alzheimer's disease brain and is incorporated into paired helical filaments (PHFs). E9 immunolabeled Alzheimer's disease neurofibrillary tangles and abnormal neurites in brain sections intensely, with increased immunoreactivity detected after pretreatment of sections with phosphatase. On immunoblots and ELISA, E9 reacted with PHF-τ and recombinant human τ but not with the high and middle molecular weight neurofilament proteins. Phosphatase treatment of PHF-τ improved the E9 immunoreactivity by 30–50%. Dephosphorylated high but not middle molecular weight neurofilament protein became reactive with E9. These results indicate that <50% of the PHF-T is phosphorylated in the subregion corresponding to residues 226–240 of τ and suggest that the phosphorylation of this region may not be essential for PHF formation. 相似文献
97.
Damage and mutagenesis of E. coli and bacteriophage λ induced by oxathiolane and aziridinyl steroids
λ-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the irradiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of λ phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage λ has been suggested. 相似文献
98.
Self-splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family. 总被引:17,自引:3,他引:14
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A. E. Gorbalenya 《Protein science : a publication of the Protein Society》1994,3(7):1117-1120
A new family of protein domains consisting of 50-80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group II introns; eubacterial genomes and plasmids; and phages. The name "EX1HH-HX3H" was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EX1HH and HX3H. The "His" motifs often alternate with amino- and carboxy-terminal motifs of a new type of Zn-finger-like structure CX2,4CX29-54[CH]X2,3[CH]. The EX1HH-HX3H domain in eubacterial E2-type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EX1HH-HX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group II introns encoding the EX1HH-HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules. 相似文献
99.
影响大肠杆菌中外源基因表达的因素 总被引:41,自引:1,他引:40
大肠杆菌已经被广泛地应用于表达各种外源基因,但是,不同的外源基因在表达效率上却有很大的差异,文章综述了影响大肠杆菌中外源基因表达的因素,这将有助于认识大肠杆菌中外源基因表达的规律,以便采取有效的方法提高外源基因在大肠杆菌中的表达效率. 相似文献
100.
Efficient extracellular production of hybrid E. coli heat-labile enterotoxin B subunits in a marine vibrio 总被引:1,自引:0,他引:1
Alessandro Marcello Arianna Loregian Giorgio Palù Timothy R. Hirst 《FEMS microbiology letters》1994,117(1):47-51
Abstract Escherichia coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides to target cells, particularly for the oral delivery of epitopes to the mucosal immune system. In this study, two extensions to the C-terminus of EtxB were genetically engineered that correspond to a well-characterized neutralising epitope of glycoprotein D from herpes simplex virus (EtxB-gD) and to the C-terminal nine amino acids from the 38 kDa subunit of HSV-encoded ribonucleotide reductase (EtxB-R2). Here we describe the extracellular secretion of the two hybrid EtxBs from a marine Vibrio harbouring a broad-host range inducible expression vector containing the hybrid genes. Large amounts of intact fusion proteins (15–20 mg per liter of culture) were secreted into the medium upon induction. These hybrid proteins maintained the receptor-binding activity of the native toxin as well as being cross-reactive with anti-EtxB and anti-heterologous peptide monoclonal antibodies. 相似文献