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81.
Ilaria?Bartalesi Ivano?BertiniEmail author Giulia?Di?Rocco Antonio?Ranieri Antonio?Rosato Murugendra?Vanarotti Paul?R.?Vasos Maria?Silvia?Viezzoli 《Journal of biological inorganic chemistry》2004,9(5):600-608
The minimal mono-heme ferricytochrome
c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.Abbreviations Bpcytc
soluble fragment of cytochrome c553 from Bacillus pasteurii
- GdmCl
guanidinium chloride
- I75A
Ile75 to Ala mutant
- I75V
Ile75 to Val mutant
- P72A
Pro72 to Ala mutant
- P72G
Pro72 to Gly mutant
- Q68K
Gln75 to Lys mutant
- WT
wild type 相似文献
82.
Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects 总被引:8,自引:0,他引:8
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D'Ovidio R Raiola A Capodicasa C Devoto A Pontiggia D Roberti S Galletti R Conti E O'Sullivan D De Lorenzo G 《Plant physiology》2004,135(4):2424-2435
83.
Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth
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Capodicasa C Vairo D Zabotina O McCartney L Caprari C Mattei B Manfredini C Aracri B Benen J Knox JP De Lorenzo G Cervone F 《Plant physiology》2004,135(3):1294-1304
Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis. 相似文献
84.
Comparison of PCR, electrochemical enzyme-linked immunosorbent assays, and the standard culture method for detecting salmonella in meat products 总被引:2,自引:0,他引:2
Croci L Delibato E Volpe G De Medici D Palleschi G 《Applied and environmental microbiology》2004,70(3):1393-1396
An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method. 相似文献
85.
Brown DR Guantieri V Grasso G Impellizzeri G Pappalardo G Rizzarelli E 《Journal of inorganic biochemistry》2004,98(1):133-143
In this paper, we report the characterization of copper(II) complexes with two prion (PrP) protein peptide fragment analogues (VNITKQHTVTTTT), one with the N-terminus acetylated and the C-terminus amidated (PrP Ac180-193NH2) and the other with both the C- and N-termini free (PrP 180-193). Such peptide sequence almost entirely encompasses the PrPC's helix 2 in the C-terminal region. The stoichiometry, the binding modes and the conformational features of the copper(II) complexes with the above mentioned two peptides were investigated by electrospray ionization-mass spectrometry (ESI-MS), UV-visible (UV-Vis) spectrometry and electron paramagnetic resonance (EPR) spectrometry as well as by circular dichroism (CD) measurements. The binding site location of copper(II) in the structured region of the protein can be here suggested on the basis of our findings that show the involvement of His 187 residue. The similarity of the EPR parameters suggests that the anchoring imidazole residue drives the copper(II) coordination environment towards a common binding motif in different regions of the prion protein. 相似文献
86.
Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays 总被引:1,自引:0,他引:1
Matsuzaki H Dong S Loi H Di X Liu G Hubbell E Law J Berntsen T Chadha M Hui H Yang G Kennedy GC Webster TA Cawley S Walsh PS Jones KW Fodor SP Mei R 《Nature methods》2004,1(2):109-111
We present a genotyping method for simultaneously scoring 116,204 SNPs using oligonucleotide arrays. At call rates >99%, reproducibility is >99.97% and accuracy, as measured by inheritance in trios and concordance with the HapMap Project, is >99.7%. Average intermarker distance is 23.6 kb, and 92% of the genome is within 100 kb of a SNP marker. Average heterozygosity is 0.30, with 105,511 SNPs having minor allele frequencies >5%. 相似文献
87.
Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection 总被引:16,自引:0,他引:16
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Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins that counteract fungal polygalacturonases, which are important virulence factors. Like many other plant defense proteins, PGIPs are encoded by gene families, but the roles of individual genes in these families are poorly understood. Here, we show that in Arabidopsis, two tandemly duplicated PGIP genes are upregulated coordinately in response to Botrytis cinerea infection, but through separate signal transduction pathways. AtPGIP2 expression is mediated by jasmonate and requires COI1 and JAR1, whereas AtPGIP1 expression is upregulated strongly by oligogalacturonides but is unaffected by salicylic acid, jasmonate, or ethylene. Both AtPGIP1 and AtPGIP2 encode functional inhibitors of polygalacturonase from Botrytis, and their overexpression in Arabidopsis significantly reduces Botrytis disease symptoms. Therefore, gene duplication followed by the divergence of promoter regions may result in different modes of regulation of similar defensive proteins, thereby enhancing the likelihood of defense gene activation during pathogen infection. 相似文献
88.
Herpes simplex virus glycoprotein K,but not its syncytial allele,inhibits cell-cell fusion mediated by the four fusogenic glycoproteins,gD, gB,gH, and gL
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A Myc epitope was inserted at residue 283 of herpes simplex virus type 1 (HSV-1) glycoprotein K (gK), a position previously shown not to interfere with gK activity. The Myc-tagged gK localized predominantly to the endoplasmic reticulum, both in uninfected and in HSV-infected cells. gK, coexpressed with the four HSV fusogenic glycoproteins, gD, gB, gH, and gL, inhibited cell-cell fusion. The effect was partially dose dependent and was observed both in baby hamster kidney (BHK) and in Vero cells, indicating that the antifusion activity of gK may be cell line independent. The antifusion activity of gK did not require viral proteins other than the four fusogenic glycoproteins. A syncytial (syn) allele of gK (syn-gK) carrying the A40V substitution present in HSV-1(MP) did not block fusion to the extent seen with the wild-type (wt) gK, indicating that the syn mutation ablated, at least in part, the antifusogenic activity of wt gK. We conclude that gK is part of the mechanism whereby HSV negatively regulates its own fusion activity. Its effect accounts for the notion that cells infected with wt HSV do not fuse with adjacent, uninfected cells into multinucleated giant cells or syncytia. gK may also function to preclude fusion between virion envelope and the virion-encasing vesicles during virus transport to the extracellular compartment, thus preventing nucleocapsid de-envelopment in the cytoplasm. 相似文献
89.
90.
Oligogalacturonides inhibit the induction of late but not of early auxin-responsive genes in tobacco
Oligogalacturonides (OGs) released from the plant cell wall regulate several defense responses, as well as various aspects of plant growth and development. In these latter effects, OGs exhibit auxin-antagonist activity. To shed light on the mechanism by which OGs antagonise auxin, we analysed the ability of these oligosaccharides to inhibit the activity of four auxin-up-regulated promoters [pGm-GH3 of soybean (Glycine max L. Merr.), pNt114 of tobacco (Nicotiana tabacum L.), and prolB and prolD of Agrobacterium rhizogenes] driving the expression of the beta-glucuronidase reporter gene (GUS) in transgenic tobacco seedlings. Our results indicate that OGs at submicromolar concentrations inhibit the activation by auxin of pNt114, prolB and prolD, but not that of pGm-GH3. Comparative analysis of the kinetics of activation of the four promoters in response to the hormone shows that, while pGm-GH3 is rapidly activated, the other three promoters exhibit a delayed activation, with a lag of at least 4 h before the appearance of GUS activity. The lack of effect of the OGs on early auxin-responsive genes was confirmed by RNA gel blot analysis of the tobacco genes Nt-GH3 and Nt-iaa2.3/2.5. Our results suggest that the auxin-antagonist action of OGs affects the expression of late but not of early auxin-responsive genes. 相似文献