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81.
The class I genes of the major histocompatibility complex (Mhc) are here investigated for the first time in a passerine bird. The great reed warbler is a rare species in Sweden with a
few semi-isolated populations. Yet, we found extensive Mhc class I variation in the study population. The variable exon 3, corresponding to the α2 domain, was amplified from genomic DNA with degenerated primers. Seven different genomic class I sequences were detected
in a single individual. One of the sequences had a deletion leading to a shift in the reading frame, indicating that it was
not a functional gene. A randomly selected clone was used as a probe for restriction fragment length polymorphism (RFLP) studies
in combination with the restriction enzyme Pvu II. The RFLP pattern was complex with 21–25 RFLP fragments per individual and extensive variation. Forty-nine RFLP genotypes
were detected in 55 tested individuals. To study the number of transcribed genes, we isolated 14 Mhc class I clones from a cDNA library from a single individual. We found eight different sequences of four different lengths
(1.3–2.2 kilobases), suggesting there are at least four transcribed loci. The number of nonsynonymous substitutions (d
N
) in the peptide binding region of exon 3 were higher than the number of synonymous substitutions (d
S
), indicating balancing selection in this region. The number of transcribed genes and the numerous RFLP fragments found so
far suggest that the great reed warbler does not have a "minimal essential Mhc" as has been suggested for the chicken.
Received: 13 May 1998 / Revised: 18 August 1998 相似文献
82.
Hofmann A Ruvinov S Hess S Schantz R Delmer DP Wlodawer A 《Protein science : a publication of the Protein Society》2002,11(8):2033-2040
The oligomeric state in solution of four plant annexins, namely Anx23(Ca38), Anx24(Ca32), Anx(Gh1), and Anx(Gh2), was characterized by sedimentation equilibrium analysis and gel filtration. All proteins were expressed and purified as amino-terminal His(n) fusions. Sequencing of the Anx(Gh1) construct revealed distinct differences with the published sequence. Sedimentation equilibrium analysis of Anx23(Ca38), Anx24(Ca32), and Anx(Gh1) suggests monomer-trimer equilibria for each protein with association constants in the range of 0.9 x 10(10)-1.7 x 10(11) M(-2). All four proteins were subjected to analytical gel filtration under different buffer conditions. Observations from this experiment series agree quantitatively with the ultracentrifugation results, and strongly suggest calcium independence of the annexin oligomerization behavior; moreover, binding of calcium ions to the proteins seems to require disassembly of the oligomers. Anx(Gh2) showed a different elution profile than the other plant annexins; while having only a very small trimer content, this annexin seems to exist in a monomer-dimer equilibrium in solution. 相似文献
83.
Experimental evidence for major histocompatibility complex-allele-specific resistance to a bacterial infection 总被引:9,自引:0,他引:9
Lohm J Grahn M Langefors A Andersen Ø Storset A von Schantz T 《Proceedings. Biological sciences / The Royal Society》2002,269(1504):2029-2033
The extreme polymorphism found at some major histocompatibility complex (MHC) loci is believed to be maintained by balancing selection caused by infectious pathogens. Experimental support for this is inconclusive. We have studied the interaction between certain MHC alleles and the bacterium Aeromonas salmonicida, which causes the severe disease furunculosis, in Atlantic salmon (Salmo salar L.). We designed full-sibling broods consisting of combinations of homozygote and heterozygote genotypes with respect to resistance or susceptibility alleles. The juveniles were experimentally infected with A. salmonicida and their individual survival was monitored. By comparing full siblings carrying different MHC genotypes the effects on survival due to other segregating genes were minimized. We show that a pathogen has the potential to cause very intense selection pressure on particular MHC alleles; the relative fitness difference between individuals carrying different MHC alleles was as high as 0.5. A co-dominant pattern of disease resistance/susceptibility was found, indicative of qualitative difference in the immune response between individuals carrying the high- and low-resistance alleles. Rather unexpectedly, survival was not higher among heterozygous individuals as compared with homozygous ones. 相似文献
84.
We compared three different molecular methods currently used for screening of Mhc variation in population studies of Atlantic salmon. Restriction fragment length polymorphism (RFLP) of the entire class II gene detected 22 haplotypes. Seventeen exon 2 sequences were obtained from individuals carrying the 22 haplotypes, two of which had not been detected by RFLP. The six alleles (27%) detected by RFLP and not by exon 2 sequencing probably resulted from sequence variation outside exon 2. Within exon 2, RFLP differentiated 88% of the sequences. Alternatively, denaturing gradient gel electrophoresis (DGGE) performed under two run conditions detected 94% of the sequence variation. Both RFLP using different probes, and the two PCR-based methods using three different primer pairs, suggest that there is only a single Mhc class II B gene in the Baltic populations of Atlantic salmon. 相似文献
85.
Hofmann A Proust J Dorowski A Schantz R Huber R 《The Journal of biological chemistry》2000,275(11):8072-8082
This work provides the first three-dimensional structure of a member of the plant annexin family and correlates these findings with biochemical properties of this protein. Annexin 24(Ca32) from Capsicum annuum was purified as a native protein from bell pepper and was also prepared by recombinant techniques. To overcome the problem of precipitation of the recombinant wild-type protein in crystallization trials, two mutants were designed. Whereas an N-terminal truncation mutant turned out to be an unstable protein, the N-terminal His-tagged annexin 24(Ca32) was crystallized, and the three-dimensional structure was determined by x-ray diffraction at 2. 8 A resolution. The structure refined to an R-factor of 0.216 adopts the typical annexin fold; the detailed structure, however, is different from non-plant annexins, especially in domains I and III and in the membrane binding loops on the convex side. Within the unit cell there are two molecules per asymmetric unit, which differ in conformation of the IAB-loop. Both conformers show Trp-35 on the surface. The loop-out conformation is stabilized by tight interactions of this tryptophan with residue side chains of a symmetry-related molecule and enforced by a bound sulfate. Characterization of this plant annexin using biophysical methods revealed calcium-dependent binding to phospholipid vesicles with preference for phosphatidylcholine over phosphatidylserine and magnesium-dependent phosphodiesterase activity in vitro as shown with adenosine triphosphate as the substrate. A comparative unfolding study of recombinant annexin 24(Ca32) wild type and of the His-tag fusion protein indicates higher stability of the latter. The effect of this N-terminal modification is also visible from CD spectra. Both proteins were subjected to a FURA-2-based calcium influx assay, which gave high influx rates for the wild-type but greatly reduced influx rates for the fusion protein. We therefore conclude that the N-terminal domain is indeed a major regulatory element modulating different annexin properties by allosteric mechanisms. 相似文献
86.
Whiteman M Chu SH Siau JL Rose P Sabapathy K Schantz JT Cheung NS Spencer JP Armstrong JS 《Cellular signalling》2007,19(4):705-714
At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H(2)O(2)) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint. 相似文献
87.
The structures of four new aliphatic compounds from the essential oil of Mentha x gentilis nm. hirtella have been elucidated as 2,4-dimethylhexane-2-ol, 5-methylnonanal, 2-butylhexanal and tetrahydrogeranyl acetate. 相似文献
88.
A biologically active acid hydrolysis product of saxitoxin. 总被引:2,自引:0,他引:2
V E Ghazarossian E J Schantz H K Schnoes F M Strong 《Biochemical and biophysical research communications》1976,68(3):776-780
The preparation, purification, and biological and chemical properties of a decarbamylated product of saxitoxin are described. 相似文献
89.
90.