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1.
The antigen-specific activation of murine nonimmunized B lymphocytes subsequently used in hybridization experiments has been investigated by using phylogenetically conserved antigens or autologous immunogens. This in vitro immunization was supported by B cell growth and differentiation factors derived from phorbol myristate acetate-stimulated EL-4 thymoma cells and mixed lymphocyte cultures (MLC). A filter immuno-plaque assay was used to evaluate the effect of different activation procedures on the number of antigen-specific plaque-forming cells (PFC). We first determined the requirement for MLC-derived lymphokines in the in vitro immunization. An optimal number of antigen-specific PFC was obtained when using 33 to 50% of the supernatant from a 48-hr MLC to support the activation. B cell growth and differentiation factors derived from EL-4 cultures were then tested for their abilities to potentiate the number of PFC by using both unseparated spleen cells and highly purified Ig-positive B cells as target cells. The combination of lymphokines found in supernatants from 25% EL-4 thymoma culture and 33% MLC yielded the highest number of PFC when used to support an in vitro immunization. This optimal factor preparation was used to determine the kinetics (4 to 7 days) and the dose response (0.01 to 10 micrograms antigen/ml) of antigen-specific B cell activation before using the immunized splenocytes as parental cells in cell fusion experiments. Mouse albumin and hemoglobin, actin (25 micrograms/ml), RNA polymerase II (5 micrograms/ml), as well as syngeneic mouse serum were used to immunize BALB/c spleen cells in vitro. We obtained antigen-specific PFC by using all of the different immunogens, including syngeneic mouse serum, and the in vitro immunized cells were then used in hybridization experiments. The specific efficiencies of each fusion that made use of cells immunized with mouse albumin, hemoglobin, syngeneic mouse serum, actin, or RNA polymerase II were 12, 31, 33, 52, and 22%, respectively, which illustrated the apparent lack of immune tolerance found when the immunization was performed in culture. 相似文献
2.
Hormonal Regulation of the Lectin Biosynthesis in Callus Culture of the Phaseolus vulgaris Plant 下载免费PDF全文
Callus cultures established from Phaseolus vulgaris seedlings were used to investigate hormonal influence on lectin biosynthesis. The plant tissue cultures were initiated using defined levels of both a cytokinin (kinetin) and an auxin (2,4-dichlorophenoxyacetic acid) and were then transferred to media containing different amounts of these hormones. The lectin content of each callus culture was determined using an enzyme immunoassay specific for the seed lectin of the P. vulgaris plant. The lectin biosynthesis was directly affected by the levels of auxin and cytokinin in the culture media and no lectin was detected in hormone-free medium. This enabled us to compose culture media yielding a maximal or minimal lectin content of the callus cultures, illustrating the ability to induce an enhancement or suppression of the in vitro lectin biosynthesis. The lectin level of callus tissue during the growth cycle of a culture was, furthermore, related to the cellular growth rate which might indicate an involvement of the lectin in cellular events during rapid cell division. 相似文献
3.
Isolation and partial characterization of a carbohydrate-binding protein from a nematode-trapping fungus 总被引:2,自引:0,他引:2 下载免费PDF全文
A developmentally regulated carbohydrate-binding protein from the capture organs of Arthrobotrys oligospora, not present on hyphae, was isolated and partially characterized. Surface structures of A. oligospora were radiolabeled with [125I]iodosulfanilic acid. The fungus was homogenized, and the homogenate was passed over an affinity column containing N-acetyl-D-galactosamine immobilized to Sepharose 6B. The bound radiolabeled protein was eluted from the affinity column with a glycine-hydrochloride buffer (pH 3.0), concentrated, and chromatographed on a metal chelate affinity gel containing Ca2+. EDTA was used as an eluant for the radiolabeled protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with autoradiography revealed a molecular weight for the carbohydrate- and cation-binding polypeptide of ca. 20,000. 相似文献
4.
Hardies SC; Martin SL; Voliva CF; Hutchison CA d; Edgell MH 《Molecular biology and evolution》1986,3(2):109-125
5.
Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies. 总被引:8,自引:1,他引:7 下载免费PDF全文
The humoral immune response to human cytomegalovirus (CMV) membrane glycoprotein gp58/116 (gB) has been studied by establishing cell lines producing specific human monoclonal antibodies. These cell lines were generated from peripheral blood lymphocytes obtained from a healthy carrier. Hybridomas producing gp58/116-specific antibodies were detected by reactivity to procaryotically expressed proteins containing the major neutralizing epitopes of this glycoprotein complex. One antibody, ITC88, which recognized an epitope located between amino acid residues 67 and 86 of gp116, potently neutralized the virus at 1 to 2 micrograms of immunoglobulin G per ml. Only four of the six human antibodies detecting the major neutralizing domain of gp58 neutralized the virus, and none of them required complement for activity. All antibodies that bound mature, processed gp58 recognized a conformational epitope involving sequences between residues 549 and 635. However, small differences existed between the antibodies in the actual minimal requirement for C- and N-terminal parts of this epitope. By peptide mapping with several of the antibodies, the epitope was shown to consist mainly of residues between amino acids 570 to 579 and 606 to 619. Despite the conformational nature of the epitope, the antibodies recognized both reduced and denatured native antigen. Presence of carbohydrates was not required for antigen binding of these gp58-specific human antibodies, but in at least one case, it greatly enhanced antigen recognition, indicating an importance of carbohydrate structures in some epitopes within the major neutralizing specificity of gp58. 相似文献
6.
A major difference between the divergence patterns within the lines-1 families in mice and voles 总被引:3,自引:0,他引:3
Vanlerberghe F; Bonhomme F; Hutchison CA d; Edgell MH 《Molecular biology and evolution》1993,10(4):719-731
L1 retroposons are represented in mice by subfamilies of interspersed
sequences of varied abundance. Previous analyses have indicated that
subfamilies are generated by duplicative transposition of a small number of
members of the L1 family, the progeny of which then become a major
component of the murine L1 population, and are not due to any active
processes generating homology within preexisting groups of elements in a
particular species. In mice, more than a third of the L1 elements belong to
a clade that became active approximately 5 Mya and whose elements are >
or = 95% identical. We have collected sequence information from 13 L1
elements isolated from two species of voles (Rodentia: Microtinae: Microtus
and Arvicola) and have found that divergence within the vole L1 population
is quite different from that in mice, in that there is no abundant
subfamily of homologous elements. Individual L1 elements from voles are
very divergent from one another and belong to a clade that began a period
of elevated duplicative transposition approximately 13 Mya. Sequence
analyses of portions of these divergent L1 elements (approximately 250 bp
each) gave no evidence for concerted evolution having acted on the vole L1
elements since the split of the two vole lineages approximately 3.5 Mya;
that is, the observed interspecific divergence (6.7%-24.7%) is not larger
than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses
showed no clustering into Arvicola and Microtus clades.
相似文献
7.
Abstract This investigation describes the detection of a component in Escherichia coli capable of binding a large proportion of human antibody variable domains including otherwise highly monospecific antibodies induced by an in vivo antibody response. This interaction is of low affinity, but cross-linking of IgG molecules by e.g. anti-immunoglobulin preparations, provides a sufficient degree of multivalency to promote a high avidity interaction. This binding which occurs both with κ and λ light chain-containing antibodies, appears to involve the variable region of human antibodies making it a superantigen-like activity. This is proposed based on the facts that: (i) different human antibodies of IgG1 isotype appear to bind to different extents suggesting that variable domain differences determine the binding activity; and (ii) addition of soluble antigen abrogates the interaction with the E. coli -derived molecule. Future studies of the nature and possible in vivo consequences of these interactions are warranted since any superantigen activity associated with this binding might affect the human immune response occurring as a consequence of E. coli infections. 相似文献
8.
Molecular phylogeny and divergence times of drosophilid species 总被引:32,自引:15,他引:17
The phylogenetic relationships and divergence times of 39 drosophilid
species were studied by using the coding region of the Adh gene. Four
genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from
Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and
Sophophora--were included. After conducting statistical analyses of the
nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA
genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila
as the outgroup. The phylogenetic tree obtained showed that the first major
division of drosophilid species occurs between subgenus Sophophora (genus
Drosophila) and the group including subgenera Drosophila and Engiscaptomyza
plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then
divided into D. willistoni and the clade of D. obscura and D. melanogaster
species groups. In the other major drosophilid group, Zaprionus first
separates from the other species, and then D. immigrans leaves the
remaining group of species. This remaining group then splits into the D.
repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila,
Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly
clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups
is monophyletic. The splitting of subgenera Drosophila and Sophophora
apparently occurred about 40 Mya, whereas the D. repleta group and the
Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the
splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya,
suggesting that Scaptomyza experienced a rapid morphological evolution. The
D. obscura and D. melanogaster groups apparently diverged about 25 Mya.
Many of the D. repleta group species studied here have two functional Adh
genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two
duplication events.
相似文献
9.
The principle of competitive binding assay in combination with an immobilized lectin (concanavalin A), in close proximity to an oxygen sensor, has been used to quantify carbohydrates and to determine association constants for lectin-carbohydrate interactions. Methyl α-d-mannopyranoside was determined down to 0.5 μg/ml. Ka (maltose) and Ka (maltotriose) was found to be 2.1 × 103 and 1.7 × 103m?1, respectively, which are comparable to values quoted in the literature of approximately 2.8 × 103m?1 for both maltose and maltotriose. Furthermore, the estimation of the bonus effect, due to multipoint attachment, for a low-molecular-weight dextran is discussed. 相似文献
10.
Sulfate reduction and S-oxidation in a moorland pool sediment 总被引:3,自引:2,他引:1
In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO4
2– reduction rates in the sediment, (2) depletion of SO4
2– in the overlying water column and (3) release of35S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO4
2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core35SO4
2– injection. Rates of SO4
2– consumption in the overlying water were estimated by changes in SO4
2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m–2 d–1. Rates of SO4
2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m–2 d–1 (November) to 0.43–1.81 mmol m–2 d–1 (July, August and April). Maximum rates of oxidation to SO4
2– in July 1990 estimated by combination of SO4
2– reduction rates and rates of in situ SO4
2– uptake in the enclosed water column were 10.3 and 10.5 mmol m–2 d–1 at an organic rich and at a sandy site respectively.Experiments with35S2– and35SO4
2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO4
2– and (2) the presence of mainly non SO4
2–-S derived from reduced S transported from the sediment into the overlying water. A35S2– tracer experiment showed that about 7% of35S2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author 相似文献