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101.
Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a Ka of 3.02 × 104 M−1 and 1.52 × 104 M−1, respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding. 相似文献
102.
Takeyuki Sugiura Kayoko Kurosawa-Ohsawa Mikiko Takahashi Hiromi B. Maruyama 《Biotechnology letters》1990,12(11):799-804
Summary The relationship between productivity and -carboxylation of recombinant protein C was studied with Chinese hamster ovary (CHO) cell line producing human protein C (huPC) by the use of two types of enzyme-linked immunosorbent assay (ELISA). Productivity could be varied by the addition of different levels of serum. An inverse correlation was found between the degree of -carboxylation and productivity of recombinant protein C. 相似文献
103.
Localization of Various Forms of the γ Subunit of G Protein in Neural and Nonneural Tissues 总被引:1,自引:0,他引:1
Tomiko Asano Rika Morishita Kayoko Ohashi Masato Nagahama Takenori Miyake Kanefusa Kato 《Journal of neurochemistry》1995,64(3):1267-1273
Abstract: For a study of the localization of various forms of the γ subunit of G proteins, antibodies were raised in rabbits against peptides that corresponded to partial amino acid sequences of bovine γ2, γ3, γ5, and γ7. Affinity-purified antibodies against γ2, γ3, and γ5 reacted specifically with γ2, γ3, and γ5, respectively, but the antibody against γ7 reacted with γ2, γ3, and a novel γ subunit, designated γs1, as well as with γ7. Because these antibodies reacted with the respective forms of the γ subunit from rat brain, we investigated the localization of γ subunits in the rat. γ2 and γ3 were abundant in all regions in the brain, whereas the concentration of γ5 and γ7 was relatively low with the single exception being a high concentration of γ7 in the striatum. The concentration of γ2 was consistently high during ontogenic development in the rat brain, whereas γ3 appeared about a week after birth and their concentrations then increased until a month after birth. In tissues other than the brain, γ3 was observed only in the pituitary gland, whereas γ2, γ5, and γ7 were found in a variety of tissues. In addition, most tissues contained relatively high concentrations of some other γ subunit, which was detected with an antibody against a γ7-derived peptide and appeared to be γs1. Among cloned cells tested, γ3 was detected only in PC12 pheochromocytoma cells. Taken together, the results indicated that γ3 was expressed specifically in neuronal cells, and γs1 was the major γ subunit in most nonneural cells. γ2, γ5, and γ7 were distributed in a variety of tissues, but γ2 was dominant in the brain. 相似文献
104.
We developed a sensitive enzyme immunoassay system specific for human lactate dehydrogenase (LDH)- B4 with antiacetylated LDH-B4 Fab-horse-radish peroxidase conjugate. The enzyme immunoassay system was not interfered with by up to 0.3 mg/tube of hemoglobin. Thus, we measured LDH-B4 concentrations in the hemolysate of seven heterozygous individuals deficient in LDH-B subunit activity and eight normal individuals. We could not find a significant difference between the LDH-B4 concentrations in heterozygous and those in normal individuals. These results demonstrate that heterozygous individuals deficient in LDH-B subunit activity produce enzymatically inactive B subunits.This work was supported in part by grants in aid for Scientific Research from the Ministry of Education, Japan (59570998), and from the Clinical Pathology Research Foundation of Japan. 相似文献
105.
Genotypic analysis of families with lactate dehydrogenase A(M) deficiency by selective DNA amplification 总被引:2,自引:2,他引:0
Summary Genomic DNA prepared from LDH-A-deficient whole blood was amplified by the polymerase chain reaction technique using two primers specific for the active human LDH-A gene. The amplified fragment was examined by direct agarose gel electrophoresis, and a deletion of 20 base pairs (bp) in exon 6 of the LDH-A gene was found. The results permitted a clear distinction between the homozygous mutant, the heterozygous mutant, and wild-type genotypes. Moreover, HinfI digestion and direct sequencing of the amplified product confirmed the results from direct agarose gel electrophoresis. Four families, including 18 individuals, were shown to contain the same mutation, that is a 20-bp deletion in exon 6. All genotypes were consistent with their biochemical phenotypes as evaluated by the ratio of LDH-B to LDH-A subunits in erythrocytes. Thus, all four known affected families in Japan have been shown to carry the same mutant gene, which may have been derived from a single mutational event. 相似文献
106.
Kayoko Yamaji Naotoshi Tsuji Takeharu Miyoshi M. Khyrul Islam Takeshi Hatta M. Abdul Alim Anisuzzaman Akio Takenaka Kozo Fujisaki 《Parasitology international》2009,58(3):232-237
We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; kcat / Km = 0.19 and 0.0023 M− 1 S− 1, respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks. 相似文献
107.
Kinbara K Goldfinger LE Hansen M Chou FL Ginsberg MH 《Nature reviews. Molecular cell biology》2003,4(10):767-776
Integrins are cell-surface receptors that mediate and coordinate cellular responses to the extracellular matrix (ECM). Cellular signalling pathways can regulate cell adhesion by altering the affinity and avidity of integrins for ECM. The Ras family of small G proteins, which includes H-ras, R-ras and Rap, are important elements in cellular signalling pathways that control integrin function. 相似文献
108.
Mitsuhiro Ishida Kayoko Kobayashi Norio Awata Fumio Sakamoto 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,727(1-2)
A simple and reproducible method for the analysis of ampicillin in human serum was developed. Serum samples were extracted using solid-phase extraction disk cartridges containing a sorbent of styrene divinyl/benzene. Extracts were separated by reversed-phase C18 high-performance liquid chromatography with UV detection at 220 nm. The mobile phase consisted of acetonitrile–10 mM NaH2PO4 (6.5:93.5, v/v). Using this extraction procedure, recovery from serum was 98.4±5.6%. The quantitation limit was 0.19 μg/ml using 0.5 ml of serum. The calibration curves from 0.19 to 9.41 μg/ml were linear with correlation coefficients of 0.999. This method is suitable for therapeutic drug monitoring of ampicillin (ABPC) after oral administration of lenampicillin hydrochloride. 相似文献
109.
A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori 总被引:4,自引:0,他引:4
Tanaka H Ishibashi J Fujita K Nakajima Y Sagisaka A Tomimoto K Suzuki N Yoshiyama M Kaneko Y Iwasaki T Sunagawa T Yamaji K Asaoka A Mita K Yamakawa M 《Insect biochemistry and molecular biology》2008,38(12):1087-1110
A genome-wide analysis of innate immunity-related genes and gene families was conducted using the silkworm, Bombyx mori. We identified orthologs for a large number of genes involved in insect immunity that have been reported from Drosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera). B. mori has a unique recognition gene and antimicrobial peptide genes that are not present in the Drosophila, Anopheles, Apis and Tribolium genomes, suggesting a lineage-specific gene evolution for lepidopteran insects. The comparative analysis of the insect immune repertoires indicated a dynamic and flexible gene expansion in recognition, modulation and effector mechanisms due to different selection pressures. Differential gene regulation by different bacterial species was found in PGRP and Serpin genes, suggesting that Bombyx has a highly selective gene regulation system depending on bacterial species. 相似文献
110.
Ubiquinone systems of Coccidioides immitis, the causative agent of coccidioidomycosis 总被引:1,自引:0,他引:1
Kazutaka Fukushima Kayoko Takizawa Kaoru Okada Yukio Maebayashi Kazuko Nishimura Makoto Miyaji Paul E. Brummer Karl V. Clemons David A. Stevens 《FEMS microbiology letters》1993,108(2):243-245
Abstract The ubiquinone (coenzyme Q) systems of eleven strains of Coccidioides immitis were determined by high performance liquid chromatography (HPLC). The ubiquinone profile of the fungi was shown to be homogeneous: in all of the strains, ubiquinone-10 (Q-10) was demonstrated to be the major component, with Q-9 as a minor component. The results imply that the ubiquinone system may serve as an additional phenotypic criterion for identifying the fungus. 相似文献