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101.
Norio Masui Tetsu Nishikawa Yumie Takagi Hiromitsu Kimura Masayuki Mori Shigeo Yokose Hidekazu Asai Toshihiko Gonda Makoto Yanabe Katsunori Sato 《Experimental Animals》2004,53(5):399-407
The congenitally athymic nude rat is used for studying cancer and transplantation owing to its hairlessness and T-cell defective function caused by the Foxn1(rnu) gene. However, NK cell activity of the nude rat is markedly increased. It is known that NK cells play a major role in rejection of xenografts and in cytotoxicity against tumor cells. Thus, the athymic nude rat with impaired NK cell activity should be a useful model for extensive studies. The DA-Lyst(bg)/Lyst(bg) rat, a model for human Chediak-Higashi syndrome (CHS) is characterized by diluted-coat color and impairment of NK cell activity. We planned to establish a combined immunodeficient double mutant rat introgressed with the Foxn1(rnu) and Lyst(bg) genes and a set of congenic strains having an identical genetic backgrounds simultaneously. Based on the phenotypic and genetic characteristics of the parental rat strains, the new strains were produced using continuous backcross and diagnosis with molecular genetic techniques. Each disease gene was diagnosed with PCR-RFLP or the long-nested PCR method. Furthermore, we used a marker-assisted congenic strategy based on scanning the genetic backgrounds of the parental rats with 461 rat microsatellite markers. We think that the newly established DA/Slc-Foxn1(rnu)/Foxn1(rnu) Lyst(bg)/Lyst(bg) double mutant will be useful as a severe disease model for human CHS, and the set of DA/Slc-Foxn1(rnu) Lyst(bg) congenic strains which have impaired NK cell activity and/or defective T cell function should be useful for studying in cancer research, xenotransplantation, immune function and other wide-ranging studies. 相似文献
102.
Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase 总被引:2,自引:0,他引:2
Summary The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angle restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time 1H–13C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally 13C-labelled sample was exploited for the stereo-specific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C, C, O) among the 25 conformers is 0.42 Å for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel -strands arranged to form two perpendicular -sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core -strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764–769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short -strands in the regions Ser15 to Glu17 and Gly25 to Glu27 flanking the so-called RT-Src loop. This loop (residues Glu17 to Leu24), together with the n-Src loop (residues Gln37 to Ser46) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His14, Tyr16, Trp41, Phe54 and Phe59). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear 15N,1H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.Abbreviations CT
constant time
- HSQC
heteronuclear single-quantum coherence
- NOE
nuclear Overhauser enhancement
- NOESY
nuclear Overhauser enhancement spectroscopy
- SH2
Src homology domain 2
- SH3
Src homology domain 3 相似文献
103.
The primary goal of cancer immunotherapy is to elicit an immune response capable of eradicating established tumors and preventing
tumor metastasis. One strategy to achieve this goal utilizes whole killed tumor cells as the primary immunogen. Killed tumor
cells provide a comprehensive source of tumor-associated antigens (TAAs), thereby eliminating the need to identify individual
antigens. Unfortunately, killed tumor cells tend to be poorly immunogenic. To overcome this limitation, we covalently conjugated
immunostimulatory CpG oligodeoxynucleotides (ODN) to apoptotic tumor cells and examined their ability to induce TAA-specific
immune responses. Results indicate that CpG conjugation enhances the uptake of cell-based vaccines by dendritic cells (DCs),
up-regulates co-stimulatory molecule expression, and promotes the production of immunostimulatory cytokines. Vaccination with
CpG-conjugated tumor cells triggers the expansion of tumor-specific cytotoxic T lymphocytes (CTL) that reduce the growth of
established tumors and prevents their metastatic spread. Thus, conjugating CpG ODN to cell-based tumor vaccines is an important
step toward improving cancer immunotherapy. 相似文献
104.
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
105.
The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics 总被引:1,自引:0,他引:1
Goda N Tenno T Takasu H Hiroaki H Shirakawa M 《Protein science : a publication of the Protein Society》2004,13(3):652-658
A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT-vector cloning, is based on a T-vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5'-sequence of the "rear" PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested. 相似文献
106.
Kazuaki Kitano Yuzo Ichimori Hidekazu Sawada Susumu Iwasa Seijiro Sasai Kyozo Tsukamoto 《Cytotechnology》1991,5(2):53-74
Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed. 相似文献
107.
Poly(3-hydroxybutyrate) depolymerase was purified to
homogeneity from the culture filtrate of Paecilomyces lilacinus D218
by column chromatography on CM-Toyopearl 650M and hydroxylapatite. The
molecular weight of the enzyme was estimated to be 48,000 by SDS-PAGE.
Maximal activity was observed near pH 7.0 and 45°C. The
K
m
and V
max values for PHB were 0.13
(mg/ml) and 3750 (U/mg protein), respectively. The enzyme hydrolyzed PHB and
p-nitrophenyl fatty acids but not polycaprolactone and triglycerides.
Received: 29 August 1996 / Accepted: 30 September 1996 相似文献
108.
Daisuke Mori Hiroyuki Kimura Hidekazu Kawashima Yusuke Yagi Kenji Arimitsu Masahiro Ono Hideo Saji 《Bioorganic & medicinal chemistry》2019,27(18):4200-4210
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that have been implicated in higher brain functions. To elucidate the functional mechanisms underlying nAChRs and contribute significantly to development of drugs targeting neurological and neuropsychiatric diseases, non-invasive nuclear medical imaging can be used for evaluation. In addition, technetium-99m (99mTc) is a versatile radionuclide used clinically as a tracer in single-photon emission computed tomography. Because A85380 is known as a potent α4β2-nAChR agonist, we prepared A85380 derivatives labeled with 99mTc using a bifunctional chelate system. A computational scientific approach was used to design the probe efficiently. We used non-radioactive rhenium (Re) for a 99mTc analog and found that one of the derivatives, Re-A-YN-IDA-C4, exhibited high binding affinity at α4β2-nAChR in both the docking simulation (?19.3 kcal/mol) and binding assay (Ki = 0.4 ± 0.04 nM). Further, 99mTc-A-YN-IDA-C4 was synthesized using microwaves, and its properties were examined. Consequently, we found that 99mTc-A-YN-IDA-C4, with a structure optimized by using computational chemistry techniques, maintained affinity and selectivity for nAChR in vitro and possessed efficient characteristics as a nuclear medicine molecular imaging probe, demonstrated usefulness of computational scientific approach for molecular improvement strategy. 相似文献
109.
Facile preparation of the N‐acetyl‐glucosaminylated asparagine derivative with TFA‐sensitive protecting groups useful for solid‐phase glycopeptide synthesis 下载免费PDF全文
Hidekazu Katayama 《Journal of peptide science》2015,21(9):696-699
In this study, a novel N‐acetyl‐glucosaminylated asparagine derivative was developed. This derivative carried TFA‐sensitive protecting groups and was derived from commercially available compounds only in three steps. It was applicable to the ordinary 9‐fluorenylmethoxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (SPPS) method, and the protecting groups on the carbohydrate moiety could be removed by a single step of TFA cocktail treatment generally used for the final deprotection step in Fmoc‐SPPS. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
110.