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61.
Using random mutagenesis, we previously obtained K33N mutant lysozyme that showed a large lytic halo on the plate coating Micrococcus luteus. In order to examine the effects of mutation of K33N on enzyme activity, we prepared K33N and K33A mutant lysozymes from yeast. It was found that the activities of both the mutant lysozymes were higher than those of the wild-type lysozyme based on the results of the activity measurements against M. luteus (lytic activity) and glycol chitin. Moreover, 3D structures of K33N and K33A mutant lysozyme were solved by X-ray crystallographic analyses. The side chain of K33 in the wild-type lysozyme hydrogen bonded with N37 involved in the substrate-binding region, and the orientation of the side chain of N37 in K33 mutant lysozymes were different in the wild-type lysozyme. These results suggest that the enhancement of activity in K33N mutant lysozyme was due to an alteration in the orientation of the side chain of N37. On the other hand, K33N lysozyme was less stable than the wild-type lysozyme. Lysozyme may sacrifice its enzyme activity to acquire the conformational stability at position 33. 相似文献
62.
Yuzo Ogawa Keiji Matsumoto Takashi Maeda Riyoko Tamai Takashi Suzuki Hironobu Sasano Ross T Fernley 《The journal of histochemistry and cytochemistry》2002,50(6):821-827
We have previously demonstrated by immunohistochemistry the presence of secreted carbonic anhydrase (CA VI) in the acinar cells of the rat lacrimal glands. In this study we purified the sheep lacrimal gland CA VI to homogeneity and demonstrated by Western analysis that it has the same apparent subunit molecular weight (45 kD) as the enzyme isolated from saliva. RT-PCR analysis showed that CA VI mRNA from the lacrimal gland was identical to that of the parotid gland CA VI mRNA. An RIA specific for sheep CA VI showed the lacrimal gland tissue concentration of the enzyme to be 4.20 +/- 2.60 ng/mg protein, or about 1/7000 of the level found in the parotid gland. Immunohistochemistry (IHC) and in situ hybridization (ISH) showed that lacrimal acinar cells expressed both immunoreactivity and mRNA for CA VI. Moreover, CA VI immunoreactivity was occasionally observed in the lumen of the ducts. Unlike the parotid gland, in which all acinar cells expressed CA VI immunoreactivity and mRNA, only some of the acinar cells of the lacrimal gland showed expression. These results indicate that the lacrimal gland synthesizes and secretes a very small amount of salivary CA VI. In tear fluid, CA VI is presumed to have a role in the maintenance of acid/base balance on the surface of the eye, akin to its role in the oral cavity. 相似文献
63.
Xia Li Masashi Inoue Danielle R. Reed Taufiqul Huque Ralph B. Puchalski Michael G. Tordoff Yuzo Ninomiya Gary K. Beauchamp Alexander A. Bachmanov 《Mammalian genome》2001,12(1):13-16
The Sac (saccharin preference) locus affecting mouse behavioral and neural responsiveness to sweeteners has been mapped to distal
Chr 4. A putative sweet taste receptor, T1R1, has been recently cloned, and the gene encoding it, Gpr70, has also been mapped to mouse distal Chr 4. To assess Gpr70 as a candidate gene for Sac, we compared the Gpr70 sequences of C57BL/6ByJ and 129P3/J mouse strains with different alleles of Sac. Using Gpr70 sequence variation between the C57BL/6ByJ and 129P3/J strains, we conducted a high-resolution analysis of the chromosomal
localization of the Gpr70 and Sac loci in the F2 hybrids and 129.B6-Sac partially congenic mice originating from these two strains. The Gpr70 gene maps proximal to Sac, which demonstrates that they are different loci.
Received: 24 April 2000 / Accepted: 14 September 2000 相似文献
64.
Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions. 相似文献
65.
66.
Ken Igarashi Jun’ichi Oda Yuzo Inouye Minoru Ohno 《Bioscience, biotechnology, and biochemistry》2013,77(5):811-812
About 30 dipeptides and some tripeptides were led to new benzimidazole derivatives by incorporating their carboxyl groups into benzimidazole ring by the reaction with o-phenylenediamine. The ring closure to benzimidazole was well achieved by heating mildly at a moderate temperature in acetic acid.Some benzimidazole derivatives of peptides had remarkable phytotoxicities. 相似文献
67.
Yuzo Azuma Katsuzi Kawamura Hiroshi Isogai Emiko Isogai 《Microbiology and immunology》1993,37(4):325-329
A 2-year-old mongrel dog developed neurological signs following tick bite. These included astasia, persistent tonic convulsions and hyper-reflexia. Both serum IgG and IgM antibody titers against Borrelia burgdorferi were positive in enzyme-linked immunosorbent assay (ELISA). The neurological signs subsided after high-dose penicillin and streptomycin treatment. A strain of spirochetes (P427a) was isolated from the midgut of Ixodes persulcatus feeding on the dog. Morphological characteristic, immunological property and protein profile revealed that the isolate was B. burgdorferi. Similarly, a 2-year-old Labrador retriever dog developed neurological signs after tick bite and showed a positive IgG antibody titer against B. burgdorferi. Antibiotic treatment was effective also in this case. These findings suggest that neurological symptoms shown in both dogs were caused by infection with B. burgdorferi. 相似文献
68.
Efficient transformation of pBR322 and its derived
plasmids, which have been widely used as cloning vectors in Escherichia
coli, was observed in Pseudomonas avenae (K1), the pathogen of
leaf blight disease in cereals. Moreover, there was a 10- to 50-fold
transformation efficiency (1.3–3.0 × 106/μg DNA) in the
proline-auxotrophic mutant (Pr47), whose virulence to rice seedlings
decreased. Similar enhancement of the frequency of transfer by mobilization
of RSF1010, a broad host range plasmid, was observed in the recipient Pr47
strain in mating with donor Pseudomonas syringae. The plasmids
harbored in these strains were maintained very stably after subcultures.
Thus, a highly efficient transformation system with pBR322-derived plasmids
used as a vector and Pseudomonas as a host bacterium was developed.
Received: 13 July 1996 / Accepted: 26 August 1996 相似文献
69.
Yuichi Kunori Masahiro Koizumi Tsukio Masegi Hidenori Kasai Hiroshi Kawabata Yuzo Yamazaki Akiyoshi Fukamizu 《European journal of biochemistry》2002,269(23):5921-5930
Although the alpha-chymases of primates and dogs are known as chymotrypsin-like proteases, the enzymatic properties of rodent alpha-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase-like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases (rMCP-2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent alpha-chymases have unique biological activity different from the chymases of other species. 相似文献
70.