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961.
Mori T Wada T Suzuki T Kubota Y Inagaki N 《The Journal of biological chemistry》2007,282(27):19884-19893
Although neuronal functions depend on their robust polarity, the mechanisms that ensure generation and maintenance of only a single axon remain poorly understood. Using highly sensitive two-dimensional electrophoresis-based proteomics, we identified here a novel protein, single axon-related (singar)1/KIAA0871/RPIPx/RUFY3, which contains a RUN domain and is predominantly expressed in the brain. Singar1 expression became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Singar1 was diffusely localized in hippocampal neurons and moderately accumulated in growth cones of minor processes and axons. Overexpression of singar1 did not affect normal neuronal polarization but suppressed the formation of surplus axons induced by excess levels of shootin1, a recently identified protein located upstream of phosphoinositide-3-kinase and involved in neuronal polarization. Conversely, reduction of the expression of singar1 and its splicing variant singar2 by RNA interference led to an increase in the population of neurons bearing surplus axons, in a phosphoinositide-3-kinase-dependent manner. Overexpression of singar2 did not suppress the formation of surplus axons induced by shootin1. We propose that singar1 ensures the robustness of neuronal polarity by suppressing formation of surplus axons. 相似文献
962.
963.
Hatakeyama T Unno H Kouzuma Y Uchida T Eto S Hidemura H Kato N Yonekura M Kusunoki M 《The Journal of biological chemistry》2007,282(52):37826-37835
CEL-III is a Ca(2+)-dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/methyl alpha-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained beta-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca(2+) located at the subdomains 1alpha, 1gamma, 2alpha, 2beta, and 2gamma, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl alpha-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca(2+) ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2alpha and 2beta. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein. 相似文献
964.
965.
Kobayashi T Shiratori M Nakano H Eguchi C Shirai M Naka D Shibui T 《Biotechnology letters》2007,29(7):1065-1073
C-Terminal protein labeling allows non-radioactive detection of proteins by using fluorescent puromycin derivatives and cell-free
translation systems. However, yields of some labeled proteins are low. Here, we report that the yield of labeled protein mainly
depends on the C-terminal amino acid sequence. The short peptide tag sequence, RGAA, at the C-terminus increased not only the labeling efficiency (more than 80%) but also the synthesis yield of labeled proteins. To
examine the relationship between the C-terminal amino acid sequence and the yield of labeled proteins, we synthesized C-terminally labeled glutathione S-transferase (GST) containing four identical amino acid residues at the C-terminus. The results demonstrated that 4 × Ala, 4 × His, 4 × Gln, and 4 × Cys produced over 200% of the yield of wild-type
GST. In addition, the two Ala residues produced almost the same synthesis activity as 4 × Ala and RGAA. Similar results were
obtained with various proteins and cell-free translation systems. 相似文献
966.
Kim Daekyung; Nakashima Takuji; Matsuyama Yukihiko; Niwano Yoshimi; Yamaguchi Kenichi; Oda Tatsuya 《Journal of plankton research》2007,29(3):241-247
Raphidophycean flagellates, Chattonella marina and C. ovata,are harmful red tide phytoplankters; blooms of these phytoplanktersoften cause severe damage to fish farming. Previous studieshave demonstrated that C. marina and C. ovata continuously producereactive oxygen species (ROS) such as superoxide anion (O2)hydrogen peroxide (H2O2) under normal growth conditions, andan ROS-mediated toxic mechanism against fish and other marineorganisms has been proposed. Although the exact mechanism ofROS generation in these phytoplankters still remains to be clarified,our previous study suggested that NADPH oxidase-like enzymelocated on the cell surface of C. marina may be involved inO2 generation. To investigate the localization of O2and H2O2 generation in C. marina and C. ovata, we employed 2-methyl-6(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-oneand 5-(and-6)-carboxy-2',7'-dichlorodihydrodihydrofluoresceindictate, acetyl ester, which are specific fluorescent probefor detecting O2 and H2O2, respectively. Observationby fluorescence microscopy of live phytoplankters incubatedwith each probe revealed that O2 is mainly generatedon the cell surface, whereas H2O2 is generated in the intracellularcompartment in these phytoplankters. When the cells were rupturedby ultrasonic treatment, O2 levels of C. marina and C.ovata decreased significantly, whereas a few times higher levelsof H2O2 were detected in the ruptured cell suspensions whencompared with the levels of the live cell suspension. In immunoblottinganalysis, the protein recognized by anti-human gp91 phox wasdetected in both species. These results suggest that, in bothphytoplankters, the underlying mechanisms of O2 and H2O2generation may be distinct and such systems are independentlyoperating in the cells. 相似文献
967.
Tatsuya Ueki Koki Shintaku Yuki Yonekawa Nariaki Takatsu Hiroshi Yamada Toshiyuki Hamada Hiroshi Hirota Hitoshi Michibata 《Biochimica et Biophysica Acta (BBA)/General Subjects》2007
Several species of ascidians, the so-called tunicates, accumulate extremely high levels of vanadium ions in their blood cells. We previously identified a family of vanadium-binding proteins, named Vanabins, from blood cells and blood plasma of a vanadium-rich ascidian, Ascidia sydneiensis samea. The 3-dimensional structure of Vanabin2, the predominant vanadium-binding protein in blood cells, has been revealed, and the vanadium-binding properties of Vanabin2 have been studied in detail. Here, we used Far Western blotting to identify a novel protein that interacts with Vanabin2 from a blood cell cDNA library. The protein, named Vanabin-interacting protein 1 (VIP1), was localized in the cytoplasm of signet ring cells and giant cells. Using a two-hybrid method, we revealed that VIP1 interacted with Vanabins 1, 2, 3, and 4 but not with Vanabin P. The N-terminal domain of VIP1 was shown to be important for the interaction. Further, Vanabin1 was found to interact with all of the other Vanabins. These results suggest that VIP1 and Vanabin1 act as metal chaperones or target proteins in vanadocytes. 相似文献
968.
Miyazono K Watanabe M Kosinski J Ishikawa K Kamo M Sawasaki T Nagata K Bujnicki JM Endo Y Tanokura M Kobayashi I 《Nucleic acids research》2007,35(6):1908-1918
Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction–modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel β-sheet that forms a ‘half pipe’. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg2+. These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes. 相似文献
969.
Jun Aoyama Sam Wouthuyzen Michael J. Miller Yuki Minegishi Mari Kuroki Sasanti R. Suharti Tatsuya Kawakami Krunaen O. Sumardiharga Katsumi Tsukamoto 《Environmental Biology of Fishes》2007,80(4):445-452
A research cruise was conducted in the eastern Indian Ocean off west Sumatra, Indonesia, in June 2003 to learn about the spawning
and larval ecology of the tropical freshwater eels of the genus Anguilla in the region. A total of 43 anguillid leptocephali were collected during the cruise and they were genetically identified
as 41 Anguilla bicolor bicolor, 1 Anguilla marmorata, and 1 Anguilla interioris. A. bicolor bicolor leptocephali were 44.1–55.5 mm TL and most of them were at the fully grown stage. Reexamination of the historical data of
Jespersen (1942) also suggested a relatively low abundance of small size leptocephali (<40 mm) of this species off west Sumatra. Although
the study area has long been considered to be a spawning site of A. bicolor bicolor, the distributions of leptocephali from the two surveys and the patterns of ocean currents in the region suggest the possibility
that the main spawning area of this species is located farther offshore. 相似文献
970.
Ishibashi H Hisajima T Hu W Yamaguchi H Nishiyama Y Abe S 《Microbiology and immunology》2007,51(5):501-506
A simple method to establish a murine esophageal candidiasis model that displayed characteristic symptoms of the condition was developed using the sedative agent, chlorpromazine. Mice were immunosuppressed with prednisolone and were given tetracycline hydrochloride. One day later, the mice received chlorpromazine to keep them in a sedated state for about 3 hr. Under the sedated condition, they were infected with 4 x 10(7) viable cells of Candida albicans by intra-esophageal injection with a round-head needle on syringe. From day 3 to day 6 post inoculation, 10(5)-10(6) colony forming units of C. albicans were recovered from the esophageal tube of each mouse and whitish, curd-like patches were observed on most of the inner surface of the tube. Histological examination showed that C. albicans in esophageal lesions grew mainly in mycelial form. In this experimental model, intragastric administration of an itraconazole oral solution (20 mg/kg/day) was clearly effective. This model would provide a useful tool to investigate the pathogenesis of C. albicans esophageal infection and the efficacy of various antifungal agents microbiologically and symptomatically. 相似文献