全文获取类型
收费全文 | 532篇 |
免费 | 25篇 |
专业分类
557篇 |
出版年
2022年 | 6篇 |
2021年 | 5篇 |
2020年 | 3篇 |
2019年 | 3篇 |
2018年 | 8篇 |
2017年 | 2篇 |
2016年 | 11篇 |
2015年 | 16篇 |
2014年 | 28篇 |
2013年 | 23篇 |
2012年 | 32篇 |
2011年 | 22篇 |
2010年 | 18篇 |
2009年 | 18篇 |
2008年 | 37篇 |
2007年 | 27篇 |
2006年 | 25篇 |
2005年 | 34篇 |
2004年 | 38篇 |
2003年 | 28篇 |
2002年 | 29篇 |
2001年 | 4篇 |
2000年 | 13篇 |
1999年 | 7篇 |
1998年 | 10篇 |
1997年 | 10篇 |
1996年 | 4篇 |
1995年 | 4篇 |
1994年 | 12篇 |
1993年 | 6篇 |
1992年 | 6篇 |
1991年 | 4篇 |
1990年 | 6篇 |
1989年 | 7篇 |
1988年 | 2篇 |
1987年 | 4篇 |
1986年 | 5篇 |
1985年 | 4篇 |
1984年 | 4篇 |
1983年 | 6篇 |
1982年 | 3篇 |
1981年 | 2篇 |
1980年 | 2篇 |
1979年 | 2篇 |
1978年 | 2篇 |
1977年 | 3篇 |
1976年 | 5篇 |
1975年 | 3篇 |
1974年 | 2篇 |
1967年 | 1篇 |
排序方式: 共有557条查询结果,搜索用时 15 毫秒
51.
Kazumichi Yonenaga Satoru Nishizawa Miki Akizawa Yukiyo Asawa Yuko Fujihara Tsuyoshi Takato Kazuto Hoshi 《Cytotechnology》2010,62(6):539-545
In cartilage tissue engineering, viable cell numbers should be correctly counted in the collagenase digest of the biopsied cartilage. However, this is a difficult task due to the presence of matrix debris, cell ghosts and their aggregates. To search for the correct cell counting method in this situation, we evaluated the utility of an automatic cell counting device, the NucleoCounter, and compared it with conventional staining using the LIVE/DEAD® kit. We first measured the cell numbers of a standard chondrocyte sample by the NucleoCounter, which showed a high accuracy (R2 = 0.9999) and reproducibility (%CV: 2.00–8.66). We then calculated the cell numbers and viability in some collagenase digests of native cartilage using either the NucleoCounter or LIVE/DEAD® kit, revealing that the total cell numbers, viable ones and viability were highly correlated between them (R2 = 0.9601, 0.9638 and 0.917, respectively). However, both the intrapersonal and interpersonal variabilities in the NucleoCounter was significantly decreased to about 1/20–1/5, compared to that of the LIVE/DEAD® kit. The NucleoCounter was regarded as a useful tool for simple, rapid, and highly reproducible cell counts, which may not only provide constant experimental data in a certain laboratory, but also contribute to the high reproducibility of the clinical results of cartilage tissue engineering among multiple institutions. 相似文献
52.
Bandow K Nishikawa Y Ohnishi T Kakimoto K Soejima K Iwabuchi S Kuroe K Matsuguchi T 《Journal of cellular physiology》2007,211(2):392-398
Constant mechanical stress is essential for the maintenance of bone mass and strength, which is achieved through the cooperative functions of osteoblasts and osteoclasts. However, it has not been fully elucidated how these cell types mediate mechanical signals. Low-intensity pulsed ultrasound (LIPUS) therapy is a recently developed method for application of mechanical stress, and is used clinically to promote bone fracture healing. In the present study, we applied LIPUS to osteoblasts at different stages of maturation and analyzed their chemokine and cytokine expression. In comparison with their immature counterparts, mature osteoblasts expressed significantly higher levels of mRNAs for the receptor activator of nuclear factor kappa B ligand (RANKL), monocyte chemoattractant protein (MCP)-1, and macrophage-inflammatory protein (MIP)-1beta after a few hours of LIPUS treatment. Intriguingly, protein and mRNA expression of angiotensin II type 1 receptor (AT1), a known mechanoreceptor in cardiomyocytes, was detected in osteoblasts, and the level of expression increased significantly during cell maturation. Furthermore, LIPUS-induced extracellular signal-regulated kinase (ERK) phosphorylation and RANKL/chemokine expression was abrogated by a specific AT1 inhibitor. Thus, AT1 may play one of the essential roles in bone metabolism as a mechanoreceptor of osteoblasts. 相似文献
53.
In vitro screening of potato against water-stress mediated through sorbitol and polyethylene glycol 总被引:1,自引:0,他引:1
With the objective to develop a practical and effective method of screening potato for drought tolerance, shoot and root growth
in microtuber-derived plantlets was studied in vitro in three genotypes with known root mass production under field conditions.
Different levels of water-stress were induced using five concentrations of either sorbitol or polyethylene glycol (PEG) in
MS medium. Water potential of various media ranged from −0.80 MPa to −2.05 MPa. Water-stress in culture adversely affected
plantlet growth, and genotypes differed for their responses. Genotype IWA-1 was less affected than IWA-3 and IWA-5. At the
same level of water potential, sorbitol had lower adverse effect than PEG; the latter being sticky. Genotype × sorbitol and
genotype × PEG interactions were significant. At 0.2 M sorbitol and 0.003 M PEG, IWA-1 had significantly more roots with higher
total root length, root volume, as well as root-dry weight than those of IWA-3 and IWA-5, whereas the latter two genotypes
were at par for all these characters. This pattern was similar to the reported pattern of these genotypes for root-dry weight
under field conditions. It is concluded that in vitro screening of potato under specific and limited water-stress conditions
may provide a system for effectively differentiating the genotypes for their expected root mass production under field conditions. 相似文献
54.
Here we report a new method for isolating antigen-specific antibody-secreting cells (ASCs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ASCs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. This protocol can be completed in less than 7 h, including 3 h of cell culture. The method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (ELISPOT) but it also overcomes the limitations of ELISPOT in recovering ASCs that can be used to produce antigen-specific human monoclonal antibodies. This method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis. 相似文献
55.
Background
Our previous study has shown that prenatal exposure to X-ray irradiation causes cerebral hypo-perfusion during the postnatal development of central nervous system (CNS). However, the source of the hypo-perfusion and its impact on the CNS development remains unclear. The present study developed an automatic analysis method to determine the mean red blood cell (RBC) speed through single microvessels imaged with two-photon microscopy in the cerebral cortex of rats prenatally exposed to X-ray irradiation (1.5 Gy).Methodology/Principal Findings
We obtained a mean RBC speed (0.9±0.6 mm/sec) that ranged from 0.2 to 4.4 mm/sec from 121 vessels in the radiation-exposed rats, which was about 40% lower than that of normal rats that were not exposed. These results were then compared with the conventional method for monitoring microvascular perfusion using the arteriovenous transit time (AVTT) determined by tracking fluorescent markers. A significant increase in the AVTT was observed in the exposed rats (1.9±0.6 sec) as compared to the age-matched non-exposed rats (1.2±0.3 sec). The results indicate that parenchyma capillary blood velocity in the exposed rats was approximately 37% lower than in non-exposed rats.Conclusions/Significance
The algorithm presented is simple and robust relative to monitoring individual RBC speeds, which is superior in terms of noise tolerance and computation time. The demonstrative results show that the method developed in this study for determining the mean RBC speed in the spatial frequency domain was consistent with the conventional transit time method. 相似文献56.
Imanishi H Hattori K Wada R Ishikawa K Fukuda S Takenaga K Nakada K Hayashi J 《PloS one》2011,6(8):e23401
Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously expressed a highly metastatic potential, mitochondrial respiration defects, and ROS overproduction. Since mitochondrial respiratory function is controlled by both mtDNA and nuclear DNA, it is possible that nuclear DNA mutations contribute to the mitochondrial respiration defects and the highly metastatic potential found in MDA-MB-231 cells. To examine this possibility, we carried out mtDNA replacement of MDA-MB-231 cells by normal human mtDNA. For the complete mtDNA replacement, first we isolated mtDNA-less (ρ(0)) MDA-MB-231 cells, and then introduced normal human mtDNA into the ρ(0) MDA-MB-231 cells, and isolated trans-mitochondrial cells (cybrids) carrying nuclear DNA from MDA-MB-231 cells and mtDNA from a normal subject. The normal mtDNA transfer simultaneously induced restoration of mitochondrial respiratory function and suppression of the highly metastatic potential expressed in MDA-MB-231 cells, but did not suppress ROS overproduction. These observations suggest that mitochondrial respiration defects observed in MDA-MB-231 cells are caused by mutations in mtDNA but not in nuclear DNA, and are responsible for expression of the high metastatic potential without using ROS-mediated pathways. Thus, human tumor cells possess an mtDNA-mediated metastatic pathway that is required for expression of the highly metastatic potential in the absence of ROS production. 相似文献
57.
The α-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus thermophilus was over 40%. The α-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 °C and pH 7. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moiety but also other substrates. 相似文献
58.
59.
Evolution of the pregnane x receptor: adaptation to cross-species differences in biliary bile salts 总被引:4,自引:0,他引:4
Krasowski MD Yasuda K Hagey LR Schuetz EG 《Molecular endocrinology (Baltimore, Md.)》2005,19(7):1720-1739
The pregnane X receptor (PXR) regulates the metabolism and elimination of bile salts, steroids, and xenobiotics. The sequence of the PXR ligand-binding domain diverges extensively between different animals, suggesting interspecies differences in ligands. Of the endogenous ligands known to activate PXR, biliary bile salts vary the most across vertebrate species, ranging from 27-carbon (C27) bile alcohol sulfates (early fish, amphibians) to C24 bile acids (birds, mammals). Using a luciferase-based reporter assay, human PXR was activated by a wide variety of bile salts. In contrast, zebrafish PXR was activated efficiently only by cyprinol sulfate, the major zebrafish bile salt, but not by recent bile acids. Chicken, mouse, rat, and rabbit PXRs were all activated by species-specific bile acids and by early fish bile alcohol sulfates. In addition, phylogenetic analysis using maximum likelihood demonstrated evidence for nonneutral evolution of the PXR ligand-binding domain. PXR activation by bile salts has expanded from narrow specificity for C27 bile alcohol sulfates (early fish) to a broader specificity for recent bile acids (birds, mammals). PXR specificity for bile salts has thus paralleled the increasing complexity of the bile salt synthetic pathway during vertebrate evolution, an unusual example of ligand-receptor coevolution in the nuclear hormone receptor superfamily. 相似文献
60.
The recent design strategy of zinc finger peptides has mainly focused on the alpha-helix region, which plays a direct role in DNA recognition. On the other hand, the study of non-DNA-contacting regions is extremely scarce. By swapping the beta-hairpin regions between the Sp1 and GLI zinc fingers, in this study, we investigated how the beta-hairpin region of the C(2)H(2)-type zinc finger peptides contributes to the DNA binding properties. Surprisingly, the Sp1 mutant with the GLI-type beta-hairpin had a higher DNA binding affinity than that of the wild-type Sp1. The result of the DNase I footprinting analyses also showed the change in the DNA binding pattern. In contrast, the GLI zinc finger completely lost DNA binding ability as a result of exchanging the beta-hairpin region. These results strongly indicate that the beta-hairpin region appears to function as a scaffold and has an important effect on the DNA binding properties of the C(2)H(2)-type zinc finger peptides. 相似文献