全文获取类型
收费全文 | 236篇 |
免费 | 26篇 |
专业分类
262篇 |
出版年
2021年 | 2篇 |
2018年 | 2篇 |
2016年 | 5篇 |
2015年 | 9篇 |
2014年 | 14篇 |
2013年 | 6篇 |
2012年 | 5篇 |
2011年 | 13篇 |
2010年 | 15篇 |
2009年 | 8篇 |
2008年 | 14篇 |
2007年 | 19篇 |
2006年 | 12篇 |
2005年 | 12篇 |
2004年 | 14篇 |
2003年 | 11篇 |
2002年 | 12篇 |
2001年 | 5篇 |
2000年 | 8篇 |
1999年 | 5篇 |
1998年 | 3篇 |
1996年 | 2篇 |
1995年 | 2篇 |
1994年 | 2篇 |
1993年 | 5篇 |
1992年 | 4篇 |
1991年 | 5篇 |
1990年 | 4篇 |
1989年 | 2篇 |
1988年 | 6篇 |
1986年 | 2篇 |
1985年 | 2篇 |
1984年 | 4篇 |
1981年 | 1篇 |
1980年 | 1篇 |
1979年 | 1篇 |
1977年 | 2篇 |
1975年 | 2篇 |
1974年 | 1篇 |
1955年 | 1篇 |
1954年 | 1篇 |
1950年 | 1篇 |
1949年 | 1篇 |
1940年 | 2篇 |
1934年 | 1篇 |
1931年 | 1篇 |
1929年 | 1篇 |
1927年 | 4篇 |
1926年 | 1篇 |
1913年 | 1篇 |
排序方式: 共有262条查询结果,搜索用时 15 毫秒
1.
2.
During the investigation of an outbreak of diarrhea in suckling rats, a virus morphologically identical to but antigenically distinct from rotaviruses was identified. The disease was characterized clinically by erythema and cracking and bleeding of the perianal skin associated with the excretion of poorly formed fecal pellets, liquid, and gas. Light microscopy-observable changes consisted of small intestinal villous atrophy, villous epithelial necrosis, and villous epithelial syncytial cell formation. The cytoplasm of the epithelial syncytial cells contained large numbers of 80-nm viral particles that were often associated with reticular aggregates of electron-dense material. Viral infection principally involved the luminal one-fourth to one-third of the intestinal villi as determined by indirect immunofluorescence. This rotavirus-like agent contained 11 double-stranded RNA segments; however, the migration pattern of these segments in polyacrylamide gels differed from the electrophoretic pattern which is characteristic of the typical rotaviruses. The agent had a buoyant density in CsCl of 1.36 to 1.4 g/cm3 and was labile at pH 3 and at 56 degrees C; however, infectivity of viral inocula was not altered by extensive treatment with ether or by pH 5 buffers. This disease, which we have named infectious diarrhea of infant rats, is the first recognized viral diarrhea of rats and appears to be a good model for the study of the recently recognized group of atypical rotaviruses. 相似文献
3.
In the present study, the bombesin-induced changes in cytosolic free Ca2+ ([Ca2+]i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca2+ transients. The average frequency of the Ca2+ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min−1 (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca2+ transient followed by a sustained plateau or by a decrease to basal levels. In Ca2+- free medium, bombesin caused only one or two Ca2+ transients and withdrawal of extracellular Ca2+ abolished the Ca2+ transients. The voltage-dependent Ca2+ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca2+ transients and stopped the Ca2+ transients in some cells. Thapsigargin caused a sustained rise in [Ca2+]i) in the presence of extracellular Ca2+ while in its absence the rise in [Ca2+]i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca2+], by about 50%. Depletion of intracellular Ca2+ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca2+-free medium caused an agonist-independent increase in [Ca2+]i) when extracellular Ca2+ was restored, which was larger than in control cells that had been incubated in Ca2+-free medium for the same period of time. This rise in [Ca2+]i and the thapsigargin-induced increase in [Ca2+]i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca2+ pool enhances Ca2+ influx through VDCC and voltage-independent Ca2+ channels (VICC). In conclusion, the bombesin-induced Ca2+ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca2+ transients. Intracellular Ca2+ is mobilized during each Ca2+ transient, but Ca2+ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca2+ response. Ca2+ influx in whole or part is activated by a capacitative Ca2+ entry mechanism. 相似文献
4.
In the present study, unilamellar liposomes coated with the crystalline bacterial cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were used as matrix for defined binding of functional molecules via the avidin- or streptavidin-biotin bridge. The liposomes were composed of dipalmitoyl phosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4 and they had an average size of 180 nm. For introducing specific functions into the S-layer lattice without affecting substances encapsulated within the liposomes, crosslinking and activation reagents had to be identified which did not penetrate the liposomal membrane. Among different reagents, a hydrophilic dialdehyde generated by periodate cleavage of raffinose and a sulfo-succinimide activated dicarboxylic acid were found to be impermeable for the liposomal membrane. Both reagents completely crosslinked the S-layer lattice without interfering with its regular structure. Biotinylation of S-layer-coated liposomes was achieved by coupling p-diazobenzoyl biocytin which preferably reacts with the phenolic residue of tyrosine or with the imidazole ring of histidine. By applying this method, two biotin residues accessible for subsequent avidin binding were introduced per S-layer subunit. As visualized by labeling with biotinylated ferritin, an ordered monomolecular layer of streptavidin was formed on the surface of the S-layer-coated liposomes. As a second model system, biotinylated anti-human IgG was attached via the streptavidin bridge to the biotinylated S-layer-coated liposomes. The biological activity of the bound anti-human IgG was confirmed by ELISA. 相似文献
5.
A Gualberto D LePage G Pons S L Mader K Park M L Atchison K Walsh 《Molecular and cellular biology》1992,12(9):4209-4214
6.
7.
Wolfgang Sommeregger Bernhard Prewein David Reinhart Alexander Mader Renate Kunert 《Cytotechnology》2013,65(5):811-818
Nucleic acid quantification is a relevant issue for the characterization of mammalian recombinant cell lines and also for the registration of producer clones. Quantitative real-time PCR is a powerful tool to investigate nucleic acid levels but numerous different quantification strategies exist, which sometimes lead to misinterpretation of obtained qPCR data. In contrast to absolute quantification using amplicon- or plasmid standard curves, relative quantification strategies relate the gene of interest to an endogenous reference gene. The relative quantification methods also consider the amplification efficiency for the calculation of the gene copy number and thus more accurate results compared to absolute quantification methods are generated. In this study two recombinant Chinese hamster ovary cell lines were analysed for their transgene copy number using different relative quantification strategies. The individual calculation methods resulted in differences of relative gene copy numbers because efficiency calculations have strong impact on gene copy numbers. However, in context of comparing transgene copy numbers of two individual clones the influence of the calculation method is marginal. Therefore especially for the comparison of two cell lines with the identical transgene any of the relative qPCR methods was proven as powerful tool. 相似文献
8.
Background
Two-component signal transduction systems are one means of bacteria to respond to external stimuli. The LiaFSR two-component system of Bacillus subtilis consists of a regular two-component system LiaRS comprising the core Histidine Kinase (HK) LiaS and the Response Regulator (RR) LiaR and additionally the accessory protein LiaF, which acts as a negative regulator of LiaRS-dependent signal transduction. The complete LiaFSR system was shown to respond to various peptide antibiotics interfering with cell wall biosynthesis, including bacitracin.Methodology and Principal Findings
Here we study the response of the LiaFSR system to various concentrations of the peptide antibiotic bacitracin. Using quantitative fluorescence microscopy, we performed a whole population study analyzed on the single cell level. We investigated switching from the non-induced ‘OFF’ state into the bacitracin-induced ‘ON’ state by monitoring gene expression of a fluorescent reporter from the RR-regulated liaI promoter. We found that switching into the ‘ON’ state occurred within less than 20 min in a well-defined switching window, independent of the bacitracin concentration. The switching rate and the basal expression rate decreased at low bacitracin concentrations, establishing clear heterogeneity 60 min after bacitracin induction. Finally, we performed time-lapse microscopy of single cells confirming the quantitative response as obtained in the whole population analysis for high bacitracin concentrations.Conclusion
The LiaFSR system exhibits an immediate, heterogeneous and graded response to the inducer bacitracin in the exponential growth phase. 相似文献9.
Conrad DM Hanniman EA Watson CL Mader JS Hoskin DW 《Journal of cellular biochemistry》2004,92(2):387-399
Ryanodine receptors (RyR) are involved in regulating intracellular Ca(++) mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR-selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor-mediated activation events and cytokine-dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)-2 by T lymphocytes responding to mitogenic anti-CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation-induced expression of the alpha chain (CD25) of the high affinity IL-2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL-2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL-2-dependent proliferation of CTLL-2 T cells. These data indicate that RyR are involved in regulating IL-2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR-associated Ca(++) signaling regulates T cell proliferation by promoting both IL-2 synthesis and IL-2-dependent cell cycle progression. 相似文献
10.
In response to significant environmental health challenges in Southeast Texas, a National Institute for Environmental Health Sciences Center at the University of Texas Medical Branch at Galveston was created to promote and conduct inter-disciplinary research in the areas of: (1) the molecular biology of DNA repair, replication and mutagenesis, (2) asthma pathogenesis in response to oxidative stress and viral exposures, and (3) environmental toxicant biotransformation. In addition, the NIEHS Center maintains close ties with neighboring communities through an active Community Outreach & Education Program (COEP) that develops and disseminates translational materials for use in environmental health awareness outreach, toxicology consultation, K-12 curriculum enrichment and in developing site-specific Community Partnership projects. The COEP core service divisions include: Environmental Arts & Sciences, Asthma Outreach & Education, Theater Outreach & Education, and Public Forum & Toxics Assistance. Public Forums focus on the use of Augusto Boal's Forum Theater dramaturgy to include the voices and local knowledge of communities within the process of Participatory Research. Forums create the preconditions for significant partnerships that link the hazardous risk perceptions and environmental health needs of communities with the expertise of NIEHS Center investigators and translational services provided through COEP outreach programs. The Forum process also creates leadership cores within environmentally challenged communities that facilitate the ongoing translational process and maintain the vital linkage between the health needs of communities and the analytic tools and the field and clinical technologies of the environmental sciences. 相似文献