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181.
Zhi Tang Erika Bereczki Haiyan Zhang Shan Wang Chunxia Li Xinying Ji Rui M. Branca Janne Lehti? Zhizhong Guan Peter Filipcik Shaohua Xu Bengt Winblad Jin-Jing Pei 《The Journal of biological chemistry》2013,288(22):15556-15570
Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function. 相似文献
182.
Shivani Ahuja Nicole Jahr Sang-Choul Im Subramanian Vivekanandan Nataliya Popovych Stéphanie V. Le Clair Rui Huang Ronald Soong Jiadi Xu Kazutoshi Yamamoto Ravi P. Nanga Angela Bridges Lucy Waskell Ayyalusamy Ramamoorthy 《The Journal of biological chemistry》2013,288(30):22080-22095
Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. 相似文献
183.
Rui Zhang Baozhan Huang Dan Du Xiurong Guo Guang Xin Zhihua Xing Yong Liang Younan Chen Qianming Chen Yang He Wen Huang 《Steroids》2013,78(11):1064-1070
Thrombosis in coronary or cerebral arteries is the major cause of morbidity and mortality worldwide. Diosgenin and total steroidal saponins extracted from the rhizome of Dioscorea zingiberensis C.H. Wright are demonstrated to have anti-thrombotic activity. However, few studies describe the anti-thrombotic activity of the diosgenyl saponin monomer. In the present study, a simple and convenient method for the preparation of a new disaccharide saponin, diosgenyl β-d-galactopyranosyl-(1 → 4)-β-d-glucopyranoside (3), is described. We evaluated the anti-thrombotic effects of diosgenin and four diosgenyl saponins by measuring the bleeding time; the results showed that compound 3 exhibits outstanding efficiency in prolonging the bleeding time. Furthermore, we assessed whether compound 3 could alter platelet aggregation in vitro and in vivo. In addition, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), coagulation factors and protection rate in mice were measured to evaluate the anti-thrombotic effect of compound 3. The results show that compound 3 inhibited platelet aggregation, prolonged APTT, inhibited factor VIII activities in rats, and increased the protection rate in mice in a dose-dependent manner. Taken together, these findings suggested that diosgenyl saponins, especially compound 3, had anti-thrombotic activity. It may execute anti-thrombotic activity through inhibiting factor VIII activities and platelet aggregation. 相似文献
184.
Jun Li Aiying Wang Fengmei Zhu Rui Xu Xiao Song Hu 《Indian journal of microbiology》2013,53(3):352-358
To clarify the mechanism of microbial inactivation by supercritical carbon dioxide (SCCO2), membrane damage of Rhodotorula mucilaginosa was investigated within specific pressure (10 Mpa), temperature (37 °C), and treatment time (10–70 min) ranges, including cell morphological structure, membrane permeability and fluidity. SEM and TEM observations showed morphological changes in the cell envelope and intracellular organization after SCCO2 treatment. Increase of membrane permeability was measured as increased uptake of the trypan blue dye with microscopy, and leakage of intracellular substances such as UV-absorbing materials and ions by determining the change of protein and electrical conductivity. The SCCO2 mediated reduction in CFU ml−1 was 0.5–1 log higher at 37 °C and 10 MPa for 60 min in Rose Bengal Medium containing 4 % sodium than a similar treatment in Rose Bengal Medium. Membrane fluidity analyzed by fluorescence polarization method using 1,6-diphenyl-1,3,5-hexatriene showed that the florescence polarization and florescence anisotropy of the SCCO2-treated cells were increased slightly and gently compared with the untreated cells. The correlation between membrane damage and death of cells under SCCO2 was clear, and the membrane damage was a key factor induced the inactivation of cells. 相似文献
185.
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187.
Xinbo Guo Rui Hai Liu Xueqing Fu Xiaofen Sun Kexuan Tang 《Plant Cell, Tissue and Organ Culture》2013,114(2):225-236
Epidemiological studies have shown that regular consumption of fruits and vegetables is associated with reduced risk of chronic diseases. Vegetables can provide vitamins, phenolics, flavonoids, minerals and dietary fibers for optimal health benefits. However, some nutrients contained in many fruits and vegetables cannot meet of the complete nutrition need in the human body. Biotechnology has the potential to improve the nutritional value of crops. Considering the high consumption of romaine lettuce in human diet worldwide, the objective of study is to enhance the contents of vitamin C, phenolics and antioxidant activity in lettuce leaves by genetic engineering techniques. The gene expression level, vitamin C content, total phenolics, as well as total and cellular antioxidant activities were analyzed by real-time PCR, HPLC, Folin–Ciocalteu, Hydro-PSC and CAA methods, respectively. The bio-fortification of genetically engineered lettuce increased vitamin C up to 48.94 ± 1.34 mg/100 g FW following the increased over-expression of At GLDH. This is almost a 3.2-fold increase as the content when compared with wild type lettuce (p < 0.05). In addition, phenolic compounds in transgenic lettuce contained 120.4 ± 1.62 mg GA equiv./100 g FW, almost double the phenolic content of the wild type. Total antioxidant activities were 735.4 ± 47.7 μmol vitamin C equiv./100 g FW, cellular antioxidant activities were 7.33 ± 0.86 μmol quercetin equiv./100 g FW (PBS wash) and 18.14 ± 0.68 μmol quercetin equiv./100 g FW (No PBS wash) in transgenic lettuce, respectively, 1.5, 4 and twofold increases when compared with the wild type. This study suggests that bio-fortification by genetic engineering has great potential to improve vitamin C, phenolic contents and antioxidant activity in lettuce. 相似文献
188.
João Vidigal Mafalda M. Dias Fabiana Fernandes Marco Patrone Cláudia Bispo Cláudia Andrade Rui Gardner Manuel J.T. Carrondo Paula M. Alves Ana P. Teixeira 《Journal of biotechnology》2013
Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells. 相似文献
189.
Yumei Li Yuwei Jiang Yiyun Chen Umesh Karandikar Kristi Hoffman Abanti Chattopadhyay Graeme Mardon Rui Chen 《Developmental biology》2013
optix, the Drosophila ortholog of the SIX3/6 gene family in vertebrate, encodes a homeodomain protein with a SIX protein–protein interaction domain. In vertebrates, Six3/6 genes are required for normal eye as well as brain development. However, the normal function of optix in Drosophila remains unknown due to lack of loss-of-function mutation. Previous studies suggest that optix is likely to play an important role as part of the retinal determination (RD) network. To elucidate normal optix function during retinal development, multiple null alleles for optix have been generated. Loss-of-function mutations in optix result in lethality at the pupae stage. Surprisingly, close examination of its function during eye development reveals that, unlike other members of the RD network, optix is required only for morphogenetic furrow (MF) progression, but not initiation. The mechanisms by which optix regulates MF progression is likely through regulation of signaling molecules in the furrow. Specifically, although unaffected during MF initiation, expression of dpp in the MF is dramatically reduced in optix mutant clones. In parallel, we find that optix is regulated by sine oculis and eyes absent, key members of the RD network. Furthermore, positive feedback between optix and sine oculis and eyes absent is observed, which is likely mediated through dpp signaling pathway. Together with the observation that optix expression does not depend on hh or dpp, we propose that optix functions together with hh to regulate dpp in the MF, serving as a link between the RD network and the patterning pathways controlling normal retinal development. 相似文献
190.
Jerome Irianto Joe Swift Rui?P. Martins Graham?D. McPhail Martin?M. Knight Dennis?E. Discher David?A. Lee 《Biophysical journal》2013,104(4):759-769
Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce chromatin condensation, while hypoosmotic conditions (100 mOsm/kg) cause decondensation. Large density changes (p < 0.001) occur over a very narrow range of physiological osmolalities, which suggests that chondrocytes likely experience chromatin condensation and decondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p < 0.01) and chromatin condensation (p < 0.001) also differed between chondrocytes in monolayer culture and three-dimensional agarose, suggesting a role for cell adhesion. The relationship between condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the chromatin condensation (<20 s), reveals the basic physicochemical nature of the process. Alterations in chromatin structure are expected to influence gene expression and thereby regulate chondrocyte activity in response to osmotic changes. 相似文献