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991.
992.
Stefan Zdraljevic Drew Wagner Kevin Cheng Laura Ruohonen Jussi J?ntti Merja Penttil? Orna Resnekov C. Gustavo Pesce 《Applied and environmental microbiology》2013,79(24):7569-7582
Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell''s pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways. 相似文献
993.
994.
Xiao-Hong Liu Hong Yan Man Xu Yong-Li Zhao Li-Min Li Xi-Hui Zhou Ming-Xu Wang Le Ma 《Neurochemistry international》2013
Neonatal hypoxia–ischemia (HI) produces neurodegeneration and brain injury, and leads to behavioral and cognitive dysfunction. Hyperbaric oxygen (HBO) treatment may potentially be neuroprotective in HI injury. The aim of this study was to examine any neuroprotection by HBO treatment on long-term neurological function in the rat model of neontatal HI. Seven-day-old rats were subjected to HI or sham surgery. HBO treatment was administered (2.5 ATA for 90 min) 1 h after hypoxia exposure. Sensorimotor (grip test and rota-rod) and cognitive tests (inhibitory avoidance and Morris water maze) were performed at postnatal day 28 to day 60. The extent of brain damage was determined by histological evaluation. Apoptosis, caspase-3 and apoptosis inducing factor (AIF) expression were assessed by immunohistochemistry 12, 24, and 48 h after HI. HI-treated animals had significantly worse sensorimotor and cognitive performances than those in the Sham group. HBO treatment led to significant improvements in neurobehavioral functions compared to the HI group, especially for cognitive performances. Morphological evaluation revealed a remarkable recovery of brain injury in the HBO group. Furthermore, the improvements in neurobehavioral impairments were correlated with the reduction in lesion size of the hippocampus and cerebral cortex. The proportion of apoptotic cells significantly increased with time after HI, and HBO significantly inhibited apoptotic cell death. The proportion of caspase-3 positive cells and nuclear AIF translocation increased and peaked at 24 h after HI injury. HBO-treated rats showed decreased expression of these proteins compared to HI-treated animals. In conclusion, our results suggested that HBO treatment was effective in promoting long-term functional and histological recovery against neonatal HI brain injury. HBO-induced neuroprotection was associated with suppression of apoptosis by inhibiting caspase-3 and AIF-mediated pathways. Further studies evaluating its associated molecular and cellular mechanism are needed. 相似文献
995.
Jian Xu Hiroaki Mon Takahiro Kusakabe Zhiqing Li Li Zhu Kazuhiro Iiyama Atsushi Masuda Takumi Mitsudome Jae Man Lee 《Applied microbiology and biotechnology》2013,97(24):10435-10444
The double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is widely employed in silkworm and its tissue-derived cell lines for gene function analysis. Baculovirus expression vector system (BEVS) has an advantage for large-scale protein expression. Previously, combining these useful tools, we improved traditional AcMNPV-Sf9 BEVS to produce modified target glycoproteins, where the ectopic expression of Caenorhabditis elegans systemic RNAi defective-1 (SID-1) was found to be valuable for soaking RNAi. In current study, we applied CeSID-1 protein to a Bombyx mori NPV (BmNPV)-hypersensitive Bme21 cell line and investigated its properties both in soaking RNAi ability and recombinant protein expression. The soaking RNAi-mediated suppression in the Bme21 cell enables us to produce modified glycoproteins of interest in BmNPV–Bme21 BEVS. 相似文献
996.
997.
Gijsbers L Man HY Kloet SK de Haan LH Keijer J Rietjens IM van der Burg B Aarts JM 《Analytical biochemistry》2011,(1):77-83
Phosphatidylinositol (PtdIns) is phosphorylated at D-3, D-4, and/or D-5 of the inositol ring to produce seven distinct lipid second messengers known as phosphoinositides (PIs). The PI level is temporally and spatially controlled at the cytosolic face of the cellular membrane. Effectors containing PI-binding domains (e.g., PH, PX, FYVE, ENTH, FERM) associate with specific PIs. This process is crucial for the localization of a variety of cell-signaling proteins, thereby regulating intracellular membrane trafficking, cell growth and survival, cytoskeletal organization, and so on. However, quantitative assessments of protein–PI interactions are generally difficult due to insolubility of PIs in aqueous solution. Here we incorporated PIs into a lipid–protein nanoscale bilayer (nanodisc), which is applied for studying the protein–PI interactions using pull-down binding assay, fluorescence polarization, and nuclear magnetic resonance studies, each facilitating fast, quantitative, and residue-specific evaluation of the protein–PI interactions. Therefore, the PI-incorporated nanodisc could be used as a versatile tool for studying the protein–lipid interactions by various biochemical and biophysical techniques. 相似文献
998.
Yam AY Wang X Gao CM Connolly MD Zuckermann RN Bleu T Hall J Fedynyshyn JP Allauzen S Peretz D Salisbury CM 《Biochemistry》2011,50(20):4322-4329
Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases. 相似文献
999.
During the early blastula period of zebrafish embryos, the outermost blastomeres begin to undergo a significant thinning in the apical/basolateral dimension to form the first distinct cellular domain of the embryo, the enveloping layer (EVL). During this shape transformation, only the EVL-precursor cells generate a coincidental series of highly restricted Ca(2+) transients. To investigate the role of these localized Ca(2+) transients in this shape-change process, embryos were treated with a Ca(2+) chelator (5,5'-difluoro BAPTA AM; DFB), or the Ca(2+) ionophore (A23187), to downregulate and upregulate the transients, respectively, while the shape-change of the forming EVL cells was measured. DFB was shown to significantly slow, and A23187 to significantly facilitate the shape change of the forming EVL cells. In addition, to investigate the possible involvement of the phosphoinositide and Wnt/Ca(2+) signaling pathways in the Ca(2+) transient generation and/or shape-change processes, embryos were treated with antagonists (thapsigargin, 2-APB and U73122) or an agonist (Wnt-5A) of these pathways. Wnt-5A upregulated the EVL-restricted Ca(2+) transients and facilitated the change in shape of the EVL cells, while 2-APB downregulated the Ca(2+) transients and significantly slowed the cell shape-change process. Furthermore, thapsigargin and U73122 also both inhibited the EVL cell shape-change. We hypothesize, therefore, that the highly localized and coincidental Ca(2+) transients play a necessary role in initiating the shape-change of the EVL cells. 相似文献
1000.
Man Luo Qingsong Sun Hongmei Zhao Jiali Tao Dongsheng Yan 《Journal of cellular physiology》2020,235(1):105-113
The biological function of long noncoding RNA NEAT1 has been revealed in a lot of diseases. Nevertheless, it is still not yet clear whether NEAT1 can modulate the process of myocardial ischemia–reperfusion injury (M-I/R). Here, we reported that NEAT1 was able to sponge miR-495-3p to contribute to M-I/R injury through activating mitogen-activated protein kinase 6 (MAPK6). First, elevated expression of NEAT1 was revealed in M-I/R injury mice, meanwhile, lactate dehydrogenase (LDH) and creatine kinase-muscle/brain (CK-MB) were also upregulated in the serum. Meanwhile, as previously reported, miR-495 serves as a tumor suppressor or an oncogenic miRNA in different types of cancer. Currently, we found miR-495-3p was remarkably reduced in M-I/R mice. Additionally, NEAT1 was significantly induced whereas miR-495-3p was greatly reduced by H2O2 treatment in H9C2 cells. Moreover, loss of NEAT1 in H9C2 cells could repress the viability and proliferation of cells. For another, overexpression of NEAT1 exhibited an opposite phenomenon. Furthermore, LDH release and caspase-3 activity were obviously triggered by upregulation of NEAT1 while suppressed by NEAT1 knockdown. miR-495-3p was indicated and validated as a target of NEAT1 using the analysis of bioinformatics. Interestingly, we observed that miR-495-3p mimics repressed tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-18 protein expression while their levels were enhanced by the inhibition of miR-495-3p in H9c2 cells. Subsequently, it was manifested that MAPK6 was a target of miR-495-3p, which could exert a lot in the NEAT1/miR-495-3p-mediated M-I/R injury. Overall, our results implied that NEAT1 contributed to M-I/R injury via the modulation of miR-495-3p and MAPK6. 相似文献