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Expression and Purification of the luciferase enzyme and in Vivo ATP Assay
Saman Hosseinkhani,mojtaba mortazavi,aboarhman emamzadeh
Physiology and Pharmacology , 2008,
Abstract: Introduction: Gene expression and purification of luciferases from the firefly, Lampyris turkestanicus, and optimization of cellular ATP measurements were performed. Methods: cDNA encoding luciferases from Lampyris turkestanicus was transferred from pQE30 vector into pET28a expression vector and pLtu28 was built. Newly constructed vector was expressed in E. coli XL1 Blue and the recombinant luciferase was purified using Ni-NTA Sepharose column. Enzymatic properties (Km and Vmax) for ATP were measured using luminescence assay. Standard curve of ATP was obtained by Promega ATP detection kit and designed method based on the L. turkestanicus luciferase and ATP serial dilution. Moreover bacterial ATP was measured by Promega kit and designed method using L. turkestanicus luciferase. Results: Results showed that ligation of L. turkestanicus luciferase encoding cDNA into pET28a and transformation of recombinant vector into competent cells was performed efficiently. Using luciferin, positive colonies were screened and cultured. SDA-PAGE showed that recombinant luciferase was efficiently purified by Ni-NTA Sepharose column. ATP standard curve and measurement of bacteria, using Promega and designed method by L. turkestanicus luciferases showed high identity. Conclusion: comparison of developed assay with promega kits in identification of bacterial concentration show its high quality and potent ability in ATP detection.
Increased Level of Extracellular ATP at Tumor Sites: In Vivo Imaging with Plasma Membrane Luciferase  [PDF]
Patrizia Pellegatti, Lizzia Raffaghello, Giovanna Bianchi, Federica Piccardi, Vito Pistoia, Francesco Di Virgilio
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002599
Abstract: Background There is growing awareness that tumour cells build up a “self-advantageous” microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP. Methodology/Principal Findings Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours. Conclusions/Significance Our results show that ATP in the tumour interstitium is in the hundrends micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.
Intracellular ATP Concentration Contributes to the Cytotoxic and Cytoprotective Effects of Adenosine  [PDF]
Shujue Li, Xiaofen Li, Haiping Guo, Shouting Liu, Hongbiao Huang, Ningning Liu, Changshan Yang, Ping Tang, Jinbao Liu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0076731
Abstract: Extracellular adenosine (Ade) interacts with cells by two pathways: by activating cell surface receptors at nanomolar/micromolar concentrations; and by interfering with the homeostasis of the intracellular nucleotide pool at millimolar concentrations. Ade shows both cytotoxic and cytoprotective effects; however, the underlying mechanisms remain unclear. In the present study, the effects of adenosine-mediated ATP on cell viability were investigated. Adenosine treatment was found to be cytoprotective in the low intracellular ATP state, but cytotoxic under the normal ATP state. Adenosine-mediated cytotoxicity and cytoprotection rely on adenosine-derived ATP formation, but not via the adenosine receptor pathway. Ade enhanced proteasome inhibition-induced cell death mediated by ATP generation. These data provide a new pathway by which adenosine exerts dual biological effects on cell viability, suggesting an important role for adenosine as an ATP precursor besides the adenosine receptor pathway.

Wang Dong Qu Yinbo Gao Peiji,

微生物学报 , 1996,
Abstract: The intracellular ATP contents in 4 strains of mycelial fungi, grown on the glucose-inorganic salts medium, were determined by luciferin-luciferase system. The results showed that the extracellular cellulase (FPA) of the fungi was synthesized only when the intracellular ATP level was lower than 10-8mg/ml. No matter what carbon source was used, cellulase synthesis was repressed once the intracellular ATP level was over 10-7mg/ml. The cellulase synthesis of the fungi had a negative relativity to their intracellular ATP contents. The cAMP content of the fungi was determined by HPLC. The fungal cellulase synthesis was increased by exogenous cAMP under depression conditions. However, exogenous cAMP could not relax the repression of cellulase synthesis when it had happened. The levels of intracellular ATP and cAMP are the essential factors in the regulation of cellulase synthesis in mycelial fungi.
Release of ATP from Marginal Cells in the Cochlea of Neonatal Rats Can Be Induced by Changes in Extracellular and Intracellular Ion Concentrations  [PDF]
Yating Peng, Jie Chen, Shan He, Jun Yang, Hao Wu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0047124
Abstract: Background Adenosine triphosphate (ATP) plays an important role in the cochlea. However, the source of ATP and the mechanism by which it is released remain unclear. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats. Methods Sprague-Dawley rats aged 1–3 days old were used for isolation, in vitro culture, and purification of marginal cells. Cultured marginal cells were verified by flow cytometry. Vesicles containing ATP in these cells were identified by fluorescence staining. The bioluminescence assay was used for determination of ATP concentration in the extracellular fluid released by marginal cells. Assays for ATP concentration were performed when the ATP metabolism of cells was influenced, and ionic concentrations in intracellular and extracellular fluid were found to change. Results Evaluation of cultured marginal cells with flow cytometry revealed the percentage of fluorescently-labeled cells as 92.9% and 81.9%, for cytokeratin and vimentin, respectively. Quinacrine staining under fluorescence microscopy revealed numerous green, star-like spots in the cytoplasm of these cells. The release of ATP from marginal cells was influenced by changes in the concentration of intracellular and extracellular ions, namely extracellular K+ and intra- and extracellular Ca2+. Furthermore, changes in the concentration of intracellular Ca2+ induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells. Conclusion We confirmed the presence and release of ATP from marginal cells of the stria vascularis. This is the first study to demonstrate that the release of ATP from such cells is associated with the state of the calcium pump, K+ channel, and activity of enzymes related to the phosphoinositide signaling pathway, such as adenylate cyclase, phospholipase C, and phospholipase A2.
Intracellular ZnO Nanorods Conjugated with Protoporphyrin for Local Mediated Photochemistry and Efficient Treatment of Single Cancer Cell
Kishwar S,Asif MH,Nur O,Willander M
Nanoscale Research Letters , 2010,
Abstract: ZnO nanorods (NRs) with high surface area to volume ratio and biocompatibility is used as an efficient photosensitizer carrier system and at the same time providing intrinsic white light needed to achieve cancer cell necrosis. In this letter, ZnO nanorods used for the treatment of breast cancer cell (T47D) are presented. To adjust the sample for intracellular experiments, we have grown the ZnO nanorods on the tip of borosilicate glass capillaries (0.5 μm diameter) by aqueous chemical growth technique. The grown ZnO nanorods were conjugated using protoporphyrin dimethyl ester (PPDME), which absorbs the light emitted by the ZnO nanorods. Mechanism of cytotoxicity appears to involve the generation of singlet oxygen inside the cell. The novel findings of cell-localized toxicity indicate a potential application of PPDME-conjugated ZnO NRs in the necrosis of breast cancer cell within few minutes.
Differential intracellular distribution of DNA complexed with polyethylenimine (PEI) and PEI-polyarginine PTD influences exogenous gene expression within live COS-7 cells  [cached]
Doyle Stephen R,Chan Chee
Genetic Vaccines and Therapy , 2007, DOI: 10.1186/1479-0556-5-11
Abstract: Background Polyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors. Methods PEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression. Results PEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependant, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression. Conclusion PEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel g
ATP-induced autophagy is associated with rapid killing of intracellular mycobacteria within human monocytes/macrophages
Debasis Biswas, Omar S Qureshi, Wing-Yiu Lee, Joanne E Croudace, Manuela Mura, David A Lammas
BMC Immunology , 2008, DOI: 10.1186/1471-2172-9-35
Abstract: We report that 3 mM ATP induces rapid cell autophagy in THP1 cells and monocyte-derived macrophages within 30 minutes post-treatment, as revealed by the expression of LC3-II bands on western blot analysis. Using Ca2+-free media and selective P2X7 agonists and antagonists, ATP-induced cell autophagy was shown to be Ca2+ and P2X7 receptor-dependent. Electron microscopy of ATP-treated, BCG-infected MDMs revealed the presence of the bacteria within characteristic double-membraned autophagosomes. Confocal analysis further confirmed that pharmacological inhibition of autophagy by wortmannin or pre-treatment of macrophages with anti-P2X7 antibody blocked ATP-induced phago-lysosomal fusion. Induction of cell autophagy with ATP was also temporally associated with a fall in intracellular mycobacterial viability, which was suppressed by treatment with wortmannin or the selective P2X7 antagonist, oxidized ATP (oATP).We provide the first evidence that ATP/P2X7-mediated killing of intracellular mycobacteria involves the induction of cell autophagy. The findings support the hypothesis that autophagy plays a key role in the control of mycobacterial infections.Tuberculosis continues to be a leading cause of human mortality and morbidity, with WHO figures estimating a global prevalence exceeding 14 million and mortality of approximately 1.6 million in the year 2005 [1]. The continuing global crisis has become further complicated by the emergence of multi-drug resistant strains of the bacteria and an increasing population of HIV-infected patients around the world. In view of this escalating clinical challenge, there is a growing need to develop novel therapeutic alternatives employing potent mycobactericidal mechanisms. Hence, there has been a lot of interest in cell autophagy as a potential immune defense mechanism against a number of bacterial pathogens, including mycobacteria [2-5]. Physiological or pharmacological induction of autophagy via cell starvation or treatment with rapamy
Rapid Granulation Tissue Regeneration by Intracellular ATP Delivery-A Comparison with Regranex  [PDF]
Jeffrey D. Howard, Harshini Sarojini, Rong Wan, Sufan Chien
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0091787
Abstract: This study tests a new intracellular ATP delivery technique for tissue regeneration and compares its efficacy with that of Regranex. Twenty-seven adult New Zealand white rabbits each underwent minimally invasive surgery to render one ear ischemic. Eight wounds were then created: four on the ischemic and four on the normal ear. Two wounds on one side of each ear were treated with Mg-ATP encapsulated lipid vesicles (ATP-vesicles) while the two wounds on the other side were treated with Regranex. Wound healing time was shorter when ATP-vesicles were used. The most striking finding was that new tissue growth started to appear in less than 1 day when ATP-vesicles were used. The growth continued and covered the wound area within a few days, without the formation of a provisional matrix. Regranex-treated wounds did not have this growth pattern. In wounds treated by ATP-vesicles, histologic studies revealed extremely rich macrophage accumulation, along with active proliferating cell nuclear antigen (PCNA) and positive BrdU staining, indicating in situ macrophage proliferation. Human macrophage culture suggested direct collagen production. These results support an entirely new healing process, which seems to have combined the conventional hemostasis, inflammation, and proliferation phases into a single one, thereby eliminating the lag time usually seen during healing process.
Intracellular delivery mechanism and brain delivery kinetics of biodegradable cationic bovine serum albumin-conjugated polymersomes
Pang Z, Gao H, Chen J, Shen S, Zhang B, Ren J, Guo L, Qian Y, Jiang X, Mei H
International Journal of Nanomedicine , 2012, DOI: http://dx.doi.org/10.2147/IJN.S32514
Abstract: tracellular delivery mechanism and brain delivery kinetics of biodegradable cationic bovine serum albumin-conjugated polymersomes Original Research (2572) Total Article Views Authors: Pang Z, Gao H, Chen J, Shen S, Zhang B, Ren J, Guo L, Qian Y, Jiang X, Mei H Published Date July 2012 Volume 2012:7 Pages 3421 - 3432 DOI: http://dx.doi.org/10.2147/IJN.S32514 Received: 01 April 2012 Accepted: 17 May 2012 Published: 06 July 2012 Zhiqing Pang,1,2 Huile Gao,1,2 Jun Chen,1,2 Shun Shen,1,2 Bo Zhang,3 Jinfeng Ren,1,2 Liangran Guo,1,2 Yong Qian,1,2 Xinguo Jiang,1,2 Heng Mei3 1Department of Pharmaceutics, School of Pharmacy, 2Key Laboratory of Smart Drug Delivery, Ministry of Education and PLA, Fudan University, Shanghai, 3Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People's Republic of China Background: A novel brain drug delivery system using cationic bovine serum albumin (CBSA)-conjugated biodegradable polymersomes (CBSA-PO) was prepared, and its intracellular delivery mechanism and brain delivery kinetics were evaluated. Methods and results: Biodegradable poly(ethylene glycol)-poly(ε -caprolactone) (PEG-PCL) was used to prepare the polymersomes, and thiolated CBSA was conjugated with the surface of the polymersome. Transmission electron microscopy and dynamic light scattering showed that the CBSA-PO had a round and vesicle-like shape, with a mean diameter of around 100 nm. Coupling of CBSA with polymersomes was confirmed by X-ray photoelectron spectroscopy. Uptake of CBSA-PO by bEnd.3 cells was significantly higher than that of unconjugated polymersomes, but was inhibited by low temperature, free CBSA, and poly-L-lysine, indicating that endocytosis was energy-driven and absorptive-mediated. Cell viability assays confirmed the good safety profile of biodegradable CBSA-PO. Pharmacokinetic results demonstrated that the polymersomes had long circulation times, and CBSA conjugation on the polymersomes significantly increased the blood–brain barrier permeability surface area product by 3.6-fold and the percentage of injected dose per gram brain (% ID/g brain) by 2.1-fold. Capillary depletion experiments showed that CBSA-PO was distributed into the brain parenchyma in a time-dependent manner, with few polymersomes detected, indicating that conjugation of polymersomes with CBSA significantly improved their transcytosis across the brain–blood barrier. Conclusion: These results suggest that CBSA-PO is a promising drug brain delivery carrier with low toxicity.
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