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71.
72.
Background
Electrochemotherapy and gene electrotransfer are novel promising treatments employing locally applied high electric pulses to introduce chemotherapeutic drugs into tumor cells or genes into target cells based on the cell membrane electroporation. The main focus of this paper was to calculate analytically and numerically local electric field distribution inside the treated tissue in two dimensional (2D) models for different plate and needle electrode configurations and to compare the local electric field distribution to parameter U/d, which is widely used in electrochemotherapy and gene electrotransfer studies. We demonstrate the importance of evaluating the local electric field distribution in electrochemotherapy and gene electrotransfer. 相似文献73.
Jelica Gluhak-Heinrich Sumin Gu Dubravko Pavlin Jean X. Jiang 《Cell communication & adhesion》2006,13(1):115-125
Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption. 相似文献
74.
75.
Restriction enzymes have proven to be among the most valuable tools in molecular biology. In this work, we demonstrate that the cleavage of fluorescently labeled, PCR-amplified DNA can be used as a simple and highly sensitive technique for detection of sequences present in a percentage as low as 0.6% in a DNA pool. Due to the fact that fluorescent labeling of DNA fragments enables such sensitive detection and quantification of restriction enzyme cleavage, the method was further exploited in monitoring of the enzymatic digestion completeness and in determination of factors that influence restriction enzyme effectiveness. We analyzed the activity of six restriction endonucleases; the percentage of uncleaved DNA fragments predominantly ranged between 2.0 and 2.5 and the highest value was 8.00%. We conclude that, since the enzymatic digestion completeness may not always be assured, each assay based on restriction enzyme cleavage that is intended to be used in investigations of heterogeneity in a DNA pool should be constructed so that the presence of cleaved sequences is the indication of pool nonuniformity. When the presence of uncleaved sequences indicates pool heterogeneity, the results could be misleading due to possible incompleteness of enzymatic cleavage. 相似文献
76.
Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase the permeability
of a cell membrane and enables transfer of genes. Poor plasmid mobility in tissues is one of the major barriers for the successful
use of gene electrotransfer in gene therapy. Therefore, we analyzed the effect of electrophoresis on increasing gene electrotransfer
efficiency using different combinations of high-voltage (HV) and low-voltage (LV) pulses in vitro on CHO cells. We designed
a special prototype of electroporator, which enabled us to use only HV pulses or combinations of LV + HV and HV + LV pulses.
We used optimal plasmid concentrations used in in vitro conditions as well as lower suboptimal concentrations in order to
mimic in vivo conditions. Only for the lowest plasmid concentration did the electrophoretic force of the LV pulse added to
the HV pulse increase the transfection efficiency compared to using only HV. The effect of the LV pulse was more pronounced
for HV + LV, while for the reversed sequence, LV + HV, there was only a minor effect of the LV pulse. For the highest plasmid
concentrations no added effect of LV pulses were observed. Our results suggest that there are different contributing effects
of LV pulses: electrophoretically increased contact of DNA with the membrane and increased insertion of DNA into permeabilized
cell membrane and/or translocation due to electrophoretic force, which appears to be the dominant effect. 相似文献
77.
Gene electrotransfer is a promising nonviral method that enables transfer of plasmid DNA into cells with electric pulses.
Although many in vitro and in vivo studies have been performed, the question of the implied gene electrotransfer mechanisms
is largely open. The main obstacle toward efficient gene electrotransfer in vivo is relatively poor mobility of DNA in tissues.
Since cells are mechanically coupled to their extracellular environment and act differently compared to standard in vitro
conditions, we developed a three-dimensional (3-D) in vitro model of CHO cells embedded in collagen gel as an ex vivo model
of tissue to study electropermeabilization and different parameters of gene electrotransfer. For this purpose, we first used
propidium iodide to detect electropermeabilization of CHO cells embedded in collagen gel. Then, we analyzed the influence
of different concentrations of plasmid DNA and pulse duration on gene electrotransfer efficiency. Our results revealed that
even if cells in collagen gel can be efficiently electropermeabilized, gene expression is significantly lower. Gene electrotransfer
efficiency in our 3-D in vitro model had similar dependence on concentration of plasmid DNA and pulse duration comparable
to in vivo studies, where longer (millisecond) pulses were shown to be more optimal compared to shorter (microsecond) pulses.
The presented results demonstrate that our 3-D in vitro model resembles the in vivo situation more closely than conventional
2-D cell cultures and, thus, provides an environment closer to in vivo conditions to study mechanisms of gene electrotransfer. 相似文献
78.
Cécile Faurie Matej Rebersek Muriel Golzio Masa Kanduser Jean‐Michel Escoffre Mojca Pavlin Justin Teissie Damijan Miklavcic Marie‐Pierre Rols 《The journal of gene medicine》2010,12(1):117-125
Background
Electroporation is a physical method used to transfer molecules into cells and tissues. Clinical applications have been developed for antitumor drug delivery. Clinical trials of gene electrotransfer are under investigation. However, knowledge about how DNA enters cells is not complete. By contrast to small molecules that have direct access to the cytoplasm, DNA forms a long lived complex with the plasma membrane and is transferred into the cytoplasm with a considerable delay.Methods
To increase our understanding of the key step of DNA/membrane complex formation, we investigated the dependence of DNA/membrane interaction and gene expression on electric pulse polarity and repetition frequency.Results
We observed that both are affected by reversing the polarity and by increasing the repetition frequency of pulses. The results obtained in the present study reveal the existence of two classes of DNA/membrane interaction: (i) a metastable DNA/membrane complex from which DNA can leave and return to external medium and (ii) a stable DNA/membrane complex, where DNA cannot be removed, even by applying electric pulses of reversed polarity. Only DNA belonging to the second class leads to effective gene expression.Conclusions
The life‐time of DNA/membrane complex formation is of the order of 1 s and has to be taken into account to improve protocols of electro‐mediated gene delivery. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献79.
Brian C. Perez John W. DayJr. Dubravko Justic Robert R. Lane Robert R. Twilley 《Hydrobiologia》2011,658(1):41-54
A 3-month field study was conducted to examine the effects of Atchafalaya River discharge on nitrogen and phosphorus concentrations
in the Fourleague Bay system, to document patterns with salinity variation, to evaluate stoichiometric nutrient ratios of
nitrogen and phosphorus in the river and bay, and to examine the relationship between estuarine freshwater residence time
and export of total nitrogen (TN) and total phosphorus (TP) to the Gulf of Mexico. During spring peak discharge of the Atchafalaya
River, nutrient ratios in lower Fourleague Bay indicate potential phosphorus limitation with an average dissolved inorganic
nitrogen (DIN) to dissolved inorganic phosphorus (DIP) ratio of 32:1, primarily a result of high concentrations of nitrogen
entering the northern bay from the Atchafalaya River and of fairly stable phosphorus concentrations. Ratios of DIN to phosphorus
in the river were much higher (54:1), indicating a significant loss of nitrogen within the Fourleague Bay system. Freshwater
residence time averaged approximately 7 days during the study and ranged from 2 to 100 days. TN export averaged 57% over the
study and ranged from less than 3% at long residence times to greater than 80% at short residence times. TN export to the
coastal ocean with respect to residence time is considerably less than has been shown in other studies. Nitrate + nitrite
export averaged 49% for the 3-month study. Percentages of TP export were greater than TN, averaging 82% for the study period.
By examining the Atchafalaya River delta as a natural analog for controlled river diversions, which are currently being used
as coastal restoration tools, this study shows that discharging river water into highly productive shallow coastal estuarine
and wetland systems can significantly reduce the amount of nitrogen exported to the Gulf of Mexico. 相似文献
80.
Mojca Pavlin Sa?a Haberl Matej Reber?ek Damijan Miklav?i? Ma?a Kandu?er 《Journal of visualized experiments : JoVE》2011,(55)
Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause destabilization of cell membrane, making it permeable to small molecules and allows transfer of large molecules such as DNA. It represents an alternative to viral vectors, due to its safety, efficacy and ease of application. For gene electrotransfer different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field direction and orientation during the pulse delivery. Changing electric field direction and orientation increase the membrane area competent for DNA entry into the cell. In this video, we demonstrate the difference in gene electrotransfer efficacy when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation. For this purpose tip with integrated electrodes and high-voltage prototype generator, which allows changing of electric field in different directions during electric pulse application, were used. Gene electrotransfer efficacy is determined 24h after pulse application as the number of cells expressing green fluorescent protein divided with the number of all cells. The results show that gene transfection is increased when the electric field orientation during electric pulse delivery is changed.Download video file.(27M, mov) 相似文献