| 1. |
- Kurczy, Michael, 1980, et al.
(författare)
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Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation
- 2010
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:7, s. 2751-2756
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry imaging has been used here to suggest that changes in membrane structure drive lipid domain formation in mating single-cell organisms. Chemical studies of lipid bilayers in both living and model systems have revealed that chemical composition is coupled to localized membrane structure. However, it is not clear if the lipids that compose the membrane actively modify membrane structure or if structural changes cause heterogeneity in the surface chemistry of the lipid bilayer. We report that time-of-flight secondary ion mass spectrometry images of mating Tetrahymena thermophila acquired at various stages during mating demonstrate that lipid domain formation, identified as a decrease in the lamellar lipid phosphatidylcholine, follows rather than precedes structural changes in the membrane. Domains are formed in response to structural changes that occur during cell-to-cell conjugation. This observation has wide implications in all membrane processes.
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| 2. |
- Kurczy, Michael, 1980, et al.
(författare)
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Nanotome Cluster Bombardment to Recover Spatial Chemistry After Preparation of Biological Samples for SIMS Imaging
- 2010
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 21:5, s. 833-836
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Tidskriftsartikel (refereegranskat)abstract
- A C-60(+) cluster ion projectile is employed for sputter cleaning biological surfaces to reveal spatio-chemical information obscured by contamination overlayers. This protocol is used as a supplemental sample preparation method for time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of frozen and freeze-dried biological materials. Following the removal of nanometers of material from the surface using sputter cleaning, a frozen-patterned cholesterol film and a freeze-dried tissue sample were analyzed using ToF-SIMS imaging. In both experiments, the chemical information was maintained after the sputter dose, due to the minimal chemical damage caused by C-60(+) bombardment. The damage to the surface produced by freeze-drying the tissue sample was found to have a greater effect on the loss of cholesterol signal than the sputter-induced damage. In addition to maintaining the chemical information, sputtering is not found to alter the spatial distribution of molecules on the surface. This approach removes artifacts that might obscure the surface chemistry of the sample and are common to many biological sample preparation schemes for ToF-SIMS imaging.
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| 3. |
- Makos, MA, et al.
(författare)
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Development and Characterization of a Voltammetric Carbon-Fiber Microelectrode pH Sensor
- 2010
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 26:12, s. 10386-10391
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Tidskriftsartikel (refereegranskat)abstract
- This work describes the development and characterization of a modified carbon-fiber microelectrode sensor capable of measuring real-time physiological pH changes in biological microenvironments. The reagentless sensor was fabricated under ambient conditions from voltammetric reduction of the diazonium salt Fast Blue RR onto a carbon-fiber surface in aprotic media. Fast-scan cyclic voltammetry was used to probe redox activity of the p-quinone moiety of the surface-bound molecule as a function of pH. In vitro calibration of the sensor in solutions ranging from pH 6.5 to 8.0 resulted in a pH-dependent anodic peak potential response. Flow-injection analysis was used to characterize the modified microelectrode, revealing sensitivity to acidic and basic changes discernible to 0.005 pH units. Furthermore, the modified electrode was used to measure dynamic in vivo pH changes evoked during neurotransmitter release in the central nervous system of the microanalytical model organism Drosophila melanogaster.
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| 4. |
- Makos, MA, et al.
(författare)
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In Vivo Electrochemical Measurements of Exogenously Applied Dopamine in Drosophila melanogaster.
- 2009
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Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 81:5, s. 1848-1854
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Tidskriftsartikel (refereegranskat)abstract
- Carbon-fiber microelectrodes coupled with electrochemical detection have been used extensively for the analysis of biogenic amines. In order to determine the functional role of these amines, in vivo studies have primarily used rats and mice as model organisms. Here, we report on the development of these microanalytical techniques for in vivo electrochemical detection of dopamine in the adult Drosophila melanogaster central nervous system (CNS). A triple-barrel micropipet injector was used to exogenously apply three different concentrations of dopamine, and a cylindrical carbon-fiber microelectrode was placed in the protocerebral anterior medial brain area where dopamine neurons are densely populated. Background-subtracted fast-scan cyclic voltammetry was used to measure dopamine concentration in the fly CNS. Distinct differences are shown for the clearance of exogenously applied dopamine in the brains of wild type flies versus fumin (fmn) mutants lacking a functional dopamine transporter. The current response due to oxidation of dopamine increased significantly from baseline for wild type flies following cocaine incubation. Interestingly, the current remained unchanged for mutant flies under the same conditions. These data confirm the accepted theory that cocaine blocks dopamine transporter function and validates the use of in vivo electrochemical methods to monitor dopamine uptake in Drosophila. Furthermore, after incubation with tetrodotoxin (TTX), a sodium channel blocker, there was a significant increase in peak oxidation current in the wild type flies; however, the current did not significantly change in the fmn mutant. These data suggest that factors that affect neuronal activity via ion channels such as TTX also influence the function of the dopamine transporter in Drosophila.
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| 5. |
- Makos, MA, et al.
(författare)
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Using in Vivo Electrochemistry To Study the Physiological Effects of Cocaine and Other Stimulants on the Drosophila melanogaster Dopamine Transporter
- 2010
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 1:1, s. 74-83
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Tidskriftsartikel (refereegranskat)abstract
- Dopamine neurotransmission is thought to play a critical role in addiction reinforcing mechanisms of drugs of abuse. Electrochemical techniques have been employed extensively for monitoring in vivo dopamine changes in the brains of model organisms including rats, mice, and primates. Here, we investigated the effects of several stimulants on dopamine clearance using recently developed microanalytical tools for in vivo electrochemical measurements of dopamine in the central nervous system of Drosophila melanogaster. A cylindrical carbon-fiber microelectrode was placed in the protocerebral anterior medial region of the Drosophila brain (an area dense with dopamine neurons) while a micropipette injector was positioned to exogenously apply dopamine. Background-subtracted fast-scan cyclic voltammetry was carried out to quantify changes in dopamine concentration in the adult fly brain. Clearance of exogenously applied dopamine was significantly decreased in the protocerebral anterior medial area of the wild-type fly following treatment with cocaine, amphetamine, methamphetamine, or methylphenidate. In contrast, dopamine uptake remained unchanged when identical treatments were employed in fumin mutant flies that lack functional dopamine transporters. Our in vivo results support in vitro binding affinity studies that predict these four stimulants effectively block normal Drosophila dopamine transporter function. Furthermore, we found 10 µM to be a sufficient physiological cocaine concentration to significantly alter dopamine transporter uptake in the Drosophila central nervous system. Taken together, these data indicate dopamine uptake in the Drosophila brain is decreased by psychostimulants as observed in mammals. This validates the use of Drosophila as a model system for future studies into the cellular and molecular mechanisms underlying drug addiction in humans.
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| 6. |
- Omiatek, Donna M, et al.
(författare)
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Only a Fraction of Quantal Content is Released During Exocytosis as Revealed by Electrochemical Cytometry of Secretory Vesicles
- 2010
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 1:3, s. 234-245
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Tidskriftsartikel (refereegranskat)abstract
- The primary method for neuronal communication involves the release of chemical messengers that are packaged intracellularly in vesicles. Although experiments measuring release at single cells have classically been thought to assess the entire content of vesicles, there is evidence in the literature that suggests that the total transmitter stored in vesicles is not expelled during exocytosis. In this work, we introduce a novel technology using a microfluidic-based platform to electrochemically probe individual PC12 cell vesicles isolated from the cell environment. We measure the total vesicular content using methodology that circumvents the biophysical processes of the cell associated with exocytosis. Direct comparisons of amperometric data from release experiments at single PC12 cells versus our cell-free model reveal that on average vesicles release only 40% of their total transmitter load. The data support the intriguing hypothesis that the average vesicle does not open all the way during the normal exocytosis process, resulting in incomplete distention of the vesicular contents. In addition, we have shown that vesicular catecholamine levels can be altered with pharmacological manipulation and variances observed from these treatments can be resolved at the single vesicle level in a high-throughput manner, a process that we have termed electrochemical cytometry. Upon establishing that release in exocytotic processes proceeds in an incomplete manner, we related electrochemical data quantified from both single cell release experiments and electrochemical cytometry of vesicles to vesicular volume from electron microscopy measurements to investigate the location of intravesicular catecholamine stores retained postfusion.
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