Biomedical subjects

Dana D Wise

Publications and source records attributed to Dana D Wise.

4 recordsLinked to original sources

PubMed2005

Quantitation of nicotinamide and serotonin derivatives and detection of flavins in neuronal extracts using capillary electrophoresis with multiphoton-excited fluorescence.

Capillary electrophoresis (CE) with multiphoton-excited fluorescence detection (CE-MPE) allows low-background analysis of spectrally distinct fluorophores using a single long-wavelength laser. Extracts were prepared from immortalized rat raphe nuclei neurons, and were analyzed by CE-MPE. Native fluorescence was detected from reduced nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), riboflavin, serotonin, and 5-hydroxytryptophan (5HTrp). Quantitation of exogenous serotonin (taken up by cells) and endogenous NADH and 5HTrp was possible using internal standards or standard addition. This system should be useful to study monamine oxidase inhibitors (MAOIs) and selective serotonin reuptake inhibitors (SSRIs).

Electrophoresis, Capillary↗

PubMed2005

Catalytic three-dimensional protein architectures.

We demonstrate a strategy for microfabricating catalytically active, three-dimensional matrixes composed of cross-linked protein in cellular and microfluidic environments. In this approach, a pulsed femtosecond laser is used to excite photosensitizers via multiphoton absorption within three-dimensionally defined volumes, a process that promotes cross-linking of protein residue side chains in the vicinity of the laser focal point. In this manner, it is possible to fabricate protein microparticles with dimensions on the order of the multiphoton focal volume (less than 1 microm(3)) or, by scanning the position of a laser focal point relative to a specimen, to generate surface-adherent matrixes or cables that extend through solution for hundreds of micrometers. We show that protein matrixes can be functionalized either through direct cross-linking of enzymes, by decoration of avidin matrixes with biotinylated enzymes, or by cross-linking biotinylated proteins that then are linked to biotinylated enzymes via an avidin couple. Several formats are explored, including microparticles that can be translocated to desired sites of action (including cytosolic positions), protein pads that generate product gradients within cell cultures, and on-column nanoreactors for microfluidic systems. These biomaterial fabrication technologies offer opportunities for studying a variety of cell functions, ranging from single-cell biochemistry and development to perturbation and analysis of small populations of cultured cells.

Animals↗

PubMed2004

Tracking variations in nicotinamide cofactors extracted from cultured cells using capillary electrophoresis with multiphoton excitation of fluorescence.

Nicotinamide cofactors play numerous roles in cellular metabolic and biosynthetic reactions and intracellular signaling events. Recently, nicotinamide cofactors have been implicated in the function of cellular biological clocks. To gain insight into the possible roles of nicotinamide cofactors in complex time-related events, we have developed a rapid and sensitive method for extraction of NAD(P)(H) from cultured cells, separation of analytes by capillary electrophoresis, and detection by multiphoton excitation of fluorescence. Extraction and quantitation steps have been systematically characterized for optimal pH, detergent, temperature, sonication, filtration, efficiency, accuracy, and reproducibility. The method is suitable for extractions at 2- to 3-h intervals over 1 day or more or as frequently as every hour for shorter durations. Natively fluorescent NAD(P)H are assayed directly, and nonfluorescent NAD(P) are enzymatically reduced to their fluorescent counterparts before analysis. The method yields accurate values for cellular NADP, NADPH, and total NAD(H) levels and relative information on cellular NADH concentration; modification of the procedure allows full quantitation of all relevant species. We conclude that these assays are more suitable than any yet published for tracking variations in nicotinamide cofactor levels over periods of 1 day or more.

Animals↗

PubMed2002

Internal standard method for the measurement of choline and acetylcholine by capillary electrophoresis with electrochemical detection.

An internal standard method has been developed for the determination of the neurotransmitter acetylcholine and/or its metabolic precursor choline. This approach couples the high separation efficiency of capillary electrophoresis with the sensitivity and selectivity of electrochemical detection at an enzyme-modified electrode. Indirect electrochemical detection is accomplished at a 25 microm platinum electrode modified by cross-linking the enzymes choline oxidase and acetylcholinesterase with glutaraldehyde. Although in this simple form of electrode fabrication there is a gradual loss of response from the electrochemical detector with time, accurate quantitation is achieved by the addition of butyrylcholine, which is also a substrate for acetylcholinesterase, as an internal standard. A linear response is achieved between 0 and 125 microM with a limit of detection of 2 microM (25 fmol). The utility of this method was demonstrated by monitoring the kinetics of choline uptake in synaptosomal preparations.

Acetylcholine↗