carbocyanines and resazurin

Electron transfer between membrane spanning oxidoreductase enzymes controls vital metabolic processes. Here we studied for the first time with single molecule resolution the function of P450 oxidoreductase (POR), the canonical membrane spanning activator of all microsomal cytochrome P450 enzymes. Measurements and statistical analysis of individual catalytic turnover cycles shows POR to sample at least two major functional states. This phenotype may underlie regulatory interactions with different cytochromes P450 but to date has remained masked in bulk kinetics. To ensure that we measured the inherent behavior of POR, we reconstituted the full length POR in "native like" membrane patches, nanodiscs. Nanodisc reconstitution increased stability by ∼2-fold as compared to detergent solubilized POR and showed significantly increased activity at biologically relevant ionic strength conditions, highlighting the importance of studying POR function in a membrane environment. This assay paves the way for studying the function of additional membrane spanning oxidoreductases with single molecule resolution.

Topics: Biomimetic Materials; Carbocyanines; Chromatography, Gel; Electron Transport; Electrophoresis, Polyacrylamide Gel; Enzymes, Immobilized; Escherichia coli; Fluorescent Dyes; Lipid Bilayers; Models, Biological; Mutation; NADPH-Ferrihemoprotein Reductase; Nanostructures; Oxazines; Protein Conformation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Time Factors; Xanthenes

The European Commission White Paper, "Strategy for a future chemicals policy" (EC, 2001) is estimated to require the testing of approximately 30,000 "existing" chemicals by 2012. Recommended in vitro tests require validation. As the White Paper (EC, 2001) requires neurotoxic data, this study evaluated an in vitro testing strategy for predicting in vivo neurotoxicity. The sensitivities of differentiated PC12 cells and primary cerebellum granule cells (CGC) were compared to undifferentiated PC12 cells which can indicate basal cytotoxicity. Cytotoxicants and neurotoxicants selected for testing covered a range of mechanisms and potencies. Neurotoxicants were not distinguished from cytotoxicants despite significantly different cell system responses using all endpoints; cell viability/activity, ATP depletion, MMP depolarisation, ROS production and cytoskeleton modifications. For all chemicals tested, neuronal-like cell systems were generally less sensitive than undifferentiated PC12 cells. Acute oral rodent LD(50) values correlated with cytotoxicity IC(50) values for the respective chemicals tested in each cell system. This study concluded that although simple non-specific assays are required to distinguish basal cytotoxicity from specific neurotoxicity by using different cell systems with different states of neuronal differentiation, further work is required to determine suitable combinations of cell systems and endpoints capable of distinguishing neurotoxicants from cytotoxicants.

Topics: Adenosine Triphosphate; Animals; Benzimidazoles; Carbocyanines; Cell Line; Cell Separation; Cerebellum; Cytoskeleton; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Indicators and Reagents; Lethal Dose 50; Neurofilament Proteins; Neurons; Neurotoxicity Syndromes; Neurotoxins; Oxazines; PC12 Cells; Rats; Xanthenes

Mitochondrial dysfunction has long been implicated in the death of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) and its experimental models. Here we further analyzed changes in the mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Deltapsi(m)) of striatal synaptosomes after the infusion of 1-methyl-4-phenylpyridinium (MPP+) into rat striatum. MPP+ (40 nmol) treatment produced decreases in mitochondrial REDOX activity and Deltapsi(m) at 18 h, as measured by fluorometric analysis with both Alamar blue and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) dyes. At this time point, tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels were not altered, but both decreased at 7 days after MPP+ (40 nmol) infusion. Both measures of mitochondrial dysfunction induced by MPP+ (40 nmol) at 18 h were attenuated, at least in part, by pretreatment with a selective dopamine uptake inhibitor GBR-12909 (1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl) piperazine). In addition, GBR-12909 partially attenuated MPP+ (40 nmol)-caused a loss of striatal nerve terminal as indicated by decreases in TH and DAT immunoreactivities as well as dopamine and its metabolites levels. The present study indicates that decreases in mitochondrial REDOX activity and Deltapsi(m) may play a role in MPP+ -induced dopaminergic neurotoxicity, and further provides that improvement of mitochondrial dysfunction may be a better way to slow progressive dopaminergic neurodegeneration commonly associated with PD.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Benzimidazoles; Carbocyanines; Coloring Agents; Corpus Striatum; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; Fluorometry; Male; Membrane Glycoproteins; Membrane Potentials; Membrane Transport Proteins; Mitochondria; Nerve Tissue Proteins; Oxazines; Oxidation-Reduction; Parkinsonian Disorders; Piperazines; Presynaptic Terminals; Rats; Rats, Wistar; Sodium Cyanide; Synaptosomes; Tyrosine 3-Monooxygenase; Xanthenes