A role for reactive oxygen species in endotoxin-induced elevation of MOR expression in the nervous and immune systems.

This study examined the mechanism by which exposure to lipopolysaccharide (LPS) alters mu-opioid receptor (MOR) expression in immune and neuronal cells using an in vitro conditioned medium model system. We found that LPS stimulated the intracellular accumulation of reactive oxygen species (ROS) and MOR expression in macrophage-like TPA-HL-60 cells. Conditioned medium from the LPS-stimulated TPA-HL-60 cells increased MOR expression in SH-SY5Y cells, a neuronal cell model, through actions mediated by TNF-α and GM-CSF. These data suggest that the endotoxin, LPS, modulates MOR expression in nervous and immune cells via ROS signaling, and demonstrates the crosstalk that exists within the neuroimmune axis.

Methamphetamine-mediated modulation of MOR expression in the SH-SY5Y neuroblastoma cell line.

Methamphetamine (METH) has been shown to induce oxidative stress in SH-SY5Y cells, a neuroblastic, dopaminergic cell line model. In neuronal cells, oxidation of dopamine by auto-oxidative or enzymatic mechanisms leads to the production of reactive oxygen species (ROS). Neuronal cells treated with METH accumulate dopamine, which can ultimately lead to increased levels of ROS. ROS has been shown to mediate the expression of the mu-opioid receptor (MOR). The goal of this in vitro study was to examine the effects of METH on the accumulation of intracellular ROS in SH-SY5Y cells, which could, in turn, modulate MOR expression. Confocal laser scanning microscopy (CLSM) indicated that METH induced intracellular accumulation of ROS, detected as increased fluorescence of rhodamine 123, in a dose- and time-dependent manner. Moreover, accumulation of ROS preceded METH-induced expression of the MOR, which was attenuated by the free radical chelator, vitamin E. Additionally, increased MOR expression was noted following hydrogen peroxide treatment, indicating a role for ROS in mediating MOR expression. Taken together, our data show that METH's effect on MOR expression is dependent upon sublethal levels of intracellular ROS, which suggests a possible coupling of METH- and opiate-mediated intracellular signaling.

Escherichia coli acetyl-coenzyme A carboxylase: characterization and development of a high-throughput assay.

Bacterial acetyl-coenzyme A carboxylase (ACCase) is a multicomponent system composed of AccA, AccD, AccC, and AccB (also known as BCCP), which is required for fatty acid biosynthesis. It is essential for cell growth and has been chemically validated as a target for antimicrobial drug discovery. To identify ACCase inhibitors, a simple and robust assay that monitors the overall activity by measuring phosphate production at physiologically relevant concentrations of all protein components was developed. Inorganic phosphate production was demonstrated to directly reflect the coupled activities of AccC and AccA/D with BCCP cycling between the two half-reactions. The K(m) apparent values for ATP, acetyl-coenzyme A, and BCCP were estimated to be 60+/-14 microM, 18+/-4 microM, and 39+/-9 nM, respectively. The stoichiometry between the two half-reactions was measured to be 1:1. Carboxy-biotin produced in the first half-reaction was stable over the time course of the assay. The assay was adapted to a high-throughput screen (HTS) 384-well format using a modified published scintillation proximity method. The optimized HTS assay has acceptable Z' factor values and was validated to report inhibitions of either AccC or AccA/D. The assay is not susceptible to signal quenching due to colored compounds.

Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts.

Kinases are an important therapeutic target for drug discovery, and many cancer chemotherapeutic agents have been derived from natural product sources. Natural product samples, however, have the likelihood of assay interference, particularly at elevated test concentrations. The authors developed a competitive fluorescence polarization (FP) assay using red-shifted fluorophores for the AKT kinase and demonstrated utility for testing concentrated natural product extracts. A set of 7 actinomycetes cultures containing indolocarbazoles, known nonselective kinase inhibitors, and a control set of 22 nonproducing indolocarbazole cultures were evaluated. Using red-shifted dyes (Cy3B or Cy5), the authors identified active samples with minimal interference up to the extract concentrations that are 3 times nonextracted culture levels. In contrast, a significant number of interferences were observed using either a fluorescein competitive FP assay or a [33P]ATP Flashplate assay. This work demonstrates that one can screen natural product extracts at high concentrations successfully using FP technology with red-shifted dyes.