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Several lines of evidence suggest that a characteristic of the neuropathology of Alzheimer's disease (AD) is the aggregation of the amyloid beta peptides (Aβ), fragments of the human amyloid precursor protein (hAPP). The dominating species are the Aβ40 and Aβ42 fragments with 40 and 42 amino acids, respectively. Aβ initially forms soluble oligomers that continue to expand to protofibrils, suggestively the neurotoxic intermediates, and thereafter turn into insoluble fibrils that are markers of the disease. Using the powerful tool of pharmacophore simulation, we selected small molecules not known to possess central nervous system (CNS) activity but that might interact with Aβ aggregation, from the NCI Chemotherapeutic Agents Repository, Bethesda, MD. We assessed the activity of these compounds on Aβ aggregation using the thioflavin T fluorescence correlation spectroscopy (ThT-FCS) assay. Förster resonance energy transfer-based fluorescence correlation spectroscopy (FRET-FCS) was used to characterize the dose-dependent activity of selected compounds at an early stage of Aβ aggregation. Transmission electron microscopy (TEM) confirmed that the interfering substances block fibril formation and identified the macrostructures of Aβ aggregates formed in their presence. We first found three compounds generating protofibrils with branching and budding never observed in the control. One compound generated a two-dimensional sheet structure and another generated a double-stranded filament. Importantly, these compounds generating protofibrils with altered macrostructure protected against Aβ-induced toxicity in a cell model while showing no toxicity in a model of cognition in normal mice. The data suggest that the active compounds act as decoys turning the aggregation into nontoxic trajectories and pointing toward novel approaches to therapy.

Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOPwt ), and its naturally occurring isoform (MOPN40D ) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble-averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside-enriched domains and partial association with cholesterol-enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor-specific. KOP-containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOPN40D . Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOPwt , whereas this effect was not observed for MOPN40D .

Self-organization of cell-surface receptors in structurally distinct domains in the plasma membrane is of vital interest for correct cellular signaling. However, this dynamic process is difficult to study in cells with sufficiently high temporal and spatial resolution. We present here two quantitative high-resolution methods with single-molecule sensitivity, Fluorescence Correlation Spectroscopy (FCS) and pair-correlation Photoactivated Localization Microscopy (pcPALM), which enable nondestructive study of receptor diffusion and lateral organization at the nanoscale level. We introduce here the methods and review their application in studies of lateral organization of G Protein-Coupled Receptors (GPCRs). Examples from our own work on opioid receptor lateral organization are presented in order to illustrate the most recent advances in the field. [Projekat Ministarstva nauke Republike Srbije, br. 172015 i br. 45001]

Asimple, rapid and accurate procedure for the quantitative determination of quercetin in its pure form and in formulations has been developed. The method is based on the spectrophotometric determination of a complex formed between quercetin and potassium titanyloxalate in 50 % ethanolic solutions. To characterize the quercetin titanyloxalato complex, the stability constants of the complex were determinated potentiometrically and spectrophotometrically at different temperatures (T = 26.0oC, 34oC and 39.0oC), as well as at different ionic strengths (I = 5.0x10-4 mol dm-3, 3.0x10-2 mol dm-3 and 6.0x10-2 mol dm-3) and the thermodynamic parameters were calculated. As quercetin is usually conjugated to vitamin C in pharmaceutical formulations, two procedures for the quantitative determination of quercetin by this complexing reaction were tested - both in the absence and presence of ascorbic acid. In both procedures, the Beer law was obeyed over the same concentration range of quercetin, i.e., 0.85 μg mL-1 - 16.9 μg mL-1. In the first procedure in the absence of ascorbic acid the molar absorptivity coefficient of the quercetin-titanyloxalate complex is a=2.49 x 104 mol-1 dm3 cm-1, Sandells sensitivity of the method is S = 1.35 x 10-2 μg cm-2 and the detection limit is d = 0.67 μg mL-1. Whereas, in the presence of ascorbic acid (second procedure) a = 3.04 x 104 mol-1 dm3 cm-1, S = 1.11 x 10-2 μg mL-1. The proposed method was verified for the determination of quercetin in pharmaceutical dosage forms. .

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant K d app = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: K d ,   Fentanyl app = (80 ± 70) nM), K d , Morphine app = (200 ± 70) nM, K d ,   Codeine app = (100 ± 70) nM and K d ,   Oxycodone app = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP–5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain. [ABSTRACT FROM AUTHOR]