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A newly synthesized nitroxide-based probe containing a fluorophore with high quantum yield at relatively long wavelengths can detect endogenously derived lipid-derived radicals and has therapeutic potential in a model of hepatocellular carcinoma. Lipids and their metabolites are easily oxidized in chain reactions initiated by lipid radicals, forming lipid peroxidation products that include the electrophiles 4-hydroxynonenal and malondialdehyde. These markers can bind cellular macromolecules, causing inflammation, apoptosis and other damage. Methods to detect and neutralize the initiating radicals would provide insights into disease mechanisms and new therapeutic approaches. We describe the first high-sensitivity, specific fluorescence probe for lipid radicals, 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen). NBD-Pen directly detected lipid radicals in living cells by turn-on fluorescence. In a rat model of hepatic carcinoma induced by diethylnitrosamine (DEN), NBD-Pen detected lipid radical generation within 1 h of DEN administration. The lipid radical scavenging moiety of NBD-Pen decreased inflammation, apoptosis and oxidative stress markers at 24 h after DEN, and liver tumor development at 12 weeks. Thus, we have developed a novel fluorescence probe that provides imaging information about lipid radical generation and potential therapeutic benefits in vivo .

Ketamine is a dissociative anesthetic drug that is abused by teenagers and young adults, commonly for recreational purposes in dance clubs, to generate euphoria and dissociation, and sometimes employed as a date-rape drug. Herein, a highly sensitive, and environmentally friendly spectrofluorimetric method was developed for detection of ketamine in pharmaceutical and plasma samples. The technique is based on a nucleophilic substitution reaction occurring between ketamine and NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole), resulting in the formation of a fluorescent derivative that exhibits detectability at a remarkable level. The resultant derivative demonstrates its maximum emission intensity at 543 nm upon excitation at a wavelength of 481 nm. Upon optimizing the reaction parameters, a linear relationship was established between the fluorescence intensity of the formed derivative and the concentrations of ketamine within the specified range of 10 - 250 ng/mL. The method demonstrated exceptional sensitivity with detection and quantitation limits of 2.37 ng/mL and 7.18 ng/mL, respectively, allowing for the detection of ketamine in both pharmaceutical and plasma samples. The method was precise (%RSD < 1) accurate (%R = 99.74%) and robust (% R ± SD from 98.00 ± 1.11 to 99.97 ± 0.75). Furthermore, the developed method was compared to the reported method, with the results indicating that the suggested method is more sensitive and ecologically benign.

A nitrobenzoxadiazole-based fluoroprobe (NBD-Bu) is designed to probe cellular metabolic activity in cancer and normal cells. NBD-Bu shows a significant fluorescence enhancement upon selective binding to the transport protein serum albumin in PBS buffer at ambient conditions. Encouraged by this finding, the site- specificity of NBD-Bu has been explored through a competitive displacement assay in the presence of site-specific markers such as warfarin and ibuprofen. Notably, even at micromolar concentrations, the probe possesses the ability to displace the site marker drug ibuprofen, efficiently. Subsequently, high-resolution fluorescence imaging results consolidated the potential of NBD-Bu for detection of abnormal cellular metabolic activity.

A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry; but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported lipid bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald ripening, the smaller domains in one leaflet erode away whereas the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.

Cancer cells exhibit an altered metabolism characterized by enhanced glycolysis and glucose consumption. In glucose-addicted cancer cells upregulation of glucose transport across the plasma membrane is mediated by a family of facilitated glucose transporter proteins, particularly glucose transporter 1 (GLUT1). The aim of the present study was to investigate the impact of GLUT1 expression on glucose uptake and viability of FTC-133 and 8305c thyroid cancer cells growing in hypoglycemic, normoglycemic and hyperglycemic conditions. The results showed that the total expression of GLUT1 was higher in the two cell types growing in low glucose compared to cells growing in normoglycemia or hyperglycemia and this was correlated with AKT Ser473 phosphorylation but not with the expression of hypoxia inducible factor α (HIF1α). However, the membrane expression of GLUT1 was correlated with HIF1α expression. HIF1α expression was positively correlated with the glucose concentration in FTC-133 cells, whereas this expression was inversely correlated in 8305c cells. Glucose uptake was dependent on the membrane level of GLUT1 but not total GLUT1 expression. Downregulation of GLUT1 expression by RNAi in FTC-133 cells caused a reduction in glucose uptake but did not significantly affect cell viability. In the case of 8305c cells showing low endogenous GLUT1 expression and lack of HIF1α expression in normoxic conditions GLUT1 RNAi impacted cell viability. These data suggested that GLUT1 may be part of an AKT1-dependent mechanism allowing cells to survive in low levels of glucose. Glucose concentration inversely affected HIF1α expression and the level of GLUT1 in membrane as well as glucose uptake in FTC-133 and 8305c cells. The extent of GLUT1 impact on cell viability was also cell-type-dependent.

During the last several years, intracellular lipid droplets have become the focus of intense study. No longer an inert bystander, the lipid droplet is now known as a dynamic organelle contributing lipids to many cellular events. However, while the dynamics of cholesterol efflux from both the plasma membrane and lipid droplets have been studied, less is known regarding the efflux of sphingomyelin from these membranes. In order to address this issue, sphingomyelin efflux kinetics and binding affinities from different intracellular pools were examined. When compared to the plasma membrane, lipid droplets had a smaller exchangeable sphingomyelin efflux pool and the time required to efflux that pool was significantly shorter. Fluorescence binding assays revealed that proteins in the plasma membrane and lipid droplet pool bound sphingomyelin with high affinity. Further characterization identified adipose differentiation-related protein (ADRP) as one of the sphingomyelin binding proteins in the lipid droplet fraction and revealed that ADRP demonstrated saturable binding to 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoyl)sphingosyl-phosphocholine (NBD-sphingomyelin) and also 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-phosphatidylcholine) with binding affinities in the nanomolar range. Taken together, these results suggest that lipid droplet associated proteins such as ADRP may play a significant role in regulating the intracellular distribution of phospholipids and lipids in general. Overall, insights from the present work suggest new and important roles for lipid droplets and ADRP in phospholipid metabolism.

The molecular species of sphingoid bases were tagged with the fluorescent amino group reagent, 4-fluoro-7-nitrobenzofurazan (NBD-F). The NBD-sphingoid bases were analyzed by a highly selective and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) technique capable of reliable detection of several fmol of the derivatives. Lipid extracts from plant samples were derivatized with NBD-F, and all nine species of free sphingoid bases present in plant sphingolipids were separated and quantified for the first time; a complete baseline resolution was achieved for cis -8 and trans -8 isomers of sphingoid bases by reversed phase HPLC on a C 18 column. The extraction and derivatization procedures and LC–MS/MS method can facilitate the progress of the studies for seeking the active components of sphingoid bases species in response to biological challenges.

The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A 2 (PLA 2 ) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C 12 -NBD-PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra- and inter-day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD-labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA-SbQ) material, from the plane of which, the sn -2 acyl chain of the phospholipid stands out and is accessible by PLA 2 . Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA 2 . Mass spectrometry showed that only C 12 -NBD-FA, the PLA 2 hydrolysis product, was detected in the reaction mixture, indicating that PLA 2 recognizes PVA-SbQ/C 12 -NBD-PtdCho as a surface to perform catalysis.

Brownian ratchets use a time-varying asymmetric potential that can be applied to separate diffusing particles or molecules. A new type of Brownian ratchet, a geometrical Brownian ratchet, has been realized. Charged, fluorescently labeled phospholipids in a two-dimensional fluid bilayer were driven in one direction by an electric field through a two-dimensional periodic array of asymmetric barriers to lateral diffusion fabricated from titanium oxide on silica. Diffusion spreads the phospholipid molecules in the orthogonal direction, and the asymmetric barriers rectify the Brownian motion, causing a directional transport of molecules. The geometrical ratchet can be used as a continuous molecular sieve to separate mixtures of membrane-associated molecules that differ in electrophoretic mobility and diffusion coefficient.

A high-performance liquid chromatography method in which fluorescence detection is used for the simultaneous determination of 21 amino acids is proposed. Amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and then separated on a monolithic silica column (MonoClad C18-HS, 150 mm × 3 mm i.d.). A mixture of 25 mM citrate buffer containing 25 mM sodium perchlorate (pH 5.5) and acetonitrile was used as the mobile phase. We found that the most significant factor in the separation was temperature, and a linear temperature gradient from 30 to 49°C was used to control the column temperature. The limits of detection and quantification for all amino acids ranged from 3.2 to 57.2 fmol and 10.8 to 191 fmol, respectively. The calibration curves for the NBD-amino acid had good linearity within the range of 40 fmol to 40 pmol when 6-aminocaproic acid was used as an internal standard. Using only conventional instruments, the 21 amino acids could be analyzed within 10 min. This method was found to be suitable for the quantification of the contents of amino acids in mouse plasma and adrenal gland samples.