Identification and quantitation of fatty acid ethyl esters in biological specimens

Fatty acid ethyl esters, recently described as enzymatic products of nonoxidative ethanol metabolism in the heart, may represent a mediator or marker of ethanol-induced organ pathology such as alcoholic cardiomyopathy. This study was designed to develop a method for the extraction, quantitation, and definitive identification of fatty acid ethyl esters formed both in biological specimens and during enzymatic incubations. First, several potential sources of error were identified and characterized. Tissue extraction with alcohols led to the time, temperature, and concentration-dependent nonenzymatic formation of fatty acid alcohol esters. Contamination of both substrates, [14C]ethanol and 14C-fatty acid, used to measure enzymatically mediated fatty acid ethyl ester synthesis, could be removed by purification. Accurate quantitation of fatty acid ethyl esters in tissue was achieved using acetone as an extraction solvent, after which isolated lipids were thin-layer chromatographed on silica gel developed with an apolar solvent system (petroleum ether:diethyl ether:acetic acid, 75:5:1). Gas chromatography and mass spectroscopy identified individual fatty acid ethyl esters. The reproducibility of this assay was high, as assessed by quintuplicate determinations of fatty acid ethyl esters formed in liver and heart homogenates, a method with standard deviations 4 to 11% of the mean.     

Manufacturing and <Emphasis Type="Italic">in vivo</Emphasis> inner ear visualization of MRI traceable liposome nanoparticles encapsulating gadolinium

Background  

Treatment of inner ear diseases remains a problem because of limited passage through the blood-inner ear barriers and lack of control with the delivery of treatment agents by intravenous or oral administration. As a minimally-invasive approach, intratympanic delivery of multifunctional nanoparticles (MFNPs) carrying genes or drugs to the inner ear is a future therapy for treating inner ear diseases, including sensorineural hearing loss (SNHL) and Meniere's disease. In an attempt to track the dynamics and distribution of nanoparticles in vivo, here we describe manufacturing MRI traceable liposome nanoparticles by encapsulating gadolinium-tetra-azacyclo-dodecane-tetra-acetic acid (Gd-DOTA) (abbreviated as LPS+Gd-DOTA) and their distribution in the inner ear after either intratympanic or intracochlear administration.     

Interaction of fusidic acid with lipid membranes: Implications to the mechanism of antibiotic activity

We have studied the effects of cholesterol and steroid-based antibiotic fusidic acid (FA) on the behavior of lipid bilayers using a variety of experimental techniques together with atomic-scale molecular dynamics simulations. Capillary electrophoretic measurements showed that FA was incorporated into fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes. Differential scanning calorimetry in turn showed that FA only slightly altered the thermodynamic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, whereas cholesterol abolished all endotherms when the mole fraction of cholesterol (X(chol)) was >0.20. Fluorescence spectroscopy was then used to further characterize the influence of these two steroids on DPPC large unilamellar vesicles. In the case of FA, our result strongly suggested that FA was organized into lateral microdomains with increased water penetration into the membrane. For cholesterol/DPPC mixtures, fluorescence spectroscopy results were compatible with the formation of the liquid-ordered phase. A comparison of FA and cholesterol-induced effects on DPPC bilayers through atomistic molecular dynamics simulations showed that both FA and cholesterol tend to order neighboring lipid chains. However, the ordering effect of FA was slightly weaker than that of cholesterol, and especially for deprotonated FA the difference was significant. Summarizing, our results show that FA is readily incorporated into the lipid bilayer where it is likely to be enriched into lateral microdomains. These domains could facilitate the association of elongation factor-G into lipid rafts in living bacteria, enhancing markedly the antibiotic efficacy of FA.     

Biophysical properties of lipids and dynamic membranes

The lipid bilayer is a 3D assembly with a rich variety of physical features that modulate cell signaling and protein function. Lateral and transverse forces within the membrane are significant and change rapidly as the membrane is bent or stretched and as new constituents are added, removed or chemically modified. Recent studies have revealed how differences in structure between the two leaflets of the bilayer and between different areas of the bilayer can interact together with membrane deformation to alter the activities of transmembrane channels and peripheral membrane binding proteins. Here, we highlight some recent reports that the physical properties of the membrane can help control the function of transmembrane proteins and the motor-dependent elongation of internal organelles, such as the endoplasmic reticulum.     

Protein-oxidized phospholipid interactions in cellular signaling for cell death: From biophysics to clinical correlations

Oxidative stress is associated with several major ailments. However, it is only recently that the developments in our molecular level understanding of the consequences of oxidative stress in modifying the chemical structures of biomolecules, lipids in particular, are beginning to open new emerging insights into the significance of oxidative stress in providing mechanistic insights into the etiologies of these diseases. In this brief review we will first discuss the role of lipid oxidation in controlling the membrane binding of cytochrome c, a key protein in the control of apoptosis. We then present an overview of the impact of oxidized phospholipids on the biophysical properties of lipid bilayers and continue to discuss, how these altered properties can account for the observed enhancement of formation of intermediate state oligomers by cytotoxic amyloid forming peptides associated with pathological conditions as well as host defense peptides of innate immunity. In the third part, we will discuss how the targeting of oxidized phospholipids by i) pathology associated peptides and ii) host defense peptides can readily explain the observed clinical correlations associating Alzheimer's and Parkinson's diseases with increased risk for type 2 diabetes and age-related macular degeneration, and the apparent protective effect of Alzheimer's and Parkinson's diseases from some cancers, as well as the inverse, apparent protection by cancer from Alzheimer's and Parkinson's diseases. This article is part of a Special Issue entitled: Oxidized phospholipids-Their properties and interactions with proteins.     

α-Lipoic acid modulates thiol antioxidant defences and attenuates exercise-induced oxidative stress in standardbred trotters

Several micronutrient supplementation strategies are used to cope with oxidative stress, although their benefits have recently been questioned. The aim of the present study was to examine the effects of DL-α-lipoic acid (LA) in response to acute exercise and during recovery in horses. Six standardbred trotters were tested on the treadmill before and after 5-week LA supplementation (25 mg/kg body weight/day). According to electron paramagnetic resonance measurements, strenuous aerobic exercise increased significantly free radical formation in the gluteus medius muscle, which was prevented by LA supplementation. The activities of thioredoxin reductase and glutathione reductase in muscle were significantly increased in LA-treated horses, but neither LA nor exercise affected muscle thioredoxin activity. LA increased the concentration of total glutathione in muscle at rest and during recovery. Treatment with LA blunted the exercise-induced increase in plasma oxygen radical absorbance capacity and decreased the post-exercise levels of lipid hydroperoxides in plasma and malondialdehyde in plasma and in muscle. These findings suggest that LA enhances thiol antioxidant defences and decreases exercise-induced oxidative stress in skeletal muscle.     

1-Palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine, an oxidized phospholipid, accelerates Finnish type familial gelsolin amyloidosis in vitro

Finnish type familial amyloidosis (FAF) is a neurodegenerative disease, which involves the deposition of D187N or -Y mutant gelsolin fragments as amyloid in various tissues, accompanied by dermatologic, neurologic, and ophthalmologic disorders. Like the other amyloid diseases, FAF is associated with oxidative stress. The latter results in an extensive chemical modification of biomolecules, such as the formation of a myriad of phospholipids with oxidatively modified acyl chains containing various functional groups. Here we demonstrate that 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde moiety at the end of its truncated sn-2 acyl chain, accelerates amyloidogenesis of FtG(179-194) (i.e., the core amyloidogenic segment of residues 179-194 of FAF gelsolin) as revealed by thioflavin T (ThT) fluorescence and electron microscopy. These techniques and Trp fluorescence show that the accelerated conversion of FtG(179-194) into amyloid fibrils consists of distinct consecutive phases. PoxnoPC at a close to critical micelle concentration (~22.5 μM) causes a maximal increase in ThT fluorescence and the K(app) for fibril formation. The rates of fibril elongation and nucleation were proportional to PoxnoPC concentration, while the rates of nucleation were different below and above the critical micelle concentration. Our data also suggest an initial rapid formation of a 1:1 complex by PoxnoPC and FtG(179-194). The latter could involve a transient Schiff base and reside at the membrane hydrocarbon-water interface in the proximity of the phosphocholine headgroup. Subsequently, these profibrils insert into a more hydrophobic milieu and undergo a slow structural transition and assemble into amyloid fibers. Different phases can be expected when proteins aggregate on the phospholipid membrane surfaces, underlying the importance of a detailed kinetic analysis to fully understand the effects of oxidized phospholipids on amyloidogenesis. This study represents the first comprehensive analysis of the kinetics and mechanisms of amyloid formation in the presence of an oxidized phospholipid.     

The cooperation of FGF receptor and Klotho is involved in excretory canal development and regulation of metabolic homeostasis in Caenorhabditis elegans

FGFs have traditionally been associated with cell proliferation, morphogenesis, and development; yet, a subfamily of FGFs (FGF19, -21, and -23) functions as hormones to regulate glucose, lipid, phosphate, and vitamin D metabolism with impact on energy balance and aging. In mammals, Klotho and beta-Klotho are type 1 transmembrane proteins that function as obligatory co-factors for endocrine FGFs to bind to their cognate FGF receptors (FGFRs). Mutations in Klotho/beta-Klotho or fgf19, -21, or -23 are associated with a number of human diseases, including autosomal dominant hypophosphatemic rickets, premature aging disorders, and diabetes. The Caenorhabditis elegans genome contains two paralogues of Klotho/beta-Klotho, klo-1, and klo-2. klo-1 is expressed in the C. elegans excretory canal, which is structurally and functionally paralogous to the vertebrate kidney. KLO-1 associates with EGL-15/FGFR, suggesting a role for KLO-1 in the fluid homeostasis phenotype described previously for egl-15/fgfr mutants. Altered levels of EGL-15/FGFR signaling lead to defects in excretory canal development and function in C. elegans. These results suggest an evolutionarily conserved function for the FGFR-Klotho complex in the development of excretory organs such as the mammalian kidney and the worm excretory canal. These results also suggest an evolutionarily conserved function for the FGFR-Klotho axis in metabolic regulation.     

Monitoring the growth and drug susceptibility of individual bacteria using asynchronous magnetic bead rotation sensors

Continuous growth of individual bacteria has been previously studied by direct observation using optical imaging. However, optical microscopy studies are inherently diffraction limited and limited in the number of individual cells that can be continuously monitored. Here we report on the use of the asynchronous magnetic bead rotation (AMBR) sensor, which is not diffraction limited. The AMBR sensor allows for the measurement of nanoscale growth dynamics of individual bacterial cells, over multiple generations. This torque-based magnetic bead sensor monitors variations in drag caused by the attachment and growth of a single bacterial cell. In this manner, we observed the growth and division of individual Escherichia coli, with 80-nm sensitivity to the cell length. Over the life cycle of a cell, we observed up to a 300% increase in the rotational period of the biosensor due to increased cell volume. In addition, we observed single bacterial cell growth response to antibiotics. This work demonstrates the non-microscopy limited AMBR biosensor for monitoring individual cell growth dynamics, including cell elongation, generation time, lag time, and division, as well as their sensitivity to antibiotics.     

Latitudinal clines in the timing and temperature-sensitivity of photoperiodic reproductive diapause in Drosophila montana

Reproductive diapause is a primary mechanism used by arthropods to synchronize their life cycle with seasonal changes in temperate regions. Our study species, Drosophila montana, represents the northern insect species where flies enter reproductive diapause under short day conditions and where the precise timing of diapause is crucial for both survival and offspring production. We have studied clinal variation in the critical day length for female diapause induction (CDL) and their overall susceptibility to enter diapause (diapause incidence), as well as the temperature sensitivity of these traits. The study was performed using multiple strains from four latitudinal clines of the species – short clines in Finland and Alaska and long clines in the Rocky Mountains and the western coast of North America – and from one population in Kamchatka, Russia. CDL showed strong latitudinal clines on both continents, decreasing by one hour per five degrees decline in latitude, on average. CDL also decreased in all populations along with an increase in fly rearing temperature postponing the diapause to later calendar time, the effects of temperature being stronger in southern than in northern population. Female diapause incidence was close to 100% under short day/low temperature conditions in all populations, but decreased below 50% even under short days in 19°C in the southern North American western coast populations and in 22°C in most populations. Comparing a diversity of climatic data for the studied populations showed that while CDL is under a tight photoperiodic regulation linked with latitude, its length depends also on climatic factors determining the growing season length. Overall, the study deepens our understanding of how spatial and environmental parameters affect the seasonal timing of an important biological event, reproductive diapause and helps to estimate the evolutionary potential of insect populations to survive in changing climatic conditions.