Macrophage mitochondrial damage from StAR transport of 7-hydroperoxycholesterol: Implications for oxidative stress-impaired reverse cholesterol transport

StAR family proteins in vascular macrophages participate in reverse cholesterol transport (RCT). We hypothesize that under pathophysiological oxidative stress, StARs will transport not only cholesterol to macrophage mitochondria, but also pro-oxidant cholesterol hydroperoxides (7-OOHs), thereby impairing early-stage RCT. Upon stimulation with dibutyryl-cAMP, RAW264.7 macrophages exhibited a strong time-dependent induction of mitochondrial StarD1 and plasma membrane ABCA1, which exports cholesterol. 7α-OOH uptake by stimulated RAW cell mitochondria (like cholesterol uptake) was strongly reduced by StarD1 knockdown, consistent with StarD1 involvement. Upon uptake by mitochondria, 7α-OOH (but not redox-inactive 7α-OH) triggered lipid peroxidation and membrane depolarization while reducing ABCA1 upregulation. These findings provide strong initial support for our hypothesis.     

Nonradiative Energy Losses of Plasmon-Polariton in a Metallic Nano-chain Deposited on a Semiconductor Substrate

Plasmonics - Propagation of plasmon-polariton in a metallic nano-chain in a dielectric surroundings is almost undamped and irradiation-less and such nano-chains are considered as quasi-perfect...     

Proteins are polyisoprenylated in Arabidopsis thaliana

Isoprenoid lipids were found to be covalently linked to proteins of Arabidopsis thaliana. Their identity (polyprenols: Prenol-9-11 with Pren-10 dominating and dolichols: Dol-15-17 with Dol-16 dominating) was confirmed by means of HPLC/ESI-MS with application of the multiple reaction monitoring technique as well as metabolic labeling of Arabidopsis plants with [³H]mevalonate and other precursors. The occurrence of typical farnesol-, geranylgeraniol-, and phytol-modified proteins was also noted. Radioisotopic labeling allowed detection of several proteins that were covalently bound to mevalonate-derived isoprenoid alcohols. A significant portion of polyisoprenylated proteins was recovered in the cytosolic/light vesicular fraction of Arabidopsis cells upon subfractionation. Taken together our data prove that a subset of plant proteins is polyisoprenylated.     

Spontaneous transfer of phospholipid and cholesterol hydroperoxides between cell membranes and low-density lipoprotein: assessment of reaction kinetics and prooxidant effects

Under oxidative pressure in the vascular circulation, erythrocytes and phagocytic cells may accumulate membrane lipid hydroperoxides (LOOHs), including cholesterol- and phospholipid-derived species (ChOOHs, PLOOHs). LOOH translocation from cells to low-density lipoprotein (LDL) might sensitize the latter to free radical-mediated oxidative modification, an early event associated with atherogenesis. To test this, we examined the spontaneous transfer kinetics of various ChOOH species (5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, 7 alpha/7 beta-OOH) and various PLOOH groups (PCOOH, PEOOH, PSOOH, SMOOH) using photoperoxidized erythrocyte ghosts as model donors and freshly prepared LDL as an acceptor. LOOH departure or uptake was monitored by reverse-phase HPLC with reductive electrochemical detection. Mildly peroxidized ghost membranes transferred overall ChOOH and PLOOH to LDL with apparent first-order rate constants approximately 60 and approximately 35 times greater than those of the respective parent lipids. Individual ChOOH rate constants decreased in the following order: 7 alpha/7 beta-OOH > 5 alpha-OOH > 6 alpha-OOH > 6 beta-OOH. Kinetics for reverse transfer from LDL to ghosts followed the same trend, but rates were significantly higher for all species and their combined activation energy was lower (41 vs 85 kJ/mol). PLOOH transfer rate constants ranged from 4- to 15-fold lower than the composite ChOOH constant, their order being as follows: PCOOH approximately PEOOH approximately PSOOH > SMOOH. Similar PLOOH transfer kinetics were observed when LDL acceptor was replaced by unilamellar liposomes, consistent with desorption from the donor membrane being the rate-limiting step. The susceptibility of transfer LOOH-enriched LDL to Cu2+-induced chain peroxidative damage was assessed by monitoring the accumulation of conjugated dienes and products of free radical-mediated cholesterol oxidation. In both cases, transfer-acquired LOOHs significantly reduced the lag time for chain initiation relative to that observed using nonperoxidized ghosts. These findings are consistent with the idea that LDL can acquire significant amounts of "seeding" LOOHs via translocation from various donors in the circulation.     

Analysis of mRNA for class I HLA on human gametogenic cells

We have studied mRNA expression for Class I HLA (human leukocyte antigen) on male germ cells by amplification of gene fragments in PCR techique and by Northern hybridization. RNA was extracted from fractionated gametogenic cells (isolated from testis) and reversely transcribed. Then, cDNA was amplified for specific HLA sequence (1151 bp) representing whole-length coding sequence (HLA, -A, -B, -C). The specificity of this product was confirmed in “nested” PCR of 400 bp gene fragment coding for alpha 2 domain, alpha 3 domain, and the transmembrane portion of Class I HLA. The results indicate minimal expression of classical Class I HLA on gametogenic cells. Northern hybridization with 669 bp cDNA fragment (spanning for alpha 3 domain, transmembrane, cytoplasmic, and 3′ untraslated region) resulted in a low intensity signal from gametogenic cell fractions and confirmed our findings obtained by PCR. The minimal expression of classical HLA antigens may create a neutral cover for the male reproductive system, thereby preventing an immunological response during germ cell differentiation. © 1994 Wiley-Liss, Inc.     

A Preliminary Study Showing the Impact of Genetic and Dietary Factors on GC–MS-Based Plasma Metabolome of Patients with and without PROX1-Genetic Predisposition to T2DM up to 5 Years Prior to Prediabetes Appearance

Risk factors for type 2 diabetes mellitus (T2DM) consist of a combination of an unhealthy, imbalanced diet and genetic factors that may interact with each other. Single nucleotide polymorphism (SNP) in the prospero homeobox 1 (PROX1) gene is a strong genetic susceptibility factor for this metabolic disorder and impaired β-cell function. As the role of this gene in T2DM development remains unclear, novel approaches are needed to advance the understanding of the mechanisms of T2DM development. Therefore, in this study, for the first time, postprandial changes in plasma metabolites were analysed by GC–MS in nondiabetic men with different PROX1 genotypes up to 5 years prior to prediabetes appearance. Eighteen contestants (12 with high risk (HR) and 6 with low risk (LR) genotype) participated in high-carbohydrate (HC) and normo-carbohydrate (NC) meal-challenge tests. Our study concluded that both meal-challenge tests provoked changes in 15 plasma metabolites (amino acids, carbohydrates, fatty acids and others) in HR, but not LR genotype carriers. Postprandial changes in the levels of some of the detected metabolites may be a source of potential specific early disturbances possibly associated with the future development of T2DM. Thus, accurate determination of these metabolites can be important for the early diagnosis of this metabolic disease.     

High-performance liquid chromatography with mercury cathode electrochemical detection: application to lipid hydroperoxide analysis

Lipid hydroperoxide species can be analyzed with high sensitivity and specificity, using reversed-phase high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop cathode [HPLC-ED(Hg)]. The purpose of this study was to examine different variables in the operation of HPLC-ED(Hg) and to select optimal conditions for the analysis of several biologically relevant peroxides, including species derived from cholesterol, cholesteryl linoleate, oleate, linoleate, and two synthetic phosphatidylcholines. Parameters such as operating potential and mobile-phase solvent proportions, electrolyte composition, and ionic strength were evaluated for each peroxide class. Under optimal conditions, we have achieved baseline separation of four cholesterol hydroperoxide species, not only from one another, but also from phospholipid hydroperoxides; detection limits were <0.3 pmol and <30 pmol for the cholesterol and phospholipid hydroperoxides, respectively.     

Membrane actions of water-soluble fusogens: Effect of dimethyl sulfoxide,glycerol and sucrose on lipid bilayer order and fluidity

The effect of three water-soluble fusogens: dimethyl sulfoxide (DMSO), glycerol and sucrose on the structural properties of model lipid membranes has been studied by electron spin resonance (ESR) using 5-doxylstearic acid as a spin probe and by fluorescence spectroscopy using pyrene as an excimer forming fluorescent probe. All three fusogens tested produce a marked increase in the order parameter of the region close to the polar surface of the lipid bilayer. The ordering effect of DMSO, but not of glycerol and sucrose, is much stronger with respect to membranes prepared from acidic than from neutral phospholipids. The membrane-perturbing action of glycerol and sucrose manifests itself also in the reduced lateral mobility of membrane incorporated pyrene, indicating thus a decreased fluidity of the bilayer hydrophobic region. The structural perturbations produced in model membranes by DMSO, glycerol and sucrose are discussed in relation to the mechanism by which these substances promote cell fusion.