Clotilde B. Angelucci

A multiplex PCR-based DNA assay for the detection of paraoxonase gene cluster polymorphisms

Paraoxonase (PON) is a high-density lipoprotein (HDL) associated protein which is supposed to protect low-density lipoprotein (LDL) against oxidation and to play a role in the development of atherosclerosis. Interindividual variability in serum PON activity is attributable to common variants in components of the PON gene cluster on chromosome 7. We describe experimental conditions that permit the simultaneous determination of three common PON polymorphisms (PON1-192, PON1-55 and PON2-311) that are tightly associated with an increased risk of atherosclerosis. We used a multiplex PCR-based DNA assay using mismatch primers that introduce a unique recognition site for the endonuclease HinfI in the PCR products in case of presence of the R allele of PON 1-192, of the L allele of PON1-55 and of the S allele of PON2-311. The restriction analysis with HinfI allows to identify an electrophoretic band pattern which is specific for the combination of the three polymorphisms. This technique could be applied in the association studies aimed at assessing the role of PON and their polymorphisms in many clinical settings. In a preliminary study on a small population sample from south Italy about 10% of chromosomes exhibited the presumed risk-related haplotype R(192)/L(55)/S(311).

Membrane lipids are key modulators of the endocannabinoid-hydrolase FAAH

Lipid composition is expected to play an important role in modulating membrane enzyme activity, in particular if the substrates are themselves lipid molecules. A paradigmatic case is FAAH (fatty acid amide hydrolase), an enzyme critical in terminating endocannabinoid signalling and an important therapeutic target. In the present study, using a combined experimental and computational approach, we show that membrane lipids modulate the structure, subcellular localization and activity of FAAH. We report that the FAAH dimer is stabilized by the lipid bilayer and shows a higher membrane-binding affinity and enzymatic activity within membranes containing both cholesterol and the natural FAAH substrate AEA (anandamide). Additionally, co-localization of cholesterol, AEA and FAAH in mouse neuroblastoma cells suggests a mechanism through which cholesterol increases the substrate accessibility of FAAH.

A novel role for iron in modulating the activity and membrane-binding ability of a trimmed soybean lipoxygenase-1

Lipoxygenases (LOXs) are iron‐containing enzymes that play critical roles in plants and animals. As yet, metal atom extraction, reconstitution, and substitution have not been successfully applied to soybean LOX‐1 [Glycine max (L.) Merrill], a prototype member of the LOX family that is widely used in structural and kinetic studies. Here, tryptic digestion of native LOX‐1, used as a control, allowed us to isolate the 60‐kDa C‐terminal region (termed miniLOX), that retains the catalytically active iron in a more accessible position. Then, iron was removed to obtain an unprecedented apo‐miniLOX, which was reconstituted and substituted with different metal ions. These forms of miniLOX were characterized vs. native LOX‐1 by kinetic analysis, near UV circular dichroism, steady‐state fluorescence, and fluorescence resonance energy transfer. MiniLOX showed a 2‐fold increase in the membrane‐binding affinity compared with native LOX‐1 and a remarkable 4‐fold increase compared with apominiLOX (Kd=9.2±1.0, 17.9±2.0, and 45.4±4.3 μM, respectively). Furthermore, miniLOX reconstituted with Fe(II) or Fe(III) partially recovered its membrane‐binding ability (Kd=21.4±2.4 and 18.9±5.5 μM, respectively), overall supporting a novel noncatalytic role for iron in the LOX family.—Dainese, E., Angelucci, C. B., Sabatucci, A., De Filippis, V., Mei, M., Maccarrone, M. A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1. FASEB J. 24, 1725–1736 (2010). www.fasebj.org

Structural Properties of Plant and Mammalian Lipoxygenases. Temperature-Dependent Conformational Alterations and Membrane Binding Ability †

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases with major health and political relevance (atherosclerosis, osteoporosis). The crystal structures of various lipoxygenase-isoforms have been reported, and X-ray coordinates for enzyme-ligand complexes are also available. Although the 3D-structures of plant and animal lipoxygenase-isoforms are very similar, recent small-angle X-ray scattering data suggested a higher degree of motional flexibility of mammalian isozymes in aqueous solutions. To explore the molecular basis for these differences we performed dynamic fluorescence measurements that allowed us to study temperature-induced conformational changes arising from three-dimensional fluctuations of the protein matrix. For this purpose, we first investigated the impact of elevated temperature on activity, secondary structure, tertiary structure dynamics and conformational alterations. Applying fluorescence resonance energy transfer we also tested the membrane binding properties of the two lipoxygenase-isoforms, and compared their binding parameters. Taken together, our results indicate that the rabbit 12/15-lipoxygenase is more susceptible to temperature-induced structural alterations than the soybean enzyme. Moreover, the rabbit enzyme exhibits a higher degree of conformational flexibility of the entire protein molecule (global flexibility) and offers the possibility of augmented substrate movement at the catalytic center (local flexibility).

Domain mobility as probed by small-angle X-ray scattering may account for substrate access to the active site of two copper-dependent amine oxidases

Amine oxidases are a family of dimeric enzymes that contain one copper(II) ion and one 2,4,5-trihydroxyphenyalanine quinone per subunit. Here, the low-resolution structures of two Cu/TPQ amine oxidases from lentil (Lens esculenta) seedlings and from Euphorbia characias latex have been determined in solution by small-angle X-ray scattering. The active site of these enzymes is highly buried and requires a conformational change to allow substrate access. The study suggests that the funnel-shaped cavity located between the D3 and D4 domains is narrower within the crystal structure, whereas in solution the D3 domain could undergo movement resulting in a protein conformational change that is likely to lead to easier substrate access.

Impact of embedded endocannabinoids and their oxygenation by lipoxygenase on membrane properties.

N-Arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol are the best characterized endocannabinoids. Their biological activity is subjected to metabolic control whereby a dynamic equilibrium among biosynthetic, catabolic, and oxidative pathways drives their intracellular concentrations. In particular, lipoxygenases can generate hydroperoxy derivatives of endocannabinoids, endowed with distinct activities within cells. The in vivo interaction between lipoxygenases and endocannabinoids is likely to occur within cell membranes; thus, we sought to ascertain whether a prototypical enzyme like soybean (Glycine max) 15-lipoxygenase-1 is able to oxygenate endocannabinoids embedded in synthetic vesicles and how these substances could affect the binding ability of the enzyme to different lipid bilayers. We show that (i) embedded endocannabinoids increase membrane fluidity; (ii) 15-lipoxygenase-1 preferentially binds to endocannabinoid-containing bilayers; and that (iii) 15-lipoxygenase-1 oxidizes embedded endocannabinoids and thus reduces fluidity and local hydration of membrane lipids. Together, the present findings reveal further complexity in the regulation of endocannabinoid signaling within the central nervous system, disclosing novel control by oxidative pathways.

5-Lipoxygenase and cyclooxygenase-2 in the lungs of pigs naturally affected by enzootic pneumonia and porcine pleuropneumonia

Enzootic pneumonia by Mycoplasma hyopneumoniae and pleuropneumonia by Actinobacillus pleuropneumoniae are among the most common and economically relevant pulmonary diseases in swine herds. We herein investigated the activity and expression of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) in healthy and diseased porcine lungs, by means of immunohistochemical, immunochemical and biochemical assays. Diseased lungs showed a significantly higher activity and expression of 5-LOX and COX-2 in a wide range of cell types, thus suggesting the likely involvement of both enzymes in the pathogenesis of bacterial porcine pneumonia. Consistently, increased enzyme activities were paralleled by increased leukotriene B(4) (LTB(4)), a 5-LOX product and prostaglandin E(2) (PGE(2)), a COX-2 product, content in diseased versus healthy lungs.

Vitamin E-Related Inhibition of Monocyte 5-Lipoxygenase and Cardiovascular Outcome in Maintenance Hemodialysis Patients

A daily supplement of vitamin E is recommended for the secondary prevention of cardiovascular events in end-stage renal disease patients on maintenance hemodialysis. Vitamin E has been entrusted with therapeutic properties against cardiovascular disease for more than 60 years. Several epidemiological studies and intervention trials have been performed with vitamin E, and some of them showed that it prevents atherosclerosis. For a long time, vitamin E was assumed to act by decreasing the oxidation of low-density lipoproteins, a key step in atherosclerosis initiation. However, at the cellular level vitamin E interferes with smooth muscle cell proliferation, platelet aggregation, monocyte adhesion, and oxidized low-density lipoproteins uptake and cytokine production, all reactions implied in the progression of atherosclerosis. Recent research points out that these effects may be not only the result of the antioxidant activity of vitamin E but also of its distinct molecular actions. These biological properties of vitamin E may allow to design better strategies for primary and secondary prevention of cardiovascular disease, with a potential exploitation of vitamin E supplements in primary and secondary prevention of major adverse cardiovascular events in all uremic patients. In this review, we also outline relevant patents on vitamin E and lipoxygenase inhibitors.

A Multiplex PCR‐Based Assay for the Detection of Genetically Modified Soybean

Abstract The detection of nucleotide sequences specific for genetically modified organisms (GMOs) in raw and processed food is based on different technological strategies, such as the extraction of DNA and the amplification by polymerase chain reaction (PCR), which allow to obtain qualitative and quantitative information. We developed a multiplex PCR‐based DNA assay for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready™). Internal control target (lectin gene) was included both to assess the efficiency of all reactions and eliminating any false negatives. The post‐PCR analysis was carried out by 2.5% agarose gel electrophoresis followed by ethidium bromide staining and densitometric analysis. The multiplex PCR method, showing high sensitivity and specificity, was tested on DNA extracted from certified reference samples containing GM soybean, and from food samples (feeds, food supplements, etc.). Comparison of this method with a quantitative evaluation, carried out by real‐time PCR, suggests a possible utilization of the multiplex approach for semi‐quantitative determinations. The method reported in this work can considerably reduce the time and the costs of the GM soybean detection, especially in the screening of a large number of food samples. †This paper is dedicated to Prof. Corradino Motti sorrowfully missed on May 1, 2001.

DNA uptake in swine sperm: Effect of plasmid topology and methyl-beta-cyclodextrin-mediated cholesterol depletion

Sperm‐mediated gene transfer (SMGT), the ability of sperm cells to spontaneously incorporate exogenous DNA and to deliver it to oocytes during fertilization, has been proposed as an easy and efficient method for producing transgenic animals. SMGT is still undergoing development and optimization to improve the uptake efficiency of foreign DNA by sperm cells, which is a preliminary, yet critical, step for successful SMGT. Towards this aim, we developed a quantitative, real‐time PCR‐based assay to assess the absolute number of exogenous plasmids internalized into the spermatozoon. Using this technique, we found that the circular form of the DNA is more efficiently taken up than the linearized form. We also found that DNA internalization into the nucleus of porcine sperm cells is better under specific methyl‐β‐cyclodextrin (MCD)‐treated conditions, where the plasma membrane properties were altered without significantly compromising sperm physiology. These results provide the first evidence that membrane cholesterol depletion by MCD might represent a novel strategy for enhancing the ability of sperm to take up heterologous DNA. Mol. Reprod. Dev. 79: 853–860, 2012.