Paraoxonase 1 in endothelial cells impairs vasodilation induced by arachidonic acid lactone metabolite

IntroductionParaoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated lactonase, which is known for its antiatherogenic properties. Previous studies in PON1 knockout (PON1KO) mice revealed that PON1KO mice have low blood pressure, which is inversely correlated with the renal levels of the cytochrome P450 -derived arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET). Our previous studies revealed that 5,6-EET is unstable, transforming to the δ-lactone isomer 5,6-δ-DHTL, an endothelium-derived hyperpolarizing factor (EDHF) that mediates vasodilation, and it is a potential substrate for PON1.AimTo elucidate the role of PON1 in the modulation of vascular resistance via the regulation of the lactone-containing metabolite 5,6-δ-DHTL.ResultsIn mouse resistance arteries, PON1 was found to be present and active in the endothelial layer. Vascular reactivity experiments revealed that 5,6-δ-DHTL dose-dependently dilates PON1KO mouse mesenteric arteries significantly more than wild type (w.t.) resistance arteries. Pre-incubation with HDL or rePON1 reduced 5,6-δ-DHTL-dependent vasodilation. FACS analyses and confocal microscopy experiments revealed that fluorescence-tagged rePON1 penetrates into human endothelial cells' (ECs') in both dose- and time- dependent manner, accumulate in the perinuclear compartment, and retains its lactonase activity in the cells. The presence of rePON1, but not the presence of PON1 loss-of-lactonase-activity mutant, reduced the Ca²⁺ influx in the ECs mediated by 5,6-δ-DHTL.ConclusionPON1 lactonase activity in the endothelium affects vascular dilation by regulating Ca²⁺ influx mediated by the lactone-containing EDHF 5,6-δ-DHTL.     

VLDL triglycerides inhibit HDL-associated paraoxonase 1 (PON1) activity: in vitro and in vivo studies

We analyzed, for the first time, both in vitro and in vivo, the effect of very low density lipoprotein (VLDL), or of pure triglycerides, on high-density lipoprotein (HDL)-associated paraoxonase1 (PON1) catalytic activities. Incubation of serum or HDL from healthy subjects with VLDL (0-330 μg protein/mL) significantly decreased serum PON1 lactonase or arylesterase activities by up to 11% or 24%, and HDL-associated PON1 lactonase or arylesterase activities by up to 32% or 46%, respectively, in a VLDL dose-dependent manner. VLDL (0-660μg protein/mL) also inhibited recombinant PON1 (rePON1) lactonase or arylesterase activities by up to 20% or 42%, respectively. Similar inhibitory effect was noted upon rePON1 incubation with pure triglyceride emulsion. Bezafibrate therapy to three hypertriglyceridemic patients (400 mg/day, for one month) significantly decreased serum triglyceride concentration by 67%, and increased serum HDL cholesterol levels by 48%. PON1 arylesterase or paraoxonase activities in the patients' HDL fractions after drug therapy were significantly increased by 86-88%, as compared to PON1 activities before treatment. Similarly, HDL-PON1 protein levels significantly increased after bezafibrate therapy. Finally, bezafibrate therapy improved HDL biological activity, as HDL obtained after drug therapy showed increased ability to induce cholesterol efflux from J774A.1 macrophages, by 19%, as compared to HDL derived before therapy. We thus conclude that VLDL triglycerides inhibit PON1 catalytic activities, and bezafibrate therapy significantly improved HDL-PON1 catalytic and biological activities. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

The synthesis and analysis of S-nitorsylated paraoxonase 1

Post-translational modification (PTM) of proteins plays a crucial role in health and disease by affecting numerous aspects of protein structure, function, stability and subcellular localization. Protein S-nitrosylation is one type of PTM that involves the covalent modification of cysteine sulfhydryl groups with nitric oxide (NO) and has a regulatory impact similar to phosphorylation. The enzyme paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL), and is responsible for many of HDL’s antiatherogenic properties. The enzyme contains a free thiol group at Cys-284 which can also be modified covalently. As part of our effort to study the effect of PTMs on PON1 activities and properties and its implication for cardiovascular disease, we examined PON1’s ability to undergo S-nitrosylation on its free Cys-284. Recombinant (re) PON1 was trans-S-nitrosylated by several NO donors, glutathione-NO (GSNO) was found to be the most effective. The S-nitrosylated rePON1 was analyzed by Q-TOF LC/MS and by Saville–Griess assay: the two analytical methods revealed closely similar results. rePON1 was also nitrosylated by nitrosylated human serum albumin (HSA-NO) via protein–protein trans-nitrosylation. HSA-NO transferred an NO group to rePON1 much more efficiently than GSNO with the formation of a higher than 70% rePON-NO when incubated with a 40-fold excess of a HSA-NO/HSA mixture. RePON1-NO was relatively stable: storage for 3 days at 37 °C resulted in only 25% decomposition. This is the first report of PON1’s S-nitrosylation via GSNO and HSA-NO.     

Injection of paraoxonase 1 (PON1) to mice stimulates their HDL and macrophage antiatherogenicity

We analyzed, for the first time, the effects of recombinant PON1 (rePON1) intraperitoneal injection to C??BL/6 mice on their HDL and macrophage antiatherogenic properties. Thioglycolate-treated mice were injected with either saline (Control), or rePON1 (50 μg/mouse), and 20 H post injection, their blood samples and peritoneal macrophages (MPM) were collected. A significant increase in serum and HDL-PON1 arylesterase and lactonase activities was noted. Similarly, a significant increment, by 3.8 and 2.8 fold, in MPM-PON1 arylesterase and lactonase activities, respectively, as compared to the activities in control MPM was observed. The HDL from rePON1-injected mice was resistant to oxidation by copper ions as compared to control HDL. Furthermore, enrichment of the mouse HDL with rePON1 increased its ability to induce cholesterol efflux from J774A.1 macrophage cell line, and to inhibit macrophage-mediated LDL oxidation. In MPM from rePON1-injected mice vs. control MPM, there was a significant reduction in cholesterol mass, by 42%, in association with inhibition in cellular cholesterol biosynthesis rate, by 33%, and with significant stimulation, by 65%, of human HDL-mediated cholesterol efflux from the cells. We conclude that rePON1 injection to mice improved the mice HDL and MPM antiatherogenic properties, and these effects could probably lead to attenuation of atherosclerosis development.     

Two common nonsynonymous paraoxonase 1 (<Emphasis Type="Italic">PON1</Emphasis>) gene polymorphisms and brain astrocytoma and meningioma

Background  

Human serum paraoxonase 1 (PON1) plays a major role in the metabolism of several organophosphorus compounds. The enzyme is encoded by the polymorphic gene PON1, located on chromosome 7q21.3. Aiming to identify genetic variations related to the risk of developing brain tumors, we investigated the putative association between common nonsynonymous PON1 polymorphisms and the risk of developing astrocytoma and meningioma.     

Acute Coronary Syndrome Remodels the Protein Cargo and Functions of High-Density Lipoprotein Subfractions

Objectives

This study examined alterations in the functions and proteome of high-density lipoprotein (HDL) subfractions (HDL2 and HDL3) isolated from patients with acute coronary syndrome (ACS) compared with control subjects.

Methods

We measured HDL subfraction cholesterol efflux capacity, inflammatory index (HII), paraoxonase-1 (PON1) activity, and lipid hydroperoxide (LOOH) levels in both male age-matched controls and the ACS group (n = 40/group). Additionally, proteomic analysis was used to monitor changes in the HDL subfraction proteome between controls and ACS subjects.

Results

Both HDL2 and HDL3 from ACS patients had greater HII and LOOH levels compared with controls (P<0.001); PON1 activity and cholesterol efflux capacity in both HDL2 and HDL3 from the ACS group were significantly less than those of controls (P<0.001). Using proteomic analysis, we demonstrated that, compared with the control group, nine proteins were selectively enriched in HDL3 from subjects with ACS, and ras-related protein Rab-7b was decreased in HDL3. Additionally, in the ACS subjects, 12 proteins were decreased in HDL2 and 4 proteins were increased in HDL2.

Conclusions

Functional HDL subfractions shifted to dysfunctional HDL subfractions during ACS, and the functional impairment was linked to remodeled protein cargo in HDL subfractions from ACS patients.     

Subjects with Low Plasma HDL Cholesterol Levels Are Characterized by an Inflammatory and Oxidative Phenotype

Background

Epidemiological studies have shown that low plasma levels of high-density lipoprotein (HDL) cholesterol are associated with increased risk of cardiovascular disease, but the mechanisms for the possible atheroprotective effects of HDL cholesterol have still not been fully clarified, in particular in relation to clinical studies.

Objective

To examine the inflammatory, anti-oxidative and metabolic phenotype of subjects with low plasma HDL cholesterol levels.

Methods and Results

Fifteen subjects with low HDL cholesterol levels (eleven males and four females) and 19 subjects with high HDL (three males and 16 females) were recruited. Low HDL cholesterol was defined as ≤10th age/sex specific percentile and high HDL-C was defined as ≥90 age/sex specific percentile. Inflammatory markers in circulation and PBMC gene expression of cholesterol efflux mediators were measured. Our main findings were: (i) subjects with low plasma HDL cholesterol levels were characterized by increased plasma levels of CRP, MMP-9, neopterin, CXCL16 and ICAM-1 as well as low plasma levels of adiponectin, suggesting an inflammatory phenotype; (ii) these individuals also had reduced paraoxonase (PON)1 activity in plasma and PON2 gene expression in peripheral blood mononuclear cells (PBMC) accompanied by increased plasma levels of oxidized LDL suggesting decreased anti-oxidative capacity; and (iii) PBMC from low HDL subjects also had decreased mRNA levels of ABCA1 and ABCG1, suggesting impaired reverse cholesterol transport.

Conclusion

Subjects with low plasma HDL cholesterol levels are characterized by an inflammatory and oxidative phenotype that could contribute to the increased risk of atherosclerotic disorders in these subjects with low HDL levels.     

Both Paraoxonase-1 Genotype and Activity Do Not Predict the Risk of Future Coronary Artery Disease; the EPIC-Norfolk Prospective Population Study

Background

Paraoxonase-1 (PON1) is an antioxidant enzyme, that resides on high-density lipoprotein (HDL). PON1-activity, is heavily influenced by the PON1-Q192R polymorphism. PON1 is considered to protect against atherosclerosis, but it is unclear whether this relation is independent of its carrier, HDL. In order to evaluate the atheroprotective potential of PON1, we assessed the relationships among PON1-genotype, PON1-activity and risk of future coronary artery disease (CAD), in a large prospective case-control study.

Methodology/Principal Findings

Cases (n = 1138) were apparently healthy men and women aged 45–79 years who developed fatal or nonfatal CAD during a mean follow-up of 6 years. Controls (n = 2237) were matched by age, sex and enrollment time. PON1-activity was similar in cases and controls (60.7±45.3 versus 62.6±45.8 U/L, p = 0.3) and correlated with HDL-cholesterol levels (r = 0.16, p<0.0001). The PON1-Q192R polymorphism had a profound impact on PON1-activity, but did not predict CAD risk (Odds Ratio [OR] per R allele 0.98[0.84–1.15], p = 0.8). Using conditional logistic regression, quartiles of PON1-activity showed a modest inverse relation with CAD risk (OR for the highest versus the lowest quartile 0.77[0.63–0.95], p = 0.01; p-trend = 0.06). PON1-activity adjusted for Q192R polymorphism correlated better with HDL-cholesterol (r = 0.26, p<0.0001) and more linearly predicted CAD risk (0.79[0.64–0.98], p = 0.03; p-trend = 0.008). However, these relationships were abolished after adjustment for HDL (particles-cholesterol-size) and apolipoproteinA-I (0.94[0.74–1.18], p-trend = 0.3).

Conclusions/Significance

This study, shows that PON1-activity inversely relates to CAD risk, but not independent of HDL, due to its close association with the HDL-particle. These data strongly suggest that a low PON1-activity is not a causal factor in atherogenesis.     

Structure of the yeast histone H3-ASF1 interaction: implications for chaperone mechanism, species-specific interactions, and epigenetics

Background  

The histone H3/H4 chaperone Asf1 (anti-silencing function 1) is required for the establishment and maintenance of proper chromatin structure, as well as for genome stability in eukaryotes. Asf1 participates in both DNA replication-coupled (RC) and replication-independent (RI) histone deposition reactions in vitro and interacts with complexes responsible for both pathways in vivo. Asf1 is known to directly bind histone H3, however, high-resolution structural information about the geometry of this interaction was previously unknown.     

Role of PON in Anoxia-Reoxygenation Injury: A Drosophila Melanogaster Transgenic Model

Background

Paraoxonase 1 (PON1) is a protein found associated with high density lipoprotein (HDL), thought to prevent oxidative modification of low-density lipoprotein (LDL). This enzyme has been implicated in lowering the risk of cardiovascular disease. Anoxia-reoxygenation and oxidative stress are important elements in cardiovascular and cerebrovascular disease. However, the role of PON1 in anoxia-reoxygenation or anoxic injury is unclear. We hypothesize that PON1 prevents anoxia-reoxygenation injury. We set out to determine whether PON1 expression in Drosophila melanogaster protects against anoxia-reoxygenation (A-R) induced injury.

Methods

Wild type (WT) and transgenic PON1 flies were exposed to anoxia (100% Nitrogen) for different time intervals (from 1 to 24 hours). After the anoxic period, flies were placed in room air for reoxygenation. Activity and survival of flies was then recorded.

Results

Within 5 minutes of anoxia, all flies fell into a stupor state. After reoxygenation, survivor flies resumed activity with some delay. Interestingly, transgenic flies recovered from stupor later than WT. PON1 transgenic flies had a significant survival advantage after A-R stress compared with WT. The protection conferred by PON1 expression was present regardless of the age or dietary restriction. Furthermore, PON1 expression exclusively in CNS conferred protection.

Conclusion

Our results support the hypothesis that PON1 has a protective role in anoxia-reoxygenation injury, and its expression in the CNS is sufficient and necessary to provide a 100% survival protection.     

The effects and mechanism of flavonoid–rePON1 interactions. Structure–activity relationship study

Flavonoids are plant phenolic secondary metabolites that are widely distributed in the human diet. These antioxidants have received much attention because of their neuroprotective, cardioprotective, and chemopreventive actions. While a major focus has been on the flavonoids’ antioxidant properties, there is an emerging view that many of the potential health benefits of flavonoids and their in vivo metabolites are due to modulatory actions in cells through direct interactions with proteins, and not necessarily due to their antioxidant function. This view relies on the observations that flavonoids are present in the circulation at very low concentrations, which are not sufficient to exert effective antioxidant effects. The enzyme paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL), and is responsible for many of HDLs’ antiatherogenic properties. We previously showed that the flavonoid glabridin binds to rePON1 and affects the enzyme’s 3D structure. This interaction protects the enzyme from inhibition by an atherogenic component of the human carotid plaque. Here, we broadened our study to an investigation of the structure–activity relationships (SARs) of 12 flavonoids from different subclasses with rePON1 using Trp-fluorescence quenching, modeling calculations and Cu²⁺-induced low-density lipoprotein (LDL) oxidation methods. Our findings emphasize the ‘protein-binding’ mechanism by which flavonoids exert their beneficial biological role toward rePON1. Flavonoids’ capacity to interact with the enzyme’s rePON1 hydrophobic groove mostly dictates their pro/antioxidant behavior.     

Human carotid plaque phosphatidylcholine specifically interacts with paraoxonase 1, increases its activity,and enhances its uptake by macrophage at the expense of its binding to HDL

Human carotid atherosclerotic plaque is in direct contact with circulatory blood components. Thus, plaque and blood components may affect each other. The current study presents the effects of plaque chloroform:methanol (C:M) extract on the HDL-associated enzyme paraoxnase 1 (PON1). This study is part of our investigation on the mutual effects of the interactions between atherosclerotic lesions and blood components. Recombinant PON1 (rePON1) was incubated with the human carotid plaques C:M extract and PON1 activities were analyzed. Lactonase and paraoxonase activities were elevated due to C:M treatment, by 140 and by 69%, respectively. Analytical chemistry analyses revealed specific phosphatidylcholines (PCs) as the plaque active components. Tryptophan fluorescence quenching assay, together with molecular docking, shows that PON1 activity is enhanced in correlation with the level of PC affinity to PON1. Molecular docking revealed that PCs interact specifically with H2-PON1 α-helix, which together with H1 enzyme α-helix links the protein to the HDL surface. These findings are supported by additional results from the PON1 ∆20 mutant that lack its H1-α-helix. Incubation of this mutant with the plaque C:M extract increased PON1 activity by only 20%, much less than the wild-type PON1 that elevated PON1 activity at the same concentration by as much as 95%. Furthermore, as much as the affinity of the enzyme to the PC was augmented, the ability of PON1 to bind to the HDL particle decreased. Finally, PON1 interaction with PC enhance its uptake into the macrophage cytoplasm. In conclusions, Specific lesion phosphatidylcholines (PCs) present in the human carotid plaque significantly enhance PON1 catalytic activities due to their interaction with the enzyme. Such a lesion׳s PC–PON1 interaction, in turn, competes with HDL PCs and enhances PON1 uptake by macrophage at the expense of PON1 binding to the HDL.     

Improving Reconstituted HDL Composition for Efficient Post-Ischemic Reduction of Ischemia Reperfusion Injury

Background

New evidence shows that high density lipoproteins (HDL) have protective effects beyond their role in reverse cholesterol transport. Reconstituted HDL (rHDL) offer an attractive means of clinically exploiting these novel effects including cardioprotection against ischemia reperfusion injury (IRI). However, basic rHDL composition is limited to apolipoprotein AI (apoAI) and phospholipids; addition of bioactive compound may enhance its beneficial effects.

Objective

The aim of this study was to investigate the role of rHDL in post-ischemic model, and to analyze the potential impact of sphingosine-1-phosphate (S1P) in rHDL formulations.

Methods and Results

The impact of HDL on IRI was investigated using complementary in vivo, ex vivo and in vitro IRI models. Acute post-ischemic treatment with native HDL significantly reduced infarct size and cell death in the ex vivo, isolated heart (Langendorff) model and the in vivo model (-48%, p<0.01). Treatment with rHDL of basic formulation (apoAI + phospholipids) had a non-significant impact on cell death in vitro and on the infarct size ex vivo and in vivo. In contrast, rHDL containing S1P had a highly significant, protective influence ex vivo, and in vivo (-50%, p<0.01). This impact was comparable with the effects observed with native HDL. Pro-survival signaling proteins, Akt, STAT3 and ERK1/2 were similarly activated by HDL and rHDL containing S1P both in vitro (isolated cardiomyocytes) and in vivo.

Conclusion

HDL afford protection against IRI in a clinically relevant model (post-ischemia). rHDL is significantly protective if supplemented with S1P. The protective impact of HDL appears to target directly the cardiomyocyte.     

Identification of Critical Paraoxonase 1 Residues Involved in High Density Lipoprotein Interaction

Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated protein with atherosclerosis-protective and systemic anti-oxidant functions. We recently showed that PON1, myeloperoxidase, and HDL bind to one another in vivo forming a functional ternary complex (Huang, Y., Wu, Z., Riwanto, M., Gao, S., Levison, B. S., Gu, X., Fu, X., Wagner, M. A., Besler, C., Gerstenecker, G., Zhang, R., Li, X. M., Didonato, A. J., Gogonea, V., Tang, W. H., et al. (2013) J. Clin. Invest. 123, 3815–3828). However, specific residues on PON1 involved in the HDL-PON1 interaction remain unclear. Unambiguous identification of protein residues involved in docking interactions to lipid surfaces poses considerable methodological challenges. Here we describe a new strategy that uses a novel synthetic photoactivatable and click chemistry-taggable phospholipid probe, which, when incorporated into HDL, was used to identify amino acid residues on PON1 that directly interact with the lipoprotein phospholipid surface. Several specific PON1 residues (Leu-9, Tyr-185, and Tyr-293) were identified through covalent cross-links with the lipid probes using affinity isolation coupled to liquid chromatography with on-line tandem mass spectrometry. Based upon the crystal structure for PON1, the identified residues are all localized in relatively close proximity on the surface of PON1, defining a domain that binds to the HDL lipid surface. Site-specific mutagenesis of the identified PON1 residues (Leu-9, Tyr-185, and Tyr-293), coupled with functional studies, reveals their importance in PON1 binding to HDL and both PON1 catalytic activity and stability. Specifically, the residues identified on PON1 provide important structural insights into the PON1-HDL interaction. More generally, the new photoactivatable and affinity-tagged lipid probe developed herein should prove to be a valuable tool for identifying contact sites supporting protein interactions with lipid interfaces such as found on cell membranes or lipoproteins.     

Formulation of novel lipid-coated magnetic nanoparticles as the probe for in vivo imaging

Background  

Application of superparamagnetic iron oxide nanoparticles (SPIOs) as the contrast agent has improved the quality of magnetic resonance (MR) imaging. Low efficiency of loading the commercially available iron oxide nanoparticles into cells and the cytotoxicity of previously formulated complexes limit their usage as the image probe. Here, we formulated new cationic lipid nanoparticles containing SPIOs feasible for in vivo imaging.     

Arylesterase activity of paraoxonase 1 (PON1) on HDL3 and HDL2: Relationship with Q192R,C-108T,and L55M polymorphisms

BackgroundControversy exists regarding the role of the subfractions of high-density lipoproteins (HDL₂ and HDL₃) in cardiovascular disease. The functionality of these particles, and their protective role, is due in part to the paraoxonase 1 (PON1) presence in them. The polymorphisms rs662 (Q192R, A/G), rs854560 (L55 M, T/A), and rs705379 (C-108T) of the PON1 gene have been related to enzyme activity and, with the anti-oxidative capacity of the HDL. The objective was to determine the arylesterase PON1 activity in HDL₃ and HDL₂ and its relationship with the polymorphisms mentioned, in a young population.MethodsThe polymorphisms were determined through mini-sequencing (SnaPshot). The HDL subpopulations were separated via ionic precipitation, cholesterol was measured with enzymatic methods, and PON1 activity was measured through spectrophotometry.ResultsThe results show that the PON1 polymorphisms do not influence the cholesterol in the HDL. A variation between 40.02 and 43.9 mg/dL was in all the polymorphisms without significant differences. Additionally, PON1 activity in the HDL₃ subfractions was greater (62.83 ± 20 kU/L) than with HDL₂ (35.8 ± 20.8 kU/L) in the whole population and in all the polymorphisms (p < 0.001), and it was independent of the polymorphism and differential arylesterase activity in the Q192R polymorphism (QQ > QR > RR). Thus, 115.90 ± 30.7, 88.78 ± 21.3, 65.29 ± 10.2, respectively, for total HDL, with identical behavior for HDL₃ and HDL₂.ConclusionsPON1 polymorphisms do not influence the HDL_-c, and the PON activity is greater in the HDL₃ than in the HDL₂, independent of the polymorphism, but it is necessary to delve into the functionality of these findings in different populations.     

HDL-associated paraoxonase-1 can redistribute to cell membranes and influence sensitivity to oxidative stress

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a major contribution to the antioxidant capacity of the lipoprotein. In previous studies, we proposed that HDL promoted PON1 secretion by transfer of the enzyme from its plasma membrane location to HDL transiently anchored to the hepatocyte. This study examined whether PON1 can be transferred into cell membranes and retain its enzymatic activities and functions. Using Chinese hamster ovary and human endothelial cells, we found that recombinant PON1 as well as PON1 associated with purified human HDL was freely exchanged between the external medium and the cell membranes. Transferred PON1 was located in the external face of the plasma membrane of the cells in an enzymatically active form. The transfer of PON1 led to a gain of function by the target cells, as revealed by significantly reduced susceptibility to oxidative stress and significantly increased ability to neutralize the bacterial virulence agent 3-oxo-C₁₂-homoserine lactone. The data demonstrate that PON1 is not a fixed component of HDL and suggest that the enzyme could also exert its protective functions outside the lipoprotein environment. The observations may be of relevance to tissues exposed to oxidative stress and/or bacterial infection.     

Oxidized-phospholipids in reconstituted high density lipoprotein particles affect structure and function of recombinant paraoxonase 1

Paraoxonase 1 (PON1) is an HDL-associated enzyme and exhibits anti-inflammatory, anti-diabetic, and anti-atherogenic properties. Association of PON1 to HDL particles increases the stability and activity of PON1 and is important for the normal functioning of the enzyme. HDL particles are made up of lipid and protein constituents and apolipoprotein A-I (apoA-I) is a principal protein constituent of HDL that facilitates various biological activities of HDL. In many disease conditions the oxidized phospholipid (Ox-PL) content of HDL is found to be increased and an inverse correlation between the activity of PON1 and oxidation of the HDL is observed. However, the molecular details of the inhibitory action of the Ox-PL-containing HDL on the function of PON1 are not clear yet. In this study we have assembled reconstituted HDL (rHDL) particles with and without Ox-PL and compared their effect on the structure and function of ¹³C-labeled recombinant PON1 (¹³C-rPON1) by employing attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectroscopy and enzymatic assay. Our results show that the presence of the Ox-PL in the rHDL particles alters the structure of rPON1 and decreases its lactonase activity.     

Antiepileptic drugs: Impacts on human serum paraoxonase‐1

Serum paraoxonase (PON1) is a key enzyme related to high‐density lipoprotein (HDL)‐cholesterol particle. It can prevent the oxidation of low‐density lipoprotein (LDL) and HDL. The present article focuses on the in vitro inhibition role of some antiepileptic drugs (AEDs) such as valproic acid, gabapentin, primidone, phenytoin, and levetiracetam on human paraoxonase (hPON1). Therefore, PON1 was purified from human serum with a specific activity of 3976.36 EU/mg and 13.96% yield by using simple chromatographic methods. The AEDs were tested at various concentrations, which showed reduced in vitro hPON1 activity. IC₅₀ values for gabapentin, valproic acid, primidone, phenytoin, and levetiracetam were found to be 0.35, 0.67, 0.87, 6.3, and 53.3 mM, respectively. K_i constants were 0.261 ± 0.027, 0.338 ± 0.313, 0.410 ± 0.184, 10.3 ± 0.001, and 43.01 ± 0.003 mM, respectively. Gabapentin exhibited effective inhibitory activity as compared with the other drugs. The inhibition mechanisms of all compounds were noncompetitive.     

Use of in vivo-induced antigen technology (IVIAT) for the identification of Streptococcus suis serotype 2 in vivo-induced bacterial protein antigens

Background  

Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes death and disease in both humans and swine. A better understanding of SS2-host molecular interactions is crucial for understanding SS2 pathogenesis and immunology. Conventional genetic and biochemical approaches used to study SS2 virulence factors are unable to take into account the complex and dynamic environmental stimuli associated with the infection process. In this study, in vivo-induced antigen technology (IVIAT), an immunoscreening technique, was used to identify the immunogenic bacterial proteins that are induced or upregulated in vivo during SS2 infection.