Case report on two diabetic donor eyes with no retinopathy: Clinicopathological and molecular studies.

We were intrigued to analyze donor eyes of two individuals without retinopathy even after 40 years of type 2 diabetes mellitus. Targeted molecular factors associated with angiogenesis and the key antioxidant enzymes in retinal tissue were analyzed. Accordingly PEDF, Adiponectin and Paraoxonase 2 showed augmented mRNA expression in both the retina with no significant change in VEGF expression. Vitreous showed increased PEDF protein in donor 1 and Adiponectin in donor 2 with no change in VEGF protein. This study highlights the profile of specific molecular factors that contribute to the non-development of diabetic retinopathy changes in these individuals.

The epigenetic regulation of paraoxonase 1 (PON1) as an important enzyme in HDL function: The missing link between environmental and genetic regulation.

BACKGROUND: Paraoxonase 1 (PON1) is an important antiatherogenic and antioxidant enzyme in the circulation that has been associated with adverse health outcomes particularly cardiovascular disease (CVD) and other metabolic disorders. PON1 is a highly promiscuous enzyme and can hydrolyse a large variety of substrates, however, detailed structure/function studies have concluded that the natural substrates for PON1 are lipophilic lactones. The interindividual variability in PON1 activity has been mainly attributed to genetic determinants; however, it appears that the contribution of epigenetics has been ignored as a result of the lack of adequate research. CONTENT: Epigenetic processes, including the histone modifications in the PON1 gene, the methylation of CpG sites in the promoter region of the PON1 gene and the microRNA modulation of PON1 expression can be responsible for the under researched gap between the environmental and genetic regulation of PON1. Environmental factors, including diet, pollution and lifestyle-related factors widely differ between individuals and populations and can cause large differences in the distribution of PON1 and it is important to note that their effects may be exerted through the epigenetic processes. This review discusses and emphasizes the importance of the epigenetic regulation of PON1 as a less-studied subject to highlight future research landscapes. SUMMARY: Epigenetic regulation is known as an important contributor to the pathogenesis of human diseases, particularly multifactorial diseases such as CVD, which is life-threatening. Due to the importance of PON1 in the functionality of high-density lipoprotein (HDL) and its association with CVD, further explorations of its epigenetic regulation using advanced methods such as Methyl-Seq may lead to the identification of new epigenetic contributors that in turn may lead to targeted therapies.

Resveratrol exhibits an effect on attenuating retina inflammatory condition and damage of diabetic retinopathy via PON1.

Diabetic retinopathy (DR), an obstacle of the visual microvascular system, is a serious complication of diabetic patients. Paraoxonase 1 (PON1) has been extensively evaluated as a genetic candidate for diabetic microvascular complications, and PON1 is associated with DR. In this study, the biological functions of PON1 and its related proteins were determined via gene ontology (GO) enrichment analysis; we demonstrated that treatment with resveratrol alleviated retinal inflammatory activities to evaluate its protective effects on streptozotocin (STZ)-induced diabetic rats and high-glucose (HG) stimulated rat retinal endothelial cells (RRECs). The GO enrichment analysis suggested that PON1 may regulate inflammatory responses and microvascular complications in DR. In an in vivo study, resveratrol significantly recovered the insulin level and PON1 expression and activity, as well as clearly reduced the retinal vascular permeability, retinal AGEs, LDL, Ox-LDL, caspase3 activity, retinal damage, IL-1ß, IL-6, TNFα, VEGF, IFNγ and MCP-1 in STZ-diabetic rats. Moreover, resveratrol reduced the caspase3 activity and Ox-LDL expression in HG stimulated RRECs. However, its protective effect was a deficiency in PON1-silenced RRECs. PON1 is a pivotal modulator in the role of resveratrol in reversing the RREC damage induced by HG. Furthermore, we found that resveratrol exhibits an effect on attenuating the retinal inflammatory condition and damage of DR via PON1. Our study suggests that resveratrol-induced PON1 in the retina may be a promising therapeutic strategy to prevent diabetes-related retinopathy.

Activity of Antioxidant Enzymes and Their Association with Lipid Profile in Mexican People without Cardiovascular Disease: An Analysis of Interactions.

Dyslipidemia and oxidative stress are both considered to be factors involved in cardiovascular disease; however, the relationship between them has been little explored. In this work, we studied the association between the lipid profile and the activity of antioxidant enzymes such as paraoxonase-1 (PON1), superoxide dismutase 1 (SOD1), ceruloplasmin, and catalase, as well as total antioxidant capacity (the ferric-reducing ability of plasma (FRAP)), in 626 volunteers without cardiovascular disease. Their lipid profile was evaluated, and they were classified as having or not having high triglycerides (↑TG), high low-density cholesterol (↑LDLC), and low high-density cholesterol (↓HDLC), resulting in eight groups: Without dyslipidemia, ↑TG, ↑LDLC, ↓HDLC, ↑TG↑LDLC, ↑TG↓HDLC, ↑LDLC↓HDLC, and ↑TG↑LDLC↓HDLC. When comparisons by group were made, no significant differences in the activity of antioxidant enzymes were obtained. However, the linear regression analysis considering the potential interactions between ↑TG, ↑LDLC, and ↓HDLC suggested a triple interaction between the three lipid profile alterations on the activity of PON1 and a double interaction between ↑TG and ↑LDLC on ferroxidase-ceruloplasmin activity. The analysis presented in this work showed an association between the lipid profile and antioxidant-enzyme activity and highlighted the importance of considering the interactions between the components of a phenomenon instead of studying them individually. Longitudinal studies are needed to elucidate the nature of these associations.

Expression and purification of biologically active recombinant human paraoxonase 1 from a Drosophila S2 stable cell line.

Many pesticides and chemical warfare nerve agents are highly toxic organophosphorus compounds (OPs), which inhibit acetylcholinesterase activity. Human paraoxonase 1 (PON1) has demonstrated significant potential for use as a catalytic bioscavenger capable of hydrolyzing a broad range of OPs. However, there are several limitations to the use of human PON1 as a catalytic bioscavenger, including the relatively difficult purification of PON1 from human plasma and its dependence on the presence of hydrophobic binding partners to maintain stability. Therefore, research efforts to efficiently produce recombinant human PON1 are necessary. In this study, we developed a Drosophila S2 stable cell line expressing recombinant human PON1. The recombinant human PON1 was fused with the human immunoglobulin Fc domain (PON1-hFc) to improve protein stability and purification efficiency. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis compared with those of the recombinant human PON1 derived from E. coli. We observed that the recombinant human PON1-hFc is functionally more stable for OP hydrolyzing activities compared to the recombinant human PON1. The catalytic efficiency of the recombinant PON1-hFc towards diisopropyl fluorophosphate (DFP, 0.26 × 106 M-1 min-1) and paraoxon hydrolysis (0.015 × 106 M-1 min-1) was 1.63- and 1.24-fold higher, respectively, than the recombinant human PON1. Thus, we report that the recombinant PON1-hFc exerts hydrolytic activity against paraoxon and DFP.

Sex and genetic differences in the effects of acute diesel exhaust exposure on inflammation and oxidative stress in mouse brain.

In addition to increased morbidity and mortality caused by respiratory and cardiovascular diseases, air pollution may also contribute to central nervous system (CNS) diseases. Traffic-related air pollution is a major contributor to global air pollution, and diesel exhaust (DE) is its most important component. DE contains more than 40 toxic air pollutants and is a major constituent of ambient particulate matter (PM), particularly of ultrafine-PM. Limited information suggests that exposure to DE may cause oxidative stress and neuroinflammation in the CNS. We hypothesized that males may be more susceptible than females to DE neurotoxicity, because of a lower level of expression of paraoxonase 2 (PON2), an intracellular anti-oxidant and anti-inflammatory enzyme. Acute exposure of C57BL/6 mice to DE (250-300µg/m3 for 6h) caused significant increases in lipid peroxidation and of pro-inflammatory cytokines (IL-1α, IL-1ß, IL-3, IL-6, TNF-α) in various brain regions (particularly olfactory bulb and hippocampus). In a number of cases the observed effects were more pronounced in male than in female mice. DE exposure also caused microglia activation, as measured by increased Iba1 (ionized calcium-binding adapter molecule 1) expression, and of TSPO (translocator protein) binding. Mice heterozygotes for the modifier subunit of glutamate cysteine ligase (the limiting enzyme in glutathione biosynthesis; Gclm+/- mice) appeared to be significantly more susceptible to DE-induced neuroinflammation than wild type mice. These findings indicate that acute exposure to DE causes neuroinflammation and oxidative stress in brain, and suggest that sex and genetic background may play important roles in modulating susceptibility to DE neurotoxicity.

High level expression of organophosphorus hydrolase in Pichia pastoris by multicopy ophcM assembly.

The residues of organophosphorus pesticides bring serious impact on the environmental safety and people's health. Biodegradation of organophosphorus pesticides is recognized as an ideal method. An organophosphorus hydrolase (OPHCM) from Pseudomonas pseudoalcaligenes was synthesized and expressed in Pichia pastoris. The yield reached approximately 470 mg/l after a 6-d induction in shake flasks. To improve the enzyme production, we describe a novel approach to express OPHCM efficiently with a biobrick assembly method in vitro. Four recombinant plasmids containing 1-4 copies of ophcM-expressing cassettes were constructed and transformed into P. pastoris. Increasing the copy number of ophcM gene enhanced the expression level of OPHCM. The maximum yield and specific activity in P. pastoris harboring two-copy tandem ophcM-expressing cassettes reached 610 mg/l after a 6-d induction in shake flasks and 7.8 g/l in high-density fermentation with specific activity of 13.7 U/mg. The optimum pH and temperature of the recombinant OPHCM activity were 11.0 and 50 °C, respectively. In addition, the enzyme activity of recombinant OPHCM enhanced 57.6% and 30.1% in the presence of 1 mM Cd(2+) and 5% glycerol, respectively. The high expression and good properties of recombinant OPHCM provide an effective solution to solve the pollution of organophosphorus pesticides in the environment. Moreover, the approach for generating multicopy gene expressing vectors here will benefit the study for enhancing the expression level of genes of interest.

A remarkable age-related increase in SIRT1 protein expression against oxidative stress in elderly: SIRT1 gene variants and longevity in human.

Aging is defined as the accumulation of progressive organ dysfunction. Controlling the rate of aging by clarifying the complex pathways has a significant clinical importance. Nowadays, sirtuins have become famous molecules for slowing aging and decreasing age-related disorders. In the present study, we analyzed the SIRT1 gene polymorphisms (rs7895833 A>G, rs7069102 C>G and rs2273773 C>T) and its relation with levels of SIRT1, eNOS, PON-1, cholesterol, TAS, TOS, and OSI to demonstrate the association between genetic variation in SIRT1 and phenotype at different ages in humans. We observed a significant increase in the SIRT1 level in older people and found a significant positive correlation between SIRT1 level and age in the overall studied population. The oldest people carrying AG genotypes for rs7895833 have the highest SIRT1 level suggesting an association between rs7895833 SNP and lifespan longevity. Older people have lower PON-1 levels than those of adults and children which may explain the high levels of SIRT1 protein as a compensatory mechanism for oxidative stress in the elderly. The eNOS protein level was significantly decreased in older people as compared to adults. There was no significant difference in the eNOS level between older people and children. The current study is the first to demonstrate age-related changes in SIRT1 levels in humans and it is important for a much better molecular understanding of the role of the longevity gene SIRT1 and its protein product in aging. It is also the first study presenting the association between SIRT1 expression in older people and rs7895833 in SIRT1 gene.

Association between paraoxonases gene expression and oxidative stress in hepatotoxicity induced by CCl4.

Objectives. The purpose of the study is to evaluate the hepatoprotective effect of rutin in carbon tetrachloride- (CCl4-) induced liver injuries in rat model. Methods. Forty male Wistar albino rats were divided into four groups. Group I was the control group and received dimethyl sulphoxide (DMSO) and olive oil. Group II received rutin. Groups III was treated with CCl4. Group IV was administered rutin after 48 h of CCl4 treatment. Liver enzymes level, lipid profile, lipid peroxidation, and hydrogen peroxide were measured. The genes expression levels were monitored by real time RT-PCR and western blot techniques. Results. CCl4 group showed significant increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), thiobarbituric acid reactive substances (TBAR), hydrogen peroxide (H2O2), and lipid profile and a significant decrease in glutathione peroxidase (GPx), glutathione S transferase (GST), catalase (CAT), paraoxonase-1 (PON-1), paraoxonase-3 (PON-3), peroxisome proliferator activated receptor delta (PPAR-δ), and ATP-binding cassette transporter 1 (ABAC1) genes expression levels. Interestingly, rutin supplementation completely reversed the biochemical and gene expression levels induced by CCl4 to control values. Conclusion. CCl4 administration causes aberration of genes expression levels in oxidative stress pathway resulting in DNA damage and hepatotoxicity. Rutin causes hepatoprotective effect through enhancing the antioxidant genes.

Human paraoxonase 1 as a pharmacologic agent: limitations and perspectives.

Human PON1 (h-PON1) is a multifaceted enzyme and can hydrolyze (and inactivate) a wide range of substrates. The enzyme shows anti-inflammatory, antioxidative, antiatherogenic, ant-diabetic, antimicrobial, and organophosphate (OP)-detoxifying properties. However, there are certain limitations regarding large-scale production and use of h-PON1 as a therapeutic candidate. These include difficulties in producing recombinant h-PON1 (rh-PON1) using microbial expression system, low hydrolytic activity of wild-type h-PON1 towards certain substrates, and low storage stability of the purified enzyme. This review summarizes the work done in our laboratory to address these limitations. Our results show that (a) optimized polynucleotide sequence encoding rh-PON1 can express the protein in an active form in E. coli and can be used to generate variant of the enzyme having enhanced hydrolytic activity, (b) in vitro refolding of rh-PON1 enzyme can dramatically increase the yield of an active enzyme, (c) common excipients can be used to stabilize purified rh-PON1 enzyme when stored under different storage conditions, and (d) variants of rh-PON1 enzyme impart significant protection against OP-poisoning in human blood (ex vivo) and mouse (in vivo) model of OP-poisoning. The rh-PON1 variants and their process of production discussed here will help to develop h-PON1 as a therapeutic candidate.

DJ-1 interacts with and regulates paraoxonase-2, an enzyme critical for neuronal survival in response to oxidative stress.

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.

Interplay between amino acid residues at positions 192 and 115 in modulating hydrolytic activities of human paraoxonase 1.

Human paraoxonase 1 (h-PON1) is a Ca(2+)-dependent serum enzyme that catalyzes the hydrolysis of different types of substrates. The crystal structure of h-PON1 is not solved yet and the molecular details of how the enzyme catalyzes different types of reactions are not clear. Literature suggests that the amino acid residues at positions 192 and 115 are important for various hydrolytic activities of h-PON1. It is proposed that catalytic residue H115 (and H134) mediates the lactonase and the arylesterase activities of the enzyme while the amino acid residue at position 192 modulates various other hydrolytic activities of the enzyme. However, the relationship between these two residues in the hydrolytic activities of h-PON1 is not studied in detail. In this study, we have expressed and purified the wild-type recombinant h-PON1 (rh-PON1(wt)) and its point mutants differing in the amino acid residues at positions 192 and/or 115 using an Escherichia coli expression system. The hydrolytic activities of the purified enzymes were compared using enzymatic assays. Our results, for the first time, show that (a) the presence of a particular amino acid residue at position 192 differentially alters the effect of the H115W substitution, and (b) H115 residue is not always needed for the lactonase and arylesterase activities of the enzyme. The results also suggest that the amino acid residues at position 192 and 115 act in conjunction in modulating the hydrolytic activities of the enzyme.

Lack of paraoxonase 1 alters phospholipid composition, but not morphology and function of the mouse retina.

PURPOSE: Biochemical and genetic analyses established a contribution of lipid metabolism to AMD pathology. Paraoxonase 1 (PON1) is an antioxidative protein involved in high density lipoprotein (HDL) function and was found to be associated with AMD. Here, we used Pon1(-/-) mice to study the influence of PON1 on retinal physiology and to reveal the potential impact of PON1 on AMD etiology. METHODS: Laser capture microdissection served to isolate single retinal layers. Retinal function was assessed by ERG. Retinal and RPE morphology were monitored by fundus imaging, fluorescein angiography, light and transmission electron microscopy, and immunofluorescence microscopy. Levels of mRNA and composition of phospholipid species were determined by real-time PCR and LC-MS, respectively. RESULTS: Adult (8 weeks old) Pon1(-/-) mice displayed normal retinal function and morphology, but their retinas contained reduced amounts of lysophosphatidylcholines (LPCs) compared to controls. Aged (12 months old) Pon1(-/-) animals did not show any morphologic or molecular signs of photoreceptor or RPE degeneration, or of accelerated aging. Photoreceptors of Pon1(-/-) and control mice were similarly susceptible to light damage. CONCLUSIONS: Results indicated that PON1 is not essential for normal development, function, ageing, and the defense against light damage of the mouse retina. Reduced levels of LPCs in eyes of Pon1(-/-) mice may reflect a decreased activity of phospholipase A2 or altered antioxidative activity in aged eyes.

Stabilization studies on bacterially produced human paraoxonase 1 for improving its shelf life.

Human paraoxonase 1 (h-PON1) is a ~40 kDa multi-tasking enzyme that plays a major role in determining individual susceptibility towards various disease conditions. It is a strong candidate for the development of therapeutic intervention for various diseases and other conditions in humans. However, purified h-PON1 is unstable and there is a need to find condition(s) that can increase the shelf life of the enzyme. In this report, we present the results of our investigation on the effect of excipients on the stability of bacterially produced human PON1 when stored under different storage conditions. Our results show that (a) glycine and serine are most effective in stabilizing the enzyme when stored in aqueous buffer at 25 °C for 30 days, and (b) trehalose, maltose, and BSA exerted maximum stabilization effect when the enzyme was stored in freeze-dried form at 25 °C for 60 days. Results of this study can be used to increase the shelf life of purified h-PON1 enzyme.

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 3days at 37°C resulted in only 25% decomposition. This is the first report of PON1's S-nitrosylation via GSNO and HSA-NO.

The rrnA promoter as a tool for the improved expression of heterologous genes in cyanobacteria.

The regulatory sequence of ribosomal RNA A (rrnA) operon from Synechococcus PCC7942 was characterized using green fluorescent protein gene (gfp) as a reporter. The PR promoter (nt. -83 to +2) including upstream promoter element and P1 promoter of rrnA exhibited GFP fluorescence intensity about 30-fold higher than full length sequence (nt. -147 to +79). The effects of PR promoter arranged in tandem with consensus-σ(70) promoter (PS) of Escherichia coli on the expression of gfp and opd gene encoding organophosphorus hydrolase (OPH) in Synechococcus were investigated. The PS-PR tandem promoter was superior to all of the other promoters; its GFP fluorescence intensity was a 1.8-fold increase when compared to PR-PR tandem promoter, a 2.5-fold, 9.5-fold and a 15-fold increase compared to PR, PS and promoter of tRNA(pro), respectively. The GFP from PS-PR tandem promoter accounted for about 12% of its total extracted proteins. OPH activity of Synechococcus harboring opd gene under the control of PS-PR tandem promoter was 738 ± 128 units/OD730. We demonstrated that the tandem promoters remarkably enhanced the GFP and OPH production which were detected on SDS-PAGE stained with Coomassie blue. The promoter system in this study could be generally applied to production of valuable organic products from cyanobacteria.

Integrated glycoproteomics demonstrates fucosylated serum paraoxonase 1 alterations in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer, and the detection of SCLCs at an early stage is necessary for successful therapy and for improving cancer survival rates. Fucosylation is one of the most common glycosylation-based modifications. Increased levels of fucosylation have been reported in a number of pathological conditions, including cancers. In this study, we aimed to identify and validate the aberrant and selective fucosylated glycoproteins in the sera of patients with SCLC. Fucosylated glycoproteins were enriched by the Aleuria aurantia lectin column after serum albumin and IgG depletion. In a narrowed down and comparative data analysis of both label-free proteomics and isobaric peptide-tagging chemistry iTRAQ approaches, the fucosylated glycoproteins were identified as up- or down-regulated in the sera of limited disease and extensive disease stage patients with SCLC. Verification was performed by multiple reaction monitoring-mass spectrometry to select reliable markers. Four fucosylated proteins, APCS, C9, SERPINA4, and PON1, were selected and subsequently validated by hybrid A. aurantia lectin ELISA (HLE) and Western blotting. Compared with Western blotting, the HLE analysis of these four proteins produced more optimal diagnostic values for SCLC. The PON1 protein levels were significantly reduced in the sera of patients with SCLC, whereas the fucosylation levels of PON1 were significantly increased. Fucosylated PON1 exhibited an area under curve of 0.91 for the extensive disease stage by HLE, whereas the PON1 protein levels produced an area under curve of 0.82 by Western blot. The glycan structural analysis of PON1 by MS/MS identified a biantennary fucosylated glycan modification consisting of a core + 2HexNAc + 1Fuc at increased levels in the sera of patients with SCLC. In addition, the PON1 levels were decreased in the sera of the Lewis lung carcinoma lung cancer mouse model that we examined. Our data suggest that fucosylated protein biomarkers, such as PON1, and their fucosylation levels and patterns can serve as diagnostic and prognostic serological markers for SCLC.

Regulatory regions of the paraoxonase 1 (PON1) gene are associated with neovascular age-related macular degeneration (AMD).

Physiological stress response and oxidative damage are factors for aging processes and, as such, are thought to contribute to neovascular age-related macular degeneration (AMD). Paraoxonase 1 (PON1) is an enzyme that plays an important role in oxidative stress and aging. We investigated association of DNA sequence variants (SNP) within the upstream regulatory region of the PON1 gene with neovascular AMD in 305 patients and 288 controls. Four of the seven tested SNPs (rs705379, rs705381, rs854573, and rs757158) were more frequently found in AMD patients compared to controls (P = 0.0099, 0.0295, 0.0121, and 0.0256, respectively), and all but one (SNP rs757158) are in linkage disequilibrium. Furthermore, haplotype TGGCCTC conferred protection (odds ratio (OR) = 0.76, (CI) = 0.60-0.97) as it was more frequently found in control individuals, while haplotype CGATGCT increased the risk (OR = 1.55, CI = 1.09-2.21) for AMD. These results were also reflected when haplotypes for the untranscribed and the 5'untranslated regions (5'UTR) were analyzed separately. To assess haplotype correlation with levels of gene expression, the three SNPs within the 5'UTR were tested in a luciferase reporter assay. In retinal pigment epithelium-derived ARPE19 cells, we were able to measure significant differences in reporter levels, while this was not observed in kidney-derived HEK293 cells. The presence of the risk allele A (SNP rs705381) caused an increase in luciferase activity of approximately twofold. Our data support the view that inflammatory reactions mediated through anti-oxidative activity may be relevant to neovascular age-related macular degeneration.

Yerba mate (Ilex paraguariensis) enhances the gene modulation and activity of paraoxonase-2: in vitro and in vivo studies.

OBJECTIVE: Paraoxonase-2 (PON-2) is an intracellular antioxidant enzyme that can be modulated by polyphenols. The aim of this study was to verify whether yerba mate (Ilex paraguariensis), a plant species rich in phenolic compounds, modulates gene expression and the activity of PON-2 in macrophages in vitro and in monocytes from peripheral blood and monocyte-derived macrophages obtained after the ingestion of green or roasted yerba mate infusions by healthy subjects. METHODS: THP-1 macrophages were incubated with increasing amounts of yerba mate extracts or chlorogenic and caffeic acids (1-10 µmol/L). The in vivo effects of yerba mate or water (control) intakes were evaluated acutely (2 h after ingestion) and in the short term (after daily ingestion for 7 d) in 20 healthy women. RESULTS: In general, there was no difference between the two kinds of yerba mate studied. Yerba mate extracts or chlorogenic acid at 1 and 3 µmol/L increased PON-2 relative gene expression in THP-1 macrophages (P < 0.05), whereas higher concentrations (5 and 10 µmol/L) increased the activity only. Caffeic acid induced PON-2 activity only. The acute ingestion of yerba mate infusions increased relative gene expression and PON-2 activity in monocytes (P < 0.05), whereas the consumption of yerba mate for 7 d increased PON-2 relative gene expression (P < 0.05) and had a tendency to increase PON-2 activity in monocytes and monocyte-derived macrophages. CONCLUSION: It is suggested that green or roasted yerba mate modulates positively the mRNA relative expression and activity of the PON-2 enzyme in monocytes and macrophages, which may prevent cellular oxidative stress.

ß-carotene reverses the IL-1ß-mediated reduction in paraoxonase-1 expression via induction of the CaMKKII pathway in human endothelial cells.

Interleukin-1 beta (IL-1ß) induces endothelial dysfunction and reduces nitric oxide (NO) production. IL-1ß also enhances adhesion molecule expression and induces arteriosclerosis. Conversely, high-density lipoprotein (HDL) induces endothelial NO synthase (eNOS), paraoxonase-1 (PON-1) activity, and maintains vascular health. Diet-derived ß-carotene prevents arteriosclerosis, but its mode of action is not understood. The purpose of this study was to examine the HDL-like mechanisms of ß-carotene in endothelial cells. We added IL-1ß and/or ß-carotene to cultured human endothelial cells and examined its effects on the regulation of HDL signal transduction pathways using RT-PCR, real-time PCR, Western blot (WB), and endothelial-U937 adhesion analysis. IL-1ß decreased the expression of Ca2+/calmodulin-dependent kinase II (CaMKII), eNOS, PON-1, phosphatidylinositol 3-kinase (PI3K), PSD-95/Dlg/ZO-1 (PZK1), and liver kinase B1 (LKB1). Conversely, it increased the expression of intercellular adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein 1 (MCP-1). In contrast, ß-carotene increased the expression of CaMKKII, PI3K, PZK1, LKB1, eNOS, PON-1, and reduced the expression of ICAM-1 and MCP-1. ß-carotene also induced phospho-AMP-activated protein kinase (p-AMPK), phospho-eNOS and PON-1 proteins. Importantly, ß-carotene upregulated the IL-1ß-mediated decrease of CaMKKII, PZK1, LKB1, eNOS and PON-1. ß-carotene inhibited IL-1ß-mediated cell adhesion of U937 to endothelial cells. The effect of ß-carotene was reversed by a CaMKK inhibitor, STO-609. These findings indicate that ß-carotene regulates the expression of PON-1, eNOS and adhesion molecules via CaMKK pathway activation. ß-carotene may contribute to the functional maintenance of vascular endothelial cells in a manner similar to HDL, protecting them against stimuli such as IL-1ß.