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The mammalian paraoxonases (PONs) have been linked to protection against oxidative stress. However, the physiological roles of members in this family (PON1, PON2, and PON3) are still being characterized. PON2 and PON3 are expressed in the aldosterone-sensitive distal nephron of the kidney and have been shown to negatively regulate expression of the epithelial sodium channel (ENaC), a trimeric ion channel that orchestrates salt and water homeostasis. To date, the nature of this phenomenon has not been explored. Therefore, to investigate the mechanism by which PON2 regulates ENaC, we expressed PON2 along with the ENaC subunits in fisher rat thyroid (FRT) cells, a system that is amenable to biochemical analyses of ENaC assembly and trafficking. We found that PON2 primarily resides in the endoplasmic reticulum (ER) in FRT cells, and its expression reduces the abundance of each ENaC subunit, reflecting enhanced subunit turnover. In contrast, no effect on the levels of mRNAs encoding the ENaC subunits was evident. Inhibition of lysosome function with chloroquine or NH4Cl did not alter the inhibitory effect of PON2 on ENaC expression. In contrast, PON2 accelerates ENaC degradation in a proteasome-dependent manner and acts before ENaC subunit ubiquitination. As a result of enhanced ENaC subunit ubiquitination and degradation, both channel surface expression and ENaC-mediated Na+ transport in FRT cells were reduced by PON2. Together, our data suggest that PON2 functions as an ER chaperone to monitor ENaC biogenesis and redirects the channel for ER-associated degradation.

Background and Aim: The type and amount of dietary protein has become a topic of renewed interest in light of their involvement in metabolic diseases, atherosclerosis and thrombosis. However, little attention has been devoted to the effect of avian proteins despite their wide human consumption. The aim was to investigate the influence of chicken and turkey as sources of protein compared with that of soybean on atherosclerosis and fatty liver disease., Methods and Results: To this purpose, male and female Apoe -deficient were fed purified Western diets differing in their protein sources for 12 weeks. After this period, blood, liver, aortic tree and heart base samples were taken for analyses of plasma lipids and atherosclerosis. Plasma triglycerides, non-esterified fatty acids, esterified cholesterol levels and radical oxygen species in lipoproteins changed depending on the diet and sex. Females consuming the turkey protein-containing diet showed decreased atherosclerotic foci, as evidenced by the en face atherosclerosis analyses. The presence of macrophages and smooth muscle cells in plaques were not modified, and no changes were observed in hepatic lipid droplets in the studied groups either. Paraoxonase activity was higher in the group consuming turkey protein without sex differences, but only in females, it was significantly associated with aortic lesion areas., Conclusions: Compared to soybean protein, the consumption of avian proteins depending on sex resulted in similar or lower atherosclerosis development and comparable hepatic steatosis.

Coronavirus disease 2019 (COVID-19) pandemic is affecting millions of patients worldwide. The consequences of initial exposure to SARS-CoV-2 go beyond pulmonary damage, with a particular impact on lipid metabolism. Decreased levels in HDL-C were reported in COVID-19 patients. Since HDL particles display antioxidant, anti-inflammatory and potential anti-infectious properties, we aimed at characterizing HDL proteome and functionality during COVID-19 relative to healthy subjects. HDLs were isolated from plasma of 8 severe COVID-19 patients sampled at admission to intensive care unit (Day 1, D1) at D3 and D7, and from 16 sex- and age-matched healthy subjects. Proteomic analysis was performed by LC-MS/MS. The relative amounts of proteins identified in HDLs were compared between COVID-19 and controls. apolipoprotein A-I and paraoxonase 1 were confirmed by Western-blot analysis to be less abundant in COVID-19 versus controls, whereas serum amyloid A and alpha-1 antitrypsin were higher. HDLs from patients were less protective in endothelial cells stiumalted by TNFα (permeability, VE-cadherin disorganization and apoptosis). In these conditions, HDL inhibition of apoptosis was blunted in COVID-19 relative to controls. In conclusion, we show major changes in HDL proteome and decreased functionality in severe COVID-19 patients.

Objective: To thoroughly characterize the P ylb promoter and identify the elements that affect the promoter activity., Result: The sequences flanking the - 35 and - 10 box of the P ylb promoter were divided into six segments, and six random-scanning mutant promoter libraries fused to an enhanced green fluorescent protein EGFP were made and analyzed by flow cytometry. Our results showed that the four nucleotides flanking the - 35 box could mostly influence the promoter activity, and this influence was related to the GC content. The promoters mutated in these regions were successfully used for expressing the gene ophc2 encoding organophosphorus hydrolase (OPHC2) and the gene katA encoding catalase (KatA)., Conclusion: Our work identified and characterized the sequence signatures of the P ylb promoter that could tune the promoter strength, providing further information for the potential application of this promoter. Meanwhile, the sequence signatures have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology.

To identify potential biomarkers supporting better phenotyping and to improve understanding of the pathophysiology of dilated cardiomyopathy (DCM), this study comparatively analyzed plasma protein profiles of DCM patients and individuals with low normal and normal left ventricular ejection fraction (LVEF) by mass spectrometry. After plasma depletion using a MARS Hu-6 column, global proteome profiling was performed using a LTQ-Orbitrap Velos mass spectrometer. To compare and confirm results, two different discovery sets of samples were investigated. Differentially abundant proteins are involved in lipid metabolism, coagulation, and acute phase response. Serum paraoxonase 1 (PON1), cystatin C, lysozyme C, apolipoprotein A-II, and apolipoprotein M were validated by targeted protein analysis in a third independent patient cohort. Additionally, PON1 levels were also determined by an ELISA. These data highlight PON1 as a potential marker for differentiating DCM patients not only from patients with normal LVEF, but also from heart failure patients with preserved ejection fraction. The results highlight lipid metabolism and inflammation as the major pathways being altered in DCM patients in comparison to patients presenting with suspicious myocarditis to the hospital. SIGNIFICANCE: Several studies focused on the identification of heart failure (HF) associated protein signatures in blood plasma, but only few that are largely based on only small sample series considered specific HF pathologies. Therefore, we performed a comparative global blood plasma protein profiling of a larger sample of individuals with reduced left ventricular ejection fraction (LVEF) classified as dilated cardiomyopathy patients and individuals with normal LVEF but presenting with suspicious myocarditis. DCM patients displayed altered levels of proteins involved in lipid metabolism, coagulation, and acute phase response. The most reliable candidates, such as serum paraoxonase 1 (PON1), cystatin C, lysozyme C, apolipoprotein A-II, and apolipoprotein M were validated by targeted protein analysis in an independent patient cohort. PON1 levels were also determined by an ELISA. These data highlight PON1 as a potential marker for differentiating DCM patients not only from patients with normal LVEF, but also from heart failure patients with preserved ejection fraction., (Copyright © 2019 Elsevier B.V. All rights reserved.)

BACKGROUND Polycystic ovary syndrome (PCOS) is associated with infertility or subfertility due to impaired ovulation. Clomiphene citrate is a first-line treatment option for the induction of ovulation in women with PCOS. The study aimed to compare markers of oxidative stress or the total oxidative status (TOS), total antioxidant status (TAS), and levels of paraoxonase-1 (PON-1) before and after day 21 of the menstrual cycle in women with PCOS treated with clomiphene citrate to induce ovulation. MATERIAL AND METHODS The study included 75 women who were divided into a control group (n=25) that included healthy untreated women, untreated women with PCOS (n=24) who had spontaneous ovulation, and women with PCOS who were treated with clomiphene citrate for subfertility or infertility (n=26) (the PCOS-CC group). The study group was treated for five days with clomiphene citrate (50 mg/day). Peripheral venous blood was sampled on day 3 and day 21 of the menstrual cycle from women in all three groups, and TAS, TOS, and PON-1 levels were measured. RESULTS In all three groups, TAS and PON levels were significantly reduced and TOS values were significantly increased on day 21 of the menstrual cycle. Comparison of TAS, TOS, and PON-1 levels between the three study groups on day 3 and day 21 of the menstrual cycle showed no significant difference (p=0.600, p=0.223, p=0.956, respectively). CONCLUSIONS This study showed that spontaneous ovulation occurs in association with an oxidative state in healthy women and women with PCOS, and women with PCOS following treatment with clomiphene citrate.

Purpose: We examined the effect of intracameral administration of cefuroxime on oxidative stress and endothelial apoptosis in rat corneal tissue., Methods: In total, 30 rats were divided into 3 groups of 10 rats each (intracameral administration of cefuroxime 0.1 mg/0.01 mL (cefuroxime group); intracameral administration of balanced salt solution 0.01 mL (control group); or absence of intracameral injection (sham group). Corneal endothelial apoptosis was assessed by immunohistochemical analysis using caspase-3 and caspase-8. Total oxidant status, total antioxidant status, oxidative stress index, and paraoxonase and arylesterase levels were examined in corneal endothelial tissue and serum., Results: Paraoxonase levels in the serum were significantly different between the sham and cefuroxime groups (p=0.027). A significant difference was also observed in total oxidant status levels between the cefuroxime and balanced salt solution groups (p=0.023). In addition, there were significant differences in total antioxidant status levels in corneal tissue between the cefuroxime and sham groups (p<0.001) and between the cefuroxime and balanced salt solution groups (p<0.001). Furthermore, significant differences were also observed in oxidative stress index levels between the cefuroxime and balanced salt solution groups (p=0.001) and between the cefuroxime and sham groups (p=0.026). According to the immunohistochemical staining results, a significant association with caspase-3 activity existed between the cefuroxime and balanced salt solution groups (p=0.007), while no significant difference was found with caspase-8 activity (p=0.541). Caspase-3 activity exhibited a significant relationship between the sham and balanced salt solution groups (p=0.018), but no relationship was found with caspase-8 activity (p=0.623)., Conclusion: Immunohistochemical examination revealed that intracameral cefuroxime increased apoptosis when compared to the sham and balanced salt solution groups. Moreover, intracameral cefuroxime increased oxidative stress in the cornea and simultaneously induced apoptosis.

The objective of this study was to compare changes in serum concentrations of acute phase proteins (APPs) and paraoxanase (PON-1) in response to two treatments in dogs with leishmaniosis (CanL). For this purpose, 20 dogs with CanL were assigned to two treatment groups: antimonial plus allopurinol (Group G, n=12) and miltefosine plus allopurinol (Group M, n=8). Serum concentrations of PON-1 and APPs including C-reactive protein, haptoglobin (Hp), ferritin (Ft) and albumin were monitored over a period of 3 months after treatment. At the beginning of the study (day 0), most of the dogs had APP abnormalities. None of the variables differed significantly between groups in the first or subsequent visits. There was a significantly higher reduction in serum Ft in Group G than in Group M from day 0 to day 30 (P=0.0085), and also from day 0 to day 90 (P=0.0214). There was a higher increase in serum PON-1 in Group G than in than Group M from day 0 to day 30 (P=0.0039), and also from day 0 to day 90 (P=0.0404). This is the first report of APPs in dogs with natural clinical leishmaniosis treated with miltefosine. There was faster resolution of serum APP concentrations in dogs treated with antimonials (P<0.05)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Organophosphorus-degrading enzymes show high hydrolysis efficiency and provide an environmentally friendly solution to the pollution of organophosphorus compound. However, poor enzyme stability and tedious purification process have limited practical applications. Spore-based display system can provide many advantages, such as safety, low cost, easy preparation and high resistance to harsh conditions. Recently, we have constituted the recombinant spore displaying organophosphorus hydrolase and organophosphorus acid anhydrolase. In the spore display systems, recombinant spores could be reliably produced and normal sporulation was not affected; the activities of recombinant spores were 15.81 and 10.67 U/mg spores (dry weight) respectively; furthermore, the recombinant spores exhibited significantly enhanced resistance to various harsh conditions compared to free-form enzymes. These results indicated that the spore display could contribute to the practical application of organophosphorus-degrading enzymes and provide a promising solution to bioremediation of organophosphorus compounds., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Nitro-oxidative stress (NOS) has been implicated in the pathophysiology of psychiatric disorders. The activity of the polymorphic antioxidant enzyme paraoxonase 1 (PON1) is altered in diseases where NOS is involved. PON1 activity may be estimated using different substrates some of which are influenced by PON1 polymorphisms., Objectives: 1) to review the association between PON1 activities and psychiatric diseases using a standardized PON1 substrate terminology in order to offer a state-of-the-art review; and 2) to review the efficacy of different strategies (nutrition, drugs, lifestyle) to enhance PON1 activities., Methods: The PubMed database was searched using the terms paraoxonase 1 and psychiatric diseases. Moreover, the database was also searched for clinical trials investigating strategies to enhance PON1 activity., Results: The studies support decreased PON1 activity as determined using phenylacetate (i.e., arylesterase or AREase) as a substrate, in depression, bipolar disorder, generalized anxiety disorder (GAD) and schizophrenia, especially in antipsychotic-free patients. PON1 activity as determined with paraoxon (i.e., POase activity) yields more controversial results, which can be explained by the lack of adjustment for the Q192R polymorphism. The few clinical trials investigating the influence of nutritional, lifestyle and drugs on PON1 activities in the general population suggest that some polyphenols, oleic acid, Mediterranean diet, no smoking, being physically active and statins may be effective strategies that increase PON1 activity., Conclusion: Lowered PON1 activities appear to be a key component in the ongoing NOS processes that accompany affective disorders, GAD and schizophrenia. Treatments increasing attenuated PON1 activity could possibly be new drug targets for treating these disorders., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Konak M, Minici M, Tarakçı N, Altunhan H, Toker A, Örs R. Effects of the storage of breast milk at different temperatures on total antioxidant capacity, total oxidant status, and paraoxonase-1 level. Turk J Pediatr 2019; 61: 1-6. Breast milk is a well-balanced ideal nutritional source with high bioavailability for infants. As being a fresh, biological and dynamic product, changes in the breast milk during these storage periods have been the subject of ongoing research. This study aims to evaluate total antioxidant capacity (TAC), total oxidant status (TOS), and paraoxonase-1 (PON-1) levels of fresh and freezestored breast milk. Ten cc of breast milk was obtained from the mothers as the days between 10 and 15 in the morning within a 1-hour period. TAC, TOS, and PON-1 levels were evaluated in the fresh breast milk. Collected breast milk samples were divided into two groups for storage at -20°C or -80°C. Stored samples were tested for TAC, TOS, and PON-1 levels after 72 hours. The highest TAC level was detected in fresh breast milk (p < 0.05). The TOS levels of fresh breast milk showed a statistically significant reduction in rate after storage. The TOS levels at -20°C and -80°C were significantly lower at -80°C (p < 0.05). Our study results show that oxidant and antioxidant activities are at the maximum level in the fresh breast milk. In terms of antioxidant status the effect of freezing temperatures hasn`t been determined. We conclude that it is more convenient to store the breast milk at -80°C than to store at -20°C in terms of preserving the storage TOS level.

Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis- p -nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with α- or β-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.

Background: Paraoxonase (PON) is a family of calcium-dependent hydrolases, which is related to many diseases. Elucidation of PON physiological roles, active center and all applications in medical fields are dependent on its substrates., Objective: The reports about PON substrates scattered in a long span of period are collected to afford clue for drug design, diagnosis of PON status and other academic purposes., Method: PON substrates from 133 references are classified and compared. Structurally, PON substrates are generally classified as organic phosphorous esters, lactones and arylesters. Some phosphoramidates, organophosphorous obidoximes, aryl carboxylic acid amides and special fatty alcohol esters as PON substrates are also included., Results: The electron nature, steric hindrance and hydrophilicity of substrate substituents affecting the PON catalytic ability, binding ability and specificities are discussed. Drugs, prodrugs and naturally endogenous molecules in life processes activated or inactivate by PON are reviewed. Interestingly, some organophosphate and lactone substrates are preferably hydrolyzed by one of the PON1R192Q allozymes, and such a substrate is generally essential for differentiating the three PON1192R phenotypes by using a dual-substrate method. Intricately, some chiral substrates are hydrolyzed by PON stereoselectively., Conclusion: As more substrates are synthesized and characterized, more facts about PON structure and catalytic properties (including PON active center and catalytic mechanism) will be revealed, and therefore the use of PON as a drug target or as an accurate disease marker will be achieved., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

This work aimed to describe the activity of paraoxonase 1 (PON1) in serum, follicular fluid and seminal plasma of sheep. Average serum PON1 activity was 286.8 ± 96.2 U/ml in females and 237.6 ± 18.9 U/ml in males. There was a positive correlation between PON1 activity in serum and follicular fluid in females, being twice higher in serum than in follicular fluid (148.8 ± 15.7 U/ml). PON1 activity in males' serum was 10-fold higher than in seminal plasma (21.18 ± 14.2 U/ml), and there was no correlation between PON1 activity in both compartments. Finally, this work suggests that PON1 activity of in sheep is higher compared to other mammalian species, and there is an association between PON1 in serum and follicular fluid only., (© 2017 Blackwell Verlag GmbH.)

Background: The aim of this study was to investigate seminal oxidant-antioxidant activity in idiopathic and varicocele infertility in men., Methods: Total anti-oxidant capacity (TAC), total oxidant status (TOS), paraoxonase (PON1), aryl esterase (ARE), and total thiol levels (TTL) were measured in seminal plasma with an autoanalyzer. The TOS/TAC ratio was determined as the oxidative stress index (OSI). A histopathological evaluation of the sperm was performed in the andrology laboratory of the hospital. Number, motility, morphology, volume, pH, and leukocytes were evaluated in all samples according to World Health Organization criteria. The three study groups were as follows: G1, males with idiopathic infertility; G2, males with varicocele infertility; and G3, normal healthy males (had fathered a child in the last 2 years). Each group was composed of 36 men (age, 25 - 40 years). The Rel Assay Diagnostics kit was used to determine the levels of the parameters. The study was conducted according to the principles of the declaration of Helsinki and was approved by Sakarya University Medicine Faculty Ethic Committee (e.n: 16214662/050.01.04/07). Statistical significance was assumed if p < 0.05. All statistical evaluations were performed using SPSS (version 20.0 for Windows; SPSS, Inc., Chicago, IL, USA)., Results: No differences were detected between the mean values of antioxidant parameters among the three groups (Kruskal-Wallis test). The p-values of the test parameters (TAC, TOS, PON1, ARE, TTL, OSI) are respectively: 0.494, 0.548, 0.068, 0.151, 0.202, 0.873. The antioxidant parameters of all subjects were compared using the MannWhitney U-test in both groups as fertile (G3) and infertile (G1 + G2). The PON1 levels in infertile subjects were significantly higher than those in fertile subjects. There was a statistically significant difference (p = 0.042). The other antioxidant parameters had no statistically significant difference (p > 0.05). The ARE was not performed in group 3 (control) due to a methodological problem., Conclusions: PON1 levels in infertile subjects were significantly higher than those of fertile subjects.

Purpose: Paraoxonase-1 (PON-1) is a high-density lipoprotein-associated (HDL) enzyme, which has been shown to reduce susceptibility to low-density lipoprotein (LDL) peroxidation. Epicardial adipose tissue (EAT) is a marker of atherosclerosis. The aim of this study was to determine the relationship between PON-1 activity and EAT in hemodialysis (HD) patients., Methods: This is a cross-sectional study conducted on 72 (43 males) HD patients with end-stage renal disease. Serum levels for lipid profiles, C-reactive protein, calcium, phosphate, and parathyroid hormone were measured. PON-1 activity was also measured and compared to the rate of enzymatic hydrolysis of paraoxon to p-nitrophenol. Echocardiography was used to measure EAT thickness (EATT). The correlation between PON-1 and EATT was assessed, while independent predictors of EATT in HD patients were similarly assessed using multivariate regression analysis., Results: There was a significant low mean value of PON-1 activity in HD patients compared with the control group (82.1 ± 31.6 vs. 164.3 ± 61.5 U/l, p = 0.0001) and significant high mean value of EATT in HD patients, compared with controls (6.2 ± 1.7 vs. 3.9 ± 1.1 mm, p = 0.0001). In addition, there was a significant negative correlation between PON-1 activity and EATT (r = -0.484, p = 0.0001) and a significant positive correlation between PON-1 activity and HDL-C (r = 0.417, p = 0.0003). PON-1, total cholesterol, triglycerides, LDL, HDL, age, and body mass index were found to be independent predictors of EATT., Conclusion: Our study demonstrated that PON-1 activity was significantly lower in HD patients compared with healthy controls and that PON-1 activity was inversely correlated with EATT in this population.

Objective: To investigate the malondialdehyde (MDA) level and paraoxonase-1 (PON-1) activity in the serum and seminal plasma of infertile men with chronic viral hepatitis and their influence on the semen parameters of the patients., Methods: We collected serum and semen samples from 42 infertile men, 45 infertile males with chronic viral hepatitis, and 50 healthy fertile men as controls. We measured the MDA level in the serum and seminal plasma by spectrophotometry, detected the PON-1 activity by spectrophotometry, and determined the sperm DNA fragmentation index (DFI) by acridine orange fluorescence staining., Results: The MDA level was significantly higher but the PON-1 activity remarkably lower in the serum and seminal plasma of the infertile males with chronic viral hepatitis than in the healthy controls and infertile patients (P <0.01 or P <0.05). Total sperm motility and sperm survival rate were significantly lower while the sperm DFI markedly higher in the former than in the latter two groups (P <0.01 or P <0.05). No statistically significant difference was found among the three groups in sperm concentration (P >0.05). The WBC counts in the semen of the infertile and infertile with chronic viral hepatitis groups were significantly higher than that in the health controls (P <0.05). The MDA level and PON-1 activity in the seminal plasma were positively correlated with those in the serum in the infertile males with chronic viral hepatitis (r=0.57 or 0.48, P <0.01)., Conclusion: Virus-induced chronic active hepatitis enhances oxidative stress in the reproductive system, aggravates sperm damage, and affects sperm quality parameters.

This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON-1, extracellular) and type 2 (PON-2, intracellular) in boar semen. To evaluate PON-1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10 min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON-1 and total cholesterol levels. Seminal-plasma PON-1 concentration ranged from 0.961 to 1.670 ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal-plasma PON-1 concentration and enzymatic activity were negatively correlated (r = -0.763; P < 0.01). The activity of seminal-plasma PON-1 negatively correlated with ejaculate volume (r = -0.726, P < 0.05), but positively correlated with sperm concentration (r = 0.654, P < 0.05). Total seminal-plasma cholesterol concentration positively correlated with PON-1 activity (r = 0.773; P < 0.01), but negatively correlated with PON-1 concentration (r = -0.709; P < 0.05). The presence of intracellular PON-2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON-2 localised to the post-acrosomal area of the sperm head and principal piece of the tail in membrane-intact spermatozoa. In summary, PON is present in boar semen, with PON-1 at low levels in seminal plasma and PON-2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal-plasma parameters., (© 2014 Wiley Periodicals, Inc.)

Objectives: To determine the serum and tissue levels of markers of impaired oxidative metabolism and correlate these levels with the histopathology and Alvarado score of acute appendicitis patients., Method: Sixty-five acute appendicitis patients (mean age, 31.4±12.06 years; male/female, 30/35) and 30 healthy control subjects were studied. The Alvarado score was recorded. Serum samples were obtained before surgery and 12 hours postoperatively to examine the total antioxidant status, total oxidant status, paraoxonase, stimulated paraoxonase, arylesterase, catalase, myeloperoxidase, ceruloplasmin, oxidative stress markers (advanced oxidized protein products and total thiol level) and ischemia-modified albumin. Surgical specimens were also evaluated., Results: The diagnoses were acute appendicitis (n = 37), perforated appendicitis (n = 8), phlegmonous appendicitis (n = 12), perforated+phlegmonous appendicitis (n = 4), or no appendicitis (n = 4). The Alvarado score of the acute appendicitis group was significantly lower than that of the perforated+phlegmonous appendicitis group (p = 0.004). The serum total antioxidant status, total thiol level, advanced oxidized protein products, total oxidant status, catalase, arylesterase, and ischemia-modified albumin levels were significantly different between the acute appendicitis and control groups. There was no correlation between the pathological extent of acute appendicitis and the tissue levels of the markers; additionally, there was no correlation between the tissue and serum levels of any of the parameters., Conclusions: The imbalance of oxidant/antioxidant systems plays a role in the pathogenesis acute appendicitis. The Alvarado score can successfully predict the presence and extent of acute appendicitis.

Citric acid is a weak organic acid found in the greatest amounts in citrus fruits. This study examined the effect of citric acid on endotoxin-induced oxidative stress of the brain and liver. Mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 200 μg/kg). Citric acid was given orally at 1, 2, or 4 g/kg at time of endotoxin injection and mice were euthanized 4 h later. LPS induced oxidative stress in the brain and liver tissue, resulting in marked increase in lipid peroxidation (malondialdehyde [MDA]) and nitrite, while significantly decreasing reduced glutathione, glutathione peroxidase (GPx), and paraoxonase 1 (PON1) activity. Tumor necrosis factor-alpha (TNF-α) showed a pronounced increase in brain tissue after endotoxin injection. The administration of citric acid (1-2 g/kg) attenuated LPS-induced elevations in brain MDA, nitrite, TNF-α, GPx, and PON1 activity. In the liver, nitrite was decreased by 1 g/kg citric acid. GPx activity was increased, while PON1 activity was decreased by citric acid. The LPS-induced liver injury, DNA fragmentation, serum transaminase elevations, caspase-3, and inducible nitric oxide synthase expression were attenuated by 1-2 g/kg citric acid. DNA fragmentation, however, increased after 4 g/kg citric acid. Thus in this model of systemic inflammation, citric acid (1-2 g/kg) decreased brain lipid peroxidation and inflammation, liver damage, and DNA fragmentation.

Background: Paraoxonase 1 (PON1) activity is lower in renal failure patients. We hypothesize that part of the salutatory effect of hemodialysis on PON1 activity that we have previously found is due to HDL remodeling and shift of PON1 among HDL particles., Methods: A total of 42 patients (18 females and 24 males, 63 ± 12 yr) on long-term HD, with a mean dialysis course of 6.4 yr (range 1-19 yr), were recruited. PON1 arylesterase and lactonase activities, PON1 and apolipoprotein distribution in HDL subclasses were measured by gel gradient electrophoresis and western blotting., Results: The 3 different activities of PON1 we measured were significantly lower in patients as compared to control subjects; lactonase by 11%, triesterase by 19% and arylesterase by 20%, p<0.01. HDL increased slightly by 4.6%. LDL increased by 13% and VLDL decreased by 30%. These data are compatible with enhanced lipolysis of VLDL that is transformed into LDL. VLDL-PON1 activity increases significantly by 60%. PON1 activity increases by 16% in HDL2 whereas by this approach we could determine a 10% increase in the total area under the curve corresponding to total HDL. Changes in total lactonase activity were associated with changes in VLDL-PON1 and HDL2. In parallel with PON1 activation and shifts among particles, there are significant changes in apoE which increases notably in HDL2, paralleling the changes in PON1. No significant changes in apoAI or apoA-II the main structural HDL apolipoproteins were apparent after dialysis., Conclusions: HD produces an activation of PON1 that can be predicted in part (30%) by efficiency of dialysis and in part (25%) by PON1 shifts to HDL2or VLDL (p<0.01). The removal of inhibitors and the change in the environment of PON1 in the micro-heterogeneity of HDL subclasses optimizes PON1 activity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Oxidative stress and inflammation underpin most diseases; their mechanisms are inextricably linked. Chronic inflammation is associated with oxidation, anti-inflammatory cascades are linked to decreased oxidation, increased oxidative stress triggers inflammation, and redox balance inhibits the inflammatory cellular response. Whether or not oxidative stress and inflammation represent the cause or consequence of cellular pathology, they contribute significantly to the pathogenesis of noncommunicable diseases (NCD). The incidence of obesity and other related metabolic disturbances are increasing, as are age-related diseases due to a progressively aging population. Relationships between oxidative stress, inflammatory signaling, and metabolism are, in the broad sense of energy transformation, being increasingly recognized as part of the problem in NCD. In this chapter, we summarize the pathologic consequences of an imbalance between circulating and cellular paraoxonases, the system for scavenging excessive reactive oxygen species and circulating chemokines. They act as inducers of migration and infiltration of immune cells in target tissues as well as in the pathogenesis of disease that perturbs normal metabolic function. This disruption involves pathways controlling lipid and glucose homeostasis as well as metabolically driven chronic inflammatory states that encompass several response pathways. Dysfunction in the endoplasmic reticulum and/or mitochondria represents an important feature of chronic disease linked to oxidation and inflammation seen as self-reinforcing in NCD. Therefore, correct management requires a thorough understanding of these relationships and precise interpretation of laboratory test results.

Adipocyte differentiation is a complex developmental process forming adipocytes from various precursor cells. The murine 3T3-L1 preadipocyte cell line has been most frequently used in the studies of adipocyte differentiation. Differentiation of 3T3-L1 preadipocytes includes a medium containing fetal bovine serum (FBS) with hormonal induction. In this study, we observed that differentiation medium containing adult bovine serum (ABS) instead of FBS did not support differentiation of preadipocytes. Impaired adipocyte differentiation was due to the presence of a serum protein factor in ABS that suppresses differentiation of preadipocytes. Using a proteomic analysis, alpha-2-macroglobulin and paraoxonase/arylesterase 1, which were previously shown to suppress differentiation of preadipocytes, were identified as anti-adipogenic proteins. Although their functional mechanisms have not yet been elucidated, the anti-adipogenic effects of these proteins are discussed.

Background: We developed a practical method for analysis of PON-1 enzymatic activity in HDL subclasses., Methods: The assay uses 4-12% polyacrylamide gradient gels, phenylacetate as substrate coupled with densitometric phenol detection using 4-aminoantipyrine. The measurement PON-1 activity in situ across the HDL subclasses has a strong correlation with the kinetic microplate assay for total PON-1 activity, r=0.91, p<0.001., Results: The same HDL-C level, healthy subjects (n=33) display a large difference in the ratio of PON-1 activity in small vs. large HDL. Since PON-1 activity is larger in HDL(3) we propose that this difference has a potent predictive value for clinical risk assessment and therapeutic choice. Our method also offers the advantage of assessing the distribution of up to six different HDL apolipoproteins in the same gel after transfer., Conclusions: We seek to further dissect the cause of a different distribution of PON-1 activity in HDL subclasses by employing this method that permits practical, inexpensive analysis of antioxidant function of HDL subclasses and has the potential for application in clinical chemistry and to shed some light on the importance of PON-1 distribution., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study was designed to determine the semen quality and seminal plasma biochemical parameters in White Leghorn layer breeders in the early phase of maturity. Individual ejaculates from 25 males were analysed for the determination of volume, sperm concentration, dead sperm percentage (DS) and sperm motility. Seminal plasma was separated and analysed for total oxidant status (TOS), total antioxidant capacity (TAC), paraoxonase (PON1), arylesterase, ceruloplasmin, triiodothyronine, thyroxine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Our findings showed a significant (p < 0.05) positive correlation between TOS and DS in layer breeders. The values of TAC were significantly negatively correlated with TOS and DS, while positively correlated with PON1. Conversely, AST showed significant negative correlation with motility and DS. Significantly, negative correlation was also observed between ALT and sperm concentration. In conclusion, these parameters provide some valuable basal data that may help better understanding the semen quality characteristics of White Leghorn layer breeders., (© 2011 Blackwell Verlag GmbH.)

The aim of the current study was to measure circulating metabolic and inflammation-related biochemical analytes in obese cats before and after weight loss. Thirty-seven overweight neutered cats were studied, median body weight 6.85 kg (range, 4.70 to 10.30 kg), representing a range of ages and both sexes. An individualized weight-loss program was devised for each cat and monitored until completion. Body fat mass was determined by dual-energy x-ray absorptiometry, whereas plasma concentrations of acute-phase proteins (APPs; eg, haptoglobin and serum amyloid A), hormones (eg, insulin, IGF-1, and adiponectin), and enzymes (eg, butyrylcholinesterase and paraoxonase type 1 [PON-1]) associated with inflammation and metabolic compounds (eg, glucose) were also measured. No significant changes were found in APPs after weight loss (P > 0.3), but significant increases in plasma adiponectin (P = 0.021) and IGF-1 (P = 0.036) were seen, whereas insulin (P < 0.001) and homeostasis model assessment (P = 0.005) decreased significantly. Plasma concentrations before weight loss of PON-1 (P = 0.004), adiponectin (P = 0.02), and IGF-1 (P = 0.048) were less in cats that failed to complete weight loss than cats that were successful, whereas glucose concentration was greater. Finally, multivariable linear regression analysis showed that lean tissue loss during weight management was associated with percentage weight loss (greater weight loss, greater lean tissue loss; R = 0.71, P < 0.001) and plasma adiponectin concentration before weight loss (lesser adiponectin, more lean tissue loss; R = -0.52, P = 0.023). In conclusion, various metabolic abnormalities occur in feline obesity, and these can be linked to outcomes of weight-loss programs. The changes that occur with weight loss suggest an improved metabolic status., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cardiovascular diseases are the main cause of mortality in the world, diabetics and patients with coronary artery diseases in particular. In fact, the increase of cardiovascular risk was established in many epidemiological and clinical studies. The aim of this work is to study both the lipid profile and the enzymatic activity of PON1 in diabetics and coronary patients from Morocco (Casablanca region) along with the cardiovascular risk factors in this population. Three groups of Moroccan subjects were investigated: 36 patients with coronary artery diseases, 110 diabetic patients and 100 healthy subjects (control group). Total cholesterol (TC), triglycerides (TG) and high-density lipoprotein cholesterol (c-HDL) levels were evaluated using colorimetric methods. Low-density lipoprotein cholesterol (c-LDL) was calculated according to the Friedewald's formula. Serum activity of PON1 was measured by spectrophotometry. Compared to healthy subjects, we noted a significant decrease of PON1 activity in coronary artery disease (285 U/mL ± 180 U/mL; P < 0.05) and in diabetic (167 U/mL ± 71 U/mL; P < 0.05) patients. In addition, we found that diabetic patients recorded significantly elevated LDL, TG and TC levels. In parallel, coronary artery disease patients scored TG level. The present study revealed an abnormal lipoprotein profile associated with hypertriglyceridemia, low levels of c-HDL, high levels of c-LDL and significant decrease of PON1 activity. These findings confirm the high risk of cardiovascular diseases in diabetic and coronary artery disease patients.

Objectives: To investigate the effects of dilution on high-density lipoprotein (HDL) associated paraoxonase-1 (PON1) activity., Design and Methods: HDL was prepared by ultracentrifugation, diluted with TRIS buffer pH 8.2 (0-128 fold) and assayed spectrophotometrically for PON1 activity using paraoxon and phenyl acetate., Results: HDL-PON1 activity increased progressively with dilution to over 200% of the undiluted activity by 128 fold dilution (P<0.001). Conversely HDL size decreased., Conclusion: These results indicate the urgent need to standardise conditions for measuring PON1 activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Inflammation and oxidative stress are associated with cardiovascular diseases and underlying atherosclerosis. The high density lipoprotein (HDL)-associated paraoxonase-1 (PON1) enzyme is known to be involved in the protection of serum lipids from such oxidation. Nonetheless, the disturbances of lipid profile during nematode-infected model have not yet been studied. Therefore, we aimed to explore the effects of Nippostrongylus brasiliensis infection in male Wistar rats, a model of human gastrointestinal nematode infections, on hepatic PON1 synthesis and the levels of lipid parameters., Materials and Methods: Nippostrongylus brasiliensis-infected rats fed standard and high-fat diets. Serum paraoxonase and arylesterase activities were measured on day 0, 2, 4, 7, and 14 post-infection (PI). Hepatic PONs and pro-inflammatory cytokines mRNA expression levels were evaluated in a standard diet-fed groups, and the disturbances in lipid profile as well as the levels of thiobarbituric acid reactive species (TBARS) and oxidized-LDL (Ox-LDL) were measured in high-fat diet-fed groups., Results: We found that N. brasiliensis-infected rats fed the standard diet show a significant reduction in serum PON1 activity and down-regulation of hepatic PON1 mRNA expression as well as up-regulation of hepatic IL-1β, IL-β receptor (R), TNF-α, and TNFR1 mRNA expressions in association with hepatic recruitments of Kupffer cells and neutrohils. In the presence of the high-fat diet, N. brasiliensis infection increases serum triglycerides, total cholesterol, LDL/VLDL, TBARS and Ox-LDL as well as decreases serum HDL coinciding with a maximum serum PON1 reduction., Conclusions: Nippostrongylus brasiliensis infection can induce atherogenic lipid profile and reduce serum PON1 activity., (© 2010 The Authors. European Journal of Clinical Investigation © 2010 Stichting European Society for Clinical Investigation Journal Foundation.)

Background: Skin autofluorescence (skin AF) is determined in part by accumulation of advanced glycation end products. Increased skin AF was shown previously to predict cardiovascular events independently of conventional risk factors. We determined the association of carotid artery intima media thickness (IMT), a marker of subclinical cardiovascular disease, with skin AF in subjects without diabetes or clinically manifest cardiovascular disease., Methods: In a cross-sectional observational study, IMT, skin AF, lipids and apolipoproteins, C-reactive protein (CRP), insulin resistance and paraoxonase-1 activity were measured in 59 non-smoking, non-obese subjects without diabetes mellitus and cardiovascular disease (32 women; 12 subjects with metabolic syndrome (MetS))., Results: In univariate analyses, skin AF was correlated with IMT (r = 0.265, P = 0.042), but not significantly with clinical factors, (apo)lipoproteins, CRP, insulin resistance and paraoxonase-1. In multiple linear regression analyses, IMT was determined independently by age (beta = 0.549, P < 0.001), apo B (beta = 0.236, P = 0.022) and skin AF (beta = 0.216, P = 0.035). IMT was also associated with skin AF (beta = 0.213, P = 0.046) in a model which included the presence of MetS., Conclusions: IMT is positively related to skin AF, independently of clinical factors, (apo)lipoproteins and MetS, suggesting that skin AF represents a determinant of subclinical atherosclerosis. Increased skin AF may reflect early abnormalities in processes involved in atherosclerosis development.

Background/aim: Paraoxonase 1 (PON1) is an esterase, exclusively synthesized by liver. The present study has two objectives: to determine the PON1 activity status in various disorders associated with hepatocellular damage and to correlate the changes of PON1 activity with the standard liver function and fasting lipid profile tests in these disorders., Patients and Methods: The study groups consisted of 95 patients with liver diseases including acute viral hepatitis (14), cirrhosis with portal hypertension (33), leptospirosis (14), sepsis and multi organ failure (15), left ventricular failure (9), and falciparum malaria (10); and 53 healthy controls. Serum PON1 activity was measured manually using spectrophotometer. Liver function test parameters and fasting lipid profile were performed in clinical chemistry auto analyzer (Hitachi 912)., Results: The serum PON1 activity in patients with acute viral hepatitis and sepsis decreased significantly ( P < 0.001) and moderately in falciparum malaria ( P < 0.05). However, in patients with cirrhosis, leptospirosis and left ventricular patients, its activity did not change significantly. On applying Pearson correlation, serum PON1 activity correlated positively with high-density lipoprotein-cholesterol (HDL-C) in patients with sepsis (r=0.633, P < 0.05), left ventricular failure patients (r=0.814, P < 0.05) and negatively with acute viral hepatitis patients (r=-0.528, P <0.05)., Conclusion: PON1 activity has decreased significantly in acute viral hepatitis, sepsis with multi organ failure and falciparum malaria patients. Determination of PON1 activity may serve as a useful additional test in assessing these conditions.

Enzymes are powerful biocatalysts capable of performing specific chemical transformations under mild conditions, yet as catalysts they remain subject to the laws of thermodynamics, namely, that they cannot catalyze chemical reactions beyond equilibrium. Here we report the phenomenon and application of using extracytosolic enzymes and medium conditions, such as pH, to catalyze metabolic pathways beyond their intracellular catalytic limitations. This methodology, termed "integrated bioprocessing" because it integrates intracellular and extracytosolic catalysis, was applied to a lactonization reaction in Pseudomonas putida for the economical and high-titer biosynthesis of 4-valerolactone from the inexpensive and renewable source levulinic acid. Mutant paraoxonase I (PON1) was expressed in P. putida, shown to export from the cytosol in Escherichia coli and P. putida using an N-terminal sequence, and demonstrated to catalyze the extracytosolic and pH-dependent lactonization of 4-hydroxyvalerate to 4-valerolactone. With this production system, the titer of 4-valerolactone was enhanced substantially in acidic medium using extracytosolically expressed lactonase versus an intracellular lactonase: from <0.2 g liter(-1) to 2.1 +/- 0.4 g liter(-1) at the shake flask scale. Based on these results, the production of 4-hydroxyvalerate and 4-valerolactone was examined in a 2-liter bioreactor, and titers of 27.1 g liter(-1) and 8.2 g liter(-1) for the two respective compounds were achieved. These results illustrate the utility of integrated bioprocessing as a strategy for enabling production from novel metabolic pathways and enhancing product titers.

Schistosomiasis is one of the most important parasitic diseases in Egypt and chemotherapy is considered the most effective method of control. This study was conducted to assess the effectiveness of zinc administration against Schistosoma mansoni infection by evaluating the activities of arylesterase and paraoxonase (PON1) enzymes, and the degree of liver damage. One hundred and twenty albino mice were divided into two groups; one was an infected control and the other a treated group which was further subdivided into three according to the praziquantel and zinc supplementation given. Blood and liver samples, collected 10 weeks post-infection, were subjected to parasitological, histopathological, and enzyme assays, and immunological studies. The results showed that dietary zinc supplementation led to marked reduction in worm load, and egg deposition in the liver and intestine. Histopathological examination showed marked reduction in the number and diameter of hepatic granulomas in the treated groups. The activity of arylesterase and PON1 enzymes were partially restored in infected animals receiving zinc. IL-10 mRNA expression was higher in the treated groups than in the infection control group. In conclusion, zinc administration could be a promising adjuvant therapy for S. mansoni infection.

Objective: High-density lipoprotein (HDL) antiatherogenic functions seem to be diminished during inflammatory conditions such as rheumatoid arthritis (RA). The aim of this study was to investigate the effects of tumour necrosis factor (TNF) inhibition on the antioxidative capacity of HDL in RA., Methods: Plasma lipids and paraoxonase (PON-1) activity were investigated in 45 RA patients, before and during 6 months of anti-TNF therapy. In addition, HDL was isolated and tested for its ability to inhibit copper-induced oxidation of low-density lipoprotein in vitro., Results: Plasma HDL concentrations did not change considerably after 6 months of therapy. However, stable increases of PON-1 activities were observed throughout the same period (p<0.03). The increases were more obvious when related to HDL or apolipoprotein AI concentrations. HDL total antioxidative capacity significantly improved 6 months after the initiation of anti-TNF therapy (p = 0.015). The initial improvement of PON-1 activity paralleled a decrease in the inflammatory status, whereas specific TNF blockade was likely to be responsible for the long-term effects., Conclusions: Anti-TNF therapy with infliximab has beneficial effects on lipids through changes in HDL antioxidative capacity, which might be clinically relevant and contribute to the reported protective effect of anti-TNF on cardiovascular morbidity in RA. This emphasises the importance of HDL antiatherogenic capacity for cardiovascular risk in chronic inflammatory conditions.

Research into the paraoxonase (PON) gene family has flourished over the past few years. In the 1970s and 1980s, only PON1 was known, and the investigations were conducted, essentially, by toxicologists focusing on protection against organophosphate poisoning. Since then, two new members of the family, PON2 and PON3, have been identified, both being shown to play antioxidant and anti-inflammatory roles. Evidence exists indicating that the PON family is central to a wide variety of human illnesses such as cardiovascular disease, diabetes mellitus, metabolic syndrome, obesity, non-alcoholic steatohepatitis, and several mental disorders. However, research is hampered considerably by the methods currently available to measure the activity of these enzymes. In this review, we summarize the state of knowledge on PON biochemistry and function, the influence of genetic variations, and the involvement of PON in several diseases. The problems associated with PON measurement, such as sample acquisition, lack of reference methods, and variety of substrates, will be presented. Also, we cover some of the present lines of research and propose some others for future progress in this field.

Small libraries for directed evolution can be obtained by neutral drifts that maintain the protein's original function, yielding highly polymorphic, stable and evolvable variants. We describe methods for preparing such libraries, using serum paraoxonase (PON1). An optimized GFP variant fused to PON1 reported levels of soluble, functional enzyme, enabling selection by flow cytometry and identification of enzyme variants exhibiting improved specific and total activities toward several substrates, including toxic organophosphates.

Schistosoma mansoni (S. mansoni) eggs trapped in the host liver elicit a chain of oxidative processes that may be, at least in part, responsible for the pathology and progression of fibrosis associated with schistosomal hepatitis. This study was designed to assess the protective effect of the antioxidant coenzyme-Q10 (Co-Q10) against experimental S. mansoni-induced oxidative stress in the liver, and its potential role as an adjuvant to praziquantel (PZQ) therapy. The oxidative stress and overall liver function were improved under Co-Q10 therapy as evidenced by significant reduction in oxidative stress markers and preservation of antioxidant factors. Liver fibrosis was also reduced with a positive impact on liver function. Moreover, addition of Co-Q10 to PZQ therapy caused: significant reduction of liver egg load, significant improvement of the redox status, and lastly decreased liver fibrosis.

Objective: Oxidative mechanisms are currently discussed as playing a crucial role in the genesis of inflammatory lung diseases. We aimed to evaluate the oxidant-antioxidant balance in the pathogenesis and activity of sarcoidosis and to search if the change in the level of PON can be taken as an activity marker., Methods: 26 active sarcoidosis subjects aged 41.3+/-12.9 years, 37 inactive subjects aged 39.6+/-11.7 years and 48 control subjects aged 48.9+/-2.5 years were recruited in our study. Malondialdehyde (MDA), paraoxonase1 (PON1) and oxidized low density lipoprotein (oxLDL) levels in serum were analyzed by spectrophotometric, kinetic, and ELISA methods, respectively., Results: PON1 levels were significantly lower in the active disease state than both the inactive form and control groups. MDA levels were significantly higher in active sarcoidosis than both the inactive disease and control groups, and oxLDL levels were significantly higher in the active disease group than the inactive group and control group. The level of PON1 in the inactive disease group is not significantly different from the control group while the oxLDL and MDA levels of inactive group is significantly higher than the control group (p<0.001). There was a negative correlation between the PON1 activities and MDA values in both active and inactive groups (p=0.008)., Conclusion: Oxidative stress increases in sarcoidosis might be due to both increase in lipid peroxidation and decrease in antioxidant status (PON1) and the relationship between oxidative status and the activation of the disease should be discussed by comparing the previously known activation criteria.

Objective: Four recent studies report a genetic association of the paraoxonase locus with sporadic amyotrophic lateral sclerosis (ALS). We tested the hypothesis that this association correlates with functional changes in paraoxonase 1 (PON1, MIM 168820)., Methods: Sera from 140 ALS participants; 153 age-, race-, and sex-matched controls; and 30 matched CSF samples were tested for paraoxonase, diazoxonase, and arylesterase activities. Participants with ALS were genotyped using tagging single nucleotide polymorphisms across the PON locus. Survival data and enzyme activity were correlated with genotype., Results: There was a trend toward increased paraoxonase activity in ALS compared with controls (mean control paraoxonase 701.9 +/- 469.7 U/L, mean ALS 792.5 +/- 574.1 U/L; p = 0.066 after correction) which correlated with increased frequency of the homozygous arginine (RR) variant of PON1(Q192R) (p = 0.004). There was no significant difference in PON1 protein levels, or arylesterase or diazoxonase activities. Organophosphate hydrolysis rates had no effect on ALS survival., Conclusions: Contrary to expectations, PON1 protein, paraoxonase, diazoxonase, and arylesterase activities were not reduced in amyotrophic lateral sclerosis (ALS). The increase in PON1(R192) frequency in ALS in our study supports previous genetic susceptibility studies. Our findings suggest that the influence of PON1 polymorphisms on ALS susceptibility is not due to reduced organophosphate hydrolysis.

Aim: The aim of the current report was to generate and explore new hypotheses into how, in a pathophysiological sense, atherosclerosis and periodontitis could be linked., Material and Methods: Two different biomedical informatics techniques were used: an association-based technique that generated a ranked list of genes associated with the diseases, and a natural language processing tool that extracted the relationships between the retrieved genes and lipopolysaccharide (LPS)., Results: This combined approach of association-based and natural language processing-based literature mining identified a hit list of 16 candidate genes, with PON1 as the primary candidate., Conclusions: Further study of the literature prompted the hypothesis that PON1 might connect periodontitis with atherosclerosis in both an LPS-dependent and a non-LPS-dependent manner. Furthermore, the resulting genes not only confirmed already known associations between the two diseases, but also provided genes or gene products that have only been investigated separately in the two disease states, and genes or gene products previously reported to be involved in atherosclerosis. These findings remain to be investigated through clinical studies. This example of multidisciplinary research illustrates how collaborative efforts of investigators from different fields of expertise can result in the discovery of new hypotheses.

Many effects that oestrogens and progestrogens used in oral contraceptive (OC) have on enzyme physiology are of importance on homeostasis. This study was carried out in order to determine the in vivo effect of three oral contraceptives containing ethinyl estradiol in combination with desogestrel and levonorgestrel on the paraoxonase (PON), catalase (CAT) and carbonic anhydrase (CA) activities in mice, which are model organisms for humans. Serum and liver paraoxonase activities were determined spectrophotometrically by using paraoxan as a substrate according to the methods of Gan et al. and Gil et al., respectively. Catalase and carbonic anhydrase activities were determined from erythrocytes used Aebi and Maren methods, respectively. For these studies, a group of ten mice (25+/-2 g) was selected for oral administration for 21 d of each drug (0.15 mg desogestrel+0.03 mg ethinylestradiol (A); 0.15 mg levanogestrel+0.03 mg ethinylestradiol (B) and 0.15 mg desogestrel+0.02 mg ethinylestradiol (C)). A group of ten mice was included in the study for a control group, which were not subject to drug administration. For each drug, a mean of the serum and liver paraoxonase activity and erythrocytes catalase and carbonic anhydrase activities were determined and compared to the control groups. While mouse liver PON activity showed a statistically significant decrease for all three drugs, serum PON activity increased. Erythrocytes catalase activity was significantly decreased by all contraceptives used. On the other hand, these contraceptives did not change the erythrocytes carbonic anhydrase activity.

The antiatherogenic properties of pomegranate juice (PJ) were attributed to its antioxidant potency and to its capacity to decrease macrophage oxidative stress, the hallmark of early atherogeneis. PJ polyphenols and sugar-containing polyphenolic anthocyanins were shown to confer PJ its antioxidant capacity. In the present study, we questioned whether PJ simple or complex sugars contribute to the antioxidative properties of PJ in comparison to white grape juice (WGJ) sugars. Whole PJ decreased cellular peroxide levels in J774A.1 macrophage cell-line by 23% more than PJ polyphenol fraction alone. Thus, we next determined the contribution of the PJ sugar fraction to the decrease in macrophage oxidative state. Increasing concentrations of the PJ sugar fraction resulted in a dose-dependent decrement in macrophage peroxide levels, up to 72%, compared to control cells. On the contrary, incubation of the cells with WGJ sugar fraction at the same concentrations resulted in a dose-dependent increment in peroxide levels by up to 37%. The two sugar fractions from PJ and from WGJ showed opposite effects (antioxidant for PJ and pro-oxidant for WGJ) also in mouse peritoneal macrophages (MPM) from control as well as from streptozotocin-induced diabetic Balb/C mice. PJ sugar consumption by diabetic mice for 10 days resulted in a small but significant decrement in their peritoneal macrophage total peroxide levels and an increment in cellular glutathione content, compared to MPM harvested from control diabetic mice administrated with water. In contrast, WGJ sugar consumption by diabetic mice resulted in a 22% increment in macrophage total peroxide levels and a 45% decrement in cellular glutathione content. Paraoxonase 2 activity in macrophages increases under oxidative stress conditions. Indeed, macrophage paraoxonase 2 activity was decreased after PJ sugars supplementation, but increased after WGJ sugars supplementation. We conclude that PJ sugar fraction, unlike WGJ sugar fraction, decreases macrophage oxidative state under normal and under diabetic conditions. These antioxidant/antiatherogenic effects could be due to the presence of unique complex sugars and/or phenolic sugars in PJ.

The gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed on the cell surface of Saccharomyces cerevisiae MT8-1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C-terminal region of OPH. Immunofluorescence microscopy confirmed the localization of OPH on the cell surface, and fluorescence intensity measurement of cells revealed that 1.4 x 10(4) molecules of OPH per cell were displayed. Seventy percent of OPH whole-cell activity was detected on the cell surface by protease accessibility assay. The activity of OPH was highly dependent on cell growth conditions. The maximum activity was obtained when cells were grown in a synthetic dextrose medium lacking tryptophan (SD-W) buffered by 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, 200 mM, pH 7.0) at 20 degrees C, and cobalt chloride was added at 0.1 mM. S. cerevisiae MT8-1 displaying OPH which exhibited a higher activity than Escherichia coli displaying OPH using the ice nucleation protein (INP) anchor. The use of S. cerevisiae MT8-1, which has a "generally regarded as safe (GRAS)" status, as a host for the easy expression of the OPH gene provides a new biocatalyst useful for simultaneous detoxification and detection of organophosphorus pesticides.

Organophosphorus hydrolase (OPH, also known as phosphotriesterase) is a bacterial enzyme that is capable of degrading a wide range of neurotoxic organophosphate nerve agents. Directed evolution has been used to generate one variant (22A11) with up to 25-fold improved hydrolysis of methyl parathion. Surprisingly, this variant also degraded all other substrates (paraoxon, parathion and coumaphos) tested 2- to 10-fold faster. Since only one mutation (H257Y) is directly located in the active site, site-directed mutagenesis and saturation mutagenesis were used to identify the role of the other distal substitutions (A14T, A80V, K185R, H257Y, I274N) on substrate specificity and activity. Sequential site-directed mutagenesis indicated that K185R and I274N are the most important substitutions, leading to an improvement not only in the hydrolysis of methyl parathion but also the overall hydrolysis rate of all other substrates tested. Using structural modeling, these two mutations were shown to favor the formation of hydrogen bonds with nearby residues, resulting in structural changes that could alter the overall substrate hydrolysis.

Human serum paraoxonase (PON1) has been implicated to play an important role in cardiovascular disease and diabetes. Studies in the literature indicate that PON1 has two different enzyme activities, i.e., esterase and hydroperoxide reducing activities. The objective of this study was to establish the importance of these two activities and to distinguish between them. As the addition of copper immediately inactivated the enzyme, we used auto-oxidation as the model system. Auto-oxidation of HDL resulted in more than 80% reduction of the esterolytic activity, which was protected by antioxidants, Vitamin E (50%) and PDTC (95%) and completely by 1 M glucose. In contrast, the hydroperoxide reducing activity, using unesterified hydroperoxides remained unaffected with time. We also used pNPHPODE (novel substrate) to establish that hydrolysis might be a prerequisite for the enzyme to act on the esterified hydroperoxide. The results indicated that the hydrolysis of the substrate was inhibited under oxidizing conditions with no reduction of the hydroperoxide. Overall, our findings suggest that protecting the esterolytic activity of PON1 by antioxidants might be important in preserving its action on phospholipid peroxides and a concerted reaction involving the esterolytic and hydroperoxide reducing activities might be suggested for the action of PON1.

We have developed a sensor surface for optical detection of organophosphates based on reversible inhibition of organophosphorus hydrolase (OPH) by copper complexed meso-tri(4-sulfonato phenyl) mono(4-carboxy phenyl) porphyrin (CuC1TPP). OPH immobilized onto glass microscope slides retains catalytic activity for more than 232 days. CuC1TPP is a reversible, competitive inhibitor of OPH, binding at the active site of the immobilized enzyme. The absorbance spectrum of the porphyrin-enzyme complex is measured via planar waveguide evanescent wave absorbance spectroscopy using a blue LED as a light source and an Ocean Optics USB2000 as the spectrophotometer. The characteristics of the absorbance spectrum of CuC1TPP are specific and different when the porphyrin is bound to the enzyme or is bound non-specifically to the surface of the slide. Addition of a substrate of OPH such as one of the organophosphates paraoxon, coumaphos, diazinon, or malathion displaces the porphyrin from the enzyme resulting in reduced absorbance intensity at 412 nm. Absorbance changes at 412 nm show log-linear dependence on substrate concentration. Paraoxon concentrations between 7 parts per trillion (ppt) and 14 parts per million (ppm) were investigated and a 3:1 S/N detection limit of 7 ppt was determined. Concentrations of 700 ppt to 40 ppm were investigated for diazinon, malathion, and coumaphos with detection limits of 800 ppt, 1 part per billion, and 250 ppt, respectively. This optical technique does not require the addition of reagents or solutions other than the sample and absorbance spectra can be collected in less than 6 s.

Background: Turkish adults have a high incidence of coronary artery disease (CAD)., Design: The role of lipoprotein(a) (Lp[a]) as a risk factor for CAD and its association with paraoxonase-1 (PON1) activity and lipid peroxidation indicators were evaluated in Turkish subjects living in Antalya., Methods: Paraoxonase-1 activity, Lp(a), conjugated dienes (CD) and thiobarbituric acid-reactive substances (TBARS) levels were measured in 45 healthy controls and 72 CAD patients., Results: In CAD patients, decreased PON1 activity and increased Lp(a), CD and TBARS levels were observed. Risk factors associated with CAD included TBARS, age and CD., Conclusions: Our results indicate an association between PON1 activity and Lp(a) level in CAD patients.

Background: Paraoxonase (PON1) associated with HDL can be regarded as a cardio- and vasoprotective enzyme. However, because HDL is not a homogeneous fraction, it is important to investigate in which subgroups of HDL active PON1 is located. It would also be useful to determine density profiles of the HDL apolipoproteins (Apo) E and J., Methods: We investigated the density range of HDL (rho = 1.063-1.256 kg/L) in healthy individuals, using the ultracentrifugation reference method and a newly introduced automated fractionation method. Profiles of PON1 activity and ApoA-I, ApoA-II, ApoE, ApoJ, and cholesterol concentrations were obtained by use of various density gradients., Results: PON1 activity was highest in the more dense HDL(3) and VHDL fractions where PON1 was not dissociated from the particles during centrifugation. The fraction in density range 1.175-1.185 kg/L showed not only the highest PON1 activity, but also the highest specific activity (activity per HDL particle). This fraction was the least-dense fraction containing both ApoE and ApoJ. Only the Q192R polymorphism had an effect on the distribution profile of PON1 activity. In contrast, L55M and the T(-107)C polymorphisms (determined by a novel nonradioactive method) were without effect on the density distribution of PON1 activity., Conclusion: The HDL(3) fraction, which is important in reverse cholesterol transport, also carries the highest PON1 activity.

A new fluorogenic substrate for the specific detection of organophosphatase (OPase) activity has been designed and evaluated. Our results indicate that 7-diethylphospho-6,8-difluor-4-methylumbelliferyl (DEPFMU) is hydrolyzed specifically by the OPases, mammalian serum paraoxonase and bacterial organophosphorus hydrolase (OPH). The apparent K(m) of DEPFMU is 29 microM for OPH and 91 and 200 microM for the PON1 L(55)R(192) and PON1 L(55)Q(192) isoforms of human paraoxonase, respectively. DEPFMU-based assay systems are 10-100 times more sensitive for OPH and mammalian paraoxonase detection than existing methods. Importantly, DEPFMU is poorly hydrolyzed by both serum and cellular phosphatases and, therefore, may be used as part of a robust and sensitive assay for detecting not only purified, but also highly impure, preparations of OPase such as blood samples. The superior sensitivity of DEPFMU makes it potentially useful in the search for new enzymes that may hydrolyze nerve poisons such as sarin, soman, and VX, monitoring the decontamination of organophosphates (OPs) by OPH and determining serum paraoxonase activity which appears to be important for protection against atherosclerosis, sepsis, and OP toxicity.