Relationship between the paraoxonase (PON1) L55M and Q192R polymorphisms and obesity in a Mexican population: a pilot study

The aim of this study was to examine the relationship between the L55M and Q192R paraoxonase (PON1) polymorphisms and obesity in a population of adult Mexican workers. The study population included 127 adult individuals from the Universidad Autónoma del Estado de Morelos, ranging in age from 20 to 56 years and representing both sexes. Based on body mass index, 63 individuals were classified as obese and 64 as normal weight. The PON1-Q192R and PON1-L55M polymorphisms were determined by restriction fragment length polymorphism PCR analysis. Both arylesterase and paraoxonase activity levels were similar in both groups, whereas systolic pressure, triglyceride, total cholesterol, low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, glucose, and insulin levels were higher in the obese group than in the normal-weight group (P < 0.05). An exception was the high-density lipoprotein cholesterol (HDL-C) levels, which were lower in the obese group (P < 0.05). Although the PON1-Q192R polymorphism was not associated with either group, the frequency of the homozygous L genotype for the PON1-L55M polymorphism was higher in the obese group than in the normal-weight group (P < 0.05). In conclusion, this study established a positive association between the PON1-L55M homozygous L genotype and obesity.     

Paraoxonase activity against nerve gases measured by capillary electrophoresis and characterization of human serum paraoxonase (PON1) polymorphism in the coding region (Q192R)

An analytical method for determining paraoxonase activity against sarin, soman and VX was established. We used capillary electrophoresis to measure directly the hydrolysis products: alkyl methylphosphonates. After enzymatic reaction of human serum paraoxonase (PON1) with nerve gas, substrate was removed with dichloromethane, and alkyl methylphoshphonates were quantified by capillary electrophoresis of reversed osmotic flow using cationic detergent and sorbic acid. This method was applied to the characterization of human serum PON1 polymorphism for nerve gas hydrolytic activity in the coding region (Q192R). PON1-192 and PON1-55 genotypes were determined by their gel electrophoretic fragmentation pattern with restriction enzymes after polymerase chain reaction (PCR) of blood leukocyte genomic DNA. Frequencies of genotypes among 63 members of our institutes with PON1-192 and PON1-55 were 9.5% (¹⁹²QQ), 30.1% (¹⁹²QR) and 44.4% (¹⁹²RR), and 82.5% (⁵⁵LL), 17.5% (⁵⁵LM) and 0% (⁵⁵MM), respectively. ¹⁹²Q and ¹⁹²R enzymes were purified from the respective genotype human plasma, using blue agarose affinity chromatography and diethyl amino ethane (DEAE) anion exchange chromatography. V_max and K_m were measured using Lineweaver-Burk plots for hydrolytic activities against sarin, soman and VX at pH 7.4 and 25 °C. For sarin and soman, the V_max for ¹⁹²Q PON1 were 3.5- and 1.5-fold higher than those for ¹⁹²R PON1; and k_cat/K_m for ¹⁹²Q PON1 were 1.3- and 2.8-fold higher than those for ¹⁹²R PON1. For VX, there was little difference in V_max and k_cat/K_m between ¹⁹²Q and ¹⁹²R PON1, and VX hydrolyzing activity was significantly lower than those for sarin and soman. PON1 hydrolyzed sarin and soman more effectively than paraoxon.     

Homology modeling of human serum paraoxonase1 and its molecular interaction studies with aspirin and cefazolin

Human serum paraoxonase1 (HuPON1) belongs to the family of A-esterases (EC.3.1.8.1). It is associated with HDL particle and prevents atherosclerosis by cleaving lipid hydroperoxides and other proatherogenic molecules of oxidized low density lipoproteins (LDL). Since the precise structure of HuPON1 is not yet available, the structure-function relationship between HuPON1 and activators/inhibitors is still unknown. Therefore, a theoretical model of HuPON1 was generated using homology modelling and precise molecular interactions of an activator aspirin and an inhibitor cefazolin with PON1 were studied using Autodock software. The ligand binding residues were found to be similar to the predicted active site residues. Both cefazolin and aspirin were found to dock in the vicinity of the predicted active sites of PON1; cefazolin bound at residues N166, S193 and Y71, while aspirin at residues N309, I310 and L311. Binding region in the PON1 by prediction (3D2GO server) and docking studies provide useful insight into mechanism of substrate and inhibitor binding to the enzyme active site.     

Impacts of some antibiotics on human serum paraoxonase 1 activity

Some enzymes are known to be drug target inhibitions of which can be critical for organisms. PON has a critical role to prevent atherogenesis by inhibiting lipid peroxidation. It is well known that paraoxonase 1 (PON1) plays an important function on high-density lipoprotein (HDL) structure to prevent lipid oxidation not only of low-density lipoprotein, but also of HDL itself. We investigated in vitro effects of some medical drugs on PON1 activity from human serum. K_i constants for oxytetracycline hydrochloride, netilmycin sulfate, lincomycin hydrochloride, clindamycin phosphate, and streptomycin sulfate were found as 0.2, 3.73, 18.30, 35.80, and 56.30 mM, respectively. Our results indicate that these commonly used drugs inhibit the activity of the enzyme at very low doses with different inhibition mechanisms.     

Chromatographic study of magnesium and calcium binding to immobilized human serum albumin

The use of immobilized human serum albumin (HSA) as a stationary phase in affinity chromatography has been shown to be useful in resolving optical antipodes or to investigate interactions between drugs and protein. However, to our knowledge, no inorganic ion binding has been studied on this immobilized protein type. To do this, the human serum albumin stationary phase was assimilated to a weak cation-exchanger by working with a mobile phase pH equal to 6.5. A study of the eluent ionic strength effect on ion retention was carried out by varying the buffer concentrations and the column temperatures. The thermodynamic parameters for magnesium and calcium transfer from the mobile to the stationary phase were determined from linear van’t Hoff plots. An enthalpy–entropy compensation study revealed that the type of interaction was independent of the mobile phase composition. A simple model based on the Gouy–Chapman theory was considered in order to describe the retention behavior of the test cations with the mobile phase ionic strength. From this theoretical approach, the relative charge densities of the human serum albumin surface implied in the binding process were estimated at different column temperatures.     

Breed differences in dogs sensitivity to human points: A meta-analysis

The last decade has seen a substantial increase in research on the behavioral and cognitive abilities of pet dogs, Canis familiaris. The most commonly used experimental paradigm is the object-choice task in which a dog is given a choice of two containers and guided to the reinforced object by human pointing gestures. We review here studies of this type and attempt a meta-analysis of the available data. In the meta-analysis breeds of dogs were grouped into the eight categories of the American Kennel Club, and into four clusters identified by Parker and Ostrander [Parker, H.G., Ostrander, E.A., 2005. Canine genomics and genetics: running with the pack. PLoS Genet. 1, 507-513] on the basis of a genetic analysis. No differences in performance between breeds categorized in either fashion were identified. Rather, all dog breeds appear to be similarly and highly successful in following human points to locate desired food. We suggest this result could be due to the paucity of data available in published studies, and the restricted range of breeds tested.     

Application of aqueous two-phase partition to the production of homogeneous preparations of fluorescently labelled human serum albumin

The development of a traceable molecular probe was investigated for the monitoring of partition behaviour of biomolecules in aqueous two-phase systems. This work was based upon the selective labelling of the free thiol group of human serum albumin (i.e. Cys34) with the fluorophore N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic acid. The preparation of homogeneously labelled protein required purification operations. A succession of five processes was successfully applied, comprising two size-exclusion chromatographic operations by gel filtration and a series of three appropriately manipulated aqueous two-phase systems comprising PEG 1450 and phosphate salt. Aqueous two-phase partitioning is herein presented as an alternative to difficult separation and could be applied for ‘fine’ purifications.     

The influence of ethnic differences upon human responses due to thermal environment

1. 1. The influence of ethnic differences is discussed with reference to the following issues.

2. 2. It has been found that total numbers of active sweat glands increase in tropical populations compared with people from northern latitudes.

3. 3. It has also been observed that the active sweat glands of Eskimos are fewer than those of Caucasians.

4. 4. The rate of the evaporated sweat loss was calculated by measuring body weight loss and it was found that the evaporated sweat loss of Caucasians is larger than that of Japanese in the same climate.

5. 5. Meteorological factors might have been responsible for the smaller loss in Japanese compared with that of Japanese-Americans.

6. 6. Under the same experimental conditions, it was observed that there were little or no differences between the Caucasians and Negros.

New mathematical approach for the evaluation of drug binding to human serum albumin by high-performance liquid affinity chromatography

A novel mathematical approach for investigation of drug–human serum albumin (HSA) interactions by means of high-performance liquid affinity chromatography is developed. The model is based on the assumption that two types of competitive binding sites exist on the HSA molecule. The widely used single-site binding equation is extended and a proper mathematical analysis is proposed allowing the determination of the major parameters characterizing the multisite binding (cobinding) process. The utility of the new approach is proved by competitive studies on HSA binding of two model drugs, diazepam and diclofenac.     

Antioxidant protection of human serum albumin by chitosan

Inhibition of protein oxidation by reactive oxygen species (ROS) would confer benefit to living organisms exposed to oxidative stress, because oxidized proteins are associated with many diseases and can propagate ROS-induced damage. We measured the ability of 2800Da chitosan, D-glucosamine and N-acetyl glucosamine to protect human serum albumin from oxidation by peroxyl radicals derived from 2,2'-azobis(2-amidinopropane)dihydrochloride and N-centered radicals from 1,1'-diphenyl-2-picrylhydrazyl and from 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). Comparison with the antioxidant action of vitamin C showed that, on a molar basis, chitosan was equally effective in preventing formation of carbonyl and hydroperoxide groups in human serum albumin exposed to peroxyl radicals. It was also a potent inhibitor of conformational changes in the protein, assessed by absorption spectrum and intrinsic fluorescence. D-glucosamine was much less effective and N-acetyl glucosamine was not a useful antioxidant. Protection of the albumin from peroxyl radicals was achieved by scavenging of peroxyl radical. Chitosan was also a good scavenger of N-centered radicals, with glucosamine and N-acetyl glucosamine much less effective. The results suggest that administration of low molecular weight chitosans may inhibit neutrophil activation and oxidation of serum albumin commonly observed in patients undergoing hemodialysis, resulting in reduction of oxidative stress associated with uremia.     

Role of genetic variants in ADIPOQ in human eating behavior

The beneficial effects of adiponectin and its negative correlation with BMI are well described. Adiponectin serum levels are altered in eating disorders such as anorexia nervosa, bulimia nervosa or binge eating. Here, we tested the hypothesis that (1) adiponectin serum levels correlate with human eating behavior factors and (2) that genetic variants of the ADIPOQ locus influence both serum levels and eating behavior. We analyzed 11 SNPs within ADIPOQ and in the 5′ UTR and measured serum adiponectin levels in 1,036 individuals from the German Sorbs population. The German version of the three-factor eating questionnaire (FEV) was completed by 548 Sorbs. For replication purposes, we included an independent replication cohort from Germany (N = 350). In the Sorbs, we observed positive correlations of restraint with adiponectin serum levels (P = 0.001; r = 0.148) which, however, did not withstand adjustment for covariates (P = 0.083; r = 0.077). In addition, four SNPs were nominally associated with serum adiponectin levels (all P < 0.05). Of these, two variants (rs3774261; rs1501229, all P < 0.05) were also related to disinhibition. Furthermore, three variants were exclusively associated with hunger (rs2036373, P = 0.049) and disinhibition (rs822396; rs864265, all P < 0.05). However, none of these associations withstood Bonferroni corrections for multiple testing (all P > 9.3 × 10⁻⁴). In our replication cohort, we observed similar effect directions at rs1501229 for disinhibition and hunger. A meta-analysis resulted in nominal statistical significance P = 0.036 (Z score 2.086) and P = 0.017 (Z score 2.366), respectively. Given the observed relationship of the SNPs with adiponectin levels and eating behavior, our data support a potential role of adiponectin in human eating behavior. Whether the relationship with eating behavior is mediated by the effects of circulating adiponectin warrants further investigations.     

Polymer–protein interactions in aqueous two phase systems: fluorescent studies of the partition behaviour of human serum albumin

This study describes the partitioning of fluorescent macromolecules in aqueous two-phase systems (ATPS) comprising phosphate salt and poly(ethylene glycol) of three different molecular masses (i.e. 1000, 1450 and 2000 Da). The impact of system assembly was studied with fluorescent macromolecules introduced in contact with either (i) first salt, then polymer or (ii) first polymer, then salt, or (iii) with both salt and polymer simultaneously. Native human serum albumin (HSA) and derivatives labelled with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic acid (1,5-IAEDANS) were partitioned using selected ATPS. Partitioning behaviour was characterised by molecular rotational studies of recovered proteins based upon changes of depolarisation. Measurements were undertaken by steady-state fluorescence or time-decay fluorescence using a single-photon counting system. In addition, circular dichroism was used as a tool for the study of macromolecular secondary structure. Two discrete categories of stable molecular structure have been identified that exist irrespective of the phase environment. The findings form the basis for a discussion of polymer–protein interactions and the molecular micro-environment of proteins in ATPS.     

Studies of phenytoin binding to human serum albumin by high-performance affinity chromatography

High-performance affinity chromatography was used to study the binding of phenytoin to an immobilized human serum albumin (HSA) column. This was accomplished through frontal analysis and competitive binding zonal elution experiments, the latter of which used four probe compounds for the major and minor binding sites of HSA injected into the presence of mobile phases containing known concentrations of phenytoin. It was found that phenytoin can interact with HSA at the warfarin-azapropazone, indole-benzodiazepine, tamoxifen, and digitoxin sites of this protein. The association constants for phenytoin at the indole-benzodiazepine and digitoxin sites were determined to be 1.04 (+/-0.05) x 10(4)M(-1) and 6.5 (+/-0.6) x 10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Both allosteric interactions and direct binding for phenytoin appear to take place at the warfarin-azapropazone and tamoxifen sites. This rather complex binding system indicates the importance of identifying the binding regions on HSA for specific drugs as a means for understanding the transport of such substances in blood and in characterizing their potential for drug-drug interactions.     

人类细胞色素P450与药物氧化代谢遗传多态性分子机制的研究现状

近年,在表型及基因型上均发现存在药物氧化代谢多态性,特别是对于人类细胞色素Ｐ４５０氧化酶与药氧化代谢遗传多态性的关系进行了深入的研究。有关ＣＹＰ２Ｄ６、ＣＹＰ２Ｃ１９等的突变已大多被鉴定;ＣＹＰ１Ａ１、ＣＹＰ１Ａ２等在表型存在多态性而确切的遗传机制尚不清楚。     

Heparin inhibits SMC growth in the presence of human and fetal bovine serum

Heparin (HP) has antiproliferative as well as anticoagulant properties, but not all HP preparations are equally antiproliferative. A recent report found that HP lost its total antiproliferative activity when fetal bovine serum (FBS) was replaced with human serum (HS) in culture media. This observation led to the investigation of our most potent antiproliferative Upjohn HP preparation effects on bovine pulmonary artery smooth muscle cells (PASMC) and systemic SMC growth stimulated in the presence of either FBS or HS. Bovine PASMC, human PASMC, and bovine aortic SMC were treated with 10 microg/ml Upjohn HP in either 15% FBS or 15% HS and the cell number was determined by a Coulter counter. We found that Upjohn HP significantly inhibited bovine PASMC and systemic SMC proliferation in both HS and FBS. The antiproliferative activity of the above HP preparation in HS may lead to an effective treatment of pulmonary vascular and systemic remodeling.     

Baculovirus-mediated expression and purification of human serum paraoxonase 1A

Human paraoxonase 1 (hPON1) is a lipid-associated enzyme transported on HDL. There is considerable interest in hPON1 because of its putative antioxidative/antiatherogenic properties. We have created a recombinant baculovirus (BV) to generate hPON1A in large quantities for structure-function studies and here describe the method for production and isolation of the enzyme. A high level of recombinant hPON1 type A (rPON1A) was produced by Hi-5 insect cells (40 mg/l); a fraction ( approximately 10 mg/l) was secreted into the cell culture medium, but the majority ( approximately 30 mg/l) remained associated with the host insect cells. Cell-associated rPON1A was purified by detergent extraction (Tergitol NP-10) followed by three simple chromatography steps (DEAE-Sepharose, Sephacryl S-200, and concanavalin A). The purified enzyme bound to concanavalin A and was converted to a lower molecular mass by endoglycosidase H digestion, suggesting that rPON1A contained high-mannose N-glycan chains. There was a significant decrease in arylesterase activity (>99%) concomitant with enzymatic deglycosylation. rPON1A was dependent on Ca(2+) for arylesterase activity, exhibiting kinetic parameters similar to native hPON1A (K(m) = 3.8 +/- 2.1 vs. 3.7 +/- 2.0 mM and V(max) = 1,305 +/- 668 vs. 1,361 +/- 591 U/mg protein, rPON1A and hPON1A, respectively). Both rPON1A and hPON1A efficiently inhibited lipoxygenase-mediated peroxidation of phospholipid. In contrast to the arylesterase activity, which was sensitive to endoglycosidase H treatment, enzymatic deglycosylation did not inhibit the antioxidant activity of rPON1A. In conclusion, our BV-mediated PON1A expression system appears ideally suited for the production of relatively large quantities of rPON1A for structure-function studies.     

Measurement of homocysteine thiolactone hydrolase activity using high-performance liquid chromatography with fluorescence detection and polymorphisms of paraoxonase in normal human serum

We developed a non-radioactive and sensitive assay method for measurement of the HTL hydrolase (HTLase) activity in biological samples, using OPA as a fluorescent post-labeling agent, l-homocysteine thiolactone (L-HTL) as the substrate, and HPLC to achieve rapid and selective separation of the substrate and product. The method was applied to measure the activity of HTLase in human, rabbit, rat and mouse serum samples. In addition, the correlation between the serum HTLase activity and PON1 polymorphisms in Japanese subjects was also investigated. The serum HTLase activity in humans, as determined by measurement of the enzyme activity in 22 subjects, was found to be in the range of 0.89-2.06 nmol/min mg protein, with a mean activity of 1.44 nmol/min mg protein.     

A solid-phase adsorption method for PEGylation of human serum albumin and staphylokinase: preparation,purification and biochemical characterization

A solid-phase adsorption method was developed to circumvent the disadvantage of the conventional liquid-phase PEGylation, i.e. the heterogeneity of the PEGylated products. The model proteins, human serum albumin (HSA) and staphylokinase (SAK), were adsorbed on the ion exchange chromatography media, followed by PEGylation with succinimidyl carbonate (SC)-mPEG5K and salt elution. Since PEGylation with SC-PEG5K alters the positive charge of the proteins, Q-Sepharose Big Beads and DEAE Sepharose Fast Flow were used for adsorption of HSA and SAK, respectively. Size exclusion chromatography and SDS-PAGE studies demonstrated that solid-phase PEGylation of proteins generate monoPEGylated proteins with the yield of 35–47%. Circular dichroism and intrinsic fluorescence studies showed that solid-phase PEGylation led to little conformational change of the proteins. Solid-phase PEGylation resulted in 35% loss in the biological activity of SAK, which is lower than the liquid-phase PEGylation (70%).     

Intrinsic tryptophan fluorescence of human serum proteins and related conformational changes

The unfolding of human serum proteins (HSP) was studied by measuring the intrinsic fluorescence intensity at a wavelength of excitation corresponding to tryptophan's or typosine's fluorescence and surface hydrophobicity. The maxima emission wavelengths (_max) of human serum albumin (HSA) and human serum globulin (HSG) before beer consumption (BC) were 336.0 and 337.0 nm and after BC shifted to 335.0 and 334.0 nm, respectively. The surface hydrophobicity slightly increased after BC. In a solution of 8 M urea the _max of BSA shifted to 346.4 and that of BSG to 342.5 nm. In contrast, in the same solution but after BC the _max positions of HSA and HSG shifted to 355.9 and 357.7 nm, respectively. A decrease in fluorescence intensity, a shift in the maximum of emission, and an increase in surface hydrophobicity which reflected unfolding of proteins were observed. Here we provide evidence that the loosening of the HSP structure takes place primarily in various concentrations of urea before and after beer consumption. Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption.