Single-nucleotide polymorphisms and activity analysis of the promoter and enhancer of the pig lactase gene

Lactose intolerance in northern Europeans is strongly associated with a single-nucleotide polymorphism (SNP) located 14 kb upstream of the human lactase gene: − 13,910*C/T. We examined whether SNPs in the 5′ flanking region of the pig lactase gene are similar to those in the human gene and whether these polymorphisms play a functional role in regulating pig lactase gene expression. The 5′ flanking region of the lactase gene from several different breeds of pigs was cloned and analyzed for gene regulatory activity of a luciferase reporter gene. One SNP was found in the enhancer region (− 797*G/A) and two were found in the promoter region (− 308*G/C and − 301*A/G). The promoter C_− 308,G_− 301(Pro-CG) strongly promotes the expression of the lactase gene, but the promoter G_− 308,A_− 301(Pro-GA) does not. The enhancer A_− 797(Enh-A) genotype for Pro-GA can significantly enhance promoter activity, but has an inhibitory effect on Pro-CG. The Enhancer G_− 797(Enh-G) has a significant inhibitory effect on both promoters. In conclusion, the order of effectiveness on the pig lactase gene is Enh-A + Pro-GA > Enh-A/G + Pro-CG > Enh-G + Pro-GA.     

Pituitary adenylate cyclase-activating polypeptide plays an anti-inflammatory role in endotoxin-induced airway inflammation: in vivo study with gene-deleted mice

The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in capsaicin-sensitive peptidergic sensory nerves, inflammatory and immune cells suggest its involvement in inflammation. However, data on its role in different inflammatory processes are contradictory and there is little known about its functions in the airways. Therefore, our aim was to examine intranasal endotoxin-induced subacute airway inflammation in PACAP gene-deficient (PACAP^−/−) and wild-type (PACAP^+/+) mice. Airway responsiveness to inhaled carbachol was determined in unrestrained mice with whole body plethysmography 6 h and 24 h after LPS. Myeloperoxidase (MPO) activity referring to the number of accumulated neutrophils and macrophages was measured with spectrophotometry and interleukin-1β (IL-1β) concentration with ELISA from the lung homogenates. Histological evaluation and semiquantitative scoring were also performed. Bronchial responsiveness, as well as IL-1β concentration and MPO activity markedly increased at both timepoints. Perivascular edema dominated the histological picture at 6 h, while remarkable peribronchial granulocyte accumulation, macrophage infiltration and goblet cell hyperplasia were seen at 24 h. In PACAP^−/− mice, airway hyperreactivity was significantly higher 24 h after LPS and inflammatory histopathological changes were more severe at both timepoints. MPO increase was almost double in PACAP^−/− mice compared to the wild-types at 6 h. In contrast, there was no difference between the IL-1β concentrations of the PACAP^+/+ and PACAP^−/− mice. These results provide evidence for a protective role for PACAP in endotoxin-induced airway inflammation and hyperreactivity.     

Ceruloplasmin has two nearly identical sites that bind myeloperoxidase

Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp–MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp–MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30 nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp’s crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp–MPO binding and rescues MPO activity. Collectively, our results confirm Cp–MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120 degrees, which, in turn, exerts tension on MPO and results in conformational change.     

The free amino acid tyrosine enhances the chlorinating activity of human myeloperoxidase

A key function of neutrophil myeloperoxidase (MPO) is the synthesis of hypochlorous acid (HOCl), a potent oxidizing agent that plays a cytotoxic role against invading bacteria and viruses at inflammatory sites and in phagosomes. MPO displayed a chlorinating activity preferably at acidic pH but at neutral pH MPO catalyzes mainly reactions of the peroxidase cycle. In the present work effects of tyrosine on the chlorinating activity of MPO were studied. At pH 7.4 we detected an increased HOCl production in the presence of tyrosine not only by the MPO-H₂O₂-Cl^- system but also in suspensions of zymosan-activated neutrophils. An excess of H₂O₂ is known to cause an accumulation of compound II of MPO blocking the generation of HOCl at neutral pH. As evidenced by spectral changes, tyrosine-induced activation of MPO to synthesize HOCl was due to the ability of tyrosine to reduce compound II back to the native state, thus accelerating the enzyme turnover. MPO-induced oxidation of tyrosine is relevant to what can be in vivo; we detected MPO-catalyzed formation of dityrosine in the presence of plasma under experimental conditions when tyrosine concentration was about three magnitudes of order less than the Cl⁻ concentration. At acidic pH formation of compound II was impaired in the presence of chloride and dityrosine couldn't be detected in plasma. In conclusion, the ability of tyrosine to increase the chlorinating activity of MPO at neutral pH and enhanced values of H₂O₂ may be very effective for the specific enhancement of HOCl production under acute inflammation.     

Kinetic evidence for rapid oxidation of (-)-epicatechin by human myeloperoxidase

Apocynin has been reported to require dimerization by myeloperoxidase (MPO) to inhibit leukocyte NADPH oxidase. (-)-Epicatechin, a dietary flavan-3-ol, has been identified as a ‘prodrug’ of apocynin-like metabolites that inhibit endothelial NADPH oxidase activity and elevate the cellular level of nitric oxide. Since (-)-epicatechin has tentatively been identified as substrate of MPO, we studied the one-electron oxidation of (-)-epicatechin by MPO. By using multi-mixing stopped-flow technique, we demonstrate that (-)-epicatechin is one of the most efficient electron donors for heme peroxidases investigated so far. Second order rate constants for the (-)-epicatechin-mediated conversion of MPO-compound I to compound II and compound II to resting enzyme were estimated to be 1.9 × 10⁷ and 4.5 × 10⁶ M⁻¹ s⁻¹, respectively (pH 7, 25 °C). The data indicate that (-)-epicatechin is capable of undergoing fast MPO-mediated one-electron oxidation.     

Inactivation of human myeloperoxidase by hydrogen peroxide

Human myeloperoxidase (MPO) uses hydrogen peroxide generated by the oxidative burst of neutrophils to produce an array of antimicrobial oxidants. During this process MPO is irreversibly inactivated. This study focused on the unknown role of hydrogen peroxide in this process. When treated with low concentrations of H₂O₂ in the absence of reducing substrates, there was a rapid loss of up to 35% of its peroxidase activity. Inactivation is proposed to occur via oxidation reactions of Compound I with the prosthetic group or amino acid residues. At higher concentrations hydrogen peroxide acts as a suicide substrate with a rate constant of inactivation of 3.9 × 10⁻³ s⁻¹. Treatment of MPO with high H₂O₂ concentrations resulted in complete inactivation, Compound III formation, destruction of the heme groups, release of their iron, and detachment of the small polypeptide chain of MPO. Ten of the protein’s methionine residues were oxidized and the thermal stability of the protein decreased. Inactivation by high concentrations of H₂O₂ is proposed to occur via the generation of reactive oxidants when H₂O₂ reacts with Compound III. These mechanisms of inactivation may occur inside neutrophil phagosomes when reducing substrates for MPO become limiting and could be exploited when designing pharmacological inhibitors.     

Formation of cholesterol ozonolysis products through an ozone-free mechanism mediated by the myeloperoxidase-H2O2-chloride system

The presence of the cholesterol ozonolysis products, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (atheronal-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (atheronal-B) in human atherosclerotic tissues was recently reported as evidence for the generation of ozone by activated human neutrophils. However, the mechanism for the formation of atheronals in atherosclerotic tissues is unknown. In this study, we found that atheronals were formed by the reaction of cholesterol with human myeloperoxidase (MPO) in the presence of its substrates H₂O₂ and Cl⁻. The omission of either H₂O₂ or Cl⁻ from the MPO-H₂O₂-Cl⁻ system resulted in a significant reduction in yields. The formation of atheronals by the MPO-H₂O₂-Cl⁻ system was inhibited by an inhibitor of MPO and scavengers of reactive oxygen species such as sodium azide, methionine, β-carotene, and vinylbenzoic acid. Our results suggest that MPO produces atheronals at least partly through an ozone-free mechanism, via the reaction of cholesterol with singlet oxygen generated from HOCl and H₂O₂.     

Resveratrol Confers Protection against Rotenone-Induced Neurotoxicity by Modulating Myeloperoxidase Levels in Glial Cells

Myeloperoxidase (MPO) functions as a key molecular component of the host defense system against diverse pathogens. We have previously reported that increased MPO levels and activity is a distinguishing feature of rotenone-exposed glial cells, and that either overactivation or deficiency of MPO leads to pathological conditions in the brain. Here, we provide that modulation of MPO levels in glia by resveratrol confers protective effects on rotenone-induced neurotoxicity. We show that resveratrol significantly reduced MPO levels but did not trigger abnormal nitric oxide (NO) production in microglia and astrocytes. Resveratrol-induced down-regulation of MPO, in the absence of an associated overproduction of NO, markedly attenuated rotenone-triggered inflammatory responses including phagocytic activity and reactive oxygen species production in primary microglia and astrocytes. In addition, impaired responses of primary mixed glia from Mpo ^−/− mice to rotenone were relieved by treatment with resveratrol. We further show that rotenone-induced neuronal injury, particularly dopaminergic cell death, was attenuated by resveratrol in neuron-glia co-cultures, but not in neurons cultured alone. Similar regulatory effects of resveratrol on MPO levels were observed in microglia treated with MPP⁺, another Parkinson’s disease-linked neurotoxin, supporting the beneficial effects of resveratrol on the brain. Collectively, our findings provide that resveratrol influences glial responses to rotenone by regulating both MPO and NO, and thus protects against rotenone-induced neuronal injury.     

New approaches to the measurement of the concentration and peroxidase activity of myeloperoxidase in human blood plasma

A novel method for spectrophotometrical measurement of myeloperoxidase (MPO) activity in plasma with o-dianisidine (DA) as a substrate is proposed. We have determined the optimal conditions, including the pH and hydrogen peroxide concentration, under which MPO is the main contributor to DA oxidation in plasma. Specific MPO inhibitors, salicylhydroxamic acid or 4-aminobenzoic acid hydrazide, are added to measure the activity of other heme-containing peroxidases (mainly hemoglobin and its derivatives) and subtract their contribution from the total plasma peroxidase activity. Plasma MPO concentrations are quantified by a new enzyme-linked immunosorbent assay (ELISA) developed by us and based on the use of antibodies raised in rats and rabbits. The sensitivity of this ELISA is high: 0.2–250 ng/ml. A direct and significant (P < 0.0001) correlation was observed between the MPO activities measured spectrophotometrically and the MPO level determined by ELISA in blood samples from 38 healthy donors. The proposed approaches to MPO measurement in plasma can be used to evaluate the enzyme activity and concentration, as well as the efficacy of mechanisms by which MPO is regulated under physiological conditions and against the background of various inflammatory diseases.     

(-)-Epicatechin enhances the chlorinating activity of human myeloperoxidase

The heme-containing enzyme myeloperoxidase (MPO) accumulates at inflammatory sites and is able to catalyse one- and two-electron oxidation reactions. Here it is shown that (-)-epicatechin, which is known to have numerous beneficial health effects, in low micromolar concentration enhances the degradation of monochlorodimedon (MCD) or the chlorination of taurine in a concentration-dependent bell-shaped manner whereas at higher concentrations it sufficiently suppresses the release of hypochlorous acid. Presented reaction mechanisms demonstrate the efficiency of micromolar concentrations of the flavan-3-ol in overcoming the accumulation of compound II that does not participate in the chlorination cycle. In case of MCD the mechanism is more complicated since it also acts as peroxidase substrate with very different reactivity towards compound I (3 × 10⁵ M⁻¹ s⁻¹) and compound II (8.8 M⁻¹ s⁻¹) at pH 7. By affecting the chlorinating activity of myeloperoxidase (-)-epicatechin may participate in regulation of immune responses at inflammatory sites.     

Heterotrimeric Gαi proteins are regulated by lipopolysaccharide and are anti-inflammatory in endotoxemia and polymicrobial sepsis

Previous studies have implicated a role of heterotrimeric Gα_i proteins in lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Toll-like receptor (TLR) signaling regulates Gα_i proteins, which are anti-inflammatory in endotoxemia and polymicrobial sepsis. RAW 264.7 cells were stimulated with LPS and the Gα_i-GTP protein complex was immunoprecipitated with a Gα_i protein activation assay. In subsequent in vivo studies, the Gα_i protein inhibitor pertussis toxin (PTx) or G_i protein agonist mastoparan (MP-7) were administrated prior to endotoxemia. LPS-induced pro-inflammatory cytokines and mortality were determined. To examine the role of Gα_i2 in sepsis, Gα_i2 (−/−) and wildtype (WT) mice were subjected to cecal ligation and puncture (CLP) and monitored every 24 h for 120 h. Other mice were sacrificed 24 h after CLP. Peritoneal fluid, blood, and tissue samples were collected. Plasma pro-inflammatory cytokine production, bacterial load in peritoneal fluid, blood and lung tissue, myeloperoxidase (MPO) activity in lung and liver and different immune cell populations in spleen were studied. We found that Gα_i proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that Gα_i proteins are modulated by TLR signaling. In following studies, PTx augmented LPS-induced plasma TNFα, IL-6, whereas MP-7 suppressed LPS-induced TNFα and decreased LPS-induced mortality. In sepsis studies, the survival rate post-CLP was significantly decreased in the Gα_i2 (−/−) mice compared to WT mice. CLP-induced plasma TNFα, IL-6, bacterial load in peritoneal fluid, blood and lung tissue and lung and liver MPO activity were significantly increased in Gα_i2 (−/−) compared to WT mice. Gα_i2 (−/−) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, Gα_i proteins are activated by LPS and negatively regulate endotoxemia and sepsis. Understanding the role of Gα_i2 protein in regulation of the inflammatory response in sepsis may provide novel targets for treatment of sepsis.     

CTCF-dependent enhancer blockers at the upstream region of the chicken alpha-globin gene domain

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5′ flanking region of the chicken α-globin domain, 11–16 kb upstream of the embryonic α-type π gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two α-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific α-globin gene domain located in a permanently open chromatin area.     

Measuring Myeloperoxidase Activity in Biological Samples

Background

Enzymatic activity measurements of the highly oxidative enzyme myeloperoxidase (MPO), which is implicated in many diseases, are widely used in the literature, but often suffer from nonspecificity and lack of uniformity. Thus, validation and standardization are needed to establish a robust method that is highly specific, sensitive, and reproducible for assaying MPO activity in biological samples.

Principal findings

We found conflicting results between in vivo molecular MR imaging of MPO, which measures extracellular activity, and commonly used in vitro MPO activity assays. Thus, we established and validated a protocol to obtain extra- and intracellular MPO from murine organs. To validate the MPO activity assays, three different classes of MPO activity assays were used in spike and recovery experiments. However, these assay methods yielded inconsistent results, likely because of interfering substances and other peroxidases present in tissue extracts. To circumvent this, we first captured MPO with an antibody. The MPO activity of the resultant samples was assessed by ADHP and validated against samples from MPO-knockout mice in murine disease models of multiple sclerosis, steatohepatitis, and myocardial infarction. We found the measurements performed using this protocol to be highly specific and reproducible, and when performed using ADHP, to be highly sensitive over a broad range. In addition, we found that intracellular MPO activity correlated well with tissue neutrophil content, and can be used as a marker to assess neutrophil infiltration in the tissue.

Conclusion

We validated a highly specific and sensitive assay protocol that should be used as the standard method for all MPO activity assays in biological samples. We also established a method to obtain extra- and intracellular MPO from murine organs. Extracellular MPO activity gives an estimate of the oxidative stress in inflammatory diseases, while intracellular MPO activity correlates well with tissue neutrophil content. A detailed step-by-step protocol is provided.     

Serum malondialdehyde levels,myeloperoxidase and catalase activities in patients with nephrotic syndrome

Abstract

Objectives

Some studies have indicated the pathophysiological importance of reactive oxygen species (ROS) in patients with nephrotic syndrome. Myeloperoxidase (MPO) is a leukocyte-derived enzyme-generating ROS that has been proposed to exert a wide array of pro-atherogenic effects throughout all stages of the atherosclerotic process. The aim of this study was to investigate the serum malondialdehyde (MDA) levels, MPO and catalase activities in patients with adult nephrotic syndrome.

Patients and Methods

Twenty-four patients with nephrotic syndrome and 24 healthy controls were enrolled. Serum MPO activity, catalase activity, and MDA levels were assessed.

Results

Serum MPO activity and MDA levels were signi?cantly higher in patients with nephrotic syndrome than controls (both, P < 0.001), while catalase activity was signi?cantly lower (P < 0.001). Serum catalase activity was found to be significantly correlated with MPO activity (r = ?0.417, P = 0.003) and MDA levels (r = ?0.532, P = 0.007). The serum MDA levels were also found to be significantly correlated with MPO activity (r = 0.419, P = 0.003).

Conclusions

We concluded that serum MPO activity and oxidative stress were increased and that serum catalase activity was decreased in patients with adult nephrotic syndrome. In addition, these results indicate that increased MPO activity is associated with an oxidant–antioxidant imbalance that may contribute to atherosclerosis in patients with adult nephrotic syndrome.     

GATA-1 modulates the chromatin structure and activity of the chicken alpha-globin 3' enhancer

Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken α-globin silencer-enhancer elements located 3′ of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken α-globin enhancer plays a central role in its capacity to differentially regulate α-globin gene expression during erythroid differentiation and development.     

Comparative studies on alveolar macrophages and polymorphonuclear leukocytes. III. Difference spectra of their cellular and subcellular fractions

The respiratory pigments of rabbit polymorphonuclear leukocytes (PMN) and alveolar macrophages (A-MO) were compared in a spectrophotometric study conducted at liquid nitrogen temperature. In the difference spectra of the KCN-treated minus the untreated states and of the reduced minus the oxidized states, the cellular and granular fractions of PMN showed sharp absorption maxima identical with the absorption bands of myeloperoxidase (MPO). Even though A-MO revealed some guaiacol-peroxidation activity, the difference spectra of cellular and subcellular A-MO fractions did not show an MPO spectrum. The enzyme catalyzing the guaiacol peroxidation by A-MO appears to be an enzyme other than the MPO found in PMN. Despite the absence of the MPO spectrum, the difference spectra of A-MO granules when measured with KCN, dithionite, and succinate showed characteristic absorption bands of mitochondrial cytochromes, in contrast to the difference spectra of PMN, which showed far smaller cytochrome absorption bands. The results obtained suggest that the phagocytic metabolism of rabbit A-MO depends on mitochondrial respiration for its energy supply.     

Influence of ceruloplasmin and lactoferrin on the chlorination activity of leukocyte myeloperoxidase assayed by chemiluminescence

We demonstrate that addition of H₂O₂ to a mixture of myeloperoxidase (MPO), chloride and luminol immediately evokes a short intense flash of chemiluminescence (CL). This flash is diminished in the absence of MPO or chloride, and in the complete system it is suppressed by an MPO inhibitor azide, hypochlorite scavengers taurine or methionine, or an MPO peroxidase-cycle substrate guaiacol. Hence, this CL is mostly due to the MPO halogenation function; a measure of this activity is provided by the integral CL. With three independent methods (CL, taurine chlorination, and peroxidase assay) it is shown that MPO activity is suppressed by ceruloplasmin (Cp). Lactoferrin has no effect either on MPO or on the MPO-Cp complex. It is also shown that peroxidase inhibition by Cp is the stronger the larger is the MPO substrate, which suggests steric hindrances to substrate binding in the MPO-Cp complex. Importantly, the conventional chlorination and peroxidase assays detect MPO inhibition by Cp only at a large excess of the latter, whereas the CL assay reveals it at stoichiometric ratios characteristic of the naturally occurring protein complexes.