S. N. Cherenkevich

Influence of neopterin on the generation of reactive oxygen species in human neutrophils.

Neopterin is synthesized by human monocyte‐derived macrophages primarily upon stimulation with the cytokine interferon‐γ. We studied the influence of neopterin on the generation of reactive oxygen species (ROS) in human peripheral blood neutrophils. Radical formation was measured using a biochemiluminometer. Neutrophils were isolated from peripheral blood of healthy donors. The generation of ROS by neutrophils suspended in Earls solution (pH=7.4) at 37°C was investigated by monitoring of chemiluminescence using luminol and lucigenin as light emitters. Neopterin induced chemiluminescence in suspensions of neutrophils in the presence of luminol, but not of lucigenin. Neopterin affected only adhesive cells. Addition of neopterin into the suspension of the cells involving D‐mannitol, L‐histidine and diazabicyclo[2.2.2]octane (DABCO) decreased luminol‐dependent chemiluminescence (LDCL) of the neutrophils. The action of superoxide dismutase (SOD) and 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO) reduced neopterin‐induced LDCL of neutrophils. Data suggest that neutrophils respond on exposure to neopterin with additional generation of singlet oxygen, hydroxyl radical and nitric oxide by nicotinamide adenine dinucleotide phosphate (NADPH)‐independent pathways.

Intracellular redox state: towards quantitative description

Redox state is a widely used term for the description of redox phenomena in biological systems. The regulating mechanisms responsible for maintaining the redox state are not yet fully known. But it was shown that changes in the redox state might lead to a cascade of intracellular events, beneficial or deleterious to the cell. There are several methods for the description of the intracellular redox state. These methods are based on using measured intracellular concentrations of reduced and oxidized glutathione in the Nernst equation. However, glutathione is not always a basic redox component in biological fluids, organelles, cells or tissues. As a result, changes in the intracellular redox state are not always accompanied by considerable changes of glutathione concentration. In this work it was proposed to use the concept of effective reduction potential for the quantitative characteristic of the intracellular redox state. The effective reduction potential was substantiated on the basis of a thermodynamic description. A new equation for the calculation of the effective reduction potential was derived. This equation summarizes the contribution of different oxidizing and reducing agents in the formation of an effective redox potential. The theoretical estimation of the effective reduction potential values for the different biological fluids and cells was carried out with the use of a method developed.

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.

Viscum album agglutinin-induced aggregation of blood cells and the lectin effects on neutrophil function.

Extracts from mistletoe enjoy a large popularity in central Europe as an unconventional treatment modality for cancer, warranting scientific efforts with defined components to delineate any potential benefit. The galactose-specific lectin from Viscum album (VAA), known to exhibit immunomodulatory and ensuing antitumoral capacities in animal model systems, was shown to aggregate human blood cells in the following order: neutrophils, mononuclear cells--thrombocytes and erythrocytes. To contribute to the analysis of lectin effects on individual aspects of the host defence system, two parameters of neutrophils were quantitatively assessed, namely the aggregating activity of VAA as a measure of strength of interaction with cell surface ligands and the effect of lectin on oxidative metabolism (H2O2 release) of these cells. It was found that whole lectin and its carbohydrate-binding B-subunit possessed the capacity to induce cell aggregation and H2O2 release, which were blocked by D-galactose and lactose. Both effects displayed similar dependence on the lectin concentration in the range 0.1-25 micrograms/ml. The toxic A-subunit displayed detectable activity only in high doses (50 micrograms/ml) while the bovine heart galaptin (14 kDa; galectin-1) failed to affect neutrophils. The role of oxidative metabolism in regulation of neutrophil aggregation induced by VAA was studied using metabolic inhibitors and controlled heating at 46 degrees C leading to inhibition of plasma membrane NADPH-oxidase system. Trifluoperazine and menadione inhibited the neutrophil aggregation in a dose-dependent manner in comparison with such inhibitors as amiloride and theophylline.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential potency of two crosslinking plant lectins to induce formation of haptenic‐sugar‐resistant aggregates of rat thymocytes by post‐binding signaling

To evaluate the significance of post‐binding events for stable aggregate formation, the aggregation/dissociation of rat thymocytes initiated by two crosslinking plant lectins, namely concanavalin A (Con A) and Solanum tuberosum agglutinin (STA), were comparatively studied. Despite intimate cell contacts in the aggregates only Con A led to establishment of haptenic‐sugar‐resistant (HSR) complexes. The presence of inhibitor II of diacylglycerol kinase, a dual calmodulin antagonist/protein kinase C inhibitor (trifluoperazine), and a sulfhydryl group reagent (N‐ethylmaleimide) impaired this process. The obtained results indicate that the formation of HSR cellular contacts is not an automatic response to lectin‐dependent cell association. In contrast to STA, Con A binding elicits this reaction with involvement of diacylglycerol kinase, protein kinase C and/or calmodulin as well as thiol level perturbation, as inferred by the application of target‐selective inhibitors.

Functional Activity of Neutrophils in Diabetes Mellitus and Coronary Heart Disease: Role of Myeloperoxidase in the Development of Oxidative Stress

We performed a comparative analysis of functional activity of neutrophils in patients with type 2 diabetes mellitus with and without symptoms of CHD. Enhanced H2O2 production by neutrophils in response to N-formyl-Met-Leu-Phe (fMLP) was found in patients with type 2 diabetes mellitus. In patients with type 2 diabetes mellitus associated with CHD, fML-Pinduced release of myeloperoxidase from azurophilic granules of neutrophils was reduced and plasma myeloperoxidase level was elevated. Increased peroxidase activity of myeloperoxidase, reduced plasma catalase activity, and increased levels of TBA-reactive lipid peroxidation products and oxidized glutathione were detected in patients of both groups. Since myeloperoxidase is an important neutrophilic mediator of oxidative stress, its increased activity in the blood can be an additional marker of oxidative stress and cardiovascular risk in patients with diabetes mellitus.

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca(2+), and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

Summary Myeloperoxidase (MPO) is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist). In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca2+ through enhancement of store-operated Ca2+ entry (SOCE). Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.

Regulation of the functional and mechanical properties of platelet and red blood cells by nitric oxide donors

The effect of NO donors (sodium nitroprusside, S-nitrosoglutathione, dinitrosyl-iron complexes) on the functional and mechanical properties of human platelets and red blood cells has been investigated. It has been established by atomic force microscopy that NO donor-induced platelet disaggregation is accompanied by changes in the elastic properties of cells. It has been shown that, in the presence of NO donors, the detergent-induced hemolysis of red blood cells is delayed, and the elasticity modulus of these cells decreases. The results obtained indicate that NO donors regulate the structural and functional properties of platelets and red blood cells.

Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils.

The gp91phox subunit of flavocytochrome b(558) is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b(558). gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin-gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H(2)O(2) generation by human neutrophils treated with the lipid raft disrupting agent methyl-β-cyclodextrin (MβCD). MβCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MβCD treatment either stimulated or inhibited H(2)O(2) production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins.