Agnieszka Bronowicka-Szydełko

Effect of tocopherol on biochemical blood parameters in pleuritis-induced rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

The aim of this study was to evaluate the effect of tocopherol on pleuritis-induced rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Rats were treated with a single TCDD dose of 5 μg/kg body weight (b.w.) and then for 3 weeks they were daily supplemented with tocopherol at a dose of 30 mg/kg b.w. The inflammation was initiated by intrapleural injection of a single dose of 1% carrageenin solution in a volume of 0.15 ml. Changes in biochemical blood parameters were measured three times at the 24th, 72nd and 120th hour of pleuritis and the blood was collected from 20 animals of each group of rats (group with the control inflammation; group treated with TCDD and with control inflammation; group treated with TCDD, supplemented with tocopherol and with the inflammation). The following biochemical parameters were measured: tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-2, IL-4, IL-6, procollagen, telopeptide, fibrinogen, cholesterol, urea, creatinine, aspartate aminotransferase (AspAT) and alanine aminotransferase (AlAT). Daily supplementation of tocopherol caused significant changes in the level of TNF, IL-1, IL-4, IL-6, urea, creatinine, AspAT and AlAT. According to the results of these studies, we suggest that tocopherol supplementation in high doses could act as a protective treatment to improve liver metabolism.

Influence of dioxin intoxication on the human system and possibilities of limiting its negative effects on the environment and living organisms

INTRODUCTION AND OBJECTIVE Despite the restrictive legal regulations related to the reduction of dioxins emission, their concentration in the environment is still too high. Mainly, this is related to the illegal utilisation of electronic equipment and combustion of wastes, and also to intensified activity and maintenance of ships, especially in developing countries. The most important remaining source in Europe is the metal industry. Studies on the mechanism of impact of dioxins are still being carried out. This review points at new possibilities for limiting the molecular mechanisms of dioxins activity, inter alia, through the application of high doses of tocopherol and acetylsalicylic acid while treating dioxins intoxication. BRIEF DESCRIPTION OF THE STATE OF KNOWLEDGE: Apart from the knowledge of dioxins affinity to the aryl hydrocarbon receptor (AhR), the multi-stage radical-form actions and the pro-inflammatory mechanism associated with cyclooxygenase-II enzyme (COX-2) are under intense investigation at the moment. Due to the high affinity of dioxins to animals adipose tissue and their ability to accumulate in it, they can enter the food chain. Furthermore, high dioxin doses can cause poisoning manifested as advanced clinical symptoms, whereas in smaller doses, when cumulated, can cause metabolic changes which are often difficult to associate with their presence. Recently, some serious food contaminations by dioxins have been demonstrated. Sea fish and products from contaminated aqueducts still constitute potential sources of dioxins pollution. CONCLUSION According to recent studies, dioxins are present in different concentrations in the environment and cause specific and long-time effects. These effects could be limited by the use of tocopherol and acetylsalicylic acid.

Mechlorethamine (NTG) effects on the erythrocytic and leukocytic blood parameters during experimentally induced pleuritis in rats

BACKGROUND According to cytotoxic and mutagenic properties, nitrogranulogen (NTG) changes the character of inflammatory reactions. Our previous studies have shown that NTG can enhance immunological defense reactions, because of its high affinity to DNA, and causes disorders in the synthesis of acute phase proteins (e.g., haptoglobin, transferrin, fibrinogen and complement protein C3) [15]. The aim of the current studies was to determine the influence of three different NTG doses: 5 μg/kg b.w. (body weight), 50 μg/kg b.w. and 600 μg/kg b.w. (cytotoxic dose) on the values of hematological blood parameters: RBC, HGB, HCT, RDW, MCV, MCH, MCHC, PLT, MPV, PCT, PDW, WBC, NEUT, LYMPH, MONO, EOS and BASO in pleuritis-induced rats. METHODS The animals were randomized into five groups: Group I - control group; Group II - IP (induced pleuritis) group; Group III - NTG5 group; Group IV - NTG50 group; Group V - NTG600 group. The blood was collected from all the groups at the 24(th) h, 48(th) h, and 72(nd) h after the initiation of the carrageenin-induced inflammatory reaction. RESULTS These investigations have revealed that NTG administered at the dose of 5 μg/kg b.w. caused the drop of the leukocyte and lymphocyte numbers and the rise of the neutrophil number at the 72(nd) h of the experimental-induced inflammatory reaction. Moreover, the dose of: 5 μg/kg b.w. was an immunomodulatory property and it also increased the erythrocytic parameters. On the contrary, NTG applied at the doses of 50 μg/kg and 600 μg/kg b.w. contributed to the drop of both: the erythrocytic and leukocytic parameters during the whole time of the inflammatory reaction. CONCLUSIONS The results suggest that nitrogranulogen affects the erythropoiesis.

Correction to: Advanced glycation end products as a source of artifacts in immunoenzymatic methods

The original version of this article unfortunately contained a mistake in the author group section. Author A. Bronowicka-Szydełko’s surname was inadvertently interchanged to “Szydełko-Bronowicka”.

Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases

Human α- and β-enolases are highly homologous enzymes, difficult to differentiate immunologically. In this work, we describe production, purification and properties of anti-α- and anti-β-enolase polyclonal antibodies. To raise antibodies, rabbits were injected with enolase isoenzymes that were purified from human kidney (α-enolase) and skeletal muscle (β-enolase). Selective anti-α- and anti-β-enolase antibodies were obtained by affinity chromatography on either α- or β-enolase-Sepharose columns. On Western blots, antibodies directed against human β-enolase, did not react with human α-isoenzyme, but recognized pig and rat β-enolase. To determine what makes these antibodies selective bioinformatic tools were used to predict conformational epitopes for both enolase isoenzymes. Three predicted epitopes were mapped to the same regions in both α- and β-enolase. Peptides corresponding to predicted epitopes were synthesized and tested against purified antibodies. One of the pin-attached peptides representing α-enolase epitope (the C-terminal portion of the epitope 3 - S262PDDPSRYISPDQ273) reacted with anti-α-enolase, while the other also derived from the α-enolase sequence (epitope 2 - N193VIKEKYGKDATN205) was recognized by anti-β-enolase antibodies. Interestingly, neither anti-α- nor anti-β-antibody reacted with a peptide corresponding to the epitope 2 in β-enolase (G194VIKAKYGKDATN206). Further analysis showed that substitution of E197 with A in α-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A198 with E in peptide representing β-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E197 is essential for preserving immunologically active conformation in epitope 2 peptidic homolog, while it is not crucial for this epitopes antigenic activity in native β-enolase.

PREPARATION OF BOVINE SERUM ALBUMIN MONOMER FOR CONJUGATION EXPERIMENTS BY USING DIFFERENT TYPES OF CHROMATOGRAPHY MEDIA

The presence of some sugar contaminants and high molecular mass (HMW) derivatives detected in commercially available bovine serum albumin (BSA) preparations precluded the use of such reagents for basic molecular biology experiments. In this study the effectiveness of three separation methods of monomeric BSA from contaminants were examined: gel filtration on Sephadex G-200, HW-55S resin, and ion-exchange chromatography on DEAE-Sephadex A-50. Only chromatography on HW-55S allowed obtaining protein lacking HMW derivates and freeing of sugar contaminants, which was confirmed in SDS/PAGE and phenol/sulfuric acid analysis, respectively. Moreover, the determination of protein particle size by dynamic light scattering (DLS) at a level of 7 nm was in accordance with data for BSA monomeric molecules previously reported. Finally, the monomer BSA was subjected to modification with natural core oligosaccharide derived from Shigella lipopolysaccharide. The SDS/PAGE analysis of reaction products demonstrated different amounts of glycoconjugates compared to values obtained for commercial BSA. Only the pure protein modification permitted correct appraisal of obtained products. We propose gel filtration chromatography on HW-55S resin as simple and effective method of monomer BSA separation from commercial reagent before basic and applied research.