DIABETES MELLITUS: CLINICAL SIGNIFICANCE OF LIPID DISTURBANCES

DIABETES MELLITUS: CLINICAL SIGNIFICANCE OF LIPID DISTURBANCES

The purpose of this study was to investigate diagnostic relevance of disturbances in lipid metabolism, which is suitable for predictive coronary risk assessment in diabetes mellitus (DM). The studied subjects were the control group (136 healthy subjects) and the experimental group (188 DM patients). The DM patients were divided in 4 groups: M1 – 54 non-insulin dependent DM patients (NIDDM) on diet; M2- 68 NIDDM patients on oral antidiabetes therapy; M3 –32 NIDDM patients on insulin; M4 –34 insulin dependent patients (IDDM).We examined: levels of total cholesterol (TCH), triglycerides (TG), HDL-c, LDL-c, VLDL-c, the index of atherosclerosis IA (LDL-c/HDL-c) and established risk factors RF (TCH/HDL-c). While HDL-c was found to be significantly lower in all patients with DM, LDL-c was significantly higher in M4 and M2 group, but the difference wasn΄ t significant in M1 and M3 in comparison to controls. Values of TCH were not significant in all groups of DM regardless to their therapy. The values of TG were significantly higher in all groups (p< 0,05); expect in group of NIDDM on insulin in comparison to control group. Values of IA and RF were indicated a high risk of atherosclerosis in all groups of DM (p< 0,01). These data suggest that low levels of HDL-c are good predictors of atherosclerotic risk in all groups of DM, regardless to their therapy.

ALTERATIONS IN ERYTHROCYTE TRANSMEMBRANE ANION TRANSPORT OF OPERATORS IN RADIO AND TV STATIONS

Margarita KOUZMANOVA¹, Miroslava HRISTOVA¹, Katja VANGELOVA<sup>2

1</sup>Department of Biophysics and Radiobiology, Biological Faculty, Sofia University, 8 Dragan Tzankov blvd.,1164 Sofia, Bulgaria

² National Center of Hygiene, Medical Ecology and Nutrition, 15 D. Nestorov blvd., 1341 Sofia, Bulgaria

kouzmanova@biofac.uni-sofia.bg

High frequency electromagnetic fields (EMF) are widely used for transmitting of radio and TV signals, in wireless communications, etc. More and more people are exposed to EM radiation not only while at work but also at home. This study was designed to investigate the changes in anion transport across the erythrocyte membrane of the staff of radio and TV stations.

The blood samples were taken from workers at different radio and TV transmitting stations. Each of these stations has some antennas emitting on various frequencies in MHz and GHz range. Three experimental groups have been set according to working time. The people, working on shifts (24 hours on work, 3 days away) were divided in two groups. In the first group blood samples were taken at the beginning of the next shift. The people from the second group were tested right after the end of the 24-hours workday. The third group included people that work 8 hours/day and the blood was taken at the beginning of work time. The measurements were performed using the modified pH-metric method of Glaser. The results obtained from the pH-test fit closely to sigmoidal logistic (dose-response) curve. This curve is defined by four parameters, which can be compared to Student-Fisher’s t-test.

Statistically significant alterations in the calculated parameters were registered among the three groups working at one and the same station, as well as between the groups from different stations. The alterations observed at the end of 24-hours shift are temporary and reversible. They were not detected at the beginning of the next shift after 72 hours away. These alterations are probably a result not only from the action of EM radiation, but also from long-lasting sleeplessness and exhausting of the organism. The exposure to EM radiation with various frequencies and intensities leads to variations in the investigated parameters among the workers in the different radio and TV stations.

The obtained results show that working for years in environment with EM radiation could cause permanent alteration in the ion transport across the erythrocyte membrane.

P224

A SITE-DIRECTED MUTANT OF THE DEHYDROQUINATE SYNTHASE SHOWS A DECREASE IN ENZYMATIC ACTIVITY BY IN VIVO COMPLEMENTATION

A. Günel-Özcan¹ and K.A. Brown<sup>2

1</sup>Kırıkkale University, Medical School Medical Biology Department, Kırıkkale, 71100 Turkey; ² Imperial College London, Department of Biological Sciences, Centre for Molecular Microbiology and Infection, London SW7 2AZ, UK

DHQS (5-dehydroquinate synthetase) is the second enzyme of the shikimate pathway and catalyzes the conversion of DHAP to DHQ (dehydroquinate). DHQS requires both NAD⁺ and Zn²⁺. In this study, we carried out site-directed mutagenesis on the Salmonella typhimurium DHQS encoded by the aroB gene .One of the highly conserved regions of DHQS contains the following motıf GHXXGHAXEXXXXXXXXXHG which includes residues important for Zn²⁺-binding. Based upon the crystal structure of DHQS from Aspergillus nidulans, two histidine residues (247, and 264) are predicted to form part of the zinc-binding site. A site-directed mutant was created which replaced the histidine side-chain at position 247 to a leucine in S. typhimurium DHQS. A PCR-based method was used requirıng four primers and three PCR reactions. The amplification primer carried three mismatches compared to the wild-type template sequence. Two of these mismatches were silent mutations which created an extra restriction enzyme site that could be used to screen for the presence of the mutation. The generated restriction enzyme site was StuI. Pfu polymerase was the enzyme of choice for the PCR. This site-directed mutant was cloned into the pET21d expression vector. The aroB gene encoding wild-type S. typhimurium DHQS was also cloned into the pET21d expression vector. Both of these contructs were transformed into an E.coli aroB^-(DE3) strain for protein production. Wild-type S. typhimurium DHQS, which is 93.9% identical to E. coli DHQS, grew on minimal medium lacking aromatic amino acid supplementation. This result indicated that the S. typhimurium DHQS could complement E. coli DHQS activity. However, the H247L mutant failed to complement E. coli DHQS activity suggesting that this histidine side chain is essential for correct folding and/or activity of the S. typhimurium enzyme.

Vesna KOJIĆ¹, Dimitar JAKIMOV^1, Aleksandar ĐORĐEVIĆ², Gordana BOGDANOVIĆ¹, Mirjana VOJINOVIĆ-MILORADIV².

Water-soluble fullerene C₆₀ derivatives - fullernols have attracted much attention due to their numerous biological characteristics. Most of these biological features are based on the ability of fullerenols to scavenge free radicals.Investigated antitumor drugs have various mechanisms of action. For most of them toxic activity can be explained by formation of free radicals.

The aim of this paper was to investigate activity of fullerol C₆₀(OH)₂₀O₂H₂ on growth of human breast cancer cell lines and its modulatory effect on Adriamycin (ADR), Cisplatin (cis-Pt), Taxol, and Thiazofurine - induced cytotoxicity on the same cell lines. Growth inhibition was evaluated by colorimetric SRB essay.

The cell growth was investigated on two cell lines: MCF7 (human breast adenocarcinoma; estrogen receptor positive (ER+)) and MDA-MB-231 (human breast adenocarcinoma, estrogen receptor negative (ER-)). Cell lines were treated with fullerenol at concentrations 0.9-3.9g/ml. Fullerol was given alone and in combination with cytostatics at various concentrations ranging from 10^-4 to 10^-8 M during 2 hours. Cytotoxic effect was evaluated 24h or 48 h after treatment .

Fullerol mildly inhibits growth of both cell lines (3-15%). Combination of fullerenol and cytostatics, given simultaneously, resulted in various growth inhibition depending on fullerenol concentration, type of antitumor drug and cell line as well.

Fullerol differently modulates cytotoxic effects of given cytostatics.The protective action of the cytostatic drugs was more pronounced in comparison to Taxol,whose action is based on formation of free radicals.

Key Wordts: fullerol C₆₀(OH)₂₀O₂H_2, Cytotoxicity, Adriamycin, Cisplatin, Taxol, Thiazofurine, Breast cancer cell line.

P226

DETERMINATION OF STEADY-STATE LEVELS OF 8-OxoGuanine IN CALF THYMUS DNA BY MEANS OF FPG PROTEIN

Beran YOKU޹, Naime CANORUDz, Dilek Ülker ÇAKIR², YILDIZ ATAMER², Abdurrahman KAPLAN², Sabri BATUN²

7,8-dihydro-8-oxoguanine (8-Oxoguanine or 8-OxoGua), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to translation of GC→AT . DNA adduct are lethal if not repaired. The primary function of Base excision repair (BER) enzymes are known to recognise various types of base damage: oxidised purine, pyrimidine damages and remove these ox datively damaged bases from DNA, protecting cells from the mutagenic and lethal effects of oxidative DNA damage. Escherichia coli Fpg protein (also known as formamidopyrimidine-DNA glycosylase) is a combined DNA glycosylase-AP lyase that removes the damaged bases (fapy-pyrimidine and 8-OxoGua lesions). The oxidized DNA base 8-OxoGua has been commonly measured by enzymatic hydrolysis of DNA followed by reverse phase HPLC-EC. There has been recently a debate surrounding the validity of this approach, from which it has become clear that artifactual oxidation of the native base to 8-OxoGua that can occur at numerous stages in sample preparation.

Hence, we developed an alternative/modified method to traditional enzymatic digestion of DNA, which based on the use of the base excision repair enzymes (Fpg protein) and limits the potential for artifactual oxidation and speeds up the assay. In addition, we showed that substrate specifity of fpg protein.

All chemicals purchased from Sigma. Calf Thymus DNA was dissolved in 20 mM TE buffer (pH 7,4),. Different concentrations of the calf thymus DNA was incubated with 16 μl Fpg protein 37° C for 2 h and hydrolysate was analysed by HPLC for 8-OxoGua using electrochemical detection (Decade, Antec-Leyden). Guanine was detected with UV/Visible spectrophotometric (Shimadzu) detector.

Results were given as 8OxoGua / Gua. Retention time of 8-OxoGua was 4,8. Km value 7 nm as calculated from the Lineweaver-Burk plot. In conclusion, excision enzymes have proved useful tools for the determination of the yield.

Key words: 8-OxoGuanine, 8-OxoGua, Oxidative DNA damage, Free radicals, DNA repair, Fpg protein

Şevki ARSLAN, Orhan ADALI, Emel ARINÇ

Formation of catechols from benzene and nitrobenzene has been implicated in the carcinogenic activity of these chemicals. p-Nitrophenol, an intermediate of p-nitrobenzene, is used in the production of acetaminophen, parathion insecticides, fungicides and dyestuffs. p-Nitrophenol is metabolized by the cytochrome P450 dependent mixed function oxidases (MFO) specially known as CYP2E1-associated p-nitrophenol hydroxylase and the product 4-nitrocatechol is oxidized to semiquinone and quinone. These substances are bioactive metabolites that have the ability of binding to macromolecules such as DNA and proteins, which ultimately cause cellular necrosis, mutagenesis and malignant transformation. The aim of this work is to determine the effect of diabetus mellitus on rabbit liver p-nitrophenol hydroxylase activity.

p-Nitrophenol 4-Nitrocatechol CONJUGATION