13th balkan biochemical biophysical days & meeting on metabolic disorders’ programme & abstracts

naciyekurtul@hotmail.com

We have found that serum SA levels were higher in smokers (p<0.001) and alcohol drinkers (p < 0.005) than those in non-smokers and non-drinkers (control) subjects. There was no statistically significant difference in saliva TSA levels between smokers and non-smokers (p > 0.05), whereas we have observed that saliva TSA levels were higher in alcohol drinkers than those in controls (p < 0.05). We have determined that there was no statistically significant difference in urine TSA levels between smokers and non-smokers (p > 0.05) but urine TSA levels were much higher in alcohol drinkers than those in healthy subjects (p<0.001) and smokers (p < 0.001).

We have observed that serum GGT activities were high in smokers (p < 0.005) and alcohol drinkers (p < 0.001) and there was no statistically significant difference in serum AST levels between smokers and non-smokers (p > 0.05) and also serum AST levels were higher in alcohol drinkers than those in control subjects (p < 0.001) and smokers (p < 0.01). We have determined that serum ALT levels were higher in smokers (p < 0.001) and alcohol drinkers (p < 0.01) than those in healthy subjects.

Our results indicate that serum TSA were affected by, and possibly related to, smoking, and that serum GGT, AST, ALT and also serum, urine, and saliva TSA can be used as a marker for monitoring of alcohol abuse.

Ioan Creanga*, Ana Andreea Arteni**, Vlad Artenie**

The effects of ionizing radiation in tree seedlings biochemistry were studied. Catalase acitivity and nucleic acid content were measured by iodometric titration and respectively by spectral measurements. Oak and black locust seedlings, 3 months and 6 months old were used. Radiation exposure was carried out by means of a Cobalt laboratory source with very low radioactivity (10 mCi).

In 3 months old oak seedlings the catalase activity is rapidly enhanced (to the exposure time enhancing) while for 6 months old oak seedlings it is linearly diminished. Two major effects of radiation may be invoked: direct action upon enzyme hydrogen bonds that are partially destroyed and indirect action, mediated by the water radicals generated throgh water radiolysis – possible agents of biochemical reaction rate influencing. Living cell being able to adapt their biochemical functions (self adjusting phenomena known as feed-back reactions), we might say that small radiation doses stimulate catalase biosynthesis even when part of molecules are inactivated by direct action. In 6 monyhs oak seedlings the direct destruction action is dominating. For relatively short exposure time a significant decrease of nucleic acid content was obtained revealing the damages of radiations at the level of biomolecule secondary structure.

In 3 months old black locust seedlings catalase activity did not present a clear dependence on the exposure time while peroxidase activity (supplementarly assayed by spectrophotometric measurement) was considerably amplified for the longest exposure time. In 6 months old seedlings of black locust catalase activity as well as the nucleic acid content were exponentially degraded to the increase of the exposure time. This means that self-adjusting phenomena responsible to the recovering of losses produced by radiation action are not sufficiently activated to compensate damages caused by radiation at molecular level.

So, low radiation doses, may influence cell biochemistry depending on the tree age and species.

P232

NONLINEAR DYNAMICS IN ERG SIGNAL OF INSECT EYE

Dorina Emilia CREANGA

The purpose of the experimental investigation was to reveal putative metabolic disorders induced by microwaves in the dynamics of invertebrate visual system by means of semi-quantitative computational tests able to characterize the system dynamics. Analysis of visual system dynamics was carried out using ERG (electroretinographic) signal recorded in Drosophila melanogaster adults that were exposed to low power density microwaves. In high intensity light abnormal small amplitude of the ERG component named receptor potential or exaggerated high amplitude of pre-potential were observed while high amplitude of lamina-on-transient appeared in other situations. Specialized soft package utilized for the characterization of ERG dynamics revealed qualitative and quantitative differences between normal and microwave exposed insects. The state space portrait appears as a double loop in normal flies while in exposed flies a single, fuzzy loop appears. The auto-correlation dimension is enhanced and Lyapunov exponent and auto-correlation time are also modified in the exposed fly lot in comparison to the control. The shape of probability distribution histogram changed its symmetry and auto-correlation function is damping more rapidly to zero. Protein kinase C being responsible for the eye adaptation to light intensity, it is possible that microwaves affected its activity either influencing its tertiary structure or influencing directly phosphoinositide metabolism, since protein kinase C is controlled by diacil glycerol, resulted from 4,5 phosphoinositol diphosphate. Never the less, protein kinase C is controlled by calcium ions delivered by intracellular stores attached to endoplasmic reticule. These calcium ions circulate by ion channels activated by 1,4,5 inositol triphosphate resulted also by 4,5 phosphoinositol diphosphate decomposition. This is why it is possible to assign the modifications observed in the visual system dynamics in high intensity light to the perturbation of phosphoinositide cascade. Since microwave exposure affected the eye response to high intensity light, phosphoinositide metabolism that is controlling protein kinase C kinetics (responsible for light intensity adaptation), seems to be troubled during electromagnetic treatment.

Losanka POPOVA and Russina YORDANOVA

Effects of soil flooding on important physiological processes and on the antioxidative capacity of barley plants were studied. Barley plants (Hordeum vulgare L., cv. Alfa) were grown for two weeks in soil in a growth chamber: irradiance 160 mmol m^-2s^-1 PAR, 12 h - photoperiod, temperature 24 ^oC, and relative humidity of 60%. When the plants were at the second to third - leaf stage half of the plants were flooded in the early morning by placing the pots inside larger glass containers filled with tap water to 25 mm above the level of the soil surface. Control plants remained well watered (60 % soil moisture) during the period of the experiment. Samples were taken 72, 96 and 120 h after the start of flooding treatment Seventy two to 120 h of soil flooding decreased the rate of C0₂ assimilation, chlorophyll and leaf protein content, activity of RuBP carboxylase, and of the photorespiratory enzymes phosphoglycolate phosphatase and glycolate oxidase. The activity of PEP carboxylase increased in all flooded plants. Soil flooding increased stomatal resistance without appreciably changing c_i values. A decrease in the level of RuBP carboxylase and its two subunits was also observed, the effect being more pronounced on the small subunit. Although no abundant expression of specific proteins was observed, we detected that soil flooding led simultaneously to down regulation of the expression of several proteins and up-regulation of others. The hypoxic treatment of barley roots caused an oxidative stress in their leaves. Increased levels of H₂0_2, electrolyte leakage and lipid peroxidation was found in flooded plants. Antioxidant capacity and the rate of ROS scavenging enzymes: SOD (superoxide dismutase), CAT (catalase), POD (peroxidase), APX (ascorbate peroxidase) and GR (glutathione reductase) were studied. Seventy two to 120 h of soil flooding decreased the activity of SOD and that decrease was mainly due to progressive reduction in the activity of Fe-containing SOD located in chloroplasts. The activity of slow moving and the most active isoperoxidases remarkably increased and 120 h after the start of flooding their total activity was 2 fold higher than the control. The changes in the activity of catalase followed the same tendency as for POD activity. Soil flooding differently affected the activity of GR and APX. Seventy two h of soil flooding the activity of GR was slightly increased and then gradually declined during the next 96 and 120 h. Both total soluble and thylakoid-bound APX activity increased in all flooded plants. Regardless of the increased activity of H₂O₂ scavenging enzymes, flooding treatment caused a substantial rise in total peroxide content. It is suggested that root oxygen deficiency caused photoxidative damage on barley leaves via an increased generation of active oxygen species.

İncilay SİNİCݹ Michael B TROPAK², Don J MAHURAN², H. Asuman ÖZKARA¹

DNA sequence to which RNA polymerase binds to initiate transcription of a gene is called promoter. A human cytomegalovirus (HCMV) immediate-early regulatory DNA sequence, termed the CMV promoter, was shown to be capable of significantly increasing the expression of a wide variety of genes. In our study, cDNA of beta-subunit of Hexosaminidase (Hex) were cloned into three different sets of expression vectors: pIRES2-EGFP and pCDNA3.1D/V5-His-TOPO vectors, carrying CMV promoter; pEFNEO vector having elongation factor (EF) promoter. All the cloned vectors were permanently and stable transfected to CHO and COS cells. Although low reporter gene, enhanced green fluorescent protein (EGFP), expression could be observed in the stabile transfected CHO cells with pIRES2-EGFP, expression of wild and mutant Hex could not be detected in Western Blot analysis. Following pcDNA3.1D-V5-His-TOPO transfection, mutant Hex still could not be detected, only barely detectable levels of wild Hex were observed. To examine the influence of different promoters on expression of Hex, pEFNEO construct was prepared. Western Blot analysis of pEFNEO wild and mutant Hex stabile transfected CHO cells clearly demonstrated high levels expression of the proteins. These observations suggest that extremely low levels of expression observed in CHO cells transfected using pIRES2-EGFP and pcDNA3.1D-V5-His-TOPO vectors may be due to weak CMV promoter. Although, reports testify to the utility and efficacy of constructs that carry CMV promoter, a previous report proved the instances of inexplicable failure to establish cell lines, having inducible expression of the cDNA under study, by using CMV promoter (1). As a conclusion, pIRES2-EGFP and pCDNA3.1D/V5-His-TOPO vectors carrying CMV promoter are not suitable enough for expression Hex. Vectors having EF promoter are useful to obtain high expression of Hex. For establishing Hex cell lines this may be taken account.

References:

1. RV Gopalkrishnan, KA Christiansen, NI Goldstein, RA DePinho, PB Fisher. Use of the human EF-1 promoter of expression can significantly increase success in establishing stable cell lines with consistent expression: a study using the tetracycline-inducible system in human cancer cells. Nucleic Acids Res, 1999; 27(24): 4775-4782.

EFFECT OF TEMPERATURE ON ELECTRICAL, MECHANICAL, FATIGUE AND RECOVERY POST-FATIGUE IN ISOLATED RAT DIAPHRAGM MUSCLE

Mustafa EMRE İsmail GÜNAY Servet KAVAK

Department of Biophysics Çukurova University Medical Faculty, Balcalı-Adana- Turkey

The aim of this work was to study the effects of temperature on electrical, mechanical fatigue and recovery post-fatigue characteristics of diaphragm muscle. Diaphragm muscles were placed in 10 ml organ-bath containing Krebs Hanseleit solution. Isometric peak twitch tension (P_t) and peak tetanic tension (P_o) parameters of the diaphragm muscle were recorded in vitro direct stimulation and at different temperatures (between 22 and 37 ºC). Fatigue was elicited by using 40 Hz fatigue protocol. Membrane and action potential parameters (amplitude, overshoot, depolarization time and half-repolarization time) were recorded and measured by using the conventional microelectrode technique. When temperature was increased, the membrane resting potential (MRP) of diaphragm muscle didn’t change. The MRP was about –75.22.4 mV and –73.82.7 mV at 22 and 37 degrees C, respectively. The amplitude (V_max) and overshoot (V_os) of action potential markedly reduced (6% V_max and 18% V_os, between 22 and 37 degrees C). Similarly, depolarization time (DT) and half– repolarization time (1/2 RT) of action potential markedly decreased with increasing temperature from 22 ºC to 37 ºC (65 % DT and 64 % ½ RT between 22 and 37 degrees C). The isometric contractile parameters [Peak twitch tension (P_t), contraction time (CT), half-relaxation time (1/2RT), peak rate of tension development (+dp/dt) and decline (-dp/dt)] of rat diaphragm muscle produced a significant (p<0.01) reduction with increasing temperature. The isometric contractile functions and recovery post-fatigue were stable with time at 22 and 25 ºC but decreased with time as a function of bath temperature above 25 degrees C. The decrease of P_t and P_o at temperatures above 35 ºC may in part be due to inadequate O₂ diffusion for the oxidative requirements of the muscle at the higher temperature. In addition, increase of protein degradation may be faster than synthesis with in vitro muscle preparations at temperatures above 35 ºC.

THE EFFECTS OF GINKGO BILOBA EXTRACT ON TISSUE OXIDANT/ANTIOXIDANT SYSTEM IN CISPLATIN NEPHROTOXICITY

İsmail TEMEL¹, Mukaddes GÜLEǹ, Ramazan YILMAZ², Mustafa IRAZ³, Sadık SÖĞÜT⁴, Hüseyin ÖZYURT⁵, Ömer AKYOL¹

Departments of ¹Biochemistry and ³Pharmacology, Inonu University Medical Faculty, Malatya, ²Department of Medical Biology and Genetics, Suleyman Demirel University Medical Faculty, Isparta, ⁴Department of Biochemistry, Mustafa Kemal University Medical Faculty, Hatay, ⁵Department of Biochemistry, Gaziosmanpasa University Medical Faculty, Tokat

mukaddesgul@hotmail.com

Cisplatin (CDDP) is a broad-spectrum antineoplastic agent. It has not commonly been used as a therapeutic agent because of its nephrotoxicity risk. Because the main underlying mechanism in nephrotoxicity has been attributed to reactive oxygen species (ROS), considering the use of antioxidant agent in those pathological processes seems to be a reasonable approach. Gingko biloba extract (GBE, Egb 761) has been shown to be effective on some organ and tissue pathologies induced by ROS. The aim of this experimental study was to determine whether antioxidant GBE has a preventive effect on ARF induced by CDDP through oxidative damage.

Male Sprague Dawley rats (60 days) were used in the experiments performed in accordance with “Guide for the Care and Use of Laboratory Animals, DHEW Publication No. (NIH) 85-123, 1985. Rats were randomly assigned to one of four groups: control untreated rats (n=7); rats treated with i.p. injection in a single dose of 7 mg/kg body wt CDDP (Cisplatin, Ebewe) (n=8); rats treated with CDDP plus i.p. injection of 10 mg/kg body wt vit E (Evigen-Aksu, Turkey) (n=9); and rats treated with CDDP plus oral administration of GBE in the dose of 100 mg/kg body wt (n=7). After 10 days of experimental procedure, animals were killed by bleeding, kidneys were removed and the oxidant and antioxidant parameters were studied to determine the effects of agents applied.

CDDP was found to lead statistically significant increases in plasma BUN and creatinine levels as well as urine N-acetyl--D-glucosaminidase leading ARF in rats. Antioxidant enzyme activities were found to be decreased in the kidney tissues of CDDP-treated rats. In the case of vit E application together with CDDP, glutathione peroxidase (GSH-px) and plasma creatinine levels were improved. GBE had no effect on the parameters studied after CDDP application. To find out the definite therapeutic effect of GBE on CDDP-induced neprotoxicity, further studies with different doses, different time interval, and more animal number are needed.

ASSESSMENT OF MAGNESIUM STATUS IN DIABETES MELLITUS

D.Pap¹,S.Bugarinovic

Psychiatric Hospital, Kovin,Serbia and Montenegro

¹Clinical-biochemical Laboratory, Medical Center Sremska Mitrovica, Serbia and Montenegro

There is emerging evidence that low intake of Mg or abnormal Mg metabolism are associated with etiologic factors in various metabolic diseases as well as diabetes mellitus. Mg²⁺ is a co-factor in more than 325 enzymes systems in cells and is the second most abundant intracellular cation. Decrements in the enzymatic activities of several metabolic pathways are seen in diabetes mellitus (DM) as a result of magnesium deficiency. We decided to test changes of total Mg²⁺concentration in plasma of 40 healthy subjects and 40 patients with DM. Patients were divided in two groups: NIDDM on oral hypoglycemic therapy and NIDDM on insulin. Glucose levels were measured with standard enzymatic methods. Mg²⁺ in plasma was measured by calmagit assay. Statistically significant differences in Mg plasma levels and glucose concentrations were detected between controls and patients, with significantly lower in patients than controls (p 0,01). The obtained results demonstrated an inverse relationship between plasma Mg levels and fasting blood glucose levels in both DM patients regardless to their therapy. There was no correlation between Mg and glucose concentrations in both groups. Neither age nor sex influenced these results in both groups. The results suggest potential mechanism whereby low Mg status may contribute to the pathogenesis of DM while Mg may beneficially alter outcomes in DM patients and interest in Mg supplementation is in the hopes of preventing long –term complications of diabetes.

P239

KINETİCS OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM SİX-MONTH-OLD LAMB KİDNEY CORTEX

Ulusu N.N., Tandoğan B., Tezcan E.F.

Hacettepe University, Faculty of Medicine, Department of Biochemistry, 06100 Ankara/Turkey

Glucose can be metabolised by a pathway other than glycolysis, and this is the pentose phosphate pathway, also known as the hexose monophosphate shunt. The role of pentose phosphate pathway is the generation of NADPH and ribose-5-phosphate. Glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate: NADP⁺ oxidoreductase EC 1.1.1.49) is the key regulatory enzyme of the pentose phosphate pathway and the products of this enzyme are NADPH and 6-phosphogluconate. In this study, the kinetic properties of glucose-6-phosphate dehydrogenase were examined. The enzyme was purified from lamb kidney cortex, about 3640 fold with an overall yield 26.32 % using a simple and rapid method. The kinetic assays were done at 37°C in 100 mM Tris/HCl buffer, pH 8.0, containing 10 mM MgCl₂, 11.25 mM KCl and various concentrations of NADP⁺ as varied substrate and glucose-6-phosphate as fixed substrate or glucose-6-phosphate as varied substrate and NADP⁺ as fixed substrate. Analysing the data by using Statistica Module Switcher shown that, the behaviour of the enzyme does not fit Ping-Pong Bi Bi mechanism and it fits sequential mechanisms (such as Ordered Bi Bi, Theorell Chance or Random Bi Bi). To identify the mechanism of the enzyme, product inhibition studies were performed since in the absence of products, the velocity equations of these three mechanisms are the same for the forward reaction in steady-state. From the product inhibition studies it was found that the enzyme follows ‘‘Ordered Bi Bi’’ kinetics. The kinetic constants have been obtained as K_ia = 0.029 mM, K_mA = 0.039 mM and K_mB = 0.018 mM.

Key words: Glucose-6-phosphate dehydrogenase, lamb kidney cortex, product inhibition and kinetics

P240

TAXOL’S EFFECT ON AGONIST INDUCED PLATELET AGGREGATION

*A.Seda KANMAZ-DEMİRCİOĞLU, *Derya ÖZSAVCI, *Şermin TETİK, *Rabia OBA, **Mehmet AĞIRBAŞLI, *Turay YARDIMCI

*Marmara University, Faculty of Pharmacy, Department of Biochemistry

**Marmara University, Faculty of Medicine, Department of Cardiology

deryaozsavci@hotmail.com

Taxol is an anti-cancer drug which stabilizes microtubules and blocks Go\G1 cell cycle cell during mitosis and inhibits cell proliferation.

In recent years Taxol has been used in intraarterial stent coating and inhibits neointimal hyperplasia and subacute thrombosis which are the cause of restenosis after stent implantation.

Platelet aggregation and activation are important in the formation of subacute thrombosis. In this study, we have examined the Taxol’s effect on platelet aggregation by aggregometer. Taxol’s effects on ADP, collagen and adrenalin induced platelet aggregation were studied in the presence of different Taxol concentrations (30, 70, 140, 280 M). From six healthy subjects whole blood samples were taken into 1\9 citrated tubes. PRP (platelet rich plasma) samples were prepared from whole blood by santrifugation at 150g for 15 minutes. PPP (platelet poor plasma) was prepared by santrifugation at 300g for 15 minutes. PPP was used to calibrate the aggregometer. For platelet aggregation studies PRP samples were used. To observe agonist induced platelet aggregation, ADP, collagen and adrenalin induced platelet aggregation was measured with and without Taxol. Taxol was used in increased concentrations.

As a result Taxol (especially 140, 280 M concentrations of Taxol) inhibited collagen, Adrenaline and ADP induced primary and secondary platelet aggregation (p<0.01).

Our results support Taxol’s inhibition effect on subacute thrombosis after Taxol coated stent implantation.

P241

TAXOL’S EFFECT ON PLATELET FIBRINOJEN RECEPTOR AND PLATELET ACTIVATION DETECTED BY FLOW CYTOMETRY

Derya OZSAVCI**, A.Seda KANMAZ-DEMİRCİOĞLU**, Gülderen YANIKKAYA-DEMİREL*, Nezih HEKİM* and Turay YARDIMCI**

**Marmara University, Faculty of Pharmacy, Department of Biochemistry, Istanbul/TURKEY, *Dr. Pakize I. Tarzi Laboratory, İstanbul/ TURKEY

deryaozsavci@hotmail.com

Taxol is an anti-cancer drug which inhibits cell processes that are dependent on microtubule turnover, including mitosis, cell proliferation and cell migration.

In recent years Taxol has been used in intraarterial stent coating and inhibitsneointimal hyperplasia and subacute thrombosis which are the cause of restenosis after stent implantation. Platelet aggregation and activation are important in the inhibition of subacute thrombosis. In this study, we have examined Taxol’s effects on fibrinogen receptor GpIIb\IIIa and platelet activation in whole blood and PRP by using flow cytometry. In recent years, flow cytometry has permitted the detection of activation antigens on platelets.

Citrated whole blood samples were obtained from seven healthy subjects and PRP (platelet rich plasma) was prepared by santrifugation at 150g for 15 minutes. Whole blood and PRP samples were incubated with Taxol for 10 minutes .To study the platelet activation, CD42b (GpIb) , to examine the effect of Taxol on platelet GpIIb/IIIa, CD61(GpIIIa) antibodies added to the samples and incubated for 15 minutes. All samples were fixed by PFA and analyzed on flow cytometer.

In different Taxol concentration (0, 0.01, 0.245, 0.5, 2.5, 5 mM), CD61 percentage in whole blood was found to decrease significantly (0.5, 2.5, 5.0 mM Taxol), whereas CD42 percentage also decreased.In PRP, CD61 and CD42b percentages decreased after incubation with different concentrations of Taxol. In our preliminary studies, in addition to Taxol’s effect on platelet activation, we have observed Taxol’s apoptotic effect on platelets (unpublished results).

In this study, the striking observation was that while Taxol was increasing the platelet activation, it decreased CD61 binding to platelets. We have also observed that when platelet activation with Taxol increased, the fibrinogen binding sites (GpIIIa) might have been suppressed by Taxol. These findings suggest that the binding sites of Taxol on platelets might be GpIIb-IIIa.

P242

NEUROCHEMICAL MONITORING: PLATELET MONOAMINE OXIDASE (MAO) ACTIVITY AS A MARKER OF BRAIN FUNCTION

S. Bugarinovic ,D.Pap¹ ,G. Prtenjak ²

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