Dragana Stanić1, Milan Nikolić2 and Vesna Niketić2

² ICTM - Center for Chemistry, 11001, Belgrade/ Serbia and Montenegro

It is generally accepted that lipids extracted from intact red blood cells (RBC) originate from red cell membrane. However, literatures values reported for the total cholesterol (Ch) and phospholipid (PL) content in normal human RBC varies greatly, which prompted us to conduct this study with aim to provide further data concerning the RBC lipid content. Fifty young healthy male subjects were screened twice in one year, at summer and winter time. Plasma and total RBC lipids, lipids from RBC membrane and hemolysates (supernatants from which membranes were carefully separated) were evaluated at each season and were compared. Our results demonstrate that in contrast to Ch and PL contents of RBC membrane which are confined to a narrow range, the lipid levels estimated in intact RBCs showed more variation: the lowest individual values for RBC lipids corresponded to those found in membrane, whereas in RBCs with higher lipid contents the “excess” was found in hemolysates. We found that “an excess” of cholesterol (associated with phospholipid) strongly binds to hemoglobin (Hb), yielding Hb-lipid adduct (Hb-Ch). Significantly higher levels of Hb-Ch in winter comparing to those in summer that parallel plasma cholesterol levels and positive correlation between %Hb-Ch and HDL-Ch levels, point to the direct influence of plasma lipoprotein metabolism on the formation of Hb-Ch. Our in vitro studies demonstrated that Hb-Ch could be formed upon incubation of (lipid-free) hemoglobin with cholesterol-phospholipid mixture as well as upon the exposure of RBCs to the excess of cholesterol-phospholipid dispersion with FCh/PL ≤ 1. It is tempting to speculate that Hb-Ch represents a new form of cholesterol in circulation, which contributes to the permanent removal of the “excess“ of unesterified cholesterol from circulation. This implies that RBCs may represent a part of the mechanisms involved in the first line “defense” against “an excess“ of free cholesterol (FCh) in circulation.

P66

INCREASING THE STABILITY of ALGINATE BEADS CROSSLINKED WITH 1,6-DIAMINOHEXANE

One of the most common method for immobilization of proteins, enzymes, whole microbial, plant and animal cells are based on their entrapment in calcium-alginate gel beads. Alginate is a polysaccharide extracted from brown algae. Alginates are linear copolymers of -L-guluronate and -D-mannuranate. Their gelling properties derive from the cooperative binding of divalent cations localized between homopolymeric blocks of guluronate residues(termed G-blocks). Ca ions are located into electronegative cavities, like eggs in a egg-box, from this similitude arises the term egg-box model. The ionic interactions between guluronate blocks and Ca ions cause the formation of a strong termostable gel which properties largely depend on the characteristics of the polymer and the preparation method. The beads are made of to drop alginate solution to CaCl₂ solution. However calcium alginate beads are very porous and present a low retention capacity of entrapped molecules. Polyelectrolite solutions like chitosan, polyethyleimine and polypropyleneimine can be used surface-coating materials by dropping alginate into them. Although chemical stability in some solutions increase but the mechanical stability and conformational changes may occur.

To prevent these problems the cross-linking agents can be used for surface-covering of Ca-alginate beads. In this study we used 1,6-Diaminohexane(HDA) for this aim. The calcium alginate beads were activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and coupled with HDA to occur a surface covering. To test the beads, pH stability and release of Bovine Serum Albumine (BSA) from the beads under different conditions were investigated.

P67

Marina T. NECHIFOR, Diana DINU

The aim of this study was to investigate the mechanism of catalase photoinactivation induced by ultraviolet radiation. Hence, the major questions regarding catalase photoinactivation may be formulated as follows: does the photoinactivation of cellular catalase proceed via the same pathways as photoinactivation of pure catalase? Does photoinactivation of pure catalase occur in the absence of cellular photosensitizers? To answer these questions, the effect of in vitro UVA (320-400 nm) irradiation on catalase activity in cellular homogenates and pure catalase solutions, was studied.

Samples were irradiated at various intervals of time and then assayed for enzymatic activity. Photoinactivation of purified liver catalase and of catalase from liver homogenate was found to be similar, and the kinetics of such photoinactivation obey first order processes. To evaluate a possible involvement of cellular photosensitizers in the photoinactivation mechanisms we determined the basic parameters, i.e. the photoinactivation constants and the half-life of catalase activity. The constant of photoinactivation was significantly higher, and the half-life of enzymatic activity was significantly lower for catalase in liver homogenate (k_i = 0.42 h^-1, t_1/2 = 99.2 min) as compared to the purified catalase (k_i = 0.0647 h^-1, t_1/2 = 630 min). These data show the enhancement of catalase photosensitivity in the presence of cellular components. A decrease of the heme Soret absorption peak at λ=405 nm was noticed, suggesting that photoinactivation is caused by the destruction of heme, as a consequence of direct absorbtion of light. Polyacrylamide gel electrophoresis analysis under denaturing conditions shows photooxidative changes in the apoprotein structure, i.e. formation of intersubunit cross-links.

In conclusion, we have shown that in vitro UVA irradiation induces a decrease of enzymatic activity of catalase. Catalase from cellular homogenate is more photosensitive than purified catalase because of the presence of endogenous photosensitizers. The photoinactivation seems to involve the destruction of the porphyrin ring, but also the modification of apoprotein.

Andreea Iren SERBAN-CAPATINA¹, Eduard CONDAC², Elena GANEA³

In diabetes and aging collagen is non-enzymatically modified by reducing sugars. The major initial product is a fructose-lysine compound resulted from the glycation of ε-amino groups. In subsequent Maillard reactions, products known as advanced glycation end products (AGEs) are formed. These AGE products include structurally characterized adducts such N- carboxymethyl lysine (CML), pentosidine and chemically unidentified compounds which induce protein binding, browning, fluorescence, and cross- linking. In the present work we detect and quantify the AGEs formed by glycation with glucose in calf tendon and aortic collagen by various assays. Collagen samples were extracted from calf aorta and tendon by delipidation followed by extensive pepsin digestion. Extracted collagen was incubated in 0.01M PBS, pH 7.4 in the presence of 0.5 M glucose for 0, 2 and 4 weeks at 37° C. The free sugar was removed by dialysis and collagen samples were lyophilised. Collagen – linked fluorescence was measured at 370/460 nm and 335/385 nm. The relative fluorescence level in tissue glycated collagen was higher then the unglycated collagen. Fluorescence measured at 335/385 nm indicated that pentosidine level was three fold higher in glycated tendon collagen than in glycated aortic collagen. SDS-PAGE analysis showed the formation of high molecular weight compounds in aortic and tendon collagen after 2 and 4 weeks of glycation. The chromatographic pattern (FPLC, Superdex 200 column) of the aortic and tendon collagen showed, after 2 and 4 weeks of incubation with glucose the appearance of new peaks comparing with native collagen; these peaks correspond to higher molecular weight, namely 86.14 kDa for glycated aortic collagen and 130.17 kDa for glycated tendon collagen. The present study supports the hypothesis that sustained hyperglycaemia and aging can induce the formation of advanced glycation end compounds, which has deleterious consequences, such as structural modifications of the extracellular matrix.

Fahri Yilmaz¹, Suleyman Dasdag², Zulkuf Akdag², Nihal Kilinc¹

A large family of genes that regulate apoptosis has been identified and these genes can be remembered as a series of three-letter words beginning with b. The first antiapoptotic gene identified, bcl-2 is a member of a large family of homodimerizing and heterodimerizing proteins, some of which inhibit apoptosis (such as bcl-2 itself and bcl-xL), whereas others (such as bax, bad, and bcl-xS) favor programmed cell death. Although the bcl-2 family of genes plays an important role in regulating apoptosis, at least two other cancer-associated genes are also intimately connected with apoptosis: the p53 gene and the protooncogene c-myc. Because of this importance of bcl-2 we investigated the accumulation of bcl-2 in rat brain and testes after whole-body exposure to radiation emitted from 900 MHz mobile phones.

Sixteen Sprague-Dawley rats were separated into two groups of eight, one sham and one experimental. The rats were confined in Plexiglas cages (20 ´10.5 ´10 cm) with ventilation holes, and the cellular phones were placed 0.5 cm under cages. Exposure began approximately 10 minutes after transferring into the exposure cages, a period of time when rats settled down to a prone position and selected a fixed location inside the cage spontaneously. For the experimental group, the phones were in the speech condition for 20 minutes per day for 1 month. By speech condition, we mean that the phone is sending a tape of human speech to the base station. The same phones were place under sham group rats, but the phones were turned off. Immunohistochemical staining of bcl-2 was performed according to the standardized avidin-biotin complex method.

The results of this study showed that no bcl-2 accumulation was observed in the brain and testes of rats exposed to the radiation emitted from mobile phones. Finally, we emphasize that there is not any adverse effect of radiation emitted from 900 MHz mobile phones in terms of the first antiapoptotic gene, which is identified bcl-2.

Svetlana DINIĆ, Mirjana MIHAILOVIĆ, Desanka BOGOJEVIĆ, Svetlana IVANOVIĆ-MATIĆ, Goran POZNANOVIĆ

Haptoglobin (Hp) is a plasma acute phase (AP) glycoprotein with the prominent role in the binding and clearance of hemoglobin. Though it was classified as a gene expressed in the liver only after birth, its expression is initiated during embryogenesis. Hp gene expression is primarily controled at the transcriptional level, depending on interactions between specific cis-acting DNA sequences and hepatocyte-enriched trans-acting regulatory factors, such as C/EBP proteins. It also appears that interactions between nuclear matrix proteins and specific DNA sequences of AP protein genes are involved in modulation of their expression. Therefore, factors controling transcriptional regulation of the Hp gene during rat liver development were assessed by the binding afinity of nuclear matrix and nuclear extract proteins to the Hp gene hormone responsive cis-element (-165/-56). South-Western analysis revealed DNA binding affinity of a common set of proteins in the 35-29 kD region in both nuclear fractions isolated from embryonal (19-days old) and postnatal (1, 3, 7, 14 and 21 day old) rat livers. Using Immuno-Western analysis, 35 kD protein was identified as an isoform of C/EBP protein in both nuclear fractions. While in the nuclear matrix fraction C/EBP was present throughout development, in the nuclear extract it was detected from day 14 of postnatal development. Our results lend further support to the assertion that the nuclear matrix takes active part in transcriptional regulation of gene expression during differentiation.

Orhan DEĞER¹,Meltem ÇOLAK¹,Yaşam BARLAK¹,Yavuz TEKELİOĞLU²,Fahri UÇAR³

Bee-collected polen and propolis are apicultural products which are composed of nutritionally valuable substances and contain considerable amounts of polyphenol substances which may act as potent antioxidants. Elastase is primarily located in the azurophil granules and is an active component of the phagocytic system of neutrophils.We wanted to show if elastase secretion from KML-62 cancer cell lines could be influenced when incubated with pollen and propolis extracts or not.Pollen and propolis extracts at concentrations of 50,25,12.5 and 0 mg/ml were prepared by dimethyl sulfoxide. KML-62 cell cultures and lymphocyte cultures by preparing peripheral blood as control cells were incubated with extracts for 24 h. Elastase secretion was determined by CellProbe reagent (RGES elastase, Beckman Coulter) by using flow-cytometric fluorescence analysis. While about 85% fluorescence positivity was obtained with 0 concentrations for both KML-62 and lymphocyte cell cultures, fluorescence positivity decreased (between 1.7 and 6.9%) as concentrations of both propolis and polen extracts increased for KML-62 cell culture, but unchanged (between 55 and 76%) for lymphocyte cell culture. It was concluded that pollen and propolis extracts inhibit elastese secretion from cancer cell lines probably by their antioxidant potentials.

Jordanka STENEVA, ¹ Albena. JORDANOVA ³, Zdravko LALCHEV, ², Samuil. NINIO ³,Tania NEICHEVA, ³ and Diana PETKOVA, ³*.

Anestisiology and Intensive Care, University Hospital Queen Giovanna , Sofia, Bulgaria ; ² Biochemistry, Sofia University St. Kliment Ohridski, Sofia, Bulgaria and ³ Lipid-protein Interactions, Institute of Biohpysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

The aim of this study is to evaluate the application of phosphatidylcholine liposomes (PL) in HCl - induced ARDS in rabbits. Acute respiratory distress syndrome (ARDS) was induced by administration of 0.2 N HCl via intratracheal instillation for 45 min. After induced ARDS animals under artificial lung ventilation were retreated with PL for 60 min. Arterial blood gas analysis was performed at 15, 30, 45 and 60 min after PL application. Untreated animals were ventilated for the same time. Rabbits were killed with thiopental and bronhoalveolar lavage fluid (BALF) was investigated for lipid and specific surfactant protein content. The equilibrium surface tension and dymanic surface tension characteristics of monolayers obtained from BALF was determined by Wilhelmy balance.

Gabriela MARINESCU, Cristina BABEANU, Elena GLODEANU, Georgeta CIOBANU

Investigations of low magnetic field effects on biological systems have drawn attention of space biologists due to the planning of long-term space flights to other planets. Yet, data are gradually accumulating and pointing to the influence of low magnetic field at different levels of living systems organization.

The purpose of this paper is to improve the general knowledge of the response mechanism of living systems to low magnetic field in the presence of other physical stimuli such as screened geoelectric field, negative thermal shock and ferrofluid presence applied as modulators of peroxidase system. In this study we exposed Nigella damascena seeds to an extremely low magnetic field (200 nT) for short periods (1 to 60 min) and to a conjugated action of two or even three stimuli such as: a) 200 nT and –196°C; b) screened geoelectric field and –196°C; c) 200 nT, -196°C and ferrofluid.

Seedlings growth has been observed over a 35-day period. Aerial part of seedlings has been assayed for isoperoxidases pattern and activity as well as for chlorophylls and carotene levels determination. Measured parameters were germination grade, plantlets’ viability, and growth rate.

The peroxidase cationic and anionic components have shown different sensibilities to low magnetic field and other investigated factors and a correlation with the exposure time has been found. Meanwhile low-temperature shock, geoelectric field cancellation and ferrofluid treatment have induced a net effect on the individual isoforms determining a clear variation in the cationic: anionic distribution, the effect of the very low magnetic field has been manifested especially by attenuating large variations produced by the other factors. Positive correlation has also been found between foliar organogenesis and the anionic component and between growth rate and the cationic component. Significant alterations in chlorophylls and carotene level accompanied these changes in the isoperoxidases’ pattern and activity.