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

Angelina STOYANOVA

Chromium(VI) is a strong oxidizing agent and possesses high toxicity to humans and animals due to its carcinogenic and mutagenic properties. That is why the determination of chromium in environmental and biological samples is of great interest.

In this work a catalytic spectrophotometric method for the determination of chromium(VI) is proposed. The method is based on the catalytic effect of chromium(VI) on the oxidation of sulphanilic acid (SA) by hydrogen peroxide in the presence of p-aminobenzoic acid (PABA) as an activator.

The reaction was followed spectrophotometrically by tracing the formation of the reaction product at 360 nm after 15 minutes of mixing the reagents.

On the bases of the investigations made, the optimum reaction conditions were established:

4.0x10^-3 mol l^-1 SA, 0.57 mol l^-1 H₂O₂ , 1x10^-3 mol l^-1 PABA and 0.04 mol l^-1 acetic acid – boric acid - orthophosphoric acid buffer solution (pH 6.6), at 50 ^oC.

The linear range of the calibration graph was up to 140 ng ml^-1 and the detection limit was 10 ng ml^-1. Interferences of Cu(II) and Cr(III) ions were masked. The method was applied to the analysis of Cr(VI) in industrial water with recoveries of 95.2 - 104.3 % and a mean RSD (n=6) of 5.6%.

Keywords: chromium(VI), catalytic method, sulphanilic acid, p-aminobenzoic acid, industrial water

Mladen IOVCHEV¹, Plamen KODROV¹, Adrian WOLSTENHOLME², William L. PAK³ and Eugene SEMENOV¹

The recent identification and characterization of two genes, encoding histamine-gated chloride channel subunits from Drosophila melanogaster, has confirmed that histamine is a major neurotransmitter in the visual system of the fruitfly. One of the cloned genes, hclA, corresponds to ort (ora transientless), mutations in which affect histaminergic synaptic transmission in the Drosophila visual system. We identified a mutational change (a null mutation) in the genomic and RNA copies of hclA derived from flies with the ort¹ allele. This correlates with new phenotypes observed in the mutant strain. We found hypersensitivity to neurotoxins of the avermectin group in both the ort¹ adult flies and third instar larvae compared to Oregon R wild-type animals. In contrast, the mutation makes male and female adults more resistant to treatment with diethyl ether, and the animals show substantially prolonged recovery from paralysis after diethylether anaesthesia, as well as an impaired recovery from paralysis after mechanical shock, as revealed by the bang sensitivity test. The examination of several other alleles ort (with identified mutations in hclAs) in the same tests revealed the allele-specific responses. Altogether, our data give direct evidence that in vivo a HCLA subunit-containing receptor has a distinct role in the response to general anaesthesia and the neurotoxins, as well as indicate that its function is not limited by the frames of the visual system.

TOPICS: 1) Ion channels and membrane trafficing;
2) Metabolic disorders; 3) Molecular structure and function

OCTOBER 15, 2003 – WEDNESDAY

HALL B

LECTURE 1

INACTIVATION OF MELANOMA CELLS IRRADIATED WITH GAMMA RAYS AND LOW ENERGY PROTONS

Aleksandra RISTIC-FIRA¹, Ivan PETROVIC¹, Danijela TODOROVOC¹, Miroslava VUJCIC¹, Lela KORICANAC¹, Sabera RUZDIJIC², Miroslav DEMAJO¹, Giacomo CUTTONE³

Radiotherapy and particularly proton therapy is very efficient in eliminating malignant growths, but it is also very delicate, since healthy tissue surrounding ill tissue should not be affected at all or very little by the irradiation. The main characteristics of protons, such as their well defined range, relatively small lateral scattering, and high energy deposition density, just before the end of the range, make them particularly suitable when malignant growths are deeply embedded or are close to critical organs, where there is a high demand to minimize the destruction of the neighbouring and overlaying tissue.

In order to obtain better results in eliminating malignant cells, the aim of this in vitro study was to investigate the difference in response of HTB63 human melanoma cells to irradiation with either gamma rays or protons considering dynamics of cell growth. Single irradiation with gamma rays using doses from 2 to 20 Gy exhibited weak inactivation of human melanoma cells in vitro. The best effect, 26% of growth inhibition was obtained after single irradiation with gamma ray using dose of 16 Gy. Using the same doses of proton irradiation, with energy at the target of 22.6 MeV, significant melanoma cell growth inhibition was induced. Doses of 12 and 16 Gy provoked growth inhibition of 48.9 and 51.2% respectively. Estimated RBEs for inactivation of HTB63 cells ranged from 1.02 to 2.22. The electrophoretical analyses of DNA samples and flow cytometric evaluation have shown a small percentage of apoptotic cells after both types of irradiation.

The inhibitory effect of protons on melanoma growth, in contrast to gamma rays, can be explained considering specific physical properties of protons, especially taking in account good dose distribution.

Dušanka JANEŽIČ

Among the main theoretical methods of investigation of the dynamic properties of biological macromolecules, such as proteins, are molecular dynamics (MD) simulation and harmonic analysis. MD simulation is a technique in which the classical equation of motion for all atoms of a molecule is integrated over a finite period of time. The resulting trajectory is used to compute time-dependent properties of the system. Harmonic analysis is a direct way of analyzing vibrational motions. Harmonicity of the potential function is a basic assumption in the normal mode approximation used in harmonic analysis. This is known to be inadequate in the case of proteins because anharmonic effects, which MD has shown to be important in protein motion, are neglected. When anharmonic effects are incorporated quasiharmonic analysis may be applied. In this method, the MD simulation is utilized to obtain effective modes of vibration from the atomic fluctuations about an average structure. These modes include the anharmonic effects neglected in a normal mode calculation [1].

The role of low frequency normal modes involving global conformation changes and which have been theoretically determined for several proteins is emphasized. Low frequency modes of proteins are particularly interesting because theyare related to functional properties. The analysis of these motions in the limit of harmonic dynamics lends insight into the behavior and flexibility of these molecules. The modes presented here include the lowest modes of Bovine Pancreatic Trypsin Inhibitor (BPTI) [2, 3].

Harmonic analysis also proved useful in developing efficient symplectic MD integration methods. Symplectic integration methods are often the right way of integrating the Hamilton equations of motion. Recent advances in development of SISM

(Split Integration Symplectic Method) and HANA (Hydrogens ANAlyticaly) for combined analytical and numerical solution of the Hamiltonian system based on a factorization of the Liouville propagator are presented [4, 5].

SISM and HANA use an analytical treatment of high frequency motions within a second order generalized leap-frog scheme. The computation cost per integration step for both methods is approximately the same as that of commonly used algorithms, and they allow an integration time step up to an order of magnitude larger than can be used by other methods of the same order and complexity. SISM and HANA have been tested on a variety of examples. In all cases they posses long term stability and the ability to take larger time steps while being economical in computer time.

The approach developed here is general, but illustrated at present by application to the MD integration of the model system of linear chain molecules and a box of water molecules.

OCTOBER 15, 2003 – WEDNESDAY

HALL B

ORAL PRESENTATION 1

IMPLEMENTATION OF A NEUROVASCULAR COUPLING MODEL

There has been tremendous achivements in brain functional imaging techniques, namely in fMRI, MRS and more recently in functional optical imaging. However quantification and determination of the physiological and biochemical mechanisms in the neurovascular system and in neural activation remain to be challenging. For our studies in metabolic disorders and functional optical imaging, we have implemented a software package which uses Aubert and Costalat’s [1] model of neurovascular coupling in the brain. The reasons of selecting this model are that it is a compact form of the neurovascular coupling, it is recent, and it takes into account the previous models.

The model is essentially a coupling model between brain electrical activity, metabolism, and hemodynamics. This model combines the interactions of the following parameters and mechanisms: (i) cerebral blood flow, (ii) intracellular sodium (iii) glycolysis (iv) ATP, PCr, and mitochondrial respiration (v) blood–brain barrier exchanges and (vi) the Balloon model. The model attempts to model the relationships between the above-mentioned parameters by means of 15 differential equations. Differential equations’ parameters were obtained basically with the use of fMRI (functional magnetic resonance imaging) and MRS (magnetic resonance spectroscopy) measurements.

Our software package is developed in Matlab 6.0 environment (in MS Windows XP) and it has a user-friendly graphical user interface. Basically, it solves 15 differential equations and enables us to get the biochemical responses of the brain under different metabolic conditions.

We have essentially generated a simulation environment for neurovascular coupling model of Aubert and Costalat. We plan to use it for the study of metabolic disorders as well as for the comparison of the responses measured by functional optical imaging technique to the simulation results generated by the implemented model.

ORAL PRESENTATION 2

Vassilij Goltsev, Ivelina Zaharieva¹, Petar Lambrev¹, Petko Chernev, Chavdar Slavov, Ivan Yordanov², Reto J. Strasser³

The contribution of different components of delayed fluorescence (DF) dark decay during the induction period of dark to light adaptation was analyzed. Using phosphoroscope fluorometer with high speed digitalization a prompt chlorophyll fluorescence signal was registered simultaneously with a series of dark relaxation kinetics of DF, recorded at different moments during the induction period. The dark relaxation of DF between 0.35 and 5 ms is poly-exponential and can be approximated by 3 components with life-times of about τ₁ ~ 0.6, τ₂ ~ 3.5 ms and τ₃ ≥ 20 ms. Both the amplitudes and the life-times of the DF components drastically changed during the induction. Тhe contribution of the millisecond components with lifetimes 0.6 and 2 – 4 ms predominated during the first second of the induction period, and later the amplitudes of the tree components became approximately equal. The contribution of DF components was highly dependent on registration temperature. At low temperature (5 ºC) the main contribution in the fast phase of DF induction curve had the millisecond component, at high (38 ºС) – the sub-millisecond and at room temperature (22 ºС) the amplitudes of the both components were approximately equal. On the basis of kinetic models describing the redox reactions in the donor and acceptor side of Photosystem II, the participation of different redox states of the reaction center in the formation of separate components of DF dark decay is discussed.

Acknowledgments. This work was financially supported by the Swiss National Science Foundation (SCOPES 2000–2003 grant № 7BUPJ062408.00/1).

ORAL PRESENTATION 3

DEVELOPMENT OF AN ANALOGUE MODEL SIMULATING THE PORTAL VEIN

It is known that the force generated by muscle cells depends on the stiffness of the cross -bridges. In addition, experiments have shown that the passive tissue mechanics modulate the force. Therefore, to understand the interaction between the force on the cross bridges and the passive tissue mechanics, mechanical models (Maxwell and Voigt) have been used. The present experiment was performed in order to find an analog model to simulate smooth muscles of the portal vein. For this purpose five different combination of Maxwell and Voigt models were designed and the stiffness-force relations of these models were obtained theoretically. Also, the stiffness-force relations of the portal vein were obtained experimentally for 7 preload levels. Stiffness was measured by applying constant amplitude 5 Hz sinusoidal length perturbations continuously to the contracting muscle preparation. It was found that during isometric contractions of the muscle, the stiffness increased linearly with the isometric force; and the slope of the stiffness-force relation is 1.26 0.08 (1/mm) and the line intersects the ordinate at 0.250.13 g/mm (n=10). In addition, it was observed that the slope of the stiffness-force relationship depends on the preload applied to the muscle. When the experimental results obtained from the portal vein were compared with the theoretical results calculated from the models, it was seen that none of the models could fully represent the portal vein. However Maxwell model can be used if it is assumed that series elastic element of the model has a preload depended stiffness properties.

This study was supported by the Scientific and Technical Research Council of Turkey (SBAG-1206).

ORAL PRESENTATION 4

PURIFICATION AND CHARACTERIZATION OF A PHOSPHATE SPECIFIC TRANSPORTER HYPERALKALINE PHOSPHATASE FROM THERMUS THERMOPHILUS