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Flux RSS. In addition to environmental factors, genetic predisposition has a significant role in the ethiopathogenesis of the disease. Apart from the two dominantly inherited syndromes FAP and HNPCC several low penetrance genes were implicated in the initiation of colorectal cancerogenesis.

A total of patients with CRC, of which six patients with multiple adenomatous polyposis, and a control group of newborns and aged individuals were included in this study.

Also, we suggest that aberrant splicing of this gene is a probable mechanism in etiopathogenesis of the multiple adenomatous polyposis phenotype.

Microsatellite instability was present in The frequency of the T RI 6A polymorphism was identical among patients and controls thus excluding this variant as a tumor susceptibility allele in our population.

A statistically significant difference in the frequency of the CCND1 polymorphism was found in the group of patients less than 60 years of age with MSI tumors, indicating that CCND1 polymorphism may influence the age at onset of colorectal cancer in young patients only when their tumors exhibit an MSI phenotype.

Ozand MD,Ph. The objective of this presentation is to present the novel strategies adopted to prevent genetic diseases in Saudi Arabia.

The genetic diseases either due to chromosome defects or to alterations in single gene structure produce infants with usually severe diseases.

These diseases, particularly those inherited single gene disorders plague the communities with consanguineous marriages.

Based on our experience in the Kingdom, we have adopted several strategies to combat this public health problem.

The methods to combat genetic diseases include: 1 if nothing else is possible to perform a neonatal screening program for treatable diseases.

This must be done within days after birth. All of these diseases are either difficult to manage requiring the devotion of many clinical hours by the physician or their procedures of management or are extremely costly.

Applying the mutations on a DNA chip and screening the extended family as well as population for carriers of these diseases.

This presentation will detail these approaches and our preliminary results. Andrey Karshikoff Experimental measurements of ionisation equilibria of titratable groups cannot provide quantitative information for the electrostatic interactions in proteins.

Moreover, in some cases an accurate prediction of electrostatic interactions is needed in order to give an adequate interpretation of the experimental observations.

This is mainly due to the cooperative ionisation behaviour of the titratable groups in proteins, referred here to as irregular titration.

Experimentally, irregular titration cannot be distinguished from the sum of the ionisation equilibria of more than one independent sites.

This may lead to misleading interpretation of the experimental data. The analysis of pH dependence enzymatic activity is also sensitive to how experimental data are interpreted.

An illustration for such a case is the understanding of the mechanism of proton abstraction from alcohol substrate of alcohol dehydrogenase.

Three hypotheses have been proposed to explain the pHdependence of this process. All of them assume that a group from the active site of the enzyme has a pK of about 7.

Non of the groups proposed by these hypotheses have been experimentally detected. The comprehensive analysis of electrostatic interactions suggested that irregular titration of the groups in active site occurs.

On the basis of this theoretical observation, a completely different molecular mechanism of the enzymatic activity of alcohol dehydrogenase can be given.

Although it is an important trace element for life, it may be toxic of only moderately higher levels of intake depending on their chemical forms.

Deficiency of element in animals makes them susceptible to injury by certain types of oxidative stress correlated diseases such as; cancer, HIV infection, renal and heart disease.

Selenium species in the living body can be in the form of selenoproteins, Secontaining proteins, inorganic selenium selenite, selenate , methylated selenium and selenoamino acids.

Many factors can influence the selenium contents of biological fluids and therefore values can vary in a significant way from one person to another depending on various parameters and pathological conditions.

Because the selenium content of biological fluids are very low, sensitive analytical techniques are needed to measure it.

Some analytical methods have been reported which the most of them require expensive instrumentation and time consuming sample preparation methods.

Taking into account the characteristics of the sample and the resources available in every laboratory we tried to modify a simple and rapid spectrophotometric method for the determination of selenium in biological materials.

The modified method is based on the reaction of Selenium Se IV with potassium iodide in acidic medium to liberate iodine which the absorbance decrease is directly proportional to selenium concentration by the use of thionin dye Calibration graph was maintained at 0.

For the preparation of plasma samples direct dilution and enzymatic digestion methods were performed and compared. Variation coefficients and standard deviations were calculated for five replicate determinations.

Furthermore, as well as the accuracy, application of the method to the biological materials was investigated by using a commercial drug Dietary Selenium Supplement and plasma samples.

Selenium in water, soil, plant materials, cosmetics, etc. These properties, purely understood so far, can be measured by a direct application of the electrochemical adhesion sensor which we have developed based on our fundamental studies in the fields of surface electrochemistry and biophysics.

Newly postulated biophysical processes of biopolymer selforganization into microparticles and mechanism of solgel phase transitions are held responsible for transformation of dissolved biopolymers and microparticles to macroscopic phases.

The macroscopic gel phase appears, episodically in Northern Adriatic, as large aggregates within the water column or covering tens of square kilometres of seasurface.

Current views leave no doubt on phytoplankton production and bacterial transformation of polysaccharides as main constituents of the gel matrix.

The phenomenon has so far been specific for Northern Adriatic but with the global climatic changes and increasing nutrient load it could be anticipated to spread over other coastal seas of Mediterranean.

There is by now accumulated evidence ranging from satellite observations to microbiological studies on sudden and dramatic changes of dispersed state microparticles to macroscopic gel-phase.

We introduce a simple electrochemical technique to detect microparticle precursors and to follow the transition to the gel phase.

When micrparticles attain critical concentration Nc our present estimate centers around Nc 5x L-1 the largescale phase transition from the dispersed to gel state takes place.

AFM is introduced to image 3-D structure of the gel matrix and biopolymer molecules. Thus, the goal of this study was to examine the effects on rat brain tissue of the nonenzymatic antioxidant hormone melatonin with Fourier Transform Infrared Spectroscopy FTIR.

Furthermore, a significant increase is observed in the frequency values of the PO2- symmetric stretching mode. Higher blood glucose levels induce metabolic disorders that initiate a sequence of events including renal, arterial, cardiac and retinal disorders.

Diabetes mellitus increases oxidative stress in tissues of both humans and animals. Increased oxidative stress might play a role in the development of diabetic complications.

The temperature dependent studies suggest that, at lower temperatures melatonin causes a decrease in the CH2 symmetric streching vibrational mode frequency, which indicates an increase in the order and thus the trans-gauche ratio of the crude brain membrane in a probable gel phase.

Then after about qC, it sets off an increase in the same frequency, indicating an increase in the number of gauche conformers leading to an increase in the disorder at a likely liquid-crystalline phase.

It is very important to note that body temperature is among the temperatures where melatonin induced an increase in disorder. In the present study, 8 male Wistar rats 4 diabetic and 4 controls were used.

Liver microsomal fractions were isolated. Protein amount in microsomal fractions of diabetic samples were significantly decreased as determined by the method of Bradford.

However, the CH2 symmetric stretching bandwidth did not differ significantly. The intensity of cm-1 band was increased for diabetic samples at different temperatures.

Secondary structure conformational changes have been investigated from the analysis of amide I band cm-1 using curve fitting procedure.

The observation of the disordering effect of melatonin at body temperature was also supported by the increases in the frequencies of both the CH3 and PO2- asymmetric stretching modes.

Taking into account that the CH2 symmetric, CH3 asymmetric and PO2- asymmetric strecthing modes give information about the order of the interior, deep interior and head-group regions of the lipid bilayer, respectively, the results suggest that melatonin induces a disordering effect on the lipid bilayer at physiological temperatures.

This seems rational since it is believed that a higher disorder of membrane lipids might make the interaction of antioxidants with lipid radicals more efficient.

This technique can be used in early diagnosis of several diseases by probing different functional groups belonging to different macromolecules such as lipids and proteins.

Ethanol was administered by a modified liquid diet with 4. Control rats were pair fed with an isocaloric liquid diet not containing ethanol.

In a parallel set of experiments the same plasmids were incubated in repair competent HEK protein extracts and the GGR repair rates were determined.

From the two sets of data the TCR rates were calculated. We found out that the four lesions were repaired with very similar efficiency by TCR pathway and with quite different efficiency by GGR.

In the latter case the repair rates generally corresponded to the degree of DNA helix distortion at the sites of damage.

Multiple host cell reactivation assay was carried out to study the competition of the different lesions for the TCR damage recognition factors in vivo.

We found out that the different lesions did not compete for the damage recognition factors, but that they compete with the transcribed genes for the transcription initiation factors.

A conclusion was drawn that the stalled RNA polymerase II and not the lesions themselves are the subject of recognition in the damage recognition step of TCR, and the distortion of the DNA helix does not play a role in the process.

Cognitive functions were evaluated by step-down passive avoidance test system for sec cut-off time in three individual groups of ethanoladministered, ethanol withdrawn 24th h withdrawal and control rats.

The daily ethanol consumption of the rats ranged from ChE activity was found significantly increased from 3rd day of ethanol 4. Serum ChE activities of the rats receiving ethanol 7.

Blood ethanol levels were measured as and 2. Passive avoidance latency was found significantly reduced in the groups that just before and 24th h of ethanol withdrawal as compared to control rats.

Xylulose 5-phosphate phosphoketolase EC 4. In presence of inorganic phosphate, this enzyme catalyses conversion of xylulose 5phosphate X5P into glyceraldehyde 3-phosphate and acetylphosphate.

So far, a limited number of molecular data are available for phosphoketolases, particularly for those from lactic acid bacteria LAB.

Their protein products are identified, and the pathway is reconstructed in vitro in considerable details. NER can remove a huge variety of lesions among them most of the DNA lesions caused by environmental agents by anticancer drugs.

There are two subclasses of nucleotide excision repair. One is the global genomic repair GGR which removes lesions throughout the genome regardless of whether any specific sequence is transcribed or not.

It can be studied in vitro, in cell free systems where no transcription of the repair substrate is taking place. There is no general way to measure TCR in vitro so far.

To study TCR we applied the following approach. Phosphoketolase gene of L. The bp long sequence was then transferred into a prokaryotic expression vector.

Optimized expression led finally to a soluble protein, which was purified using an affinity based approach.

Using a spectrophoptometric, coupled assay, the preliminary kinetic analysis was also performed. Altogether, these features define X5P phosphoketolase of L.

RP causes night blindness, a gradual loss of peripheral visual field and eventual loss of central vision. The disease can be inherited in autosomal dominant, autosomal recessive, X-linked and digenic modes.

Twentysix genes have been identified or cloned for RP and additional 14 genes mapped but not yet identified.

Five loci have been mapped for XLRP. ORF15 mutations have also been detected in simplex RP males.

These patients were the only ones showing the disease phenotype in their families. The main aims of this study were: to determine the prevalence of Y microdeletions among the infertile males from the Republic of Macedonia; to determine the breakpoints and the size of the deletions; to correlate the genotype with the phenotype; and to introduce the screening for Y chromosome microdeletions in the routine clinical practice.

Fifty fertile males and infertile males were included in the study. The breakpoints and the size of the deletions were determined by analysis of additional STS flanking markers.

Four of the 6 patients with AZFc microdeletion carried an identical deletion with a size of 3. The Y microdeletions were detected in six patients with azoospermia and one with severe oligozoospermia.

The prevalence of Y microdeletions among the infertile males from the Republic of Macedonia is 6. Testicular volumes were reduced in the patients with Y microdeletions, FSH concentrations were high and LH and testosterone were within the normal range.

Results showed heavy methylation of the rRNA genes. As judged from the almost total lack of digestion with Hpa II, in the repeating rDNA units there were either no methylation free regions or just a few were observed.

The methylation pattern of IGS was not changed upon hormonal treatment of cotyledons DNase I assay with isolated nuclei revealed defined regions of DNase I hypersensitivity in IGS which coincide with the regulatory elements.

No appreciable differences in DNase I hypersensitivity of IGS were found after hormone treatment of cotyledons nor in differentiated leaves of intact seedlings.

The obtained results are discussed with respect to the possible molecular mechanisms of cytokinin regulation of rRNA gene transcription.

Conclusion: These findings suggest that the eNOS polymorphism might influence the development of renal failure in Fabry disease.

Briefly, RNA from tumor samples was isolated according the method of Chomczynski and Sacchi ; total RNA was reverse transcribed with oligo dT primer; PCR was performed using the specific primers positioned in exons 4 and 6.

PCR bp product were analyzed on polyacrilamide gel electrophoresis. Level of expression was quantified densitometricaly and relatively ranged from 1 to 3.

Another disease in which the kidneys are dramatically affected is the monogenic X-linked Fabry disease. The Latter is characterized by progressive accumulation of lipids due to the deficiency of alphagalactosidase A activity preferentially in vascular endothelial cells EC which become activated.

Aim: The purpose of the study was to analyze whether the gene encoding endothelial markers such as endothelial nitric oxide synthase eNOS , and angiotensin converting enzyme ACE influence the development of nephropathy in DM and Fabry disease.

CYP2E1 is also involved in chemical carcinogenesis, both in the metabolic activation of small molecular weight organic chemicals and in the generation of ROS.

NDMA is known to be carcinogenic in liver, kidney and lung. Pyridine, constituent of tobacco and tobacco smoke, has tumor-promoting and teratogenic activities.

Following in vitro pyridine treatment of rabbits, NDMA Ndemethylase activity of both liver and lung is stimulated by 6.

In addition, experiments carried out in our laboratory demonstrated that in the experimentally induced diabetic rabbits, both CYP2E1 and NDMA N-demethylase activity have been increased about two-fold in kidney and liver of rabbits.

Therefore, it is expected that the incidence of tumor formation due to exposure to NDMA will be more pronounced in diabetic state because of CYP2E1 induction.

The majority of the organic carcinogens is not reactive as such but must undergo enzymatic reactions to form electrophilic species.

The first step in biotransformation is usually the oxidative step, catalyzed by microsomal cytochrome P CYP dependent monooxygenases.

Upto now more than P genes are sequenced and P families have been identified and 18 of them are found in humans. Thus induction of CYP1A1 is used to measure both exposure and resulting toxic carcinogenic effects of these types of organic pollutants.

Licensing of DNA for replication takes place only after completion of mitosis and ensures once per cell cycle replication.

Cdt1, a central licensing factor conserved across evolution, is tightly controlled in human cells, so that it is present only in the G1 phase, when licensing is legitimate.

Overexpression of Cdt1 leads to genomic instability. We have shown that ubiquitin dependent proteolysis ensures that Cdt1 is destroyed as soon as S-phase starts.

In contrast, cancer cells over-express both these proteins. Good correlation between smoking and lung cancer is established.

Individuals with a high inducible phenotype CYP1A1 show high risk of lung cancer. Gene-environment interactions are more pronounced at lower levels of cigarette exposure in which the susceptibility to lung cancer increased in the case of patients with either CYP1A1 mutated alleles and GSTM1 null gene.

CYP1A2 exhibits polymorphisms and inducibility. Aflatoxin B1 AFB1 must be activated to a reactive epoxide prior to exerting carcinogenic effects.

Time-lapse microscopy and fluorescence lifetime imaging microscopy FLIM allows us to identify the location and timing of protein-protein and protein-DNA interactions as cells progress through the cell cycle.

By the screening of the ABCA4 gene in various types of inherited retinal degenerations, more than two hundred disease-associated mutations reported in different populations.

It has been proposed that a nonsteroidal hormone such as insulin may exert an influence on steroid receptors and alter the cell responses to steroid hormone action.

Diabetes mellitus is very frequent metabolic disorder that also has implications on steroid hormone action. The main goal of the presented study was to elucidate the effects of insulin treatment and insulin deficiency on steroid receptors.

The experiments were performed on 3-months-old male Wistar rats. Protein contents of estrogen ER , progesterone PR , androgen AR and glucocorticoid receptors GR in liver were analyzed by immunoblotting method using appropriate specific antibodies.

The obtained results point out that streptozotocin did not cause significant changes in protein content of any kind of analyzed steroid receptors.

Insulin injection significantly decreased the AR protein content in intact rats, while there were no changes in protein content of other three analyzed steroid receptors in rat liver.

Insulin given to insulindeficient animals did not cause changes in steroid receptor content compare to intact rats. However, in comparison to diabetic rats there was the significant decrease in PR content, whereas protein contents of other steroid receptors were unchanged.

Further studies will be helpful in understanding of complex genotype- phenotype relationship in ABCA4 gene in our population.

The levels of PR and AR were not influenced by streptozotocin treatment, while, depend on endogenous level of insulin, hormone treatment decreased their content.

Grant number Idiopathic PD is the most common form of parkinsonism, but its etiology is still unknown.

Mitochondrial dysfunction that generates oxidative stress contributes to the etiology of idiopathic PD. The reduced complex I activity, that is one of the basic abnormalities responsible for mitochondrial dysfunction, has been reported in PD not only in the substantia nigra but also in peripheral tissues.

However, there are contraversing studies about the decrease in complex I activity in peripheral tissues of idiopathic PD, since methodological factors such as the use of homogenates or purified mitochondria or age differences between patients and control group affect those enzyme activities.

In order to eliminate these factors, in this study, we purified mitochondria from leukocytes of idiopathic PD patients and analyzed mitochondrial complex I and complex IV enzyme activities in these purified mitochondrial suspensions to evaluate the functional activity of the mitochondrial respiratory enzymes.

But, there was no significant correlation between these enzyme activities and age, age of onset and duration of the disease.

The observed respiratory chain enzyme deficiency supports the hypothesis that systemic mitochondrial dysfunction is important in the pathogenesis of idiopathic PD.

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.

In addition, we showed that substrate specifity of fpg protein. All chemicals purchased from Sigma. Retention time of 8OxoGua 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.

IC3 also inhibited the binding of I-galanin to GalR1. This suggests that the N-terminal part of IC3 defines the coupling of GalR1 to Gi1 and consequently, to the signal transduction cascade.

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.

Psichari, E. High activity of serum response factor in the mesenchymal transition of epithelial tumor cells is regulated by RhoA signaling.

Zoumpourlis, V. High levels of phosphorylated c-Jun, Fra-1, Fra-2 and ATF-2 proteins correlate with malignant phenotypes in the multistage mouse skin carcinogenesis model.

Oncogene 19, Knowledge from the presented and other studies was used to design the receptor derived synthetic peptides with the potential to regulate some physiological processes, as demonstrated by ex vivo functional studies on the isolated tissues.

Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor EGFR using a probability weighteddynamic Monte Carlo simulation.

By using a realistic multi compartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics.

Our new model also allows the incorporation of physio-chemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments.

To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor EGF and transforming growth factor- alpha TGF-a.

Our simulations predict that when EGFR is activated with TGF-a, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias towards the endosomal compartment.

Experiments confirm these predictions from our model and simulations. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude.

Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by highthroughput approaches, such as quantitative image analysis.

Roberts M. Constantinou Avenue, 35 Athens, Greece. Constitutively active forms of Ras are found in a variety of tumours 1 suggesting an important role for this pathway in cancer.

We have developed conditionally mouse and human cell systems of activated V12 mutant Harvey Ras oncogene expression.

Here we report for the first time that in the absence of growth factors initial cellular exposure to oncogenic Ras only transiently activated the same pathway in the nucleus by a mechanism which involves the phosphotyrosine phosphatase MKP-1 2.

We have also investigated the impact of transient nuclear MAPK activation on the cell cycle as well as to changes in global gene expression profiles by using high density microarray analysis.

We have also compared early events in Ras signalling with late nuclear effects of Ras associated with cell transformation 3, 4.

The interplay between proliferative and apoptotic signals mediated by Ras are going to be discussed. Bos, J. Cancer Res. Plows, D. In the present study we demonstrate that the post-translational processing proteolysis and covalent dimerization observed with the cysteineless recombinant human interferon-gamma rhIFNJ is tightly associated with its in vivo glycation.

Antiviral assay showed that the proteolysis lowered, while the covalent dimerization completely abolished the antiviral activity of rhIFN-J.

Bonchev Str. A series of 9 derivative genes obtained by systemic deletion of 3 codons was constructed and expressed in E. It was shown that the yield of recombinant protein gradually decreased and the solubility gradually increased with truncation of the Cterminus.

To avoid artifacts related with the imperfect folding of the proteins during purification, the biological activity of the hIFNJ proteins was measured in clear cell lysates containing the soluble fractions only.

The deletion of the C-terminus had a two step effect on both hIFNJ antiviral and antiproliferative activities. Whereas the removal of the last 3, 6 and 9 C-terminal amino acids led to a gradual increase up to 10 times in biological activity of hIFNJ, the deletion of more than 9 amino acids had an opposite effect.

The truncation of the whole unstructured C-terminal domain resulted in a fold decrease but not in a complete loss in biological activity of hIFNJ.

The latter was sequestered upon deletion of 24 amino acids, three of which belonged to the D-helical domain F. Conventional oximeters for whole blood do not assay for MetHb.

Liposome encapsulated hemoglobin LEH was used as a synthetic hemoglobin fluids. In addition, the effect of thermal incubation at 40qC on LEH was determined.

Our experiments stated that significant loss of OxyHb occurred after 4 hrs and all OxyHB was lost after 24 hrs. Also, singlet oxygen quenchers such as imidazole, sodium azide, and ascorbic acid were not protective against thermal incubation.

But radical scavenger sodium formate and antioxidant agents -tocopherol caused protection against thermal effect when they were added to the lipid mixture.

However, in a recent study Mironova, R. This approach can employ to determine LEH substitute, containing known amounts of the hemoglobin derivatives.

For Scws no enzymatic attachment to wall polysaccharides was postulated and the adsorption may simply be due to chemical properties of E-1,3 glucan which reacts with many proteins by hydrogen bonding.

All mutants were viable, however, some of them like scw4scw10 and scw4scw10bgl2, showed significantly increased fraction of dead cells in the culture.

Additional scw11 mutation suppressed the observed phenotype indicating an antagonistic behaviour of Scw11p to Scw4p, Scw10p and Bgl2p.

The SPSS program was used in the statistical evaluations. Successive mutations of PIR genes led to changes in cell morphology and stability and also to increased mortality.

Young cells seem to be more affected. Microscopic investigation showed an increased fraction of cells with more than one bud and in most cases daughter cells still attached to their mothers stained with methylene blue.

The results show that more training courses should be arranged about HbA1c and the complications of DM.

The other advantage of this study may be the initiation of the standardization of HbA1c measurements in Turkey. Some potential medical and biotechnological applications of the obtained results will be discussed.

This medium was serum free. Catabolic or anabolic agents were included in the medium. The media were changed every days and after each change of media, the used medium from each bone culture tube was analyzed individually for calcium release from the bone into the medium.

Bones were fixed with formalin and processed for histological examination. Bones without fixing with formalin were hydrolyzed and analyzed for hydroxyproline.

Amount of calcium measured from the bone organ culture medium after prostaglandine E2 PGE2 or human parathyroid hormone h-PTH fraction was included in the medium.

Both test substances cause calcium release or bones resorption. The consequences depend on the electric field features amplitude, frequency, spatial distribution and on the cell electrical characteristics conductivity and permittivity of the media which compound the cell.

On the other hand, the electrical potential induced across the cellular membranes is able to produce local reversible increase of the permeability electroporation allowing to the exogenous chemical species to diffuse into the cell.

Calcium release from the bone into the medium can be observed by using a calcium ion-selective electrode for the purpose of observation of resorption process.

This method is absolutely exact. The electro-mechanical techniques dielectrophoresis, electro-rotation and electro-orientation will be reviewed addressing the mechanisms, the theoretical models and the applications.

The multi-shell model and its use to the description of the yeast cells mechanical behavior in an external electric field will be discussed.

Also other techniques rely on the cell electro-mechanical behavior will be described. The resorption process in bone organ culture can be measured and evaluated by only measuring the calcium concentration amount in the medium.

Further analysis is not required. The results, thus, indicate that calcium can be considered as an independent index of bone resorption.

Both substances stimulate osteogenesis. In this case, increased calcium release into the medium did not occur. The electroporation of the artificial and natural membrane will be analyzed in connection with the mechanism of the phenomenon reversibility.

The diffusion of the different kind of molecules will be discussed. Measuring of calcium release from the bone into the medium using a calcium ion-selective electrode for observation and estimation of formation process is insufficient.

The combination of these effects with other techniques, as laser trapping of the cell, produced other complex noninvasive methods for cells investigation and manipulation that will be briefly presented.

A complex method of High Pressure Liquid Chromatography with Fluorescence detection HPLC-FD of hydroxyproline biomarker of colagen synthesis in bone matrix or time-consuming and expensive histological examination for osteoides observation are necessary.

However, both methods can be substituted with simple, reliable and practical method of biogravimetry, as established by Sofic and Goldhaber in Light intensified inhibition effect.

This fenomenon can be observed and estimated by conducting a calcium analysis. A potential molecular target is vascular cell adhesion molecule-1 VCAM-1 which is over-expressed in vivo by activated endothelial cells EC covering the developing atheromatous plaque and has an significant role in leukocyte adhesion to these cells.

Taking advantage that VCAM-1 can be induced in cultured EC in the presence of inflamatory cytokines and endotoxins, we searched for the mechanisms of interaction between activated EC and liposomes targeted to VCAM-1 expressed on the cell surface.

For binding studies the cells were incubated with fluorescently labelled liposomes for 2h at 4oC. To follow the fate of liposomes after binding to the cell's surface we analyzed the uptake and the transmigration of liposomes and the subsequently induced intracellular changes using radioactively labelled liposomes.

As methods, flow cytometry, liquid scintilation counting, fluorescence microscopy and fluorimetry were employed. Results: The data showed that: i liposome coupled to anti-VCAM-1 bind selectively to activated EC; ii the immunoliposomes are taken up by specific e.

Conclusion: The data suggest that VCAM-1 may be an appropriate molecular target for specific delivery of drugs to activated EC using immunoliposomes.

For the biosensors developed in order to optimize working conditions some optimization studies such as determination of optimum pH, temperature, the most suitable buffer system and concentration were done.

In the characterization studies of the biosensors substrate specificity, reproducibility, determination of interference effects of some substances, operational and storage stability experiments were done.

Results obtained in the optimization and the characterization studies for both two biosensors were also compared eachother.

That is why the determination of chromium in environmental and biological samples is of great interest. 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.

Biosensors are defined as an analytical device incorporating a biological sensing element such as enzyme, tissue, microorganism, cell, and DNA with a suitable transducer.

On the bases of the investigations made, the optimum reaction conditions were established: 4. The linear range of the calibration graph was up to ng ml-1 and the detection limit was 10 ng ml The method was applied to the analysis of Cr VI in industrial water with recoveries of 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.

Using the same doses of proton irradiation, with energy at the target of Doses of 12 and 16 Gy provoked growth inhibition of 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.

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 allelespecific 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.

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.

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 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 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 model is essentially a coupling model between brain electrical activity, metabolism, and hemodynamics. The model attempts to model the relationships between the above-mentioned parameters by means of 15 differential equations.

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.

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.

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.

In all cases they posses long term stability and the ability to take larger time steps while being economical in computer time. The dark relaxation of DF between 0.

Both the amplitudes and the life-times of the DF components drastically changed during the induction. The contribution of DF components was highly dependent on registration temperature.

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.

The enzyme exhibited an optimal pH of approximately Other cations, EDTA, pyrophosphate, vanadate and molybdate markedly inhibited the enzyme activity.

NaF, tartrate and okadaic acid had a less potent or not at all inhibitory effect. Its apparent Km for pnitrophenyl phosphate was 0. N-terminally characterization of protein exhibited high degree of similarity with the mature chain of alkaline phosphatases of PSTS family while partially internal sequence analysis showed that the protein showed similarities with ATPases involved.

Based on the above data and the enzyme substrates specificities that work as ATPase and phosphodiesterase activity we can conclude that this enzyme belongs to the phosphate specific transporter system PSTS which probably participate in the DNA repair.

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.

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.

Pinar T. Ozand MD, Ph. Saudi Arabia has an inordinately large number of autosomal recessive diseases. Hence primary TSH screening is the preferred method for screening in Turkey.

An average physician in this part of the world is not well aware of this disease and missed diagnoses lead to death and crippling.

While successful treatments are available. Homocystine is an end product of methionine metabolism.

It is toxic and will destroy Fibrillin. This leads to the dolicostenomelic features and cataracts of lens. It also damages vascular endothelium causing thrombosis.

There are two systems that get rid of it: 1 conversion to cystathionine, 2 conversion into methionine by methionine synthetase.

The deficiency of cystathionine synthetase, the first pathway, leads to the accumulation of very high levels of homocystine.

The toxic effects start appearing at years of age with classic clinical picture of HCU. The deficiency of methionine synthetase system leads usually to very early symptomatology mainly characterized by failure of the development of CNS.

This usually is caused by the deficiency of the cofactors of methionine synthetase, methylene-tetrahydrofolic MTHF acid and methylcobalamine.

These latter forms have milder elevations of homocystine and very low levels of methionine and are almost always missed.

The existence of a tandem MS has changed the prognosis of both forms of this disease. Early detection of CH through newborn screening proved to be one of the great successes of preventive medicine.

The screening should be oriented to detection of primary hypothyroidism. Simultaneous measurement of both T4 and TSH in dried blood spots is the most sensitive method to that effect, however it is not cost-effective.

Profiling of amino acids and acylcarnitines in a single analysis has enabled newborn screening programs to expand testing to include up to 30 treatable inborn error of metabolism IEM.

Besides the increase in the number of diseases covered, tandem MS has also improved testing from an analytical point of view.

The false positive rates are lowered because disorders are identified not only on the basis of quantification of metabolites but also by the screening for a pattern of metabolite abnormalities as opposed to screening for a single metabolite and also by measuring metabolite ratios.

The metabolic pathways and rationale for treatment of HCU will be discussed. Within the first group, three babies with PKU and one with Citrullinemia were identified.

In the latter group, we identified 8 amino acid disorders, 4 urea cycle defects, 7 organic acidemias and 1 fatty acid oxidation defect.

Early diagnosis and treatment prevents brain damage and the ensuing mental retardation. The recall rate was quite high at 2.

We identified 15 amino acid disorders and 13 organic acidemias The conclusions we can deduct from our experience with screening for IEM by tandem mass spectrometry are 1.

The overall frequency of IEM is high in our country and newborn screening for these disorders at least in a selected high risk group will be cost effective both for the family and for the society in the long run.

Quite a number of treatable IEM can be rapidly diagnosed from a very simple sample , namely a dried blood spot which is both easy to obtain, to tranport and to store.

This advantage should be made use of for screening IEM especially in states of emergency and in cases where laboratories capable of performing advanced metabolic tests are not readily available.

Some of these areas are: Pre implantation genetics, detection of metabolic diseases of foetus and screening for treatable metabolic diseases of new born.

Also, at the time, approach to disease detection is to look for mutations on the gene, but there are variations of mutations in each and every country even in the same country at different locations and sequence analysis is not a screening test.

This area is named foetal biochemistry and many biochemistry laboratories are shifting interest to this area. Without any doubt, the diagnosis of the diseases and abortion after the diagnosis will be within the frame of prenatal rules.

Foetal biochemistry will be the starting point for in utero genetic treatments in near future. We have aimed to share our experiment and knowledge on foetal biochemistry with our colleagues with this presentation.

Laboratory medicine is an important discipline in health care with its remarkable effect on risk assessment, diagnosis of health and disease state and especially from newborn screening approach with its, retest, recall and follow-up procedures.

This real life trial, emphasizes the need of split sample design evaluation of newly opened test kits. Quantitative measurement of phenylalanine and nTSH neonatal thyroid stimulating hormone were performed in both of the laboratories.

After validation of calibrations were performed in the laboratory that used these industrially prepared screening kits for the first time, the same real newborn blood spot samples were analysed for phenylalanine and nTSH measurements in both of the laboratories and the obtained results were compared non parametrically and examined by the Deming regression graph and by the difference plot.

However, nTSH values were found to be significantly higher in the laboratory that used the nTSH kit for the first time. This work implies that acceptable comparability of split sample design analysis is strictly needed for testing the analytical performance of the industrially prepared tests kits and this can be achieved only by certified reference materials.

These disorders which are mainly seen in the newborn period and early childhood are characterized by the high levels of one or more than one amino acids in the plasma or urine due to the enzyme deficiency.

In the light of these points, accurate, sensitive and prompt amino acid analysis in biological fluids is very important.

Blood, urine, cerebrospinal fluid, vitreous fluid and amnion fluid are used for the diagnosis of disorders of amino acid metabolism.

The analytical techniques for the measurement of amino acids can be investigated in two parts as screening tests and quantitative methods.

Screening tests including Guthrie test, thin layer chromatography, paper chromatography, photometric methods and spot tests in the urine.

In the recent years a world wide and important technique, that could screen many metabolic diseases in a single analytical step named tandem mass spectrometry has been used for this purpose.

Among the tests those are used for the quantitative measurement of amino acids are capillary electrophoresis, gas-liquid chromatography, high pressure liquid chromatography, ion-exchange liquid chromatography amino acid analayzer and tandem mass spectrometry.

High resolution nuclear magnetic resonance spectroscopy and molecular analysis are also used in amino acid measurements.

Tandem mass spectrometry TMS is an analytical technique that can be implemented in the analysis of blood spots taken shortly after birth. Rather than testing the blood for the presence of just one compound e.

MCAD deficiency and some organic acidemias e. One of the most recent and effective technic of those is tandem mass spectrometry.

Tandem mass spectrometry is a very important analytical technique that could determine many metabolic diseases from one blood sample in a very short time in a single analytical step.

Phenylketonuria, hyperphenylalaninemia, maple syrup urine disease, tyrosinemia type I and II, homocystinuria, hypermethioninemia are the disorders of amino acid metabolism those could be determined by tandem mass spectrometry.

In addition to amino acid disorders fatty acid oxidation disorders, organic acidemias and urea cycle defects could also be determined by tandem mass spectrometry.

This technique detects well amino acids and acylcarnitines; at this moment however it is still impossible to screen for congenital hypothyroidism or biotinidase deficiency, and there is only a limited experience in screening for congenital adrenal hyperplasia using this technique.

In the newborn period, screening tests were begun by the screening of phenylketonuria which is a kind of bacterial inhibition test progressed by Robert Guthrie.

Thin layer chromatography and paper chromatography are chromatographic methods those are used for the separation and determination of amino acids.

The sensitivity of the screening by TMS is high but the specificity can be rather low with a high rate of false positives resulting in a high number of retests and recalls: e.

A broad screening program also involves the detection of non-diseases e. MCAD def. In quantitative amino acid analysis with high pressure liquid chromatography the main steps of the method are the pre-column derivatization of the amino acids with phenylisothiocyanate, o-phthalaldehyde and other similar compounds, separation in reversed-phase column and detection with either ultraviolet or fluorescence detectors.

In amino acid analayzer the main part of the system is ionexchange column, followed by gradient elution. Long period of investigation is a disadvantage for these methods.

The great danger in my opinion of the technique of TMS is that labs that have no experience whatsoever with screening, will take over the newborn mass screening solely on the base that they possess such an instrument, Amino acid levels in body fluids are influenced by a number of factors, such as age, physiological changes, nutritional status, diseases, medications and toxins.

Also the factors such as collection time, transportation and keeping of the samples are very important. As a conclusion to gain success in the treatment and to prevent permanent sequels due to disorders of amino acid metabolism which makes up a great part of metabolic diseases, early diagnosis and treatment is highly important.

Polycystic ovary syndrome PCOS is the most common reproductive endocrinopathy of women in their childbearing years.

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