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

ORAL PRESENTATION 2

Güvenç Görgülü*, Sevgi Görgülü**, Ökkeş Yılmaz***, Tülin Güray* and Feride Severcan**

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.

In the present study, 8 male Wistar rats (4 diabetic and 4 controls) were used. Diabetes was induced by an injection of streptozotocin (STZ) (50 mg/kg i.p.) after a 24-h fast. Liver microsomal fractions were isolated. Lipid peroxidation in microsomes was analyzed both by TBA-RS test and FT-IR study. Protein amount in microsomal fractions of diabetic samples were significantly decreased as determined by the method of Bradford. This was further supported by FT-IR study measuring the lipid-to-protein ratio from CH₂ symmetric/CH₃ symmetric bands. The results of FT-IR spectral analysis of absorption and second derivative signals revealed that significant increase in wavenumber of the CH₂ asymmetric (p<0.005) and the CH₂ symmetric (p<0.05) stretching vibrations were obtained, indicating an increase in disorder in diabetic rat liver microsomes. The bandwidth of the CH₂ asymmetric stretching band significantly increased for diabetic samples (p<0.05). However, the CH₂ symmetric stretching bandwidth did not differ significantly. Olefinic band (=CH at 3012 cm^-1) was analyzed by measuring the intensity of this band, which shows the degree of lipid peroxidation in the system. The intensity of 3012 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 (1653 cm^-1) using curve fitting procedure.

Use of FT-IR spectroscopy for diagnostic purposes has been an increasing demand by researchers. 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.

Key Words: FTIR spectroscopy, diabetes, liver tissues, microsomal membranes, lipid peroxidation

ORAL PRESENTATION 3

S. Banu AKKA޹, Faruk ZORLU² and Feride SEVERCAN¹

The brain is highly vulnerable to free radical attack since it is rich in polyunsaturated fatty acids and consumes very high amounts of oxygen. Thus, the goal of this study was to examine the effects on rat brain tissue of the non-enzymatic antioxidant hormone melatonin with Fourier Transform Infrared Spectroscopy (FTIR).

The results of the present study reveal that melatonin induces a significant increase in the relative lipid to protein ratio of the melatonin treated samples. Furthermore, a significant increase is observed in the frequency values of the PO₂^- symmetric stretching mode.

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 29-32C, 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.

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.

ORAL PRESENTATION 4

Erdinç ÇAKIR¹, Turgay. ÇELİK², Hakan KAYIR², Cumhur BİLGݹ, and I. Tayfun UZBAY²

The aim of the present study was to investigate if the chronic ethanol administration by liquid diet to rats may be a dementia model.

Female Wistar rats (188-244 g) were used in the study. Ethanol was administered by a modified liquid diet with 4.8% (v/v) ethanol for 3 days followed by 25 days on a liquid diet in which the ethanol concentration was increased to 7.2%. Control rats were pair fed with an isocaloric liquid diet not containing ethanol. Serum ChE activity and blood ethanol concentration were measured at the end of the 4.8% ethanol consumption and after 35 days of ethanol (7.2%) feeding and, just before, 24^th and 72^nd hours ethanol witdrawal period. Cognitive functions were evaluated by step-down passive avoidance test system for 150 sec (cut-off time) in three individual groups of ethanol-administered, ethanol withdrawn (24^th h withdrawal) and control rats. The data was evaluated by one-way analysis of variance followed by Tukey’s test for post-hoc comparison.

The daily ethanol consumption of the rats ranged from 11.5 to 14.9 g/kg. ChE activity was found significantly increased from 3^rd day of ethanol (4.8%) consumption. Serum ChE activities of the rats receiving ethanol (7.2%) also increased significantly as compared to ethanol (4.8%) ingesting rats. Blood ethanol levels were measured as 200 and 2.2 mg/dl at 35^th days of ethanol consumption (just before ethanol withdrawal) and 24^th h of ethanol withdrawal, respectively. Passive avoidance latency was found significantly reduced in the groups that just before and 24^th h of ethanol withdrawal as compared to control rats.

Our results suggest that serum ChE activity increased by chronic ethanol consumption in rats and chronic ethanol caused some marked impairments on the cognitive functions Overall the data indicated that chronic ethanol feeding might be a model for evaluation cognitive functions in rats.

OCTOBER 14 , 2003 - TUESDAY

HALL A

LECTURE 1

GLOBAL GENOMIC AND TRANSCRIPTION-COUPLED DNA REPAIR RATES IN HUMAN CELLS

George Russev, Boyko Atanassov, Emil Mladenov, Anelyia Velkova, Boyka Anachkova

The most general and for historical reasons the best-studied DNA repair pathway is the human nucleotide excision repair (NER) pathway. It includes 7 genes designated XPA through XPG. 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. The other is transcription-coupled repair (TCR), which removes lesions only from DNA sequences, which are transcribed. There is no general way to measure TCR in vitro so far. To study TCR we applied the following approach. Human HEK 293 cells were transfected with pEGFP and pEYFP plasmids treated with UVC light, cis-DDP, Angelicin and Trioxsalen and 24 hours later the restored fluorescence was measured and used to calculate the combined TCR+GGR rates. In a parallel set of experiments the same plasmids were incubated in repair competent HEK293 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.