Volume 40, Issue 2

Objective: The lack of success with classical targeted therapies in cancer treatment has forced researchers to employ either combined therapies or agents that interfere with multiple pathways.In this study, a proteomic evaluation was performed to understand the in vitro antiproliferative effect of dietary compounds against leukemia cells in proteome level. Typical human cell models of leukemia (i.e., wild K562 cells and multi-drug resistant leukemia cells K562/R) were treated with rosemary (Rosmarinus officinalis L.) extracts rich in polyphenols and a comparative proteomic study was performed to identify up- or down-regulated proteins by in vitro antiproliferative effect of polyphenols. Methods: The protein fraction from the polyphenol-treated and control K562 and K562/R cells were separated through two dimensional polyacrylamide gel electrophoresis (2DE) and their proteomic profiles compared by image analysis. The proteins identified to be overexpressed or underexpressed in K562 and K562/R cells after the polyphenol treatment were identified by MALDI-TOF/TOF MS. Results: The results in this study were evaluated to understand the mode of action of these dietary constituents against leukemia cell proliferation. Dietary polyphenols extracted from rosemary bring about the up regulation or down regulation of different proteins on leukemia cells. These proteins are related to tumorigenesis, cancer proliferation and antioxidant activity. Conclusion: Our results revealed that the studied rosemary extract rich in polyphenols induced the down regulation of adenine phosphoribosyl transferase and annexin A1 in K562/R cell lines and tubulin alpha-1C chain in K562 cell lines. According to the expression proteomics results in this study, it could be concluded that the rosemary polyphenols shows in vitro antiproliferative activity in K562 and K562/R leukemia cell lines.

Objective: Disasters such as leakages and accidental falls are the main causes of environmental pollution by the petroleum industry product. Since no commercial yeast strain with biodegradation capacity is available, we aimed to isolate and characterize hydrocarbon degrading yeasts. Methods: Yeast isolates used in the study were isolated from samples of wastewater, active sludge and crude oil, which was obtained from a petroleum refinery as well as from soil samples, which were contaminated with crude oil. Yeast isolates used in the study were isolated from wastewater, active sludge and petroleum samples obtained from petroleum refinery and soil samples contaminated with petroleum. Degradation of naphthalene, phenanthrene, pyrene and crude petroleum by yeasts were determined using a microtiter plate-based method. Molecular characterization was achieved by performing a sequence analysis of the ITS1-5.8S rRNA-ITS2 and 26S rRNA regions. Results: In total, 100 yeast isolates were obtained from four different samples. Following the incubation in media containing different polycyclic aromatic hydrocarbon compounds (naphthalene, phenanthrene, pyrene) and crude petroleum, 12 yeast isolates were detected to degrade more than one polyaromatic hydrocarbon. Sequence analyses of rRNA regions revealed that the identified yeasts represented 10 species belonging to 6 genera. The isolates were identified as Candida parasilopsis, Candida sinolaborantium, Cryptococcus albidus, Cryptococcus diffluens, Cryptococcus uzbekistanensis, Pichia kudriavzevii, Rhodosporidium diobovatum, Rhodotorula glutinis, Rhodotorula muciloginosa and Saccharomyces cerevisiae. Conclusion: Yeast strains that are capable of degrading more than one polycyclic aromatic hydrocarbon compound have the potential of being utilized in future research.

Objective: Ciprofibrate (2-[4-(2,2-dichloro cyclopropyl) phenoxyl]-2-methyl propinoic acid) is a lipid-lowering drug used in the treatment of various metabolic diseases, especially hyperlipidemias. Human carbonic anhydrase II (hCA II) and glucose 6 phosphate dehydrogenase (G6PD) are crucial enzymes associated with many metabolic pathways. The aim of this study was to reveal the in vitro effect of ciprofibrate on the hydratase and esterase activities of hCAII and G6PD enzyme activity. Methods: hCA II hydratase activity was measured using the modified Wilbur-Anderson method, while esterase activity was determined using the method described by Armstrong et al. G6PD activity was determined using the Beutler method. Results: Different concentrations of ciprofibrate increased hCAII hydratase activity but exhibited no effects on hCA II esterase or G6PD activities. Conclusion: Since activation of hCA II is important in neurodegenerative diseases, such as Alzheimer’s, in which carbonic anhydrase activity decreases, the use of ciprofibrate may be proposed in the treatment of such diseases. However, this now needs to be confirmed by in vivo studies.

Objective: The purpose of the present study was to purify and characterize an esterase from a thermophilic bacterium Thermus sp. NCCB 100425T and to check its suitability for industrial applications. Methods: Thermus sp. NCCB 100425 T esterase was purified by using ammonium sulphate precipitation and hydrophobic interaction chromatography and then characterized biochemically. Results: The purity of the enzyme was observed as a single band on native- and SDS- PAGE. In the presence of p-nitrophenyl acetate (pNPA) as a substrate, the optimum pH and temperature of the enzyme were found to be 7.5 and 60°C, respectively. K m and V max values are calculated as 18.32 mM and 96.15 U/mg protein, respectively, with pNPA. pH stability was investigated in the range of pH 4.0-9.0 at 4°C and 60°C. After 7 days incubation, activity of pure enzyme was retained 85-90% for all pH at 4°C and 59±5.2% for pH 8.0 at 60°C. It was determined that approximately 80% of enzyme activity was retained between 30-60°C after 7 days incubation. In the presence of 10% ethanol and DMSO, the enzyme activity was retained 96±2.7% and 78±2.5%, respectively. Additionally, it was detected that some metal ions affect the enzyme activity at different ratios. Conclusion: It is clear that Thermus sp. NCCB 100425 T esterase might have advantages for industrial and/or clinical applications in terms of especially its high thermal- and pH-stability.

Objective: Amaranthus lividus is consumed as popular vegetable in West Black Sea Region of Turkey. In this study, we aimed to evaluate the protective and antioxidant effects of A. lividus on carbon tetrachloride (CCl 4 ) induced oxidative stress and acute liver injury in rats. Methods: Male albino Wistar rats were divided into 7 groups: Normal control, A. lividus control, silymarin control, CCl 4 , A. lividus (250 mg/kg)+CCl 4 , A. lividus (500 mg/kg)+CCl 4 , silymarin+CCl 4 . Rats were orally pretreated with A. lividus (250 and 500 mg/kg) or silymarin (25 mg/kg) daily for 9 days before administration of CCl 4 (1.5 mL/kg, 1:1 in olive oil, i.p.). Results: Pretreatment of rats with A. lividus, significantly prevented the CCl 4 induced elevation in the levels of serum alanine aminotransferase, aspartate aminotransferase, bilirubin and hepatic lipid peroxidation and myeloperoxidase. In addition, pretreatment with A. lividus significantly prevented the CCl 4 induced depletion in the activities of antioxidant enzymes such as catalase, glutathione-S-transferase, glutathione peroxidase, glutathione reductase, superoxide dismutase and glutathione level in liver. It has been observed that the hepatoprotective effect of A. lividus was comparable to that of silymarin, a standard drug. Histopathological evaluation of the liver also revealed that A. lividus at 250 mg/kg dose partially suppressed the CCl 4 induced liver damage in rats. Conclusion: Our results indicated that A. lividus has a protective effect against CCl 4 induced acute hepatotoxicity in rats, and this effect might be related to its antioxidant activity.

Objective: The objective of the study was to examine the nutritive value of Helix lucorum meat and to create awareness about fatty acid composition of phospholipid subclasses of the total body and cephalopedal tissues of the snail. Methods: Thin layer chromatography plates contained lipid samples placed in the chromatography tank containing: chloroform/ethanol/water/triethylamine. The phospholipid subclasses were dissolved in about 5 ml of methanol and 5 drop of sulfuric acid. The mixture was refluxed for 2 h to form fatty acid methyl esters at 85 °C. Fatty acids were detected by Gas chromatography. Results: The most noteworthy result was the high level of C20:2ω6 in PE (10.49%-11.35%) and PC (17.33%-12.96%). Appreciable quantity of essential fatty acid C18:2ω6 was determined in PC (20.85%-17.46%) and PE (16.88%-17.53%) from both tissues. Precursor of eicosanoids, C20:4ω6 was found apparently high in PI, PS and PE of the total body. The highest level of ΣPUFA was 63.90% in PE from total body whereas the highest level of ΣSFA was 60.79% in PI from the cephalopedal. ΣMUFA level was pretty low in PI, PS and PE. Conclusion: The study is a guide for biochemical and nutritional value of edible snails and can be useful for further investigation on physiological and systematic studies of other species

Objective: The aim of this study was to evaluate heavy metals (Cr, Fe, Ni, Cu, Zn, As, Cd, Hg, Pb) and selenium levels in digestive gland and gill tissues of wild Mediterranean mussel Mytilus galloprovincialis (Lamarck, 1819) collected from four locations in Izmir Bay. Methods: A total of 180 samples of the wild Mediterranean mussel M. galloprovincialis with shell length of 50-60 mm were collected from four locations. Inductively coupled plasmamass spectroscopy was used to determine heavy metals and selenium concentrations in mussel samples after microwave digestion process. Results: The highest values (mg kg -1 dry weight) obtained for digestive gland and gills were 0.17 and 0.15 for Cr, 28.62 and 29.49 for Fe, 0.25 and 0.29 for Ni, 2.53 and 1.78 for Cu, 18.52 and 22.03 for Zn, 1.26 and 1.08 for As, 0.04 and 0.04 for Cd, 0.02 and 0.02 for Hg, 0.19 and 0.16 for Pb, 0.40 and 0.48 for Se, respectively. Statistically significant differences among digestive gland and gills (p<0.05) were found for Cr, Ni, Zn, As, Se and Cd. In general, the levels of the studied elements were higher for the Karşıyaka than for other three stations. Conclusion: The low levels of toxic metals (Cd, Hg, Pb, Cr, Ni) found in tissues of M. galloprovincialis, comparing with the other studies including taken place in the Inner of Izmir Bay. M. galloprovincialis can be used as a sensitive biomonitor for the availabilities of studied elements in the Inner Bay of Izmir, Turkey.

Objective: Diabetes mellitus (DM) is a significant health problem and its treatmet has taken herbal and syntetic remedies as well as insulin therapy. The aim of this study was to evaluate the therapeutic effects of caffeic acid phenil ester (CAPE) in rats with streptozotocin (STZ)-induced diabetes using hystopathological and biochemical methods. Methods: 22 adult Wistar Albino strain male rats were divided into three groups: control group (n=8), diabetic group (n=6) and the diabetic+CAPE group (n=8). Two groups were injected intraperitoneally (i.p.) with 50 mg/kg STZ to induce DM. Rats with blood glucose levels of 270 mg/dL or greater and with siagns of polyuria and polydipsia after 3 days were considered to be diabetic. Diabetic+CAPE group was given ip CAPE (10 μmol/kg/during 60 days) for treatment. Results: Liver malondialdehyde (MDA) level was significantly higher in the diabetic group. The higher liver MDA was supported by higher enzyme levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). MDA levels in kidney were not significantly different between groups and superoxide dismutase (SOD) levels were increased in CAPE group. Additionally, blood urea nitrogen (BUN) and creatinine enzyme levels were mostly stable in serum samples. At the end of the experiment, pancreatic tissue MDA in the experimental groups decreased compared with the control group. The kidney, liver and pancreas had almost normal histological structure in the CAPE group when compared to the diabetic group. Considering blood glucose levels, CAPE treatment maintained blood glucose at the same level with controls, compared to diabetic group. Conclusion: Liver tissue might be the most affected by oxidative stress caused DM. A single injection of STZ decreased the level of MDA in pancreatic tissue at the end of experiment. This decrease might be exhausting MDA due to disruption in tissue integrity. Consequently, it was observed that CAPE suppressed oxidative stress and decreased glucose levels in STZ-induced diabetic rats. Thus, it may be useful to use as remedies, the antioxidant properties, in addition to anti-diabetic drugs in DM.

Objective: The aim of this study is to investigate the association of serum chemerin levels with metabolic parameters of women with polycystic ovary syndrome. Chemerin is an adipocytokine associated with adiposity, insulin resistance and metabolic syndrome, all of which are frequently observed in polycystic ovary syndrome. Methods: This is a prospective case-control study. We compared the metabolic, biochemical and anthropometric parameters of 35 women with polycystic ovary syndrome and 30 body mass index and age matched controls. Results: Chemerin levels of women with polycystic ovary syndrome were similar to that of the control group. Chemerin levels correlated positively with DHEAS (r=0.427, p=0.011) and LH (r=0.407, p=0.015) levels in women with polycystic ovary syndrome. There was a negative correlation between chemerin and lipoprotein-a levels in normal weight women with polycystic ovary syndrome (r=-0.426, p=0.043) and there was a negative correlation between chemerin and triglyceride levels in overweight women with polycystic ovary syndrome (r=-0.669, p=0.024). In obese control group chemerin correlated positively with C-reactive protein levels (r=0.738, p=0.037). Conclusion: In this study we could not detect a definitive role of chemerin in the pathophysiology of polycystic ovary syndrome, but its probable role in the development of the syndrome should be investigated with more detailed studies in the future.

Objective: Manganese superoxide dismutase (MnSOD,SOD2), the only known superoxide scavenger in mitochondria, may be particularly important for antioxidant defense because mitochondria are the major sites for cellular metabolism and hence production of reactive oxygen species. Methods: In this study, 440 Turkish individuals were genotyped for polymorphisms of SOD2 gene. The distribution of these polymorphisms in this population was examined using a PCRRFLP method. Results: In the present study, a total of 440(females: 201, 46% and males: 239, 54%) healthy individuals were studied. The mean age of the study population was 54,41±5,76 years (males, 55,34±5,76; females, 53,12±7,16). The observed genotype frequencies of SOD2 were 17.5, 50.5 and 32.0% for CC, CT and TT, respectively. Conclusion: This study provides basic information about the allele and genotype frequency distributions of polymorphisms of rs4880 in the SOD2 gene studied. These frequencies may be useful parameters as a reference for future studies on genetic basis of various diseases and cancer susceptibility.

Objective: The susceptibility of plasma adrenocorticotropic hormone (ACTH) to enzymatic degradation has limited the stability of ACTH. Many non-routine tests require the collection of blood on special proteinase inhibitor cocktails to preserve the integrity of the sample. This may provide benefits in sample collection and routine clinical practice. This study examined if a proteinase inhibitor, aprotinin, mixed with blood at the time of collection, would change the stability of ACTH in plasma at various temperatures for several hours and days using immunoassay. Methods: Sequential human blood samples were collected from 22 patients in EDTA collection tubes and EDTA plus aprotinin tubes, respectively. Plasma obtained was stored for 4 days at 4°C and on the other hand a second aliquot at room temperature. ACTH was measured immediately and after storage times of 1, 2, 4, 24 h, and 96 h at 4°C and other aliquots at room temperature. Results: Aprotinin plus K3EDTA-plasma samples centrifuged and stored at 4°C showed the best stability of ACTH up to 96 h compared to the ACTH concentration collected into K3EDTA tubes. Also, aprotinin plasma stored at room temperature showed no significant change according to baseline values. ACTH in plasma samples without aprotinin centrifuged and stored at 4°C declined in 2 h. Conclusion: The addition of aprotinin showed an improvement of the stability at 4°C/RT. Blood samples for ACTH measurement could be taken into blood tubes containing K3EDTA + aprotinin and without the need of keeping the sample on ice. These data indicate that aprotinin has a profound preservative effect upon plasma ACTH level and that it may be possible to submit unfrozen samples collected with this inhibitor to appropriate referral laboratories for analysis of ACTH immunoassay. From a practical standpoint, referral laboratories may wish to prepare collection tubes containing both EDTA and aprotinin for distribution to other laboratories.

Objective: Tuta absoluta is one of the most important insects that destroy tomato plants. The larvae of T. absoluta infest tomato plants leading to significant reduction in tomato crop yield. It has been reported that several insecticides exert their effects by inducing oxidative stress with stimulation of reactive oxygen species formation. The aims of this study were to determine oxidative status of two different field populations and to analyse detoxification mechanisms in T. absoluta field populations under the oxidative stress conditions by using biochemical methods. Methods: Total thiol levels and enzymatic activities of esterases, cytochrome P450 monooxygenases and glutathione S-transferases were determined. Results: The thiol content was decreased in Adana (19% less) and Antalya (25% less) field populations compared to a susceptible population. While CYP450s did not show any significant activity increase, acetylcholinesterases did not show any significant activity decrease in both the field populations. However, a significant increase of 2.3-fold in EST-PNPA activity in Adana population was determined. Furthermore, GST-DCNB activity showed 2.7-fold and 1.8-fold increase compared to susceptible population in Adana and Antalya field populations, respectively. Conclusion: Consequently, GSTs seem to have an important role in overcoming oxidative stress in tested field populations. As acetylcholinesterases and CYP450s did not show significant changes, it was concluded that they are possibly not involved in causing oxidative stress in field populations of T. absoluta. In addition, a significant increase of 2.3-fold in EST-PNPA activity in Adana population indicating that ESTs could play a role in causing oxidative stress in Adana field population.

Objective: Proteolytic enzymes are one of the well-known components of animal venom. The aim of this study was to identify protease from the venom gland of Scolopendra subspinipes dehaani. Methods: After total RNA extraction, a cDNA encoding a serine protease was identified by RT-PCR and was subsequently sequenced. The sequence was analysed by multiple alignment and homology modelling. Results: A cDNA encoding the precursor of a centipede venom serine protease was identified. The full-length cDNA was 1,014 nucleotides with 780 nucleotides of open reading frame. The precursor nucleotide sequence encoded a signal peptide of 19 residues and a mature protein of 260 residues. It belonged to clan PA of serine proteases in the MEROPS database classification. Catalytic residues were identified by multiple alignments, including an unusual hydrogen bond network in the catalytic site. Secondary structure prediction and homology modelling revealed a chymotrypsin-like fold, which is characteristic of PA clan proteases. Conclusion: The sequence of the identified serine protease showed unusual features, making this centipede venom serine protease an interesting candidate to investigate. A better understanding of the molecular mechanism of venom components will be useful for further applications and help to improve envenomation treatment.

Objective: The aim of this study is to investigate the in vitro antioxidant activity, the total phenolic and flavonoid contents and the possible protective effects of seven cultivars of olive on lipid peroxidation induced by iron in mice liver. Methods: Antioxidant activity was evaluated by using different assays, including thiobarbituric acid reactive species (TBARS), total antioxidant, 2,2-diphenlyl-1-picrylhydrazyl (DPPH) radical and metal ion-chelating activities. Results: The aqueous extracts of fruit showed inhibition against thiobarbituric acid reactive species (TBARS) induced by pro-oxidant (10 μM FeSO4) in mice liver. The order of the antioxidant activity among different cultivars on lipid peroxidation assay is Maurino>Mission> Picholine>Ascolano>Carolea>Itrana>Pedolina. Moreover, the free radical scavenging activities of the extracts was evaluated by the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (IC 50 , 85.9 μg/ml (Ascolano), 82.1 μg/ml (Mission), Pedolino and Carolea >200 μg/ml, 85.1 μg/ml (Picholine), 143.6 μg/ml (Maurino) and 150 μg/ml (Itrana). Principal component analysis showed that phenolics and flavonoids contributed to the antioxidant activity of olives. Conclusion: The oxidative stress in liver can be potentially managed/prevented by the dietary intake of olive fruit.