Tülin Güray

Interindividual variability of coumarin 7-hydroxylation in a Turkish population.

One hundred healthy Turkish volunteers (70 male, 30 female) aged from 19 to 56 years were given 5 mg coumarin p.o. after an overnight fast. Urine samples were collected before and 2, 4 and 8 h after drug administration. The extent and rate of formation of 7-OH-coumarin (7OHC) was determined by the urinary excretion of the metabolite as measured with the fluorometric method.On average, 80% of 7OHC formed was excreted in 2 h. The total amount of 7OHC formed was 59.8% (21.5%) (mean and SD, n=100, range 17–100%) of the given dose. The percentage of 7OHC excreted during the first 2 h compared with the 7OHC excretion at 8 h was a constant and stable individual characteristic for the rate of the formation of 7OHC (‘2 h coumarin test’).Although four individuals had relatively slow coumarin test values (34–40%), no clear-cut polymorphism in the rate of 7OHC formation was found. However, 7OHC formation was lower in males and in cigarette smokers.

Effect of vitamin C and lipoic acid on streptozotocin-induced diabetes gene expression: mRNA and protein expressions of Cu–Zn SOD and catalase

The involvement of oxidative stress in the pathogenesis of diabetes mellitus has been confirmed by numerous studies. In this study, the expression of two antioxidant enzymes, superoxide dismutase (SOD), and catalase which are involved in the detoxification of reactive oxygen species was studied in the streptozotocin-induced diabetic rat liver tissues. The enzyme assays showed a significant decrease in both enzymes activities compared to control animals. The RT-PCR and Western-blot analysis results demonstrated that this decrease in activity is regulated at the level of gene expression, as both catalase and Cu–Zn SOD mRNA and protein expressions were also suppressed. Supplementing the animals with vitamin C, a powerful antioxidant increased both SOD and catalase activities with no change in both mRNA and protein expressions suggesting a role of post-translational modification. However, even though mRNA expressions of both catalase and Cu–Zn SOD were not changed, the protein levels increased in parallel to activities in the case of another antioxidant, α-lipoic acid. An increase in the rate of translation, without changing the rate of transcription indicates a translational effect of lipoic acid in changing the activities of antioxidant enzymes to prevent the oxidative damage in diabetes.

Gene expressions of Mn-SOD and GPx-1 in streptozotocin-induced diabetes: effect of antioxidants

Increased oxidative stress and impaired antioxidant defense mechanisms are believed to be the important factors contributing to the pathogenesis and progression of diabetes mellitus. In this study, we have reported the effects of the streptozotocin-induced diabetes on the gene expression and the activities of two antioxidant enzymes, manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPx). We also studied the effects of two antioxidants, vitamin C and DL-α-lipoic acid (LA), on the system. Our results showed no significant change in both enzymes activities in diabetic animals compared to controls. Similarly, mRNA and protein profiles of MnSOD showed no change. Though the mRNA expression of GPx did not show any change, Western-blot analysis results demonstrated that protein expression is increased. LA, which is a water- and lipid-soluble antioxidant, decreased the protein expression of MnSOD, though mRNA levels and activities remained unchanged. LA treatment increased the GPx activities in diabetic tissues, significantly, and RT-PCR and Western-blot analysis results demonstrated that this increase in activity is not regulated at the gene level, as both mRNA and protein levels did not change. Supplementing the animals with vitamin C, a powerful water-soluble antioxidant, increased the mRNA expression of MnSOD, though the protein expression and the activity did not change statistically. On the other hand GPx activity increased significantly through post-translational modifications, as both mRNA and protein expressions did not change. These results together with our previous findings about the gene expressions of catalase and Cu–Zn SOD indicate the presence of very intricate control mechanisms regulating the activities of antioxidant enzymes in order to prevent the damaging effects of oxidative stress.

Changes in expression profiles of antioxidant enzymes in diabetic rat kidneys

In diabetes mellitus, increased formation of reactive oxygen species due to high level of glucose in both blood plasma and tissues creates oxidative stress and damages the tissues. Antioxidants together with the antioxidant enzymes are very important in order to protect the cells against oxidative damage.

Stimulatory effects of benzene on rabbit liver and kidney microsomal cytochrome P-450 dependent drug metabolizing enzymes

Treatment of rabbits with benzene (880 mg/kg/day), s.c. for 3 consecutive days, caused 3.8- and 5.7-fold increases in aniline 4-hydroxylation rates of liver and kidney microsomes, respectively. Benzene treatment markedly enhanced hydroxylation rates ofp-nitrophenol by liver and kidney by 7.2- and 4.2-fold, respectively. Both of these enzymes are associated with cytochrome P-450 LM3a. In contrast, the activity of benzphetamine N-demethylase, associated with P-450 LM2, was not altered significantly in either liver or kidney microsomes. Although the total cytochrome P-450 contents of liver and kidney microsomes were not altered significantly by the benzene treatment, in the case of liver microsomes, formation of a new cytochrome P-450 with an apparent Mr of 51,400 was observed on SDS-PAGE. On the other hand, in the kidney microsomes, the intensity of the bands corresponding to approximate Mr of 50000 and 51400 was markedly increased. The results of the present work, in combination with those of the previous work (Arinç et al. 1988), indicate the existence of tissue specificity in the induction of rabbit P-450 isozyme by benzene.

Isolation of a chitosan degrading fungus, Penicillium spinulosum, and chitosanase production by the isolate*

A chitosan degrading fungus was isolated from chitosan and then identified as Penicillium spinulosum by the International Mycological Institute, England. The isolate was used for the production of chitosanase in a salts medium with either chitosan or Rhizopus cell walls as the sole carbon source. Chitosanase was produced under all the conditions tested, however better yields were obtained by using Rhizopus cell walls.

Abstract 1125: Eukaryotic Elongation Factor 2 Kinase (eEF-2K) is a novel therapeutic target in BRCA1+ mutated breast cancer

BReast CAncer 1 and 2 (BRCA1/2) tumor suppressor genes are frequently mutated in familial breast and ovarian cancers. Recent clinical trials in breast cancers with BRCA1/2 mutations indicate that 50-75% of patients may not have response to poly (ADP-ribose) polymerase inhibitors (PARP) inhibitors. Thus, identification of novel molecular targets and therapeutic interventions are needed to improve poor clical outcome and patient survival. Here we report that Eukaryotic Elongation Factor-2 Kinase (EF2K), an atypical kinase, is highly upregulated in BRCA1+ mutated cell lines and involved in colony formation, migration/ invasion and enhances the effect of PARP inhibitors. Furthermore, in vivo therapeutic targeting of EF2K by once a week systemic injections with magnetic nanoparticles (MNPs) incorporating EF2K siRNA significantly inhibits growth of BRCA1+ tumors in orthotopic xenograft models in mice by inhibition of tumor cell proliferation, angiogenesis and induction of robust apoptosis, which were associated with in vivo inhibition of clinically relevant molecular pathways including, proliferation, survival/drug resistance (PI3K/Akt, c-Myc), cell cycle (Cyclin D1), motility/invasion (Src/FAK/Paxillin), translation (4E-BP1), angiogenesis (VEGF) and stem-cell markers. No toxicity was detected during the study and by postmortem analysis of major organs, indicating that targeting EF2K is safe. The combination of PARP inhibitor (AZD2281, Oleparib) with MNP-EF2K siRNA enhanced significantly growth inhibition in MDA-MB-436 cells indicting EF2K siRNA sensitize BRCA1 + mutation breast cancer cells to PARP inhibitors. Collectively, our results suggest, for the first time, that EF2K is involved in tumorigenesis and progression and may serve as a potential therapeutic target in BRCA1+ mutated breast cancers. Citation Format: Elif Asik, Nermin Kahraman, Tulin Guray, Murvet Volkan, Gabriel Lopez-Berestein, Bulent Ozpolat. Eukaryotic Elongation Factor 2 Kinase (eEF-2K) is a novel therapeutic target in BRCA1+ mutated breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1125. doi:10.1158/1538-7445.AM2017-1125

Sheep Tissue Acetyl Coenzyme A-Dependent Arylamine N-Acetyltransferases

Acetyl coenzyme A-dependent arylamine N-acetyltransferases (NATs), EC 2.3.1.5. were measured in sheep tissues (N = 14) using p-aminobenzoic acid (PABA) as a substrate. Specific activities of liver, kidney, and lung NATs were 5.3 x 10(-3) +/- 1.4 (mean +/- SE) nmoles.min-1.mg protein-1, 3.4 x 10(-3) +/- 1.1 nmoles.min-1.mg protein-1, 2.5 x 10(-3) +/- 1.2 nmoles.min-1.mg protein-1, respectively. Km values were 53 +/- 3 microM for liver, 34 +/- 2 microM for kidney, and 28 +/- 2 microM for lung tissue. Optimum pH for the acetylation reaction was found as 7.5. The enzyme activity was stable for at least 6 months in all tissues, when stored at -70 degrees C. The enzyme from sheep tissues were quite heat-stable. Inhibition studies showed that N-ethylmaleimide was a potent inhibitor of sheep tissue NAT enzymes.