Purification of lactoperoxidase from creek-water buffalo milk and investigation of kinetic and antibacterial properties

Water buffalo lactoperoxidase (WBLP) was purified with Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skim milk. All purification steps of the WBLP were shown with SDS-PAGE and Rz (A412/A280) controlled the purification degree of the enzyme. Rz value for the purified WBLP was 0.8. To determine purification steps and kinetic properties, the activity of enzyme was measured by using 2,2-azino-bis-(3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH=6. Km, Vmax, optimum pH, and optimum temperature for the WBLP were found by means of graphics for ABTS as substrates. Optimum pH and optimum temperature of the WBLP were 6 and 60 degrees C, respectively. Km value at optimum pH and optimum temperature for the WBLP was 0.82 mM. Vmax value at optimum pH and optimum temperature was 13.7 micromol/mL x min. Km value at optimum pH and 25 degrees C for the WBLP was 0.77 mM. Vmax value at optimum pH and 25 degrees C was 4.83 micromol/mL x min. The purified WBLP was found to have high antibacterial activity in a thiocynate-H2O2 medium for some pathogenic bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginose, Shigella sonnei, Staphylococcus saphrophyticus, Staphylococcus epidermidis, and Shigella dysenteriae and compared with well known antibacterial substances such as tetracycline, penicillin, and netilmicine.     

Combined point mutations in codon 12 and 13 of KRAS oncogene in prostate carcinomas

Prostate cancer is a common malignancy that develops by structural mutation(s) and/or other genetic alterations in specific genes.The G to T transversions in codon 12 and C to T transitions in codon 13 of KRAS proto-oncogene are predominant point mutations that occur in about 20% of different cancers in human. In the current study it was aimed to investigate the prevalence and predictive significance of KRAS mutations in patients with prostate carcinomas. In a total of 30 fresh tumoural tissue specimens were investigated in patients with prostate carcinoma. All tumoural specimens were histo-pathologically diagnosed and genotyped for codon 12, 13 KRAS point mutations by reverse hybridisation and direct sequencing methods. KRAS mutations were found in 12 (40%) samples with 29 samples deriving from adenocarcinomas and 1 sample was small cell prostate carcinoma. In 1 (3.44%) sample codon 12 was found to be mutated and in 2 (6.8%) samples codon 13 and in 9 (31%) samples combined codon 12 and 13 were found to be mutated particularly in higher grade of tumoural tissues. Our study, based on representative collection of human prostate tumours, indicates that combined mutations in codons 12 and 13 KRAS are relatively infrequent and most commonly occur in prostate carcinomas.     

Gastroprotective,cytoprotective and antioxidant effects of Oleum cinnamomi on ethanol induced damage

Peptic ulcer disease is a gastrointestinal disorder defined by mucosal damage and free oxygen radicals associated with peptic ulcer and gastritis. Cinnamon is a traditional herb used for many diseases and it has also effects as an antioxidant, anti-inflammatory, antispasmodic and anti-ulcerative. Our research is based on oxidative stress and effects of Oleum cinnamomi on stomach, liver and kidney disorders induced by ethanol. In our experiment, 2–3 month old male Sprague–Dawley rats were used. One hour before the mucosal damage induced by 70 % ethanol, O. cinnamomi (2.5 ml/kg) was added into the groups. Gastric pH, analysis of gastric mucus and ulcer index were calculated from samples obtained from the stomach. Superoxide dismutase (SOD), malondialdehyde and catalase (CAT) levels were determined in stomach, liver and kidney homogenates and erythrocyte hemolysate. Histopathological examination of stomach, liver and kidney were determined with H&E staining. The non-treated ulcerative group showed higher scores than the control group which was treated with O. cinnamomi, when ulcer scores, gastric mucus and pH level of stomach are compared. Increased lipid peroxidation levels were observed in the liver, kidney and erythrocyte hemolysate. SOD activity was decreased in liver whereas increased in stomach of ethanol treated ulcerative groups. CAT levels were increased in stomach and liver of ethanol treated rats. Histopathological findings showed that ethanol treatment cause multiply organ damage such as stomach, liver and kidney injury. O. cinnamomi treatment protected these tissues from ethanol-induced damage. Consequently, the current investigation shows that O. cinnamomi has protective effects on ethanol-induced oxidative and mucosal damage.     

Anoxybacillus sp. SO B1–Immobilized Amberlite XAD-16 for Solid-Phase Preconcentration of Cu(II) and Pb(II) and Their Determinations by Flame Atomic Absorption Spectrometry

A new method for the determination of Cu(II) and Pb(II) by flame atomic absorption spectrometry (FAAS) after preconcentrating on a column containing Anoxybacillus sp. SO B1–immobilized Amberlite XAD-16 was developed. The functional groups of Anoxybacillus sp. SO B1 immobilized on Amberlite XAD-16 were characterized in KBr tablets by Fourier transform infrared (FT-IR) spectrometry. Various parameters such as pH, amount of the adsorbent, eluent type and volume, and flow rate of the sample solution were studied. The optimum pH values of quantitative sorption for Cu(II) and Pb(II) were found to be pH 7.0 and 5.0 and Cu(II) and Pb(II) ions could be quantitatively eluted with 5.0 ml of 1.0 mol L^?1 HCI and 10.0 ml of 0.25 mol L^?1 HNO₃, respectively. Recoveries of Cu(II) and Pb(II) were found to be 100.9 ± 1.57% and 100.3 ± 0.49% (N = 5), the limits of detection of Cu(II) and Pb(II) in the determination by FAAS (3 s, N = 10) were found to be 0.8 and 1.6 μg L^?1, respectively. The proposed enrichment method was applied for metal ion determination from water samples such as two parts of Tigris River water in Diyarbak?r and Elaz??, Lake of Hazar in Elaz??, and tap water in Diyarbak?r. Furthermore, the accuracy of the proposed method was verified by studying the analytical recovery and by analyzing certified reference material (NCS-DC 73350 leaves of poplar).     

Antimicrobial activity of extracts of chemical races of the lichen Pseudevernia furfuracea and their physodic acid, chloroatranorin, atranorin, and olivetoric acid constituents

The antimicrobial activity and the MIC values of the ethanol, chloroform, diethyl ether, and acetone extracts of the chemical races of Pseudevernia furfuracea (var. furfuracea and var. ceratea) and their physodic acid, chloroatranorin, atranorin, and olivetoric acid constituents have been investigated against some microorganisms. Nearly all extracts of both chemical races showed antimicrobial activity against Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica, Candida albicans, Candida glabrata, Alternaria alternata, Ascochyta rabiei, Aspergillus niger, Fusarium culmorum, Fusarium moniliforme, Fusarium oxysporum, Fusarium solani, and Penicillium notatum. There was no antimicrobial activity of the extracts against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas syringae, Salmonella typhimurium, Alternaria citri, Alternaria tenuissima, and Gaeumannomyces graminis. Chloroatranorin and olivetoric acid were active against the same microorganisms with few exceptions. Physodic acid was active against about the same bacteria and yeasts and inactive against all of the filamentous fungi tested. Also no activity of atranorin against the filamentous fungi was observed.     

Lithium-Induced Hypothyroidism: Oxidative Stress and Osmotic Fragility Status in Rats

The present study was conducted to explore the possible effects of different doses of lithium carbonate on thyroid functions, erythrocyte oxidant–antioxidant status, and osmotic fragility. Twenty-four Wistar-type male rats were equally divided into three groups: groups I and II received 0.1 and0.2 % lithium carbonate in their drinking water, respectively, for 30 days. The rats in group III served as controls, drinking tap water without added lithium. At the end of the experimental period, the erythrocyte osmotic fragility and the levels of triiodothyronine (T₃), thyroxine (T₄), thyroid-stimulating hormone (TSH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were measured in blood samples. Compared to controls, there was a statistically significant increase of TSH but decreases of the T₃ and T₄ levels in group II. Both experimental groups showed a statistically significant increase of the maximum osmotic fragility limit. The minimum osmotic fragility values of the animals in group II were statistically higher than those of controls. The standard hemolytic increment curve of both experimental groups was shifted to the right when compared to the curve obtained from the controls. Also, relative to controls, the activities of MDA and SOD were significantly higher and the GSH level lower in group II, but not so in group I. The results of the present study show that treatment with lithium carbonate may result in thyroid function abnormalities, increased oxidative damage, and possible compromise of the erythrocyte membrane integrity resulting from increased osmotic fragility.     

Effects of curcumin on bleomycin-induced apoptosis in human malignant testicular germ cells

Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a frequently used drug for the treatment of several malignancies and is part of the chemotherapy protocols in testicular cancer. Bleomycin causes an increase in oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has attracted interest because of its anti-inflammatory and chemopreventive activities. However, no study has been carried out so far to elucidate its interaction with bleomycin in testicular cancer cells. In this study, we investigated the effects of curcumin and bleomycin on apoptosis signalling pathways and compared the effects of bleomycin with H₂O₂ which directly produces reactive oxygen species. We measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and Cyt-c levels in NCCIT cells incubated with curcumin (5 μM), bleomycin (120 μg/ml), bleomycin + curcumin, H₂O₂ (35 μM), and H₂O₂ + curcumin for 72 h. Curcumin, bleomycin, and H₂O₂ caused apoptosis indicated as increases in caspase-3, caspase-8, and caspase-9 activities and Bax and cytoplasmic Cyt-c levels and a decrease in Bcl-2 level. Concurrent use of curcumin with bleomycin decreased caspase activities and Bax and Cyt-c levels compared to their separate effects in NCCIT cells. Our findings suggest that concurrent use of curcumin during chemotherapy in testis cancer should be avoided due to the inhibitory effect of curcumin on bleomycin-induced apoptosis.