Identification of bacterial species in milk by MALDI-TOF and assessment of some oxidant-antioxidant parameters in blood and milk from cows with different health status of the udder.

This study aimed to identify bacterial pathogens in milk samples from dairy cows with subclinical and clinical mastitis as well as to assess the concentrations of oxidant-antioxidant parameters [malondialdehyde (MDA), reduced glutathione (GSH), and total GSH levels] in both blood and milk samples. From a total of 200 dairy cows in 8 farms, 800 quarter milk samples obtained from each udder were tested in the laboratory for the presence of udder pathogens. Cultivated bacteria causing intramammary infection from milk samples were identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). In addition, from tested animals 60 cows were selected including 20 healthy cows that were CMT negative, 20 cows with subclinical mastitis (SM), and 20 cows with clinical mastitis (CM) for detection of MDA, GSH, and total GSH levels in blood and milk samples. Three hundred and eighty (47.5%; 380/800), 300 (37.5%; 300/800), and 120 (15%; 120/800) of milk samples, respectively were CMT positive or SM and CM, and those positives were cows from different farms. We observed that 87.4% (332/380), 25.3% (76/300), and 34.2% (41/120) of cows with CMT positive, CMT negative, and CM had bacterial growth. The most predominantly identified bacteria were Staphylococcus chromogenes (18.7%) obtained mainly from SM and Staphylococcus aureus (16.7%) as the most frequent cause of CM. According to our results, dairy cows with CM had the highest MDA levels, the lowest GSH, and total GSH levels in both blood and milk samples however, high MDA levels and low GSH levels in milk samples with SM were observed. Based on our results, lipid oxidant MDA and antioxidant GSH could be excellent biomarkers of cow's milk for developing inflammation of the mammary gland. In addition, there was no link between nutrition and MDA and GSH levels.

Role of different types of potassium channels in the relaxation of corpus cavernosum induced by resveratrol.

BACKGROUND: Resveratrol (RVT), one of the most commonly employed dietary polyphenol, is used in traditional Japanese and Chinese medicine for treatment of cardiovascular diseases. Recently, we have shown that RVT has a potent relaxant effect on rat corpus cavernosum via endothelium-dependent and -independent mechanisms. OBJECTIVE: The present study addressed the question whether different types of potassium channels are involved in the endothelium-dependent and -independent mechanism of corpus cavernosum relaxation induced by RVT. MATERIALS AND METHODS: Strips of corpus cavernosum from rats were mounted in an organ-bath system for isometric tension studies. RESULTS: RVT (1-100 µmol/L) produced concentration-dependent relaxation responses in rat corpus cavernosum pre-contracted by phenylephrine. The non-selective potassium channels blocker tetraethylammonium chloride (TEA, 10 mmol/L), ATP-sensitive potassium (KATP) channels blocker glibenclamide (10 µmol/L), and inward rectifier potassium (Kir) channels inhibitor barium chloride (BaCl2, 30 µmol/L) caused a significant inhibition on the relaxation response to RVT, whereas voltage-dependent potassium channels inhibitor 4-aminopyridine (4-AP, 1 mmol/L), and large conductance calcium-activated potassium (BKCa) channels inhibitor iberiotoxin (IbTX, 0.1 µmol/L) did not significantly alter relaxant responses of corpus cavernosum strips to RVT. In addition, relaxant responses to RVT did not significantly inhibited by the combination of selective inhibitors of small and intermediate conductance BKCa channels (0.1 µmol/L charybdotoxin and 1 µmol/L apamin, respectively). CONCLUSION: These results demonstrated that endothelial small and intermediate conductance BKCa channels are not thought to be an important role in RVT-induced endothelium-dependent relaxation of corpus cavernosum. The endothelium-independent corpus cavernosum relaxation induced by RVT is seems to largely depend on Kir channels and KATP channels in corporal tissue.

The potent relaxant effect of resveratrol in rat corpus cavernosum and its underlying mechanisms.

The aim of this study was to evaluate the relaxant effect of resveratrol (RVT), one of the most commonly employed dietary polyphenols, in rat corpus cavernosum, and to further investigate the contribution of possible underlying mechanisms. Strips of corpus cavernosum were used in organ baths for isometric tension studies. RVT (10(-6)-10(-4) M) produced concentration-dependent relaxation responses in rat corpus cavernosum precontracted by phenylephrine. The relaxant responses to RVT partially, but significantly inhibited by removal of endothelium. Nitric oxide (NO) synthase (NOS) blocker N-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) or soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10(-5) M) caused a significant inhibition on relaxation response to RVT, whereas cyclooxygenase inhibitor indomethacin (10(-5) M) did not significantly alter relaxant responses of corpus cavernosum strips to RVT. Corpus cavernosum contractions induced by stepwise addition to Ca2+ to high KCl solution with no Ca2+ were significantly inhibited by RVT incubation. The treatment of corpus cavernosum tissues with non-specific potassium channel inhibitor tetraethylammonium (TEA, 10(-2) M) did also significantly affect the relaxant activity of RVT. Otherwise, the relaxation response of corpus cavernosum induced by the phosphodiesterase-5 inhibitor sildenafil increased significantly in the group pretreated with 10(-5) M RVT. These results demonstrated that RVT has a potent relaxant effect on rat corpus cavernosum via endothelium-dependent and -independent mechanisms. Endothelium-dependent relaxation of corpus cavernosum to RVT is thought to be mediated primarily through NO/cGMP signaling pathway, and possibly an additional mechanism, endothelium-dependent hyperpolarization factor (EDHF). The residual endothelium-independent corpus cavernosum relaxation induced by RVT is uncertain but seems to depend on the interactions of RVT with Ca2+ entry mechanism from the extracellular space and also other undefined direct effects in this tissue.

Effect of melatonin and time of administration on irradiation-induced damage to rat testes.

The effect of ionizing irradiation on testes and the protective effects of melatonin were investigated by immunohistochemical and electron microscopic methods. Eighty-two adult male Wistar rats were divided into 10 groups. The rats in the irradiated groups were exposed to a sublethal irradiation dose of 8 Gy, either to the total body or abdominopelvic region using a 60Co source at a focus of 80 cm away from the skin in the morning or evening together with vehicle (20% ethanol) or melatonin administered 24 h before (10 mg/kg), immediately before (20 mg/kg) and 24 h after irradiation (10 mg/kg), all ip. Caspace-3 immunoreactivity was increased in the irradiated group compared to control (P < 0.05). Melatonin-treated groups showed less apoptosis as indicated by a considerable decrease in caspace-3 immunoreactivity (P < 0.05). Electron microscopic examination showed that all spermatogenic cells, especially primary spermatocytes, displayed prominent degeneration in the groups submitted to total body and abdominopelvic irradiation. However, melatonin administration considerably inhibited these degenerative changes, especially in rats who received abdominopelvic irradiation. Total body and abdominopelvic irradiation induced identical apoptosis and testicular damage. Chronobiological assessment revealed that biologic rhythm does not alter the inductive effect of irradiation. These data indicate that melatonin protects against total body and abdominopelvic irradiation. Melatonin was more effective in the evening abdominopelvic irradiation and melatonin-treated group than in the total body irradiation and melatonin-treated group.

Effect of melatonin and time of administration on irradiation-induced damage to rat testes

The effect of ionizing irradiation on testes and the protective effects of melatonin were investigated by immunohistochemical and electron microscopic methods. Eighty-two adult male Wistar rats were divided into 10 groups. The rats in the irradiated groups were exposed to a sublethal irradiation dose of 8 Gy, either to the total body or abdominopelvic region using a 60Co source at a focus of 80 cm away from the skin in the morning or evening together with vehicle (20% ethanol) or melatonin administered 24 h before (10 mg/kg), immediately before (20 mg/kg) and 24 h after irradiation (10 mg/kg), all ip. Caspace-3 immunoreactivity was increased in the irradiated group compared to control (P < 0.05). Melatonin-treated groups showed less apoptosis as indicated by a considerable decrease in caspace-3 immunoreactivity (P < 0.05). Electron microscopic examination showed that all spermatogenic cells, especially primary spermatocytes, displayed prominent degeneration in the groups submitted to total body and abdominopelvic irradiation. However, melatonin administration considerably inhibited these degenerative changes, especially in rats who received abdominopelvic irradiation. Total body and abdominopelvic irradiation induced identical apoptosis and testicular damage. Chronobiological assessment revealed that biologic rhythm does not alter the inductive effect of irradiation. These data indicate that melatonin protects against total body and abdominopelvic irradiation. Melatonin was more effective in the evening abdominopelvic irradiation and melatonin-treated group than in the total body irradiation and melatonin-treated group.

The effect of experimental diabetes on the circadian pattern of adenosine deaminase and myeloperoxidase activities in rat liver.

This study investigated time-dependent variations in the activities of adenosine deaminase (ADA), an adenosine-metabolizing enzyme, and myeloperoxidase (MPO), an oxidation reaction-catalyzing enzyme, in control and streptozotocin (STZ)-induced diabetic rat liver. The animals were sacrificed at six different times of day (1, 5, 9, 13, 17 and 21 hours after lights on - HALO). The hepatic activity of ADA did not change depending on the STZ treatment whereas MPO activity was significantly higher in the diabetics than in the controls. Hepatic ADA activity was dependent on the time of sacrifice with the lowest activity at 21 HALO and the highest activity at 5 HALO. Both enzyme activities failed to show any significant interaction between STZ treatment and time of sacrifice, which means that diabetes does not influence the 24 h pattern of these activities. Since MPO, a heme protein localized in the leukocytes, is involved in the killing of microorganisms, increased MPO activity in diabetic rat liver may reflect leukocyte infiltration secondary to diabetes. A reduction in ADA activity during the dark (activity/feeding) period will presumably lead to high concentrations of adenosine in the liver, possibly contributing to changes in some metabolic processes, such as glycogen turnover and oxygen supply.

The effect of experimental diabetes on the twenty-four-hour pattern of the vasodilator responses to acetylcholine and isoprenaline in the rat aorta.

The aim of this study was to investigate whether time-dependent variations in the relaxant effect of acetylcholine, an endothelium-dependent vasorelaxant via muscarinic receptors, and isoprenaline, a nonselective beta-adrenoceptor agonist in rat aorta, are influenced by streptozotocin (STZ)-induced experimental diabetes. Adult male rats were divided randomly into two groups: control and STZ-induced (STZ, 55 mg/kg, intraperitoneal) diabetes. The animals were synchronized to a 12:12 h light-dark cycle (lights on 08:00 h) and sacrificed at six different times of day (1, 5, 9, 13, 17, and 21 hours after lights on; HALO) eight weeks after STZ injection. The in vitro responsiveness of thoracic aorta rings obtained from control and diabetic rats to acetylcholine (10(-9)-10(-5) M) and isoprenaline (10(-10)-10(-3) M) was determined in six different times. EC(50) (the concentration inducing half of the maximum response) values and maximum responses were calculated from cumulative concentration-response curves of the agonists and were analyzed with respect to time and STZ treatment. Treatment, time, and interactions between treatment and time were tested by two-way analysis of variance (ANOVA). To analyze differences due to biological time, one-way ANOVA was used. STZ treatment did not significantly change EC(50) values or maximum responses for both agonists. There were statistically significant time-dependent variations in the EC(50) values for isoprenaline and maximum responses for both acetylcholine and isoprenaline in control groups by one-way ANOVA, but significant time-dependent variations disappeared in the aortas isolated from STZ-induced diabetic rats. The vasodilator responses to acetylcholine and isoprenaline failed to show any significant interaction (treatmentxtime of study) between STZ treatment and time of sacrifice in both EC(50) values and maximum responses by two-way ANOVA. These results indicate there is a basic temporal pattern in the responses to acetylcholine and isoprenaline in rat aorta which continues in diabetes. It is shown for the first time that experimental diabetes does not change the 24 h pattern of responses to acetylcholine and isoprenaline, and that time-dependent variations in the responses to these agonists disappear in diabetic animals. Although further studies are required to define the underlying mechanism(s) of these findings, results suggest that experimental diabetes can modify the time-dependent vasorelaxant responses of rat aorta. This may help to understand the circadian rhythms in cardiovascular physiology and pathology or in drug effects in diabetes.

Circadian variations in the activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in the liver of control and streptozotocin-induced diabetic rats.

The aim of this study was to examine: the 24 h variation of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin-induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light-dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day--1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)--and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6-phosphogluconate dehydrogenase activity was measured by substituting 6-phosphogluconate as substrate. Glucose-6-phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two-way ANOVA revealed that 6-phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6-phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose-6-phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6-phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one-way ANOVA). Time-dependent variation in glucose-6-phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ-treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH-generating enzymes exhibit 24 h variation, which is not influenced by diabetes.

Isolation of Campylobacter jejuni and Campylobacter coli from the gall bladder samples of sheep and identification by polymerase chain reaction.

In this study, 100 gall bladder samples of sheep slaughtered at an abattoir in Elazig province were examined for Campylobacter jejuni and Campylobacter coli by culture and polymerase chain reaction (PCR). Preston Campylobacter Agar supplemented with 7% horse blood and Preston Selective Supplement (Oxoid, Hampshire, UK) were used for isolation of the agents. Campylobacter spp. were isolated in 66 samples, and they were identified as 34% C. jejuni and 32% C. coli. A multiplex PCR based upon the use of ceuE gene-specific primers was applied on DNA samples extracted from C. jejuni and C. coli isolates. All C. jejuni and C. coli strains that were positive by culture were also detected to be positive by PCR. This study shows that PCR can be used an alternative, rapid and sensitive test for the identification of C. jejuni and C. coli which threaten human and animal health.