Olcay Hisar

Effects of low molecular weight plasma inhibitors of rainbow trout (Oncorhynchus mykiss) on human erythrocyte carbonic anhydrase-II isozyme activity in vitro and rat erythrocytes in vivo.

The effects of low molecular weight plasma inhibitors from rainbow trout (Oncorhynchus mykiss) (RT) were investigated on the carbonic anhydrase enzyme (CA) activities in in vitro human and in in vivo Sprague–Dawley rat erythrocytes. The RT blood was used as extracellular fluid (plasma) source and plasma inhibitors were obtained by dialysis of the plasma. For the in vitro study, human carbonic anhydrase-II (HCA-II) isozyme was obtained by Sepharose 4B-l-tyrosine-sulfanylamide affinity chromatography with an overall purification of about 646-fold. The enzyme (specific activity of 7750 EU/mg protein) was obtained with a yield of 71.1% and SDS-PAGE showed a single band. From in vitro studies, the I50 value for RT plasma inhibitors obtained was 0.37 mg/ml. From in vivo studies on rat erythrocytes, CA activity was significantly inhibited by the inhibitors from the extracellular fluid of RT for up to 3 h (p<0.05) following intraperitoneal administration.

The in vitro and in vivo inhibitory effects of some sulfonamide derivatives on rainbow trout (Oncorhynchus mykiss) erythrocyte carbonic anhydrase activity.

The in vitro and in vivo inhibitory effects of 5-(3α, 12α-dihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3α, 7α, 12α-trihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3α, 7α, 12α-triacetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3) and acetazolamide on rainbow trout (Oncorhynchus mykiss) (RT) erythrocyte carbonic anhydrase (CA) were investigated. The RT erythrocyte CA was obtained by affinity chromatography with a yield of 20.9%, a specific activity of 422.5 EU/mg protein and a purification of 222.4-fold. The purity of the enzyme was confirmed by SDS-PAGE. Inhibitory effects of the sulfonamides and acetazolamide on the RT erythrocyte CA were determined using the CO2-Hydratase method in vitro and in vivo studies. From in vitro studies, it was found that all the compounds inhibited CA. The obtained I50 value for the sulfonamides (1), (2) and (3) and acetazolamide were 0.83, 0.049, 0.82 and 0.052 μM, respectively. From in vivo studies, it was observed that CA was inhibited by the sulfonamides (1), (2) and (3) and acetazolamide.

Effect of Melatonin on Glucose-6-Phosphate Dehydrogenase from Rainbow Trout (Oncorhynchus mykiss) Erythrocytes in vitro and in vivo

Abstract Beydemir, Ş. Gülçin, İ., Hisar, O., Küfrevioğlu, Ö.İ. and Yanik, T. 2005. Effect of melatonin on glucose-6-phosphate dehydrogenase from rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo. J. Appl. Anim. Res., 28: 65–68. The in vitro and in vivo effects of melatonin on rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase (G6PD) were investigated. G6PD was purified with a specific activity of 16.7 EU/mg protein and 1,852-folds in a yield of 60.6% by using ammonium sulphate precipitation and 2′,5′-ADP Sepharose 4B affinity gel. In vitro studies showed that G6PD enzyme activity increased up to 0.22 mM melatonin concentration and was inhibited at higher levels. In vivo studies showed that though initial G6PD activity was 8.33±1.13 EU g1 Hb, it was inhibited (P<0.05) 3h after injection of 10 mg kg1 melatonin. It is recommended that intramuscular dose of melatonin for rainbow trout should be less than 0.22 mM.

Kinetic Properties of Carbonic Anhydrase Purified from Gills of Rainbow Trout (Oncorhynchus mykiss)

Abstract Hisar, O., Beydemir, Ş., Bülbül, M. and Yanik, T. 2006. Kinetic properties of carbonic anhydrase purified from gills of rainbow trout (Oncorhynchus mykiss). J. Appl. Anim. Res., 30: 185–188. Kinetic behavior and some properties of carbonic anhydrase enzyme purified from gills of rainbow trout (Oncorhynchus mykiss) were studied at 4C. The purification steps included high-speed centrifugation, sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography and dialysis. Yield and specific activity of the enzyme were 40% and 55.56 EU/mg protein, respectively. The overall purification was 104.8-fold. The molecular mass was approximately estimated as 28 kDa by SDS polyacrylamide gel electrophoresis and as 27 kDa by gel filtration column chromatography. Optimal pH, stable pH and optimal temperature of the enzyme were 9.5, 8.2 in 0.025 M boric acid buffer and 17.5C, respectively. KMand Vmax values of the enzyme for the substrate (p-nitrophenylacetate) were 8.13 mM and 2.10 μmol/mg protein/min, respectively. The dissociation constant of the enzyme inhibitor complex (Ki) value was 3.93±0.65 μM for sulfanilamide and sulfanilamide inhibited the enzyme in a noncompetitive manner. Data from present study showed that optimum kinetic properties of gill CA enzyme were different from other vertebrate CAs.

Inhibitory effects of ammonia and urea on gill carbonic anhydrase enzyme activity of rainbow trout (Oncorhynchus mykiss)

The effects of ammonia and urea on branchial carbonic anhydrase (CA) enzyme which plays a key role in ionoregulation, osmoregulation and acid-base balance of rainbow trout (Oncorhynchus mykiss) were investigated. CA activity of the control group for ammonia and urea was determined as 1285.7 ± 67.9 and 1261.7 ± 60.8EU/mg protein, respectively. The CA enzyme activities of the other groups were measured at 1, 2 and 3h after ammonia and urea applications. The corresponding activities of ammonia were 774.9 ± 68.8, 732.1 ± 48.6 and 768.1 ± 59.5EU/mg protein, respectively and that of urea were 769.3 ± 58.9, 638.2 ± 47.7 and 1108.1 ± 61.1EU/mg protein, respectively. The differences between the initial CA activities for the controls was not significantly (P > 0.01). The CA activities were significantly (P < 0.01) inhibited both in ammonia and urea group. However, the ammonia inhibited more than urea since there was significant differences between final values of gill CA activities.

Preliminary Assessment of Dietary Mannanoligosaccharides on Growth Performance and Health Status of Gilthead Seabream Sparus auratus

A feeding trial was performed to assess the potential beneficial effect of two levels of mannanoligosaccarides (MOS) on the growth performance, feed utilization, hematological parameters, and liver histopathology of gilthead seabream Sparus auratus (also known as gilthead bream). Mannanoligosaccarides were added at the rates of 2 and 4 g/kg to a fish-meal-based control diet, and each diet was given (twice daily [midmorning and midafternoon] to apparent satiation) to triplicate groups of gilthead seabream growers (mean weight = approximately 170 g) in sea cages. The trial lasted 12 weeks, and the average ambient water temperature ranged from 19.6 degrees C to 24.7 degrees C during the experimental period. At the end of the experiment, fish attained market size (350-450 g) and their health status was evaluated by blood analysis and liver histology. There were no differences in survival rates among fish fed experimental diets. However, there were significant improvements in both growth and feed utilization among fish fed diets supplemented with MOS. Hemoglobin (Hb) and hematocrit (Ht) levels and erythrocyte, leukocyte, and thrombocyte (Thr) counts were unaffected by any dietary MOS. The levels of Hb (g/dL; mean +/- SD) and Ht (%; mean +/- SD) were 11.0 +/- 2.5 and 45.6 +/- 6.7 for the control group, 11.1 +/- 1.7 and 39.3 +/- 8.0 for the 2-g/kg group, and 11.2 +/- 1.9 and 40.2 +/- 8.4 for the 4-g/kg group. The mean Thr count ranged from 47.6 to 53.8 x 10(3)/mm3. Despite the apparently higher Thr counts for fish fed diets supplemented with MOS, these differences were not significant. Moreover, no histopathological differences were observed in liver tissue cross sections between control and treatment groups. These results suggest that supplementation of diets with MOS had no significant effects on general fish health.

Metals in Two Species of Fish in Karasu River

In this study, cadmium (Cd), copper (Cu), iron (Fe), nickel (Ni), lead (Pb) and zinc (Zn) levels were determined in the tissues (muscle, liver and gills) of two fish species, Capoeta capoeta umbla and Chalcalburnus mosullensis, collected from three stations of the Karasu River. The lowest metal accumulation was detected in the muscle tissues. Moreover there was some variability in the metal concentrations measured in the same tissues from samples obtained from the three different stations. A positive correlation was observed for the concentration of metal pairs Fe–Cu, Fe–Zn, Ni–Pb, Pb–Zn. These findings were also compared with national and international food standards, and Pb and Cd concentrations were determined to be above the level set by the standards. In conclusion, it was supposed that excessive consumption of these two fish species, which already occurs in this region, might pose a public health risk.

Inhibitory Effects of Some Antibiotics on Activity of Carbonic Anhydrase from Rainbow Trout Erythrocytes in Vitro and in Vivo

Abstract Carbonic anhydrase (CA) from erythrocytes of rainbow trout Oncorhynchus mykiss was purified by affinity chromatography through a process comprising hemolysate preparation, sepharose- 4B-l-tyrosine-sulfanylamide affinity gel chromatography, and dialysis. Then the effects of three antibiotics—sodium ampicillin, streptomycin, and trimethoprim plus sulfamethoxazole—on this enzyme were investigated in vitro and in vivo. Inhibition effects were determined in vitro by means of the Lineweaver–Burk and activity–antibiotic regression graphs. The dissociation constant of the enzyme inhibitor complex (Ki ) and 50% inhibitory values were 2.16 ± 0.40 and 1.87 mM for sodium ampicillin, 7.40 ± 0.310 and 5.21 mM for streptomycin, and 18.50 ± 0.35 and 9.74 mM for trimethoprim–sulfamethoxazole. The in vivo experiment demonstrated that CA was significantly inhibited by trimethoprim–sulfamethoxazole but not by sodium ampicillin and streptomycin.

Discrimination of Penaeid Shrimps with PCR-RFLP Analysis

Abstract A rapid polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis was designed to examine genetic differentiation of some penaeid shrimps. Three hundred and fifty-six bp of cytochrome-b gene, a specific part of mitochondrial genome, was amplified with PCR to figure out differences between shrimp species Penaeus semisulcatus, Penaeus kerathurus, Parapenaeus longirostris, and Metapenaeus monoceros. PCR products were digested with different restriction endonucleases (DdeI, MboI, MboII, and MseI.). None of the tested enzymes, alone, was able to distinguish between the four shrimp species, but with the combination of the results of two digestions, all of the species could be differentiated. It was shown that PCR-RFLP method can be used to expose fraudulent substitutions of processed shrimps prior to marketing.

Effects of environmental hypercapnia on hemato-immunological parameters, carbonic anhydrase, and Na+, K+-ATPase enzyme activities in rainbow trout (Oncorhynchus mykiss) tissues

In this study, the effects of environmental hypercapnia on hemato-immunological parameters and the activities of respiratory enzymes such as carbonic anhydrase (CA) and Na+, K+-ATPase were investigated in rainbow trout (Oncorhynchus mykiss) tissues (gill, liver and kidney). Batches of 12 fish were exposed to 4.5 mg L−1 (control) and 14 mg L−1 CO2. No mortalities occurred during the 14 days of the experimental period. Red blood cell (RBC), hemoglobin (Hb), and hematocrit (Ht) levels, and innate immune parameters such as nitro blue tetrazolium (NBT), lysozyme, and myeloperoxidase activities, and the melano-macrophage frequency were negatively affected by elevated CO2 levels. Patterns of change in CA activity differed among the gill, liver, and kidney. Compared with the activities of CA in the control group, the CA enzyme was significantly stimulated at day 7 in the gill tissue, whereas it was stimulated at day 14 of the experiment in the liver tissue of fish exposed to 14 mg L−1 CO2 (P < 0.05). In contrast to the pattern of CA enzyme activities, the Na+, K+-ATPase enzymes were stimulated significantly in the liver after day 7 but inhibited in the kidney and gill (P < 0.05). These results suggest that a subchronic exposure to hypercapnia of rainbow trout tissues may lead to adaptive changes in the respiratory enzymes and negatively affects hemato-immunological parameters.