Mehmet A. Kilic

Stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein.

The stability of Rhodobacter capsulatus bacterioferritin, a 24‐meric homopolymer, toward denaturation on variation in pH and temperature, and increasing concentrations of urea and guanidine. HCl was investigated with native PAGE, and CD and fluorescence spectroscopies. With temperature and urea, the wild‐type protein denatured without discernible intermediates in the equilibrium experiments, but with guanidine.HCl (Gnd.HCl) one or more intermediate species were apparent at relatively low Gnd.HCl concentrations. Dissociated subunit monomers, or aggregates smaller than 24‐mers containing the high α‐helical content characteristic of the native protein were not obtained at any pH without a high proportion of the 24‐mer being present, and taken together with the other denaturation experiments and the construction of stable subunit dimers by site‐directed mutagenesis, this observation indicates that folding of the bacterioferritin monomer could be coupled to its association into a dimer. Glu 128 and Glu 135 were replaced by alanine and arginine in a series of mutants to determine their role in stabilizing the 24‐meric oligomer. The Glu128Ala, Glu135Ala and Glu135Arg variants retained a 24‐meric structure, but the Glu128Ala/Glu135Ala and Glu128Arg/Glu135Arg variants were stable subunit dimers. CD spectra of the Glu135Arg, Glu128Ala/Glu135Ala, and Glu128Arg/Glu135Arg variants showed that they retained the high α‐helical content of the wild‐type protein. The 24‐meric Glu135Arg variant was less stable than the wild‐type protein (Tm, [Urea]50% and [Gnd.HCl]50% of 59°C, 4.9 M and 3.2 M compared with 73°C, ∼8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (Tm, [Urea]50% and [Gnd.HCl]50% of 43°C, ∼3.2 M and 1.8 M, respectively). The differences in stability are roughly additive, indicating that the salt‐bridges formed by Glu 128 and Glu 135 in the native oligomer, with Arg 61 and the amino‐terminal amine of neighboring subunits, respectively, contribute equally to the stability of the subunit assembly. The additivity and assembly states of the variant proteins suggest that the interactions involving Glu 128 and Glu 135 contribute significantly to stabilizing the 24‐mer relative to the subunit dimer.

The effects of orchidectomy on toxicological responses to dietary ochratoxin A in Wistar rats

Ochratoxin A (OTA) causes pathological lesions in the organs of animals. Males are more sensitive to OTA exposure than females but the reasons for this are unknown. The objective of this study was to explore the role of testosterone in male rats with OTA-related pathogenesis. To test the effect of testosterone on OTA toxicity, the testes of a group of rats were surgically removed. Male and female rats (approximately 300 and 200 g) were fed with OTA-contaminated feed (initially approximately 300 μg kg(-1) b.w. per day) for 24 weeks. The organs of all the animals were collected and their organ lesion pathology, caspase-3 expression, OTA plasma and organ concentrations and total plasma testosterone concentrations were evaluated. OTA treatment created serious lesions in the kidney, liver and testes of rats. The major histopathological changes in the kidney and liver were karyomegaly, hemorrhages and vacuolization. In the testes, there was a marked decrease in the amount of spermatozoon. The degrees of organ lesion were evaluated and the castrated males had the lowest kidney and liver lesion scores, indicating that testosterone reduction in males dramatically reduces OTA-related organ damage. The plasma OTA levels for the intact males, the castrated and the females were 6.34, 8.42 and 12.5 μg ml(-1), respectively. In conclusion, despite the similar plasma OTA levels of the intact and castrated males, OTA is less toxic in the castrated males. Therefore, the well-known gender specific toxicity of OTA seems to be related to the testosterone levels of rats.

Contribution of Organ Vasculature in Rat Renal Analysis for Ochratoxin A: Relevance to Toxicology of Nephrotoxins

Assumptions surrounding the kidney as a target for accumulation of ochratoxin A (OTA) are addressed because the contribution of the toxin in blood seems invariably to have been ignored. Adult rats were maintained for several weeks on toxin-contaminated feed. Using standard perfusion techniques, animals were anaesthetised, a blood sample was taken, one kidney was ligated, and the other kidney perfused with physiological saline in situ under normal blood pressure. Comparative analysis of OTA in pairs of kidneys showed marked reduction in the perfused organ in the range 37%–98% (mean 75%), demonstrating the general efficiency of perfusion supported also by histology, and implying a major role of blood in the total OTA content of kidney. Translation of OTA values in plasma to whole blood, and its predicted contribution as a 25% vascular compartment in kidney gave values similar to those in non-perfused kidneys. Thus, apparent ‘accumulation’ of OTA in kidney is due to binding to plasma proteins and long half-life in plasma. Attention should be re-focused on whole animal pharmacokinetics during chronic OTA exposure. Similar principles may be applied to DNA-OTA adducts which are now recognised as occurring in blood; application could also extend to other nephrotoxins such as aristolochic acid. Thus, at least, quantitative reassessment in urological tissues seems necessary in attributing adducts specifically as markers of potentially-tumourigenic exposure.

Diabetogenic Effects of Ochratoxin A in Female Rats

In this study, the diabetogenic effects of long term Ochratoxin A (OTA) administration in rats were investigated, and its role in the etiology of diabetes mellitus (DM) was examined utilizing 42 female Wistar rats for these purposes. The rats were divided into three different study and control groups according to the duration of the OTA administration. The rats received 45 μg OTA daily in their feed for 6, 9 and 24 weeks, respectively. Three control groups were also used for the same time periods. Blood and pancreatic tissue samples were collected during the necropsy at the end of the 6, 9 and 24 weeks. The plasma values of insulin, glucagon and glucose were determined for the study and control groups. Pancreatic lesions were evaluated via histopathological examination and insulin and glucagon expression in these lesions was subsequently determined using immunohistochemical methods. Statistically significant decreases in insulin levels were observed, in contrast to increases in blood glucagon and glucose levels. Histopathological examinations revealed slight to moderate degeneration in Langerhans islet cells in all OTA-treated groups. Immunohistochemistry of pancreatic tissue revealed decreased insulin and increased glucagon expression. This study demonstrated that OTA may cause pancreatic damage in the Langerhans islet and predispose rats to DM.

The role of Escherichia coli bacterioferritin in stressed induced conditions

Escherichia coli possesses at least two iron storage proteins: FtnA (ferritin) and Bfr (Bacterioferritin). FtnA is shown to be a major iron storage protein in E. coli . Although Bfr resembles the ferritin in many of its structural and functional features, the role of Bfr in E. coli is not clear. The aim of this study was to investigate the role of E. coli Bfr in stress induced conditions. In this study, E. coli bfr - (lacking the chromosomal bfr gene) and E. coli bfr + (the same as the former but containing the E. coli bfr overexpression vector) mutants were used. In order to examine the role of E. coli Bfr in stress induced conditions, E. coli cells were grown in the presence of excess iron. In the case of hydrogen peroxide toxicity, the cells were grown in an agar plate containing hydrogen peroxide discs and the results of toxicity were expressed as the size of the cell death zone. In order to find out whether Bfr supports cell growth under iron and phosphate starvation, the E. coli cells were grown in iron and phosphate depleted media. In E. coli the overproduced Bfr did not support the growth of cells under iron and phosphate deficient conditions. The results showed that overproduced Bfr in E. coli does not contribute to the survival of the cell in iron and phosphate starvation. Furthermore, it does not enhance the resistance of E. coli against iron and hydrogen peroxide toxicity.