Clinical Features and Outcome of Children With Hereditary Spherocytosis.

OBJECTIVE: The aim of this study was to evaluate the demographics, clinical, and laboratory findings and treatment responses of patients with hereditary spherocytosis (HS). MATERIALS AND METHODS: Data of children with HS were examined. Diagnosis was based on clinical history, physical examination, family history, presence of spherocytes on peripheral blood smear, and osmotic fragility test. RESULTS: A total of 101 patients were included. The median (range) age at diagnosis was 38.0 (1 to 188) months. Mild, moderate, and severe forms of HS were present in 29 (28.7%), 15 (14.9%), and 57 (56.4%) patients, respectively. Family history was available in 73 patients and 56 of these (76.7%) had a positive family history for HS. Forty-five (44.5%) patients needed regular transfusions and all of these had severe disease. Although most patients did not require transfusion postsplenectomy, 2 of 45 (4.4%) patients continued to require transfusion. Transfusion dependence was significantly (P<0.001) higher in patients with severe spherocytosis. CONCLUSIONS: In HS, splenomegaly, pallor, and jaundice are the most common clinical features. Splenectomy dramatically reduces hemolysis in most cases and virtually abolishes further requirement for transfusion.

Identification of heavy metal origins related to chemical and morphological soil properties using several non-destructive X-ray analytical methods.

Soils are complex mixtures of organic, inorganic materials, and metal compounds from anthropogenic sources. In order to identify the pollution sources, their magnitude and development, several X-ray analytical methods were applied in this study. The concentrations of 16 elements were determined in all the soil samples using energy dispersive X-ray fluorescence spectrometry. Soils of unknown origin were observed by scanning electron microscopy equipped with a Si(Li) X-ray detector using Monte Carlo simulation approach. The mineralogical analyses were carried out using X-ray diffraction spectrometry. Due to the correlations between heavy metals and oxide compounds, the samples were analyzed also by electron probe microanalyzer (EPMA) in order to have information about their oxide contents. On the other hand, soil pH and salinity levels were identified owing to their influence between heavy metal and soil-surface chemistry. Moreover, the geoaccumulation index (I (geo)) enables the assessment of contamination by comparing current and pre-industrial concentrations.

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.

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 alpha-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 alpha-helical content of the wild-type protein. The 24-meric Glu135Arg variant was less stable than the wild-type protein (T(m), [Urea](50%) and [Gnd.HCl](50%) of 59 degrees C, 4.9 M and 3.2 M compared with 73 degrees C, approximately 8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T(m), [Urea](50%) and [Gnd.HCl](50%) of 43 degrees C, approximately 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.

A novel protein-based anticancer drug encapsulating nanosphere: apoferritin-doxorubicin complex.

Developing a drug delivery system, which is uniform, biocompatible, stable and non-toxic, is a challenging issue in anticancer drug delivery strategies. Ferritin is a nano-size spherical protein with an internal cavity where drug molecules can be encapsulated. The apoferritin-doxorubicin complex has been formed by 'opening' and 'closing' the apoferritin sphere in the presence of doxorubicin. The doxorubicin encapsulation was carried out using direct and step-wise change of pH of the solution from 2.5 to 7.4. Non-denaturing polyacrylamide gels showed that the protein cage of the complex successfully self-assembles into its nanosphere form. It was found that up to 28 molecules of doxorubicin can be capsulated per apoferritin protein and no significant drug leakage occurs during the first two days. The apoferritin-doxorubicin complex is a promising nanocarrier for the delivery of anticancer drugs.

Protein-template-driven formation of polynuclear iron species.

Ferritins are iron-storage proteins capable of holding up to 4500 Fe(3+) ions within a single water-soluble protein shell made from 24 polypeptide chains. The Glu128Arg/Glu135Arg mutants of Escherichia coli and Rhodobacter capsulatus bacterioferritins are unable to associate into 24-meric structures, with dimers of polypeptide chains being their stable forms. The aerobic addition to these of up to 8-10 or 14-20 Fe(2+) ions per dimer, respectively, results in the oxidation of the added Fe(2+) to Fe(3+). Gel permeation chromatography and sedimentation equilibrium studies confirm that the Fe(3+) ions are associated with the polypeptide dimer, and the lack of intense EPR signals from magnetically isolated Fe(3+) ions confirms the formation of one or more antiferromagnetically coupled clusters of Fe(3+) ions. The effect of Fe(3+) chelators on iron-loaded subunit dimers is to remove the Fe(3+) from the protein, but to do so slowly, consistent with it not being merely adventitiously associated with protein. These data provide experimental support for the presence of nucleation centers for the mineral cores in bacterioferritins and indicate that these proteins are not simply acting as vessels in which hydrolysis of Fe(3+) occurs independent from the protein surface. From analyses of X-ray structures and amino acid sequence comparisons, possible nucleation sites are identified.