Validation of dHPLC for molecular diagnosis of beta-thalassemia in Southern Italy

Beta-thalassemia, the most common hereditary anemia in the Mediterranean area, results from over 200 causative mutations in the beta-globin locus. The aim of this study was to validate a denaturing high-performance liquid chromatography (dHPLC)-based assay for postnatal and prenatal molecular diagnosis of beta-thalassemia in Southern Italy. Sixty beta-thalassemic patients, affected either by thalassemia intermedia or thalassemia major, were analyzed in a blind study. We also carried out prenatal molecular diagnosis in 12 couples at-risk for having affected offspring. Chorionic villi samples were subjected to dHPLC analysis upon molecular characterization of the parental beta-globin alleles. Direct sequence analysis was used to validate each result, showing an accuracy rate of 100% for dHPLC. Overall, our protocol was able to identify the responsible mutations in all 96 analyzed subjects (including 12 prenatals in at-risk pregnancies), detecting the eight most common mutations in Southern Italy. Three rare mutations (one of which, reported here for the first time) that standard mutation detection methods failed to reveal, were also identified. dHPLC assay proved to be a reliable, rapid, and sensitive method for detecting both common and rare mutations within the beta-globin gene. Because of this property our protocol has the potential to be implemented for mutational screening in different areas of high prevalence for beta-thalassemia.     

Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces--modulation of conformation and activity,and lipid-protein interaction

Sticholysins I and II (St I and St II) are water-soluble toxins produced by the sea anemone Stichodactyla helianthus. St I and St II bind to biological and model membranes containing sphingomyelin (SM), forming oligomeric pores that lead to leakage of internal contents. Here we describe functional and structural studies of the toxins aiming at the understanding at a molecular level of their mechanism of binding, as well as their effects on membrane permeabilization. St I and St II caused potassium leakage from red blood cells and temperature-dependent hemolysis, the activation energy of the process being lower for the latter toxin. Protein intrinsic fluorescence measurements provided evidence for toxin binding to model membranes composed of 1:1 (mol:mol) egg phosphatidyl choline (ePC):SM. The fluorescence intensity increased and the maximum emission wavelength decreased as a result of binding. The changes were quantitatively different for both toxins. Circular dichroism spectra showed that both St I and St II exhibit a high content of beta-sheet structure and that binding to model membranes did not alter the toxin's conformation to a large extent. Changing the lipid composition by adding 5 mol% of negatively charged phosphatidic acid (PA) or phosphatidyl glycerol (PG) had small, but detectable, effects on protein conformation. The influence of lipid composition on toxin-induced membrane permeabilization was assessed by means of fluorescence measurements of calcein leakage. The effect was larger for ePC:SM bilayers containing 5 mol% of negative curvature-inducing lipids. Electron paramagnetic resonance (EPR) spectra of intercalated fatty acid spin probes carrying the nitroxide moiety at different carbons (5, 7, 12, and 16) evidenced the occurrence of lipid-protein interaction. Upon addition of the toxins, two-component spectra were observed for the probe labeled at C-12. The broader component, corresponding to a population of strongly immobilized spin probes, was ascribed to boundary lipid. The contribution of this component to the total spectrum was larger for St II than for St I. Moreover, it was clearly detectable for the C-12-labeled probe, but it was absent when the label was at C-16, indicating a lack of lipid-protein interaction close to the lipid terminal methyl group. This effect could be either due to the fact that the toxins do not span the whole bilayer thickness or to the formation of a toroidal pore leading to the preferential interaction with acyl chain carbons closer to the phospholipids head groups.     

A structural characterization of in situ chromatin on cell lines isolated from patients affected by ataxia telangiectasia

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by numerous clinical and cellular features. The pleiotropic nature of the AT syndrome attests to the multiple roles of ATM, the protein codified by the gene altered in AT patients. We investigated if different mutations of ATM could reflect on different alterations of nuclear architecture and chromatin organization. We selected three lymphoblastoid cell lines isolated from AT patients affected by different mutations of ATM gene and one healthy control. We characterized the in situ chromatin structure of each cell line by a biophysical approach: (1) we evaluated the rearrangements of the chromatin domains at the level of single cell by quantitative fluorescence microscopy; (2) we analysed the changes of the average chromatin condensation by differential scanning calorimetry. The results show that the three different ATM mutations produce significant modifications of both nuclear architecture and chromatin condensation.     

Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB) using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence antibody test (IFAT) results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals). S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.     

Beauveria bassiana yeast phase on agar medium and its pathogenicity against Diatraea saccharalis (Lepidoptera: Crambidae) and Tetranychus urticae (Acari: Tetranychidae)

Beauveria bassiana colonizes insect hosts initially through a yeast phase, which is common in some artificial liquid cultures, but not reported on artificial solid media. We describe a yeast-like phase for B. bassiana isolate 447 (ATCC 20872) on MacConkey agar and its virulence toward Diatraea saccharalis and Tetranychus urticae. The yeast-like cells of B. bassiana developed by budding from germinating conidia after 24-h incubation. Cells were typically 5-10 microm and fungal colonies were initially circular and mucoid, but later were covered with mycelia and conidia. Ability to produce yeast-like cells on MacConkey medium was relatively common among different B. bassiana isolates, but growth rate and timing of yeast-like cell production also varied. Metarhizium anisopliae and Paecilomyces spp. isolates did not grow as yeast-like cells on MacConkey medium. Yeast-like cells of B. bassiana 447 were more virulent against D. saccharalis than conidia when 10(7)cells/ml were used. At 10(8)cells/ml, the estimated mean survival time was 5.4 days for the yeast suspension and 7.7 days for the conidial suspension, perhaps due to faster germination. The LC(50) was also lower for yeast than conidial suspensions. Yeast-like cells and conidia had similar virulence against T. urticae; the average mortalities with yeast-like cells and conidia were, respectively, 42.8 and 45.0%, with 10(7)cells/ml, and 77.8 and 74.4%, with 10(8)cells/ml. The estimated mean survival times were 3.6 and 3.9 for yeast and conidial suspensions, respectively. The bioassay results demonstrate the yeast-like structures produced on MacConkey agar are effective as inoculum for B. bassiana applications against arthropod pests, and possibly superior to conidia against some species. Obtaining well-defined yeast phase cultures of entomopathogenic hyphomycetes may be an important step in studies of the biology and nutrition, pathogenesis, and the genetic manipulation of these fungi.     

Temporal fluctuation and reproduction of Thermocyclops decipiens (Copepoda,Cyclopoida) in an eutrophic lake of central Brazil

Lake Paranoá is an eutrophic reservoir situated in the urban region of Brasília. This study was carried out in a fixed collection station located in the Riacho Fundo branch of the reservoir. Zooplankton samples were collected at intervals of 3-5 d at 9:00 a.m. during two months in the dry and rainy seasons for two years (dry-1996, rainy-1997, dry-1997 and rainy-1998), using a 64 microns-mesh plankton net. The most predominant species was Thermocyclops decipiens (about 50% of the total zooplankton community), which during the whole period had a high reproductive rate. The highest densities were found in the dry-1996 season (1700 ind/l for nauplii), and also fluctuated widely overtime. Nauplii stages dominated during the four periods, comprising 50-75% of the total population density. Ovigerous females peaked in the dry-1996 and rainy-1997 seasons, with 20-30% of the total females. The largest peak in egg production occurred during the dry-1996 season, and the total egg production was 2.0 x 10(3) eggs/l. Mesocyclops longisetus is a first record for Lake Paranoá. The ecological factors that determine the success of T. decipiens in eutrophic systems are related to omnivorous feeding habits and prey-predator interactions.     

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.