The endocrine disruptor cadmium alters human osteoblast-like Saos-2 cells homeostasis in vitro by alteration of Wnt/ß-catenin pathway and activation of caspases.

PURPOSE: The pollutant Cadmium (Cd) is widespread in the environment and causes alterations of human health by acting as an endocrine disruptor. Bone tissue seems to be a crucial target of Cd contamination. Indeed, we have previously demonstrated that this endocrine disruptor induces osteoblast apoptosis and necrosis. Thus, aim of this study was to further evaluate the effect of Cd on osteoblasts homeostasis, investigating potential modification of the Wnt/ß-catenin intracellular pathway, the intracellular process involved in programmed cellular death and the cytoskeletal alterations. MATERIAL AND METHODS: To this purpose, human osteoblastic Saos-2 cells, a human osteosarcoma osteoblast-like cell line, were cultured and treated with Cd. RESULTS: Osteoblastic cells were treated for 6 h with 10µM Cd, which induced nuclear translocation of ß-catenin and increased expression of Wnt/ß-catenin target genes. Longer exposure to the same Cd concentration induced osteoblastic cell apoptosis. To better characterize the intracellular events involved in these Cd-induced alterations, we evaluated the effect of Cd exposure on actin filaments and proteins associated to cytoskeletal actin, characterized by the presence of LIM domains. Long (15, 24 h) exposure of osteoblasts to Cd reduced LIM proteins expression and induced actin filaments destruction and a significant caspase-3 activation after 24 h. In addition, to prove that Cd induces osteoblastic cells apoptosis after long exposure, we performed TUNEL assay which demonstrated increase of cell apoptosis after 24 h. CONCLUSION: In conclusion, our study shows that osteoblasts exposed to Cd for short intervals of time demonstrated an increase in cell proliferation through a Wnt/ß-catenin dependent mechanism, likely as a compensatory mechanism in response to cell injury. Longer exposure to the same Cd concentration induced cells apoptosis through cytoskeleton disruption-mediated mechanisms and caspase activation.

The alleles of the bft gene are distributed differently among enterotoxigenic Bacteroides fragilis strains from human sources and can be present in double copies.

Enterotoxigenic Bacteroides fragilis (ETBF) strains are associated with diarrheal disease in children. These strains produce a zinc metalloprotease enterotoxin, or fragilysin, that can be detected by a cytotoxicity assay with HT-29 cells. Recently, three different isoforms or variants of the enterotoxin gene, designated bft-1, bft-2, and bft-3, have been identified and sequenced. We used restriction fragment length polymorphism analysis of the PCR-amplified enterotoxin gene to detect the isoforms bft-1 and bft-2 or bft-3 borne by ETBF. By sequencing the portion of the bft gene corresponding to the mature toxin in some strains and applying allele-specific PCR for strains categorized as bft-2 or bft-3, we found in our collection two strains harboring bft-3, a variant that had been described for isolates from East Asia. Analysis of 66 ETBF strains from different sources showed that bft-1 is the most frequent allele, being present in 65% of isolates; it is largely predominant in isolates from feces of adults, while bft-2 is present in isolates from feces of children. This association is statistically significant (P, 0.0064). Sixteen strains were examined by Southern hybridization using, as probes, the bft and second metalloprotease genes, both included in a pathogenicity islet. Five strains were found to harbor double copies of both genes, suggesting that the whole islet was duplicated. Four of these strains, harboring bft-1 (three strains) or bft-2 (one strain), were found to produce a large amount of biologically active toxin, as determined by a cytotoxicity assay with HT-29 cells. The strains harboring bft-3, either in a single copy or in double copies, produced the smallest amount of toxin in our collection.

The region 3' to Calpha1 gene of human IG heavy chain displays a polymorphic duplicated sequence and encodes an RNA associated with polysomes.

A highly spread polymorphism flanking the 3. Calpha1 human IG heavy chain gene was identified. This polymorphism allowed the detection of an internal duplication within the 3' flanking region of both Calpha1 and Calpha2. This region has a regulatory function with four enhancer structures also present at the 3' end of the human Calpha2 as well as in that of mouse and rat single Calpha genes. The 5682-bp sequence of clone lambdapl8 described here starts 3' of Calpha1 and presents three open reading frames; one of them contains part of the tandem repeats with the 20-bp consensus described previously that is expressed in a poly(A)+ RNA and found in three dbEST clones of the human tonsillar cDNA library. Here, we demonstrate that in the CLF1 B lymphoblastoid cell line, this transcript is associated with polysomes. We also discuss the possibility of the presence of a new regulatory gene that does not encode an immunoglobulin and maps in the human IG heavy chain gene cluster.

Characterization of a dominant antigenic determinant of Par o I encoded by recombinant DNA.

BACKGROUND: The pollens from Parietaria judaica and Parietaria officinalis are a major cause of pollinosis in Europe. Par o I (13.5 kDa) and Par j I (12 kDa), the major allergens from these species, are highly crossreactive. METHODS: We have immunoscreened a P. judaica pollen cDNA expression library with a rabbit antiserum specific for Par j I and with a serum pool from allergic patients. An immunopositive clone containing a 26 bp insert was further characterized. The insert sequence was determined and the beta-galactosidase fusion protein was partially purified by electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. RESULTS: This fusion protein specifically and extensively inhibited Par o I and Par j I binding of a rabbit antiserum and of a serum pool obtained from allergic patients. The antifusion-protein antiserum obtained in a rabbit (anti 6a) specifically precipitated radioiodinated purified Par o I in the double antibody radioimmunoassay (DARIA) and competed with antibodies of sera from allergic patients for the binding to Parietaria pollen extract allergens by enzyme linked immunosorbent assay (ELISA). We investigated the prevalence of antibody response towards the 6a epitope in patients naturally sensitized to Parietaria. The presence of 6a specific IgE antibodies was assessed in the sera of 33 patients using inhibition assays. All sera had antibodies with this specificity: the extensive percentage of inhibition reached suggested that they dominated individual ab response. CONCLUSION: In conclusion, the antibody response induced by natural exposure to the pollen of Parietaria appears to be higly focused on a single linear antigenic determinant of the major allergens which may play a relevant role in the development of clinical allergy. This report is, to our knowledge, the first description of a dominant linear epitope of a major allergen.