1. The binding of sodium dodecyl sulphate to proteins by equilibrium dialysis was investigated. 2. Most of the proteins studied bound 90-100% of their weight of sodium dodecyl sulphate. 3. The glycoproteins studied bound 70-100% of their weight of sodium dodecyl sulphate, calculated in terms of the polypeptide moiety of the molecule. 4. Proteins not containing S.S groups bound about 140% of their weight of sodium dodecyl sulphate. 5. Reduction of four proteins containing S.S groups caused a rise in sodium dodecyl sulphate binding to 140% of the weight of protein. 6. The apparent micellar molecular weights of the protein-sodium dodecyl sulphate complexes were measured by the dye-solubilization method; they were all found to have approximately the same micellar molecular weight (34000-41000) irrespective of the molecular weight of the protein to which they were attached.

Grafts of tissue-engineered bone represent a promising alternative in the treatment of large and small bone defects. Current approaches are often badly tolerated by patients because of invasiveness, ethical problems, culture, and possibility of infection. Autologous grafts have been indicated as a solution to such problems. Because of tissue availability, many have proposed the use of cultured cells derived from bone marrow expanded in culture and induced to differentiate in bone tissue. Data reported in the literature show that it is possible to produce tissue substitutes in vitro indeed, but results are not always concordant regarding the in vitro produced bone quality. In the present work, we investigated bone formation in aggregates of human bone marrow-derived mesenchymal stem cells induced to differentiate in bone. After osteoinduction we characterized the mineral matrix produced using Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray powder diffraction. Cells were obtained from bone marrow, subjected to immunodepletion for CD3, CD11b, CD14, CD16, CD19, CD56, CD66b, and glycophorin A using RosetteSep and cultured in a new formulation of medium for four passages and then were allowed to form spontaneous aggregates. At the end of proliferation before aggregation, cells were analyzed by fluorescent activated cell sorting (FACS) for markers routinely used to characterize expanded mesenchymal stem cells and were found to be remarkably homogeneous for CD29 (99% ± 1%), CD73 (99% ± 1%), CD90 (95% ± 4%), CD105 (96% ± 4%), and CD133 (0% ± 1%) expression. Our results show that not only aggregated cells express the major markers of osteogenic differentiation, such as osteocalcin, osteonectin, osteopontin, and bone sialoprotein, but also the inorganic matrix is made of an apatite structurally and morphologically similar to native bone even without a scaffold.

Augmented ICAM-1 in the presence of iodide excess and low-dose IFN-gamma could induce secretion of proinflammatory cytokines and lymphocytic infiltration in the thyroid gland. Decreased Tg production in the presence of KI excess and IFN-gamma could explain the development of hypothyroidism after adding iodide in a diet of subjects that already have lymphocytic infiltration and/or mild inflammation in the thyroid gland.

No abstract

Alteration of the redox potential has been proposed as a mechanism influencing gene expression. Reduced glutathione (GSH) is one of the cellular scavengers involved in the regulation of the redox potential. To test the role that GSH may play in thyroid cells, we cultured a differentiated rat thyroid cell strain (FRTL-5) in the presence of L-buthionine-(S,R)-sulfoximine (BSO). BSO affects GSH synthesis by irreversibly inhibiting gamma-glutamylcysteine synthetase (EC 6.3.2.2), a specific enzyme involved in GSH synthesis. BSO-treated FRTL-5 cells show a great decrease in the GSH level, whereas malondialdehyde increases in the cell culture medium as a sign of lipid peroxidation. In these conditions the activity of two thyroid-specific promoters, thyroglobulin (Tg) and thyroperoxidase (TPO), is strongly reduced in transient transfection experiments. As both Tg and TPO promoters depend upon the thyroid-specific transcription factors, thyroid-specific transcription factor-1 (TTF-1) and Pax-8 for full transcriptional activity, we tested whether reduction of GSH concentration impairs the activity of these transcription factors. After BSO treatment of FRTL-5 cells, both transcription factors fail to trans-activate the respective chimerical targets, C5 and B-cell specific activating protein promoters, containing, respectively, multimerized TTF-1- or Pax-8-binding sites only as well as the Tg and TPO natural promoters. Northern analysis revealed that endogenous Tg messenger RNA (mRNA) expression is also reduced by BSO treatment, whereas endogenous TPO expression is not modified. Furthermore, the Pax-8 mRNA steady state concentration does not change in BSO-treated cells, whereas TTF-1 mRNA slightly decreases. Immunoblotting analysis of FRTL-5 nuclear extracts does not show significant modification of the Pax-8 concentration in BSO-treated cells, whereas a decrease of 25% in TTF-1 protein is revealed. Furthermore, BSO treatment decreases the DNA-binding activity to the respective consensus sequence of both transcription factors. Finally, different mechanisms seem to act on TTF-1 and Pax-8 functional impairment in BSO-treated cells. Indeed, with a lowered GSH concentration, the overexpressed Pax-8 still activates transcription efficiently, whereas, on the contrary, the overexpressed TTF-1 does not recover its transactivation capability when the respective chimerical target sequences are used (C5 and BSAP). When the natural Tg and TPO promoter sequences are used, overexpression of Pax-8 parallels the effect on both promoters observed using the chimeric target sequences, whereas overexpression of TTF-1 increases TPO promoter transcriptional activity only.

The present project has been developed because of the desire to unify the research lines in the A.S.I. 'Medicine & Biotechnology' area into one research line that could satisfy the interests of all of the collaborative groups and at the same time could pursue a relevant social goal. A 6 month feasibility study (SF) called MoMa was carried out in the ASI framework. During the SF the know-how and the tools already available in the national scientific community have been assessed, selected and evaluated even with the important contribution of Small and Medium-size Enterprises (SME) and of Italian industries already involved in Space Research. As result of the SF MoMa, all of the participants decided to combine all the efforts together and define, with all the know-how and the available technologies, one strategic topic, the "Aging" with a special attention to the Quality of Life (QoL). The space environment is a unique laboratory to study the reaction of living organisms (especially humans) to microgravity and cosmic radiation. The study of the effects of these two variables at the molecular and cellular levels will shed light on the response of cells and living organisms to adverse stimulations that are always present even on Earth and will help us able to develop the best strategies to protect the organisms from the progressive structural and functional decline related to Aging. Relevant spin-offs on Earth and also relevant industrial applications are the expected outputs of this project.

No abstract

Oct‐4A (Octamer binding protein 4, isoform A) is a key component of the molecular circuitry which regulates stem cells self‐renewal and pluripotency. Very little is known about the mechanisms through which Oct‐4A responds to complex extracellular stimuli and regulates stemness. In our studies we noted that Oct‐4 was expressed both in nucleus and cytoplasm, implicating the presence of nuclear‐cytoplamatic translocation mechanisms. To further explore the functional network of Oct‐4A, in vitro and in vivo methods were used to search interacting proteins in human Dental Pulp Marrow Similar Cells (DPMSC) and NTera2, a human embryonal carcinoma (EC) stem cell line that shares many characteristics with human embryonic stem cells. Glutathione S‐transferase pull‐down and co‐immunoprecipitation assays identified Erk1/2 as a possible Oct‐4A interactor. One mechanism by which Erk1/2 kinases ensure their specificity of action is by interacting with their substrates through docking domains, enhancing also the efficiency of phosphorylation. Oct‐4A sequence analysis evidenced the presence of a D‐domain supporting our interaction hypothesis. Further studies are needed to better understand the mechanism.