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BACKGROUND: Organogenesis of the thyroid gland requires the Pax8 protein. Absence or reduction of Pax8 results in congenital hypothyroidism in animal models and humans, respectively. This study aims at elucidating the regulatory mechanism leading to the expression of Pax8 in thyroid cells. METHODS: The murine Pax8 gene promoter was functionally dissected by mutagenesis and transfection in the thyroid cell line FRTL-5. Nuclear factors important for thyroid-specific gene expression were identified by DNA-binding assays. RESULTS: We show that Pax8 binds to and controls the expression of its own promoter. Furthermore, we identify a novel, thyroid-specific, DNA-binding activity (denominated nTTF [for novel Thyroid Transcription Factor]) that recognizes a specific region of the Pax8 promoter. CONCLUSIONS: The Pax8 promoter appears to be autoregulated, a feature that might be responsible for the haploinsufficiency displayed by this gene.

BACKGROUND: Mucosal infections are a major global health problem and it is generally accepted that mucosal vaccination strategies, able to block infection at their entry site, would be preferable with respect to other prevention approaches. However, there are still relatively few mucosal vaccines available, mainly because of the lack of efficient delivery systems and of mucosal adjuvants. Recombinant bacterial spores displaying a heterologous antigen have been shown to induce protective immune responses and, therefore, proposed as a mucosal delivery system. A non-recombinant approach has been recently developed and tested to display antigens and enzymes. RESULTS: We report that the binding subunit of the heat-labile toxin (LTB) of Escherichia coli efficiently adsorbed on the surface of Bacillus subtilis spores. When nasally administered to groups of mice, spore-adsorbed LTB was able to induce a specific immune response with the production of serum IgG, fecal sIgA and of IFN-gamma in spleen and mesenteric lympho nodes (MLN) of the immunized animals. Dot blotting experiments showed that the non-recombinant approach was more efficient than the recombinant system in displaying LTB and that the efficiency of display could be further increased by using mutant spores with an altered surface. In addition, immunofluorescence microscopy experiments showed that only when displayed on the spore surface by the non-recombinant approach LTB was found in its native, pentameric form. CONCLUSION: Our results indicate that non-recombinant spores displaying LTB pentamers can be administered by the nasal route to induce a Th1-biased, specific immune response. Mutant spores with an altered coat are more efficient than wild type spores in adsorbing the antigen, allowing the use of a reduced number of spores in immunization procedures. Efficiency of display, ability to display the native form of the antigen and to induce a specific immune response propose this non-recombinant delivery system as a powerful mucosal vaccine delivery approach.

Background Applications of bacteriocins as food preservatives have been so far limited, principally because of their low antimicrobial activity in foods. Nisin is the only bacteriocin of significant use, but applications are restricted principally because of its very low activity at neutral or alkaline pH. Thus the isolation of new bacteriocins active in foods is desirable. Results We isolated a Bacillus licheniformis thermophilic strain producing a bacteriocin with some novel features, named here bacillocin 490. This bacteriocin was inactivated by pronase E and proteinase K and was active against closely related Bacillus spp. both in aerobic and in anaerobic conditions. Bactericidal activity was kept during storage at 4°C and was remarkably stable in a wide pH range. The bacteriocin was partially purified by elution after adhesion to cells of the food-isolated strain Bacillus smithii and had a rather low mass (2 KDa). Antimicrobial activity against B. smithii was observed also when this organism was grown in water buffalo milk. Conclusions Bacillocin 490 is a novel candidate as a food anti-microbial agent since it displays its activity in milk, is stable to heat treatment and during storage, is active in a wide pH range and has bactericidal activity also at high temperature. These features may allow the use of bacillocin 490 during processes performed at high temperature and as a complementary antimicrobial agent of nisin against some Bacillus spp. in non-acidic foods. The small size suggests its use on solid foods.

Despite advances in our understanding of basic mechanisms driving post-surgical pain, treating incision-induced pain remains a major clinical challenge. Moreover, surgery has been implicated as a major cause of chronic pain conditions. Hence, more efficacious treatments are needed to inhibit incision-induced pain and prevent the transition to chronic pain following surgery. We reasoned that activators of AMP-activated protein kinase (AMPK) may represent a novel treatment avenue for the local treatment of incision-induced pain because AMPK activators inhibit ERK and mTOR signaling, two important pathways involved in the sensitization of peripheral nociceptors.

Cadherin-16 was originally identified as a tissue-specific cadherin present exclusively in kidney. Only recently, Cadherin-16 has been detected also on the plasma membrane of mouse thyrocytes. This last finding prompted us to note that the expression profile of Cadherin-16 resembles that of the transcription factor Pax8, a member of the Pax (paired-box) gene family, predominantly expressed in the developing and adult kidney and thyroid. Pax8 has been extensively characterized in the thyroid and shown to be a master gene for thyroid development and differentiation. In this study, we determined the role of the transcription factor Pax8 in the regulation of Cadherin-16 expression. We demonstrate that the Cadherin-16 minimal promoter is transcriptionally active in thyroid cells as well as in kidney cells, that Pax8 is able to activate transcription from a Cadherin-16 promoter reporter construct, and more importantly, that indeed Pax8 is able to bind in vivo the Cadherin-16 promoter region. In addition, by means of Pax8 RNA interference in thyroid cells and by analyzing Pax8 null mice, we demonstrate that Pax8 regulates also in vivo the expression of Cadherin-16. Finally, we reveal that the expression of Cadherin-16 is TSH dependent in FRTL-5 thyroid cells and significantly reduced in mouse thyroid carcinomas. Therefore, we conclude that Cadherin-16 is a novel downstream target of the transcription factor Pax8, likely since the early steps of thyroid development, and that its expression is associated with the fully differentiated state of the thyroid cell.

Fe65 interacts with the cytosolic domain of the Alzheimer amyloid precursor protein (APP). The functions of the Fe65 are still unknown. To address this point we generated Fe65 knockout (KO) mice. These mice do not show any obvious phenotype; however, when fibroblasts (mouse embryonic fibroblasts), isolated from Fe65 KO embryos, were exposed to low doses of DNA damaging agents, such as etoposide or H2O2, an increased sensitivity to genotoxic stress, compared with wild type animals, clearly emerged. Accordingly, brain extracts from Fe65 KO mice, exposed to non-lethal doses of ionizing radiations, showed high levels of gamma-H2AX and p53, thus demonstrating a higher sensitivity to X-rays than wild type mice. Nuclear Fe65 is necessary to rescue the observed phenotype, and few minutes after the exposure of MEFs to DNA damaging agents, Fe65 undergoes phosphorylation in the nucleus. With a similar timing, the proteolytic processing of APP is rapidly affected by the genotoxic stress: in fact, the cleavage of the APP COOH-terminal fragments by gamma-secretase is induced soon after the exposure of cells to etoposide, in a Fe65-dependent manner. These results demonstrate that Fe65 plays an essential role in the response of the cells to DNA damage.

Bacillus subtilis spores displaying the tetanus toxin fragment C (TTFC) on their surface have been previously shown to induce the production of specific IgG and secretory IgA in mice immunized through the oral or nasal route. Aim of this study was to analyze whether these spores were also able to induce cellular immunity, and whether such immune response was dependent on spore germination in the animal gastro-intestinal tract (GIT). We first developed a germination defective strain of B. subtilis unable to produce viable cells inside the mouse GIT. Germination-defective and congenic wild-type spores both expressing TTFC on their surface were then used to orally immunize Balb/C mice. Both types of spores induced spleen and mesenteric lymph nodes cell proliferation as well as production of IFNgamma but not of IL-4 and IL-10 in both districts. Our results indicate that recombinant spores preferentially induce a strong cell-mediated immune response with a Th1 phenotype, independently from their ability to germinate in the GIT.

Bacillus subtilis spores displaying the tetanus toxin fragment C (TTFC) on their surface have been previously shown to induce the production of specific IgG and secretory IgA in mice immunized through the oral or nasal route. Aim of this study was to analyze whether these spores were also able to induce cellular immunity, and whether such immune response was dependent on spore germination in the animal gastro-intestinal tract (GIT). We first developed a germination defective strain of B. subtilis unable to produce viable cells inside the mouse GIT. Germination-defective and congenic wild-type spores both expressing TTFC on their surface were then used to orally immunize Balb/C mice. Both types of spores induced spleen and mesenteric lymph nodes cell proliferation as well as production of IFNgamma but not of IL-4 and IL-10 in both districts. Our results indicate that recombinant spores preferentially induce a strong cell-mediated immune response with a Th1 phenotype, independently from their ability to germinate in the GIT.

The biological role of archaeal proteins, homologous to the eukaryal replication initiation factors of cell division control (Cdc6) and origin recognition complex (ORC1), has not yet been clearly established. The hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as Sso) possesses three Cdc6/ORC1- like factors, which are named Sso Cdc6-1, Cdc6-2 and Cdc6-3. This study is a report on the biochemical characterization of Sso Cdc6-1 and Cdc6-3. It has been found that either Sso Cdc6-1 or Cdc6-3 behave as monomers in solutions by gel filtration analyses. Both factors are able to bind to various single-stranded and double-stranded DNA ligands, but Sso Cdc6-3 shows a higher DNA-binding affinity. It has also been observed that either Sso Cdc6-1 or Cdc6-3 inhibit the DNA unwinding activity of the S. solfataricus homo-hexameric mini-chromosome maintenance (MCM)-like DNA helicase (Sso MCM); although they strongly stimulate the interaction of the Sso MCM with bubblecontaining synthetic oligonucleotides. The study has also showed, with surface plasmon resonance measurements, that Sso Cdc6-2 physically interacts with either Sso Cdc6-1 or Sso Cdc6-3. These findings may provide important clues needed to understand the biological role that is played by each of these three Cdc6 factors during the DNA replication initiation process in the S. solfataricus cells.

In a search for bacteria having putative probiotic activity, we screened a collection of food-isolated microorganisms for the ability to survive at low pH in the presence of bile salts and for the production of antimicrobial compounds active against a number of animal pathogens. Among these, we found a strain that we classified as a member of Lactobacillus fermentum sp., and we further investigated its features. This organism was able to adhere to human enterocyte-like (Caco-2) cells with high efficiency as compared to that of a well known indicator strain. Chromatographic analysis indicated that at least two small (less than 3 kDa) factors were involved in mediating the in vitro interaction of L. fermentum with Caco-2 cells. Adhesion activity could be abolished by mild treatment of the bacterial cells in buffer and rescued by incubating them with either the same buffer after its use in the treatment or with chromatographic fractions containing each of the two factors, which indicated that these factors were loosely associated with the cell wall and that each of them was sufficient to warrant the adhesiveness of L. fermentum to Caco-2 cells. These data are suggestive of a novel mechanism of bacterial adhesion to epithelial cells.