Giovanni Materazzi

Polypyrrole-polysaccharide thin films characteristics: electrosynthesis and biological properties.

Polypyrrole-polysaccharide thin films were electropolymerized from starting solutions containing pyrrole and a polysaccharide, namely, heparin, chondroitin-4-sulphate or hyaluronic acid. The synthesized samples showed good chemical and physicochemical properties determined by the synthesis parameters such as the current density and time. For instance, the sample morphology was strictly correlated to the current density as follows: a smooth surface morphology was observed when the current density was in the range of 100-700 microA/cm(2), whereas high current (I > 1.0 mA/cm(2)) or longer time (synthesis charge > 100 mC/cm(2)) led to rough surfaces. The presence of polysaccharide within the polymeric matrix assured proper hydrophilicity to the samples. The optimized surface chemistry due to the presence of a polysaccharide and the controllable morphology allowed positive cell/substrate interactions and these are proved by cellular tests using MC3T3-E1 osteoblast cultures.

Anti-apoptotic Bcl-2 enhancing requires FGF-2/FGF receptor 1 binding in mouse osteoblasts.

In this study, we investigated the role of prostaglandin F2α (PGF2α) in mouse osteoblast survival and the function of fibroblast growth factor 2 (FGF‐2) and fibroblast growth factor receptor 1 (FGFR1) in this process. In particular, for the first time, we demonstrated that PGF2α increased osteoblast survival in a dose‐dependent manner and we showed that the effect is correlated with an increase in Bcl‐2/Bax ratio. Furthermore, we demonstrated that PGF2α caused a decrement of the active caspases 9 and 3. By blocking FGF‐2 with the specific neutralizing antibody and by depletion of FGFR1 gene with a specific siRNA, we showed that FGFR1 and FGF‐2 are critical for the increment of Bcl‐2/Bax ratio and the decrement of the active caspases 9 and 3, induced by PGF2α. Moreover, transmission electron microscopy studies showed that PGF2α increased binding of FGF‐2 and FGFR1 and co‐localization of reactive sites at plasma membrane level. In conclusion, we report a novel mechanism in which PGF2α induces FGF‐2 binding to its specific cell surface receptor 1 leading to a cascade pathway that culminates with increased mouse osteoblast survival. J. Cell. Physiol. 214:145–152, 2008.

Sialoglycoderivatives of bovine submandibular gland identified in situ by histochemical techniques combined with lectins

SummaryWe employed sialidase procedures followed by lectin stainings combined with oxidizing and deacetylating agents to visualize the distribution and sequentiate sialoglycoconjugates in the bovine submandibular gland. In particular we evidenced in acinar and ductal cells the dishomogeneous presence of sialic acids acetylated in the polyhydroxy side chain (C7, C8, C9), whereas O-acetyl substituents at position C1 and/or C4 were not found. Sialoglycoderivatives were also differentiated by the occurrence of penultimate sugars; indeed the dimers sialic acid-(α2→3,6)-β-galactose and sialic acid-(α2→6)-α-N-acetylgalactosamine were identified. Using such technique we supported further the possibility to develop methods for the identification of the positions of Oacetyl groups and the reconstruction of terminal disaccharides within surface and cytoplasm glycoconjugates.

PGF2α increases FGF‐2 and FGFR2 trafficking in Py1a rat osteoblasts via clathrin independent and importin β dependent pathway

Previous studies showed that prostaglandin F2α (PGF2α) stimulated fibroblast growth factor‐2 (FGF‐2) and fibroblast growth factor receptor 2 (FGFR2) cytosolic and nuclear accumulation, however, the endocytic pathway has not been elucidated. This study demonstrates that although PGF2α increased the formation of clathrin‐coated structures in Py1a rat osteoblasts, they were not involved in FGF‐2 and FGFR2 trafficking. PGF2α increased binding of FGF‐2 and FGFR2 and co‐localization of reactive sites in addition to nuclear translocation at the nuclear pore complex level. FGF‐2 and FGFR2 were in close spatial correlation with importin β, further supporting nuclear import of the FGF‐2/FGFR2 complex. Immunogold and immunofluorescence techniques as well as Western blotting demonstrated increased importin β protein labeling in response to PGF2α. Similar to PGF2α, phorbol 12‐myristate 13‐acetate (PMA) also increased importin β protein. These data strongly suggest that prostaglandins may regulate osteoblast metabolism via FGF‐2/FGFR2/importin β nuclear trafficking. J. Cell. Biochem. 97: 1379–1392, 2006.

Benzyl butyl phthalate influences actin distribution and cell proliferation in rat Py1a osteoblasts.

We previously reported that transient administration of phthalates induced actin cytoskeleton disruption in Py1a osteoblasts. However, the mechanism of this transient effect was not elucidated. In this study we provided evidence that the actin cytoskeletal re‐established conditions are dependent on new actin expression and synthesis. To assess the role of phthalates in modulating the distribution of actin, confocal and electron microscopy studies were carried out. Results indicated a modification of actin distribution after phthalate administration. In addition, a relation with the nucleoskeletal component lamin A supports the hypothesis that phthalates may participate in regulatory cell processes involving actin in Py1a osteoblasts. The present study also supports the mitogenic effects of phthalates, which involve microfilament disruption, nuclear actin and lamin A. In particular, the increased levels of cyclin D3, which in mammalian cells plays a critical role in G1 to S transition and is a putative proto‐oncogene in benzyl butyl phthalate treated cells, suggested a possible effect of the endocrine disruptor in cancer processes. J. Cell. Biochem. 101: 543–551, 2007.

Specialised cell types in the chorioallantoic membrane express carbonic anhydrase during chick embryogenesis

The expression of carbonic anhydrase in the chorioallantoic membrane (CAM) of the chick embryo was investigated by means of the histochemical localisation of the enzyme catalytic sites and the immunohistochemical identification of its isoenzymatic forms. The results show that carbonic anhydrase is developmentally expressed in a subset of cells both in the ectodermal and the endodermal epithelium. The distribution patterns from both methodological approaches indicated that carbonic anhydrase is a marker of the villus cavity cells and the mitochondria‐rich cells in the ectodermal and the endodermal epithelium, respectively. Such a cell‐specific pattern of the enzyme expression provides a further contribution to characterising the heterogeneous cell population of the chick CAM and supports specific functional involvement for the distinct cell types in CAM‐mediated processes, such as calcium transport, maintenance of acid‐base balance and water and electrolyte reabsorption, during chick embryogenesis.

DEVELOPMENTAL CHANGES OF SUGAR OCCURRENCE AND DISTRIBUTION IN THE RAT SUBMANDIBULAR AND SUBLINGUAL GLANDS

 The developmental expression of salivary glycoconjugates was investigated in the rat submandibular and sublingual glands by conventional and lectin histochemistry. By the time of the first differentiation of secretory structures, in spite of similar morphological features, a different histochemical reactivity was detected, accounting for a relevant content of neutral glycoconjugates in the submandibular gland and the occurrence of both neutral and acidic glycoconjugates in the sublingual one. The use of lectins allowed the main changes of secretory components to be noted around gestational day 18. DBA and WGA lectins seemed to act as pre- and post-natal development markers while Con A lectin was indicative of post-natal differentiation. Taken together, data from lectin histochemistry indicated the transitional occurrence of glycoconjugates, probably involved in temporally restricted functions, as well as the co-existence of different secretory components that might also reflect maturational changes of single products.

Prostaglandin F2α involves heparan sulphate sugar chains and FGFRs to modulate osteoblast growth and differentiation

The present investigation extends our previous studies on PGF2α‐mediated signalling in osteoblast metabolism. In particular, the role of PGF2α as modulator of heparan sulphate proteoglycans (HSPGs), fibroblast growth factor 2 (FGF‐2) and fibroblast growth factor receptors (FGFRs) was evaluated. We hereby reported the novel observation that PGF2α was able to promote the formation of HSPGs/FGF‐2/FGFRs complexes. Moreover, our data suggested that PGF2α could induce new synthesis of heparan sulphate (HS) chains on osteoblasts by a mechanism involving a modulation of MAPK signalling and that HS is required for the regulation of FGF‐2 induced by PGF2α. Indeed, a proteolytic cleavage of HSPGs with heparinase III (Hep III) prior to PGF2α administration down‐regulated the basal expression of phospho‐p44/42, likely inhibiting FGFRs tyrosine kinase activity. Interestingly, MAPK signalling influenced syntheses and subcellular localization of FGF‐2, its specific receptor and HS. In addition, the proteolytic cleavage by Hep III and the MAPK kinase inhibition by PD‐98059 also revealed that PGF2α induced cell proliferation is dependent on HSPGs and FGF‐2 specific receptor, respectively. Of further relevance of this study, we demonstrated, by using a specific siRNA for FGFR1, that PGF2α modulates Runx2 expression by FGFR1 and HS. J. Cell. Physiol. 217: 48–59, 2008.

Cell type-specific and developmentally regulated expression of the AE1 anion exchanger in the chicken chorioallantoic membrane.

Antibodies specific for the chicken AE1 anion exchanger have been used to determine the cell-type specific pattern of expression of this electroneutral transporter in the chick chorioallantoic membrane (CAM) during embryonic development. Immunolocalisation analyses demonstrated that the AE1 anion exchanger accumulated in the basolateral membrane of a subset of cells in both the chorionic and allantoic epithelial layers. Double immunostaining indicated that the AE1-positive cells in the chorionic and allantoic epithelia were also positive for the carbonic anhydrase isoform, CAII, which serves as a marker for the villus cavity (VC) cells of the chorionic epithelium and the mitochondria-rich cells of the allantoic epithelium. Immunoelectron microscopy revealed that AE1 accumulated in extensive projections that extended from the lateral membrane of VC cells towards the adjacent capillary covering cells. These results represent the first demonstration of anion exchanger expression in the chick CAM, and they suggest a role for basolateral AE1 in bicarbonate reabsorption that is required in the embryo for maintaining acid-base balance during development.

Codistribution of lectin reactive glycoderivativesand PA-TCH-SP positive sites in rat oviduct

Specimens from rat ampulla and isthmus were stained with a battery of 10 lectin-horseradish peroxidase conjugates and lectin binding patterns were correlated with the distribution of periodate-reactive vicinal diol groups as determined by the PA-TCH-SP (periodic acid-thiocarbohydrazide-silver proteinate) sequence. The free surface of ciliated and non-ciliated cells was stained moderately to intensely by all lectins and PA-TCH-SP sequence. Binding sites for WGA, UEA I, LTA, Con A were also detected on the tight junctional regions. Secretory granules reacted with all stainings, except for LPA. The localization of certain sugars (sialic acid, fucose) appeared useful for understanding the functional meaning of positive sites.