Giulia Bernardini

Biochemical investigation of the effects of human platelet releasates on human articular chondrocytes

The aim of the present study was to demonstrate the mitogenic and differentiating properties of platelet‐rich plasma releasates (PRPr) on human chondrocytes in mono‐ and three‐dimensional cultures. In order to assess if PRPr supplementation could maintain the chondrocyte phenotype or at least inhibit the cell de‐differentiation even after several days in culture, we performed a proteomic study on several cell cultures independently grown, for different periods of time, in culture medium with FCS, human serum (HS), and releasates obtained from PRP and platelet‐poor plasma (PPP). We found that PRP treatment actually induced in chondrocytes the expression of proteins (some of which novel) involved in differentiation. J. Cell. Biochem. 108: 1153–1165, 2009.

Conformations and Biological Activities of Amyloid Beta Peptide 25-35

Amyloid-beta (Abeta) peptide is commonly found in human Alzheimers disease (AD) brain and is the main component of Alzheimer amyloid plaques. The predominant forms of Abeta in the human brain are Abeta(1-40) and Abeta(1-42), but Abeta(25-35) fragment, physiologically present in elderly people, is the more toxic region and has been recently found to play a relevant role in AD, due to its peculiar aggregation properties. In this work, we review the current understanding on the conformations and biological activity of Abeta(25-35) exploring aggregation, cytotoxic and neurodegenerative properties of this fundamental Abeta fragment, in order to provide an effective starting point to better approach a pathology spread and problematic as AD.

Modern proteomic methodologies for the characterization of lactosylation protein targets in milk

Heat treatment of milk induces the Maillard reaction between lactose and proteins; in this context, β‐lactoglobulin and α‐lactalbumin adducts have been used as markers to monitor milk quality. Since some milk proteins have been reported as essential for the delivery of microelements and, being resistant against proteolysis in the gastrointestinal tract, also contributing to the acquired immune response against pathogens and the stimulation of cellular proliferation, it is crucial to systematically determine the milk subproteome affected by the Maillard reaction for a careful evaluation of aliment functional properties. This is more important when milk is the unique nutritional source, as in infant diet. To this purpose, a combination of proteomic procedures based on analyte capture by combinatorial peptide ligand libraries, selective trapping of lactosylated peptides by m‐aminophenylboronic acid‐agarose chromatography and collision‐induced dissociation and electron transfer dissociation MS was used for systematic identification of the lactosylated proteins in milk samples subjected to different thermal treatments. An exhaustive modification of proteins was observed in milk powdered preparations for infant nutrition. Globally, this approach allowed the identification of 271 non‐redundant modification sites in 33 milk proteins, which also included low‐abundance components involved in nutrient delivery, defence response against virus/microorganisms and cellular proliferative events. A comparison of the modified peptide identification percentages resulting from electron transfer dissociation or collision‐induced dissociation fragmentation spectra confirmed the first activation mode as most advantageous for the analysis of lactosylated proteins. Nutritional, biological and toxicological consequences of these findings are discussed on the basis of the recent literature on this subject, emphasizing their impact on newborn diet.

Antiproliferative and proapoptotic activities of new pyrazolo[3,4-d]pyrimidine derivative Src kinase inhibitors in human osteosarcoma cells

Osteosarcoma is the most frequent primitive malignant tumor of the skeletal system, char acterized by an extremely aggressive clinical course that still lacks an effective treatment. Src kinase seems to be involved in the osteosarcoma malignant phenotype. We show that the treatment of human osteosarcoma cell lines with a new pyrazolo[3,4‐d]pyrimidine derivative Src inhibitor, namely SI‐83, impaired cell viability, with a half‐maximal inhibitory concentration of 12 μ.M in nonstarved cells and a kinetic different from that known for the Src inhibitor PP2. Analysis by terminal deoxynucleotidyl transferase‐mediated nick end labeling, Hoechst, and flow cytometric assay showed that SI‐83 induced apoptosis in SaOS‐2 cells. Moreover, SI‐83, by inhibiting Src phosphorylation, decreased in vivo osteosarcoma tumor mass in a mouse model. Finally, SI‐83 showed selectivity for osteosarcoma, since it had a far lower effect in primary human osteoblasts. These results show that human osteosarcoma had Src‐ dependent proliferation and that modulation of Src activity may be a therapeutic target of this new com pound with low toxicity for nonneoplastic cells—Spreafico, A., Schenone, S., Serchi, T., Orlandini, M., Angelucci, A., Magrini, D., Bernardini, G., Collodel, G., Di Stefano, A., Tintori, C., Bologna, M., Manetti, F., Botta, M., Santucci, A. Antiproliferative and proapo‐ ptotic activities of new pyrazolo[3,4‐d]pyrimidine deriv ative Src kinase inhibitors in human osteosarcoma cells. FASEBJ. 22, 1560–1571 (2008)

Linking protein oxidation to environmental pollutants: redox proteomic approaches.

Environmental pollutants, such as compounds used in agriculture or deriving from vehicles, industries and human activities, can represent major concern for human health since they are considered to contribute significantly to many diseased states with major public health significance. Besides considerable epidemiological evidence linking environmental pollutants with adverse health effects, little information is provided on the effects of these compounds at the cellular and molecular level. Though oxidative stress is generally acknowledged as one of the most important mechanisms of action for pollutant-induced toxicity, redox proteomics, the elective tool to identify post-translationally oxidized proteins, is still in its very infancy in this field of investigation. This review will provide the readers with an outline of the use of redox proteomics in evaluating pollutant-induced oxidative damage to proteins in various biological systems. Future potential applications of redox proteomic approaches from an environmental point of view will be discussed as well.

Extragastric Manifestations of Helicobacter pylori Infection: H. pylori and Extragastric Diseases

The possible role of Helicobacter pylori as a trigger for some extragastric diseases has been largely investigated in the last year. There are, in fact, several studies concerning cardiovascular diseases, neurological disorders, diabetes mellitus, ear and eyes diseases, immunological and hematological disorders, liver and bile tract diseases, gynecological and respiratory tract pathologies. Among them, idiopathic sideropenic anemia and idiopathic thrombocytopenic purpura still remain the extragastric diseases showing the most convincing results. Concerning ischemic heart disease, there are new interesting data playing in favor of the association, even though there are still some open issues to be clarified. For the other diseases, more studies are needed to clarify the reality of the proposed association.

Evaluation of antioxiodant drugs for the treatment of ochronotic alkaptonuria in an in vitro human cell model

Alkaptonuria (AKU) is a rare autosomal recessive disease, associated with deficiency of homogentisate 1,2‐dioxygenase activity in the liver. This leads to an accumulation of homogentisic acid (HGA) and its oxidized derivatives in polymerized form in connective tissues especially in joints. Currently, AKU lacks an appropriate therapy. Hence, we propose a new treatment for AKU using the antioxidant N‐acetylcysteine (NAC) administered in combinations with ascorbic acid (ASC) since it has been proven that NAC counteracts the side‐effects of ASC. We established an in vitro cell model using human articular primary chondrocytes challenged with an excess of HGA (0.33 mM). We used this experimental model to undertake pre‐clinical testing of potential antioxidative therapies for AKU, evaluating apoptosis, viability, proliferation, and metabolism of chondrocytes exposed to HGA and treated with NAC and ASC administered alone or in combination addition of both. NAC decreased apoptosis induced in chondrocytes by HGA, increased chondrocyte growth reduced by HGA, and partially restored proteoglycan release inhibited by HGA. A significantly improvement in efficacy was found with combined addition of the two antioxidants in comparison with NAC and ASC alone. Our novel in vitro AKU model allowed us to demonstrate the efficacy of the co‐administration of NAC and ASC to counteract the negative effects of HGA for the treatment of ochronotic arthropathy. J. Cell. Physiol. 225: 84–91, 2010.

Homogentisate 1,2 dioxygenase is expressed in human osteoarticular cells: Implications in alkaptonuria†

Alkaptonuria (AKU) results from defective homogentisate1,2‐dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT‐PCR, mono‐ and 2D‐Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. J. Cell. Physiol. 227: 3254–3257, 2012.

Biochemical and proteomic characterization of alkaptonuric chondrocytes

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin‐like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub‐populations were identified: cells coming from the black portion of the cartilage were referred to as “black” AKU chondrocytes, while those coming from the white portion were referred to as “white” AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro‐inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both “white” and “black” AKU cells. We then undertook a proteomic and redox‐proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post‐translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in “black” AKU chondrocytes. J. Cell. Physiol. 227: 3333–3343, 2012.

Evaluation of anti-oxidant treatments in an in vitro model of alkaptonuric ochronosis

OBJECTIVES Alkaptonuria (AKU) is a rare genetic disease associated with deficient homogentisate 1,2-dioxygenase activity in the liver. This leads to the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products in connective tissues, which in turn become characterized by the presence of melanin-like pigments (ochronosis). Since at present, further studies are necessary to support the use of drugs for the treatment of AKU, we investigated the effects of various anti-oxidants in counteracting melanin-like pigmentation and oxidative stress related to HGA and its metabolites. METHODS We set up an in vitro model using human serum treated with 0.33 mM HGA and tested the anti-oxidants ascorbic acid, N-acetylcysteine, phytic acid (PHY), taurine (TAU), ferulic acid (FER) and lipoic acid (LIP) for their ability to prevent or delay the production of melanin-like pigments, as well as to reduce oxidative post-translational modifications of proteins. Monitoring of intrinsic fluorescence of HGA-induced melanin-like pigments was used to evaluate the efficacy of compounds. RESULTS Our model allowed us to prove efficacy especially for PHY, TAU, LIP and FER in counteracting the production of HGA-induced melanin-like pigments and protein oxidation induced by HGA and its metabolites. CONCLUSIONS Our model allows the opening of new anti-oxidant therapeutic strategies to treat alkaptonuric ochronosis.