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Biomedical subjects

B Pucci

Publications and source records attributed to B Pucci.

At least 19 recordsLinked to original sources
PubMed2006

Protection against reactive oxygen species injuries in rat isolated perfused hearts: effect of LPBNAH, a new amphiphilic spin-trap derived from PBN.

Numerous strategies have been designed to protect the myocardium against reactive oxygen species (ROS). The present study was designed to test wether LPBNAH, a new amphiphilic spin-trap derived from PBN is able to protect isolated perfused rat hearts against ROS injuries. Following total glola ischemia (30 min), hearts were reperfused in the presence or not of LPBNAH (10 micromol/l), and left ventricular function was continuously monitored. The addition of LPBNAH led to a significant recovery in left ventricular developped pressure (LVDevP, control: 16.5+/- 7.5, p < 0.05). To conclude, the present results strongly suggest that the modification of previous wellknown molecules in order to facilitate their access to intracellular site of ROS production might be of interest to limit oxidative stresses.

Animals↗
PubMed2003

Synthesis and antioxidant efficiency of a new amphiphilic spin-trap derived from PBN and lipoic acid.

The synthesis of a new amphiphilic antioxidant called PBNLP and derived from both alpha-phenyl-N-tert-butyl nitrone (PBN) and lipoic acid was described. Grafting a lactobionamide moiety onto the aromatic group of the PBN provided the water solubility of this compound. In vitro preliminary biological evaluations of its antioxidant capacity were performed using the KRL biological test based on free radical-induced hemolysis. The PBNLP induces a protection of erythrocytes against exogenous free radicals higher than that measured with lipoic acid or PBN alone or with lipoic acid or PBN derivatives in admixtures.

Antioxidants↗
PubMed2003

Amphipols: polymeric surfactants for membrane biology research.

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.

Animals↗
PubMed2003

Induction of apoptosis in skeletal tissues: phosphate-mediated chick chondrocyte apoptosis is calcium dependent.

In an earlier study, we have shown that Pi induced apoptosis of terminally differentiated hypertrophic chondrocytes. To ascertain whether Ca2+ modulates Pi-induced cell death, we asked the following two questions: First, can we prevent Pi-induced apoptosis by removing Ca2+ from the culture medium; alternatively, can we potentiate cell death by increasing the Ca2+ concentration? Second, can we inhibit chondrocyte apoptosis by blocking Pi transport? We also explored the mechanism of apoptosis by evaluating mitochondrial activity and reactive oxygen species (ROS) generation in cells treated with the ion pair. We noted that EDTA and EGTA blocked Pi-induced apoptosis in a dose-dependent manner. While high levels of Ca2+ alone had little effect on chondrocyte viability, the cation enhanced Pi-dependent cell death and greatly increased Pi uptake. When Pi transport was blocked, there was complete inhibition of cell killing. The process of cell death was characterized by mitochondrial hyperpolarization; two hours following apoptogen treatment, there was a significant decrease in the mitochondrial membrane potential. Coincident with the changes in mitochondrial function, there was an increase in intracellular Ca2+ that was maintained throughout the experimental period. A raised Ca2+ signal was observed in blebs at the cell membrane. Finally, we noted that, 75 minutes after treatment with the ion pair, there was a six-fold elevation in ROS levels. This increase declined to baseline values after three hours. Based on these observations, we suggest that, at the cartilage mineralization front, an elevation in local environmental Ca2+ and Pi concentrations modulates oxidative metabolism, and triggers apoptosis of terminally differentiated chondrocytes.

Animals↗
PubMed2003

In vitro and in vivo evaluations of THAM derived telomers bearing RGD and Ara-C for tumour neovasculature targeting.

As an approach to the development of specific drug delivery systems, a new class of low macromolecular carriers called 'telomers' endowed with an antitumour agent, such as arabinofuranosylcytosine (Ara-C), RGDSK peptidic sequences, as tumour targeting moieties, and tyrosine groups labelled with 125I atoms allowing the in vivo scintigraphic follow up, were synthesized. Their tumour targeting ability was assessed in vivo in mice bearing a murine B16 melanoma. The biological results showed that the presence of RGDSK sequences onto the macromolecules leads to the selective targeting and the accumulation of telomers within the vascularized zone of the tumour. Moreover, such compounds exhibited in vitro a better IC(50) (0.015 muM) than pure Ara-C and in vivo an oncostatic index higher than 160%.

Acrylates↗
PubMed2003

An in vitro study of the sub-cellular distribution of nicotinic receptors.

Nicotinic and serotoninergic 5HT3 receptors share important sequence identities except for their cytoplasmic loop. Both ends of this loop display conserved 3D helical structures with distinct primary sequences. We decided to check whether these two helices named F and G play a role in the sub-cellular distribution of different nicotinic receptors. We systematically exchanged each helix with the equivalent sequence of neuronal nicotinic and alpha4, beta2 and alpha7 subunits in the functional chimeric alpha7-5HT3 receptor used as a model system. The new chimeras were expressed in vitro in polarized epithelial cells from pig kidney. We quantified synthesis and export of the receptors to the cell surface by measuring alpha-bungarotoxin binding sites. Immunogold labelling was used, at the electron microscope level, to determine the amount of each chimera present at either domain, apical and/or basolateral, of these cells. We noticed that in epithelial cells the majority of alpha-bungarotoxin binding sites remained sequestered in the cytoplasm as already observed in neurons in vivo. The majority of the pentamers present at the cell surface were located at the apical domain. Our results suggest that helix F and G differently regulate assembly and export to the cell surface of alpha-bungarotoxin binding receptors.

Amino Acid Sequence↗
PubMed2002

Gene expression profiling following BMP-2 induction of mesenchymal chondrogenesis in vitro.

OBJECTIVE: This study aims to apply gene expression profiling technology to gain insight into the molecular regulation of mesenchymal chondrogenesis. METHODS: The experimental system consists of micromass cultures of C3H10T1/2 cells, a murine multipotential embryonic cell line, treated with the chondroinductive growth factor, bone morphogenetic factor-2 (BMP-2). In this system, chondrogenic differentiation characterized by both morphological changes and cartilage matrix gene expression has been shown to be completely dependent upon BMP-2 treatment and the high cell plating density of micromass cultures. To identify candidate genes that may have key functional roles in chondrogenesis, we have applied subtractive hybridization to isolate genes whose expression is significantly up- or down-regulated during chondrogenesis. RNA was isolated from micromass cultures treated with BMP-2 for 24 h and analysed for representational differences by means of a subtractive hybridization screening method. RESULTS: Sixteen different genes were identified whose expression was up-regulated between two- and 12-fold by B,P-2, and twelve different genes were identified whose expression was down-regulated between two- and seven-fold by BMP-2. CONCLUSIONS: The potential of this screening methodology to identify new BMP-2 regulated genes is suggested by the fact that a majority of the identified genes are indeed novel. Identification and characterization of these genes should provide insight as to how chondrogenesis is regulated and also should provide important new markers for the study of osteoarthritis.

Animals↗
PubMed2001

A transcriptionally inactive E2F-1 targets the MDM family of proteins for proteolytic degradation.

E2F-1-activated transcription promotes cell cycle progression and apoptosis. These functions are regulated by several factors including the E2F-1-binding protein MDM2 and the retinoblastoma protein pRb. Using a yeast two-hybrid screen we have identified the MDM2-related protein, MDMX, as an E2F-1-binding protein. In these studies we find that coexpression of MDMX with E2F-1 results in degradation of the MDMX protein. Although this proteolytic degradation can be blocked by the protease inhibitors bafilomycin A(1), N-acetyl-Leu-Leu-Norleu-AL, and N-acetyl-Leu-Leu-Met-AL, MDMX degradation is not inhibited by lactacystin, suggesting that degradation occurs by a proteasome-independent mechanism. Using an E2F-1 deletion mutant (E2F-1(180-437)) we show that E2F-1-targeted degradation of MDMX does not require the E2F-1 DNA binding domain and therefore is independent of E2F-1-driven transcription. We also find that this transcriptionally inactive E2F-1 mutant is capable of degrading the MDMX-related protein MDM2 and the MDMX isoform MDMX-S. Mapping of the E2F-1 C terminus reveals that neither a previously characterized C-terminal MDM2 binding domain nor the pRb binding domain on E2F-1 is required for MDMX and MDM2 degradation.

Base Sequence↗
PubMed2001

Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2.

The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environment, following treatment with soluble human bone morphogenetic protein-2 (BMP-2). To enhance this process, the human BMP-2 cDNA was cloned into a retroviral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3HIOT1/2 cells with this retroviral construct resulted in an infection efficiency of 90-95% and was highly effective in converting cells in micromass culture to a chondrocyte phenotype, as assessed by positive Alcian blue staining for extracellular matrix proteoglycans, increased sulfate incorporation, increased expression of the cartilage marker genes collagen type II and aggrecan, and decreased expression of collagen type I. Interestingly, BMP-2 expression in the micromass cultures also induced the expression of the cell cycle inhibitory protein/differentiation factor p21/WAF1, suggesting its functional involvement in chondrogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected cells did not chondrify when co-cultured as a nonoverlapping micromass adjacent to BMP-2 expressing cells. These data indicate that retrovirally expressed BMP-2 is highly effective at inducing a chondrocyte phenotype in a multipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.

Aggrecans↗
PubMed2001

Constructed wetlands for wastewater treatment in central Italy.

The performance of 4 constructed wetlands designed by IRIDRA Srl and operating in Tuscany (central Italy) has been monitored during the last three years. The 4 treatment systems have different sizes and characteristics: one single stage secondary treatment (150 p.e.); two secondary treatment plants with effluent reuse: one small (60 p.e) and the other big (350 p.e.); a tertiary treatment of effluents from an activated sludge plant with high hydraulic load fluctuation (5-500 p.e.). Due to geographical and economic constraints the four systems have high hydraulic and organic loading rates, neverthless the systems show very good removal performance of COD (62-95%), especially the ones with higher inflow COD concentrations (87-95%). Interesting results concerning also removal percentage of MBAS (42-88%) and ammonium (42-85%) were obtained, even though NH4+ concentration in the outflows of some of the plants, doesn't always comply with Italian quality standards.

Conservation of Natural Resources↗
PubMed2000

Interaction between the lipoamide-containing H-protein and the lipoamide dehydrogenase (L-protein) of the glycine decarboxylase multienzyme system. 1. Biochemical studies.

Lipoamide dehydrogenase or dihydrolipoamide dehydrogenase (EC 1.8.1. 4) is the E3-protein component of the mitochondrial 2-oxoacid dehydrogenase multienzyme complexes. It is also the L-protein component of the glycine decarboxylase system. Although the enzymology of this enzyme has been studied exhaustively using free lipoamide as substrate, no data are available concerning the kinetic parameters of this enzyme with its physiological substrates, the dihydrolipoyl domain of the E2 component (dihydrolipoyl acyltransferase) of the 2-oxoacid dehydrogenase multienzyme complexes or the dihydrolipoyl H-protein of the mitochondrial glycine decarboxylase. In this paper, we demonstrate that Tris(2-carboxyethyl)phosphine, a specific disulfide reducing agent, allows a continuous reduction of the lipoyl group associated with the H-protein during the course of the reaction catalysed by the L-protein. This provided a valuable new tool with which to study the catalytic properties of the lipoamide dehydrogenase. The L-protein displayed a much higher affinity for the dihydrolipoyl H-protein than for free dihydrolipoamide. The oxidation of the dihydrolipoyl H-protein was not affected by the presence of structurally related analogues (apoH-protein or octanoylated H-protein). In marked contrast, these analogues strongly and competitively inhibited the decarboxylation of the glycine molecule catalysed by the P-protein component of the glycine decarboxylase system. Small unfolded proteolytic fragments of the H-protein, containing the lipoamide moiety, displayed Km values for the L-protein close to that found for the H-protein. On the other hand, these fragments were not able to promote the decarboxylation of the glycine in the presence of the P-protein. New highly hydrophilic lipoate analogues were synthesized. All of them showed Km and kcat/Km values very close to that found for the H-protein. From our results we concluded that no structural interaction is required for the L-protein to catalyse the oxidation of the dihydrolipoyl H-protein. We discuss the possibility that one function of the H-protein is to maintain a high concentration of the hydrophobic lipoate molecules in a nonmicellar state which would be accessible to the catalytic site of the lipoamide dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed2000

p27Kip1 expression is associated with clinical outcome in advanced epithelial ovarian cancer: multivariate analysis.

Few biological parameters have been shown to have a prognostic role in patients with advanced ovarian cancer. p27Kip1 is a cyclin-dependent kinase inhibitor, and its loss may contribute to tumor progression. We determined whether p27Kip1 protein expression in advanced ovarian cancer could be associated with prognosis. p27Kip1 status was assessed by immunohistochemical analysis of tissue sections from primary tumors of 99 patients with stages III-IV ovarian carcinoma and was analyzed in relation to clinicopathological variables, time to progression (TTP), and overall survival (OS). p27Kip1 expression was detected in 47 (47%) of the 99 patients. p27 expression did not correlate with any of the classical clinicopathological parameters. Loss of p27 protein was significantly associated with a short TTP (P = 0.0004) and decreased OS (P = 0.0302). The 5-year TTP rate in p27-positive patients was 50% versus 11% in p27-negative patients. p27-positive cases showed a 5-year OS rate of 53% compared with 43% of p27-negative cases. In multivariate analysis, p27 expression was an independent predictor of progression of disease (P = 0.0009) and survival (P = 0.0032) when considered together with stage of disease, presence of ascites, and residual tumor at surgery. Loss of p27Kip1 conferred poor prognosis independently of proliferative index, as assessed by proliferating cell nuclear antigen immunostaining. p27 immunoreactivity can be used to predict progression of disease and survival in patients with advanced epithelial ovarian cancer and therefore may represent a new prognostic marker.

Age Factors↗
PubMed1999

Synthesis and preliminary assessments of ethyl-terminated perfluoroalkyl nonionic surfactants derived from tris(hydroxymethyl)acrylamidomethane.

We describe the synthesis and preliminary physicochemical and biological assessments of a new class of nonionic hybrid hydrofluoro amphiphiles derived from tris(hydroxymethyl)aminomethane (THAM). The synthesis of the hydrophobic tail of these amphiphiles is based on the preparation of an asymmetrical hydrofluorocarbon derivative containing an ethyl segment, a fluorocarbon core, and an ethyl thiol moiety. This molecule led to either THAM galactosylated monoadducts or telomers. These amphiphiles exhibit neither detergency toward cell membranes nor membrane protein denaturation.

Acrylamides↗
PubMed1999

Paclitaxel induces apoptosis in Saos-2 cells with CD95L upregulation and Bcl-2 phosphorylation.

We examined the effect of paclitaxel on human osteoblastic cells Saos-2 to determine if paclitaxel can affect proliferation and apoptosis. We used a p53-negative cell line in order to mimic the loss of function frequently observed at the clinical level. Paclitaxel induced cell death in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were observed by Hoechst 33258 stain, DNA ladder formation, electron microscopy, and flow cytometry at concentrations as low as 100 nM, a concentration which can be achieved by infusion in human plasma. At 100 nM, paclitaxel induced a G2 arrest at 8 h of treatment. The cells then continued to accumulate in G2 until 72 h when the percentage of apoptotic events reached 54%. At the molecular level, Bcl-2 protein was phosphorylated at 16 h and PARP protein was cleaved, indicating the activation of caspase-3-like proteases. Caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK rescued Saos-2 cells from paclitaxel-induced apoptosis. CD95 expression was constantly high, while CD95L showed a threefold increase in expression. This suggests that, following the G2 arrest, apoptosis is induced through the CD95/CD95L system.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Synthesis and surface-active properties of glycosyl carbamates and thioureas.

Several amphiphilic glycosyl carbamates, glycosyl thiocarbamates and glycosylthioureas were prepared by addition of the anomeric hydroxyl group of acetylated glycosyl derivatives to alkyl isocyanates, or by reaction of glycosyl isothiocyanates with alcohols or amines. The solubility, critical micelle concentrations and detergent efficiency for the extraction of proteins of these compounds were evaluated and compared.

Carbamates↗
PubMed1999

Frequent loss of expression of the cyclin-dependent kinase inhibitor p27 in epithelial ovarian cancer.

p27Kip1 is a member of the Cip1/Kip1 family of cyclin-dependent kinase inhibitors and is a potential tumor suppressor gene. Low levels of p27 are associated with poor prognosis in a variety of tumors, including breast, colon, prostate, and lung carcinomas. In the present study, p27 protein expression was investigated by immunohistochemistry and Western blot analysis in a series of 82 epithelial ovarian tumors [16 classified as low malignant potential (LMP) and 66 classified as primary ovarian adenocarcinomas]. Immunohistochemical analysis revealed frequent loss of p27 expression in primary ovarian adenocarcinomas (33%), with respect to LMP tumors (6%; P = 0.0009). In addition to nuclear staining, cytoplasmic localization of p27 was noted in 45 (55%) of 82 cases. p27 levels inversely correlated with cdk2 kinase activity in a representative subset of tumors. When the clinical outcome of the patients was evaluated in relationship to p27 status, we observed a significant correlation between presence of p27 staining and a longer time to progression (P = 0.032 by log-rank test). These data indicate that loss of p27 is a frequent event in ovarian carcinomas as compared with LMP tumors, suggesting that these tumor types may have different pathogenesis. p27 levels may also represent a useful prognostic marker for predicting disease recurrence in primary ovarian carcinomas.

Adenocarcinoma↗
PubMed1998

A new class of sulfoxide surfactants derived from Tris. Synthesis and preliminary assessments of their properties.

A new class of non-ionic amphiphilic molecules suitable for biological purposes, especially extraction of membrane proteins, is reported. Such surfactants were prepared in two steps: addition of alkyl or fluoroalkyl mercaptan on Tris(hydroxymethyl)acrylamidomethane (THAM) derivatives, followed by the oxydation of sulfide group in sulfoxide moiety in order to provide water solubility to the molecule. The detergent efficiency of these new surfactants were assayed on rat liver cells.

Animals↗