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P Uboldi

Publications and source records attributed to P Uboldi.

10 recordsLinked to original sources
PubMed2005

Liver X receptor and retinoic X receptor agonists modulate the expression of genes involved in lipid metabolism in human endothelial cells.

The cooperation of liver X receptors (LXRs) alpha and beta, and retinoic X receptor (RXR) modulate the expression of several genes involved in lipid metabolism in hepatocyte and macrophages. Using cDNA microarray technology, we have shown previously that several of these genes are also expressed in endothelial cells. In the present study, we investigated whether the activation of LXR and RXR affects the expression of genes involved in lipid metabolism in human endothelial cells. Relative expression of ABCA-1, CETP, SR-B1, EL, LPL, PLTP, ApoE and LDLR was investigated in HUVECs, human fibroblasts (hFB) and HepG2 cells by quantitative real-time PCR. For CETP and EL mRNA expression, the results were HUVECs > hFB > HEPG2; for PLTP, LDLR and LPL: hFB > HUVECs > HEPG2; for SR-B1 and ApoE: HEPG2 > HUVECs > hFB; and for ABCA-1 HEPG2: > hFB > HUVECs. Incubation of HUVECs with LXR agonists as 22-(R)-hydroxycholesterol (22-(R)-HC) or T0901317-induced ABCA1 (20.1- and 17.8-fold), LPL (3.46- and 7.03-fold) and CETP (6.34- and 3.98-fold) expression; EL, LDLR and SR-B1 expression was induced only upon incubation with T0901317 (2.40-, 2.83- and 2.19-fold, respectively) while 22-(R)-HC had no effect on EL and SR-B1 expression (0.8- and 0.9-fold) and decreased LDLR expression (0.4-fold). No effect of either 22-(R)-HC or T0901317 on PLTP and ApoE expression was observed. The RXR agonist, 9-cis retinoic acid (9CRA) alone induced the expression of CETP, LPL and SR-B1 (2.8-, 8.2- and 2.4-fold). No effect of 9CRA on ABCA-1, EL, PLTP, ApoE, and LDLR expression was observed. Association of 9CRA with 22-(R)-HC or T0901317 increased the expression of CETP and LPL while no effect on ABCA-1 or LDLR was observed. Activation of LXRs and RXRs in endothelial cells represents a new target of LXR and RXR agonist in the arterial wall. Modulation of gene expression in the endothelium should be taken into account when studying the effects of LXR and RXR agonists on lipid metabolism in the arterial wall.

ATP Binding Cassette Transporter 1↗
PubMed2001

Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor.

Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), we have studied the interaction of ten different murine monoclonal antibodies (mAbs, all IgG(1)), raised against the main protein constituent of human low density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). These mAbs identify distinct domains on apoB-100, relevant to LDL-receptor interaction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: aa 1-1297) and in the middle region (mAb 6B: aa 1480-1693; mAbs 2A, 3B: aa 2152-2377; mAbs 9A, L2 and L4: aa 2657-3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recognized by all these mAbs. A rabbit anti-mouse IgG(1)-Fc antibody (RAM.Fc) was first coupled to the gold surface in order to capture one anti-human apoB-100 mAb. ApoB-100 protein was subsequently injected and allowed to react with this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over the sensing surface. The capacity of each mAb to interact with the entrapped apoB-100 in a multimolecular complex was monitored in real time by SPR. The results achieved were comparable to those obtained by western immunoblotting using the same reagents. However, SPR ensures a more detailed epitope identification, demonstrating that BIA-technology can be successfully used for mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoassays, it is significantly time saving (CNR-P.F. MADESS 2).

Animals↗
PubMed2000

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA.

We report on systemic delivery and long-term biological effects of apolipoprotein E (apoE) obtained by intramuscular (i.m.) plasmid DNA injection. ApoE plays an important role in lipoprotein catabolism and apoE knock-out mice develop severe hypercholesterolemia and diffuse atherosclerosis. We have injected apoE-deficient mice with 80 microg of a plasmid vector (pCMV-E3) encoding the human apoE3 cDNA under the control of the CMV promoter-enhancer in both posterior legs. Local expression of the transgene was demonstrated throughout 16 weeks. Human apoE3 recombinant protein reached 0.6 ng/ml serum level. After i.m. injection of pCMV-E3 expression vector the mean serum cholesterol concentrations decreased from 439 +/- 57 mg/dl to 253 +/- 99 mg/dl (P < 0.05) 2 weeks after injection and persisted at a significantly reduced level throughout the 16 weeks observation period (P < 0.005). Serum cholesterol was unaffected and reached an absolute level of 636 +/- 67 mg/dl in control groups. Finally, injection of pCMV-E3 into apoE-deficient mice resulted in a redistribution of cholesterol content between lipoprotein fractions, with a marked decrease in VLDL, IDL and LDL cholesterol content and an increase in HDL cholesterol. These results demonstrate that severe hypercholesterolemia in apoE-deficient mice can be effectively reversed by i.m. DNA injection, and indicate that this approach could represent a useful tool to correct several hyperlipidemic conditions resulting in atherosclerosis.

Analysis of Variance↗
PubMed2000

Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase. Implications for HDL function.

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL(3) by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL(3) revealed that proteolysis for up to 24 h did not alter the integrity of the alpha-migrating HDL, whereas a minor peak containing particles of smaller size with prebeta mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the alphaHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL(3) with chymase inhibited binding of Mab A-I-9 (specific for prebeta(1)HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL(3) to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL(3) by chymase failed to reduce the ability of HDL(3) to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation. This differential sensitivity of the two key functions of HDL(3) to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for prebeta(1)HDL to promote the high-affinity efflux of cellular cholesterol, but not for the alpha-migrating HDL particles to activate LCAT.

Animals↗
PubMed1996

Localization of apolipoprotein A-I epitopes involved in the activation of lecithin:cholesterol acyltransferase.

Eight murine monoclonal antibodies (Mab) to apolipoprotein A-I were characterized for their epitopes and for their ability to interfere with lecithin:cholesterol acyltransferase (LCAT) activation mediated by apo apoA-I using a synthetic substrate. Using overlapping synthetic peptides we have identified six continuous epitopes that span amino acids 1-10 (Mab A-I-19), 96-101 (Mab A-I-15), 133-141 (Mab A-I-5), 140-145 (Mab A-I-9), 144-148 (Mab A-I-8), and 167-174 (Mab A-I-57). Furthermore, antibodies A-I-11 and A-I-16 recognized discontinuous epitopes, namely amino acids 124-128 and 144-148. When antibodies were tested for their ability to inhibit LCAT activation, an inhibitory effect was observed with those whose epitopes covered the area of apoA-I encompassing amino acids 96-174. From these data we conclude that several areas of apoA-I spanning the middle region of the apolipoprotein act in concert to stimulate LCAT activity, possibly by cooperative interaction with the enzyme.

Animals↗
PubMed1993

Immunoreactivity of apo B towards monoclonal antibodies that inhibit the LDL-receptor interaction: effects of LDL oxidation.

We studied the immunochemical stability of the epitopes for six monoclonal antibodies to human apolipoprotein B-100 upon Cu(2+)-mediated (20 microM) oxidation of LDL. The antibodies used in this study, some of which are known to interfere with the interaction of LDL with their cellular receptors, recognize epitopes in the amino terminal region (Mb 19), in the middle part (6B, 2A, 7A, and 9A) and near aa 3500 (Mb 47) of native apo B. All antibodies except one (7A) recognized native and oxidized LDL (OxLDL) equally well; the immunoreactivity of the epitope for Ab 7A was markedly reduced upon LDL oxidation. Since antibodies 2A, 7A, 9A, and Mb 47 inhibit the LDL-receptor interaction and OxLDL poorly interact in vitro with the LDL receptor we conclude that: (1) various epitopes for monoclonal antibodies against native apo B are spared upon LDL oxidation; and (2) the epitopes for antibodies 2A, 9A, and Mb 47 do not define a unique domain of apo B directly involved in the binding of LDL to their receptor.

Antibodies, Monoclonal↗
PubMed1992

Characterization of a family with moderate hypercholesterolemia and binding defective low density lipoprotein.

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder presenting with hypercholesterolemia and abnormal low density lipoprotein (LDL) that binds poorly to LDL receptors. This disease appears to be caused by a mutation in the apo B gene. In the present study thirteen members of a family with moderate hypercholesterolemia (250-350 mg/dl) were investigated. Biochemical studies on cultured skin fibroblasts ruled out classical familial hypercholesterolemia (receptor deficiency). LDL from nine affected members displayed, in an "in vitro" cell binding assay, a reduced affinity (2.5 fold) for the receptor, and had normal electrophoretic mobility, size and chemical composition. Lp(a) levels in family members were comparable to those present in normolipidemics and lower than those observed in primary hypercholesterolemia. The disorder is transmitted over three generations as an autosomal codominant trait and all the affected members are heterozygotes and hypercholesterolemic.

Adolescent↗
PubMed1992

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.

Antibodies, Monoclonal↗
PubMedDate not supplied

Nucleolar protein p120 expression in oral carcinoma.

Nucleolar protein p120 is a proliferation-associated antigen expressed by cells in early G1 phase, identified by the monoclonal antibody FB-2. Its expression has been evaluated in breast and prostate cancer, and proved to be significantly correlated with other prognostic parameters. In oral pathology, p120 protein content is able to distinguish between non-neoplastic and malignant lesions. In the present study, the immunohistochemical expression of p120 protein was evaluated in fifty cases of oral squamous carcinoma and compared with histological grading, pTNM staging, DNA ploidy status and follow-up of the patients, in order to establish its prognostic value, p120 mean area was significantly correlated to all these parameters, apart from lymph node involvement, indicating the strong predictive potential of this marker. In conclusion, quantitative immunohistochemical analysis of p120 protein represents an easy and reliable method for the assessment of clinical outcome and the definition of risk groups in oral carcinoma.

Aged↗