Giuseppe Pontieri

Ganglioside Expression in Human Pancreatic Islets

Recent biochemical studies have shown that the cytoplasmic islet cell–antibody autoantigen has properties of a monosialoganglioside (GM). To characterize islet glycolipids and ascertain whether islets express unique gangliosides, we determined the pattern of ganglioside expression in whole human pancreas and isolated human islets using high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC). The major gangliosides detected in glycolipid extracts of whole human pancreas were GM3, GD3 (disialoganglioside), and in a lesser amount, a GD1a-comigrating ganglioside. In contrast to whole human pancreas, isolated human islets were found to predominantly express GM3, an acidic glycolipid comigrating with GM2, and a ganglioside with mobility between GM2 and GM1 by both HPLC and HPTLC. Quantitation of the major ganglioside UV peaks seen on HPLC gave the following results. In whole pancreas, GM3 represented 66.7% of total gangliosides detected; an asialoglycolipid comigrating with GM2, 2.0%; a ganglioside migrating between GM2 and GM1, 2.6%; GD3, 22.6%; and a GD1a-comigrating ganglioside, 6.1%. In isolated islets, these components were found at the following levels: GM3, 14.9%; GM2-comigrating glycolipid, 74.2%; a ganglioside migrating between GM2 and GM1, 9.8%; GD3, 1.1%; and the GD1a-comigrating ganglioside, not detectable.

Colocalization and Complex Formation Between Prosaposin and Monosialoganglioside GM3 in Neural Cells

Abstract: Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12‐amino acid sequence located in the NH2‐terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca2+‐independent and not disassociated during sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The association of prosaposin‐GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22‐mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide‐induced neurite outgrowth, as well as prosaptide‐enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein‐mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and right GM3‐prosaposin association on NS20Y plasma membranes. We suggest that ganglioside‐protein complexes are structural components of the prosaposin receptor involved in cell differentiation.

Monosialoganglioside GM3 induces CD4 internalization in human peripheral blood T lymphocytes

Gangliosides modulate the expression of CD4 molecules on the cell surface of T lymphocytes. We report here that treatment of human peripheral blood lymphocytes with exogenous monosialoganglioside GM3 induces a rapid down‐modulation of the CD4 molecules on the plasma membrane of CD4+ T lymphocytes, as assessed by cytofluorimetric analysis and quantitative immunoelectron microscopy. The CD4 down‐modulation was gang Hoside‐dose dependent and was already evident after 5 min of treatment, reaching the maximum after 20 min. The expression of other surface antigens was not affected by GM3 treatment. The immunoelectron microscopic analysis showed that, following GM3 addition, gold labelled CD4 molecules were rapidly redistributed on the cell surface, clustered and internalized via endocytic pits and vesicles. These results indicate that CD4 down‐modulation induced by GM3 occurs through an endocytic mechanism. A persistent low level of CD4 expression on the cell surface up to 24 h after GM3 treatment, compared with a stable expression of either CD4 in untreated cells and CD3 in GM3‐treated cells, suggests intracellular degradation of the internalized CD4 molecules.

Association of Cellular Prion Protein with Gangliosides in Plasma Membrane Microdomains of Neural and Lymphocytic Cells

In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrPc presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrPc and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrPc-enriched microdomains. In lymphoblastoid T cells PrPc molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrPc was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrPc-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrPc immunoprecipitates. The physical association of PrPc with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrPc-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.

Blood Leukotrienes in Headache: Correlation With Platelet Activity

SYNOPSIS

Determination of gangliosides by high-performance liquid chromatography with photodiode-array detection

Abstract Mixtures of gangliosides were separated by high-performance liquid chromatography (HPLC) on amino-silica columns according to their negative charge and the length of the carbohydrate portion. The use of an on-line variable wave-length diode-array detector allows the identification of gangliosides on the basis of their UV spectra with maximum absorbance at 196 nm. Accurate analytical data acquired with the diode-array detector allow the qualitative and quantitative determination of gangliosides and therefore eliminate the need for thin-layer chromatography after HPLC separation.

Increased levels of prostaglandins and nitric oxide in esophageal mucosa of children with reflux esophagitis

BACKGROUND Prostaglandin E2 (PGE2) is said to be both protective and detrimental for esophageal mucosal integrity. Nitric oxide (NO) controls several esophageal neuromuscular functions, including relaxation of the lower esophageal sphincter. The purpose of this study was to verify PGE2 and NO levels in esophageal mucosa of children with reflux esophagitis. METHODS The patients were 10 children, age range 7 to 12 years, affected by reflux esophagitis. The control subjects were 10 children, age range 6 to 11 years, with recurrent abdominal pain. Tissue fragments obtained by esophageal biopsies were placed in a culture medium and processed to obtain a cell suspension. Cells were incubated for 24 hours at 37 degrees C. Thereafter, supernatants were collected and divided into aliquots to determine the amounts of PGE2 and NO metabolites. RESULTS Esophageal cells obtained from reflux esophagitis patients synthesize and release a significantly higher (p < 0.01) amount of PGE2 and NO (PGE2 1.9 +/- 0.56 ng/10(6) cells per 24 hours; NO 124.94 +/- 18.36 microM/10(6) cells per 24 hours) than did the control group (PGE2 0.66 +/- 0.14 ng/10(6) cells per 24 hours; NO 68.03 +/- 12.3 microM/10(6) cells per 24 hours). CONCLUSIONS These results suggest that in esophageal mucosa, PGE2 and NO, in low concentrations, are protective, whereas, at high doses, they can be harmful. Higher amounts of PGE2 and NO in the esophageal mucosa of reflux esophagitis patients suggest that similar noxious stimuli trigger the inducible forms of the respective enzyme.

Autoantibodies Against Ganglioside GM3 Represent a Portion of Anti-Lymphocyte Antibodies in AIDS Patients

In this study we analysed the relationship between anti‐lymphocytic ganglioside antibodies and anti‐lymphocyte antibodies in AIDS patients. Anti‐lymphocytic ganglioside antibodies were detected by thin layer chromatography (TLC) immunostaining; three colour flow cytometry was used to analyse circulating antibodies against different lymphocyte subsets.

Identification of Blood Leukotrienes in Classical Migraine

SYNOPSIS

Association between GM3 and CD4-Ick complex in human peripheral blood lymphocytes.

The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56lck in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56lck complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56lck were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56lck complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56lck, we analyzed this association in U937, a CD4+and p56lck negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58[emsp4 ]kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56lck. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.