Constitutive localization of DR4 in lipid rafts is mandatory for TRAIL-induced apoptosis in B-cell hematologic malignancies.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts as an apoptosis inducer for cancer cells sparing non-tumor cell targets. However, several phase I/II clinical trials have shown limited benefits of this molecule. In the present work, we investigated whether cell susceptibility to TRAIL ligation could be due to the presence of TRAIL death receptors (DRs) 4 and 5 in membrane microdomains called lipid rafts. We performed a series of analyses, either by biochemical methods or fluorescence resonance energy transfer (FRET) technique, on normal cells (i.e. lymphocytes, fibroblasts, endothelial cells), on a panel of human cancer B-cell lines as well as on CD19(+) lymphocytes from patients with B-chronic lymphocytic leukemia, treated with different TRAIL ligands, that is, recombinant soluble TRAIL, specific agonistic antibodies to DR4 and DR5, or CD34(+) TRAIL-armed cells. Irrespective to the expression levels of DRs, a molecular interaction between ganglioside GM3, abundant in lymphoid cells, and DR4 was detected. This association was negligible in all non-transformed cells and was strictly related to TRAIL susceptibility of cancer cells. Interestingly, lipid raft disruptor methyl-beta-cyclodextrin abrogated this susceptibility, whereas the chemotherapic drug perifosine, which induced the recruitment of TRAIL into lipid microdomains, improved TRAIL-induced apoptosis. Accordingly, in ex vivo samples from patients with B-chronic lymphocytic leukemia, the constitutive embedding of DR4 in lipid microdomains was associated per se with cell death susceptibility, whereas its exclusion was associated with TRAIL resistance. These results provide a key mechanism for TRAIL sensitivity in B-cell malignances: the association, within lipid microdomains, of DR4 but not DR5, with a specific ganglioside, that is the monosialoganglioside GM3. On these bases we suggest that lipid microdomains could exert a catalytic role for DR4-mediated cell death and that an ex vivo quantitative FRET analysis could be predictive of cancer cell sensitivity to TRAIL.

Association of fission proteins with mitochondrial raft-like domains.

It was shown that receptor-mediated apoptosis involves a cascade of subcellular events including alterations of mitochondria. Loss of mitochondrial membrane potential that follows death receptor ligation allows the release of apoptogenic factors that result in apoptosis execution. Further important mitochondrial changes have been observed in this regard: mitochondrial remodeling and fission that appear as prerequisites for the occurrence of the cell death program. As it was observed that lipid rafts, glycosphingolipid-enriched structures, can participate in the apoptotic cascade being recruited to the mitochondria under receptor-mediated proapoptotic stimulation, we decided to analyze the possible implication of these microdomains in mitochondrial fission. We found that molecules involved in mitochondrial fission processes are associated with these domains. In particular, although hFis1 was constitutively included in mitochondrial raft-like domains, dynamin-like protein 1 was recruited to these domains on CD95/Fas triggering. Accordingly, the disruption of rafts, for example, by inhibiting ceramide synthase, leads to the impairment of fission molecule recruitment to the mitochondria, reduction of mitochondrial fission and a significant reduction of apoptosis. We hypothesize that under apoptotic stimulation the recruitment of fission-associated molecules to the mitochondrial rafts could have a role in the morphogenetic changes leading to organelle fission.

Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells.

Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.

Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis.

We previously reported that during death receptor-mediated apoptosis, cardiolipin (CL) relocates to the cell surface, where it reacts with autoantibodies from antiphospholipid syndrome sera. Here, we analysed the intracellular distribution of CL and its metabolites during the early phase of cell death signalling triggered by Fas stimulation in U937 cells and mouse liver. We found a redistribution of mitochondrial CL to the cell surface by using confocal microscopy and flow cytometry. Mass spectrometry revealed that CL and its metabolites relocated from mitochondria to other intracellular organelles during apoptosis, with a conversion into non-mitochondrial lipids. Concomitantly, cytosolic Bid relocated to the light membranes comprised in fraction P100, including the plasma membrane and associated vesicular systems. A direct Bid-CL interaction was demonstrated by the observation that CL and monolysoCL coimmunoprecipitated with Bid especially after Fas stimulation, suggesting a dynamic interaction of the protein with CL and its metabolites.

GD3 glycosphingolipid contributes to Fas-mediated apoptosis via association with ezrin cytoskeletal protein.

Efficiency of Fas-mediated apoptosis of lymphoid cells is regulated, among other means, by a mechanism involving its association with ezrin, a cytoskeletal protein belonging to the 4.1 family of proteins. In the present work, we provide evidence for a further molecule that associates to ezrin in Fas-triggered apoptosis, the disialoganglioside GD3. In fact, as an early event, GD3 redistributed in membrane-associated domains in uropods and co-localized with ezrin. Co-immunoprecipitation analyses confirmed this result, indicating a GD3-ezrin association. Altogether, these results are suggestive for a role of GD3 in Fas/ezrin-mediated apoptosis, supporting the view that uropods contain a multimolecular signaling complex involved in Fas-mediated apoptosis.

Magnetic resonance imaging of temporomandibular disorders: classification, prevalence and interpretation of disc displacement and deformation.

AIM: To analyse the prevalence of disc displacements and deformations from MRI of symptomatic temporomandibular disorders (TMD). METHODS: The study was conducted retrospectively on 192 joints of 98 patients (67 females, 31 males, mean age 29 years), who had undergone bilateral MRI (except for four who had unilateral) in the sagittal (both open and closed mouth) and coronal (closed mouth only) planes. These displacements were subdivided into static (complete anterior and posterior, partial anterolateral and anteromedial, sideways lateral and medial, anterolateral and anteromedial rotational) and dynamic (with reduction, without reduction, with incomplete reduction; non-determinable). Disc deformations were subdivided into: enlargement of the posterior band, reversed biconcave shape, biplanar (flattened) and biconvex shape. RESULTS: Eighty per cent of patients had bilateral displacement, 15% unilateral and 5% a normally positioned disc. Complete anterior displacement was the commonest and sideways the rarest. Reduction was present in 58% of disc displacements, no reduction in 26%, incomplete reduction in 4%, while in the remaining 12%, it could not be determined. Rotational displacement was the most likely to feature reduction and sideways the least. Temporomandibular joints with no reduction were closely correlated with bone lesions. The most frequent deformation was biplanar and the rarest enlargement of the posterior band. CONCLUSIONS: There was a high prevalence of displacements and deformations. While they do not appear to be significant in inducing pain, they can predispose to the onset of osteoarthrosis.

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 p56Ick in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56Ick 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 p56Ick were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56Ick complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56Ick, we analyzed this association in U937, a CD4 + and p56Ick negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56Ick. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.

Glycosphingolipid domains on cell plasma membrane.

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.

Colocalization and complex formation between prosaposin and monosialoganglioside GM3 in neural cells.

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 tight GM3-prosaposin association on NS20Y plasma membranes. We suggest that ganglioside-protein complexes are structural components of the prosaposin receptor involved in cell differentiation.

A novel mechanism of CD4 down-modulation induced by monosialoganglioside GM3. Involvement of serine phosphorylation and protein kinase c delta translocation.

In this report the molecular mechanism(s) involved in the rapid and selective endocytosis of cell surface glycoprotein CD4 induced by exogenous monosialoganglioside GM3 in human peripheral blood lymphocytes have been investigated. Inhibition of the GM3-induced CD4 down-modulation was observed in the presence of specific protein kinase C (PKC) inhibitors. Scanning confocal microscopy revealed the translocation and clustering on the cell surface of PKC isozymes delta and theta (more evidently than alpha and beta) after GM3 treatment, suggesting the involvement of these isozymes in the ganglioside-induced CD4 down-modulation. Exogenous GM3 induced phosphorylation of CD4 molecule, which then dissociated from p56(lck), as early as after 5 min. Moreover, addition of GM3 resulted in a rapid (1 min) cytosolic phospholipase A2 activation with consequent arachidonic acid release, whereas no phosphatidylinositol-phospholipase C activity was observed. Both PKC translocation and CD4 down-modulation were blocked by the trifluoromethylketone analog of arachidonic acid, a selective inhibitor of cytosolic phospholipase A2 and by mitogen-activated protein kinase inhibitor PD98059. Taken together, these findings strongly suggest that GM3 may trigger a novel mechanism of modulation of the CD4 surface expression through the activation of enzyme(s) involved in the regulation of cellular functions.

Accuracy and efficacy of nuclear scintigraphy for the detection of gastrointestinal bleeding.

OBJECTIVES: To determine the accuracy and cost-effectiveness of nuclear scintigraphy for the diagnosis and localization of gastrointestinal (GI) bleeding and to determine whether nuclear scintigraphy accurately predicts the results of angiography. DESIGN: Retrospective chart review. The following data were obtained from the medical records: age; diagnosis before scintigraphy; duration of bleeding; hemoglobin and hematocrit values; number and duration of blood transfusions; results of angiography and GI endoscopy; location of bleeding as determined by angiography, endoscopy, and nuclear scintigraphy; treatment received by patients; actual site of bleeding as documented in the medical record; and outcome of treatment. SETTING: Community hospital in a city with a population of approximately 100,000 and a catchment area of approximately 500,000. PATIENTS: One hundred fifty-five patients undergoing 161 nuclear scintigraphic examinations because of GI bleeding between January 1, 1989, and December 31, 1992. MAIN OUTCOME MEASURES: Diagnosis and location of GI bleeding as determined by nuclear scintigraphy and angiography and actual site of GI bleeding as confirmed by operative intervention or endoscopy. RESULTS: Of 114 scintigraphic examinations for which the diagnosis and localization of GI bleeding were definite, results were positive in 56 (49.1%); of these 56, a definite location of the bleeding was shown in 51 cases (91.1%), and the bleeding was localized to its exact site in 22 cases (19.3%). Of the scintigraphic examinations for which results were positive, results of angiograms were positive in 7 cases, which gives nuclear scintigraphy a 39% positive predictive value for angiography. CONCLUSION: The routine use of nuclear scintigraphy is neither accurate nor cost-effective for determining the site of GI bleeding in the community hospital.