Enrichment and localization of ganglioside G(D3) and caveolin-1 in shed tumor cell membrane vesicles.

Tumor cell ganglioside shedding has been implicated in the process of tumor formation. Previously, we identified three forms of tumor ganglioside shedding: micelles, monomers and membrane vesicles. Here, we have explored the membrane vesicle form of ganglioside shedding, using a newly identified human ovarian carcinoma cell line, CABA I. These cells synthesize and express a spectrum of gangliosides, including the disialoganglioside, G(D3). Immunostaining using the monoclonal antibody R24 confirmed G(D3) expression and its presence in the plasma membrane of these cells. Cellular gangliosides were detected in the culture supernatant by HPTLC autoradiography, confirming an active shedding rate of 3% of cellular gangliosides/24 h. CABA I cell membranes also express caveolin-1, a characteristic protein marker for caveolae, which was detected by flow cytometric analysis and by Western blotting in both the cell membranes and the isolated membrane vesicles. To further define the expression of G(D3) and caveolin-1, we used immunogold electron microscopy. This revealed localization of G(D3) in small clusters in the plasma membrane as well as enrichment and localization of ganglioside G(D3) and caveolin-1 in shed membrane vesicles, with 58-78% of vesicles carrying both G(D3) and caveolin-1. Together, these results suggest that membrane vesicle shedding originates in plasma membrane domains enriched in gangliosides and caveolin-1.

Psychiatric patients' perceptions of waiting time in the psychiatric emergency service.

Recent customer service surveys have indicated that patients in the Psychiatric Emergency Service (PES) are increasingly dissatisfied with the "waiting" time connected with expanded services. This study examined the impact of six interventions that altered the environment of waiting patients and their resulting perceptions of time spent in PES. The interventions were communication (three styles), relaxing music, educational videos, and recreational activities. Each intervention was conducted for 7 days, followed by 7 days of no intervention. Patients completed a Time Assessment Tool that measured expectations of, perceptions of, and satisfaction with waiting time. These data were compared to actual time, as reflected on the log maintained in the PES. In general, environmental interventions reduced patients' perceived time, compared to no intervention. Significantly, interacting with patients with caring and concern consistently resulted in a reduced perception of waiting time, compared to the other interventions.

[Stable transfection of DAOY cells with a GM3 synthase antisense construct and transient reduction in ganglioside content].

Tumor cell gangliosides are bioactive molecules involved in tumor-host interactions. To investigate their role in tumor formation and angiogenesis, we sought to develop an inhibitory model targeting human GM3 synthase, an essential enzyme in the ganglioside synthesis pathway, by antisense transfection. We prepared a number of transfectants from DAOY human medulloblastoma cells and isolated clones that stably expressed a 560-bp fragment of human GM3 synthase cDNA, in either sense or antisense orientation, as well as clones transfected with an empty vector. Both sense and antisense clones permanently incorporated mammalian expression vectors into their genomes. The DAOY cell clones were screened for ganglioside content using total lipid extraction, ganglioside isolation, and HPTLC. One antisense-transfected clone, 7.2A, in which total ganglioside content was reduced by 70%, was selected for further study. All sense- and sham-transfectants had ganglioside levels not different from that of untransfected DAOY cells. After 10 passages however, while antisense mRNA expression was fully maintained, the ganglioside content of 7.2A cells had reverted to normal levels. Antisense RNA transfection can sometimes have a reversible effect on the expression of a target. Possible regulatory mechanisms of this previously unrecognized process of reversion to wild type phenotype are discussed.

Cellular gangliosides promote growth factor-induced proliferation of fibroblasts.

Cell surface gangliosides have been proposed as modulators of transmembrane signaling. In this study, we used two complementary approaches to investigate the function of cellular gangliosides in the response of mammalian fibroblasts to growth factors. First, inhibition of glucosyl ceramide synthase by a new specific inhibitor of d-l-threo-1-phenyl-2-hexadecanoylamino-3 -pyrrolidino-1-propanol-HC l (glucosylceramide synthase), which depletes cellular gangliosides at a concentration of 1 microm without causing an increase in ceramide levels, blocked epidermal growth factor-stimulated proliferation of fibroblasts. Similarly, responses to several other growth factors that activate receptor tyrosine kinases, including fibroblast growth factor, insulin-like growth factor-I, and platelet-derived growth factor, were inhibited by 50-100%. Conversely, enrichment of cellular gangliosides by preincubation of the mouse and human fibroblasts with exogenously added gangliosides enhanced growth factor-elicited cell proliferation. Novel findings of this study, distinguishing it from previous similar studies, include differential effects of preincubation versus continuous incubation of cells with gangliosides on growth factor-dependent cell proliferation and the growth factor-like action of NeuNAc alpha 2-3Gal beta 1-3GalNAc beta 1-4(NeuNAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer when cells are pretreated with the ganglioside.