Incorporation of ganglioside analogs into fibroblast cell membranes. A spin-label study

Schwarzmann, Günter; Hoffmann-Bleihauer, P.; Schubert, Jöerg; Sandhoff, Konrad; Marsh, Derek

doi:10.1021/bi00290a025

Cited by 155 publications

(71 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…added to cells in vitro, the ceramide moiety of the ganglioside Radiolabeled gangliosides were isolated from lyophilized cell pellets molecule can intercalate into the membrane bilayer in a trypand conditioned medium, with 10 nmol purified human brain gangliosides added as a cold carrier, using previously described methods sin-resistant manner [6,7]. This is observed either when puri- [11].…”

Section: Ganglioside Purification and Autoradiographymentioning

confidence: 99%

“…In contrast, the ganglioside concentration in upper chamber at a concentration of 7 × 106 cells/1.5 ml. 6 the CM on the contralateral side of the membrane from the 8 × 106 YAC-1 cells (viability >95%) were recovered from the YAC-1 cells was 0.0054 + 0.0004 ~tM. This ten-fold lower conupper chamber at the end of the 24 h co-incubation period, centration of shed YAC-1 gangliosides was in part due to Several approaches established that no lymphoma cells were slow diffusion through the membrane, as supported by the present on the contralateral side of the membrane, i.e.…”

Section: Shedding Of Yac-1 Gangliosidesmentioning

confidence: 99%

See 1 more Smart Citation

Intercellular transfer of shed tumor cell gangliosides

Olshefski

Ladisch

1996

FEBS Letters

View full text Add to dashboard Cite

Three distinct steps underlie immunosuppression byis prevented. The significant finding of this study is that the tumor gangliosides: (i) their shedding by the tumor cell, (ii) shed gangliosides, detected at a concentration significantly binding to target leukocytes in the tumor microenvironment, and lower than that routinely used in ganglioside binding studies (iii) action upon the target ceil. While shedding is well [7], were incorporated into the membrane of the target cells, documented, cell to cell transfer of shed gangliosides is not. To and that this binding occurred to a degree which is likely to be address this, we employed a dual chamber culture system. In this physiologically relevant. washed six times by centrifugation in complete medium. Following the final wash, the cells were resuspended in EMEM at 3.5x106 cells/ ml. Cell viability, assessed by trypan blue exclusion, was greater than The shedding of endogenous gangliosides by tumor cells is a 98%. Human flbroblasts were cultured to pre-confluency, radiolabeled well-recognized process [1][2][3][4], and occurs at a rapid rate, i.e.for 48 h as above, washed six times with phosphate-buffered saline up to 0.5% of the membrane ganglioside content per hour [5].(PBS), and then harvested by trypsinization. Less is known about the interaction of exogenous ganglioside molecules with cells. When highly purified gangliosides are Ganglioside purification and autoradiographyadded to cells in vitro, the ceramide moiety of the ganglioside Radiolabeled gangliosides were isolated from lyophilized cell pellets molecule can intercalate into the membrane bilayer in a trypand conditioned medium, with 10 nmol purified human brain gangliosides added as a cold carrier, using previously described methods sin-resistant manner [6,7]. This is observed either when puri- [11]. The ganglioside-associated radioactivity was quantified by [3-scinfled exogenous gangliosides, which exist in micellar form or as tillation counting, and confirmed by high-performance thin-layer free molecular species, are used [8], or when gangliosides are chromatography (HPTLC) autoradiography [5]. Purified 14C-labeled incorporated in artificial membrane systems, such liposomes gangliosides were quantified by a modified resorcinol method [12] to [9]. Both approaches have shown that gangliosides can be determine specific activity.stably bound by target cells, but neither approach can address the question of whether the in vivo process of tumor cell Co-incubation studies42sk fibroblasts were cultured in 6-well plates (Costar) and grown ganglioside shedding can lead to suppression of the cellular to confluence. At the beginning of the experiment, the culture medium immune response [10]. One aspect of this problem is to deterwas removed from four plates and 2.0 ml fresh medium was added to mine whether immunosuppressive gangliosides, as shed from each of 24 wells. Upper chamber inserts containing a polycarbonate tumor ceils, bind to target cells in the tumor microenvironmembrane with 0.4 ktm pore si...

show abstract

Section: Ganglioside Purification and Autoradiographymentioning

confidence: 99%

Section: Shedding Of Yac-1 Gangliosidesmentioning

confidence: 99%

Intercellular transfer of shed tumor cell gangliosides

Olshefski

Ladisch

1996

FEBS Letters

View full text Add to dashboard Cite

show abstract

“…Cells were incubated at 37°C in 2 ml of the gangliosidecontaining medium for up to 24 h, and then washed and treated as reported [9,10,13]. All the above-described operations were carried out in the dark.…”

Section: Treatment Of Fibroblasts Wrh Fh]gml(?43) and Preparation Of mentioning

confidence: 99%

Formation of a cytosolic ganglioside‐protein complex following administration of photoreactive ganglioside GM1 to human fibroblasts in culture

et al. 1990

View full text Add to dashboard Cite

A GM 1 ganglioside derivative bearing a photoreactive nitrophenyl azide group and tritium labeled at the acetyl group of N-acetylneuraminic acid, has been administered to cultured human fibroblasts. With photolabeling experiments we found that a portion of the ganglioside in the cell cytosol was associated with a soluble protein of about 30 kDa.Photoreactive GM 1; Cytosolic complex; Ganglioside-protein interaction

show abstract

“…Exogenously added gangliosides (1) are taken up by cells in culture, and are partly inserted into the outer plasma membrane layer, thus mimicking the major localization of cellular gangliosides [1,2]. A portion of incorporated gangliosides is internalized by endocytosis [3] and then rapidly subjected to metabolic processing [1,4,5].…”

Section: Introductionmentioning

confidence: 99%