Permeabilized cells as a way of gaining access to intracellular organelles: an approach to glycosylation reactions

Lepers, Anne; Cacan, René; Verbert, André

doi:10.1016/0300-9084(90)90166-e

Cited by 31 publications

(13 citation statements)

References 52 publications

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“…Both digitonin and saponin open pores in the membrane that allow the diffusion of large molecules, such as antibodies or enzymes, into the cytoplasm (15,16,23,24). The results shown in this work indicate that in saponin-permeabilized cells, p34 cdc2 -mediated phosphorylation does create MPM-2 epitopes at well known locations of the natural targets of this kinase (e.g.…”

Section: Discussionmentioning

confidence: 49%

“…Because both centrosomes and IFs remain in a Triton X-100-insoluble fraction, it is unlikely that the "glue" proteins will be extracted by a mild permeabilization. To test the feasibility of this strategy, confluent CACO-2 epithelial cell monolayers were permeabilized with 0.05% saponin that, like digitonin, opens pores in the plasma membrane, permitting the diffusion of large molecules (15)(16)(17). The cells were incubated with exogenous recombinant active p34 cdc2 /cyclin B and 1 mM ATP.…”

Section: Phosphorylation Of Permeabilized Caco-2 Cell Monolayers Withmentioning

confidence: 99%

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p34 -mediated Phosphorylation Mobilizes Microtubule-organizing Centers from the Apical Intermediate Filament Scaffold in CACO-2 Epithelial Cells

Terán-Figueroa

Wald

Salas

2002

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 49%

Section: Phosphorylation Of Permeabilized Caco-2 Cell Monolayers Withmentioning

confidence: 99%

p34 -mediated Phosphorylation Mobilizes Microtubule-organizing Centers from the Apical Intermediate Filament Scaffold in CACO-2 Epithelial Cells

Terán-Figueroa

Wald

Salas

2002

Journal of Biological Chemistry

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“…DPM did not affect the uptake or efflux of [3H]-VBL in protein-free liposomes (data not shown) prepared as previously described (Kim et al, 1989). In flow cytometric studies, DPM did not alter the forward light scatter of KB-3-1 cells, but the membrane permeabilising agent, digitonin (Lepers et al, 1990), produced a maximal increase within 75 s of incubation (data not shown). Digitonin, but not DPM, also produced a large increase in permeability to propidium iodide as determined by measuring cellular fluorescence of this agent (data not shown).…”

Section: Effect Of Dpm On Vbl Influxmentioning

confidence: 91%

Regulation of initial vinblastine influx by P-glycoprotein

Shalinsky

Jekunen²,

Alcaraz³

et al. 1993

Br J Cancer

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Summary P-glycoprotein (PGP) is an energy-dependent efflux pump that serves to protect cells against the cytotoxicity of many natural product drugs including vinblastine (VBL). In this study we investigated the role of PGP in regulating initial VBL influx. The apparent influx of VBL, measured over the first 20 s, was 2-fold lower in KB-GRCI cells expressing a transfected mdrl gene at high level than in non-expressing parental KB-3-1 cells. Inhibition of PGP efflux function with dipyridamole increased the influx rate constant by 4.0-fold in the KB-GRC1 cells but only 2.1-fold in the KB-3-1 cells. Verapamil, another inhibitor of PGP-mediated efflux, increased the initial influx rate constant by 2.7-fold in the KB-GRCI cells but only 1.4-fold in the KB-3-1 cells. Inhibition of PGP function by depletion of ATP increased influx by 6.8-fold and 2.2-fold in the two cell types, respectively. Mutation of PGP at both ATP binding sites abolished its ability to limit initial influx. Thus, VBL is serving as an efficient substrate for the efflux pump even within the first few seconds of drug exposure, consistent with the hypothesis that PGP may directly efflux drug from the cell membrane.

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“…Permeabilization of rat luteal cells was based on published methods that describe the use of saponin to allow exogenous bioactive molecules access to the intracellular milieu with continued cell function (36,76). Luteal cells were washed with IC buffer (containing in mM: 20 HEPES, 110 KCl, 10 NaCl, 1 KH 2PO4, 5 KHCO3, and 0.03 MgCl2, pH 7.1), centrifuged, and resuspended at 1 ϫ 10 7 cells/ml with IC buffer supplemented with MgCl2 (3.3 mM), ATP (4.4 mM), and saponin (0.1 mg/ml).…”

Section: Cell Incubationsmentioning

confidence: 99%

Localized accumulation of angiotensin II and production of angiotensin-(1–7) in rat luteal cells and effects on steroidogenesis

Pepperell¹,

Németh²,

Yamada³

et al. 2006

American Journal of Physiology-Endocrinology and Metabolism

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These studies aim to investigate subcellular distribution of angiotensin II (ANG II) in rat luteal cells, identify other bioactive angiotensin peptides, and investigate a role for angiotensin peptides in luteal steroidogenesis. Confocal microscopy showed ANG II distributed within the cytoplasm and nuclei of luteal cells. HPLC analysis showed peaks that eluted with the same retention times as ANG-(1-7), ANG II, and ANG III. Their relative concentrations were ANG II >or= ANG-(1-7) > ANG III, and accumulation was modulated by quinapril, an inhibitor of angiotensin-converting enzyme (ACE), Z-proprolinal (ZPP), an inhibitor of prolyl endopeptidase (PEP), and parachloromercurylsulfonic acid (PCMS), an inhibitor of sulfhydryl protease. Phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, did not affect peptide accumulation. Quinapril, ZPP, PCMS, and PMSF, as well as losartan and PD-123319, the angiotensin receptor type 1 (AT1) and type 2 (AT2) receptor antagonists, were used in progesterone production studies. ZPP significantly reduced luteinizing hormone (LH)-dependent progesterone production (P < 0.05). Quinapril plus ZPP had a greater inhibitory effect on LH-stimulated progesterone than either inhibitor alone, but this was not reversed by exogenous ANG II or ANG-(1-7). Both PCMS and PMSF acutely blocked LH-stimulated progesterone, and PCMS blocked LH-sensitive cAMP accumulation. Losartan inhibited progesterone production in permeabilized but not intact luteal cells and was reversed by ANG II. PD-123319 had no significant effect on luteal progesterone production in either intact or permeabilized cells. These data suggest that steroidogenesis may be modulated by angiotensin peptides that act in part through intracellular AT1 receptors.

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Permeabilized cells as a way of gaining access to intracellular organelles: an approach to glycosylation reactions

Cited by 31 publications

References 52 publications

p34 -mediated Phosphorylation Mobilizes Microtubule-organizing Centers from the Apical Intermediate Filament Scaffold in CACO-2 Epithelial Cells

p34 -mediated Phosphorylation Mobilizes Microtubule-organizing Centers from the Apical Intermediate Filament Scaffold in CACO-2 Epithelial Cells

Regulation of initial vinblastine influx by P-glycoprotein

Localized accumulation of angiotensin II and production of angiotensin-(1–7) in rat luteal cells and effects on steroidogenesis

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