Identification of polypeptides whose presence correlates with retention or loss of an albumin extinguisher chromosome in rat hepatoma-mouse L cell fibroblast microcell hybrids

Laurent‐Winter, Christine; Fougère-Deschatrette, Catherine; Weiss, Mary C.

doi:10.1046/j.1432-0436.1994.5530225.x

Cited by 7 publications

(5 citation statements)

References 28 publications

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“…Two‐dimensional gel electrophoresis was essentially performed as described by Garrels (1983) with the modifications already published (Laurent‐Winter et al ., 1994): briefly 30 µl of sample corresponding to 100 µg of phagosomal proteins were loaded onto IEF 18 cm containing ampholines of pH ranging from 3.5 to 10 (Genomic solutions, Ann Harbor, MI, USA). The second dimension was carried out on 12.5% acrylamide 22 cm slab gels.…”

Section: Methodsmentioning

confidence: 99%

Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach

Marion

Laurent

Guillén

2005

Cellular Microbiology

116

119

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SummaryPhagocytosis of human cells is a crucial activity for the virulence of the human parasite Entamoeba histolytica . This protozoan invades and destroys the intestine by killing and phagocytosing epithelial cells, erythrocytes and cells from the immune system. In this study, we used magnetic beads covered with proteins from human serum as a model system to study the early events involved in phagocytosis by E. histolytica . We validated the system showing that the beads uptake triggered the activation of the actinmyosin cytoskeleton and involved a PI3-kinase as previously described for erythrophagocytosis. We purified early phagosomes from wild-type (WT) amoeba and from parasites that overproduced myosin IB (MyoIB + + + + ), the unique unconventional myosin of E. histolytica . The MyoIB + + + + cells exhibit a slower and more synchronized uptake process than the WT strain. Proteomic analysis by liquid chromatography and tandem mass spectroscopy (LC-MS/MS) of the WT and MyoIB + + + + phagosomes allowed us to identify, for the first time, molecular actors involved in the early step of the uptake process. These include proteins involved in cytoskeleton activity, signalling, endocytosis, lytic activity and cell surface proteins. Interestingly, the proteins that we found specifically recruited on the phagosomes from the MyoIB + + + + strain were previously described in other eukarytotic cells, as involved in the regulation of cortical F-actin dynamics, such as a a a a -actinin and formins. This proteomics approach allows a step further towards the understanding of the molecular mechanisms involved in phagocytosis in E. histolytica that revealed some interesting differences compared with phagocytosis in macrophages or Dictyostelium discoideum , and allowed to identify putative candidates for proteins linked to myosin IB activity during the phagocytic process

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Section: Methodsmentioning

confidence: 99%

Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach

Marion

Laurent

Guillén

2005

Cellular Microbiology

116

119

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“…Two-dimensional gel electrophoresis was performed essentially as described by Garrels [29], with the modifications of Laurent-Winter et al [30]. For the first dimension (isoelectric focusing, IEF), we used gradient gels covering the range pH4 to pH8 (ampholines, Millipore Inc.), which were run for 20,000 Vh.…”

Section: Methodsmentioning

confidence: 99%

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Prévot

Laurent‐Winter

Rodhain

et al. 2003

Malar J

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Background: Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite.

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“…After lyophilization, samples were resuspended in 30 l of sample buffer (9.95 M urea, 4% Nonidet P-40, 2% ampholyte [pH 5 to 7], and 100 mM dithiothreitol) and centrifuged for 2 min. Samples were then loaded onto the isoelectrofocusing gel (pH range, 4 to 8; Millipore Corporation) and run for 20,000 V ⅐ h. The second dimension was performed as previously described on a 12.5% acrylamide gel (15). Relative isoelectric points were determined by parallel migration of a carbamylated muscle creatine phosphokinase standard (BDH), and the relative molecular masses of the proteins were determined according to molecular weight markers applied to an adjacent slot on the same gel.…”

Section: Methodsmentioning

confidence: 99%

N- and C-Terminal Sequences Control Degradation of MAD3/IκBα in Response to Inducers of NF-κB Activity

Whiteside

Ernst

LeBail

et al. 1995

Molecular and Cellular Biology

219

207

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The proteolytic degradation of the inhibitory protein MAD3/IB␣ in response to extracellular stimulation is a prerequisite step in the activation of the transcription factor NF-B. Analysis of the expression of human IB␣ protein in stable transfectants of mouse 70Z/3 cells shows that, as for the endogenous murine protein, exogenous IB␣ is degraded in response to inducers of NF-B activity, such as phorbol myristate acetate or lipopolysaccharide. In addition, pretreatment of the cells with the proteasome inhibitor N-Ac-Leu-Leu-norleucinal inhibits this ligand-induced degradation and, in agreement with previous studies, stabilizes a hyperphosphorylated form of the human IB␣ protein. By expressing mutant forms of the human protein in this cell line, we have been able to delineate the sequences responsible for both the ligand-induced phosphorylation and the degradation of IB␣. Our results show that deletion of the C terminus of the IB␣ molecule up to amino acid 279 abolishes constitutive but not ligand-inducible phosphorylation and inhibits ligand-inducible degradation. Further analysis reveals that the inducible phosphorylation of IB␣ maps to two serines in the N terminus of the protein (residues 32 and 36) and that the mutation of either residue is sufficient to abolish ligand-induced degradation, whereas both residues must be mutated to abolish inducible phosphorylation of the protein. We propose that treatment of 70Z/3 cells with either phorbol myristate acetate or lipopolysaccharide induces a kinase activity which phosphorylates serines 32 and 36 and that these phosphorylations target the protein for rapid proteolytic degradation, possibly by the ubiquitin-26S proteasome pathway, thus allowing NF-B to translocate to the nucleus and to activate gene expression.The Rel/NF-B family of proteins (p50, p52, p65/RelA, cRel, and RelB) bind specifically as homo-or heterodimers to B motifs located in the promoters and enhancers of a large number of genes that control various aspects of the immune and inflammatory responses (including the major histocompatibility complex class I genes, the immunoglobulin light chain, interleukin 2 and its receptor, interleukins 6 and 8, granulocyte-macrophage colony-stimulating factor, beta interferon, and T-cell receptor ␤ chain) as well as those of several viruses, including human immunodeficiency virus types 1 and 2 and cytomegalovirus (for recent reviews, see references 2 and 32). In most cell types, NF-B is maintained in an inactive, cytoplasmic state in complexes with members of the IB family, which includes IB␣, IB␤, and IB␥ (only B cells and some cells of the monocyte or macrophage lineage exhibit constitutive nuclear NF-B activity). These inhibitors all contain multiple copies of a motif, the ankyrin repeat, which interact with and inhibit the nuclear localization and DNA binding of the Rel/NF-B proteins (for a review, see reference 3). NF-B is induced by a wide variety of stimuli (including phorbol myristate acetate [PMA], tumor necrosis factor, interleukin 1, lipopolysaccharide [L...

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Identification of polypeptides whose presence correlates with retention or loss of an albumin extinguisher chromosome in rat hepatoma-mouse L cell fibroblast microcell hybrids

Cited by 7 publications

References 28 publications

Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach

Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach

Untitled

N- and C-Terminal Sequences Control Degradation of MAD3/IκBα in Response to Inducers of NF-κB Activity

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