Ligation of the B cell antigen receptor (BCR) with antigen induces lipid raft coalescence, a process that occurs after crosslinking of a variety of signaling receptors and is thought to potentiate cellular activation. To investigate lipid raft dynamics during BCR signaling, we quantitatively analyzed the B cell lipid raft proteome. BCR engagement induced dissociation of the adaptor protein ezrin from lipid rafts as well as threonine dephosphorylation of ezrin and its concomitant detachment from actin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Expression of constitutively active ezrin chimeras inhibited the BCR-induced coalescence of lipid rafts. Our data demonstrate that the release of ezrin from lipid rafts acts as a critical trigger that regulates lipid raft dynamics during BCR signaling.

SummaryIn an attempt to characterize genes participating in the allergic late phase reaction, we have isolated a novel intercrine/chemokine (called MARC) from a cDNA library of the stimulated mouse mast cell line, CPlI. As measured by Northern blotting, it is strongly upregulated at the mRNA level after the physiological challenge of the cells with immunoglobulin (Ig)E plus antigen. Unstimulated cells completely lack significant, stable expression, as do a number of other, different cell lines (uninduced and induced) and mouse tissues. In contrast to the Northern blot analysis, a polymerase chain reaction (PCR) analysis, performed on CPII ceUs and on Perco11 gradient purified mouse peritoneal mast cells, revealed a basal level of transcription in the uninduced stage. After 2 h of IgE plus antigen challenge, a quantitative reverse transcriptase-PCR, using a spiked in MIMIC, showed a level of transcripts more than 100-fold higher in the CPII cells and 5-20-fold higher in purified mouse peritoneal cavity mast cells. This rapid induction after the Fc+RI challenge, the identification of the gene as a member of the chemokine family, and its upregulated expression in peritoneal mast cells, all suggest an involvement in certain acute and chronic pathological mast cell-driven diseases.I ntercrines/r are small soluble proteins that regulate the physiological tra~cking and the partial activation ofleukocytes (1). In contrast to most other known cytokines and lymphokines, chemokines show a considerable homology (identity and similarity) at the amino acid level Additionally, they are characterized by a common protein structure of two loops formed via disulfide bridges of four highly conserved cysteines. Based on the location of the two NH2-terminal cysteines, this superfamily is subdivided into a CXC (IL-8 or PF4 family) and CC (RANTES/sis family) branch (2, 3). In the last, six members in the human system and five corresponding mouse genes are currently isolated (4, 5).Spedfic sites of production in the body and the low amount produced in vivo make the direct isolation and characterization of these proteins from healthy individuals nearly impossible. A few members, like macrophage inflammatory protein (MIP)-la, MIP-IB and monocyte chemotactic protein (MCP)I, have been purified at protein level from overexpressing tumor cell lines by functional monitoring, and the corresponding genes were subsequently identified (6, 7). However, the majority in the CC branch were isolated by inductionspecific differential hybridization of cDNA libraries (see reference 4). This reflects the fact that most members are strongly upregulated at the transcriptional level after cell activation (4). Supernatants from transiently transfected cell lines were used for a detailed functional analysis afterwards (8).Type I allergic reactions are characterized at the level of mast cells by a biphasic response. In an immediate reaction (up to several minutes) preformed low molecular weight substances, like histamine and serotonin, are released after IgE plu...

To understand the nature of negative responses through the B-cell antigen receptor (BCR), we have screened an expression cDNA library for the ability to block BCR-induced growth arrest and apoptosis in the immature B-cell line, WEHI-231. We isolated multiple copies of full-length, unmutated Bcl10, a signaling adaptor molecule encoded by a gene found to translocate to the immunoglobulin heavy chain (IgH) locus in some mucosa-associated lymphoid tissue ( IntroductionProper regulation of apoptosis is critical for the normal function of the immune system and for the prevention of bloodborne malignancies. B and T lymphocytes are normally subject to the induction of apoptotic cell death in the primary lymphoid organs, as exemplified by B-cell receptor/T-cell receptor (BCR/TCR)-induced apoptosis; and in the periphery, as exemplified by Fas-or cytokine deprivationinduced cell death. 1,2 Genetic changes that cause loss of apoptotic susceptibility are likely early events in the pathogenesis of many lymphoid cancers.To understand the regulation of B-cell apoptosis, WEHI-231, a lymphoma-derived B-cell line with an immature B-cell phenotype, has been frequently studied because it exhibits growth arrest and essentially 100% apoptosis in response to BCR crosslinking. 3 A number of factors known to be involved in B-cell oncogenesis have been shown to relieve or prevent BCR-induced growth arrest and/or apoptosis in WEHI-231 cells. These include the cell-cycle regulator c-Myc 4 and the B-cell lymphoma-2 (Bcl-2) family member, Bcl-xL. 5,6 Another lymphoma-related gene, BCL6, which inhibits B-cell terminal differentiation, displays abnormality at its genomic locus in these cells. 7 BCR-induced growth arrest and apoptosis of these cells are prevented by immunologic stimuli that promote activation of normal B cells, including lipopolysaccharide stimulation via Toll-like receptor 4 (TLR4) and helper T-cell stimulation via cytokines and CD40. 8 To understand the molecular events controlling B-cell fate in this system, we have taken a function-based approach and screened a cDNA expression library for genes that can abrogate BCR-induced apoptosis of WEHI-231 cells. From this screen, we isolated multiple copies of full-length, unmutated Bcl10.The human BCL10 gene was initially discovered via its involvement in a chromosomal translocation associated with a fraction of extranodal marginal zone B-cell lymphomas of mucosaassociated lymphoid tissue (MALT) and was also found to be mutated in some lymphoid and nonlymphoid malignancies. [9][10][11][12] Paradoxically, overexpression of Bcl10 in cell lines causes apoptosis. [13][14][15][16][17] Thus, the mechanism underlying the role of Bcl10 in lymphomagenesis remains to be clarified. Surprisingly, Bcl10-deficient mice were not found to have a defect in apoptosis, but they were found to have decreased numbers of all 3 types of mature B cells and to be defective in antigen receptor-induced activation of lymphocytes. 18,19 Subsequent studies have indicated that Bcl10 functions downstream of lympho...

SummaryPlant oilseeds are a major source of nutritional oils. Their fatty acid composition, especially the proportion of saturated and unsaturated fatty acids, has important effects on human health. Because intake of saturated fats is correlated with the incidence of cardiovascular disease and diabetes, a goal of metabolic engineering is to develop oils low in saturated fatty acids. Palmitic acid (16:0) is the most abundant saturated fatty acid in the seeds of many oilseed crops and in Arabidopsis thaliana. We expressed FAT-5, a membrane-bound desaturase cloned from Caenorhabditis elegans, in Arabidopsis using a strong seed-specific promoter. The FAT-5 enzyme is highly specific to 16:0 as substrate, converting it to 16:1Δ9; expression of fat-5 reduced the 16:0 content of the seed by two-thirds. Decreased 16:0 and elevated 16:1 levels were evident both in the storage and membrane lipids of seeds. Regiochemical analysis of phosphatidylcholine showed that 16:1 was distributed at both positions on the glycerolipid backbone, unlike 16:0, which is predominately found at the sn-1 position. Seeds from a plant line homozygous for FAT-5 expression were comparable to wild type with respect to seed set and germination, while oil content and weight were somewhat reduced. These experiments demonstrate that targeted heterologous expression of a desaturase in oilseeds can reduce the level of saturated fatty acids in the oil, significantly improving its nutritional value.

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