Cytosolic phospholipase A 2 (cPLA 2 ) is activated by a wide variety of stimuli to release arachidonic acid, the precursor of the potent inflammatory mediators prostaglandin and leukotriene. Specifically, cPLA 2 releases arachidonic acid in response to agents that increase intracellular Ca 2؉. In vitro data have suggested that these agents induce a translocation of cPLA 2 from the cytosol to the cell membrane, where its substrate is localized. Here, we use immunofluorescence to visualize the translocation of cPLA 2 to distinct cellular membranes. In Chinese hamster ovary cells that stably overexpress cPLA 2 , this enzyme translocates to the nuclear envelope upon stimulation with the calcium ionophore A23187. The pattern of staining observed in the cytoplasm suggests that cPLA 2 also translocates to the endoplasmic reticulum. We find no evidence for cPLA 2 localization to the plasma membrane. Translocation of cPLA 2 is dependent on the calcium-dependent phospholipid binding domain, as a calcium-dependent phospholipid binding deletion mutant of cPLA 2 (⌬CII) fails to translocate in response to Ca 2؉ . In contrast, cPLA 2 mutated at Ser-505, the site of mitogen-activated protein kinase phosphorylation, translocates normally. This observation, combined with the observed phosphorylation of ⌬CII, establishes that translocation and phosphorylation function independently to regulate cPLA 2 . The effect of these mutations on cPLA 2 translocation was confirmed by subcellular fractionation. Each of these mutations abolished the ability of cPLA 2 to release arachidonic acid, establishing that cPLA 2 -mediated arachidonic acid release is strongly dependent on both phosphorylation and translocation. These data help to clarify the mechanisms by which cPLA 2 is regulated in intact cells and establish the nuclear envelope and endoplasmic reticulum as primary sites for the liberation of arachidonic acid in the cell.The 85-kDa cytosolic phospholipase A 2 (cPLA 2 ), 1 which selectively releases arachidonic acid from the sn-2 position of membrane phospholipids, is crucial to the initiation of the inflammatory response. cPLA 2 activity is stimulated by a wide variety of agents, including the proinflammatory cytokines interleukin 1 (1, 2) and tumor necrosis factor (3), macrophage colony-stimulating factor (4), thrombin (5, 6), ATP (5), mitogens (7-10), and endothelin (11). The release of arachidonic acid is the rate-limiting step in the generation of prostaglandins and leukotrienes, the proinflammatory eicosanoids. Cleavage of arachidonoyl-containing phospholipids also results in the release of lysophospholipid, the precursor of the inflammatory mediator platelet-activating factor (12).cPLA 2 is expressed in many cell types. Many of these are associated with the inflammatory response, such as monocytes (4), neutrophils (13), and synovial fibroblasts (14). However, cPLA 2 is also expressed in kidney, spleen, heart, lung, liver, testis, and hippocampus (15). This diverse pattern of expression is consistent with accumulating evidence tha...

The subcellular locations of prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and -2) were determined by quantitative confocal fluorescence imaging microscopy in murine 3T3 cells and human and bovine endothelial cells using immunocytofluorescence with isozyme-specific antibodies. In all of the cell types examined, PGHS-1 immunoreactivity was found equally distributed in the endoplasmic reticulum (ER) and nuclear envelope (NE). PGHS-2 immunoreactivity was also present in the ER and NE. However, PGHS-2 staining was twice as concentrated in the NE as in the ER. A histofluorescence staining method was developed to localize cyclooxygenase/peroxidase activity. In quiescent 3T3 cells, which express only PGHS-1, histofluorescent staining was most concentrated in the perinuclear cytoplasmic region. In contrast, histochemical staining for PGHS-2 activity was about equally intense in the nucleus and in the cytoplasm, a pattern of activity staining distinct from that observed with PGHS-1. Our results indicate that there are significant differences in the subcellular locations of PGHS-1 and PGHS-2. It appears that PGHS-1 functions predominantly in the ER whereas PGHS-2 may function in the ER and the NE. We speculate that PGHS-1 and PGHS-2 acting in the ER and PGHS-2 functioning in the NE represent independent prostanoid biosynthetic systems.

The ARABIDOPSIS CBF transcriptional activators bind to the CRT/DRE regulatory element present in the promoters of many cold-regulated genes and stimulate their transcription. Expression of the CBF1 proteins in yeast activates reporter genes carrying a minimal promoter with the CRT/DRE as an upstream regulatory element. Here we report that this ability of CBF1 is dependent upon the activities of three key components of the yeast Ada and SAGA complexes, namely the histone acetyltransferase (HAT) Gcn5 and the transcriptional adaptor proteins Ada2 and Ada3. This result suggested that CBF1 might function through the action of similar complexes in ARABIDOPSIS In support of this hypothesis we found that ARABIDOPSIS has a homolog of the GCN5 gene and two homologs of ADA2, the first report of multiple ADA2 genes in an organism. The ARABIDOPSIS GCN5 protein has intrinsic HAT activity and can physically interact in vitro with both the ARABIDOPSIS ADA2a and ADA2b proteins. In addition, the CBF1 transcriptional activator can interact with the ARABIDOPSIS GCN5 and ADA2 proteins. We conclude that ARABIDOPSIS encodes HAT-containing adaptor complexes that are related to the Ada and SAGA complexes of yeast and propose that the CBF1 transcriptional activator functions through the action of one or more of these complexes.

In a large kindred of German descent, we found a novel allele that segregates with deafness when present in trans with the 35delG allele of GJB2. Qualitative polymerase chain reaction-based allele-specific expression assays showed that expression of both GJB2 and GJB6 from the novel allele is dramatically reduced. This is the first evidence of a deafness-associated regulatory mutation of GJB2 and of potential coregulation of GJB2 and GJB6.

Few studies of gene-environment interactions for the serotonin transporter promoter polymorphism (5-HTTLPR), life stressors and depression have considered women separately or examined specific types of stressful life events. None have looked at depression during pregnancy. In the Pregnancy Outcomes and Community Health (POUCH) Study, women were queried about history of stressful life events and depressive symptoms at the time of enrollment (15-27 weeks gestation). Stressful life events were grouped a priori into ''subconstructs'' (e.g. economic, legal, abuse, loss) and evaluated by subconstruct, total subconstruct score and total stressful life event score. The effect of genotype on the association between stressful life events and elevated depressive symptoms was assessed in 568 white non-Hispanic participants. The relationship between exposure to abuse and elevated depressive symptoms was more pronounced in the s/s group (OR 5 24.5) than in the s/ l group (OR 5 3.0) and the l/l group (OR 5 7.7), but this significant interaction was detected only after excluding 73 (13%) women with recent use of psychotropic medications. There was no evidence of gene-environment interaction in analytic models with other stressful life events subconstructs, total subconstruct score or total stressful life events score. These data offer modest support to other reports of gene-environment interaction and highlight the importance of considering specific stressful life events.