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Cytosolic phospholipase A2 (cPLA2)–generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.

We have previously established a model of cytosolic phospholipase A2 (cPLA2)-deficient PLB-985 cells and demonstrated that cPLA2-generated arachidonic acid (AA) is essential for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and NADPH-dependent diaphorase activity. The present study focuses on the C-terminal cytoplasmic domain of gp91phox (residues 283–570), which contains the NADPH binding and flavin adenine dinucleotide-reducing center, to determine if this portion is regulated by AA. The gp91phox C-terminal reductase domain was expressed in X-CGD PLB-985 cells lacking normal gp91phox (X-CGD PLB 91CT cells) and was detected in the plasma membrane. It appears to be bound electrostatically to the plasma membrane, as it is eluted by high salt. Permeabilized, granulocyte-like X-CGD PLB 91CT cells lacking cPLA2 protein and activity, as well as AA release after stimulation, supported NADPH-dependent diaphorase activity after stimulation, similar to granulocyte-like X-CGD PLB 91CT cells. Normal translocation of p47phox and p67phox to the membrane fractions of both stimulated cell types indicated that the gp91phox C-terminal region is sufficient to anchor the cytosolic oxidase components to the membranes. cPLA2 translocated to membranes and bound the assembled oxidase in granulocyte-like X-CGD PLB 91CT cells after stimulation. Therefore, the assembled membrane-bound oxidase complex encompassing the flavin domain of gp91phox provides a docking site for cPLA2 but is not the site of AA-based regulation of oxidase activity.

Objectives: To discuss the problems encountered teaching interviewing skills to first year medical students and describe their responses to a structured workshop in interviewing skills. Methods: Focus groups and a short evaluation questionnaire filled in by 56 first year medical students before and after a workshop in interviewing skills were used. Results: All students evaluated their skills very highly prior to the workshop. After participating in the workshop, students evaluated their skills as lower than before. Discussion: When communication skills are taught in an informal, unstructured manner, medical students might view this knowledge as unspecialized, repetitive, and even boring. It is suggested that employing a structured model for teaching doctor-patient communication skills awards psycho-social issues the status of formal knowledge. This can lead to students viewing communication skills as a relevant and consistent body of knowledge. Introducing a structured model can overcome two kinds of problems: over- confident students are formally introduced to unique aspects of medical interviewing, whilst those who lack confidence are offered a lifeline in the form of a structured model. Identifying possible sources of resistance to communications training has important implications for medical education as it allows for appropriate course planning and follow up.

Our previously established model of cy- tosolic phospholipase A2 (cPLA2)-deficient, differen- tiated PLB-985 cells (PLB-D cells) was used to de- termine the physiological role of cPLA2 in eicosanoid production. Parent PLB-985 (PLB) cells and PLB-D cells were differentiated toward the monocyte or granulocyte lineages using 5 10 8 M 1,25 dihy- droxyvitamin D3 or 1.25% dimethyl sulfoxide, re- spectively. Parent monocyte- or granulocyte-like PLB cells released prostaglandin E2 (PGE2) when stimulated by ionomycin, A23187, opsonized zymo- san, phorbol 12-myristate 13-acetate, or formyl- Met-Leu-Phe (fMLP), and monocyte- or granulocyte- like PLB-D cells did not release PGE2 with any of the agonists. The kinetics of cPLA2 translocation to nu- clear fractions in monocyte-like PLB cells stimulated with fMLP or ionomycin was in correlation with the kinetics of PGE2 production. Granulocyte-like PLB cells, but not granulocyte-like PLB-D cells, secreted leukotriene B4 (LTB4) after stimulation with ionomy- cin or A23187. Preincubation of monocyte-like par- ent PLB cells with 100 ng/ml lipopolysaccharide (LPS) for 16 h enhanced stimulated PGE2 produc- tion, which is in correlation with the increased levels of cPLA2 detected in these cells. LPS preincubation was less potent in increasing PGE2 and LTB4 secre- tion and did not affect cPLA2 expression in granulo- cyte-like PLB cells, which may be a result of their lower levels of surface LPS receptor expression. LPS had no effect on monocyte- or granulocyte-like PLB-D cells. The lack of eicosanoid formation in stimulated, differentiated cPLA2-deficient PLB cells indicates that cPLA2 contributes to stimulated eico- sanoid formation in monocyte- and granulocyte-like PLB cells. J. Leukoc. Biol. 76: 000-000; 2004.