Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to ␤-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca 2؉ -ATPase). Recently, direct phosphorylation of Ca 2؉ -ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca 2؉ -ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (ϳ1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by ϳ50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca -ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on V max for calcium transport.

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Obtaining large, flat, well ordered crystals represents the key to structure determination by electron crystallography. Multilamellar crystals of Ca(2+)-ATPase are a good candidate for this methodology, and we have optimized methods of crystallization and of preparation for cryoelectron microscopy. In particular, high concentrations of glycerol were found to prevent nucleation and to reduce stacking; thus, by seeding solutions containing 40% glycerol, we obtained thin crystals that were 5-30 microns in diameter and 2-10 unit cells thick. We found that removing vesicles and minimizing concentrations of divalent cations were critical to preparing flat crystals in the frozen-hydrated state. Finally, we developed two methods for determining the number of lamellae composing individual crystals, information that is required for structure determination of this crystal form. The first method, using low magnification images of freeze-dried crystals, is more practical in our case. Nevertheless, the alternative method, involving analysis of Laue zones from electron diffraction patterns of slightly tilted crystals, may be of general use in structure determination from thin, three-dimensional crystals.

Head and neck cancer (H&N CA) patients have known depression of cell-mediated immunity. There is suggestive evidence that prostaglandin (PCE+secreting cells may be a major factor. The authors have sought to determine the role of PGEZ-releasing monocytes-macrophages in this immune depression by determining the effects of adherent cell depletion and by measuring the effects of indomethacin, a PGEl synthetase inhibitor, on selected tests of lymphocyte function. Lymphocyte stimulation with phytohem-agglutinin (PHA) (T-cell stimulant) and Staph phage lysate (SPL) (B-cell stimulant) was done in the presence of varying concentrations of indomethacin; the effect of adherent cell depletion also was determined. The study population included 45 patients with localized or locoregional squamous CA of the H&N and 40 controls. Results included the following: (I) lymphocyte stimulation responses to PHA and SPL were generally depressed in the CA patients versus controls; (2) incubation with indomethacin produced bivalent effects in both controls and CA patients, depending on the concentration of indo-methacin and lymphocyte stimulant; incubation with optimum concentrations of indomethacin generally produced augmented responses in both study groups whereas high concentrations of indomethacin were suppressive: (3) the immune potentiating effects were not observed in older patients with advanced disease; and (4) removal of adherent leukocytes (mainly monocytes) also restored depressed lymphocyte responses. Although other factors also are operative, our data suggest that PGE2-secreting monocytes-macrophages may have a major role in the immune depression of H&N CA patients. Age and host effects of the cancer and the malnutrition common to these patients probably are involved also, although their singular contribution has not been measured. This depression is largely reversible by a PCE2 synthesis inhibitor, indomethacin, which suggests the potential value of in vivo administration of indo-methacin to H&N CA patients as an adjunct. Cancer 61A62-474, 1988. ATIENTS WITH head and neck cancer have known