Purified, reconstituted cardiac Ca2+-ATPase is regulated by phospholamban but not by direct phosphorylation with Ca2+/calmodulin-dependent protein kinase.

Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to beta-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 (Ca2+-ATPase). Recently, direct phosphorylation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-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 (approximately 1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by approximately 50%, 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 Ca2+-ATPase and not with a change in Vmax. Neither the purified, reconstituted cardiac Ca2+-ATPase nor the Ca2+-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 Vmax for calcium transport.

Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography.

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.

Beneficial effects of prewarming the donor liver for rat liver transplantation.

Cold preservation of donor liver can injure the microcirculation enough to impair the function and survival of the transplanted liver. In this study, prewarming the donor liver to 10 degrees C to 12 degrees C after cold storage prior to implantation significantly increased survival rates in rats 1 week after surgery. Following 6, 8, or 9 hours of cold storage in chilled normal saline, prewarming increased survival rates from 40% to 78.6%, from 0% to 35.7%, and from 0% to 14.3%, respectively. The results suggest that pretreatment of the donor liver with warmth following cold storage and before implantation will improve the survival rate of liver transplanted rats.

Indomethacin sensitive suppressor-cell activity in head and neck cancer patients. The role of the adherent mononuclear cell.

Head and neck cancer (H&N CA) patients have known depression of cell-mediated immunity. There is suggestive evidence that prostaglandin (PGE2)-secreting cells may be a major factor. The authors have sought to determine the role of PGE2-releasing monocytes-macrophages in this immune depression by determining the effects of adherent cell depletion and by measuring the effects of indomethacin, a PGE2 synthetase inhibitor, on selected tests of lymphocyte function. Lymphocyte stimulation with phytohemagglutinin (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: (1) 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 indomethacin 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 PGE2 synthesis inhibitor, indomethacin, which suggests the potential value of in vivo administration of indomethacin to H&N CA patients as an adjunct.

Antitumor effects of a normal human serum factor (normal human globulin fraction 1) on human tumor cells in vitro.

Normal human globulin fraction 1 (NHG-1) produced cytotoxicity and/or cytostasis as well as inhibition of protein synthesis in 8 well-characterized human tumor cell lines (4 breast cancer, 1 colon cancer, 1 melanoma, and 2 leukemia) and in 2 variants of murine B-16 melanoma. NHG-1 was not cytotoxic for the Chang liver cell line, a normal kidney embryo line, or for normal lymphocytes or macrophages when used in lower concentrations but was growth inhibitory for normal cells in higher concentrations. Although lymphocyte blastogenesis with phytohemagglutinin (PHA) was inhibited by high concentrations of NHG-1, augmentation of the lymphocyte PHA response was seen at lower concentrations, suggesting a lymphokine-like effect. Preincubation with the mitogen partially nullified these NHG-1 effects (suggesting the need for cell surface binding). Although NHG-1 antitumor activity was confirmed in selected human and murine tumor cell lines, the mechanism of its activity is unknown. Occurrence of NHG-1 in the alpha 2-globulin region (an area rich in immune-regulating factors) suggests that NHG-1 may have general "cytokine"-like effects and may be capable of regulating replication of both normal and transformed cells.

Monocyte-mediated antibody-dependent cell-mediated cytotoxicity and spontaneous cytotoxicity in normals and cancer patients as assayed by human erythrocyte lysis.

The monocyte-macrophage system has long been recognized as a necessary accessory to the immune response. Recently, however, monocyte-macrophages have been shown to be important effectors of cell-mediated cellular cytotoxicity (both antibody dependent and antibody independent). In this study, monocyte-mediated cellular cytotoxicity of both types was assessed on 51Cr-labeled human erythrocytes (type B+) using autologous and standardized AB serum, and monocytes from 57 normal controls, 16 women with benign breast disease, and 175 patients with cancers of the breast (44 patients), colorectum (46 patients), head and neck (33 patients), lung (13 patients), and melanoma (39 patients). Although results were variable, many of the patients had depressed antibody-dependent cellular cytotoxicity suggesting decreased ability of their monocyte-macrophage to lyse the sensitized erythrocytes. Enhanced antibody-dependent cellular cytotoxicity was observed in patients with localized colorectal cancer, but this effect was reversed in patients with advanced disease. Serum factors did not significantly affect responses in most cases. The clinical relevance of this assay remains to be determined.

Monocyte dysfunction in human cancer.

General immunobiologic studies in cancer patients have stressed measurements of lymphocyte function and have commonly ignored the monocyte-macrophage system. A preliminary study of peripheral blood monocyte-macrophage function was undertaken in a group of 90 cancer patients (18 breast, 32 colon, 13 head and neck, 9 lung, and 18 melanoma) and 70 controls. Studies included enumeration of extractible monocytes (EM), quantitation of differentiation into macrophages (macrophage precursor test: MP), evaluation of antibody-dependent cellular cytotoxicity (ADCC), and spontaneous cellular cytotoxicity (SCC) as measured with human erythrocytes, and measurements of monocyte and serum lysozyme activity. Breast cancer patients had normal profiles. Colon cancer patients showed the most profound abnormalities with decreased EM and MP and increased ADCC and SCC. Patients with Stage I and Stage II melanoma had normal profiles, whereas those with advanced melanoma had significantly decreased MP. This defect was restored in two patients by BCG immunotherapy. Head and neck cancer and benign breast disease patients had decreased EM, whereas patients with lung cancer had increased EM. Monocyte lysozyme production was unchanged in the cancer patients compared to controls. Serum lysozyme levels, however, were significantly increased in patients with cancers of the colon, head and neck, and lung. Although patients with localized breast cancer and melanoma had normal levels, these were increased in both patient groups with advanced disease. It would appear that the source of the increased serum lysozyme is probably not the peripheral blood monocytes, but could have originated in the intra-tumoral or tissue-bound macrophages which were not examined. Selected assays of peripheral blood monocyte function were deranged in certain types of cancer patients but were fully normal in others, and did not show consistent correlations with tumor type or stage. Tissue-bound or intra-tumoral macrophages might provide a more fruitful area for study in these disease categories.

The influence of incubation time and mitogen concentration on lymphocyte blastogenic response: determination of conditions that maximize population differences.

The blastogenic responsiveness of peripheral blood lymphocytes is a commonly applied measure of immune response capability. This assay is generally run under conditions of maximum lymphocyte stimulation. The purpose of this study was to evaluate which conditions of lymphocyte culture were able to best discriminate normal from subnormal lymphocyte responses to mitogens and antigens. Mitogen or antigen concentrations and the length of incubation time were varied while other assay conditions (serum supplement, lymphocyte concentration, amount of label, and length of label pulse) were kept constant. A total of 24 cancer patients and 25 normal controls were studied. Under optimal assay conditions that provided for maximum lymphocyte stimulation, we were unable to distinguish differences in response between young and old individuals or cancer patients and normal controls. However, at suboptimal mitogen/antigen concentrations or suboptimal incubation times, significant differences were observed in these populations. It is suggested that in the evaluation of lymphocyte stimulation responses in various clinical groups, suboptimal lymphocyte culture conditions may provide for maximum discrimination among the groups.