Alteration in protein kinase C activity and subcellular distribution in sickle erythrocytes

In agreement with previous data, membrane protein phosphorylation was found to be altered in intact sickle cells (SS) relative to intact normal erythrocytes (AA). Similar changes were observed in their isolated membranes. The involvement of protein kinase C (PKC) in this process was investigated. The membrane PKC content in SS cells, measured by [3H]phorbol ester binding, was about 6-times higher than in AA cells. In addition, the activity of the enzyme, measured by histone phosphorylation was also found to be increased in SS cell membranes but decreased in their cytosol compared to the activity in AA cell membranes and cytosol. The increase in membrane PKC activity was observed mostly in the light fraction of SS cells, fractionated by density gradient, whereas the decrease in cytosolic activity was only observed in the dense fraction. PKC activity, measured in cells from the blood of reticulocyte-rich patients, exhibited an increase in both membranes and cytosol, thus explaining some of the effects observed in the SS cell light fraction, which is enriched in reticulocytes. The increase in PKC activity in the membranes of SS cells is partly explained by their young age but the loss of PKC activity in their cytosol, particularly in that of the dense fraction, seems to be specific to SS erythrocytes. The relative decrease in membrane PKC activity between the dense and the light fractions of SS cells might be related to oxidative inactivation of the enzyme.     

Inhibition ofCandida albicans andStaphylococcus aureus by phenolic compounds from the terrestrial cyanobacteriumNostoc muscorum

Phenolic compounds were determined in methanolic extract from the algal mass of aNostoc muscorum culture. Bioassays with two human pathogens,Candida albicans andStaphylococcus aureus indicated that algal phenolic compounds evoked significant growth inhibition for both species (89.1% and 88.2%, respectively). It is suggested that this strong inhibitory effect is of potential medicinal value.     

Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes

We compared the radiosensitivity of human, rat and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. For each species and dose 4-ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150 or 300 cGy. Controls were sham-irradiated. After exposure to X-rays, mononuclear leukocytes were isolated using density gradients and cultured in RPMI 1640 medium containing phytohemagglutinin to stimulate mitogenesis. At 21 h cytochalasin B was added to produce BN PBLs, and all cultures were harvested at 52 h post-initiation using a cytocentrifuge. Significant dose-dependent increases in the percentage of micronucleated cells and the number of MN per BN cell were observed in all three species. The linear-quadratic regression curves for the total percentage of micronucleated cells for the three species were similar; however, the curve for the mouse PBLs had a larger quadratic component than either of the curves for the rat or human PBLs. Although the correlation between the percentage of cells with MN and those with chromosome aberrations was high (r2 greater than 0.95), the mouse and rat PBLs were over twice as efficient as human PBLs in forming MN from presumed acentric fragments. These data indicate that the induction of MN in BN cells following ionizing radiation is similar in human, rat and mouse PBLs, but care must be taken in using the MN results to predict frequencies of cells with chromosomal aberrations.     

Characteristics of an inhibitor of the Na+/K+ pump in human cerebrospinal fluid

Human cerebrospinal fluid (CSF) inhibits the Na+/K+ pump in human red cells and the activity of purified Na+/K+-ATPase (Halperin, J. A., Shaeffer, R., Galvez, L., and Malavé, S. (1985) Proc. Natl. Acad. Sci. U.S. A. 80, 6102-6104, 1983; Halperin, J. A., Martin, A. M., and Malavé, S. (1985) Life Sci. 37, 561-566. We describe here some properties of the CSF inhibitor of the Na+/K+ pump. Active material was extracted from human CSF with 50% methanol and then concentrated and desalted by ultrafiltration. This extract inhibited, in a dose-dependent manner, the ouabain-sensitive influx of K+ into human red cells and the activity of purified Na+/K+-ATPase. Partial separation of the inhibitory activity was achieved by gel filtration and reverse-phase high performance liquid chromatography. Inhibition of both pump and enzyme was specific in that other red cell membrane transport systems or enzymes examined were not influenced by CSF extracts. Dialysis and ultrafiltration experiments indicate that the molecular weight of the inhibitor is approximately equal to 600. The inhibitory activity is sensitive to proteolytic enzymes indicating that the inhibitor might be a small peptide. In the presence of CSF extract the K0.5 for external K+ to stimulate the Na+/K+ pump increased from 1.4 to 3.1 mM, suggesting that the CSF inhibitor competes with external K+ for stimulation of the pump. We estimate that the concentration of the inhibitor in CSF might be approximately equal to 50 pg/ml, a value close to the concentration of other active peptides found in human CSF.     

Glycerated hemoglobin, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine. A new post-translational modification occurring in erythrocyte bisphosphoglyceromutase deficiency

A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined.     

Increased digitalis-like activity in human cerebrospinal fluid after expansion of the extracellular fluid volume

The present study was designed to determine whether acute expansion of the extracellular fluid volume influenced the digitalis-like activity of human cerebrospinal fluid (CSF), previously described by our laboratory. Human CSF samples, drawn before and 30 minutes after the intravenous infusion of 1 liter of either saline or glucose solutions, were assayed for digitalis-like activity by inhibition of either the 86Rb+ uptake into human erythrocytes or by the activity of a purified Na+ - K+ ATPase. The CSF inhibitory activity on both systems significantly increased after the infusion of sodium solutions but did not change after the infusion of glucose. These results indicate that the digitalis-like factor of human CSF might be involved in the regulation of the extracellular fluid volume and electrolyte content and thereby in some of the physiological responses to sodium loading.     

Compartmentalization of Ca2+ in sickle cells

Control (AA) and sickle cell anemia (SS) erythrocytes were loaded with Ca-chelator (Quin2 or Benz2) to increase the cellular exchangeable Ca2+ pool and to measure the Ca2+ exchange fluxes and the cytosolic ionized Ca2+ ([Ca]i) (Lew et al., 1982, Nature, 298, 478). The chelator incorporation induced a decrease in the ATP content which was smaller in SS than in AA cells and partially reversible upon reincubation in a chelator-free medium. The amount of trapped chelator was determined by two methods: 45Ca binding to the chelator in Ca-ionophore treated cells in Ca-EGTA buffers and [3H]Quin2 incorporation. A slight over-estimation of the chelator content was found with the second method but incorporation was the same in both types of cells. The kinetics of 45Ca equilibration and 45Ca release were used to measure Ca2+ fluxes and [Ca]i in oxygenated chelator-loaded cells. SS cells, as compared to AA cells, exhibited a moderate increase in Ca2+ fluxes (30-75%) but [Ca]i remained in the same range (about 20 nM). Thus the excess of Ca2+ found in SS cells is not available for the Ca2+ pump or the K+ channel a conclusion in agreement with that of Bookchin et al. (1984, Cell Calcium, 5, 277). Analysis of the 45Ca kinetics showed that in AA cells, exchangeable Ca2+ behaved as one compartment. In SS cells, the existence of a second slowly-exchangeable Ca2+ compartment was demonstrated. This latter (3-5 mumol/l cells) was independent of the concentration of the chelator and thus could represent exchangeable Ca2+ enclosed within the intracellular inside-out vesicles recently observed in SS cells (Williamson et al., 1984, J. Cell. Biol., 99, 430a). Alternatively, these two kinetic pools could reflect heterogeneity of the SS cell population.     

Effects of the methanol extract residue (MER) tubercle bacillus fraction on the production of antibodies in vitro. II. Effects on macrophage and lymphocyte populations

The effect of the methanol extract residue (MER) fraction of BCG tubercle bacilli on the generation of primary antibody responsiveness in vitro to sheep red blood cells (SRBC) was ascertained in cell reconstitution experiments, employing enriched populations of mouse macrophages and of T and B lymphocytes. In each of the antibody generation cultures one or another of the cell fractions had been exposed to MER, either by treatment of the donor animals or by preincubation with the agent for 48 hr in vitro. In some experiments, supernatants of MER-preincubated cells were employed in place of the cells. Macrophages and T cells that had been exposed to MER in vivo or in vitro and their supernatants demonstrated a markedly greater effect than nonexposed cells in the generation of direct specific plaque-forming cells (PFC) upon antigenic stimulation of the cultures with SRBC. In contrast, PFC production was not stimulated in B-lymphocyte populations that had been in contact with the agent.     

Differential recovery of circulating T cell subsets after nodal irradiation for Hodgkin's disease

To better understand the immunologic effects of lymphoid irradiation (LI), blood levels of T cell subsets were sequentially monitored in 15 patients before, during, and after irradiation treatment for Hodgkin's disease. Blood levels of all lymphocytes, T cells, and T cell subsets (defined by OKT4 and OKT8) fell dramatically and in similar proportions during early therapy, reaching levels less than 20 to 25% of control by the completion of mantle irradiation, and continuing at very depressed levels through the completion of therapy. Blood levels of OKT8-reactive (OKT8+) cells returned to pretreatment levels (402 +/- 38/mm3 vs 360 +/- 32/mm3 pretreatment) between 6 to 8 mo after LI, whereas blood levels of OKT4-reactive (OKT4+) cells returned to only 42% of previous values (242 +/- 22/mm3 vs 584 +/- 34/mm3 pretreatment) over the same period. The pre-LI ratio of OKT4+ to OKT8+ cells was 1.85 +/- 0.24 and fell to 0.65 +/- 0.05 between 6 to 8 mo after LI. During the recovery period, discrepancies of 208 +/- 32 cells/mm3 (3 to 5 months post LI) and 198 +/- 32 cells/mm3 (6 to 8 mo post LI) developed between the blood levels of OKT3+ cells and the sum of OKT4+ and OKT8+ cells. This suggests the emergence of OKT4+/OKT3-, OKT8+/OKT3-, and/or OKT4+/OKT8+ cells. In five patients, the sum of OKT4+ and OKT8+ cells was compared with the number of cells simultaneously co-stained by OKT4 and OKT8. It appeared that a significant proportion of the cells were OKT4+/OKT3- and OKT8+/OKT3- lymphocytes. We concluded that LI is similarly cytotoxic to peripheral blood T cell subpopulations. The reversed ratio after LI is a result of a slower repopulation of the peripheral blood by OKT4+ cells relative to OKT8+ cells. T cells after LI show a high degree of antigenic immaturity. It is postulated that the bone marrow that lies outside the fields of treatment and contains predominantly immature OKT8+/OKT3- cells is a major source of T cells repopulating the peripheral blood after LI.