Assessment and characterization of Ca2+-ATPase expression in selected isolates and clones of Plasmodium falciparum.

Ca2+-ATPase expression in 15 selected isolates from malaria patients at the University College Hospital (UCH) Ibadan and two cloned strains (W2-chloroquine resistant, D6-chloroquine sensitive) of P.falciparum was assessed using spectrophotometric assay method. The kinetics of activity of Ca2+- ATPase in three isolates (NCP 14, NCP5, NCP1) and two clones (W2, D6) also assessed. 12% SDS-PAGE analysis of total proteins in one isolate (NCP14) and two clones (W2, D6) was also investigated. All the selected isolates and the two cloned strains exhibited measurable Ca2+-ATPase activity. The Ca2+-ATPase activity in cloned strain D6 (6.50 + 0.74mmolPi/min/mg protein) was higher than in cloned strain W2 (3.93 + 0.61mmolPi/min/mg protein. The Ca2+-ATPase activity in isolates from malaria patients varied widely (1.95 + 0.74 - 21.56 +1.43mmolPi/min/mg protein). The kinetic constants obtained for the two cloned strains showed that clone W2 had a higher Vmax (Vmax = 363mmolPi/min/mg protein) than clone D6 (Vmax = 74mmolPi/min/mg protein). All the isolates and the two cloned strains showed similar affinity for ATP (Km ~ 10mM). Scan of SDS-PAGE gel of total proteins in the isolate and cloned strains showed the presence of oligopeptide bands of molecular weights range of 148-176 kDa; 116-123 kDa respectively. These suggest the presence of predicted polypeptide of Ca2+-ATPase nature of molecular weight estimate of 139 kDa. The study agrees with previous findings that Ca2+-ATPase is functionally expressed in P.falciparum, The study also indicates that Ca2+-ATPase functional expression may vary with isolate or clone but the ATP binding mechanism to the enzyme is similar in all isolates and clones of P. falciparum. The study further suggests a possible association between acquisition of chloroquine resistance and Ca2+-ATPase functional expression in P. falciparum.

Effect of toluene on erythrocyte membrane stability under in vivo and in vitro conditions with assessment of oxidant/antioxidant status.

Toluene, an organic solvent used widely in the industry, is highly lipophilic and accumulates in the cell membrane impeding transport through it. Its metabolites cause oxygen radical formation that react with unsaturated fatty acids and proteins in erythrocytes leading to lipid peroxidation and protein breakdown. In this study, we aimed to investigate the membrane stabilizing and the oxidative stress-inducing effects of toluene in human erythrocytes. Measurements of osmotic fragility, mean corpuscular volume (MCV), oxidative stress parameters and antioxidant enzyme activities were performed simultaneously both in individuals exposed to toluene professionally (in vivo) and human erythrocytes treated with toluene (in vitro). To measure osmotic fragility, erythrocytes were placed in NaCl solutions at various concentrations (0.1% [blank], 0.38%, 0.40%, 0.42%, 0.44%, 0.46%, 0.48% and 1% [stock]). Percentage of haemolysis in each solution was calculated with respect to the 100% haemolysis in the blank solution. The erythrocyte packs prepared at the day of the above-mentioned measurements were kept at -80 degrees C until the time for determination of malonyldialdehyde and protein carbonyl levels, and catalase (CAT) and glutathione peroxidase activities as indicators of oxidative stress. Toluene increased oxidative stress parameters significantly both in vivo and in vitro; it also caused a significant decrease in the activities of antioxidant enzymes. Osmotic fragility was altered only in the case of in vitro exposure. In conclusion, toluene exposure resulted in increased lipid peroxidation and protein damage both in vivo and in vitro. Although, it is natural to expect increased osmotic fragility due to oxidative properties of toluene, its membrane-stabilizing effect overcame the oxidative properties leading to decreased osmotic fragility or preventing its deterioration in vitro and in vivo toluene exposures, respectively, in the present study.

Sodium transport in hypertension: assessment of membrane-associated defects in South African black and white hypertensives.

Sodium transport was studied in red cells taken from four groups of patients, whites or blacks, and normotensive or hypertensive, with approximately ten patients in each group. There were no differences between normotensive and hypertensive whites. Normotensive blacks had higher rates of sodium-proton exchange than did the other groups, and hypertensive blacks had higher rates of efflux via the sodium-potassium ATPase than did other groups. These data support proposals that hypertension in black subjects is linked to abnormalities of control of intracellular sodium.

Erythrocytes, erythrocyte membranes, neutrophils and platelets as biopsy materials for the assessment of zinc status in humans.

During a controlled zinc depletion-repletion study, fifteen men aged 25.3 (SD 3.3) years were fed on a low-Zn diet with high phytate:Zn and phytate x calcium:Zn molar ratios for 7 weeks, followed by a 2 week repletion period when 30 mg supplemental Zn/d was given. Changes in plasma, urine, and hair Zn concentrations, taste acuity, and cellular immune response confirmed the development of mild Zn deficiency. Zn concentrations in neutrophils, platelets, erythrocytes and erythrocyte membranes, mean platelet volume, and activities of alkaline phosphatase (EC 3.1.3.1) and alpha-D-mannosidase (EC 3.2.1.24) in neutrophils did not respond to changes in Zn status. In contrast, alkaline phosphatase activity in erythrocyte membranes showed a significant decline which was consistent in all subjects (nmol product formed/min per mg protein; baseline v. 7-week Zn depletion, 0.656 (SD 0.279) v. 0.506 (SD 0.230), at 7 weeks; P < 0.05); neutral phosphatase activity remained unchanged. Alkaline phosphatase activity in erythrocyte membranes may be a potential index of Zn status in humans.

[Qualitative and quantitative assessment of blood erythrocytes of intact animals under the effect of 5-fluorouracil in solution and in microspheres made of acetylcellulose combined with short-term gas hypoxia]. / Kachestvennaia i kolichestvennaia otsenka éritrotsitov krovi intaktnykh zhivotnykh pri deistvii 5-ftoruratsila v rastvore i v mikrosferakh iz atsetiltselliulozy v sochetanii s kratkovremennoi gazovoi gipoksiei.

Resistance of erythrocyte membranes, estimated by means of ultrasound hemolysis, was altered 7 days before the cells quantitative changes after simultaneous treatment of intact animals with 5-fluorouracil, administered as solution or in acetate cellulose drug-containing granules, and short-term gas hypoxia. Simultaneous effect of 5-fluorouracil and hypoxia was less toxic for erythropoiesis, while the long-term form of the drug did not affect considerably the liver tissue functions.

[Assessment of the role of endogenous regulators in the activation of Ca-ATPase in erythrocyte membranes]. / Otsenka roli éndogennykh reguliatorov v aktivatsii Ca-ATPazy membran éritrotsitov.

The contribution of calmodulin and protein kinases A or C to the activation of membrane Ca-ATPase was studied on saponin-permeabilized rat erythrocytes. In the presence of all endogenous regulators, the dependence of the Ca-ATPase activity of Ca2+ concentration was described by a bell-shaped curve with a maximum at 2-5 microM Ca2+; K0.5 = 0.43 microM Ca2+. Washing of erythrocyte membranes with 5-10 microM Ca2+ maintained up to 75% of the ATPase activity, while washing with EGTA (2 mM) decreased the activity, on the average, 5-fold, and increased K0.5 up to 0.54-0.6 microM Ca2+. An addition of an EGTA extract to washed membranes restored up to 75% of the original ATPase activity, while calmodulin restored about 40% of the original Ca-ATPase activity and decreased K0.5 to 0.23-0.3 microM Ca2+. The calmodulin inhibitor R24571 failed to alter the Ca-ATPase activity in permeabilized erythrocytes but slightly diminished it in reconstituted membranes. The protein kinase C inhibitors H7 and polymyxin increased the Ca-ATPase activity in permeabilized red cells and suppressed it in reconstituted membranes. The data obtained suggest that in native red cell membranes Ca-ATPase is activated by regulator(s) dependent on Ca2+ and protein kinase which are other than calmodulin.

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia.

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.

Effect of simultaneous administration of vitamin E and pyridoxine on erythrocyte membrane Na+K+ ATPase activity.

Vitamin E is known to have an interaction with pyridoxine. To understand the functional significance of this interaction a study was carried out on the effect of simultaneous administration of vitamin E and pyridoxine on erythrocyte membrane Na+K+ ATPase activity. Adult male volunteers belonging to low socio-economic class with prior consent were used as subjects for the study. The results of the study have shown that administration of vitamin E alone brings about a transient increase in the total and ouabain insensitive Na+K+ ATPase. Administration of B6 along with vitamin E brought about an increase in the total and true Na+K+ ATPase activity. B6 administration alone showed a statistically insignificant increase in true ATPase activity. The results of the study are interpreted to show that administration of vitamin E along with B6 is beneficial to membrane function.

Malignant hyperthermia: characterization of erythrocyte membranes from individuals at risk.

Structural and functional characteristics of erythrocytes and isolated erythrocyte membranes from known malignant hyperthermia (MH) carriers have been examined in the hope of deriving some information concerning the underlying molecular basis of this genetic abnormality, which may represent a state of generalized membrane involvement. The increase in erythrocyte osmotic fragility which has previously been noted in porcine MH was found not to apply to the human disorder and there was evidence that in some individuals at risk osmotic fragility was in fact reduced. Although no alteration in erythrocyte membrane phospholipid profiles was detected, membrane cholesterol levels were reduced in all three definite carriers examined as well as in approximately half of the possible MH carriers investigated. No evidence for associated changes in membrane protein sulfhydryl group latency or in temperature-dependent perturbations of membrane fluidity using a stearic acid spin probe could be detected. Finally, since alterations at the level of skeletal muscle membrane -Ca++ interaction have been implicated in the pathogenesis of MH, we have examined in detail the influence of temperature on the Ca++-stimulated components of the Mg++-dependent ATPase of erythrocyte membranes from known MH carriers but no evidence of any abnormality could be found. Since MH carriers detection based solely on measurements of plasma creatine phosphokinase elevations may yield equivocal results, a decrease in erythrocyte membrane cholesterol content may provide a convenient means of identifying such individuals at risk.

Erythrocyte membrane enzyme abnormalities in two hereditary disorders of muscle.

Enzymatic properties of erythrocyte membranes in Duchenne muscular dystrophy (DMD) and malignant hyperthermia (MH), two genetically determined abnormalities of skeletal muscle, were examined. Acetylcholinesterase (AChE) and ATPase activities were chosen for investigation since alterations in these enzymes have been demonstrated in animal models of dystrophy. A significant decrease in Na+,K+-ATPase activity was noted in DMD patients and a number of possible DMD carriers, suggesting that this enzyme may provide a useful marker of the carrier state in carriers not exhibiting an elevation in plasma creatine phosphokinase activity. No abnormalities in AChE were demonstrable in any of our DMD patients, indicating that human dystrophy is biochemically distinct from certain animal models of dystrophy (e.g., dystrophic mice) where erythrocyte AChE is decreased. In contrast, evidence was found in two known MH carriers, who had normal erythrocyte ATPase activities, for the presence of an altered membrane AChE characterized by an increase in substrate affinity and a large decrease in maximal hydrolytic rate. While the exact relevance of this membrane defect, if any, to the pathogenesis of MH remains to be seen, the presence of this modified enzyme may serve to identify those individuals in a family where a positive history of MH exists who are at risk of developing a hyperthermic crisis during anesthesia.