Alterations in erythrocyte membrane protein composition in advanced non-small cell lung cancer

Cancer can be associated with hematological complications related to red blood cell (RBC) function, whose physiological roles have now been expanded since it is now known that RBC are also signalling cells. The aim of this study was to explore the alterations occurring in the protein composition of RBC in advanced non-small cell lung cancer (NSCLC). Blood samples from 21 patients with advanced (stages III-IV) NSCLC (16 squamous cell carcinomas and 5 adenocarcinomas), and from 21 healthy volunteers were used. Samples from 6 randomly selected patients and 6 controls were used for the screening of erythrocyte ghost alterations by Differential Scanning Calorimetry (DSC). Samples from 15 patients and 15 controls, different from those used in the DSC measurements, were randomly selected for analysis of the expression of glycophorin (GP) species, band 3, and glycoproteins by SDS-PAGE and Western blotting or lectin enzyme immunoassays. Additionally, 5 patients with chronic obstructive pulmonary disease (COPD) were used as a control group representative of a benign inflammatory disease. Blood samples from the COPD patients were used to analyze the expression of GPs, band 3 and syaloglycoproteins. We observed the following in NSCLC: (a) changes in GP expression levels, mainly decreases in the GPA and GPC monomers, and in the GPAB dimers; (b) a decrease in the band 3 protein level, and (c) alterations in the expression of different sialoglycoproteins. RBC from the COPD patients also showed protein abnormalities, some of them, especially at the level of band 3 and the syaloglycoproteins, being similar to those in NSCLC.     

Receptor-mediated activation of a phospholipase A2 in rabbit neutrophil plasma membrane.

Using the exogenous substrate [1-14C]oleate-labeled autoclaved Escherichia coli, we have demonstrated that the chemotactic factors fMet-Leu-Phe, complement component C5a, and leukotriene B4 [(5S,12R)-dihydroxy-6-cis,8-trans,11-trans,14-cis-icosatetraenoic acid] stimulate a phospholipase A2 of isolated plasma membranes of rabbit peritoneal neutrophils. Each of the chemotactic factors shows a biphasic concentration dependence with the optimal concentrations occurring at 1, 10, and 0.1 nM, respectively. The specific antagonists of fMet-Leu-Phe binding, carbobenzoxy-Phe-Met and t-butoxycarbonyl-Phe-Leu-Phe, effectively block the stimulation by fMet-Leu-Phe, indicating that the activation is receptor mediated. delta 6-trans-leukotriene [(5S-12R)-dihydroxy-all-trans-6,8,10,14-icosatetraenoic acid], a biologically inactive stereoisomer of leukotriene B4, does not stimulate phospholipase activity, suggesting that the enhancement by leukotriene B4 is also receptor mediated. The unstimulated and activated phospholipase exhibit a broad range of maximal activity between pH 7.0 and pH 8.5, both with an optimal pH of 8.5. The activation of the phospholipase by fMet-Leu-Phe is completely calcium dependent; no increase in activity is demonstrable if fMet-Leu-Phe is added in the absence of exogenous calcium or in the presence of EGTA. In contrast, the unstimulated plasma membrane activity of the phospholipase, as well as the activity arising after stimulation, is relatively insensitive to the concentration of calcium, being inhibited by less than 50% in the presence of 10 mM EGTA. The phospholipase hydrolyzes 1-[1-14C]palmitoyl-2-acyl-sn-glycerophosphoethanolamine to form only radioactive lysophosphatidylethanolamine as the product, indicating that the enzyme has an A2 specificity.     

Alterations in rheological properties and erythrocyte membrane proteins in cats with diabetes mellitus

Many studies have shown that diabetes mellitus is associated with increased whole and blood viscosity and decreased erythrocyte deformability. It has been suggested that these abnormalities in blood rheology may play a causative role in the pathogenesis of diabetic vascular complications. However, less is known about the content and quality of membrane proteins which may contribute to abnormalities in membrane dynamic and decreased erythrocyte deformability. In the present study we analysed various rheological parameters (blood and plasma viscosity, erythrocyte deformability, haemotological parameters), in cats with non-insulin dependent diabetes mellitus (NIDDM). We also investigated alterations in erythrocyte membrane protein content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We found that erythrocyte rigidity and plasma and whole blood viscosities were significantly higher in cats with NIDDM compared to controls. SDS-PAGE revealed that the band 5 corresponding to actin was weaker while band 4.5 corresponding to integral membrane proteins (glycophorin A, B and C) had disappeared. Also, band 4.9, which is composed of dematin (a protein with actin-bundling capacity) was lost. We suggest that the observed abnormalities in membrane proteins may play a role in reduced erythrocyte deformability associated with diabetes mellitus.     

Alterations in erythrocyte enzymes in cancer

An increase in lactate production, and in activities of phosphohexoisomerase, phosphofructokinase and pyruvate kinase was found in erythrocytes of patients with advanced cancer disease. Phosphofructokinase isolated from patients' erythrocytes showed enhanced affinity for substrate and coenzyme, diminished thermal stability and changed dependence of the activity on pH. Allosteric properties of the enzyme were modified. A decrease in glucose-6-phosphate dehydrogenase activity was observed in hemolysate. The partially purified enzyme showed decreased affinity for glucose-6-phosphate, and markedly reduced stability at 45 degrees C and in the acid and alkaline pH range. Changes in kinetic and molecular properties of the two key enzymes of glucose metabolism may contribute to hemolysis observed in many cancer patients. Incubation in vitro of normal human erythrocytes with blood sera of patients resulted in increasing of phosphofructokinase, and decreasing of glucose-6-phosphate dehydrogenase activity, indicating that an acquired enzymopathy may be present in cancer.     

Effect of gemfibrozil on erythrocyte membrane lipids in geriatric patients

Twenty geriatric patients with primary or secondary hyperlipidemia and suffering from various other diseases received for three weeks once daily 900 mg gemfibrozil. The hyperlipidemia had not been treated before, and a cholesterol-reduced diet did not succeed in lowering total cholesterol below 6.75 mmol/l (260 mg/100 ml) and serum triglycerides below 1.97 mmol/l (175 mg/100 ml). The purpose of this study was to analyze the lipid composition of the erythrocyte membrane, serum lipids and rheological parameters before and after the therapy. Mean serum total cholesterol and triglyceride content decreased significantly by 16.3% (p less than 0.05) and 35.2% (p less than 0.01) on average, respectively. Aggregation of thrombocytes and of erythrocytes, thrombin time and partial thromboplastin time slightly varied during the three weeks' treatment, but without statistical significance. The content of total long-chain saturated fatty acids in the phospholipid fraction of the erythrocyte membrane decreased slightly from 41.3% to 40.9% (p less than 0.05), whereas the total w6-unsaturated fatty acids without the precursor linoleic acid increased by about the same extent from 15.66% to 16.0% (p less than 0.05). The molar ratio of phospholipid to cholesterol content decreased significantly (p less than 0.01) due to a reduced phospholipid content at the end of the therapy. In conclusion, in addition to reducing the serum lipids, gemfibrozil slightly effects the lipid composition of erythrocytes, but the effects of the varied concentrations of long-chain saturated and long-chain w6-unsaturated fatty acids in the phospholipid fraction on membrane fluidity might be compensated, at least partly, by the decrease of the ratio of membrane phospholipid to cholesterol.     

Asynchronous synthesis of erythrocyte membrane proteins.

The synthesis of membrane proteins of the mature mouse erythrocyte is asynchronous. During erythropoiesis, synthesis of the bulk of the spectrin and actin polypeptides is completed before that of the major transmembrane glycoprotein. Synthesis of the glycoprotein ceases before that of several minor proteins found on the inner surface of the red cell membrane, and one of these minor proteins is made predominantly by reticulocytes. These findings were the result of experiments in which a normal mouse was given a single injection of [35S]methionine. The appearance of radioactivity in the membrane proteins of circulating mature erythrocytes was followed. The earliest labeled proteins to emerge into the blood represent those synthesized at the last stages of erythropoiesis.     

Effect of insulin on human erythrocyte membrane fluidity in diabetes mellitus

Summary The effect of insulin in vitro on the fluidity of the human erythrocyte membrane in Type I (insulin-dependent) diabetic patients and healthy control subjects was investigated using a fluorescence technique. It was found that the addition of 10^-9 mol/l porcine insulin significantly increased fluorescent probe lateral mobility in the membrane lipid layer but did not appear to produce any conformational changes of membrane proteins.     

Action of sodium aurothiomalate on erythrocyte membrane.

The number of sulphydryl groups on the erythrocyte membrane has been assessed as a function of nutritional status for two groups of patients, one receiving non-steroidal anti-inflammatory drugs (NSAIDs) and the other receiving sodium aurothiomalate (Myocrisin). The patients receiving NSAIDs had a significantly higher number of sulphydryl groups in both the glucose depleted and glucose activated states than the patients receiving sodium aurothiomalate. The study focuses on the hexose transport protein where there is a specific binding site for gold using the two sulphydryl residues on helices 11 and 12 of the protein. The data suggest that the strong binding of gold to the erythrocyte membrane occurs via thiol pairs rather than by isolated sulphydryl groups and that there are possibly two further binding sites for gold on the membrane, the identities of which are still unclear.     

Inflammatory events after fibrin microembolization. Alterations in alveolar macrophage and neutrophil function

We performed bronchoalveolar lavage (BAL) 0.5 to 24 h after thrombin-induced pulmonary microembolization in spontaneously breathing sheep to examine the inflammatory events that occur after pulmonary intravascular coagulation. Neutrophil alveolitis was evident as early as 0.5 h after microembolization and was maximal at 4 h (4.9 +/- 1.5% neutrophils of total BAL cells at baseline versus 26.2 +/- 2.8% at 4 h post-thrombin). Neutrophils obtained both at baseline (isolated from peripheral blood) and at 0.5 to 24 h after thrombin (isolated from BAL) did not demonstrate significant basal production of superoxide anion (O2-) and produced similar amounts of O2- upon challenge with phorbol myristate acetate (PMA) 200 micrograms/ml. The basal O2- production by alveolar macrophages was also not increased. However, alveolar macrophages recovered after fibrin microembolization produced greater amounts of O2- (29.1 +/- 6.3 nm O2-/10(6) cells at 0.5 h) after challenge with PMA compared with alveolar macrophages recovered prior to embolization (10.6 +/- 1.6 nm O2-/10(6) cells baseline), suggesting that thrombin-induced microembolization primes alveolar macrophages and enhances their O2- generation. Neutrophil chemotactic activity was detected in BAL fluid at 0.5 h post-microembolization and reached a peak level at 2 h. Alveolar macrophages were a source of the chemotactic activity since conditioned medium obtained from 2-h post-thrombin macrophages induced neutrophil chemotaxis, whereas baseline cells did not. The addition of the thrombin to macrophages did not result in the generation of chemotactic activity from baseline macrophages, indicating that macrophages were activated during the process of intravascular coagulation rather than by thrombin per se. Post-thrombin BAL fluid also stimulated O2- generation from sheep neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dietary fats on erythrocyte membrane lipid composition and membrane-bound enzyme activities.

Four different oil-based diets were used in a feeding study involving rats to assess the relationship between the fatty acid composition of the dietary fat and its influence on erythrocyte membrane (EM) lipid composition and the activities of membrane-bound enzymes. Nutritionally adequate diets containing 20% groundnut (GNO), coconut (CO), safflower (SO), or mustard oil (MO) were fed to weanling CFY rats for 4 months. EMs were analyzed for total cholesterol, phospholipids, fatty acid profiles, and sialic acid content. Activities of membrane-bound enzymes such as Na+, K(+)-adenosine triphosphatase (ATPase), Mg(2+)-ATPase, Ca2+, Mg(2+)-ATPase, and acetylcholinesterase were also assayed. The activities of all membrane-bound enzymes, except Mg(2+)-ATPase, and sialic acid content were higher in the MO-fed group than in the rest of the groups. Ca2+, Mg(2+)-ATPase activity was distinctly lower in the SO-fed group than in the other groups. Cholesterol to phospholipid molar ratio was similar in all the groups. However, SO- and MO-fed groups displayed an increased cholesterol content and a higher degree of unsaturation in the membrane fatty acid composition. The higher membrane fatty acid unsaturation in the SO-fed group was principally due to linoleic (18:2) and arachidonic (20:4) acids, while in the MO-fed group it was mainly due to oleic (18:1), eicosenoic (20:1), erucic (22:1), and linoleic (18:2) acids. These results suggest a relationship between the quality of dietary fat, EM fatty acyl composition, and the activities of membrane-bound enzymes.     

微量元素锌对大鼠红细胞膜唾液酸含量的影响

对大鼠红细胞膜唾液酸含量进行了测定，并初步观察了微量元素锌和维生素Ｄ３（ＶＤ３）对大鼠红细胞膜唾液酸含量的影响。结果表明，锌对ＶＤ３所引起的红细胞膜唾液酸含量降低具有恢复作用，提示微量元素锌对红细胞膜具有明显的保护作用。     

Alterations in erythrocyte rheology in patients with severe peripheral vascular disease: 1. Cell volume dependence of erythrocyte rigidity

The erythrocyte rigidity of patients suffering from severe peripheral vascular disease (PVD) was measured by a filtration method using 3 microns pore size filters. Filtration pressures for both PVD patients and normal subjects showed a cell volume dependency, and patient filtration pressures were normalized to cell volume to evaluate intrinsic, ie, nonvolume dependent, abnormalities in erythrocyte deformability. A significant (p less than 0.001) increase in cell rigidity was found in 44 of 54 PVD patients in comparison with volume-matched normal controls. No significant difference was found between patient mean corpusculer hemoglobin (MCH) and normal MCH at any given mean corpuscular volume (MCV), indicating that observed increases in erythrocyte rigidity are not attributable to changes in patient MCH. Therefore, the mechanism of increase in erythrocyte rigidity for PVD patients still needs further investigation into such parameters as levels of adenosine triphosphate,2,3-DPG, and membrane fluidity (calcium- and/or protein-binding membrane, cholesterol and phospholipid content of membrane, etc), as well as other aspects of erythrocyte physiology.     

Effect of erythrocyte membrane on extracellular development of the erythrocytic cycle of Plasmodium falciparum.

We have earlier reported conditions that support the axenic development in vitro of a complete asexual erythrocytic cycle of Plasmodium falciparum. Up to 30% of merozoites showed initial differentiation into trophic forms (rings) viable at 14 hr. However, only approximately 1% of the merozoites would develop further into trophozoites and early schizonts viable at 36 hr. In efforts to increase the number of late stage parasites, we have now found a significant favorable effect of the addition of erythrocyte ghosts. Doubling the quantity of erythrocyte membrane in the erythrocyte sonicate medium resulted in approximate doubling of the number of trophozoites and early schizonts. These results indicate that components of the erythrocyte membrane are essential for the complete development of the erythrocytic cycle.     

Crystal-induced neutrophil activation. II. evidence for the activation of a phosphatidylcholine-specific phospholipase D

Objective. To investigate the involvement of phospholipase D in the signaling pathways activated by 2 pathologically relevant inflammatory microcrystals, monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD). Methods. Human peripheral blood neutrophils were used throughout. Phospholipase D activity was monitored by measuring 3 separate indices: 1) the mass of phosphatidic acid, 2) the levels of alkyl-phosphatidic acid, and 3) the levels of formation, in the presence of ethanol, of phosphatidylethanol. The latter 2 parameters were measured in cells labeled with 1-0-³H–alkyl-2-acetyl-sn-glycero-3-phosphocholine. The cells were stimulated with microcrystals of triclinic morphology. Results. Both MSU and CPPD crystals induced a time- and concentration-dependent accumulation of phosphatidic acid mass and elevation in levels of alkyl-phosphatidic acid and phosphatidylethanol in prelabeled cells. The activation of phospholipase D by the microcrystals was partially sensitive to colchicine and largely resistant to pertussis toxin. Inhibition of phosphatidic acid formation by wortmannin or ethanol reduced the microcrystal-stimulated production of superoxide anions. Conclusion. These results indicate that microcrystals stimulate phospholipase D in human neutrophils and that at least some of the functional consequences of neutrophil-microcrystal interactions may be dependent on this biochemical pathway.     

Effect of complement on the viscoelastic properties of human erythrocyte membrane

Using the micropipette technique, we examined the viscoelastic properties of the red blood cell (RBC) membrane which had been strongly coated with various components of complement (primary C3b, C3d, C4b, C4d) in vitro. The membrane elastic modulus (E), the viscosity index of the initial rapid phase of deformation (eta D1), the viscosity of the later slow phase of deformation (eta D2) and the viscosity of the recovery phase (eta R) were determined. Compared to control non-complement coated RBCs, RBCs coated with C3d, either alone or with other complement components, showed significant increase in the values for elasticity and viscosities. Thus C3d fixation resulted in decreased membrane deformability. Changes in membrane viscoelasticity due to bound C3d were not enhanced by bound C4b, C4d, C5, factor Bb or p; presence on RBC membrane of the latter two complement components may partially reverse the effect of C3d fixation. Lipid fluidity of RBC membrane, examined by fluorescence depolarization, increased with fixation of all complement components except C5. These complement-induced changes in membrane viscoelastic properties have potential pathophysiological and clinical implications. The data suggest that extravascular sequestration of human RBCs may be explained in part by increased membrane rigidity resulting from C3d fixation.     

X-ray diffraction studies of human erythrocyte membrane structure.

Small angle x-ray diffraction patterns have been obtained from ordered arrays of hemoglobin-free human erthyrocyte membranes by use of improved techniques. Diffraction data have been recorded to 9 A resolution on samples whose lattice periodicity was varied (by changing humidity) from 55.5 A to 69.6 A. The observed reflections permitted tracing the intensity transform of the membranes. Phases for the reflections were assigned by the minimum wavelength principle. An electron density profile was then obtained by Fourier inversion, and yielded a symmetric membrane about 55 A in width. This structure can account for the previously reported diffuse scattering observed in other preparations (thus rendering unnecessary the proposed assignment of this scattering to a separated lipoprotein phase) and for the continuous scattering that we have recorded from isolated membranes in buffer. Lower resolution data that we have obtained from ultracentrifugally prepared lattices in buffer (and therefore without dehydration) are consistent with the above results, and support our view that we are observing diffraction from intact membranes.     

Effect of the natriuretic hormone on ion transport through erythrocyte membrane

A study of the mechanism of the natriuretic hormone action on ion transport across cellular membranes was carried out in normal donor blood erythrocytes and their shadows. The natriuretic hormone obtained from canine arterial blood changed sodium and potassium ion transport, when acting on external membrane surface. Its effect can be compared to that of strophanthin which produces a similar change in sodium and potassium transport and is only active on the outer side of closed erythrocyte shadows.     

Impaired expression of erythrocyte glycosyl-phosphatidylinositol-anchored membrane CD59 in patients with psoriatic arthritis. Relation to terminal complement pathway activation

OBJECTIVE: Complement-mediated injury is regulated by many factors; among these CD59 has been identified as a widely distributed glycoprotein that inhibits membrane C5b-9 (terminal complement component) formation. The aim of the study was to assess erythrocyte CD59 expression in patients with psoriatic arthritis in order to understand the role of CD59 in the pathogenesis. METHODS: Washed erythrocytes from 50 patients with psoriatic arthritis, 8 with cutaneous psoriasis and 24 healthy subjects were incubated with monoclonal anti-CD59 antibody followed by a second FITC conjugated antibody and fluorescence intensity analysed by FAC-Scan flow cytometer to assess their CD59 membrane expression. SC5b-9 levels were measured in the plasma by ELISA and results compared with CD59 values. Immune complexes, complement C3 and C4 and rheumatoid factor were also determined. RESULTS: Impaired expression of erythrocyte membrane-anchored CD59 was found in patients with psoriatic arthritis; the lowest levels were seen in active patients (p < 0.01). Increased SC5b-9 was seen in the plasma of patients with active disease. An inverse correlation was also found between plasma C5b-9 and the CD59 expression levels (r = -0.81, p < 0.001). CONCLUSION: The low CD59 expression on erythrocytes from patients with psoriatic arthritis may be an index of a low tissue CD59 expression. This impairment could facilitate the activation of complement pathway and increase the risk for arthritis. Membrane attack complex formation in deficient membrane bound CD59 may also exacerbate synovial cell injury and inflammation.     

Deformability of the erythrocyte membrane in patients with myelodysplastic syndromes.

One of the major determinants of erythrocyte survival is membrane deformability, and an important intrinsic parameter of membrane deformability is the shear elastic modulus (mu) with higher mu values corresponding to increased membrane rigidity. Using a micropipette technique, we determined the shear elastic modulus of erythrocytes from 21 patients with myelodysplastic syndromes (MDS). Ten thalassemic patients and 15 healthy subjects served as controls. The shear elastic modulus of MDS erythrocytes was very significantly increased in all the patients studied, reflecting the rigidity of the membrane; the value of mu was also significantly higher in comparison with thalassemic cells. These data point to a fundamental change in the mechanical properties of the erythrocyte membrane in MDS. Biochemical studies of the membrane composition are clearly needed.