Enzymatic cleavage of cell surface proteins of pig and cow erythrocytes and its effect on concanavalin-mediated agglutinability.

Study was carried out to understand and compare architecture of the proteins of erythrocyte cell surface of some mammals viz., Homo sapiens (human), Sus scorfa domestica (pig) and Bos taurus domestica (cow). In this study, we investigated the action of proteinases viz., trypsin and chymotrypsin and neuraminidase on the erythrocyte surface proteins and erythrocyte agglutination tendency with a lectin (concanavalin A). The electrophoretic pattern of membrane proteins and glycophorins (analyzed by SDS-PAGE and visualized by Coomassie brilliant blue and periodic acid-schiff stains, respectively) and concanavalin A (Con A) agglutinability revealed that: (i) There were variations in the number and molecular weights of glycophorins in human, pig and cow, (ii) trypsin action on pig and cow erythrocyte membrane proteins was similar, unlike human, (iii) glycophorins degradation by trypsin and chymotrypsin was not similar in pig, as compared to that of human and cow, (iv) erythrocytes agglutination with Con A was significantly different due to differences in membrane composition and alterations in the surface proteins after enzyme treatment, (v) a direct correlation was found between degradation of glycophorins and Con A agglutinability, and (vi) removal of erythrocyte surface sialic acids by neuraminidase specifically indicated an increase in Con A agglutinability of pig and cow erythrocytes, similar to human.

In vivo antipyretic, antiemetic, in vitro membrane stabilization, antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves

BACKGROUND: The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh. RESULTS: In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 µg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC50 value 48.978 µg/ml) activity, whereas ethyl acetate fraction showed mild (LC50 value 216.77 µg/ml) cytotoxic activity. CONCLUSIONS: Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.

The protective effect of aqueous extracts of roselle (Hibiscus sabdariffa L. UKMR-2) against red blood cell membrane oxidative stress in rats with streptozotocin-induced diabetes

OBJECTIVES: The aim of this study was to investigate the protective effects of aqueous extracts of roselle (Hibiscus sabdariffa L. UKMR-2) against red blood cell (RBC) membrane oxidative stress in rats with streptozotocin-induced diabetes. METHODS: Forty male Sprague-Dawley rats weighing 230-250 g were randomly divided into four groups (n = 10 rats each): control group (N), roselle-treated control group, diabetic group, and roselle-treated diabetic group. Roselle was administered by force-feeding with aqueous extracts of roselle (100 mg/kg body weight) for 28 days. RESULTS: The results demonstrated that the malondialdehyde levels of the red blood cell membranes in the diabetic group were significantly higher than the levels in the roselle-treated control and roselle-treated diabetic groups. The protein carbonyl level was significantly higher in the roselle-treated diabetic group than in the roselle-treated control group but lower than that in the diabetic group. A significant increase in the red blood cell membrane superoxide dismutase enzyme was found in roselle-treated diabetic rats compared with roselle-treated control rats and diabetic rats. The total protein level of the red blood cell membrane, osmotic fragility, and red blood cell morphology were maintained. CONCLUSION: The present study demonstrates that aqueous extracts of roselle possess a protective effect against red blood cell membrane oxidative stress in rats with streptozotocin-induced diabetes. These data suggest that roselle can be used as a natural antioxidative supplement in the prevention of oxidative damage in diabetic patients. .

Antioxidant effect of 4-nerolidylcatechol and -tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellata root extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

Gender differences in alcohol-induced oxidative stress and altered membrane properties in erythrocytes of rats.

Alcohol-induced oxidative stress leads to imbalance between reactive oxygen species (ROS) and the antioxidant defense system, resulting in oxidative damage to membrane components such as lipids and proteins, ultimately altering membrane properties. In this study, we assessed oxidative stress status and alterations in erythrocyte membrane properties in alcohol-administered rats with respect to gender difference. Alcohol (20% v/v) administered rats of both genders showed significant changes in plasma lipid profile with elevated nitrite/nitrate levels. Furthermore, alcohol-administration significantly decreased erythrocyte antioxidant enzymes and enhanced erythrocyte membrane lipid peroxidation, cholesterol/phospholipid (C/P) ratio and Na+/K+-ATPase activity in both males and females. Besides, anisotropic studies revealed that alcohol-administration significantly decreased erythrocyte membrane fluidity. In conclusion, alcohol- administration significantly increased oxidative stress by decreasing antioxidant status, and subsequent generation of ROS altered membrane properties by altering fluidity and Na+/K+-ATPase activity. Female rats were more vulnerable to alcohol-induced biochemical and biophysical changes in plasma and erythrocyte including oxidative stress than male rats.

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

Solubilization of human erythrocyte membranes by ASB detergents

Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 µM and ASB-16 = 10 µM) was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (Re sat) and total lysis (Re sol) were calculated, allowing the determination of the membrane binding constants (Kb). ASB-14 presented lower membrane affinity (Kb = 7050 M-1) than ASB-16 (Kb = 15610 M-1). The amount of proteins and cholesterol solubilized by both ASB detergents was higher while Re sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively) were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes.

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.

Inhibition of membrane Na(+)-K+ Atpase of the brain, liver and RBC in rats administered di(2-ethyl hexyl) phthalate (DEHP) a plasticizer used in polyvinyl chloride (PVC) blood storage bags.

Significant amounts of di(2-ethylhexyl) phthalate (DEHP) leach out into blood stored in DEHP plasticized polyvinyl chloride (PVC) bags resulting in the exposure of recipients of blood transfusion to this compound. The aim of this study was to find out whether DEHP at these low levels has any effect on the activity of membrane Na(+)-K+ ATPase, since a decrease in this enzyme activity has been reported to take place in a number of disorders like neurodegenerative and psychiatric disorders, coronary artery disease and stroke, syndrome-X, tumours etc. DEHP was administered (ip) at a low dose of 750 microg/100 g body weight to rats and the activity of membrane Na(+)-K+ ATPase in liver, brain and RBC was estimated. Histopathology of brain, activity of HMG CoA reductase (a major rate limiting enzyme in the isoprenoid pathway of which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is a product), intracellular concentration of Ca2+ and Mg2+ in RBC (which is altered as a result of inhibition of Na(+)-K+ ATPase) were also studied. (In the light of the observation of increase of intracellular Ca2+ load and intracellular depletion of Mg2+ when Na(+)-K+ ATPase is inhibited). Histopathology of brain revealed areas of degeneration in the rats administered DEHP. There was significant inhibition of membrane Na(+)-K+ ATPase in brain, liver and RBC. Intracellular Ca2+ increased in the RBC while intracellular Mg2+ decreased. However activity of hepatic HMG CoA reductase decreased. Activity of Na(+)-K+ ATPase and HMG CoA reductase, however returned to normal levels within 7 days of stopping administration of DEHP. The inhibition of membrane Na(+)-K+ ATPase activity by DEHP may indicate the possibility of predisposing recipients of transfusion of blood or hemodialysis to the various disorders mentioned above. However since this effect is reversed when DEHP administration is stopped, it may not be a serious problem in the case of a few transfusion; but in patients receiving repeated blood transfusion as in thalassemia patients or patients undergoing hemodialysis, possibility of this risk has to be considered. This inhibition is a direct effect of DEHP or its metabolites, since activity of HMG CoA reductase, (an enzyme which catalyses a major rate limiting step in the isoprenoid pathway by which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is synthesized) showed a decrease.

Oxidative damage in intermediate syndrome of acute organophosphorous poisoning.

BACKGROUND & OBJECTIVES: The main cause of morbidity due to organophosphate poisoning is intermediate syndrome (Type II paralysis) that can occur 48-72 h after poisoning. Mechanisms that underlie the intermediate syndrome are not known. This study investigates the role of oxidative damage to muscles as a possible mechanism underlying the development of the intermediate syndrome. METHODS: Nineteen patients with acute organophosphate poisoning were evaluated from admission to discharge from intensive care for the severity of poisoning and the development and duration of the intermediate syndrome. Blood cholinesterases and parameters of oxidative stress were studied daily and their temporal profiles analysed according to the severity of poisoning and the development and duration of the intermediate syndrome. RESULTS: Fifteen patients had severe poisoning and 16 developed intermediate syndrome. There was a positive association between the severity of poisoning and the occurrence of intermediate syndrome. There was no association between the organophosphate ingested and the development of intermediate syndrome. Erythrocyte membrane acetylcholinesterase and serum butyrylcholinesterase levels at admission and over the course of poisoning were significantly (P < 0.001) reduced in patients compared to controls. There were significantly (P < 0.05) higher levels of lipid peroxidation, conjugated dienes and protein thiols in erythrocyte membranes of patients who developed the intermediate syndrome compared to healthy controls, in patients who developed intermediate syndrome compared to those who did not and in patients with long compared to short duration intermediate syndrome. INTERPRETATION & CONCLUSION: In acute organophosphate poisoning, severe and prolonged acetylcholinesterase inhibition is associated with oxidative stress, detected in erythrocyte membranes, that occurs early in the course of poisoning and may contribute to the development and severity of intermediate syndrome.

Influence of ethanolic extract of Tephrosia purpurea Linn. on mast cells and erythrocytes membrane integrity.

The ethanolic extract of T. purpurea Linn. was studied for its in vitro effect on rat mast cell degranulation and erythrocyte membrane integrity in vitro. The extract in concentration of 25-200 microg/ml showed a dose-dependant inhibition of rat mast cell degranulation induded by compound 48/80 and egg albumin. T. purpurea extract was found to inhibit haemolysis of erythrocytes induced by hypotonic solution but accelerated haemolysis induced by heat at a concentration of 100 microg/ml. The studies reveal that the ethanolic extract of T. purpurea may inhibit degranulation of mast cells by a mechanism other than membrane stabilization.

Protective effect of capsaicin on the osmotic fragility of human erythrocytes.

Capsaicin exerts a stabilizing effect on erythrocytes making them more resistant to lysis under hypotonic stress. The protective action of capsaicin on osmotic fragility (OF) was not receptor mediated since no dose responsive effect was observed. The results suggest that this protective effect of capsaicin on OF is due to a direct interaction of capsaicin with the erythrocyte membrane rather than due to any alteration in the intracellular metabolism of erythrocytes.

Administration of the plasticizer di (2-ethylhexyl) phthalate alters glycoconjugate profile.

This study was undertaken to evaluate the level of glycoproteins and sialic acid in rats fed di (2-ethylhexyl)-phthalate (DEHP) in the diet for 24 weeks. Protein-bound hexose, hexosamine and sialic acid were increased in plasma and liver of rats treated with DEHP, whereas the erythrocyte membrane showed a reduction following DEHP administration. Evaluation of glycoproteins is a useful indicator of the carcinogenic process. It is suggested that profound alterations in membrane components observed in the present study may be related to the carcinogenic potential of DEHP.

Effect of Ep475 on changes induced by calcium activated neutral protease on erythrocyte membrane.

Increase in cytosolic calcium leads to activation of calcium activated neutral protease (CANP). CANP is known to cause many membrane abnormalities that aid erythrocyte destruction. An epoxy compound Ep475 causes the reversal of such changes induced by calcium and CANP. In absence of Ep475, CANP caused reduction in free sulfhydryl groups and glycoprotein content of the membrane to 61, and 50% respectively, compared to untreated membranes. Calcium ATPase and membrane associated CANP were increased 3 and 1.4 times respectively. Significant reversal of these changes by Ep475 suggests a possible role of this compound in reversing the calcium dependent alterations in RBC including the action of CANP.

Haematological and erythrocyte membrane changes induced by amiodarone, in rats.

The effect of Amiodarone (AD), a cationic amphiphilic drug, on erythrocytes and leucocytes was studied. Treatment of rats with AD showed a significant decrease in the red cell count and the level of Hemoglobin. Amiodarone altered the fluidity of the erythrocyte membrane followed by a decrease in the activities of membrane bound enzymes like (Na+, K+)-ATPase, Acetylcholine esterase and NADH dehydrogenase. A slight increase in the leucocyte count was also observed in the treated animals.

Membrane skeletal protein structure and interactions in human erythrocytes after their treatment with diamide and calcium.

To analyse the role of native structures of membrane proteins in their structural modifications induced by the elevated intracellular free Ca2+ levels, we have studied the Ca(2+)-mediated effects on membrane skeletal proteins in human erythrocytes that were loaded with Ca2+ using the ionophore A23187 after their pretreatment with the sulphydryl oxidizing agent, diamide. The diamide treatment not only induced polymerization of the major membrane skeletal protein, spectrin, in the erythrocytes, but it also promoted intersubunit crosslinking within the tetramers and dimers of this protein. Loading of these diamide-treated cells with Ca2+ failed to induce significant structural modifications of spectrin as well as polypeptide 4.1, another major membrane skeletal protein, as compared to the erythrocytes that were loaded with Ca2+ without the diamide pretreatment. These results have been interpreted to suggest that the Ca(2+)-induced membrane skeletal protein changes in erythrocytes depend on both the shape and relative orientation of these proteins within the membrane skeleton.

Membrane damaging potential of photosensitized riboflavin.

Riboflavin upon exposure to UV and visible radiations has been shown to produce active oxygen species. The present work deals with erythrocyte membrane as model system to study the damaging potential of photosensitized riboflavin. Membrane preparations (2.5 mg protein/ml) following exposure to sunlight in presence of riboflavin for different time intervals revealed significant inhibition of ATPases, p-nitrophenyl phosphatase and acetylcholinesterase. Considerable increase in lipid peroxidation was caused by the photosensitized riboflavin. Quenching studies using specific scavengers indicated remarkable inhibition. The production and identification of reactive oxygen species by photosensitized riboflavin and their possible involvement in membrane damaging effect has been discussed.

Chicken erythrocyte membrane: lipid profile and enzymatic activity under lanthanum chloride and neodymium chloride administration.

Acute single dose (ip) administration of two rare earth elements like lanthanum chloride (250 mg/kg body wt) and neodymium chloride (200 mg/kg body wt) to chicks have been found to reduce the activity of certain erythrocyte membrane bound enzymes, viz. acetylcholinesterase, NADH dehydrogenase, Mg(2+)-ATPase, p-nitrophenyl phosphatase. Erythrocyte membrane bound glycosidases e.g. beta-D-glucosidase, beta-D-galactosidase and beta-D-glucuronidase were also reduced. Other components such as cholesterol and phospholipid residues were reduced but their ratio (cholesterol/phospholipid) remaining unchanged. Membrane sulfhydryl groups were also significantly inhibited by these rare earth elements.

Effect of defoliant (butiphose) on morpho-physiological properties and enzyme systems of natural membranes.

Butiphose (Tributyltritiophosphate, (C4H9S)3PO) a commonly used defoliant in cotton growing regions of USSR, caused extensive alterations in morphological features of erythrocyte and nuclear membranes and affected the permeability properties of rat liver mitochondrial membrane. It disrupted Ca2+ transport system and other energy dependent processes in mitochondria. A reduction in the activity of cytochrome-c-oxidase and NAD.H-oxidase was also observed.

Effect of alpha-tocopherol on peroxidative membrane damage caused by doxorubicin: an in vitro study in human erythrocytes.

Level of lipid peroxidation in doxorubicin treated human erythrocytes was studied and compared with that of cells pretreated with alpha-tocopherol. Erythrocytes treated with alpha-tocopherol had reduced level of lipid peroxidation with concomitantly lowered membrane damage. The membrane damage was monitored by the levels of conjugated diene absorption, lipid hydroperoxides and lipid peroxides. alpha-tocopherol was not effective in inhibiting the conjugated diene formation, but the lipid hydroperoxides and the lipid peroxide levels were significantly decreased. Methemoglobin level was found to be increased in alpha-tocopherol pretreated cells, which protects the membrane from damage. Erythrocyte membrane lipids were found to be decreased during doxorubicin treatment and alpha-tocopherol significantly reduced the membrane lipid breakdown. Level of reduced glutathione was maintained in alpha-tocopherol pretreated cells. These results are discussed with reference to the antioxidant property of alpha-tocopherol.