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Aging, a time-dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d-galactose ( d-Gal)-induced aging model. Anion exchange capability, measured by the rate constant of SO₄²- uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d-Gal for 24 h. Our results show that: (i) in vitro application of d-Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d-Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age-related disturbances.

Hyperglycemia is considered a threat for cell homeostasis, as it is associated to oxidative stress (OS). As erythrocytes are continuously exposed to OS, this study was conceived to verify the impact of either diabetic conditions attested to by glycated hemoglobin (Hb) levels (>6.5% or higher) or treatment with high glucose (15–35 mM, for 24 h) on erythrocyte homeostasis. To this aim, anion exchange capability through the Band 3 protein (B3p) was monitored by the rate constant for SO42− uptake. Thiobarbituric acid reactive species (TBARS), membrane sulfhydryl groups mostly belonging to B3p, glutathione reduced (GSH) levels, and B3p expression levels were also evaluated. The rate constant for SO42− uptake (0.063 ± 0.001 min−1, 16 min in healthy volunteers) was accelerated in erythrocytes from diabetic volunteers (0.113 ± 0.001 min−1, 9 min) and after exposure to high glucose (0.129 ± 0.001in−1, 7 min), but only in diabetic volunteers was there an increase in TBARS levels and oxidation of membrane sulfhydryl groups, and a decrease in both GSH and B3p expression levels was observed. A combined effect due to the glycated Hb and OS may explain what was observed in diabetic erythrocytes, while in in vitro hyperglycemia, early OS could explain B3p anion exchange capability alterations as proven by the use of melatonin. Finally, measurement of B3p anion exchange capability is a suitable tool to monitor the impact of hyperglycemia on erythrocytes homeostasis, being the first line of high glucose impact before Hb glycation. Melatonin may be useful to counteract hyperglycemia-induced OS at the B3p level.

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 μM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42− uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 μM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.

Background/aims Magnesium, whose supplementation provides beneficial effects against oxidative stress-related conditions, has been here used to possibly protect Band 3 protein anion exchange capability and underlying signaling in an in vitro model of oxidative stress. Methods Whole blood samples pre-exposed to 10 mM MgCl2, were treated for 30 min with H2O2 (300 µM, 600 µM and 1 mM) chosen as oxidant molecule. In a separate protocol, NEM (0.5,1 and 2 mM), a phosphatase inhibitor and thiol-alkilant agent, has been also applied. The rate constant for SO4= uptake, accounting for Band 3 protein anion exchange capability, has been measured by a turbidimetric method, while intracellular reduced glutathione (GSH) levels and membrane -SH groups mostly belonging to Band 3 protein were spectrophotometrically quantified after reaction with DTNB (5,5'-dithiobis-(2-nitrobenzoic acid). Expression levels of Band 3 protein, phosporylated Tyrosine (P-Tyr) and tyrosine kinase (Syk) involved in signaling have been also measured. Results Our results show that Mg2+ prevented the reduction in the rate constant for SO4= uptake on H2O2-treated erythrocytes, not involving GSH levels and membrane -SH groups, unlike NEM, remaining both P-Tyr and Syk expression levels high. Conclusion Hence, i) the measurement of the rate constant for SO4= uptake is a useful tool to evaluate Mg2+ protective effect; ii) the use of two different oxidant molecules shed light on Mg2+ effect which seems not to modulate phosphorylative pathways but would putatively stabilize membrane organization; iii) the use of Mg2+ in food supplementation can be reasonably supported to protect erythrocytes homeostasis in case of oxidative stress-related diseases.

Band 3 protein (B3p), anion transporter, allows the HCO3 - /Cl- exchange across plasma membrane and plays an important role for erythrocytes homeostasis. In addition, B3p is linked to proteins cytoskeleton, thus contributing to cell shape and deformability, essential to erythrocytes adjustment within narrowest capillaries. Taking into account that erythrocytes are a suitable cell model to investigate the response of the oxidative stress effects, B3p functions, and specifically anion exchange capability, determining the rate constant for SO4 2- uptake, has been considered. As, in the latter years, rising attention has been addressed to membrane transport system, and particularly to this protein, the present mini-review has been conceived to report the most recent knowledge about B3p, with specific regard to its functions in oxidative stress conditions, including oxidative stress-related diseases.

Целью работы стало установление нарушений белкового спектра эритроцитарных мембран крупного рогатого скота в условиях технологического стресса и эффективности использования в коррекции стресса низкоинтенсивного лазерного излучения (НИЛИ). При технологическом стрессе у животных отмечено изменение содержания белков цитоскелета и интегральных белков: белка полосы 3 и гликофоринов. При воздействии низкоинтенсивного лазерного излучения с длиной волны 830 нм на фоне стресса обнаружены разнонаправленные изменения в белковом спектре эритроцитарных мембран. Эффекты зависели от места и времени воздействия. При 15 мин экспозиции НИЛИ в области холки к седьмым суткам эксперимента наблюдалось восстановление белкового состава эритроцитарных мембран до значений интактной группы, The aim of the work was to establish violations of the protein spectrum of the erythrocyte membranes of cattle in the conditions of technological stress and the effectiveness of using low-intensity laser radiation (LILR) in the correction of stress. Under technological stress, the animals showed changes in the content of cytoskeletal proteins and integral proteins: band 3 protein and glycophorins. When exposed to low-intensity laser radiation with a wavelength of 830 nm under stress, multidirectional changes in the protein spectrum of erythrocyte membranes were detected. The effects depended on the place and time of exposure. By the seventh day of the experiment, the protein composition of the erythrocyte membranes was restored to the values of the intact group at 15 minutes of exposure of the LILR in the withers region.

d-Galactose (d-Gal), when abnormally accumulated in the plasma, results in oxidative stress production, and may alter the homeostasis of erythrocytes, which are particularly exposed to oxidants driven by the blood stream. In the present investigation, the effect of d-Gal (0.1 and 10 mM, for 3 and 24 h incubation), known to induce oxidative stress, has been assayed on human erythrocytes by determining the rate constant of SO42&minus, uptake through the anion exchanger Band 3 protein (B3p), essential to erythrocytes homeostasis. Moreover, lipid peroxidation, membrane sulfhydryl groups oxidation, glycated hemoglobin (% A1c), methemoglobin levels (% MetHb), and expression levels of B3p have been verified. Our results show that d-Gal reduces anion exchange capability of B3p, involving neither lipid peroxidation, nor oxidation of sulfhydryl membrane groups, nor MetHb formation, nor altered expression levels of B3p. d-Gal-induced %A1c, known to crosslink with B3p, could be responsible for rate of anion exchange alteration. The present findings confirm that erythrocytes are a suitable model to study the impact of high sugar concentrations on cell homeostasis, show the first in vitro effect of d-Gal on B3p, contributing to the understanding of mechanisms underlying an in vitro model of aging, demonstrate that the first impact of d-Gal on B3p is mediated by early Hb glycation, rather than by oxidative stress, which may be involved on a later stage, possibly adding more knowledge about the consequences of d-Gal accumulation.

Band 3 protein (B3p) exchanging Cl− and HCO3− through erythrocyte membranes is responsible for acid balance, ion distribution and gas exchange, thus accounting for homeostasis of both erythrocytes and entire organisms. Moreover, since B3p cross links with the cytoskeleton and the proteins underlying the erythrocyte membrane, its function also impacts cell shape and deformability, essential to adaptation of erythrocyte size to capillaries for pulmonary circulation. As growing attention has been directed toward this protein in recent years, the present review was conceived to report the most recent knowledge regarding B3p, with specific regard to its anion exchange capability under in vitro oxidative conditions. Most importantly, the role of natural antioxidants, i.e., curcumin, melatonin and Mg2+, in preventing detrimental oxidant effects on B3p is considered.

This comparative study, focused on the anion kinetic of band 3 protein of pig erythrocytes, investigates in vitro the metabolic functionality of the red blood cells of this mammal which show particular characteristics of the cell membrane and significant s...

Band 3 protein efficiency in mediating Cl−/HCO3 − exchange through erythrocytes membrane is reduced by oxidative stress. The aim of the present study was to verify whether and how anion transport through band 3 protein may be useful in monitoring canine leishmaniasis (Leishmania infantum) development, a disease associated to membrane protein degradation and oxidative stress. To accomplish this aim, serological analysis to determine IFAT (immunofluorescence antibody test) titers against leishmaniasis has been performed and 1:160 and 1:540 titers, determined at diagnosis and after 6 months, were considered for experiments. Oxidative conditions have been assessed by estimating MDA (malondialdehyde) plasma levels, intracellular GSH (reduced glutathione) content, and membrane –SH groups. Band 3 protein anion exchange capability was evaluated by measuring the rate constant for SO4 = uptake, and its expression levels, along with those of P-Tyr (phosphorylated tyrosine), involved in pathways underlying band 3 protein function, have been also determined. Our results show that, in infected dogs with 1:160 IFAT titer, high MDA plasma levels and oxidation of –SH groups are associated to increased P-Tyr expression levels, leading to a reduction in anion exchange capability throughout 6 months of diagnosis. On the other hand, infected dogs with 1:540 IFAT titer, exhibited oxidative conditions associated to an impaired anion exchange capability at diagnosis, were ameliorated after 6 months. Such findings suggest that (1) band 3 protein-mediated anion transport is reduced by oxidative conditions associated to leishmaniasis, putatively via phosphorylative pathways; (2) band 3 protein efficiency may account for canine leishmaniasis development; and (3) the assessment of band 3 protein function may represent an additional tool for canine leishmaniasis diagnosis and monitoring of its development, with potential application to humans, either in case of leishmaniasis or other oxidative-related pathologies.

The beneficial effect of Melatonin (Mel), recognized as an anti-inflammatory and antioxidant compound, has been already proven to prevent oxidative stress-induced damage associated to lipid peroxidation. As previous studies modeled the impact of oxidative stress on Band 3 protein, an anion exchanger that is essential to erythrocytes homeostasis, by applying H2O2 at not hemolytic concentrations and not producing lipid peroxidation, the aim of the present work was to evaluate the possible antioxidant effect of pharmacological doses of Mel on Band 3 protein anion exchange capability. The experiments have been performed on human erythrocytes exposed to 300 &mu, M H2O2-induced oxidative stress. To this end, oxidative damage has been verified by monitoring the rate constant for SO4= uptake through Band 3 protein. Expression levels of this protein Mel doses lower than 100 &micro, M have also been excluded due to lipid peroxidation, Band 3 protein expression levels, and cell shape alterations, confirming a pro-oxidant action of Mel at certain doses. On the other hand, 100 &micro, M Mel, not provoking lipid peroxidation, restored the rate constant for SO4= uptake, Band 3 protein expression levels, and H2O2-induced cell shape alterations. Such an effect was confirmed by abolishing the endogenous erythrocytes antioxidant system. Therefore, the present findings show the antioxidant power of Mel at pharmacological concentrations in an in vitro model of oxidative stress not associated to lipid peroxidation, thereby confirming Band 3 protein anion exchange capability measurement as a suitable model to prove the beneficial effect of Mel and support the use of this compound in oxidative stress-related diseases affecting Band 3 protein.

In our previous paper, we demonstrated that Arg 901 in the C-terminal tail of human AE1 (band 3, anion exchanger 1) had a functional role in conformational change during anion exchange. To further examine how Arg 901 is involved in conformational change, we expressed various Arg 901 mutants and alanine mutants of the C-terminal tail (from Leu 886 to Val 911) on the plasma membrane of Saccharomyces cerevisiae and evaluated the kinetic parameters of sulfate ion transport. As a result, Vmax decreased as the hydrophobicities of the 901st and peripheral hydrophilic residues increased, indicating that the hydrophobicity of the C-terminal residue is involved in the conformational change. We also found the alkali and protease resistance of the C-terminal region after Arg 901 modification with hydroxyphenylglyoxal (HPG) or phenylglyoxal (PG), a hydrophobic reagent. These results suggested that the increased hydrophobicity of the C-terminal region around Arg 901 leads to inefficient conformational change by the newly produced hydrophobic interaction.

The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation–deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine–haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO2 thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane. Furthermore caffeine destabilizes the haeme–protein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silico docking and molecular dynamics studies and by what we may call the “caspase silence”; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing power.

© 2012 – IOS Press and the authors. All rights reserved, Recent evidence has shown that plasma fibrinogen, a major cardiovascular risk factor, interacts with the erythrocyte membrane and acts to influence blood flow via erythrocyte nitric oxide (NO) modulation. In the present in-vitro study, whole blood samples were harvested from healthy subjects and aliquots were incubated in the absence (control aliquots) and presence of fibrinogen at different degrees of band 3 phosphorylation, and the erythrocyte deformability was determined. The present study shows that in the presence of higher fibrinogen concentrations, similar to those found in inflammatory conditions, erythrocyte deformability is increased only when band 3 is dephosphorylated by the presence of syk inhibitor and at low shear stress. On the contrary, no changes were verified in the presence of fibrinogen when band 3 is allowed to be phosphorylated by inhibiting the phosphotyrosine phosphatase enzyme activity with calpeptin. We also observed that the presence of fibrinogen at higher concentration does not induce changes in erythrocyte deformability in the absence of modulators of the band 3 phosphorylation degree. However, the mechanisms by which fibrinogen signalling modulates erythrocyte function remain to be clarified and are currently under study.

Palytoxin (PTX) is classified as one of the most powerful marine biotoxins (of high molecular weight and no protein origin) because it is able to interact strongly with important cellular structures influencing their function in different biological processes. This study of the effects of PTX on red blood cells (RBC) extends the knowledge about its toxicity, which concerns not only the well-known action on Na(+)/K(+)-ATPase but also band 3 protein (B3 or AE1), the role of which is essential for anion transport and for the structure, function, and metabolic integrity of the erythrocyte. The effects of PTX on RBC can be summarized as follows: it alters the anionic flux and seriously compromises not only CO(2) transport but also the metabolic modulation centered on the oxy-deoxy cycle of hemoglobin; it stabilizes the plasma membrane by preventing lipid peroxidation; and its effect does not lead to activation of caspases 3 and 8. From what is reported in steps 2 and 3, and on the basis of the results obtained on hemolysis, methemoglobin levels, and phosphatase activity, an increase of the reducing power of the erythrocytes (RBC) in the presence of PTX clearly emerges. The results have enabled us to outline some metabolic adaptations induced in the RBC by PTX.

© 2011 – IOS Press and the authors. All rights reserved, Recent evidence has shown that plasma fibrinogen, a major cardiovascular risk factor, interacts with the erythrocyte membrane and acts to influence blood flow via erythrocyte nitric oxide (NO) modulation. In the present pioneer in-vitro study, whole blood samples were harvested from healthy subjects and aliquots were incubated in the absence (control aliquots) and presence of fibrinogen at different degrees of band 3 phosphorylation, and the levels of NO, nitrite, nitrate and S-nitroglutathione (GSNO) were determined. Hyperfibrinogenemia interferes with erythrocyte NO mobilization without changing its efflux in a way that seems to be dependent of the degree of band 3 phosphorylation. In presence of higher fibrinogen concentrations the NO efflux is reinforced when band 3 is phosphorylated (p < 0.001). Higher levels of nitrite, nitrate and GSNO were documented (p < 0.05). However, the mechanisms by which fibrinogen signalling modulates erythrocyte function remain to be clarified and are currently under study. These conditions may be considered an approach to be followed in blood storage for transfusions., This study was supported by grants from the FCT - Fundação para a Ciência e a Tecnologia (project reference PTDC/SAU-OSM/73449/2006)

The antioxidative activity of some natural flavonoids was analyzed against the stable free radical 2,2-diphenyl-1-picryhydrazyl. The results indicate that the scavenging power of the tested flavonols is higher than that of the synthetic antioxidants butylated hydroxyanisole and butylated hydroxytoluene; instead, the flavanones show little activity, as indicated by efficient concentration (EC50) values. Flavonoid autoxidation and interaction with Fe2+ and hydrogen peroxide were tested using erythrocyte membranes as a model. The results show that some compounds, like hesperetin, evidence a pro-oxidant activity higher than the ascorbic acid/iron reference system. The compounds with strong oxidative capability do not only influence cellular redox balance but also activate caspase-3, producing lactate dehydrogenase release and enhancing anionic exchange at the level of band 3 protein.

The cytoplasmic domain of band 3, the main intrinsic protein of the erythrocyte membrane, possesses binding sites for a variety of other proteins of the membrane and the cytoplasm, including the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase. We have studied the stoichiometry of the complexes of human band 3 protein and GAPDH and the competition by aldolase for the binding sites. In addition, we have tried to verify the existence of mixed band 3/GAPDH/aldolase complexes, which could represent the nucleus of a putative glycolytic multienzyme complex on the erythrocyte membrane. The technique applied was analytical ultracentrifugation, in particular sedimentation equilibrium analysis, on mixtures of detergent-solubilized band 3 and dye-labelled GAPDH, in part of the experiments supplemented by aldolase. The results obtained were analogous to those reported for the binding of hemoglobin, aldolase and band 4.1 to band 3: (1) the predominant or even sole band 3 oligomer forming the binding site is the tetramer. (2) The band 3 tetramer can bind up to four tetramers of GAPDH. (3) The band 3/GAPDH complexes are unstable. (4) Artificially stabilized band 3 dimers also represent GAPDH binding sites. In addition it was found that aldolase competes with GAPDH for binding to the band 3 tetramer, and that ternary complexes of band 3 tetramers, GAPDH and aldolase do exist.

A link between reactive oxygen species (ROS) and Systemic Sclerosis (SSc) development has been already described, along with an oxidative stress-induced reduction of anion exchange capability through Band 3 protein. On this basis, blood from SSc patients has been collected to measure the rate constant for SO4= uptake, which is an index for anion exchange capability through Band 3 protein. This parameter was significantly reduced in SSc patients, with or without Interstitial Lung Disease, a complication of SSc. As a reduced Band 3 protein function was observed in SSc patients with either low or high levels of malonildialdehyde (MDA), the end product of lipoperoxidation, anion exchange through erythrocytes membrane may be not univocally related to oxidative stress and, what is more, can be considered as a sensitive tool to monitor SSc development.

We constructed cDNA clones encoding N-terminal, C-terminal and internal polypeptide fragments of the human red cell anion exchanger (band 3; AE1). The internal fragments comprised between one and seven putative transmembrane spans with two or more spans deleted from both termini of the membrane domain of band 3. Sets of three, four or five complementary fragments, which together represented the complete amino acid sequence of the membrane domain, were co-expressed in Xenopus oocytes. Stilbene disulphonate-sensitive chloride uptake assays revealed that all six of the three-fragment combinations and two of the four-fragment combinations reassembled functionally in vivo. Unexpectedly, co-expression of a non-complementary pair of fragments comprising the first five and last seven putative transmembrane spans (i.e. entirely lacking spans six and seven) was also found to be sufficient to generate stilbene disulphonate-sensitive chloride uptake.

Current-voltage curves for DIDS-insensitive Cl 2 conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and dif- ferential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered sus- pensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained in- variant during cell shrinkage, indicating a homogeneous Cl 2 conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K 1 and Cl 2 were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at z 65% above 1 m M DIDS at both 25 8 C and 37 8 C. The nonlinear cur- rent-voltage curves for DIDS-insensitive net Cl 2 effluxes, induced by valinomycin or gramicidin at varied (K 1 ) o , were compared with predictions based on ( 1 ) the theory of electrodiffusion, ( 2 ) a single barrier model, ( 3 ) single occupancy, multiple barrier models, and ( 4 ) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and (K 1 ) o . Under our experimental conditions (pH 7.5, 23 8 C, 1-3 m M valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P K /P Cl is 74 6 9 with 10 m M DIDS, corresponding to 73% inhibition of P Cl . Fitting the constant field current-volt- age equation to the measured Cl 2 currents yields P Cl 5 0.13 h 2 1 with DIDS, compared to 0.49 h 2 1 without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl 2 current, however, is in- consistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well de- scribed either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 6 0.005 S/g Hb, half the channels are open at 2 27 6 2 mV, and the equivalent gating charge is 2 1.2 6 0.3.

While a considerable amount of literature deals with the structural energetics of water-soluble proteins, relatively little is known about the forces that determine the stability of membrane proteins. Similarly, only a few membrane protein structures are known at atomic resolution, although new structures have recently been described. In this article, we review the current knowledge about the structural features of membrane proteins. We then proceed to summarize the existing literature regarding the thermal stability of bacteriorhodopsin, cytochrome-c oxidase, the band 3 protein, Photosystem II and porins. We conclude that a fundamental difference between soluble and membrane proteins is the high thermal stability of intrabilayer secondary structure elements in membrane proteins. This property manifests itself as incomplete unfolding, and is reflected in the observed low enthalpies of denaturation of most membrane proteins. By contrast, the extramembranous parts of membrane proteins may behave much like soluble proteins. A brief general account of thermodynamics factors that contribute to the stability of water soluble and membrane proteins is presented.

Our study concerns the effects of exposure to lead chloride on the morphology, K+ efflux, SO4 − influx and GSH levels of the human erythrocyte. Blood was collected in heparinized tubes and washed three times. The cells were suspended at 3% hematocrit and incubated for 1 h at 25°C in a medium containing increasing concentrations of lead chloride (0, 0.3, 0.5 and 1 μM). After incubation, the suspensions were centrifuged and the erythrocyte pellets were divided into three aliquots for testing. The results show: an increase in the permeability of erythrocytes treated with lead chloride with consequent damage and cellular death, especially in the presence of high concentrations; an increase in potassium ion efflux; alterations in the morphology and membrane structure of the red blood cells; and a decrease in sulphate uptake, due either to the oxidative effect of this compound on the band 3 protein, which loses its biological valence as a carrier of sulphate ions, or to a decrease in the ATP erythrocyte concentration. In conclusion, the exposure of erythrocytes to Pb2+ ions leads to a reduction in the average lifetime of the erythrocytes and the subsequent development of anemia. These data are discussed in terms of the possible effect of lead on the reduction-oxidation systems of the cell. Oxidant agents, such as lead, are known to cross-link integral membrane proteins, leading to K/Cl-cotransport. The increased K+ efflux affects the altered redox state.

The aim of this study was to assess the effect of the different aquatic conditions on anion transport of fish erythrocytes through the measurement of the sulphate uptake operating from band 3, the determination of reduced glutathione (GSH) and oxidized glutathione (GSSG). To this purpose, blood samples of 30 Liza aurata and 30 Salmo irideus fishes were collected via caudal vein and washed with physiological buffer. Successively, erythrocytes suspended at 3% haematocrit were used to measure the SO(4) (=) influx by atomic absorption spectrophotometry at 425 nm wavelength and the GSH concentration using an immunodiagnostic assay intended for the quantitative determination of glutathione in ethylenediaminetetraacetic acid in blood. All results were analyzed using unpaired Student's t-test and a P0.05 was considered statistically significant. The results of this study showed statistically significant differences about the sulphate uptake and the glutathione levels in S. irideus with respect to L. aurata (P0.0001). In conclusion, the different aquatic conditions play an important role on anion transport in fish erythrocytes, showing that environmental differences induce perturbations in erythrocyte membrane and should be evaluated to prevent physiological damages in fishes.

Resveratrol, an important phytoalexine found in many plants, has been shown to be significantly effective in the treatment of several pathological conditions such as cancer, coronary heart disease and osteoarthritis. This study focuses on the effects of this drug on human red blood cells. In particular, we have examined the influence of resveratrol on Band 3, the anion exchanger protein, and hemoglobin as a function of the oxygenation-deoxygenation cycle. Moreover, special attention has been given to the metabolic changes imposed by caspase 3 activation. Resveratrol has proved to lower superoxide production, thereby decreasing heme-iron oxidation and saving the reducing power required for met-hemoglobin reduction. Oxygen binding experiments showed that resveratrol interacts with hemoglobin, shifting the T→R conformational transition towards the higher-affinity R state. This might contribute to altering the metabolic balance of the cell through an intensification of the pentose phosphate pathway. Moreover, at high oxygenation levels of the erythrocytic hemoglobin, resveratrol induces a significant activation of caspase 3, the action of which on Band 3 has a strong impact on cellular metabolism and anion transport.

To further clarify some peculiar molecular mechanisms related to the physiology and pathophysiology of erythrocytes with respect to oxygen binding and release, metabolism and senescence, we investigated the oxidative effects of gemfibrozil in normal and beta-thalassemic red blood cells. Our results showed that the oxidative stress promoted by the drug, through a direct interaction with hemoglobin, may lead to activation of caspase 3, which in turn influences the band 3 anion flux and glucose metabolism. In a comparative context, we also evaluated the effect on band 3 and caspase 3 activation of orthovanadate (a phosphatase inhibitor) and t-butylhydroperoxide (a known oxidant). The results support the hypothesis that gemfibrozil influences band 3 function through several mechanisms of action, centered on oxidative stress, which induces significant alterations of glucose metabolism.

The purpose of this study was to determine the expression and distribution of band 3 in the collecting duct and connecting tubules of the kidney of the marmoset monkey (Callithrix jacchus), and to establish whether band 3 is expressed in type A intercalated cells. The intracellular localization of band 3 in the different populations of intercalated cells was determined by double-labeling immunohistochemistry. Immunohistochemical microscopy demonstrated that band 3 is located in the basolateral plasma membranes of all type A intercalated cells in the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) of the marmoset. However, type B intercalated cells and non-A/non-B intercalated cells did not show band 3 labeling. Electron microscopy of the CNT, CCD and OMCD confirmed the light microscopic observation of the basolateral plasma membrane staining for band 3 in a subpopulation of interacted cells. Basolateral staining was seen on the plasma membrane and small coated vesicles in the perinuclear structure, some of which were located in the Golgi region. In addition, there was no labeling of band 3 in the mitochondria of the CNT, CCD and in OMCD cells. The intensity of the immunostaining of the basolateral membrane was less in the CNT than in the CCD and OMCD. In contrast, band 3 immunoreactivity was greater in the intracellular vesicles of the CNT. From these results, we suggest that the basolateral Cl(-)/HCO(3)(-) exchanger in the monkey kidney is in a more active state in the collecting duct than in the CNT.

In order to utilize virosomes or proteoliposomes for the delivery of drugs or macromolecules to specific pathologic target cells we elaborated a system to shuttle drugs to solid tissue (liver) as well as to the macrophages, a crucial cellular compartment of the immune system. Using virosomes prepared from the P3HR1 strain of Epstein-Barr virus, we demonstrated that these particles fused with human hepatocarcinoma cell line Li7A and therefore might be used as drug vectors. Furthermore, we report that proteoliposomes prepared by reconstituting in a cocktail of phosphatidylserine-phosphatidylcholine the anion transporter band 3 protein markedly increased the phagocytic activity of macrophages in culture. This could represent a new device to be used as a drug delivery system to enhance specific macrophagic functions.

Band 3 protein of the human erythrocyte membrane, the anion transport protein, possesses a high affinity steroid binding site. In mixed phospholipid—cholesterol monolayers, the state of occupancy of this site is positively correlated with their cholesterol and sphingomyelin content and negatively with their glycerophospholipid content. We suggest that, in the erythrocyte membrane, the binding site is an inhibitory site of anion transport and that the modulation of its state of occupancy by the membrane lipid is responsible for the negative correlation of anion transport with the membrane's content of cholesterol and sphingomyelin and the positive correlation with the phosphatidylcholine content

The anion transport protein of the human erythrocyte membrane, band 3, was incorporated into unilamellar sphingomyelin vesicles. The vesicles showed a rapid sulfate efflux which could be inhibited by specific inhibitors of the erythrocyte anion transport system. All band 3 molecules contributing to the inhibitor-sensitive flux component were arranged ‘right-side-out’. The turnover number of the transport protein for sulfate transport was virtually identical to that in phosphatidylcholine bilayers and around 6 times larger than in human erythrocyte membranes. Thus, in contrast to other claims, sphingomyelin does not inhibit the erythrocyte anion transport system.

The anion transport system of the human erythrocyte membrane was reconstituted in unilamellar phosphatidylcholine vesicles, and a vesicle subpopulation of a narrow size distribution was isolated from the sample by gel filtration. In this subpopulation, the turnover number of the transport protein (the band 3 protein) for sulfate transport was determined. It was found that, in the reconstituted system, the protein transports sulfate 5–10-times faster than in the human erythrocyte membrane.

Vesicles have been prepared from 18 : 1c/18 : 1c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, 31P-NMR and K+ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicles both exhibit exchangeable pools larger than the fraction of phosphatidylcholine in the outer monolayer, whereas in the protein-free vesicles the exchangeable pool is consistent with the outer monolayer. The results indicate that both glycophorin and the partially purified band 3 preparation enhance the transbilayer movement of phosphatidylcholine.

The band 3 protein-mediated, valinomycin-induced KCl efflux continues to increase with increasing [KCl] when the Cl−/Cl− equilibrium exchange becomes saturated. This suggests the existence of a band 3-mediated component of Cl− flux that contributes to the electrical conductance without being related to slippage; i.e., equilibration of the unloaded transport protein between the two membrane surfaces.

© Copyright Bulgarian Society of Biomechanics, The degree of erythrocyte deformability (ED) changes as a consequence of alterations on either its membrane properties, or shape or internal composition may provide information about erythrocyte functional disturbances. Several metabolic, cerebral and cardiovascular diseases are characterized by decreased ED that may compromise the blood flow in microcirculation. “In vitro” effects of compounds on ED under different shear stresses may be studied using an ektacytometer apparatus which give us the erythrocyte elongation index (EEI) as a degree of the ED ability. When phosphorylation level of erythrocyte band 3 protein was modified by specific PTK and PTP inhibitors, no ED changes were observed. Cytoskeleton protein phosphorlation levels modifications obtained with PKC inhibitor or activator decreased and increased EEI respectively. When the thiol status of the erythrocyte were preserved with dithiothreitol (DTT), no alterations on EEI were verified. ED values increased for all shear stresses when in presence of the acetylcholine at variance when submitted to the Spermine NONOate that only at high shear stresses an EEI increase was observed. Better understanding of those molecules on erythrocyte signal transduction mechanisms may bring potential targets for drug intervention on ED-dependent blood rheology., This work was supported by Fundação para a Ciência e a Tecnologia grant.

Physico-chemical, metabolic and hormonal theories regarding the pathogenesis of calcium oxalate nephrolithiasis do not sufficiently explain many features of this disease. The recent findings of an abnormally faster oxalate self-exchange and higher phosphorylation of band 3 in erythrocytes of idiopathic calcium oxalate stone formers suggest the hypothesis that nephrolithiasis may be a cellular disease, characterized by a defect in the function of the anion-exchange. The cellular anomaly seems genetically controlled. Band 3 anion exchanger function seems to be biochemically regulated through modulation of band 3 phosphorylation, which depends on cyclic AMP- and phospholipid-sensitive Ca2+ independent protein kinases. In this light, a reduced glycosaminoglycan concentration in the erythrocyte membranes of stone formers might play a role, as these molecules exert a strong inhibitory effects on band 3 phosphorylation and anion transport in vitro and in vivo. An in vivo trial was performed in which stone formers were administered glycosaminoglycans orally. A reduction in oxalate excretion, and oxalate renal clearance, and a simultaneous correction of the abnormal RBC oxalate flux and band 3 phosphorylation were observed. These data suggest the existence of a link between the erythrocyte abnormality and oxalate transport by the kidney and gut.