High Glucose Concentrations Affect Band 3 Protein in Human Erythrocytes

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.

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d-Galactose Decreases Anion Exchange Capability through Band 3 Protein in Human Erythrocytes

Antioxidants ◽

10.3390/antiox9080689 ◽

2020 ◽

Vol 9 (8) ◽

pp. 689

Author(s):

Alessia Remigante ◽

Rossana Morabito ◽

Sara Spinelli ◽

Vincenzo Trichilo ◽

Saverio Loddo ◽

...

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Anion Exchange ◽

Band 3 ◽

Human Erythrocytes ◽

Band 3 Protein ◽

Expression Levels ◽

Vitro Effect ◽

The Impact

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− 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.

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Effect of cadmium on anion exchange capability through Band 3 protein in human erythrocytes

Journal of Biological Research - Bollettino della Società Italiana di Biologia Sperimentale ◽

10.4081/jbr.2018.7203 ◽

2018 ◽

Vol 91 (1) ◽

Cited By ~ 1

Author(s):

Rossana Morabito ◽

Alessia Remigante ◽

Benedetta Arcuri ◽

Angela Marino ◽

Marco Giammanco ◽

...

Keyword(s):

Protein Expression ◽

Anion Exchange ◽

Rate Constant ◽

Band 3 ◽

Human Erythrocytes ◽

Band 3 Protein ◽

Toxic Heavy Metal ◽

Expression Levels ◽

Sh Groups ◽

The Impact

The efficiency of Band 3 protein, mediating HCO3-/Cl- exchange across erythrocytes membrane, is reduced by oxidative stress. The aim of the present study was to verify whether Band 3 protein efficiency is compromised by treatment with Cadmium (Cd2+), an extremely toxic heavy metal known to interfere with antioxidant enzymes, energy metabolism, gene expression and cell membranes. To this end, the rate constant for SO4= uptake through Band 3 protein (accounting for velocity of anion exchange) was measured along with membrane –SH groups, Malonyldialdehyde (MDA) and Band 3 protein expression levels in Cd2+ -treated human erythrocytes (300 µM, 1 mM). Our results show that Cd2+ reduced the rate constant for SO4= uptake, with a significant increase in MDA levels at both concentrations and with a reduction in –SH groups observed after 1 mM Cd2+ treatment, whereas Band 3 protein expression levels were unchanged in both conditions. In conclusion: i) Cd2+ reduces Band 3 protein efficiency via different mechanisms depending on metal concentration and with unchanged expression levels; ii) the assessment of Band 3 protein anion exchange capability is a good tool to assay the impact of heavy metals on cell homeostasis and, possibly, useful for diagnosis and monitoring of devalopment of Cd2+ toxicity-related pathologies.

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Melatonin Protects Band 3 Protein in Human Erythrocytes against H2O2-Induced Oxidative Stress

Molecules ◽

10.3390/molecules24152741 ◽

2019 ◽

Vol 24 (15) ◽

pp. 2741 ◽

Cited By ~ 9

Author(s):

Rossana Morabito ◽

Alessia Remigante ◽

Angela Marino

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Protein Expression ◽

Beneficial Effect ◽

Anion Exchange ◽

Rate Constant ◽

Cell Shape ◽

Band 3 ◽

Band 3 Protein ◽

Expression Levels

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 μM 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 µM 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 µM 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.

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Natural Antioxidants Beneficial Effects on Anion Exchange through Band 3 Protein in Human Erythrocytes

Antioxidants ◽

10.3390/antiox9010025 ◽

2019 ◽

Vol 9 (1) ◽

pp. 25 ◽

Cited By ~ 6

Author(s):

Alessia Remigante ◽

Rossana Morabito ◽

Angela Marino

Keyword(s):

Anion Exchange ◽

Natural Antioxidants ◽

Band 3 ◽

Erythrocyte Membranes ◽

Band 3 Protein ◽

Ion Distribution ◽

Beneficial Effects ◽

Cross Links

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.

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Impact of anion exchange adsorbents on regional citrate anticoagulation

The International Journal of Artificial Organs ◽

10.1177/0391398820947733 ◽

2020 ◽

pp. 039139882094773

Author(s):

Karin Strobl ◽

Stephan Harm ◽

Ute Fichtinger ◽

Claudia Schildböck ◽

Jens Hartmann

Keyword(s):

Anion Exchange ◽

Regional Citrate Anticoagulation ◽

Liver Support ◽

Anion Exchange Resins ◽

Increased Risk ◽

Extracorporeal Liver Support ◽

Target Molecules ◽

Exchange Resins ◽

The Impact

Introduction: Heparin and citrate are commonly used anticoagulants in membrane/adsorption based extracorporeal liver support systems. However, anion exchange resins employed for the removal of negatively charged target molecules including bilirubin may also deplete these anticoagulants due to their negative charge. The aim of this study was to evaluate the adsorption of citrate by anion exchange resins and the impact on extracorporeal Ca2+ concentrations. Methods: Liver support treatments were simulated in vitro. Citrate and Ca2+ concentrations were measured pre and post albumin filter as well as pre and post adsorbents. In addition, batch experiments were performed to quantify citrate adsorption. Results: Pre albumin filter target Ca2+ concentrations were reached well with only minor deviations. Citrate was adsorbed by anion exchange resins, resulting in a higher Ca2+ concentration downstream of the adsorbent cartridges during the first hour of treatment. Conclusions: The anion exchange resin depletes citrate, leading to an increased Ca2+ concentration in the extracorporeal circuit, which may cause an increased risk of clotting during the first hour of treatment. An increase of citrate infusion during the first hour of treatment should therefore be considered to compensate for the adsorption of citrate.

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Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

10.21203/rs.3.rs-330029/v1 ◽

2021 ◽

Author(s):

Jozsef Bodis ◽

Endre Sulyok ◽

Akos Varnagy ◽

Viktória Prémusz ◽

Krisztina Godony ◽

...

Keyword(s):

Gene Expression ◽

In Vitro Fertilization ◽

Mrna Expression ◽

Granulosa Cells ◽

Follicular Fluid ◽

Vitro Fertilization ◽

Expression Levels ◽

The Impact ◽

Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

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Cytosolic protein binding to band-3 protein inhibits endocytosis of isolated human erythrocyte membranes

Biochemical Journal ◽

10.1042/bj2070595 ◽

1982 ◽

Vol 207 (3) ◽

pp. 595-598 ◽

Cited By ~ 1

Author(s):

K A Cordes ◽

J M Salhany

Keyword(s):

Membrane Protein ◽

Integral Membrane Protein ◽

Band 3 ◽

Erythrocyte Membranes ◽

Band 3 Protein ◽

High Affinity ◽

Human Erythrocyte Membranes ◽

Affinity Interaction ◽

Cytoplasmic Side

Recent studies of haemoglobin binding to the cytoplasmic side of the erythrocyte membrane have shown that the predominant high-affinity interaction occurs with the major integral membrane protein known as band-3 protein and that this interaction may occur within the intact erythrocyte in a manner regulated by cell pH. We report here that haemoglobin and glyceraldehyde 3-phosphate dehydrogenase binding to band-3 protein in isolated membranes can inhibit endocytosis during vesiculation in vitro. The specificity of this effect was demonstrated by showing that myoglobin, which has an affinity for the membrane fully one to two orders of magnitude lower than that for haemoglobin, does not inhibit endocytosis.

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Cerebrospinal Fluid Pulsation Stress Promotes the Angiogenesis of Tissue-Engineered Laminae

Stem Cells International ◽

10.1155/2020/8026362 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Linli Li ◽

Yiqun He ◽

Han Tang ◽

Wei Mao ◽

Haofei Ni ◽

...

Keyword(s):

Tissue Engineering ◽

Cerebrospinal Fluid ◽

Engineering Technology ◽

Expression Levels ◽

Fluid Pulsation ◽

Endothelial Growth Factor ◽

The Impact

Background. Angiogenesis is a prerequisite step to achieve the success of bone regeneration by tissue engineering technology. Previous studies have shown the role of cerebrospinal fluid pulsation (CSFP) stress in the reconstruction of tissue-engineered laminae. In this study, we investigated the role of CSFP stress in the angiogenesis of tissue-engineered laminae. Methods. For the in vitro study, a CSFP bioreactor was used to investigate the impact of CSFP stress on the osteogenic mesenchymal stem cells (MSCs). For the in vivo study, forty-eight New Zealand rabbits were randomly divided into the CSFP group and the Non-CSFP group. Tissue-engineered laminae (TEL) was made by hydroxyapatite-collagen I scaffold and osteogenic MSCs and then implanted into the lamina defect in the two groups. The angiogenic and osteogenic abilities of newborn laminae were examined with histological staining, qRT-PCR, and radiological analysis. Results. The in vitro study showed that CSFP stress could promote the vascular endothelial growth factor A (VEGF-A) expression levels of osteogenic MSCs. In the animal study, the expression levels of angiogenic markers in the CSFP group were higher than those in the Non-CSFP group; moreover, in the CSFP group, their expression levels on the dura mater surface, which are closer to the CSFP stress stimulation, were also higher than those on the paraspinal muscle surface. The expression levels of osteogenic markers in the CSFP group were also higher than those in the Non-CSFP group. Conclusion. CSFP stress could promote the angiogenic ability of osteogenic MSCs and thus promote the angiogenesis of tissue-engineered laminae. The pretreatment of osteogenic MSC with a CSFP bioreactor may have important implications for vertebral lamina reconstruction with a tissue engineering technique.

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High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation

Blood ◽

10.1182/blood-2002-03-0767 ◽

2003 ◽

Vol 101 (5) ◽

pp. 1759-1768 ◽

Cited By ~ 110

Author(s):

Bernhard Schiedlmeier ◽

Hannes Klump ◽

Elke Will ◽

Gökhan Arman-Kalcek ◽

Zhixiong Li ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Therapeutic Window ◽

Hematopoietic Stem ◽

Expression Levels ◽

Cd34 Cells ◽

Cord Blood Cd34 ◽

The Impact

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P = .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

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