Mechanisms Underlying the Effects of Chloroquine on Red Blood Cells Metabolism.

Chloroquine (CQ) is a 4-aminoquinoline derivative largely employed in the management of malaria. CQ treatment exploits the drug's ability to cross the erythrocyte membrane, inhibiting heme polymerase in malarial trophozoites. Accumulation of CQ prevents the conversion of heme to hemozoin, causing its toxic buildup, thus blocking the survival of Plasmodium parasites. Recently, it has been reported that CQ is able to exert antiviral properties, mainly against HIV and SARS-CoV-2. This renewed interest in CQ treatment has led to the development of new studies which aim to explore its side effects and long-term outcome. Our study focuses on the effects of CQ in non-parasitized red blood cells (RBCs), investigating hemoglobin (Hb) functionality, the anion exchanger 1 (AE1) or band 3 protein, caspase 3 and protein tyrosine phosphatase 1B (PTP-1B) activity, intra and extracellular ATP levels, and the oxidative state of RBCs. Interestingly, CQ influences the functionality of both Hb and AE1, the main RBC proteins, affecting the properties of Hb oxygen affinity by shifting the conformational structure of the molecule towards the R state. The influence of CQ on AE1 flux leads to a rate variation of anion exchange, which begins at a concentration of 2.5 µM and reaches its maximum effect at 20 µM. Moreover, a significant decrease in intra and extracellular ATP levels was observed in RBCs pre-treated with 10 µM CQ vs. erythrocytes under normal conditions. This effect is related to the PTP-1B activity which is reduced in RBCs incubated with CQ. Despite these metabolic alterations to RBCs caused by exposure to CQ, no signs of variations in oxidative state or caspase 3 activation were recorded. Our results highlight the antithetical effects of CQ on the functionality and metabolism of RBCs, and encourage the development of new research to better understand the multiple potentiality of the drug.

Implication of COVID-19 on Erythrocytes Functionality: Red Blood Cell Biochemical Implications and Morpho-Functional Aspects.

Several diseases (such as diabetes, cancer, and neurodegenerative disorders) affect the morpho-functional aspects of red blood cells, sometimes altering their normal metabolism. In this review, the hematological changes are evaluated, with particular focus on the morphology and metabolic aspects of erythrocytes. Changes in the functionality of such cells may, in fact, help provide important information about disease severity and progression. The viral infection causes significant damage to the blood cells that are altered in size, rigidity, and distribution width. Lower levels of hemoglobin and anemia have been reported in several studies, and an alteration in the concentration of antioxidant enzymes has been shown to promote a dangerous state of oxidative stress in red blood cells. Patients with severe COVID-19 showed an increase in hematological changes, indicating a progressive worsening as COVID-19 severity progressed. Therefore, monitored hematological alterations in patients with COVID-19 may play an important role in the management of the disease and prevent the risk of a severe course of the disease. Finally, monitored changes in erythrocytes and blood, in general, may be one of the causes of the condition known as Long COVID.

Diaphragm Involvement in Duchenne Muscular Dystrophy (DMD): An MRI Study.

BACKGROUND: Duchenne muscular dystrophy (DMD) is characterized by progressive weakness and wasting of skeletal, cardiac, and respiratory muscles, with consequent cardiopulmonary failure as the main cause of death. Reliable outcome measures able to demonstrate specific trends over disease progression are essential. PURPOSE: To investigate MRI as a noninvasive imaging modality to assess diaphragm impairment in DMD. In particular, we sought to correlate MRI measurement of diaphragm structure and function with pulmonary function tests and with the abdominal volumes (VAB ) measured by optoelectronic plethysmography, being an index of the action of the diaphragm. STUDY TYPE: Cross-sectional study. POPULATION: Twenty-six DMD patients (17.9 ± 6.2 years) and 12 age-matched controls (17.8 ± 5.9 years). FIELD STRENGTH/SEQUENCE: 3-Point gradient echo Dixon sequence at 3T. ASSESSMENT: Images were acquired in breath-hold at full-expiration (EXP) and full-inspiration (INSP). INSP and EXP lung volumes were segmented and the diaphragm surface was reconstructed as the bottom surface of the left and the right lung. The inspiratory and the expiratory diaphragm surfaces were aligned by a nonrigid iterative closest point algorithm. On MRI we measured: 1) craniocaudal diaphragmatic excursion; 2) diaphragm fatty infiltration. STATISTICAL TESTS: Three-parameter sigmoid regression, one-way analysis of variance (ANOVA), Spearman's correlation. RESULTS: In patients, diaphragm excursion decreased with age (r2 = 0.68, P < 0.0001) and fat fraction increased (r2 = 0.51, P = 0.0002). In healthy subjects, diaphragm excursion and fat fraction had no relationship with age. Diaphragm excursion decreased with decreasing FEV1 %pred (r = 0.78, P < 0.0001) and FVC %pred (r = 0.76, P < 0.0001) and correlated with VAB (r = 0.60, P = 0.0002). Fatty infiltration increased with decreasing FEV1 %pred (r = -0.88, P < 0.0001) and FVC %pred (r = -0.88, P < 0.0001). DATA CONCLUSION: The progressive structural and functional diaphragm impairment is highly related to pulmonary function tests and to VAB . The results suggest that MRI might represent a new and noninvasive tool for the functional and structural assessment of the diaphragm. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 5 J. Magn. Reson. Imaging 2020;51:461-471.

Comparison between men and women of volume regulating hormones and aquaporin-2 excretion following graded central hypovolemia.

Central hypovolemia induced by orthostatic loading causes reno-vascular changes that can lead to orthostatic intolerance. In this study, we investigated volume regulating hormonal responses and reno-vascular changes in male and female subjects as they underwent central hypovolemia, induced by graded lower body negative pressure (LBNP). Aquaporin-2 (AQP2) excretion was measured as a biomarker for the renal system response to vasopressin. 37 young healthy subjects (n = 19 males; n = 18 females) were subjected to graded LBNP until - 40 mmHg LBNP. Under resting conditions, males had significantly higher copeptin (a stable peptide derived from vasopressin) levels compared with females. Adrenocorticotropin (ACTH), adrenomedullin (ADM), vasopressin (AVP) and brain natriuretic peptide (BNP) were not affected by our experimental protocol. Nevertheless, an analysis of ADM and BNP with the data normalized as percentages of the baseline value data showed an increase from baseline to 10 min after recovery in the males in ADM and in the females in BNP. Analysis of BNP and ADM raises the possibility of a preferential adaptive vascular response to central hypovolemia in males as shown by the normalized increase in ADM, whereas females showed a preferential renal response as shown by the normalized increase in BNP. Furthermore, our results suggest that there might be a difference between men and women in the copeptin response to alterations in orthostatic loading, simulated either using LBNP or during posture changes.

Multiparametric quantitative MRI assessment of thigh muscles in limb-girdle muscular dystrophy 2A and 2B.

INTRODUCTION: The aim of this study was to apply quantitative MRI (qMRI) to assess structural modifications in thigh muscles of subjects with limb girdle muscular dystrophy (LGMD) 2A and 2B with long disease duration. METHODS: Eleven LGMD2A, 9 LGMD2B patients and 11 healthy controls underwent a multi-parametric 3T MRI examination of the thigh. The protocol included structural T1-weighted images, DIXON sequences for fat fraction calculation, T2 values quantification and diffusion MRI. Region of interest analysis was performed on 4 different compartments (anterior compartment, posterior compartment, gracilis, sartorius). RESULTS: Patients showed high levels of fat infiltration as measured by DIXON sequences. Sartorius and anterior compartment were more infiltrated in LGMD2B than LGMD2A patients. T2 values were mildly reduced in both disorders. Correlations between clinical scores and qMRI were found. CONCLUSIONS: qMRI measures may help to quantify muscular degeneration, but careful interpretation is needed when fat infiltration is massive. Muscle Nerve 58: 550-558, 2018.

Interleukin-13 increases pendrin abundance to the cell surface in bronchial NCI-H292 cells via Rho/actin signaling.

Interleukin-13 (IL13) is a major player in the development of airway hyperresponsiveness in several respiratory disorders. Emerging data suggest that an increased expression of pendrin in airway epithelia is associated with elevated airway hyperreactivity in asthma. Here, we investigate the effect of IL13 on pendrin localization and function using bronchiolar NCI-H292 cells. The data obtained revealed that IL13 increases the cell surface expression of pendrin. This effect was paralleled by a significant increase in the intracellular pH, possibly via indirect stimulation of NHE. IL13 effect on pendrin localization and intracellular pH was reversed by theophylline, a bronchodilator compound used to treat asthma. IL13 upregulated RhoA activity, a crucial protein controlling actin dynamics, via G-alpha-13. Specifically, IL13 stabilized actin cytoskeleton and promoted co-localization and a direct molecular interaction between pendrin and F-actin in the plasma membrane region. These effects were reversed following exposure of cells to theophylline. Selective inhibition of Rho kinase, a downstream effector of Rho, reduced the IL13-dependent cell surface expression of pendrin. Together, these data indicate that IL13 increases pendrin abundance to the cell surface via Rho/actin signaling, an effect reversed by theophylline.

Negative feedback from CaSR signaling to aquaporin-2 sensitizes vasopressin to extracellular Ca2.

We previously described that high luminal Ca(2+) in the renal collecting duct attenuates short-term vasopressin-induced aquaporin-2 (AQP2) trafficking through activation of the Ca(2+)-sensing receptor (CaSR). Here, we evaluated AQP2 phosphorylation and permeability, in both renal HEK-293 cells and in the dissected inner medullary collecting duct, in response to specific activation of CaSR with NPS-R568. In CaSR-transfected cells, CaSR activation drastically reduced the basal levels of AQP2 phosphorylation at S256 (AQP2-pS256), thus having an opposite effect to vasopressin action. When forskolin stimulation was performed in the presence of NPS-R568, the increase in AQP2-pS256 and in the osmotic water permeability were prevented. In the freshly isolated inner mouse medullar collecting duct, stimulation with forskolin in the presence of NPS-R568 prevented the increase in AQP2-pS256 and osmotic water permeability. Our data demonstrate that the activation of CaSR in the collecting duct prevents the cAMP-dependent increase in AQP2-pS256 and water permeability, counteracting the short-term vasopressin response. By extension, our results suggest the attractive concept that CaSR expressed in distinct nephron segments exerts a negative feedback on hormones acting through cAMP, conferring high sensitivity of hormone to extracellular Ca(2+).

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70.

BACKGROUND/AIMS: AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. METHODS: MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. RESULTS: We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. CONCLUSION: Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation.

A new erythrocyte-based biochemical approach to predict the antiproliferative effects of heterocyclic scaffolds: The case of indolone.

BACKGROUND: The indole core is a key structural feature of many natural products and biomolecules with broad spectrum chemotherapeutic properties. Some of us have recently synthesized a library of biologically promising indolone-based compounds. The present study focuses on the effects of one of them, namely DPIT, on human erythrocytes. METHODS: We have examined the influence of DPIT on band 3 protein, intracellular ATP concentration and transport, caspase 3 activation, metabolic adaptation and membrane stability. RESULTS: Our study elucidates that DPIT, intercalated into the phospholipid bilayer, decreases the anion transport, the intracellular ATP concentration and the cytosolic pH, inducing a direct activation of caspase 3. CONCLUSIONS: Starting from the metabolic similarity between erythrocytes and cancer cells, we investigate how the metabolic derangements and membrane alterations induced by selected heterocycles could be related to the antiproliferative effects. GENERAL SIGNIFICANCE: Our work aims to propose a new model of study to predict the antiproliferative effects of heterocyclic scaffolds, pointing out that only one of the listed conditions would be unfavorable to the life cycle of neoplastic cells.

Molecular interactions of hemoglobin with resveratrol: potential protective antioxidant role and metabolic adaptations of the erythrocyte.

This article reports the role of resveratrol in the erythrocyte as a result of its interaction with hemoglobin and describes the effect of this interaction on the metabolism, the redox state, and the release of ATP. The drug crosses the erythrocyte membrane and binds to hemoglobin, altering its modulation and the release of ATP. Our study correlates the variation of the phosphorylation balance induced by resveratrol with the change in the intracellular concentration of ATP and with the decrease in ATP release from red blood cell and the consequent paracrine alteration on the vascular epithelium. Molecular docking calculations indicate larger specificity of binding for oxy-hemoglobin that correlates well with the stabilization of the R-quaternary structure and with the functional modulation of resveratrol on the protein. Finally, we locate a putative binding site at the central cavity of hemoglobin and characterize its key interacting residues with the drug. Computational results support the assumption that resveratrol may act as a protector agent against oxidative protein damage by interacting with hemoglobin.

Identification of mitochondrial coenzyme a transporters from maize and Arabidopsis.

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.

Brain Alteration Patterns in Children with Duchenne Muscular Dystrophy: A Machine Learning Approach to Magnetic Resonance Imaging.

Background: Duchenne Muscular Dystrophy (DMD) is a genetic disease in which lack of the dystrophin protein causes progressive muscular weakness, cardiomyopathy and respiratory insufficiency. DMD is often associated with other cognitive and behavioral impairments, however the correlation of abnormal dystrophin expression in the central nervous system with brain structure and functioning remains still unclear. Objective: To investigate brain involvement in patients with DMD through a multimodal and multivariate approach accounting for potential comorbidities. Methods: We acquired T1-weighted and Diffusion Tensor Imaging data from 18 patients with DMD and 18 age- and sex-matched controls with similar cognitive and behavioral profiles. Cortical thickness, structure volume, fractional anisotropy and mean diffusivity measures were used in a multivariate analysis performed using a Support Vector Machine classifier accounting for potential comorbidities in patients and controls. Results: the classification experiment significantly discriminates between the two populations (97.2% accuracy) and the forward model weights showed that DMD mostly affects the microstructural integrity of long fiber bundles, in particular in the cerebellar peduncles (bilaterally), in the posterior thalamic radiation (bilaterally), in the fornix and in the medial lemniscus (bilaterally). We also reported a reduced cortical thickness, mainly in the motor cortex, cingulate cortex, hippocampal area and insula. Conclusions: Our study identified a small pattern of alterations in the CNS likely associated with the DMD diagnosis.

Psychosocial resources and psychopathology among persons with neuromuscular disorders during the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic substantially affected the lives of persons with inherited neuromuscular disorders (INMD), causing disruption in clinical and support services. While several studies have investigated mental health, distress and psychosocial resources in the general population during the pandemic, little is known about the experience of persons with INMD. METHODS: This study was aimed to fill this gap by jointly investigating both psychopathological symptoms and psychosocial resources - specifically, resilience and perceived social support - among persons with INMD during the pandemic, taking into account demographic and clinical factors. Between April and December 2020, 59 participants with INMD (aged 15-59, 71.2% M) completed a questionnaire collecting demographic and clinical data, the Multidimensional Scale of Perceived Social Support, the Resilience Scale for Adults, and the Achenbach System of Empirically Based Assessment. RESULTS: Overall, participants showed good levels of resilience and perceived social support. A minority of participants reported clinically relevant psychopathological symptoms, 28.81% for anxiety and depression. Most psychopathological symptoms were negatively correlated with resilience (-0.347 < r < - .420), but not significantly associated with social support. Consistent with previous studies, regression analyses highlighted that participants with Duchenne muscular dystrophy were more prone to report anxious and depressive symptoms (B = 1.748, p = .028, OR = 5.744), and participants with myotonic dystrophy, attention problems (B = 2.339, p = .006, OR = 10.376). Resilience emerged as a potential predictor of lower anxious-depressive symptoms (B=-1.264, p = .012, OR = 0.283). CONCLUSIONS: The findings suggest the importance to investigate psychosocial resources in addition to psychopathology among persons with INMD, and to design interventions supporting resilience as a protective factor for mental health promotion.

Effect of roscovitine on intracellular calcium dynamics: differential enantioselective responses.

Cyclin-dependent kinases (CDKs) inhibitors have emerged as interesting therapeutic candidates. Of these, (S)-roscovitine has been proposed as potential neuroprotective molecule for stroke while (R)-roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis. In addition, (R)-roscovitine has been suggested as potential antihypertensive and anti-inflammatory drug. Dysfunction of intracellular calcium balance is a common denominator of these diseases, and the two roscovitine enantiomers (S and R) are known to modulate calcium voltage channel activity differentially. Here, we provide a detailed description of short- and long-term responses of roscovitine on intracellular calcium handling in renal epithelial cells. Short-term exposure to (S)-roscovitine induced a cytosolic calcium peak, which was abolished after stores depletion with cyclopiazonic acid (CPA). Instead, (R)-roscovitine caused a calcium peak followed by a small calcium plateau. Cytosolic calcium response was prevented after stores depletion. Bafilomycin, a selective vacuolar H(+)-ATPase inhibitor, abolished the small calcium plateau. Long-term exposure to (R)-roscovitine significantly reduced the basal calcium level compared to control and (S)-roscovitine treated cells. However, both enantiomers increased calcium accumulation in the endoplasmic reticulum (ER). Consistently, cells treated with (R)-roscovitine showed a significant increase in SERCA activity, whereas (S)-roscovitine incubation resulted in a reduced PMCA expression. We also found a tonic decreased ability to release calcium from the ER, likely via IP3 signaling, under treatment with (S)- or (R)-roscovitine. Together our data revealed that (S)-roscovitine and (R)-roscovitine exert distinct enantiospecific effects on intracellular calcium signaling in renal epithelial cells. This distinct pharmacological profile can be relevant for roscovitine clinical use.

The human gene SLC25A17 encodes a peroxisomal transporter of coenzyme A, FAD and NAD+.

The essential cofactors CoA, FAD and NAD+ are synthesized outside the peroxisomes and therefore must be transported into the peroxisomal matrix where they are required for important processes. In the present study we have functionally identified and characterized SLC25A17 (solute carrier family 25 member 17), which is the only member of the mitochondrial carrier family that has previously been shown to be localized in the peroxisomal membrane. Recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN and AMP, and to a lesser extent of NAD+, PAP (adenosine 3',5'-diphosphate) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and was inhibited by pyridoxal 5'-phosphate and other mitochondrial carrier inhibitors. It was expressed to various degrees in all of the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD+ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP. The present paper is the first report describing the identification and characterization of a transporter for multiple free cofactors in peroxisomes.

The peroxisomal NAD+ carrier of Arabidopsis thaliana transports coenzyme A and its derivatives.

The peroxisomal protein PXN encoded by the Arabidopsis gene At2g39970 has very recently been found to transport NAD+, NADH, AMP and ADP. In this work we have reinvestigated the substrate specificity and the transport properties of PXN by using a wide range of potential substrates. Heterologous expression in bacteria followed by purification, reconstitution in liposomes, and uptake and efflux experiments revealed that PNX transports coenzyme A (CoA), dephospho-CoA, acetyl-CoA and adenosine 3', 5'-phosphate (PAP), besides NAD+, NADH, AMP and ADP. PXN catalyzed fast counter-exchange of substrates and much slower uniport and was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline and tannic acid. Transport was saturable with a submillimolar affinity for NAD+, CoA and other substrates. The physiological role of PXN is probably to provide the peroxisomes with the essential coenzymes NAD+ and CoA.

Hybrid Polyelectrolyte Nanocomplexes for Non-Viral Gene Delivery with Favorable Efficacy and Safety Profile.

The development of nanovectors for precise gene therapy is increasingly focusing on avoiding uncontrolled inflammation while still being able to effectively act on the target sites. Herein, we explore the use of non-viral hybrid polyelectrolyte nanocomplexes (hPECs) for gene delivery, which display good transfection efficacy coupled with non-inflammatory properties. Monodisperse hPECs were produced through a layer-by-layer self-assembling of biocompatible and biodegradable polymers. The resulting nanocomplexes had an inner core characterized by an EGFP-encoding plasmid DNA (pDNA) complexed with linear polyethyleneimine or protamine (PEI or PRM) stabilized with lecithin and poly(vinyl alcohol) (PVA) and an outer layer consisting of medium-molecular-weight chitosan (CH) combined with tripolyphosphate (TPP). PEI- and PRM-hPECs were able to efficiently protect the genetic cargo from nucleases and to perform a stimuli-responsive release of pDNA overtime, thus guaranteeing optimal transfection efficiency. Importantly, hPECs revealed a highly cytocompatible and a non-inflammatory profile in vitro. These results were further supported by evidence of the weak and unspecific interactions of serum proteins with both hPECs, thus confirming the antifouling properties of their outer shell. Therefore, these hPECs represent promising candidates for the development of effective, safe nanotools for gene delivery.

Palytoxin induces functional changes of anion transport in red blood cells: metabolic impact.

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.