Search Results (8)

Introduction: cigarette smoking is a major source of systemic oxidative stress and a well-established risk factor for cardiovascular disease. Heated tobacco products (HTPs) are increasingly promoted as reduced-risk alternatives, yet their cellular effects remain incompletely understood. Methods: this study compared the oxidative stress-mediated effects of conventional cigarette smoking and HTP use on human erythrocytes. Erythrocytes from healthy non-smokers, conventional smokers, and HTP users were analyzed using biochemical, functional, and cytological approaches to assess redox status, membrane and cytoskeletal organization, anion exchanger 1 (AE1) function, antioxidant response, and redox-sensitive signaling pathways. Results: conventional smokers exhibited higher intracellular reactive oxygen species (ROS) levels, thiol depletion, methemoglobin and hemichrome formation, whereas HTP users showed marked lipid peroxidation despite lower ROS availability. Both groups instead displayed altered expression and distribution of key membrane and cytoskeletal proteins, including glycophorin A, AE1, spectrin, ankyrin, and band 4.1, indicating impaired membrane–cytoskeleton interactions. Functional analyses revealed an accelerated AE1-mediated anion exchange in erythrocytes from conventional smokers, whereas cells from HTP users exhibited a reduced sulfate accumulation, indicating altered transport capacity. In both groups, G6PDH activity was significantly increased, and redox-sensitive signaling pathways involving ERK, AKT, and eNOS were activated, accompanied by sex-dependent alterations in estrogen receptor expression and distribution. Conclusions: collectively, these findings identify erythrocytes as sensitive biomarkers of tobacco-related systemic damage and indicate that smoking-induced erythrocyte dysfunction, including that associated with HTP use, may actively contribute to vascular impairment. This evidence challenges the assumption that heated tobacco products confer a substantially reduced cardiovascular risk compared with conventional cigarettes. Full article

The formation of hemoglobin (Hb) and myoglobin (Mb) supramolecular complexes is examined. These key proteins for oxygen transport and storage undergo conformational transitions, some of which are induced by stress factors, particularly redox-active and toxic substances, e.g., reactive oxygen species (ROS) and reactive carbonyl compounds (RCC). These modifications can lead to partial denaturation, exposure of hydrophobic regions, and loss of stability, promoting self-assembly into high-molecular structures. Reversible associations serve regulatory roles: protein stabilization, transient functional inactivation, and generation of biological signals. Irreversible associations result in the formation of stable aggregates constituting pathological hallmarks of amyloidosis and other proteopathies. Although Hb and Mb fibrillization is not part of their physiological function, under oxidative stress, altered pH, high temperatures, or the presence of post-translational modifications, they can adopt amyloid-like structures characterized by cross-β conformation. Such aggregates exhibit high resistance to proteolysis and accumulate in tissues. Understanding molecular mechanisms behind Hb and Mb aggregation is critical for the diagnosis and timely therapy of amyloid-related diseases. The stability, regular structure, and biocompatibility of Hb and Mb fibrils make them promising for biomedical applications. Functional nanomaterials based on these fibrils are being developed for high-sensitivity biosensors, bioelectronic devices, and nanocarriers for targeted drug delivery. Full article

The calcium aluminate hydrate AFm and AFt phases formed upon hydration of Portland cement have an important role in the stabilization and solidification of hazardous chromate ions in hardened cement. AFm monochromate (Ca₄[Al(OH)₆]₂(CrO₄)·12H₂O), AFm hemichromate (Ca₄[Al(OH)₆]₂(CrO₄)_0.5(OH)·12H₂O) and the chromate-containing AFt phase, Ca₆[Al(OH)₆]₂-(CrO₄)₃·24H₂O, were synthesized and investigated by ¹H, ²⁷Al, and ⁵³Cr MAS NMR spectroscopy. ²⁷Al quadrupolar coupling parameters (C_Q, η_Q) and isotropic chemical shifts (δ_iso) were determined for the three phases, including two distinct Al sites in chromate-AFt, as observed by ²⁷Al MAS and MQMAS NMR. Two dominant peaks are apparent in the ¹H MAS NMR spectra of each of the phases. For the AFm phases, these resonances are assigned to framework hydroxyl groups (1.7–2.0 ppm) and water molecules/hydroxyls (5.0–5.5 ppm) in the interlayer. For chromate-AFt, the peaks are ascribed to framework hydroxyl groups in the [Ca₆Al₂(OH)₁₂]⁶⁺ columns (~1.4 ppm) and water molecules (~4.8 ppm) associated with the Ca ions. ⁵³Cr MAS NMR spectra acquired at 22.3 T for the samples show a narrow resonance for both chromate AFm phases, whereas indications of three distinct Cr resonances are apparent for the chromate AFt. The absence of any second-order quadrupolar effects in the ⁵³Cr NMR spectra strongly suggests that the chromate ions are highly mobile in the anionic sites of the AFm and AFt structures. The NMR data reported in this work are in agreement with the reported crystal structures for the chromate AFm and AFt phases and may be useful for identification and characterization of chromate fixation in cementitious systems, complementing information gained from conventional powder X-ray diffraction studies. Full article

Extracellular vesicles (EVs) released by different cell types play an important role in many physiological and pathophysiological processes. In physiological conditions, red blood cell (RBC)-derived EVs compose 4–8% of all circulating EVs, and oxidative stress (OS) as a consequence of different pathophysiological conditions significantly increases the amount of circulated RBC-derived EVs. However, the mechanisms of EV formation are not yet fully defined. To analyze OS-induced EV formation and RBC transformations, we used flow cytometry to evaluate cell esterase activity, caspase-3 activity, and band 3 clustering. Band 3 clustering was additionally analyzed by confocal microscopy. Two original laser diffraction-based approaches were used for the analysis of cell deformability and band 3 activity. Hemoglobin species were characterized spectrophotometrically. We showed that cell viability in tert-Butyl hydroperoxide-induced OS directly correlated with oxidant concentration to cell count ratio, and that RBC-derived EVs contained hemoglobin oxidized to hemichrome (HbChr). OS induced caspase-3 activation and band 3 clustering in cells and EVs. Importantly, we showed that OS-induced EV formation is independent of calcium. The presented data indicated that during OS, RBCs eliminated HbChr by vesiculation in order to sacrifice the cell itself, thereby prolonging lifespan and delaying the untimely clearance of in all other respects healthy RBCs. Full article

Although artemisinin-based combination therapies (ACTs) treat Plasmodium falciparum malaria effectively throughout most of the world, the recent expansion of ACT-resistant strains in some countries of the Greater Mekong Subregion (GMS) further increased the interest in improving the effectiveness of treatment and counteracting resistance. Recognizing that (1) partially denatured hemoglobin containing reactive iron (hemichromes) is generated in parasitized red blood cells (pRBC) by oxidative stress, (2) redox-active hemichromes have the potential to enhance oxidative stress triggered by the parasite and the activation of artemisinin to its pharmaceutically active form, and (3) Syk kinase inhibitors block the release of membrane microparticles containing hemichromes, we hypothesized that increasing hemichrome content in parasitized erythrocytes through the inhibition of Syk kinase might trigger a virtuous cycle involving the activation of artemisinin, the enhancement of oxidative stress elicited by activated artemisinin, and a further increase in hemichrome production. We demonstrate here that artemisinin indeed augments oxidative stress within parasitized RBCs and that Syk kinase inhibitors further increase iron-dependent oxidative stress, synergizing with artemisinin in killing the parasite. We then demonstrate that Syk kinase inhibitors achieve this oxidative enhancement by preventing parasite-induced release of erythrocyte-derived microparticles containing redox-active hemichromes. We also observe that Syk kinase inhibitors do not promote oxidative toxicity to healthy RBCs as they do not produce appreciable amounts of hemichromes. Since some Syk kinase inhibitors can be taken daily with minimal side effects, we propose that Syk kinase inhibitors could evidently contribute to the potentiation of ACTs. Full article

In red blood cells, hemoglobin iron represents the most plausible candidate to catalyze artemisinin activation but the limited reactivity of iron bound to hemoglobin does not play in favor for its direct involvement. Denatured hemoglobin appears a more likely candidate for artemisinin redox activation because it is expected to contain reactive iron and it has been described to release free heme and/or iron in erythrocyte. The aim of our study is to investigate, using three different methods: fluorescence, electron paramagnetic resonance and liquid chromatography coupled to mass spectrometry, how increasing the level of accessible iron into the red blood cells can enhance the reactive oxygen species (ROS) production derived from artemisinin. The over-increase of iron was achieved using phenylhydrazine, a strong oxidant that causes oxidative stress within erythrocytes, resulting in oxidation of oxyhemoglobin and leading to the formation of methemoglobin, which is subsequently converted into irreversible hemichromes (iron (III) compounds). Our findings confirmed, using the iron III chelator, desferrioxamine, the indirect participation of iron (III) compounds in the activation process of artemisinins. Furthermore, in strong reducing conditions, the activation of artemisinin and the consequent production of ROS was enhanced. In conclusion, we demonstrate, through the measurement of intra-erythrocytic superoxide and hydrogen peroxide production using various methods, that artemisinin activation can be drastically enhanced by pre-oxidation of erythrocytes. Full article

Cellular life is reliant upon rapid and efficient responses to internal and external conditions. The basic molecular events associated with these processes are the structural transitions of the proteins (structural protein allostery) involved. From this view, the human hemoglobin (Hb) molecule (α₂β₂) holds a special position in this field. Hb has two types of αβ interface (i.e., α₁β₁ [and α₂β₂] and α₁β₂ [and α₂β₁]). The latter α₁–β₂ (and α₂–β₁) interface is known to be associated with cooperative O₂ binding, and exhibits principal roles if the molecule goes from its deoxy to oxy quaternary structure. However, the role of the former α₁–β₁ (and α₂–β₂) interface has been unclear for a long time. In this regard, important and intriguing observations have been accumulating. A new role was attributed first as stabilizing the HbO₂ tetramer against acidic autoxidation. That is, the α₁–β₁ (and α₂–β₂) interface produces a conformational constraint in the β chain whereby the distal (E7) histidine (His) residue is tilted slightly away from the bound O₂ so as to prevent proton-catalyzed displacement of O₂^– by a solvent water molecule. The β chains thus acquire pH-dependent delayed autoxidation in the HbO₂ tetramer. The next role was suggested by our studies searching for similar phenomena in normal human erythrocytes under mild heating. Tilting of the distal (E7) His in turn triggered degradation of the Hb molecule to hemichrome, and subsequent clustering of Heinz bodies within the erythrocyte. As Heinz body-containing red cells become trapped in the spleen, it was demonstrated that the α₁–β₁ (and α₂–β₂) interface may exert delicate control of the fate (removal) of its own erythrocyte. Herein we review and summarize the related results and current interpretation of the oxidative behavior of human Hb, emphasizing the correlation between hemichrome emergence and Heinz-body formation, and specifically discuss the new roles assigned to the α₁–β₁ (and α₂–β₂) interface. The α₁–β₁ (and α₂–β₂) interface seems to adequately differentiate between the two types of function (dual roles) from physiological to cellular. Full article

Heinz bodies are intraerythrocytic inclusions of hemichrome formed as a result of hemoglobin (Hb) oxidation. They typically develop in aged red cells. Based on the hypothesis that hemichrome formation is an innate characteristic of physiologically normal Hb molecules, we present an overview of our previous findings regarding the molecular instability of Hb and the formation of hemichrome, as well as recent findings on Heinz body formation within normal human erythrocytes. Human adult Hb (HbO₂ A) prepared from healthy donors showed a tendency to produce hemichrome, even at close to physiological temperature and pH. Recent studies found that the number of Heinz bodies formed in red cells increased with increasing temperature when freshly drawn venous blood from healthy donors was subjected to mild heating above 37 °C. These findings suggest that Hb molecules control the removal of non-functional erythrocytes from the circulation via hemichrome formation and subsequent Heinz body clustering. In this review, we discuss the molecular biosensing mechanisms in the spleen, where hemichrome formation and subsequent Heinz body clustering within erythrocytes play a key role in the removal of aged and damaged red cells from the blood circulation. Full article