Erythrocyte Shape Research Articles

Эритроцит: мешок с гемоглобином или живая, активная клетка?

The review concerns the oxygen transfer in the body and the role of erythrocytes in this process. The mechanisms of regulation of hemoglobin-oxygen binding are described, as well as the effect of erythrocytes shape on blood rheology.

Sensitivity of medaka (Oryzias latipes) to 4-nonylphenol subacute exposure; erythrocyte alterations and apoptosis

The present study was undertaken to assess the effects of the endocrine-disrupting compound; 4-nonylphenol (4-NP) in medaka (Oryzias latipes). The frequencies of erythrocyte alterations, apoptosis, and micronuclei were used as biological indicators of damage. Medaka were exposed 15 days to 4-NP at three sublethal concentrations (50, 80, and 100 μg/l 4-NP) and results compared with those of a previous study using catfish as an animal model. Exposure of medaka resulted in a dose-dependent increase in the frequency of erythrocyte alterations, apoptosis and micronucleus (MN). Many morphological alterations and nuclear abnormalities were observed, including acanthocytes, lobed nucleus, eccentric nucleus, fragmented nucleus, blebbed nucleus, binuclei, deformed nucleus, notched nucleus, hemolysed cells, crenated cells, teardrop-like cells, and schistocytes. Mortality was recorded after treatment with 80 and 100 μg/l 4-NP, indicating that medaka are more sensitive than catfish to 4-NP exposure. We concluded that, 4-NP causes several malformations in the shape and number of erythrocytes in medaka, indicating its genotoxicity.

Morphometric analysis of erythrocytes from patients with thalassemia using tomographic diffractive microscopy.

Complete blood count is the most common test to detect anemia, but it is unable to obtain the abnormal shape of erythrocytes, which highly correlates with the hematologic function. Tomographic diffractive microscopy (TDM) is an emerging technique capable of quantifying three-dimensional (3-D) refractive index (RI) distributions of erythrocytes without labeling. TDM was used to characterize optical and morphological properties of 172 erythrocytes from healthy volunteers and 419 erythrocytes from thalassemic patients. To efficiently extract and analyze the properties of erythrocytes, we developed an adaptive region-growing method for automatically delineating erythrocytes from 3-D RI maps. The thalassemic erythrocytes not only contained lower hemoglobin content but also showed doughnut shape and significantly lower volume, surface area, effective radius, and average thickness. A multi-indices prediction model achieved perfect accuracy of diagnosing thalassemia using four features, including the optical volume, surface-area-to-volume ratio, sphericity index, and surface area. The results demonstrate the ability of TDM to provide quantitative, hematologic measurements and to assess morphological features of erythrocytes to distinguish healthy and thalassemic erythrocytes.

Erythrocyte characteristics of the progenies of pure and reciprocal crosses of Pangasianodon hypophthalmus (Sauvage, 1878) and Clarias gariepinus (Burchell, 1822)

In this study, erythrocyte characteristic of pure and reciprocal crosses of African catfish Clarias gariepinus (Burchell, 1822) and Asian catfish Pangasianodon hypophthalmus (Sauvage, 1878) were evaluated. Blood smear was made from the progenies of C. gariepinus (CG), P. hypophthalmus (PH), Pangapinus (♀PH × ♂CG) and in two morphotypes (Clarias-like and Panga-like) of Clariothalmus (♀CG × ♂PH) obtained from similar breeding history. Erythrocyte measurement of the cell and nuclear axis was done under the microscope, while many other parameters were calculated. Result obtained suggests a preponderant maternal influence on the erythrocytes shape and size characteristics of the reciprocal crosses. Erythrocytes from progenies with C. gariepinus maternal origin was rounded in shape (not less than 90%) while those with P. hypophthalmus maternal origin had relatively high oval shaped erythrocytes (between 76 and 64%). Also, significant size variations were recorded for the different group. Principal component analysis and biplot of component loading of the trio comparison of hybrid progenies and the pure crosses suggest the possibility of discriminating the direction of hybridization of the reciprocal crosses. However, it was impossible to describe the hybridization status of the progenies assessed in this study. This approach may be a simple and viable alternative to molecular markers which characterizes maternal inheritance in hybrid crosses provided that there is significant shape difference between the fishes. Erythrocyte characterization coupled with morphological and cytogenetic studies is recommended for clearer understanding of the nature of hybrid progenies.

Cell Axis Ratio a Standardized Approach for Normal Erythrocyte Shape Determination in Fishes

Cell Axis Ratio a Standardized Approach for Normal Erythrocyte Shape Determination in Fishes

One-year recombinant growth hormone therapy does not improve hemoglobin state and morphology of erythrocytes in growth hormone deficient children

An increase in growth rates of children suffering from growth hormone deficiency (GHD) subjected to recombinant growth hormone treatment (rGHT) was shown to be accompanied by acceleration of metabolic processes that may stimulate oxygen consumption in various organs and tissues. Therefore, oxygen-transporting properties of RBC should undergo considerable changes during the rGHT. The aim of this study was to examine the effects of rGHT on erythrocyte shape and hemoglobin state in GHD children. The level of oxyhemoglobin (Oxy-Hb) in RBC was analyzed by Raman spectroscopy. The RBC count, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and other parameters were calculated. The blood of eleven treatment-naive prepubertal children with GHD (aged 3–9, median 5.7 years) was examined and compared with control group (aged 5–7; median 6.0 years) at three time points: 0, 3 and 12 months of rGHT. Before rGHT, the MI in GHD children was higher (median 0.48 vs 0.14 p=0.0018) and the RBC count was lower (median 4.20 vs 4.96 1012 cells/L p=0.0022) than in control group. After the treatment, cell count in GHD patients did not differ significantly from the control group, but Oxy-Hb level became higher (median 0.64 vs 0.41 p=0.0075). During rGHT, MCV decreased (median 80.3 vs 83.2μm3 p=0.0231). Morphological and functional characteristics of erythrocytes in GHD children were shown to differ significantly from the healthy control group. A twelve-month rGHT partially improved some of the studied parameters but Oxy-Hb level and echinocyte count remained high.

Morphometric Variations Between Triploid and DiploidClarias gariepinus(Burchell, 1822)

AbstractSeveral scientific methods have been described in the identification of triploid fish. However, many of these methods are not applicable for routine management purposes due to their complexity and cost. In this study, the possibility of using morphological variation as a least cost and less complex method of distinguishing triploid and diploid African catfishClarias gariepinus(Burchell, 1822) was examined. Triploid catfish were produced by cold shock of fertilized eggs in 5°C for 20 mins (at approximately 3 mins after fertilization). The fish were incubated, hatched and raised for 3 months. Ploidy levels of the fish were then ascertained by observing the erythrocyte shape. Triploid erythrocyte was ellipsoidal in shape while diploid was round. Morphological characterization was then carried out on 100 samples each of triploid and diploid African catfish. Although significant differences were observed in many parameters, the principal morphometric difference between triploid and diploid African catfish could not be clearly distinguished. It was therefore concluded that morphological characteristics is not ideal for discriminating triploids and diploids of African catfish. The used of erythrocyte characteristics still remains the cheapest and relatively effective method for triploid and diploid determination in African catfish.

βIII Spectrin Is Necessary for Formation of the Constricted Neck of Dendritic Spines and Regulation of Synaptic Activity in Neurons.

Dendritic spines are postsynaptic structures in neurons often having a mushroom-like shape. Physiological significance and cytoskeletal mechanisms that maintain this shape are poorly understood. The spectrin-based membrane skeleton maintains the biconcave shape of erythrocytes, but whether spectrins also determine the shape of nonerythroid cells is less clear. We show that βIII spectrin in hippocampal and cortical neurons from rodent embryos of both sexes is distributed throughout the somatodendritic compartment but is particularly enriched in the neck and base of dendritic spines and largely absent from spine heads. Electron microscopy revealed that βIII spectrin forms a detergent-resistant cytoskeletal network at these sites. Knockdown of βIII spectrin results in a significant decrease in the density of dendritic spines. Surprisingly, the density of presynaptic terminals is not affected by βIII spectrin knockdown. However, instead of making normal spiny synapses, the presynaptic structures in βIII spectrin-depleted neurons make shaft synapses that exhibit increased amplitudes of miniature EPSCs indicative of excessive postsynaptic excitation. Thus, βIII spectrin is necessary for formation of the constricted shape of the spine neck, which in turn controls communication between the synapse and the parent dendrite to prevent excessive excitation. Notably, mutations of SPTNB2 encoding βIII spectrin are associated with neurodegenerative syndromes, spinocerebellar ataxia Type 5, and spectrin-associated autosomal recessive cerebellar ataxia Type 1, but molecular mechanisms linking βIII spectrin functions to neuronal pathologies remain unresolved. Our data suggest that spinocerebellar ataxia Type 5 and spectrin-associated autosomal recessive cerebellar ataxia Type 1 pathology likely arises from poorly controlled synaptic activity that leads to excitotoxicity and neurodegeneration.SIGNIFICANCE STATEMENT Dendritic spines are small protrusions from neuronal dendrites that make synapses with axons of other neurons in the brain. Dendritic spines usually have a mushroom-like shape, which is essential for brain functions, because aberrant spine morphology is associated with many neuropsychiatric disorders. The bulbous head of a mushroom-shaped spine makes the synapse, whereas the narrow neck transmits the incoming signals to the dendrite and supposedly controls the signal propagation. We show that a cytoskeletal protein βIII spectrin plays a key role for the formation of narrow spine necks. In the absence of βIII spectrin, dendritic spines collapse onto dendrites. As a result, synaptic strength exceeds acceptable levels and damages neurons, explaining pathology of human syndromes caused by βIII spectrin mutations.

The Effect of Endogenous and Synthetic Estrogens on Whole Blood Clot Formation and Erythrocyte Structure.

As erythrocyte and estrogens interact so closely and erythrocytes can indicate the healthiness of an individual, it is essential to investigate the effects of natural estrogens as well as synthetic estrogens on these cells. Whole blood samples were used for thromboelastography (TEG), light microscopy (LM), and scanning electron microscopy (SEM) investigation. Viscoelastic investigation with TEG revealed that estrogens affected the rate of clot formation without any significant effect on the strength or stability of the clot. Axial ratio analysis with LM showed a statistically significant increase in number of erythrocytes with decreased roundness. Morphological analysis with SEM confirmed the change in erythrocyte shape and revealed both ultrastructural membrane changes and erythrocyte interactions. As erythrocyte shape and membrane flexibility correlates to physiological functioning of these cells in circulation, these changes, indicative of possible eryptosis brought on by estrogens, when experienced by individuals with an underlying inflammatory or hematological illness, could impair erythrocyte functioning and even result in obstructions in circulation. In conclusion, we suggest that whole blood analysis with viscoelastic and morphological techniques could be used as assessment of the hematological healthiness of individuals using estrogens.

Mechanical diagnosis of human erythrocytes by ultra-high speed manipulation unraveled critical time window for global cytoskeletal remodeling

Large deformability of erythrocytes in microvasculature is a prerequisite to realize smooth circulation. We develop a novel tool for the three-step “Catch-Load-Launch” manipulation of a human erythrocyte based on an ultra-high speed position control by a microfluidic “robotic pump”. Quantification of the erythrocyte shape recovery as a function of loading time uncovered the critical time window for the transition between fast and slow recoveries. The comparison with erythrocytes under depletion of adenosine triphosphate revealed that the cytoskeletal remodeling over a whole cell occurs in 3 orders of magnitude longer timescale than the local dissociation-reassociation of a single spectrin node. Finally, we modeled septic conditions by incubating erythrocytes with endotoxin, and found that the exposure to endotoxin results in a significant delay in the characteristic transition time for cytoskeletal remodeling. The high speed manipulation of erythrocytes with a robotic pump technique allows for high throughput mechanical diagnosis of blood-related diseases.

Submicrometric Lipid Domains Play Key Roles in Erythrocyte Deformation: From Membrane Bending to Shape Restoration

Although direct evidence for lipid domains in living plasma membranes have been recently provided, their existence is still up for debate as their functions remain unclear. Vital imaging of the smooth erythrocyte plasma membrane allowed us to highlight two distinct populations of submicrometric domains with differential cholesterol and sphingomyelin enrichment. We here report direct evidence for the implication of these two domains in key steps of erythrocyte deformation, combining erythrocyte models exhibiting differential membrane curvature (healthy erythrocytes vs erythrocytes from patients with elliptocytosis and spherocytosis, two erythrocyte membrane fragility diseases), (de)stretching biophysical experiments and the decoration of endogenous cholesterol and sphingomyelin by specific fluorescent toxin fragments. Whereas cholesterol-enriched domains were implicated in the generation/maintenance of high membrane curvature areas needed for membrane bending, sphingomyelin-enriched domains were involved in calcium efflux during erythrocyte shape restoration. Our results indicate that cholesterol- and sphingomyelin-enriched domains play distinct key roles in erythrocyte deformation, as confirmed by the specific domain loss upon erythrocyte aging and erythrocyte deformability impairment upon domain abrogation by pharmacological approaches. Thus, whereas cholesterol- and sphingomyelin-enriched domains were so far highly associated, our findings give direct evidence for differential biophysical properties but also distinct physiopathological roles: (i) membrane bending modulators and/or stabilizers (cholesterol-enriched domains), (ii) platforms for protein sorting and/or activation (e.g. calcium efflux pump; sphingomyelin-enriched domains), and (iii) preferential fragility sites for membrane vesiculation (both domains).

Erythrocyte morphology of the Critically Endangered Geometric Tortoise Psammobates geometricus: effects of season, sex and age

Land transformation reduced the habitat of Psammobates geometricus to small isolated fragments, rendering the species vulnerable to extinction. Field biologists often use erythrocyte metrics to assess wildlife health but need baseline values to distinguish normal variations from disease or chronic stress. Here we characterise erythrocyte types and features of P. geometricus and assess if age, sex and environmental fluctuations influence their frequencies and metrics. We collected blood from males, females and juveniles over four seasons to prepare smears for cytological evaluation. We treated the smears with May–Grünwald–Giemsa stains and used imaging software to measure size, shape and colour intensity of erythrocyte types. The blood of all groups contained immature, mature and senescent erythrocytes, and the appearance of each cell type corresponded to descriptions for other chelonians. Season had a strong effect on the occurrence of erythrocyte types and features with few group differences. The frequency of immature erythrocytes was high in winter and spring, the wet season, reflecting increased erythropoiesis. Mature erythrocytes dominated in summer and autumn, but frequencies of senescent cells, poikilocytosis, cytoplasmic inclusions and cytoplasmic vacuoles increased in autumn, indicating that water and food shortages caused physiological stress. The small size and elongated shape of summer and autumn erythrocytes would facilitate oxygen exchange when high temperatures increase metabolic needs of ectotherms. Lower temperatures and energy demands in the wet season counter the lower oxygen capacity of round, immature cells. Our results provide baseline information on erythrocyte physiology and metrics for health assessments of geometric tortoises.

Age, sex and seasonal variation in the shape and size of erythrocytes of the alpine accentor, Prunella collaris (Passeriformes: Prunellidae)

The aim of our study was to analyse red blood cells from the high-altitude avian species alpine accentor (Prunella collaris). We caught 79 alpine accentors in the High Tatra Mountains (the West Carpathians, Slovakia) from 2001 to 2008. Blood samples were collected from the vena ulnaris cutanea. The cells and nuclei of juveniles tend to be wider and larger while adults, especially males, tend to have elongated (narrow) cells. The largest erythrocytes were found in winter and the smallest in summer, in June. Red blood cells with larger, elongated nuclei but overall smaller in size occurred at the beginning and the end of the breeding season, while larger, rounded cells with smaller nuclei were found in the middle of the breeding season. This kind of reshaping of erythrocytes of alpine accentors in summer is probably a response to hypoxia and elevated summer temperature in high mountains. As regards the length/width ratio, in August and September, erythrocytes were more elongated (both cell and nucleus) than in the other months. This study is probably the first assessment of seasonal effects on size and shape of red blood cells of bird species permanently living and breeding at high altitudes of the Palearctic mountains.

Трансформация эритроцитов на этапах криоконсервирования в криозащитных средах на основе оксиэтилированных производных глицерина со степенью полимеризации n=25 и n=30

This paper presents experimental data on the effect of oxyethylated glycerol derivatives with n=25 and n=30 polymerization degree (OEG n=25 and OEG n=30 ) on shape and diameter of human erythrocytes at the stages of cryopreservation. The shape and diameter of the cells before and after cryopreservation were studied in ‘live drop’ by means of immersion microscopy. It has been shown that the contact with the investigated cryoprotective solutions and following low temperature preservation led to erythrocyte transformation. Character of change in their shape and diameter before and after cryopreservation depended upon the polymerization degree and concentration of cryoprotectant. It was established that 40% OEG n=25 and OEG n=30 solutions at the exposure stage caused cell deformation and agglutination. The largest number of erythrocytes of discoid shape was maintained after cryopreservation in 30% OEG n=25 and OEG n=30 solutions. It was found that after cooling-warming the cells in 30% OEG n=30 solution their diameter increased, while using 30% concentration of OEG n=25 this parameter was within the normal range. Probl Cryobiol Cryomed 2016; 26(4): 349–360

In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models

Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes. According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40–50 μM memantine was required to interact with isolated phosphatidylcholine bilayers.

Ischemic Pain Testing Demonstrates Hypoxia Dependent Early Pain and Hypoxia Independent Persistent Pain Responses in Adults with Sickle Cell Anemia

The hallmark manifestation of homozygous sickle cell disease (SCD) is acute pain. The underlying etiology is unknown. It is believed that hypoxia induces changes in erythrocyte shape and produces microvasculature occlusion by sickled red cells. Vascular occlusion is believed to represent the event inducing tissue hypoxia, reperfusion injury and pain.Studies have only recently characterized pain responses to experimental stimuli in rodents and humans with SCD using quantitative sensory testing (QST). SCD is characterized by thermal and mechanical sensitivity, and there is evidence of central sensitization (heightened sensitivity to pain) from QST, temporal summation and functional MRI studies. However, the effect of hypoxia upon nociception has not been studied despite the need for hypoxia to induce sickle hemoglobin polymerization. The ischemic tourniquet pain test is a QST used to investigate nociception. It was the first QST to demonstrate opioid analgesia. While it is assumed that the resulting pain is due to hypoxia, no studies have demonstrated the degree of hypoxia needed to produce pain.Methods: Thirty adults with SCD and 30 age and sex matched 30 controls underwent the ischemic pain test. A pneumatic cuff was inflated around the upper arm to induce forearm hypoxia, and the time from cuff inflation to first reported pain (pain threshold) and until pain became intolerable (pain tolerance) was measured. Pain was quantified on a 20 point scale. Time to pain threshold and tolerance were the primary end points. Testing was repeated after a washout period in a subset of the initial subjects: 18 SCD patients and 22 controls. Repeat testing proceeded with a 15 minute observation period followed by the ischemic pain test with the time and pain score recorded at threshold and tolerance. Subjects were monitored after cuff deflation during recovery with pain scores obtained at 30 seconds, then every minute for 5 minutes and finally every 3 minutes over a total recovery period of 20 minutes. Skeletal muscle tissue oxygenation at the thenar eminence was measured continuously with a near infrared spectroscopy (NIRS) monitor (OxiplexTS; ISS, Champaign, IL).Results: SCD subjects reached the pain threshold at a mean of 6.44 versus 11.57 minutes in controls (p=0.07). Time to pain tolerance was significantly different (SS 11.29 versus controls 19.59 minutes; p=0.004). Both groups reported identical pain scores. By stepwise linear regression, SCD (p=0.002) and gender (p=0.0008) were associated with time to pain tolerance (r2=0.29), while recent opioid use and pain crisis frequency were not. Because these results demonstrated that SCD adults reach tolerance more rapidly than controls, we sought to determine if this difference was due to tissue hypoxia. Pain scores with repeat testing were not significantly different at threshold or tolerance. However, pain score curves over testing and recovery were significantly different (p=0.003, Fig 1A). SCA had pain above baseline after 11 minutes of recovery (p=0.02) suggesting a persistent pain response compared to controls. Oxyhemoglobin and deoxyhemoglobin from NIRS were not different between groups at the primary endpoints (Fig 1B and 1C). StO2 (% tissue oxygenation, Fig 1D) at both threshold and tolerance was not different between SCD and controls, suggesting that the onset and maximal level of pain during this QST is defined an StO2 thresholds 40% common to sickle cell patients and controls. However, oxyhemoglobin, deoxyhemoglobin and StO2 had already recovered to baseline by the time the pain was evident (Fig 1B, 1C and 1D).Conclusions: Adults with SCD reach hypoxia induced, experimental pain earlier than NVs. Time to maximal pain is associated with SCD and sex. NIRS showed that pain tolerance occurred at a uniform tissue oxygenation threshold indicating hypoxia is the primary determinant for sensing acute pain. However, persistent SCD pain was independent of tissue oxygenation, and could be explained by the presence of peripheral or central sensitization. These data suggest that SCD pain treatment may require improved oxygen delivery to affected tissues and analgesics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The good, the bad and the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa).

Endemic Sicilian pond turtles Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink were examined for the presence of haemogregarine parasites. The presence of haemogregarines, occurring mainly in the microgametocyte stage (13.2 ± 0.12 μm in length and 6.4 ± 0.52 μm in width), was observed in approximately 9% of the sampled E. trinacris. Based on the observed morphology and on the sequencing of nuclear 18S rDNA, we identified the parasite as Haemogregarina stepanowi Danilewsky, 1885. Morphometric study of uninfected and infected red blood cells has shown that H. stepanowi induces different changes in erythrocyte shape depending on the infective stage. The differential count of leukocytes in specimens infected with H. stepanowi showed no significant difference compared with healthy specimens. However, considering the health problems which might be induced by H. stepanowi in the closely related European pond turtle Emys orbicularis (Linneaus), monitoring of the health status of the infected Sicilian populations of E. trinacris is desirable. The restricted distribution of populations of Emys infected with haemogregarines in Sicily is quite puzzling and the possible human-mediated introduction of the parasite in Sicily is briefly discussed.

A hypothesis of target cell formation in sickle cell disease

A fraction of erythrocytes appear as target cells in stained blood smears in sickle cell disease, due to a inheritance of the hemoglobin variant Hb S, polymerizing upon deoxygenation. These cells appear in a three dimension as thin cups. A process of their formation in this disease is proposed based on a band 3-based mechanism of the erythrocyte shape control, able to explain the erythrocyte echinocytosis by glucose depletion. It indicates that their formation is due to a stomatocytogenic slow outward transport of the dibasic form of endogenous Pi with an H+ by band 3, promoted by the decrease of the Donnan ratio, which decreases cell pH and volume, attributed by a decrease of cell KCl concentration by the higher efflux of K+Cl− cotransport and Ca2+ activation of the Gardos channel. Its implications are briefly discussed with respect to target cells per se, target cell formation in other hemoglobinopathies, acquired and inherited disorders of the lipid metabolism and dehydrated hereditary stomatocytosis as well as a stomatocyte presence in a double heterozygote of Hb S and Hb C and of an involvement of the process of target cell formation in acanthocytosis in acquired and inherited disorders.

Relationship between intracellular calcium and morphologic changes in rabbit erythrocytes: Effects of the acylated and unacylated forms of E. coli alpha-hemolysin

Alpha-hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic Escherichia coli strains, whose expression correlates with the severity of the infections produced. HlyA is synthesized as a protoxin, ProHlyA, that becomes matured to the active form in the cytosol by hemolysin-C–directed fatty acylation at the ε-amino residues of Lys 564 and Lys 690, before export from the toxin-producing bacteria. This posttranslational modification is remarkable because the nature of the protein is changed by the lipidic moiety from a benign protein to a frank toxin.In the present work, we demonstrated for the first time that, despite being hemolitically inactive, ProHlyA induced cellular morphologic changes in rabbit erythrocytes. A discocyte-to-echinocyte transformation was triggered by the protoxin in the absence of any accompanying increase in intracellular-Ca2+ levels. In addition, the Ca2+-influx kinetics in HlyA-treated erythrocytes indicated that the active toxin induced an elevation in intraerythrocyte-Ca2+ content in a biphasic manner, with an initial rise in Ca2+ that depended on the association of the protein with the target membrane and a second increment corresponding to an activation of purinergic channels. The first increase was sufficient to trigger a discocyte-echinocyte-spherocyte transition in erythrocyte shape, though the subsequent rise mediated by purinergic signalling was essential for the occurrence of hemolysis. The results presented here provide new insights into the mechanism of action of this toxin.

H2O2-Induced Oxidative Stress Affects SO4= Transport in Human Erythrocytes.

The aim of the present investigation was to verify the effect of H2O2-induced oxidative stress on SO4= uptake through Band 3 protein, responsible for Cl-/HCO3- as well as for cell membrane deformability, due to its cross link with cytoskeletal proteins. The role of cytoplasmic proteins binding to Band 3 protein has been also considered by assaying H2O2 effects on hemoglobin-free resealed ghosts of erythrocytes. Oxidative conditions were induced by 30 min exposure of human erythrocytes to different H2O2 concentrations (10 to 300 μM), with or without GSH (glutathione, 2 mM) or curcumin (10 μM), compounds with proved antioxidant properties. Since SO4= influx through Band 3 protein is slower and better controllable than Cl- or HCO3- exchange, the rate constant for SO4= uptake was measured to prove anion transport efficiency, while MDA (malondialdehyde) levels and –SH groups were estimated to quantify the effect of oxidative stress. H2O2 induced a significant decrease in rate constant for SO4= uptake at both 100 and 300 μM H2O2. This reduction, observed in erythrocytes but not in resealed ghosts and associated to increase in neither MDA levels nor in –SH groups, was impaired by both curcumin and GSH, whereas only curcumin effectively restored H2O2-induced changes in erythrocytes shape. Our results show that: i) 30 min exposure to 300 μM H2O2 reduced SO4= uptake in human erythrocytes; ii) oxidative damage was revealed by the reduction in rate constant for SO4= uptake, but not by MDA or –SH groups levels; iii) the damage was produced via cytoplasmic components which cross link with Band 3 protein; iv) the natural antioxidant curcumin may be useful in protecting erythrocytes from oxidative injury; v) SO4= uptake through Band 3 protein may be reasonably suggested as a tool to monitor erythrocytes function under oxidative conditions possibly deriving from alcohol consumption, use of drugs, radiographic contrast media administration, hyperglicemia or neurodegenerative diseases.