Erythrocyte Protein Research Articles

Multifunctional protein 4.1R regulates the asymmetric segregation of Numb during terminal erythroid maturation

The asymmetric cell division of stem or progenitor cells generates daughter cells with distinct fates that balance proliferation and differentiation. Asymmetric segregation of Notch signaling regulatory protein Numb plays a crucial role in cell diversification. However, the molecular mechanism remains unclear. Here, we examined the unequal distribution of Numb in the daughter cells of murine erythroleukemia cells (MELCs) that undergo DMSO-induced erythroid differentiation. In contrast to the cytoplasmic localization of Numb during uninduced cell division, Numb is concentrated at the cell boundary in interphase, near the one-spindle pole in metaphase, and is unequally distributed to one daughter cell in anaphase in induced cells. The inheritance of Numb guides this daughter cell toward erythroid differentiation while the other cell remains a progenitor cell. Mitotic spindle orientation, critical for distribution of cell fate determinants, requires complex communication between the spindle microtubules and the cell cortex mediated by the NuMA-LGN-dynein/dynactin complex. Depletion of each individual member of the complex randomizes the position of Numb relative to the mitotic spindle. Gene replacement confirms that multifunctional erythrocyte protein 4.1R (4.1R) functions as a member of the NuMA-LGN-dynein/dynactin complex and is necessary for regulating spindle orientation, in which interaction between 4.1R and NuMA plays an important role. These results suggest that mispositioning of Numb is the result of spindle misorientation. Finally, disruption of the 4.1R-NuMA-LGN complex increases Notch signaling and decreases the erythroblast population. Together, our results identify a critical role for 4.1R in regulating the asymmetric segregation of Numb to mediate erythropoiesis.

Haematopoeitic potentials of methanolic extract of Tetrapleura Tetraptera pods in male Albino rats

Anaemia constitutes a major challenge in the livestock industry worldwide. This study was carried out to determine the haematopoietic potential of methanol extract of Tetrapleura tetraptera (T. tetraptera) in male albino rats. Twenty five rats weighing between 147 and 166 g were used for the study. Anaemia was induced by daily removal of 2ml of blood per 100g body weight for ten days. The extraction was by cold maceration. Thereafter, the animals were administered with the extract for seven days. The rats were treated with graded doses of the extract, 50mg/kg, 100mg/kg and 200mg/kg for groups A, B and C respectively while groups D (negative control) and E (positive control) received 5 ml/kg of water. Results showed successful induction of anaemia. There was significant (p<0.05) increases in the erythrocytic parameters and plasma protein after administration of the methanoloic extract of T. tetraptera. The concentration dependent increase in the PCV, HBC, RBC, MCH and MCHC in the groups treated with the extract when compared with the untreated groups indicates haematopoietic potentials of T. Tetraptera.

GWAS of Osmotic Hemolysis in 12,352 Healthy Blood Donors Identifies Red Cell Genetic Variants Associated with Steady State Hemolysis in Patients with Sickle Cell Disease

Background: The evolutionary pressure of endemic malaria has produced highly variable genes encoding erythrocyte proteins and enzymes, many of which modulate cellular structure and function. We hypothesized that a high throughput screen of red blood cell susceptibility to osmotic hemolysis, coupled with a GWAS study, would identify rare and common genetic variants associated with cellular structure and function during red blood cell storage and in hemolytic diseases like sickle cell and malaria.Methods: The REDS III RED Blood Cell Omics (RBC-Omics) study enrolled 13,403 successful blood donors at 4 major hubs. Two labs were used for red blood cell phenotyping of leukoreduced RBC unit-derived samples stored for ~42 days under blood bank conditions. Red blood cell osmotic fragility was assayed in 12,352 donors, including 1,483 African American, 1,477 Asian and 960 Hispanic donors. DNA was isolated and genotyped using a custom Affymetrix Axiom TM-Array, which contained approximately 875,000 SNPs enriched for blood and transfusion related polymorphisms.Results: Genome wide association analysis in the complete RBC-omics data set revealed 18 genes/loci to be associated with osmotic hemolysis at a genome wide significance threshold of P<5x10-8. The genes included candidates with clear internal validity such as HBB (specifically rs344 the HbS variant) and promotor regions of HBA2 (thalassemic variants), as well as SNPs in logical candidates, such as Ankyrin, Spectrin, and Aquaporin 1, mutations in which are responsible for spherocytosis and red cell membrane water transport defects, among others. To test the potential relevance of these finding to human disease, we evaluated the top 100 regions in the RBC-Omics studies in the 852 pediatric and adult patients with sickle cell disease enrolled in the PUSH and Walk-PHASST screening studies with genome SNP data, and analyzed the association of the SNPs with the intensity of steady state hemolysis, as measured by a principle component analysis of reticulocyte count, bilirubin, lactate dehydrogenase and aspartate amino transferase. After multiple testing correction, numerous genes were associated with the intensity of hemolysis in both studies, including SNPs in red cell metabolism enzymes genes (HK1, TKTL1 MTOR signaling), ion channels (PIEZO1), as well as regulatory gene (ZNF573) and less obvious genes (FLN, SBF2, GAB3, and TMEM116).Conclusion: An analysis of the genetic variability underlying normal human red cell susceptibility to osmotic stress hemolysis identified new candidate genes that are also associated with the severity of red blood cell hemolysis during the disease stress of human SCD. We hypothesize that many of these candidate SNPs may modulate the severity of red cell hemolysis in other diseases like malaria and potential outcomes after routine red blood cell storage and transfusion. These studies highlight the discovery of a number of new metabolic and ion/water transport variants that are associated with the severity of steady state hemolysis and under disease stress. DisclosuresMast:Novo Nordisk: Research Funding. Gordeuk:Emmaus Life Sciences: Consultancy.

STUDIES OF ALTERATIONS OF THE CELLULAR MEMBRANE BARRIER FUNCTION AT LARYNGEAL CANCER

Introduction. Cellular membrane barrier alterations lead to metabolic and functional disorders. However, in the case of laryngeal cancer (LC) they are insufficiently studied.&#x0D; The aim of the study – to learn the nature of the interaction of erythrocyte membranes with introduced spin probes as an indicator of changes in the barrier function of membranes at LC.&#x0D; Research Methods. Samples of the erythrocyte membranes from 40 patients with LC stages II and III and 20 healthy volunteers were probed by EPR with AdTEMPO test. Microviscosity of erythrocyte membranes was determined by the τeff and the decreasing in RSSI. The content of MWM was identified in the blood plasma and in erythrocyte. The partition coefficient between blood plasma proteins and erythrocyte glycocalyx was calculated. SCEM was evaluated by amount of unabsorbed methylene blue.&#x0D; Results and Discussion. It was established that LC patient’s endogenous intoxication is characterized by excessive accumulation of the total pool of MWM both in blood plasma and glycocalyx of erythrocyte. SCEM was significantly decreased in samples of both LC stages in comparison to control. The most apparent decline in τeff was observed prior to washing of erythrocytes for 5 min after probe insertion. The deceleration after 60 min was observed only in LC stage II. The value of τeff was at control values levels after washing of erythrocytes of LC stage II 5 min after probe insertion and was significantly reduced in stage III LC in comparison to control. RSSI in samples both stage of patients prior to and after washing of erythrocytes was on average 1.5-fold higher than that of control.&#x0D; Conclusions. It was established that the LC patient’s endogenous intoxication is characterized by excessive accumulation of the total pool of MWM both in blood plasma and glycocalyx of erythrocytes, activation of catabolic processes in plasma, redistribution of MWM between the pool of erythrocyte proteins, which corresponds to the second stage of endotoxicosis. The reduction of the SCEM is shown, which is a manifestation of pathological changes in the surface functional activity of erythrocyte membranes. The effectiveness of AdTEMPO for the eva­luation of microviscosity of erythrocyte membranes in patients with LC was confirmed.

D‐Galactose‐induced accelerated aging model on human erythrocytes and role of Band 3 protein

Aging is a physiological process that induces several changes in cells and tissues, resulting in a greater risk of disease and death. Though this process has been extensively studied both in vivo and in vitro, including human erythrocytes which are an appropriate model to study aging, the involved pathways have not been fully elucidated. D-Galactose (D-Gal) has been widely used in vitro to model the characteristics of premature aging. Nevertheless, there is few information regarding its effect on Band 3 protein (B3p) in human erythrocytes. Band 3 protein is an anion exchanger, essential to maintain homeostasis of erythrocytes and, in turn, of the whole organism. In the present study, human erythrocytes were treated with high D-Gal concentrations (25-35-50-100 mM) for 24 hours at 25°C, and the rate constant for SO42-uptake through B3p was determined to investigate anion exchange capability. As oxidative damage is associated to aging, lipid peroxidation, membrane sulfhydryl oxidation (-SH groups) and % MetHb (methemoglobin) levels after D-Gal treatment were determined. Moreover, % A1c levels (glycated hemoglobin) and expression levels of B3p have been also measured. Our results suggest that D-Gal (35-50-100 mM) reduces anion exchange capability via B3p, putatively mediated both by -SH groups oxidation and lipid peroxidation of cellular membranes; conversely, neither B3p expression levels nor % MetHb were modified. However, D-Gal increased % A1c, known to crosslink with B3p, and involved in the alteration of anion exchange capability. The present findings show that erythrocytes are a good model to study the impact of aging on cell homeostasis. The D-Gal-induced aging model provokes changes at the erythrocyte's membrane level, altering thus anion exchange capability through B3p. High D-Gal concentrations induce elevation of oxidative stress levels, and successively increase % A1c in human erythrocytes. Our findings contribute to the understanding of mechanisms underlying in vitro-induced aging and provide a useful basis for developing therapeutic strategies to counteract age-related diseases and their oxidative impact on erythrocytes. In this view, future studies are recommended to assay antioxidant molecules and their beneficial effect on B3p function during aging process.

Plasmodium yoelii Erythrocyte Binding Like Protein Interacts With Basigin, an Erythrocyte Surface Protein.

Erythrocyte recognition and invasion is critical for the intra-erythrocytic development of Plasmodium spp. parasites. The multistep invasion process involves specific interactions between parasite ligands and erythrocyte receptors. Erythrocyte-binding-like (EBL) proteins, type I integral transmembrane proteins released from the merozoite micronemes, are known to play an important role in the initiation and formation of tight junctions between the apical end of the merozoite and the erythrocyte surface. In Plasmodium yoelii EBL (PyEBL), a single amino acid substitution in the putative Duffy binding domain dramatically changes parasite growth rate and virulence. This suggests that PyEBL is important for modulating the virulence of P. yoelii parasites. Based on these observations, we sought to elucidate the receptor of PyEBL that mediates its role as an invasion ligand. Using the eukaryotic wheat germ cell-free system, we systematically developed and screened a library of mouse erythrocyte proteins against native PyEBL using AlphaScreen technology. We report that PyEBL specifically interacts with basigin, an erythrocyte surface protein. We further confirmed that the N-terminal cysteine-rich Duffy binding-like region (EBL region 2), is responsible for the interaction, and that the binding is not affected by the C351Y mutation, which was previously shown to modulate virulence of P. yoelii. The identification of basigin as the putative PyEBL receptor offers new insights into the role of this molecule and provides an important base for in-depth studies towards developing novel interventions against malaria.

The Role of the Histone Methyltransferase PfSET10 in Antigenic Variation by Malaria Parasites: a Cautionary Tale.

The virulence of the malaria parasite Plasmodium falciparum is due in large part to its ability to avoid immune destruction through antigenic variation. This results from changes in expression within the multicopy var gene family that encodes the surface antigen P. falciparum erythrocyte protein one (PfEMP1). Understanding the mechanisms underlying this process has been a high-profile research focus for many years. The histone methyltransferase PfSET10 was previously identified as a key enzyme required both for parasite viability and for regulating var gene expression, thus making it a prominent target for developing antimalarial intervention strategies and the subject of considerable research focus. Here, however, we show that disruption of the gene encoding PfSET10 is not lethal and has no effect on var gene expression, in sharp contrast with previously published reports. The contradictory findings highlight the importance of reevaluating previous conclusions when new technologies become available and suggest the possibility of a previously unappreciated plasticity in epigenetic gene regulation in P. falciparumIMPORTANCE The identification of specific epigenetic regulatory proteins in infectious organisms has become a high-profile research topic and a focus for several drug development initiatives. However, studies that define specific roles for different epigenetic modifiers occasionally report differing results, and we similarly provide evidence regarding the histone methyltransferase PfSET10 that is in stark contrast with previously published results. We believe that the conflicting results, rather than suggesting erroneous conclusions, instead reflect the importance of revisiting previous conclusions using newly developed methodologies, as well as caution in interpreting seemingly contrary results in fields that are known to display considerable plasticity, for example metabolism and epigenetics.

Anemia in patients with Covid-19: pathogenesis and clinical significance.

COVID-19 patients typically present with lower airway disease, although involvement of other organ systems is usually the rule. Hematological manifestations such as thrombocytopenia and reduced lymphocyte and eosinophil numbers are highly prevalent in COVID-19 and have prognostic significance. Few data, however, are available about the prevalence and significance of anemia in COVID-19. In an observational study, we investigated the prevalence, pathogenesis and clinical significance of anemia among 206 patients with COVID-19 at the time of their hospitalization in an Internal Medicine unit. The prevalence of anemia was 61% in COVID-19, compared with 45% in a control group of 71 patients with clinical and laboratory findings suggestive of COVID-19, but nasopharyngeal swab tests negative for SARS-CoV-2 RNA (p = 0.022). Mortality was higher in SARS-CoV-2 positive patients. In COVID-19, females had lower hemoglobin concentration than males and a higher prevalence of moderate/severe anemia (25% versus 13%, p = 0.032). In most cases, anemia was mild and due to inflammation, sometimes associated with iron and/or vitamin deficiencies. Determinants of hemoglobin concentration included: erythrocyte sedimentation rate, serum cholinesterase, ferritin and protein concentrations and number of chronic diseases affecting each patient. Hemoglobin concentration was not related to overall survival that was, on the contrary, influenced by red blood cell distribution width, age, lactate dehydrogenase and the ratio of arterial partial oxygen pressure to inspired oxygen fraction. In conclusion, our results highlight anemia as a common manifestation in COVID-19. Although anemia does not directly influence mortality, it usually affects elderly, frail patients and can negatively influence their quality of life.

Structural and Functional State of Erythrocyte Membranes at Drug Addiction

The steady growth in the number of drug addicts, especially among young people, dictates the need to find ways to prevent and treat this disease. In this regard, there is a need for a more detailed study of the mechanisms of the course of this disease using modern research methods, such as atomic force microscopy and fluorescence analysis of amino acid residues. Purpose of the work: to reveal the structural and functional state of erythrocyte membranes in drug addiction. Materials and methods. The studies were carried out on the erythrocyte membranes of 60 subjects suffering from heroin addiction. The shape and topography of the erythrocyte surface were studied, and spectral analysis of the proteins of the erythrocyte membranes was carried out. Results. The conducted AFM studies of erythrocyte membranes indicate the heterogeneity of the surface mechanical properties of the erythrocyte membranes of drug addicts. The data obtained indicate an acceleration of the aging process of erythrocytes in drug addiction, which goes in two ways: the formation of outgrowths on the plasmolemma, which subsequently die off (echinocytes) and invagination of the plasmolemma of erythrocytes (spherocytes). The fluorescence spectrum of amino acids in erythrocytes of drug addicts is characterized by a significant decrease in the intensity of almost all peaks and a shift of the fluorescence peak to the short-wave region. Findings. With drug addiction, changes in the structural integrity of red blood cells are noted. In people with drug addiction, in comparison with healthy people, there is a higher variability of the morphology of erythrocytes, which is expressed in a significant increase in the proportion of echinocytes and spherocytes against the background of a significant decrease in the number of discocytes. For the membrane proteins of erythrocytes of drug addicts, conformational changes are characteristic, manifested in a decrease in the intensity of fluorescence of aromatic amino acids, which indicates their structural modification and significant vulnerability of the hematopoietic system. They are largely determined by changes in the fluorescence intensity of tryptophan and, to a lesser extent, tyrosine, which indicates the preservation of the three-dimensional structure of the protein.

Characterization of the erythrocyte GTPase Rac1 in relation to Plasmodium falciparum invasion

Malaria is still a devastating disease with 228 million cases globally and 405,000 lethal outcomes in 2018, mainly in children under five years of age. The threat of emerging malaria strains resistant to currently available drugs has made the search for novel drug targets compelling. The process by which Plasmodium falciparum parasites invade the host cell has been widely studied, but only a few erythrocyte proteins involved in this process have been identified so far. The erythrocyte protein Rac1 is a GTPase that plays an important role in host cell invasion by many intracellular pathogens. Here we show that Rac1 is recruited in proximity to the site of parasite entry during P. falciparum invasion process and that subsequently localizes to the parasitophorous vacuole membrane. We also suggest that this GTPase may be involved in erythrocyte invasion by P. falciparum, by testing the effect of specific Rac1 inhibitory compounds. Finally, we suggest a secondary role of the erythrocyte GTPase also in parasite intracellular development. We here characterize a new erythrocyte protein potentially involved in P. falciparum invasion of the host cell and propose the human GTPase Rac1 as a novel and promising antimalarial drug target.

Biological Effects of Scattered Versus Scanned Proton Beams on Normal Tissues in Total Body Irradiated Mice: Survival, Genotoxicity, Oxidative Stress and Inflammation.

Side effects of proton therapy are poorly studied. Moreover, the differences in the method of dose delivery on normal tissues are not taken into account when proton beams are scanned instead of being scattered. We proposed here to study the effects of both modalities of proton beam delivery on blood; skin; lung and heart in a murine model. In that purpose; C57BL/6 mice were total body irradiated by 190.6 MeV proton beams either by Double Scattering (DS) or by Pencil Beam Scanning (PBS) in the plateau phase before the Bragg Peak. Mouse survival was evaluated. Blood and organs were removed three months after irradiation. Biomarkers of genotoxicity; oxidative stress and inflammation were measured. Proton irradiation was shown to increase lymphocyte micronucleus frequency; lung superoxide dismutase activity; erythrocyte and skin glutathione peroxidase activity; erythrocyte catalase activity; lung; heart and skin oxidized glutathione level; erythrocyte and lung lipid peroxidation and erythrocyte protein carbonylation even 3 months post-irradiation. When comparing both methods of proton beam delivery; mouse survival was not different. However, PBS significantly increased lymphocyte micronucleus frequency; erythrocyte glutathione peroxidase activity and heart oxidized glutathione level compared to DS. These results point out the necessity to take into account the way of delivering dose in PT as it could influence late side effects.

Underlying mechanisms of oxygen uptake kinetics in chronic post-stroke individuals: A correlational, cross-sectional pilot study.

Post-stroke individuals presented deleterious changes in skeletal muscle and in the cardiovascular system, which are related to reduced oxygen uptake ([Formula: see text]) and take longer to produce energy from oxygen-dependent sources at the onset of exercise (mean response time, MTRON) and during post-exercise recovery (MRTOFF). However, to the best of our knowledge, no previous study has investigated the potential mechanisms related to [Formula: see text] kinetics response (MRTON and MRTOFF) in post-stroke populations. The main objective of this study was to determine whether the MTRON and MRTOFF are related to: 1) body composition; 2) arterial compliance; 3) endothelial function; and 4) hematological and inflammatory profiles in chronic post-stroke individuals. Data on oxygen uptake ([Formula: see text]) were collected using a portable metabolic system (Oxycon Mobile®) during the six-minute walk test (6MWT). The time to achieve 63% of [Formula: see text] during a steady state (MTRON) and recovery (MRTOFF) were analyzed by the monoexponential model and corrected by a work rate (wMRTON and wMRTOFF) during 6MWT. Correlation analyses were made using Spearman's rank correlation coefficient (rs) and the bias-corrected and accelerated bootstrap method was used to estimate the 95% confidence intervals. Twenty-four post-stroke participants who were physically inactive took part in the study. The wMRTOFF was correlated with the following: skeletal muscle mass (rs = -0.46), skeletal muscle mass index (rs = -0.45), augmentation index (rs = 0.44), augmentation index normalized to a heart rate of 75 bpm (rs = 0.64), reflection magnitude (rs = 0.43), erythrocyte (rs = -0.61), hemoglobin (rs = -0.54), hematocrit (rs = -0.52) and high-sensitivity C-reactive protein (rs = 0.58), all p < 0.05. A greater amount of oxygen uptake during post-walking recovery is partially related to lower skeletal muscle mass, greater arterial stiffness, reduced number of erythrocytes and higher systemic inflammation in post-stroke individuals.

Molecular patterns of oxidative stress in drug-induced nephropathy

Introduction: Drug induced kidney disorder is a frequent adverse event which contributes to morbidity and even incapacitation. The discovery and development of novel biomarkers and local (renal) response mechanisms, are needed for effective prevention of drug-induced nephrotoxicity. Objectives: The main purpose of our study was to investigate the oxidative modifications of proteins in blood plasma and erythrocytes of patients with drug-induced nephropathies. Patients and Methods: Around105 patients were divided into two groups: first group was represented by patients with psychotropic drug-induced nephropathy; the second one consisted of patients received nonsteroidal anti-inflammatory drugs (NSAIDs). Advanced oxidation protein products (AOPPs) were measured by Witko-Sarsat method. Protein reactive carbonyl derivatives (PRCD) were assayed in blood plasma and erythrocytes by the Levine method. Neutrophil gelatinaseassociated lipocalin (NGAL) was determined with the use of a commercially available ELISA kit. Results: Carbonyl derivatives are significantly higher in red blood cells of the 1st and 2nd group patients compared to the control subjects. AOPP statistically increased both in patients with various types of drug nephropathy and in patients with chronic kidney disease (CKD) compared with the control group. The NGAL was significantly higher in all groups compared to the control subjects. Conclusion: The patients with drug-induced nephropathy have increased level of oxidative stress products and response NGAL reaction. The mechanisms that lead to the development of oxidative stress and the production of modified proteins are different in patients treated with different drugs. Establishing patterns of cell-molecular interaction permit the drug-induced nephropathy to be timely diagnosed and therapeutic programs to be optimized.

Host Porphobilinogen Deaminase Deficiency Confers Malaria Resistance in Plasmodium chabaudi but Not in Plasmodium berghei or Plasmodium falciparum During Intraerythrocytic Growth.

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel non-sense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited ~50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was ~10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi, which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore, affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd-deficient mice infected with P. berghei, which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20–50%) reduced levels of PBGD. Furthermore, the growth of Pbgd-null P. falciparum and Pbgd-null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd-deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum, but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection.

Hematological And Biochemical Blood Changes In Chronic Tendinitis Thoroughbred Race Horses

Tendon injury is the most important veterinary reason for wastage of thoroughbred racehorses. Clinical diagnosis of tendon injuries is confirmed by a combination of clinical, ultrasonographic or post mortem examination of the injured limb. In addition, measurement of hematological and biochemical blood parameters are an important tool that aid health assessment and decision-making in diagnosis, treatment and follow-up of the injured tendon. Therefore, the lack of information or misinterpretation of these parameters may affect the accuracy of disease diagnosis and then lead to poor treatment. The present study was conducted to evaluate the levels of some hematological and biochemical parameter in the blood of thoroughbred horses affected by chronic tendinitis and compared with normal horses. Blood samples were collected from 15 healthy thoroughbred horses (8 stallions and 7 mares) and 21 tendinitis thoroughbred horses (11 stallions and 10 mares); and the levels of 18 blood parameters were determined. The tendinitis horses had higher number of erythrocytes and thrombocytes, higher values of packed cell volume (PCV) and mean corpuscular volume (MCV); lower enzyme activity of creatine kinase (CK), lower values of lactic acid (LA), icteric index and mean corpuscular hemoglobin concentration (MCHC) and lower numbers of band neutrophils than the normal horses. The chronic tendinitis mares had higher number of thrombocytes and lower values of lactate dehydrogenase (LDH) enzyme activity, lactic acid, plasma proteins, MCHC and lower numbers of white blood cells (WBC) than the normal mares. The chronic tendinitis stallions had higher levels of lactic acid, plasma proteins, MCV, and higher numbers of erythrocytes and thrombocytes,; and lower values of icteric jaundice, MCHC, band neutrophils than the normal horses. No significant differences were reported when tendinitis mares were compared with tendinitis males. However, normal mares showed higher levels of plasma proteins than normal stallions. The results obtained by this study can be used as useful index to diagnose and treat chronic tendinitis in horses.

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

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.

Novel Insights into Mercury Effects on Hemoglobin and Membrane Proteins in Human Erythrocytes.

Mercury (Hg) is a global environmental pollutant that affects human and ecosystem health. With the aim of exploring the Hg-induced protein modifications, intact human erythrocytes were exposed to HgCl2 (1–60 µM) and cytosolic and membrane proteins were analyzed by SDS-PAGE and AU-PAGE. A spectrofluorimetric assay for quantification of Reactive Oxygen Species (ROS) generation was also performed. Hg2+ exposure induces alterations in the electrophoretic profile of cytosolic proteins with a significant decrease in the intensity of the hemoglobin monomer, associated with the appearance of a 64 kDa band, identified as a mercurized tetrameric form. This protein decreases with increasing HgCl2 concentrations and Hg-induced ROS formation. Moreover, it appears resistant to urea denaturation and it is only partially dissociated by exposure to dithiothreitol, likely due to additional protein–Hg interactions involved in aggregate formation. In addition, specific membrane proteins, including band 3 and cytoskeletal proteins 4.1 and 4.2, are affected by Hg2+-treatment. The findings reported provide new insights into the Hg-induced possible detrimental effects on erythrocyte physiology, mainly related to alterations in the oxygen binding capacity of hemoglobin as well as decreases in band 3-mediated anion exchange. Finally, modifications of cytoskeletal proteins 4.1 and 4.2 could contribute to the previously reported alteration in cell morphology.

Epb41l5 interacts with Iqcb1 and regulates ciliary function in zebrafish embryos.

Erythrocyte protein band 4.1 like 5 (EPB41L5) is an adaptor protein beneath the plasma membrane that functions to control epithelial morphogenesis. Here we report a previously uncharacterized role of EPB41L5 in controlling ciliary function. We found that EPB41L5 forms a complex with IQCB1 (previously known as NPHP5), a ciliopathy protein. Overexpression of EPB41L5 reduced IQCB1 localization at the ciliary base in cultured mammalian epithelial cells. Conversely, epb41l5 knockdown increased IQCB1 localization at the ciliary base. epb41l5-deficient zebrafish embryos or embryos expressing C-terminally modified forms of Epb41l5 developed cilia with reduced motility and exhibited left-right patterning defects, an outcome of abnormal ciliary function. We observed genetic synergy between epb41l5 and iqcb1. Moreover, EPB41L5 decreased IQCB1 interaction with CEP290, another ciliopathy protein and a component of the ciliary base and centrosome. Together, these observations suggest that EPB41L5 regulates the composition of the ciliary base and centrosome through IQCB1 and CEP290.

Evaluation of the Effect of Selected Brominated Flame Retardants on Human Serum Albumin and Human Erythrocyte Membrane Proteins.

Brominated flame retardants (BFRs) have been using to reduce the flammability of plastics contained in many products, such as household articles, furniture, mattresses, textiles or insulation. Considering the fact that these compounds may be released into the environment leading to the exposure of living organisms, it is necessary to study their possible effects and mechanisms of action. Proteins play a crucial role in all biological processes. For this reason, a simple model of human serum albumin (HSA) was chosen to study the mechanism of BFRs’ effect on proteins. The study determined interactions between selected BFRs, i.e., tetrabromobisphenol A (TBBPA), tetrabromobisphenol S (TBBPS), 2,4-dibromophenol (2,4-DBP), 2,4,6-tribromophenol (2,4,6-TBP) and pentabromophenol (PBP), and HSA by measurement of fluorescence of intrinsic tryptophan and absorbance of circular dichroism (CD). In addition, in order to understand the possible effect of these compounds in their native environment, the effect of BFRs on membrane proteins of human erythrocytes (red blood cells, RBCs) was also assessed. Among bromophenols, PBP had the strongest oxidative effect on RBC membrane, and 2,4-DBP demonstrated the weakest fluorescence-quenching effect of both membrane tryptophan and HSA. By contrast to PBP, 2,4-DBP and 2,4,6-TBP caused spatial changes of HSA. We have observed that among all analyzed BFRs, TBBPA caused the strongest oxidation of RBC membrane proteins and the model HSA protein, causing reduction of fluorescence of tryptophan contained in them. TBBPA also changed albumin conformation properties, leading to impairment of the α-helix structure. However, TBBPS had the weakest oxidative effect on proteins among studied BFRs and did not affect the secondary structure of HSA.

Proteomic analysis reveals pathogen-derived biomarkers of acute babesiosis in erythrocytes, plasma, and urine of infected hamsters

Babesiosis among humans is on the rise in North America. Current diagnostic assays for the screening of babesiosis require blood collection by venipuncture, which is an invasive method. Urine on the other hand is a desirable biospecimen for biomarker analysis of Babesia microti infections because it can be collected periodically and non-invasively. Our group uses a new class of biomarker harvesting nanocage technology, which, when combined with mass spectrometry (MS), can determine the presence of parasite proteins shed in different bodily fluids of mammalian hosts, including urine. Using the hamster model of babesiosis, our nanoparticle-MS approach identified several B. microti proteins in erythrocytes, plasma, and urine samples. Surface and secreted antigens previously shown to elicit host immune responses against the parasite were particularly abundant in erythrocytes and plasma compared to other proteins. Two of these antigens, BmSA1 and BMR1_03g00947, showed different localization patterns by immunofluorescence of infected erythrocytes. Hamster urine samples from parasitemic animals harbored lower numbers of B. microti proteins compared to erythrocytes and plasma, with glycolytic enzymes, cytoskeletal components, and chaperones being the most frequently detected proteins. By applying novel nanoparticle-MS methods, a high level of analytical sensitivity can be achieved to detect multiple B. microti proteins in blood and urine. This is generally difficult to obtain with other techniques due to the masking of parasite biomarkers by the complex biomolecular matrix of bodily fluids from the host.