Influence of Anticholinesterase Drugs on Activity and Properties of Na+,K+-ATPase in Rat Erythrocytes under Stress Caused by Intense Physical Exercise.

We studied the effect of intramuscular injection of physostigmine and neostigmine on Na+,K+-ATPase activity in erythrocytes of rats subjected to intense physical exercise. Both anticholinesterase drugs had a significant effect on the development of the stress response, which was expressed in a decrease in the manifestation of its individual components such as the concentration of ascorbic acid in the adrenal glands, stress-related erythrocyte polycythemia, and LPO indicators. Anticholinesterase drugs reverse the stress-induced decrease in Na+,K+-ATPase activity, as well as changes in its magnesium-dependent properties. There were no changes in the activity of the studied enzyme in the erythrocyte ghosts. We associate the observed differences with the correction of the functions of the cholinergic components of the hypothalamic-pituitary-adrenal axis leading to the development of a hypoergic type stress reaction.

Single-Step Purification of Catalase Enzyme From Human Blood Erythrocytes Using Affinity Chromatography Technique.

In this study, we aimed to isolate and purify catalase from human blood erythrocytes by using a newly synthesized affinity gel. The synthesized ω-amino hexyl agarose-1,2,3-triazole-5-carboxylic acid affinity gel was analyzed by FT-IR. Then, different buffer, pH, and ionic strength parameters were optimized to determine the equilibration, washing, and elution buffer conditions. The catalase was purified from human blood erythrocytes with a specific activity of 45.58 EU/mg, purification fold of 529.50, and a yield of 0.416% using the synthesized new affinity gel. The purity and molecular weight of the enzyme were analyzed by SDS-PAGE, and a single band at 60 kDa was observed for catalase. The optimum reaction temperature of the catalase was found to be 30°C, while the thermal stability temperature was 60°C. The Km and Vmax of the enzyme for hydrogen peroxide were calculated at 0.125 mM and 2500 U mL-1, respectively.

[Proinflammatory activity of monocytes and activity of glutathione-dependent enzymes in red blood cells in women with depressive conditions]. / Provospalitel'naya aktivnost' monotsitov i aktivnost' glutation-zavisimykh fermentov v eritrotsitakh krovi u zhenshchin s depressivnymi sostoyaniyami.

OBJECTIVE: To search for possible connections between the anti-inflammatory activity of monocytes (PAM) and the activity of glutathione metabolic enzymes: glutathione reductase (GR) and glutathione-S-transferase (GT) in patients with depressive states (DS) within various mental pathologies, as well as between the studied biological parameters and clinical condition of patients. MATERIAL AND METHODS: Sixty-one women, aged 18-56 years, with DC were examined before and after treatment. Symptom severity was assessed using the Positive and Negative Syndrome Scale (PANSS) and the Hamilton Depressive Symptom Rating Scale (HDRS-21). The control group included 23 women of the corresponding age without mental pathology. Biological parameters were assessed in the peripheral blood of patients and healthy people. RESULTS: Patients with a high level of PAM compared to the control (p<0.001) (subgroup 1, n=31) and with a low (at the control level) level (subgroup 2, n=30) were identified. In the subgroup 1, the values of GR and GT were significantly lower than in patients of subgroup 2 (p<0.05 and p<0.01, respectively). Negative correlations between the level of PAM before treatment and GR before and after treatment were revealed in patients who responded to treatment (r=-0.67; p=0.0041; r=-0.76; p=0.0001). CONCLUSION: The results may indicate the inverse relationship between the level of PAM and the activity of GR and GT, which are involved in the pathogenesis of DC, and can also serve as criteria for assessing the response of patients to treatment.

Soluble adenylyl cyclase is an acid-base sensor in rainbow trout red blood cells that regulates intracellular pH and haemoglobin-oxygen binding.

AIM: To identify the physiological role of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout. METHODS: We used: (i) super-resolution microscopy to determine the subcellular location of sAC protein; (ii) live-cell imaging of RBC intracellular pH (pHi) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid-base regulation; (iii) spectrophotometric measurements of haemoglobin-oxygen (Hb-O2) binding in steady-state conditions; and (iv) during simulated arterial-venous transit, to determine the role of sAC in systemic O2 transport. RESULTS: Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pHi regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pHi recovery after an acid-base disturbance. RBC pHi recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO3 --dependent sAC signaling. Inhibition of sAC decreased Hb-O2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O2 binding. During in vitro simulations of arterial-venous transit, sAC inhibition decreased the amount of O2 that is unloaded by ~11%. CONCLUSION: sAC represents a novel acid-base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pHi and blood O2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates.

Differences in endothelial function between patients with Type 1 and Type 2 diabetes: effects of red blood cells and arginase.

The mechanisms underlying endothelial dysfunction in Type 1 and Type 2 diabetes (T1DM and T2DM) are unresolved. The red blood cells (RBCs) with increased arginase activity induce endothelial dysfunction in T2DM, but the implications of RBCs and the role of arginase inhibition in T1DM are unexplored. We aimed to investigate the differences in endothelial function in patients with T1DM and T2DM, with focus on RBCs and arginase. Thirteen patients with T1DM and twenty-six patients with T2DM, matched for HbA1c and sex were included. In vivo endothelium-dependent and -independent vasodilation (EDV and EIDV) were assessed by venous occlusion plethysmography before and after administration of an arginase inhibitor. RBCs were co-incubated with rat aortic segments for 18h followed by evaluation of endothelium-dependent (EDR) and -independent relaxation (EIDR) in isolated organ chambers. In vivo EDV, but not EIDV, was significantly impaired in patients with T2DM compared with patients with T1DM. Arginase inhibition resulted in improved EDV only in T2DM. RBCs from patients with T2DM induced impaired EDR but not EIDR in isolated aortic segments, whereas RBCs from patients with T1DM did not affect EDR nor EIDR. The present study demonstrates markedly impaired EDV in patients with T2DM in comparison with T1DM. In addition, it highlights the divergent roles of RBCs and arginase in mediating endothelial dysfunction in T1DM and T2DM. While endothelial dysfunction is mediated via RBCs and arginase in T2DM, these phenomena are not prominent in T1DM thereby indicating distinct differences in underlying mechanisms.

Managing hemolysis in serum neuron-specific enolase measurements - an automated algorithm for routine practice.

Neuron-specific enolase (NSE) derived from neurons and peripheral neuroendocrine cells is a biomarker for neuroendocrine tumors and for prognostication in comatose cardiac arrest survivors. However, as platelets and erythrocytes contain NSE, hemolysis causes falsely elevated NSE. We used native serum and hemolysate derived from the same patients to make serial dilutions, and subsequently measured NSE (mNSE) and hemolytic index (HI) in each dilution. An algorithm suitable for the laboratory information system was developed based on the mNSE, HI and the estimated gradient of hemolytic interference from 30 patients. We estimated the associated uncertainty of the corrected NSE (cNSE) results based on the observed range of the gradient and derived an equation for cNSE for samples with limited hemolysis (i.e. 5 < HI ≤ 30): cNSE = mNSE - HI × (0.34 ± 0.23) µg/L. By semi-quantitatively grading the contribution from limited hemolysis, a texted result noting the hemolysis-associated degree of uncertainty can accompany the cNSE result. The major challenge of hemolysis when using serum NSE as a biomarker can be managed using an automated algorithm for correction of NSE results based on degree of hemolysis. However, laboratorians and clinicians should be aware of the limitations associated with in vivo hemolysis.

Acetylcholinesterase purification from human erythrocytes using magnetic nanoparticles containing procainamide.

This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification.

Study of the antigenic characteristics of red blood cells units and their sickle cell disease recipients and the G6PD activity of transfused red blood cells units.

INTRODUCTION: Transfusion has a central place in the treatment of patients with sickle cell disease (SCD). Matching blood groups of red blood cell (RBC) units with the blood groups of the patient is essential to prevent alloimmunization and delayed hemolytic transfusion reaction. African ancestry donors have the best phenocompatibility with patients of the same origin, however their RBCs may present characteristic that can alter quality of the unit such as glucose-6-phosphate dehydrogenase (G6PD) deficiency. The objective is to analyze transfusion protocol, immunization rate and mismatch situations of SCD recipients and to evaluate the frequency of G6PD deficiency in RBCs units from African ancestry donors. METHODS: Samples of units transfused to SCD patients were analyzed. Transfusion data were collected from institutional databases. The activity of G6PD was measured in the segment of the RBC units. RESULTS: A total of 98 segments of units transfused to 37 SCD recipients in 41 transfusions episodes was collected. Among patients, 35.1% (n = 13) had no antibodies; 10.8% (n = 4) had antibodies against Fya/Fyb, Jka/Jkb, M/N, S/s; 21.6% (n = 8) against RH/K antigens. In all cases, the protocols were in line with the recommendations. G6PD deficiency was observed in 9 units, that were all collected from Afro-Caribbean donors. CONCLUSION: The transfusion protocol is established to prevent immunological reactions due to disparities in blood group antigens between donors and SCD recipients. However, the units of African ancestry donors, which allowed the best compatibility, displayed a high rate of G6PD deficiency. The storage and recovery impact of this deficiency must be evaluated.

An investigation of gill and blood carbonic anhydrase characteristics in three basal actinopterygian species: alligator gar (Atractosteus spatula), white sturgeon (Acipenser transmontanus) and Senegal bichir (Polypterus senegalus).

Many teleosts possess a unique set of respiratory characteristics allowing enhanced oxygen unloading to the tissues during stress. This system comprises three major components: highly pH sensitive haemoglobins (large Bohr and Root effects), rapid red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of membrane-bound plasma-accessible carbonic anhydrase (paCA; absence in the gills). The first two components have received considerable research effort; however, the evolutionary loss of branchial paCA has received little attention. In the current study, we investigated the availability of branchial membrane-bound CA, along with several other CA-related characteristics in species belonging to three basal actinopterygian groups: the Lepisosteiformes, Acipenseriformes and Polypteriformes to assess the earlier hypothesis that Root effect haemoglobins constrain branchial paCA availability. We present the first evidence suggesting branchial membrane-bound CA presence in a basal actinopterygian species: the Senegal bichir (Polypterus senegalus) and show that like the teleosts, white sturgeon (Acipenser transmontanus) and alligator gar (Atractosteus spatula) do not possess branchial membrane-bound CA. We discuss the varying respiratory strategies for these species and propose that branchial paCA may have been lost much earlier than previously thought, likely in relation to the changes in haemoglobin buffer capacity associated with the increasing magnitude of the Bohr effect. The findings described here represent an important advancement in our understanding of the evolution of the unique system of enhanced oxygen unloading thought to be present in most teleosts, a group that encompasses half of all vertebrates.

Red cell adenylate kinase deficiency in China: molecular study of 2 new mutations (413G > A, 223dupA).

BACKGROUND: Adenylate kinase (AK) is a monomolecular enzyme widely found in a variety of organisms. It mainly catalyses the reversible transfer of adenosine nucleotide phosphate groups and plays an important role in maintaining energy metabolism. AK deficiency is a rare genetic disorder that is related to haemolytic anaemia. Chronic haemolytic anaemia associated with AK deficiency is a rare condition, and only 14 unrelated families have been reported thus far. Moreover, only 11 mutations have been identified in the AK1 gene, with only 3 cases of psychomotor impairment. CASE PRESENTATION: The patient was a 3-year-old boy with severe haemolytic anaemia and psychomotor retardation. A molecular study of the patient's AK gene revealed 2 different mutations: a heterozygous missense mutation in exon 6 (c.413G > A) and a heterozygous frameshift mutation in exon 5 (c.223dupA). Molecular modelling analyses indicated that AK gene inactivation resulted in a lack of AK activity. The patient recovered after regular blood transfusion therapy. CONCLUSIONS: AK1 deficiency was diagnosed on the basis of low enzymatic activity and the identification of a mutation in the AK1 gene located on chromosome 9q. Here, we report the first case of moderate red cell AK1 deficiency associated with chronic nonspherocytic haemolytic anaemia (CNSHA) in China. The genetic mutations were confirmed by Sanger sequencing. The variants were classified as pathogenic by bioinformatics tools, such as ACMG/AMP guidelines, Mutation Taster, SIFT, MACP, REVEL and PolyPhen2.2. Based on our evidence and previous literature reports, we speculate that the site of the AK1 gene c.413G > A (p.Arg138His) mutation may be a high-frequency mutation site and the other mutation (c.223dupA) might be related to the neuropathogenicity caused by AK1 deficiency. NGS should be a part of newborn to early childhood screening to diagnose rare and poorly diagnosed genetic diseases as early as possible.