Ecological aspects of bivalve adaptation to salinity fluctuations on the example of Anadara Kagoshimensis

Impact of salinity stress on the ark clam (Anadara kagoshimensis (Tokunaga, 1906)) hemocyte functions were investigated using flow cytometry and light scattering technique. In control group water salinity was 18 ppm and experimental groups were carried at 14 ppm, 8 ppm, 35 ppm and 45 ppm. Hemolymph osmolarity decreased at hypoosmotic conditions and increased after hyperosmotic treatment. Osmotic stress induced changes in osmotic fragility of the ark clam hemocytes. Salinity 14 ppm did not affect the functional parameters of hemocytes. Incubation of ark clams at salinity and 35 ppm did not influence on the mitochondrial membrane potential of hemocytes but led to a decrease in hemocyte reactive oxygen species (ROS) production by 30 % compared to control. An increase in water salinity to 45 ppm and its decrease to 8 ppm induced substantial changes in the ROS production and mitochondrial membrane potential of hemocytes. Hyposalinity (8 ppm) led to an increase in ROS production by hemocytes (up to 2.4 times) and mitochondrial membrane potential (up to 1.3 times). An increase of salinity level from 18 ppm to 45 ppm decreased the total ability of hemocytes to produce ROS by 11% and increased mitochondrial potential of hemocytes by 150%.

Different Involvement of Band 3 in Red Cell Deformability and Osmotic Fragility—A Comparative GP.Mur Erythrocyte Study

GP.Mur is a clinically important red blood cell (RBC) phenotype in Southeast Asia. The molecular entity of GP.Mur is glycophorin B-A-B hybrid protein that promotes band 3 expression and band 3–AQP1 interaction, and alters the organization of band 3 complexes with Rh/RhAG complexes. GP.Mur+ RBCs are more resistant to osmotic stress. To explore whether GP.Mur+ RBCs could be structurally more resilient, we compared deformability and osmotic fragility of fresh RBCs from 145 adults without major illness (47% GP.Mur). We also evaluated potential impacts of cellular and lipid factors on RBC deformability and osmotic resistivity. Contrary to our anticipation, these two physical properties were independent from each other based on multivariate regression analyses. GP.Mur+ RBCs were less deformable than non-GP.Mur RBCs. We also unexpectedly found 25% microcytosis in GP.Mur+ female subjects (10/40). Both microcytosis and membrane cholesterol reduced deformability, but the latter was only observed in non-GP.Mur and not GP.Mur+ normocytes. The osmotic fragility of erythrocytes was not affected by microcytosis; instead, larger mean corpuscular volume (MCV) increased the chances of hypotonic burst. From comparison with GP.Mur+ RBCs, higher band 3 expression strengthened the structure of RBC membrane and submembranous cytoskeletal networks and thereby reduced cell deformability; stronger band 3–AQP1 interaction additionally supported osmotic resistance. Thus, red cell deformability and osmotic resistivity involve distinct structural–functional roles of band 3.

Profile of blood of weaner boars fed Tetrapleura tetraptera pod pulp meal

A study was conducted to determine the effect of Tetrapleura tetraptera pod pulp meal on haematology, red blood cell osmotic fragility and serum chemistry values of weaned boars. A total of 18 weaner boars of Large White x Duroc crossbreeds, aged 6 weeks and weighing 9.4kg on the average, were used for the study, which lasted 56 days. The piglets were divided into 3 equal groups, each of which were assigned 0.0%, 2.5% or 5.0% TPM per kg, respectively, of a basal weaner pig diet, in a completely randomized design. Data sets were collected on haematology, serum chemistry and lipid profile. Red blood cells (6.74, 6.03,12 5.57) x 10 /l, packed cell volume (46.33, 39.33, 37.35) %, haemoglobin concentration 9 (13.77, 11.43, 10.37) g/L and the platelet counts (37.90, 33.97, 33.83) x 10 /L were significantly lower (P<0.05) in the sera of boars fed TPM than in those fed zero TPM. Mean cell haemoglobin and mean cell haemoglobin concentration were significantly (P<0.05) lowered by addition of TPM in a dose-dependent fashion. Addition of TPM increased the fragility of the erythrocytes in sodium chloride solution of varying concentrations. Serum glucose increased progressively (P<0.05) with increase in TPM. Albumin was significantly reduced only at 5.0% TPM while globulin and urea were reduced (P<0.05) at both levels of TPM. Total protein and ALT were significantly reduced with increase in TPM. HDL was progressively lowered (P<0.05) with addition of TPM while the reverse was the case with LDL. VLDL and TG were significantly increased only at 5.0% TPM. It is concluded that TPM depressed the haematological indices, compromised the stability of the erythrocytes even in normal saline solution, reduced serum albumin, globulin, total protein, ALT and HDL but increased serum glucose, LDL, VLDLand TG. Cet étude a été menée pour déterminer l'effet de la farine de pulpe de gousse de aTetrapleuratetraptera sur l'hématologie, la fragilité osmotique des globules rouges et lesvaleurs de chimie sérique des verrats sevrés. Au total, 18 verrats sevrés issus de croisements Large White x Duroc, âgés de 6 semaines et pesant en moyenne 9,4 kg, ont été utilisés pour l'étude, qui a duré 56 jours. Les porcelets ont été divisés en 3 groupes égaux, chacun ayant reçu 0,0%, 2,5% ou 5,0% de TPM par kg, respectivement, d'un régime de base pour porcelet sevré, dans un plan complètement randomisé. Des ensembles de données ont été collectés sur l'hématologie, la chimie du sérum et le profil lipidique. Globules rouges (6,74, 6,03, 5,57) x 1012/l, hématocrite (46,33, 39,33, 37,35) %, concentration en hémoglobine (13,77, 11,43, 10,37) g/L et numération plaquettaire (37,90, 33,97, 33,83) x 109/L étaient significativement plus faibles (P<0,05) dans les sérums de verrats nourris au TPM que dans ceux nourris sans TPM. L'hémoglobine cellulaire moyenne et la concentration moyenne d'hémoglobine cellulaire ont été significativement (P < 0,05) abaissées par l'ajout de TPM d'une manière dose-dépendante. L'ajout de TPM a augmenté la fragilité des érythrocytes dans une solution de chlorure de sodium de concentrations variables. La glycémie a augmenté progressivement (P<0,05) avec l'augmentation de la TPM. L'albumine n'était significativement réduite qu'à 5,0 % de TPM tandis que la globuline et l'urée étaient réduites (P< 0,05) aux deux niveaux de TPM. La protéine totale et l'ALT ont été significativement réduites avec l'augmentation de la TPM. Le HDL a été progressivement abaissé (P<0,05) avec l'ajout de TPM alors que l'inverse était le cas avec le LDL. Les VLDL et les TG n'ont augmenté de manière significative qu'à 5,0 % de TPM. Il est conclu que le TPM a diminué les indices hématologiques, a compromis la stabilité des érythrocytes même dans une solution saline normale, a réduit l'albumine sérique, la globuline, les protéines totales, l'ALT et le HDL mais a augmenté le glucose sérique, le LDL, le VLDLet le TG

Mutations in the Hemoglobin Binding Domain of Band 3 Perturb Cellular Metabolism and Alter the Sickling of SS Erythrocytes

Under oxygenated conditions, 4 glycolytic enzymes that perform the terminal steps of glycolysis (phospho-fructoKinase [PFK], lactate dehydrogenase [LDH], aldolase [ALD] and glygeraldehyde 3 phosphate dehydrogenase [GAPDH]) bind to the cytoplasmic domain of band 3. Under deoxy conditions deoxy hemoglobin (Hb) is bound to band 3 and PFK, LDH, ALD and GAPDH are displaced (Campanella et al. PNAS 102, 2005; Blood 112, 2008). We generated transgenic mice in which the sequence encoding the first 35 amino acids of the wild type human band 3 cytoplasmic domain replaced the endogenous mouse band 3 sequences in the Slc4a1 gene, a mutant line in which human amino acids 12-21 were deleted removing the deoxy Hb binding site (-Hb) and a third line in which amino acids 1-11 were deleted creating a high affinity binding site for deoxyHb (++Hb). Erythrocytes from the mutant lines were insensitive to Oxygen concentration resulting in changes in oxygen dependent deformability and other physical properties compared to the wild type line (Chu et al. Blood 128, 2016, Zheng et al. JBC 294, 2019, Zhou et al. Sci. Adv. 5, 2019). We crossed our humanized band 3 mouse strains to the Townes Sickle Cell Disease (SCD) mouse model, maintaining both the human βA and βS alleles to generate human AA, AS and SS mice homozygous for each of the human band 3 cytoplasmic domain sequences. Using an assay in which SS red cells in phosphate buffer are deoxygenated to 6% oxygen over time (Dunkelberger et al., J. Phys. Chem. B 122, 2018), we observed that -Hb band 3/SS mice showed an accelerated rate of sickle cell formation and a higher percent of sickled cells than wild type band 3/SS mice (p&lt;0.01). Conversely, ++Hb band 3/SS mice showed an inhibition of both the rate of sickling and the precent of sickled cells compared to wild type band 3/SS mice (p&lt;0.05). We hypothesized that the inability of the glycolytic enzymes to reversibly bind to band 3 in the mutant mice were responsible for the differences in sickling. To test this hypothesis, we analyzed a panel of 28 cellular metabolites in 12 mice (6 female, 6 male) of each genotype: wild type band 3/AA, -AS and -SS, -Hb band 3/AA, -AS, -SS and ++Hb/AA, -AS, -SS. The metabolites were quantified by LC-MS/MS using an API 4500 triple quadrupole mass spectrometer (AB Sciex), with chromatographic resolution enabled on a polymeric amino column (apHera by Supelco) under alkaline mobile phase conditions (pH ~9.3). Stable isotope dilution and 8pt calibration curves allowed the absolute quantification of each metabolite. Consistent with the constitutive binding of the terminal glycolytic enzymes to band 3 in -Hb erythrocytes, glycolysis was inhibited after the phosphoenol pyruvate step, as evidenced by significant accumulation of the intermediates at top of the glycolysis pathway, including fructose 1,6 biphosphate (FBP; p&lt;0.01), dihydroxyacetone phosphate/ glyceraldehyde-3-phosphate (G3P; p&lt;0.01), and 3-phosphoglycerate/2-phosphoglycerate (PG; p&lt;0.01). In the ++Hb mutant where the terminal glycolytic enzymes are constitutively displaced from band 3, significantly lower levels of FBP, G3P and PG were observed (p&lt;0.01). The levels of these metabolites in wild type band 3/SS erythrocytes were intermediate between the two mutant strains. We hypothesized that the accumulation of FBP, G3P and PG contributed to the increased rate of sickling in the -Hb band 3/SS mice. To test this, we incubated wild type band 3/SS cells with either FBP or PG. Both intermediates increased the rate of sickle cell formation and percentage of sickled cells in a dose dependent fashion with no alteration in any RBC indices including MCV and osmotic fragility. We next hypothesized that reduction of the levels of glycolytic intermediates would have an antisickling effect. To test this, we incubated wild type band 3/SS cells with 2,3 diphosphoglycerol (DPG), which is a potent inhibitor of glycolysis. We found that DPG treatment led to a dose dependent decrease in the rate of sickle cell formation and percentage of sickled cells, again with no alteration in any RBC indices including MCV and osmotic fragility. We conclude that the accumulation of glycolytic intermediates leads to increased sickle cell formation. We propose that reduction in the levels of glycolytic intermediates either by accelerating the terminal stages of glycolysis or by redirection to the pentose phosphate pathway may offer a means to treat SCD. Disclosures No relevant conflicts of interest to declare.

Invitro Membrane Stabilizing Potentials Of Fractionates Of Ethanolic Extract of Carica Papaya Leaf

Invitro membrane-stabilizing potentials of fractionates of ethanolic extract of Carica Papaya leaf was investigated in this study. The soxhlet extraction method was used to extract the plant, fractionated with 6 different solvents to give 6 different fractions (hexane, ethyl acetate, chloroform, butanol, methanol benzene). Hbss red blood cells samples were obtained from non-crises state sickle cell patients from Eku Baptist hospital Abraka Delta State, Nigeria. These tests involved the use of positive (p-hydroxy benzoic acid 5ug/ml) and negative controls (normal saline) for membrane stability experiments. Hbss blood was treated with 2mg/ml to 10mg/ml in seven groups with leaf fractionates. Data was analyzed using ANOVA test. The results shows that osmotic fragility was reduced by the introduction of the leaf fractionate, with the highest rate of reduction noticed in the hexane 1 fractionate. PHBA reversal rate and osmotic fragility effect was normal at low doses, but as concentration increases, reversal rate and percentage reduction of sickling decreases. It was concluded that Carica papaya leaf extract fractions, just as its crude extracts, have as much osmotic fragility activities, and this is dose-dependent and has no negative effect on tested blood samples as compared with the treatments with PHBA.

Effect of Diabetes Mellitus and Hypertension on Osmotic Fragility and Hemorheological Factors in Male Wistar Rats

Diabetes mellitus is a common risk factor for erythrocyte osmotic stress. This study was aimed at exploring the effect of streptozotocin (STZ)-induced diabetes mellitus and salt-induced hypertension on osmotic fragility and hemorheological variables in male Wistar rats. Thirty male rats were grouped into five groups of six animals each as follows: negative control (zero salt in diet); positive control (normal salt diet - 0.3% salt); high salt diet (8% salt) (HSD only); STZ induced diabetes and normal salt diet (STZ only); STZ induced diabetes and high salt diet (STZ + HSD). At the end of a 4 weeks period, hematological variables, osmotic fragility, rheology and cardiovascular responses were assessed. There was an increase (p<0.05) in the mean arterial pressure and heart rate of HSD, STZ and HSD + STZ groups indicating a salt induced hypertension. There was a decrease in the body weight of STZ and HSD +STZ groups. There was significant increase (p<0.05) in the haematocrit, platelets estimates and fibrinogen concentrations in the experimental groups when compared with the controls. The STZ and STZ + HSD groups showed a reduced clotting time which corresponded to the increased platelet estimates and fibrinogen concentration. The increase in haematocrit, platelet and plasma protein resulted in the increased blood viscosity and a decreased flow rate. The osmotic fragility test was also observed to be increased (p<0.05) in HSD, STZ only and STZ + HSD groups. Diabetes mellitus and hypertension increase the rate of hemolysis of erythrocyte, as well as increase blood viscosity.

Association between the osmotic fragility of erythrocytes and the course of acute myocardial infarction

Aim. To investigate the relationship between the osmotic fragility of erythrocytes and the course of acute myocardial infarction (MI).Methods. An analysis of the osmotic fragility of erythrocytes was conducted using beta-blocker-based osmotic fragility test in sixty-two patients within the first 6 hours after onset of MI symptoms.Results. The results revealed that the patients with increased erythrocyte osmotic fragility experienced more complications after acute MI, such as left ventricular failure and cardiac arrhythmias (ventricular extrasystoles and ventricular tachycardia) (p = 0.026). Moreover, these patients exhibited greater myocardial injury - the concentration of biomarkers of myocardial necrosis, such as creatine phosphokinase, creatine phosphokinase MB and Troponin I was increased - p = 0.009, p = 0.032 and p = 0.001, respectively. In addition to that, the patients with high osmotic fragility had a larger number of hypokinetic and akinetic segments, high impaired myocardial contractility index, and low ejection fraction. The impaired myocardial contractility index was significantly higher in patients with increased erythrocyte osmotic fragility (1.5 (1.22; 1.75) vs 1.12 (1.0; 1.56), U = 157.5, p = 0.032).Conclusion. Increased erythrocyte osmotic fragility in patients was associated with greater myocardial injury, manifesting through the higher concentration of biomarkers of myocardial necrosis in blood, as well as higher number of hypokinetic segments.

Assessment of Nanosilver Hemocompatibility in Prehypertensive Salt-Induced Animal Model

Aim: There are Conflicting reports on safety profile of nanoparticles on biological cells. This study evaluated the impact of nanosilver on hemocompatibility on salt-loaded rats. Materials and Methods: Sprague-Dawley rats [(inbred) (120-140 g)] randomly divided into of 4 groups, (n = 6) were studied. Group 1(control) received normal rat chow and tap water, Group 2 received rat chow containing 8% NaCl [(salt-loaded rats (SLRs)]. Group 3 received rat chow + Nanosilver Solution (NS) 0.18 mL 10 ppm/kg/day. Group 4 comprised SLRs + NS. After 6 weeks oral gavage treatments, measurements of Blood pressure (Bp) and Heart Rate (HR) were by pressure transducer via cannulation of left common carotid artery following anaesthesia with urethane. HR was computed by the number of arterial pulse per 60 seconds. 5 ml of blood for WBC, PLATELETS, RBC, PCV, HB, MCH, MCHC and MCV analyses using automated haematology analyser and Osmotic fragility reactivity with standard spectrophotometer at 540 nm wavelength. Results: Exposure of nanosilver to normotensive rats resulted in significantly lower RBC level compared with control, whereas RBC level in Salt-Loaded Co-Treated Nanosilver (SCNS) was comparable with the SLRs. The tenet was the same for HB, PCV, MCH and MCHC. Nanosilver induced leukopenia in normotensive compared with control and prevented WBC elevation in SCNS. Platelets significantly increased in Nanosilver-Treated Normotensive Rats (NTNRs) compared with control and decreased in SCNS. Osmotic burst resistance increased in NTNRs and decreased in cells from treated groups. Conclusion: Chronic exposure of nanosilver to salt loaded rats alters haematological parameters which may worsen circulatory function and activate risk factors of cardiovascular disorders.