OBJECTIVETo study the toxicity of methomyl to acetylcholinesterase (AChE) in different regions.

METHODSThe optimal temperature and time for measurement of AChE activity were determined in vitro. The dose- and time-response relationships of methomyl with AChE activity in human erythrocyte membrane, rat erythrocyte membrane, cortical synapses, cerebellar synapses, hippocampal synapses, and striatal synapses were evaluated. The half maximal inhibitory concentration (IC50) and bimolecular rate constant (K) of methomyl for AChE activity in different regions were calculated, and the type of inhibition of AChE activity by methomyl was determined.

RESULTSAChE achieved the maximum activity at 370 °C, and the optimal time to determine initial reaction velocity was 0-17 min. There were dose- and time-response relationships between methomyl and AChE activity in the erythrocyte membrane and various brain areas. The IC50 value of methomyl for AChE activity in human erythrocyte membrane was higher than that in rat erythrocyte membrane, while the Ki value of methomyl for AChE activity in rat erythrocyte membrane was higher than that in human erythrocyte membrane. Among synapses in various brain areas, the striatum had the highest IC50 value, followed by the cerebellum, cerebral cortex, and hippocampus, while the cerebral cortex had the highest Ki value, followed by the hippocampus, striatum, and cerebellum. Lineweaver-Burk diagram demonstrated that with increasing concentration of methomyl, the maximum reaction velocity (Vmax) of AChE decreased, and the Michaelis constant (Km) remained the same.

CONCLUSIONMethomyl is a reversible non-competitive inhibitor of AChE. AChE of rat erythrocyte membrane is more sensitive to methomyl than that of human erythrocyte membrane; the cerebral cortical synapses have the most sensitive AChE to methomyl among synapses in various brain areas.

Acetylcholinesterase ; metabolism ; Animals ; Cerebellum ; drug effects ; Cerebral Cortex ; drug effects ; Erythrocyte Membrane ; drug effects ; enzymology ; Hippocampus ; drug effects ; Humans ; Inhibitory Concentration 50 ; Methomyl ; toxicity ; Rats ; Synapses ; drug effects ; Toxicity Tests

This study was aimed to investigate various factors influencing the proceduction of Cu(II) crossing human erythrocyte membrane, including concentration of Cu²⁺, pH value of the medium, temperature and time of incubation, and to derive kinetic equation of Cu(II) crossing human erythrocyte membrane. Suspension red blood cells were incubated by Cu²⁺, then content of Cu²⁺ crossed human erythrocyte membrane was determined by atomic absorption spectrometry under various conditions after digestion. The results showed that content of Cu²⁺ crossed human erythrocyte membrane increased with the increase of extracellular Cu²⁺ and enhancement of incubation temperature, and the content of Cu²⁺ crossed human erythrocyte membrane showed a increasing tendency when pH reached to 6.2-7.4, and to maximum at pH 7.4, then gradually decreased at range of pH 7.4-9.2. It is concluded that the Cu²⁺ crossing human erythrocyte has been confirmed to be the first order kinetics characteristics within 120 min, and the linear equation is 10³ × Y = 0.0497t +6.5992.
Copper ; pharmacology ; Erythrocyte Membrane ; drug effects ; Humans ; Hydrogen-Ion Concentration ; Kinetics ; Temperature

This study was purposed to evaluate the effect of trehalose-loading on physiological and biochemistry properties of red blood cell (RBC) membrane. The samples were divided into the control group (RBC without trehalose loading) and the test group (RBC with trehalose loading). Osmotic fragility reaction was used to determine the osmotic fragility change of loaded RBC membrane in NaCl solution of different osmotic concentration. Flow cytometry and deformeter were used to assay the integrality and deformability of the RBC, respectively. The results showed that the NaCl solution osmotic concentrations were 160 mOsm and 121.4 mOsm, respectively when the haemolysis rate was 50% of the control group and the test group. Flow cytometry data demonstrated that incubation of RBC in a hypertonic trehalose solution resulted in a fraction of cells with different complexity that attached to little Annexin V-FITC, and that it could be removed by washing and resuspending the RBC in an iso-osmotic (300 mOsm PBS) medium. The deformability of the loaded RBC descend, the statistical difference was significant between control and test groups (P < 0.01). It is concluded that the membrane physiological and biochemistry stability and membrane integrality of RBC in a hyper osmotic pressure can be retained after trehalose loading.
Blood Preservation ; methods ; Cryopreservation ; methods ; Erythrocyte Membrane ; drug effects ; Erythrocytes ; drug effects ; Humans ; Osmotic Fragility ; drug effects ; Trehalose ; pharmacology

OBJECTIVETo investigate the effect of Yisui Shengxue Granule (, YSSXG), a complex Chinese medicine, on the oxidative damage of erythrocytes from patients with hemoglobin H (HbH) disease.

METHODSTwenty-two patients with HbH disease and 22 healthy volunteers were observed. YSSXG was given to patients with HbH disease for 3 months. Before and after the 3-month treatment, blood parameters [hemoglobin (Hb), red blood cells (RBCs), and reticulocyte percent (Ret)] were examined; inclusion bodies in erythrocytes were observed by transmission electron microscopy (TEM); activities of antioxidant defense enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (Cat)] and erythrocyte membrane malondialdehyde (MDA) concentrations were determined.

RESULTSIn patients with HbH disease, measured values of RBC and Hb obtained from the first to the third months after treatment with YSSXG were significantly higher than before treatment (P<0.01). Measured values of Ret from the second to the third months after treatment were significantly lower than before treatment (P<0.05 and P<0.01, respectively). Prior to treatment with YSSXG, TEM images of RBCs showed the presence of numerous inclusion bodies. After treatment with YSSXG, the amount and volume of inclusion bodies decreased. Treatment with YSSXG also led to a significant increase in SOD activity (P<0.01), a decrease in Cat activity (P<0.01), and no significant differences in GSHPx activity (P>0.05) or MDA concentration (P>0.05). However, compared with the healthy counterparts, SOD, GSH-Px, and Cat activities presented at high levels (P<0.01) both before and after treatment.

CONCLUSIONSYSSXG could improve the degree of hemolysis and anemia in patients with HbH disease. The mechanism may be related to its antioxidative effects, which could elevate the activity of total SOD in erythrocytes and efficiently inhibit the oxidative precipitation of β-globin chains.

Adolescent ; Adult ; Catalase ; metabolism ; Child ; Child, Preschool ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Erythrocyte Membrane ; drug effects ; metabolism ; ultrastructure ; Erythrocytes ; drug effects ; enzymology ; pathology ; ultrastructure ; Female ; Glutathione Peroxidase ; metabolism ; Humans ; Inclusion Bodies ; drug effects ; ultrastructure ; Male ; Malondialdehyde ; metabolism ; Oxidative Stress ; drug effects ; Superoxide Dismutase ; metabolism ; Young Adult ; alpha-Thalassemia ; blood ; drug therapy ; pathology

The present paper was aimed to explore the effect of Shuxuetong on the membrane viscoelasticity of erythrocyte taken from the acute phase patients suffering from chronic pulmonary heart disease. The membrane viscoelasticity of erythrocyte was taken from the acute phase patients suffering from chronic pulmonary heart disease. The changes of membrane viscoelasticity of erythrocyte after treated with shuxuetong were detected by micropipette aspiration technique. The results showed that the Shuxuetong of certain concentration could cause the decrease of membrane elastic modulus and viscous coefficients in acute phase patients suffering from chronic pulmonary heart disease. The study offers experimental evidences that the comprehensive treatment of pulmonary heart disease should involve the drug or measure to improve the erythrocyte deformability.
Aged ; Blood Viscosity ; drug effects ; Chronic Disease ; Drugs, Chinese Herbal ; therapeutic use ; Elasticity ; drug effects ; Erythrocyte Deformability ; physiology ; Erythrocyte Membrane ; physiology ; Erythrocytes ; physiology ; Female ; Humans ; Male ; Middle Aged ; Phytotherapy ; Pulmonary Heart Disease ; blood ; drug therapy

OBJECTIVEDanshen, as a traditional Chinese medicine for promoting blood circulation and removing blood stasis, is widely applied in improving human erythrocyte deformability in clinics. But its direct effect on erythrocyte membranes is still unclear.

METHODIn this essay, the confocal Raman technique was adopted to measure the changes in Raman spectra of human erythrocytes before and after the administration of Danshen injection.

RESULTThe results showed slight changes in group conformations corresponding to erythrocyte membranes after the administration of danshen injection. Specifically, 1064, 1126 cm(-1) spectral lines attributed to phospholipid molecule acyl C-C skeleton anti-conformation were obviously weakened, whereas 1091 cm(-1) spectral line attributed to phospholipid molecule acyl C-C skeleton guache conformation notably intensified. Besides, the longitudinal order-parameter in chains (Strans) of phospholipids was reduced significantly.

CONCLUSIONDanshen injection can transfer erythrocyte membrane phospholipid molecule acyl C-C skeleton anti-conformation to guache conformation, indicating the increase in liquidity of erythrocyte membrane phospholipids and erythrocytes. Danshen injection's effect in improving erythrocyte membrane structure and function may be the intrinsic mechanism for its pharmacological effect as a traditional Chinese medicine.

Adult ; Blood Proteins ; chemistry ; Erythrocyte Membrane ; chemistry ; drug effects ; Female ; Humans ; Injections ; Male ; Molecular Conformation ; Phospholipids ; chemistry ; Salvia miltiorrhiza ; Spectrum Analysis, Raman ; methods ; Young Adult

In order to explore the activity of a peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment (RES2 derivative) in vitro, the peptide (Leu-Ala-Ala-Met-Glu-Asp-Glu-Pro-Ser-Asn-Asp-Asn-Gly-Gly-Gly-Ser) was synthesized by peptide synthesizer with Fmoc method and purified by high performance liquid chromatography (HPLC). Its binding activity was identified by radioligand-receptor binding assay (RRA) and its bioactivity was measured by erythrocyte (86)Rb uptake. The results of saturation binding experiment and competitive binding experiment showed that the synthesized RES2 derivative had the capability to bind to (3)H-ouabain. The dissociation constant (K(d)) was 38.46 nmol/L and IC(50) was 6.353 nmol/L. Erythrocyte (86)Rb uptake experiment showed that the RES2 derivative blocked the inhibitory effect of ouabain on the sodium pump on erythrocyte membrane in a dose-dependent manner. The results showed that the RES2 derivative is capable of binding to ouabain and improving the activity of sodium pump on erythrocyte membrane, suggesting that the RES2 derivative might become an effective antihypertensive drug in the future.
Amino Acid Sequence ; Animals ; Chromatography, High Pressure Liquid ; Erythrocyte Membrane ; drug effects ; Ouabain ; pharmacology ; Peptide Fragments ; metabolism ; Rats ; Sodium-Potassium-Exchanging ATPase ; metabolism

Though high concentration of glucose can benefit the survival of lyophilized human red blood cells, the high concentration of glucose can result in serious damage of red blood cells. This study was aimed to investigate the inhibitory effect of trehalose on damage of red blood cells induced by high concentration of glucose. After incubation with the high concentration of glucose buffer containing different concentrations of trehalose for three hours at 37 degrees C, the phosphatidylserine exposure and the osmotic fragility of cells were analyzed by flow cytometry and the lipid peroxidation of membrane was evaluated by TBA method. The results showed that the high concentration of glucose could lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage which were dependent on the glucose concentrations and incubation temperature. However, trehalose could effectively prevent the phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage induced by high concentration glucose. With increase of the trehalose concentrations. As the trehalose concentration increases, the phosphatidylserine exposure, maloni-aldehyde concentration and cell debris rate decreased gradually. In conclusion, the high concentration of glucose can lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage of red blood cells. However, trehalose can inhibit the damaging effects of high concentration of glucose on red blood cells, which may be useful for the application of sugars to lyophilization of red blood cells.
Blood Preservation ; methods ; Erythrocyte Membrane ; drug effects ; metabolism ; Erythrocytes ; drug effects ; Flow Cytometry ; Glucose ; adverse effects ; Humans ; Lipid Peroxidation ; drug effects ; Membrane Lipids ; metabolism ; Osmotic Fragility ; drug effects ; Phosphatidylserines ; pharmacology ; Trehalose ; pharmacology

This study was aimed to evaluate the effect of benzyl alcohol on trehalose-loading red blood cells (RBCs). The RBCs were incubated in 10, 30, 50 and 100 mmol/L concentrations of benzyl alcohol-trehaloe solution at 4 degrees C for 24 hours. The hemolysis rate of loaded RBCs was detected by using cyanohemoglobin kit, the intracellular trehalose level were assayed by sulfate anthrone method. The results showed that the intracellular trehalose concentration in group with 100 mmol/L benzyl alcohol was 72 +/- 12.98 mmol/L, compared with that in groups of 10, 30 and 50 mmol/L, the statistical difference were significant (p = 0.000); the hemolysis rate of loaded RBCs in group with 100 mmol/L of benzyl alcohol was 17.99 +/- 3.75%, as compared with groups of 10, 30 and 50 mmol/L, the statistical difference was significant (p = 0.000). It is concluded that benzyl alcohol can enhance the intracellular trehalose concentration. As concentration of benzyl alcohol ascends, the intracellular trehalose concentration increases. 100 mmol/L benzyl alcohol may be proper for loading RBCs.
Benzyl Alcohol ; pharmacology ; Blood Preservation ; methods ; Erythrocyte Membrane ; metabolism ; Erythrocytes ; drug effects ; metabolism ; Freeze Drying ; methods ; Humans ; Trehalose ; metabolism ; pharmacology

The key points for better protection of trehalose in freeze-drying red blood cells (RBCs) are to resolve non-osmosis of trehalose to red blood cells and to make cytoplasmic trehalose to reach effective concentration. This study was aimed to investigate the regularity of loading RBCs with trehalose, screen out optimal loading condition and evaluate the effect of trehalose on physico-chemical parameters of RBCs during the period of loading. The cytoplasmic trehalose concentration in red blood cells, free hemoglobin and ATP level were determined at different incubation temperatures (4, 22 and 37 degrees C), different trehaolse concentrations (0, 200, 400, 600, 800 and 1000 mmol/L) and different incubation times (2, 4, 6, 8 and 10 hours), the cytoplasmic trehalose, free hemoglobin (FHb), hemoglobin (Hb) and mean corpuscular volume (MCV) in fresh RBCs and RBCs stored for 72 hours at 4 degrees C were compared, when loading condition was ensured. The results showed that with increase of incubation temperature, time and extracellular trehalose concentration, the loading of trehalose in RBCs also increased. Under the optimal loading condition, cytoplasmic trehalose concentration and free hemoglobin level of fresh RBCs and RBCs stored for 72 hours at 4 degrees C were 65.505 +/- 6.314 mmol/L, 66.2 +/- 5.002 mmol/L and 6.567 +/- 2.568 g/L, 16.168 +/- 3.922 g/L respectively. It is concluded that the most optimal condition of loading trehalose is that fresh RBCs incubate in 800 mmol/L trehalose solution for 8 hours at 37 degrees C. This condition can result in a efficient cytoplasmic trehalose concentration. The study provides an important basis for long-term preservation of RBCs.
Biological Transport, Active ; drug effects ; Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; metabolism ; pharmacology ; Erythrocyte Membrane ; metabolism ; Erythrocytes ; Freeze Drying ; Humans ; Osmotic Fragility ; Temperature ; Time Factors ; Trehalose ; metabolism ; pharmacology