BACKGROUND: The eosin-5'-maleimide (EMA) binding test using flow cytometry is a common method to measure reduced mean channel fluorescence (MCF) of EMA-labeled red blood cells (RBCs) from patients with red cell membrane disorders. The basic principle of the EMA-RBC binding test involves the covalent binding of EMA to lysine-430 on the first extracellular loop of band 3 protein. METHODS: In the present study, the MCF of EMA was analyzed for samples derived from 12 healthy volunteers (controls) to determine the stability (i.e., the percentage decrease in fluorescence) of EMA over a period of 1 year. RESULTS: Comparison of periodical MCF readings over time, that is, at 2-month intervals, showed that there were no significant changes in mean channel fluorescence for up to 6 months; however, there was a significant decrease in MCF at 8 months. CONCLUSION: For optimal dye utilization, EMA remained stable only for up to 6 months. Therefore, we recommend reconstitution of the dye every 6 months when implementing this test and storage at -80degrees C in dark conditions.
Anion Exchange Protein 1, Erythrocyte ; Cell Membrane* ; Erythrocytes ; Flow Cytometry ; Fluorescence ; Healthy Volunteers ; Humans ; Reading

OBJECTIVETo study the clinical features of two families with distal renal tubular acidosis (dRTA) and mutations in the pathogenic gene SLC4A1.

METHODSFamily investigation, medical history collection, and measurement of biochemical parameters were performed to analyze the clinical phenotype and genetic characteristics of dRTA. Direct sequencing was used to detect SLC4A1 gene mutations.

RESULTSThree patients in these two families (two of them were mother and son) were diagnosed with dRTA with typical clinical features, including short stature, metabolic acidosis, alkaline urine, hypokalemia, and nephrocalcinosis. SLC4A1 gene analysis showed that all the three patients had a pathogenic missense mutation R589H (c.1766G>A). The child in family 1 had a de novo mutation of SLC4A1, and the child in family 2 had an SLC4A1 gene mutation inherited from the mother, which met the characteristic of autosomal dominant inheritance.

CONCLUSIONSThis study reports the R589H mutation in SLC4A1 gene in families with hereditary dRTA for the first time in China. Clinical physicians should perform gene detection for patients suspected of hereditary dRTA to improve the diagnosis and treatment of this disease.

Acidosis, Renal Tubular ; genetics ; Anion Exchange Protein 1, Erythrocyte ; genetics ; Child ; Humans ; Male ; Mutation

AIM AND METHODSThe purpose of this investigation was to determine the changes of erythrocyte deformability, band-3 protein and actin in the definite volume of erythrocyte membrane after high intensity running training and recovery in rats.

RESULTSLong-term training could significantly improve erythrocyte deformability and the quantity of membrane proteins. Erythrocyte deformability, band-3 protein and actin decreased transitorily at varying degrees after inadaptable high intensity exercise. One and two week training could improve erythrocyte deformability, the quantity of band-3 protein and actin after recovery.

CONCLUSIONAlterations of erythrocyte membrane protein after high intensity training could cause the change in erythrocyte membrane structure and hence influenced erythrocyte deformability. That was maybe one of mechanisms of training effecting erythrocyte deformability.

Actins ; metabolism ; Animals ; Anion Exchange Protein 1, Erythrocyte ; metabolism ; Erythrocyte Deformability ; Erythrocyte Indices ; Erythrocyte Membrane ; chemistry ; metabolism ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS). METHODS: Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique. Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed. The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology. RESULTS: Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing. CONCLUSION The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient, fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.
Anion Exchange Protein 1, Erythrocyte ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA Primers ; Heterozygote ; Humans ; Mutation ; Spherocytosis, Hereditary ; genetics

OBJECTIVETo explore the hemolytic mechanism of glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes in the view of phosphorylation of membrane protein.

METHODSThe alternation of membrane protein phosphorylation and the effect of dithiothreitol (DTT) on protein phosphorylation were analysed by Western blot technique. The activity of phosphotyrosine phosphatase (PTPs) was determined by using p-nitrophenyl phosphate as substrate.

RESULTSTyrosine phosphorylation of band 3 protein was obviously enhanced in G6PD-deficient erythrocytes. The activity of PTPs was low compared to the normal erythrocytes. The level of phosphotyrosine in G6PD-deficient erythrocytes incubated with DTT was almost the same as in those without DTT. The results were consistent with the activity of PTPs.

CONCLUSIONSPTPs activity reduction and tyrosine phosphorylation enhancement induced by oxidation in G6PD deficiency play an important role in erythrocytes hemolysis. However, the alternation of thiol group is not the only factor affecting the activity of PTPs in G6PD-deficient erythrocytes.

Anion Exchange Protein 1, Erythrocyte ; metabolism ; Blotting, Western ; Erythrocyte Membrane ; metabolism ; Glucosephosphate Dehydrogenase Deficiency ; enzymology ; metabolism ; Humans ; Phosphorylation ; Protein Tyrosine Phosphatases ; metabolism ; Tyrosine ; metabolism

Hereditary spherocytosis (HS) is caused by mutations in the SPTA1, SPTB, ANK1, SLC4A1, and EPB42 genes, all of which encode erythrocyte membrane proteins. Mutations in SLC4A1, which encodes band 3 protein, have rarely been reported as the causative factor among Korean patients with HS. Here, we report two Korean patients with HS carrying mutations in SLC4A1. Patient 1 was a 3-year-old girl with unremarkable past and family histories and was evaluated for anemia that was detected after a complete blood count. She was suspected of having HS considering the spherocytosis of her peripheral blood smear, increased osmotic fragility, hemolytic features in blood chemistry tests, and splenomegaly. Sequence analysis revealed that the patient harbored a single heterozygous missense mutation, c.2278C>T (p.Arg760Trp) in exon 17 of SLC4A1. Patient 2 was a 23-year-old man who had a prior history of intermittent jaundice. Although the patient did not have anemia, a genetic test for HS was performed due to evidence of hemolytic features in the blood chemistry test, splenomegaly, and a family history of HS. The test confirmed a single heterozygous missense mutation, c.2423G>T (p.Arg808Leu) in exon 18 of SLC4A1.
Anemia ; Anion Exchange Protein 1, Erythrocyte ; Blood Cell Count ; Chemistry ; Child, Preschool ; Erythrocyte Membrane ; Exons ; Female ; Humans ; Jaundice ; Mutation, Missense ; Osmotic Fragility ; Sequence Analysis ; Splenomegaly ; Young Adult

Anion Exchange Protein 1, Erythrocyte ; metabolism ; Antiporters ; metabolism ; Child ; Chordoma ; metabolism ; pathology ; radiotherapy ; surgery ; Female ; Follow-Up Studies ; Humans ; Skull Base Neoplasms ; metabolism ; pathology ; radiotherapy ; surgery ; Vimentin ; metabolism

Anion Exchange Protein 1, Erythrocyte ; metabolism ; Breast ; metabolism ; pathology ; surgery ; Cadherins ; metabolism ; Female ; Fibrocystic Breast Disease ; metabolism ; pathology ; surgery ; Follow-Up Studies ; Humans ; Middle Aged ; S100 Proteins ; metabolism

In developing rat kidney, apoptosis plays an important role in the morphogenesis of renal papilla, and hyperosmolality is well known to be one of regulating factor in apoptosis. The aim of this study was to determine the effect of hypoosmolality induced furosemide in neonate on cell proliferation and apoptosis in renal papilla. One-dayold pups were given two times a day, at 12 hour intervals, subcutaneous injection of furosemide (10 mg/kg BW) or saline for 4 days or 7days. We identified thick ascending limb by labeling with antibody to BSC1 (bumetanide-sensitive Na/K/2Cl cotransporter) and type A intercalated cell in collecting duct by labeling with antibody to band 3 protein. We also inspected apoptosis with TdT-mediated dUTP nick end labeling (TUNEL) method and cell proliferation with immunostaining for proliferating cell nuclear antigen (PCNA). The effect of hypoosmolality induced furosemide on neonatal rat kidney. 1) In furosemide-treated animals, body and kidney weights were reduced compare to control groups. the length of renal papillae of furosemide-treated groups were shortened compare to control groups. 2) Transformation from a cuboidal epithelium of thick ascending limb to a squamous epithelium of ascending thin limb in renal papilla was delayed in furosemide-treated groups. 3) In the inner medullary collecting duct of furosemide-treated groups, elimination of type A intercalated cells was delayed compared to control groups. 4) Furosemide treatment reduced the apoptotic index in transforming thick ascending limb of Henle's loop and collecting duct in renal medulla. 5) PCNA-positive cells in the transforming thick ascending limb of Henle's loop and the collecting duct in renal medulla were decreased in number in furosemide-treated groups compared to control groups. These finding suggest that renal hypoosmolality induced furosemide treatment decrease not only apoptosis but also cell proliferation and may retard renal papilla growth at least in neonatal rat kidney.
Animals ; Anion Exchange Protein 1, Erythrocyte ; Apoptosis ; Cell Proliferation ; Epithelium ; Extremities ; Furosemide* ; Humans ; Infant, Newborn ; Injections, Subcutaneous ; Kidney ; Morphogenesis* ; Proliferating Cell Nuclear Antigen ; Rats* ; Weights and Measures

OBJECTIVETo study the clinical and pathological characteristics of pulmonary epithelioid hemangioendothelioma.

METHODSFour cases of pulmonary epithelioid hemangioendothelioma were studied by histopathologic and immunohistochemical examination of lung biopsy specimens.

RESULTSThere were 3 female and 1 male, age 28 to 40 years. Clinically the tumor presented as multiple bilateral small nodules in the lung. Histologically, crown-like clusters of epithelioid tumor cells were obtained which filled in the alveoli locating at the periphery of the tumor nodules, while the central part of the nodules contained myxoid to hyaline matrix. The overall architecture of the lung was still preserved. Additionally, intracytoplasmic vacuoles were seen in tumor cells within which red blood cells were sometimes identified. Tumor cells generally lacked pleomorphism, mitotic activity and necrosis. They were immunohistochemically positive for CD31 and CD34. AE1/AE3 staining was positive in some cases.

CONCLUSIONSPulmonary epithelioid hemangioendothelioma often occurs in a middle-aged woman and represents a distinct clinical pathological entity.

Adult ; Anion Exchange Protein 1, Erythrocyte ; analysis ; Antigens, CD34 ; analysis ; Antiporters ; analysis ; Female ; Hemangioendothelioma, Epithelioid ; immunology ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Lung ; pathology ; Lung Neoplasms ; immunology ; metabolism ; pathology ; Male ; Platelet Endothelial Cell Adhesion Molecule-1 ; analysis