Alanine solution as enzyme reaction buffer used in A to O blood group conversion / 中国实验血液学杂志

The aim of this study was to investigate the effect of alanine solution as α-N-acetylgalactosaminidase enzyme reaction buffer on the enzymatic activity of A antigen. The binding ability of α-N-acetylgalactosaminidase with RBC in different reaction buffer such as alanine solution, glycine solution, normal saline (0.9% NaCl), PBS, PCS was detected by Western blot. The results showed that the efficiency of A to O conversion in alanine solution was similar to that in glycine solution, and Western blot confirmed that most of enzymes blinded with RBC in glycine or alanine solution, but few enzymes blinded with RBC in PBS, PCS or normal saline. The evidences indicated that binding of enzyme with RBC was a key element for A to O blood group conversion, while the binding ability of α-N-acetylgalactosaminidase with RBC in alanine or glycine solution was similar. It is concluded that alanine solution can be used as enzyme reaction buffer in A to O blood group conversion. In this buffer, the α-N-acetylgalactosaminidase is closely blinded with RBC and α-N-acetylgalactosaminidase plays efficient enzymatic activity of A antigen.

Removal of αGal xenotransplantation antigen by a novel α-galactosidase / 中国实验血液学杂志

αGal, a xenotransplantations antigen (XTA), can lead to hyper acute reaction (HAR) in xenotransplantation. α-Galactosidase from B. fragilis is a novel galactosidase belong to CAZy GH110 which can clear the terminal αGal from branched and linear oligosaccharides. This study was purposed to investigate the removal effect of a novel α-galactosidase on α-Gal XTA on surface of red blood cells. The αGal XTA from the red blood cells of cattle, pig, dog and rabbit was digested by using recombinant α-galactosidase; the α-Gal antigens on surface of cells was detected by flow cytometry. The results showed that the XTA was disappeared completely or mainly. It is concluded that the novel α-galactosidase is a potential enzyme to remove the XTA on the surface of xenotransplants and can be used to overcome the HAR in xenotransplantation.

A reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human blood type B→O conversion / 中国实验血液学杂志

This study was aimed to prepare a reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human B to O blood group conversion. Based on the construction of recombinant E. Coli (DE3) which can express α-galactosidase, the inducing time and inducer concentration were optimized for high expression of α-galactosidase. Then, the expression products in supernatant were purified by cation and anion exchange column chromatography. The purified α-galactosidase was used to treat B group red blood cells in phosphate buffer (pH 6.8) for 2 hours to prepare O group red blood cells. The results showed that the optimal inducing conditions for α-galactosidase expression were IPTG 0.1 mmol/L, 37°C and 2 hours. The specific enzyme activity of purified protein increased from 0.42 U/mg to 2.1 U/mg as compared with pre-purification. And, the conditions of B to O blood group conversion were 26°C, pH 6.8 (neutral pH condition) and 2 hours. Moreover, 225 µg of the enzyme could converse 1 ml B red blood cells to O completely. It is concluded that the technology of expression and purification of recombinant α-galactosidase has been established, and the purified protein can converse B red blood cells to O completely, which means that an effective enzyme conversing B red blood cells to O has been obtained.

Signal pathways of eryptosis-review / 中国实验血液学杂志

Erythrocytes are devoid of nuclei and mitochondria which are the crucial elements of apoptosis, so their programmed suicidal death is called eryptosis. Eryptosis is characterized by cell shrinkage, membrane blebbing, activation of proteases, and phosphatidylserine exposure. Prostaglandin E(2) (PGE(2)) activates nonselective cation channels that increase cytosolic Ca(2+) activity and platelet-activating factor (PAF) activates a sphingomyelinase which lead to formation of ceramide. Either can lead to membrane scrambling with subsequent phosphatidylserine exposure. Exposed phosphatidylserine is recognized by macrophages that engulf and degrade the injured cells. As such, eryptosis can clear the injured red blood cells and avoid the release of hemoglobin. The signaling of eryptosis includes PGE(2), cation channels, PAF, ceramide, protein kinase C, and in some instances, caspases. In this review, the PGE(2), PAF and protein kinase pathways, erythrocyte surface receptor-mediated effects, oxidative stress and caspase effects, the inhibitory factors of eryptosis and the clinical eryptosis-related diseases are discussed.

Correlation between DNA methylation of the ABO gene promoter CpG island and leukemia / 中国实验血液学杂志

Recent studies have found that ABO blood group antigen is also closely related to the onset and development of many diseases. More and more attention is being paid to the decrease of A/B blood group antigen caused by some tumors. This study was purpose to investigate the correlation between DNA methylation of the ABO gene promoter CpG island and leukemia. The relative contents of ABH antigen on the surface of RBC from kinds of blood disease patients and healthy individuals were detected by using flow cytometry and confocal laser scanning microscopy. The DNA sequences and CpG methylation of ABO gene promoter in patients with hematopathy and healthy individuals, as well as the -102 site methylation of ABO gene promoter in patients with hematopathy and healthy individuals were detected by PCR and MSP-PCR respectively. The results showed that RBC from leukemia patients displayed different degree of A/B antigen decrease. The sequences of ABO gene promotor of patients with hematopathy were not different from healthy individuals indicating high conservation of promoter sequences. Comparison of sequences between patients with hematopathy and healthy individual indicated that CpG islands on ABO gene promoter either from blood disease patients or from healthy individual had no methylated site in AA patients, but C residues at position -102, -101, -100, -99 and -97 on the promoter of ABO gene in AML, CML, ALL and some MDS patients were methylated. It is concluded that methylation of CpG islands in promoter of ABO gene may result in AB antigen decrease in patients with leukemia. The methylation sites -102, -101, -100, -99 and -97 may be specific for leukemia. The methylation of site -102 can be used as a molecular marker in differential diagnosis for leukemias.

B to O erythrocyte conversion by the recombinant alpha-galactosidase / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell.</p><p><b>METHODS</b>alpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O.</p><p><b>RESULTS</b>The purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe.</p><p><b>CONCLUSION</b>ECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.</p>

Hyperthermia enhanced the killing effect of 5-fluorocytosine on human colon cancer cell line transfected with cytosine deaminase gene / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate whether hyperthermia can enhance the killing effect of 5- fluorocytosine (5- FC) on human colorectal carcinoma cell lines SW480 transfected with carcinoembryonic antigen (CEA) tissue- specific cytosine deaminase (CD) gene in vitro,and study its mechanism.</p><p><b>METHODS</b>Human colorectal carcinoma cell lines SW480 transfected with G1CEACDNa were cultured. The proliferated colonies were treated with the combined therapy of 5-FC and hyperthermia at a temperature of 43 degrees C for 30 min. After eight days, MTT was used to calculate the cellular survival rate,to analyze the killing effect of 5-FC combined with hyperthermia on SW480 cells transfected with CD gene. Flow cytometry was performed to analyze the cellular cycle and transmission electron microscope was used to observe the morphologic changes of SW480 cells after thermochemotherapy.</p><p><b>RESULTS</b>Hyperthermia combined with 5-FC had an enhanced killing effect on SW480-CEACD cells than 5-FC alone (P< 0.05, t =2.403, n=9). Flow cytometry revealed that the proportion of S stage cell increased in the group treated with hyperthermia and 5- FC (P< 0.001, t =7.158, n=6). Transmission electron microscope showed apoptosis after thermo- chemotherapy.</p><p><b>CONCLUSIONS</b>Hyperthermia can improve the anti- tumor effect of 5- FC on human colorectal carcinoma cell lines SW480 transfected with CD gene, and the cells were blocked at S stage of cellular cycle and apoptosis was induced following thermochemotherapy.</p>

Preparation of transfusable human universal red blood cell with recombinant alpha-galactosidase / 中国实验血液学杂志

In order to meet the demand for safe transfusion in special conditions and to utilize the donated blood supply efficiently, technology has been developed to convert erythrocytes from type A, B, or AB to "universal donor" blood. Conversion of blood type B to O was performed by means of recombinant alpha-galactosidase digestion. The results showed that blood type B to O was converted successfully, 1 transfusion unit of red cells of group B (100 ml totally) could converted to universal blood cells in the optimal conditions including pH 5.6, 26 degrees C, 2 hours, obturation and sterilization. It is concluded that the universal red blood cells converted from group B to group O are conformed to demand of identification rules of biological products, no harmful effects of alpha-galactosidase on cell structure and function are observed. The converted red cells can stored in 4 degrees C for 21 days.