UNLABELLEDObjective： To study the effect of PD98059, a specific inhibitor of Ras/Raf/MEK/ERK signaling pathway, on the proliferation and apoptisis of bone marrow CD71(+), CD235a(+) nucleated erythrocytes in patients with high altitude polycythemia (HAPC) and the pathogenesis of HAPC.

METHODSThe CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients and controls (patients with simple obsolete stracture) were sorted by using the immunemagnetic beads, then were added with 5, 10, 20 µmol/L of PD98059 and DMSO (as control) and were cultured for 72 h under hypoxia. The cell apoptosis was detected by flow cytometry with Annexin V and PI double staining, the cell proliferation was detected by CCK8 method, at same time the erythroid colong-formation ability of bone marrow mononuclear cells (BMMNC) treated with 5, 10, 20 µmol/L of PD98059 and DMSO was observed.

RESULTSWith the increase of PD98059 concentration, the apoptosis rate of bone marrow CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients was enhanced (r=0.807,P<0.01), while the proliferation rate of CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients dereased (r=0.502,P<0.01). The erythroid colong-formation ability of BMMNC in HAPC patients decreased with the increase of PD98059 concentration (r=0.504,P<0.01). There were statistic differences among different groups at 7 and 14 d.

CONCLUSIONThe MEK specific inhibitor PD98059 can inhibit the proliferation and promote the apoptosis of CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients, then inhibit the excessive accumulation of erythrocytes.

Altitude Sickness ; Apoptosis ; Bone Marrow ; Bone Marrow Cells ; Cell Proliferation ; Erythroblasts ; Erythrocyte Count ; Erythrocytes ; Flavonoids ; Flow Cytometry ; Glycophorin ; Humans

OBJECTIVETo detect the base sequences of all exons and part of introns in the GYPA gene of the glycophorin GPA and to investigate the polymorphism of M, N alleles in Chinese population.

METHODSA total of 225 blood sample were randomly colleeted from unrelated Chinese volunteers and were detected by serology techniques. The primers were designed by self, the seguencing of GYPA gene related with sample exon 1-7 full length sequences of bases and intron-1-7 partial sequence was performed, the polymorphism of M, N gene mutation in mucleotide sequence was analysed.

RESULTSThe results of M and N genotyping were in agreement with the results of serological detection. The 23rd base of intron-2, the 55th base of intron-3, the 63rd base of intron-4, the 55th, 189th and 190th base of intron-6, the 712th base variation of exon-7 in the gene M and N were used to subdivide the gene M and N into the mutant M103, M201, M202, N101, N102, N103, N104, and N201. At the same time, it was found that 42th and 54th base were mutated, the base T was inserted between 59th and 60th base in the intron-2, the new mutations occurred in the alleles 28, 29, 65 and 102 in intron-3 in this study.

CONCLUSIONThe polymorphism of the the Chinese population's GYPA gene occurs in all the exons and partly in the introns. The gene polymorphism of M and N blood group in Chinese population might provide the theoretical basis for the studies of clinical blood transfusion, human population genetics and molecular biology.

Alleles ; Asian Continental Ancestry Group ; Blood Group Antigens ; Blood Grouping and Crossmatching ; Exons ; Genotype ; Glycophorin ; Humans ; Introns ; Polymorphism, Genetic

This study was purposed to investigate the molecular polymorphism of gypa gene in association with MN human blood group in Chinese Han population. The MN phenotypes of 202 random samples from unrelated Chinese Han volunteers were identified by serology techniques. The primer for gypa gene exon 2 were designed and synthesized according to reference sequences of NG-007470 gene from GenBank, the DNA of 202 samples was amplified by PCR, at the same time, the amplified products were analyzed by direct DNA sequencing. The results showed that all samples had 2 base substitutions at 1st and 56th nt of gypa exon 2, among them the MN phenotype heterozygote exited mainly in the form of 1A > C, 22T/C, 34A/G, 35T/G, 56T > C; the MM phenotype homozygote exited mainly in the form of 1A > C, 22C, 34G, 35T, 56T > C; the NN phenotype homozygote exited mainly in the form of 1A > C, 22T, 34A, 35G, 56T > C. It is concluded that the polymorphism of gypa gene in associated with MN blood group in Chinese Han population is decided by 5 nucleotide sites of 1, 22, 34, 35 and 56. The bases of 1 and 56 are non-functional gypa single nucleotide polymorphism.
Asian Continental Ancestry Group ; genetics ; Base Sequence ; Exons ; Genotype ; Glycophorin ; genetics ; Humans ; MNSs Blood-Group System ; genetics ; Molecular Sequence Data ; Polymorphism, Genetic ; Sequence Analysis, DNA

OBJECTIVETo study the relationship of GYPC and TRIP3 gene expression and the prognosis of acute lymphoblastic leukemia (ALL) in children in order to explore the molecular biological mechanisms of recurrence and remission of ALL.

METHODSThirty-eight newly diagnosed ALL children were enrolled. Of the 38 patients, 31 achieved complete remission (CR) and 12 relapsed. Semi-quantitative RT-PCR was employed to measure blood GYPC and TRIP3 gene expression. Twenty blood samples from normal children were used as controls.

RESULTSBlood GYPC expression in newly diagnosed ALL children was significantly higher than that in the control group (p<0.01) and the CR group (p<0.01). The expression of GYPC gene in the CR group was similar to that in the control group. Other than the control group (p<0.01) and the CR group (p<0.01), the GYPC expression of the relapse group was significantly higher than that in the newly diagnosed group (p<0.01). The CR group showed lower GYPC gene expression than the nonjremission group before treatment (p<0.05). Blood expression of TRIP3 gene in the newly diagnosed and the relapse groups was significantly lower than that in the control group (p<0.05). The CR group had increased TRIP3 gene expression compared with the control group (p<0.01) as well as the newly diagnosed and the relapse groups (p<0.01). Of the 38 newly diagnosed ALL children, the patients with positive TRIP3 expression showed higher remission rate than those with negative TRIP3 (p<0.05). The TRIP3 gene expression before treatment in patients who achieved CR was higher than that in non-remission patients (p<0.05).

CONCLUSIONSA high GYPC gene expression is associated with an unfavorable outcome, in contrast, a high TRIP3 gene expression is associated with a favorable outcome in childhood ALL.

Adolescent ; Child ; Child, Preschool ; Female ; Glycophorin ; genetics ; Humans ; Infant ; Male ; Mediator Complex Subunit 1 ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; mortality ; Prognosis ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factors ; genetics

OBJECTIVETo analyze related factors which affect GPA mutation frequency of workers exposed to benzene, with the Glycophorin A (GPA) mutation assay and explore the possibility of GPA mutation frequency as an index of predicting the risk of benzene poisoning.

METHODSThe erythrocytes were bound with fluorescent-labeled monoclonal antibody after isolated and fixed from the peripheral blood, and then the GPA mutation assay was performed using the flow cytometry (FCM). The related factors of GPA mutation frequency were analyzed by statistical methods.

RESULTSThe GPA mutation frequency of chronic benzene poisonings was significantly higher than that of their controls (P < 0.05). Significant direct correlation was found between age, length of service, accumulative exposure score and the GPA mutation frequency of workers exposed to benzene (P < 0.01). However, there was significantly inverse correlation between the 3AB index and the GPA mutation frequency (GPAN0: r(s) = -0.589, P < 0.01, GPANN: r(s) = -0.615, P < 0.01). In the multiple factor regression analysis on GPA mutation frequency, benzene exposure and individual susceptibility both entered model of multiple factors analysis, the coefficient of determination of benzene-exposed workers was 0.819.

CONCLUSIONExposure to benzene and individual susceptibility are the most important factors that affect GPA mutation frequency. GPA mutation frequency increases with the benzene exposure and individual susceptibility.

Benzene ; poisoning ; Glycophorin ; genetics ; Humans ; Mutation ; Mutation Rate ; Occupational Exposure

OBJECTIVETo explore the association of the glycophorin A(GPA) gene mutation in peripheral erythrocytes and chronic benzene poisoning.

METHODSSixty-three patients with chronic benzene poisonings and 45 benzene-exposed workers who were engaged in the same job title were investigated. Fluorescence immunolabeling technique and flow cytometry were used to detect GPA mutation frequency in peripheral read blood cell.

RESULTSA significant decrease in WBC count and neutrophil count was found in patients with chronic benzene poisoning compared with control individuals (P<0.01). The WBC count and neutrophil count both decreased along with the GPA-NN frequency, and the trends were significant(P<0.05).Both WBC counts and neutrophil counts decreased as the frequency, and trends were significant(P<0.05). GPA-NN frequency increased along with the accumulative exposure score, and the trend was significant (P = 0.0026). There was no significant trend between the GPA-Nphi frequency and the accumulative exposure score (P = 0.2037).

CONCLUSIONA decrease in WBC count and neutrophil count is found in patients with chronic benzene poisoning, which can arise from genetic damage in bone marrow stem cells, namely gene-duplicating mutations (NN) at the GPA locus in bone marrow cells of MN-heterozygous subjects, GPA-NN mutagens contributed to the pathogenesis of chronic benzene poisoning.

Adult ; Benzene ; poisoning ; Bone Marrow Cells ; pathology ; Case-Control Studies ; Erythrocytes ; pathology ; Female ; Genetic Variation ; Glycophorin ; genetics ; Humans ; Leukocyte Count ; Male ; Middle Aged ; Mutation Rate ; Neutrophils ; pathology

OBJECTIVETo identify the binding site on glycophorin A (GPA) for EBA-175 to provide clue for developing short peptide vaccine and therapeutic agents against Plasmodium falciparum.

METHODSWith the recombinant protein of EBA-175 as the target molecule, the mimetic peptides of GPA were screened from a 12-mer random peptide library. Three rounds of biopanning were carried out, and enzyme-linked immunosorbent assay (ELISA), competitive ELISA, Dot-ELISA and Western blotting used to evaluate the binding between the phage-borne peptides and EBA-175. The insert DNA sequences of positive clones were determined and their amino acid sequences deduced.

RESULTSThirty clones from the third round were randomly selected, of which 27 were found positive by sandwich ELISA. Competitive ELISA proved that most of the phage-borne peptides could competitively inhibit the binding of antibody (EBA-175 Ab) with EBA-175. Analysis of DNA and amino acid sequences indicated that 24 positive phage clones contained the conservative sequence of IRR, which was highly homologous with the 114-116 amino acids of GPA.

CONCLUSIONThese phage-displayed peptides can bind with EBA-175, and the amino acid sequence IRR might play an important role in the binding between EBA-175 and GPA.

Antigens, Protozoan ; metabolism ; Binding Sites ; Enzyme-Linked Immunosorbent Assay ; Glycophorin ; chemistry ; Humans ; Peptide Library ; Plasmodium falciparum ; Protozoan Proteins ; metabolism ; Sequence Analysis, DNA ; Sequence Analysis, Protein

BACKGROUND: The Polycomb-group gene Bmi-1 is known to be a molecular regulator of self-renewal of normal and leukemic stem cells and be involved in various aspects of cellular proliferation, differentiation, and survival. METHODS: This study evaluated the effects of overexpression of Bmi-1 on human cord blood CD34+ cells. Bmi-1 was introduced into CD34+ cells through lentivirus transduction. Bmi-1 expressing CD34+ cells were applied to colony forming assay, stromal co-culture, and cytokine-stimulatied culture. RESULTS: Ectopic expression of Bmi-1 resulted in the increased number of erythroid colonies in primary and secondary colony forming assay in an erythropoietin dependent manner. In stromal co-culture, Bmi-1-expressing postnatal hematopoietic stem cells seemed to lose the ability of self-renewal, as determined by week 5 cobblestone area-forming cell assay and by week 5 secondary colony assay. In cytokine-stimulated suspension culture of Bmi-1-transduced CD34+ cells, we observed increased erythropoiesis marked by Glycophorin A expression. CONCLUSION: Our data suggest that ectopic expression of Bmi-1 in human hematopoietic stem/progenitor cells may result in the differentiation to the erythroid lineage rather than promoting self-renewal.
Cell Proliferation ; Coculture Techniques ; Erythropoiesis ; Erythropoietin ; Fetal Blood ; Glycophorin ; Hematopoietic Stem Cells ; Humans* ; Lentivirus ; Stem Cells

Erythroleukemic blast crisis of chronic myeloid leukemia (CML) is very rare. We report two cases of erythroleukemic blast crisis of CML resistant to imatinib treatment. Both patients made a rapid progression to blast crisis 6 and 4 months after diagnosis while being treated with imatinib 400 mg/day. Bone marrow aspiration revealed predominant erythroid precursors with 65.4% and 54.8% each. There were significant proportions (more than 20%) of myeloblasts among non-erythroid cells. Immunophenotyping revealed expression of glycophorin A confirming erythroleukemic blast crisis. The karyotyping result of patient 1 was 46,XX,t(9;22)(q34;q11.2)[3]/52,idem,+8,+12,+18,+21,+22,+der(22)t(9;22)[17] and that of patient 2 was 46,XX,inv(3)(q21q26.2),t(9;22)(q34;q11.2)[20]. Patient 1 showed no response to imatinib and BMS-354825 in the following bone marrow study. She died of septic shock as a complication of an infection after 69 days of blast crisis. Patient 2 received allogeneic bone marrow transplantation (BMT) in the cytogenetically no response state, but she also died of graft-versus-host disease 9 weeks after BMT. The poor prognosis and rapid progression of disease in both cases were correspondent to most of the reported cases. During the course of the disease of the two patients, we monitored the BCR-ABL chimeric mRNA with real-time quantitative polymerase chain reaction (RT-PCR), and it was found useful in predicting the imatinib response and progression to blast crisis of CML. Although both of our cases showed the typical bad prognosis and findings of erythroleukemic blast crisis of CML, the karyotypes were different from the expected type of t(3;21)(q26;q22). But the relationship between additional changes of EVI1 on chromosome 3q26 shown in case 2, and progression to the erythroleukemic blast crisis need further investigation.
Blast Crisis* ; Bone Marrow ; Bone Marrow Transplantation ; Diagnosis ; Glycophorin ; Graft vs Host Disease ; Granulocyte Precursor Cells ; Humans ; Immunophenotyping ; Karyotype ; Karyotyping ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive* ; Polymerase Chain Reaction ; Prognosis ; RNA, Messenger ; Shock, Septic ; Dasatinib ; Imatinib Mesylate

BACKGROUND: In this study, we attempted to generate RBCs from CD34+ cells in cord blood using a 3-step culture protocol and also evaluated a change in immunophenotypic characteristics and expression profile according to erythropoietin (EPO) concentrations and culture duration. METHODS: Using mini-MACS columns, CD34+ cells were isolated from cord blood. The culture procedure comprised three steps. For each step, cells were cultured sequentially for 7 days in a serum free liquid medium with a specific combination of growth factors for 21 days. [1st step: Flt3-ligand (Flt3-L), thrombopoietin and stem cell factor (SCF); 2nd step: IGF-1, SCF and EPO; and 3rd step: IGF-1 and EPO] To evaluate the effect of EPO on proliferation and differentiation, cells were cultured with different EPO concentrations (0, 3, 10 & 20 U/mL). Cell count and morphology were monitored during the culture. For phenotyping, antibodies to CD34, CD38, CD45 and glycophorin A (GPA) were used. The expression profile of cultured cells was analyzed by 17, 000-gene microarray analysis. RESULTS: As EPO concentration increased, cell expansion was also increased, showing a maximum expansion at 20 U/mL. The cell population showed a gradual decrease in expression of CD34 and CD45, whereas the expression of GPA was not prominent in any conditions. However, we observed increased expression in some genes associated with erythropoiesis (e.g. glycophorin A, rhesus blood group CcEe antigens). CONCLUSIONS: This study shows that erythropoietin enhances the proliferation of hematopoietic progenitor cells. Our culture system did not achieve pure production of RBCs, but induced expression changes that indicated erythroid differentiation.
Antibodies ; Cell Count ; Cells, Cultured ; Erythropoiesis ; Erythropoietin ; Fetal Blood* ; Glycophorin ; Hematopoietic Stem Cells ; Humans ; Insulin-Like Growth Factor I ; Intercellular Signaling Peptides and Proteins ; Microarray Analysis ; Stem Cell Factor ; Thrombopoietin