[Microsequencing, analysis of molecular weight and amino acid composition for pyrimidine 5'-nucleotidase I of human erythrocytes].

To further explore the mechanism of congenital pyrimidine 5'-nuleotidase I (P5'N-I) deficiency, on the basis of purification of the protein, the molecular weight and amino acid composition were analysed by mass-spectrograph and amino-acid analyzer, microsequencing and bioinformation analysis of P5'N-I were performed after it was hydrolysed by trypsin. The results showed that three fractions were found in the purified P5'N-I and their molecular weights were 26,952.9, 55,476 and 110,938, respectively. The sequence from one of the peptide fragments was I-E-G-P-T-I-R-Q-I-E. The homologous sequence was not found after comparision with the ten-amino-acid sequence in GenBank by blast procedure. Amino acid analysis indicated that P5'N-I was composed of 18 amino acids at least, and 243 amino acid residues. In conclusion, the enzyme might be an allosteric enzyme, there might be homologous dimer or tetramer in physiological status of normal human erythrocyte, the microsequence could be designed as the probe for fishing the genes of interest. The composition of amino acid might be an important information in determination of its protein primary structure.

[Combination of mycophenolate mofetil with cyclosporine A and methotrexate for the prophylaxes of acute graft versus host disease in allogeneic peripheral stem cell transplantation].

OBJECTIVE: To evaluate the efficacy of combination of mycophenolate mofetil (MMF) with cyclosporine (CsA) and methotrexate (MTX) on prophylaxes of acute graft-versus-host disease (GVHD) in HLA-matched allogeneic peripheral blood stem cell transplantation (allo-PBSCT). METHODS: Thirty-nine patients with acute leukemia (n = 21) and chronic myeloid leukemia (n = 17) and severe aplastic anemia (n = 1) were treated with allo-PBSCT from HLA matched siblings (n = 36) or unrelated donors (n = 3). Twenty-six patients were in CsA + MTX group. CsA was given at a dosage of 2 mg.kg(-1).d(-1) by continuous intravenous injection for 24 h, since on day(-1) and injection of CsA was changed to oral administration of CsA around day 18. CsA was tapered by 10% per week after day + 90. MTX was given at the dosage of 15 mg at day + 1, and 10 mg at day + 3, + 6 and + 11, respectively. Thirteen patients were included in MMF + CsA + MTX group with the same dosage of CsA and MTX as above but omitted at day + 11. MMF of 2 g/day was added orally from day + 1 to day + 28 post transplantation. RESULTS: All patients in both groups were successfully engrafted. The days of recovery of neutrophils and platelets were not significantly different between two groups (P > 0.05). The incidence of acute GVHD in MMF + CsA + MTX group (7.6%) was significantly lower than that in CsA/MTX group (46.2%, P < 0.05). Incidence of grade II approximately IV GVHD in MMF group was 0 while that in control group was 23.0%. The incidence of severe mucositis was lower in MMF group (15.4%) than in the control group (30.8%) (P < 0.05). CONCLUSION: The regimen of MMF + CsA + MTX for prevention of acute GVHD in allo-PBSCT is more efficient than that of CsA + MTX, without adversely affecting the engraftment and relapse rate.

[Establishment of Quantitative Assay for Human Erythrocyte Pyrimidine 5'-Nucleotidase Content by Double Antibody Sandwich ELISA]

For exploring pathogenesis of pyrimidine 5'-nucleotidase (P5'N) deficiency, a quantitative assay method for human erythrocyte pyrimidine 5'-nucleotidase was established. The specific substrate uridine monophosphate (UMP) of P5'N was used as ligand. The UPM-ADH-Sepharose 4B affinity column was prepared. P5'N of human erythrocyte was purified by ammonium sulfate fractionation and precipitation, ion chromatography and affinity chromatography. Rabbit anti-human P5'N antibody was acquired by immunizing rabbits with purified P5'N. Using rabbit anti-human antibody as the coated anti-body and HRP-rabbit anti-human antibody as demonstrated anti-body, the double antitody sandwich ELISA for quantitative assay of human erythrocyte P5'N was established after square rank trial, antigen blocked trial and antigen substituted test. Results showed that the titer of rabbit anti-human erythrocyte was 1:4 and the sensitivity of double antibody sandwich ELISA was 5 ng/ml. Its blocking rate was more than 95% and the rate of substitution less than 30%. The content of P5'N was (71.77 + 10.98) ng/mg NHP in normal human erythrocyte. A new ELISA method for quantitative determination of human erythrocyte P5'N was first established. It not only had high specificity and sensitivity but also could assay the minimun content of P5'N as 5 - 20 ng/ml. It could be a suitable method for large sample in clinics.

[Fetal Bone Marrow Stromal Cells in Cooperation with Exogenous Cytokines Support In Vitro Expansion of Cord Blood CD34(+) Cells]

To elucidate the role of fetal bone marrow stromal cells (FBMSC) in cooperation with exogenous cytokines in supporting the in vitro expansion of cord blood CD34(+) cells which were purified by negative immunomagnetic selection, FBMSCs were cultured with different combinations of cytokines including SCF, IL-3, IL-6, FL, G-CSF and EPO in a 14-day liquid culture system. The results showed FBMSC plus SCF, IL-3, IL-6, FL and EPO was the most effective combination which increased total nucleated cells, CFU-GM, BFU-E and CD34(+) cells by (692.4 +/- 52.7) fold, (237.1 +/- 106.6) fold, (114.8 +/- 32.8) fold and (25.3 +/- 10.1) fold, respectively. Our studies indicated that fetal bone marrow stromal cells combined with above-mentioned cytokines can efficiently expand cord blood CD34(+) cells.

[Advance in Reseach on Human Immunodeficiency Virus Type I Vector]

Human immunodeficiency virus type-I (HIV-I), one kind of lentiviruses, was characterized by a complex genome that encodes two regulatory proteins and four accessory proteins in addition to the common gag, pol and env gene products. So far, a few of different types of replication-defective vectors were constructed, the highest viral titer from one of which was above 10(7) TU/ml. Several studies on packaging cell line found that eliminating the four accessory genes would have few effect on transduction ability of vector and split-genome package system could reduce the possibility of producing replication-competent virus. There are two kinds of characters on HIV-I vectors. Firstly, it was highly efficient in transducing CD34(+) human hematopoietic stem/progenitor cells; secondly, repeated injections of the HIV-I vector into animal did not elicit the rejection response. HIV-I vector had an extensive host range.