OBJECTIVETo explore the clinical significance of genetic detection and changes of red cell enzyme activities of pyrimidine 5' nucleotidase (P5'N), pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G-6-PD) in patients with α-thalassaemia (α-thal).

METHODSThree α-thal patients were further processed to gene detection by PCR-trans-dot blot and gap-PCR, and red cell enzymes activities by absorbance at 260 and 280 nm (A) for P5'N and fluorescence spot test for PK and G-6-PD.

RESULTSRed cells in 3 α-thal cases were microcytic hypochromic with obvious augmented target cells and basophilic stippling erythrocytes. Two patients had anemia, splenomegaly, hyperbilirubinemia and augmented LDH. HbH was positively identified by hemoglobin electrophoresis and hemoglobin cellulose acetate membrane electrophoresis; the other patient had no such abnormalities. Genotypes of 3 patients were of (-α(3.7)/--(SEA)), (αα(QS)/--(SEA))and (--(SEA)), respectively. The activity of P5'N (but not for PK and G-6-PD) in red cell reduced.

CONCLUSIONSThis is the first documented α-thal with P5'N deficiency. Genetic detection might be clinical significant for the diagnosis and pedigree screening of α-thal.

5'-Nucleotidase ; deficiency ; Adolescent ; Adult ; Erythrocytes ; enzymology ; metabolism ; Female ; Humans ; Male ; Middle Aged ; alpha-Thalassemia ; enzymology ; genetics

The study was purposed to investigate the expression of CD73 on bone marrow nucleated cells (BMMNC) in various leukemia subtypes and its relationship with cell differentiation of leukemia. Immunocytochemistry staining and Wright-Giemsa staining of BMMNC from 75 cases of leukemia, 11 cases of myelodysplastic syndrome (MDS), 13 cases of non-leukemic patients and 9 healthy adults were performed, and the CD73(+) ratio in BMMNC and its relationship with differentiation of leukemia cells were analyzed. The results showed that the ratios of CD73(+) in BMMNC of com-B ALL, pre-B ALL and PLL were significantly higher than those in B-CLL (p < 0.05). CD73(+) ratios in AML subtypes of M(1), M(2a), t (8; 21), t (15; 17), M(4) and M(5) were markedly higher than those in MDS respectively, but in M(6) and MDS were lower and had no statistical difference between them. CD73(+) ratios in T-ALL, B-CLL, M(6), MDS, non-leukemia patients and healthy adults were close to each other and all of them were lower than those in B-ALL and other AML subtypes. It is concluded that the expression of CD73 is associated with leukemia subtype, differentiation and development. The higher differentiation of leukemia cells, the lower of CD73 expression in myeloid and B lymphoid leukemia, but T-ALL does not meet this pattern.
5'-Nucleotidase ; metabolism ; Adolescent ; Adult ; Cell Differentiation ; Humans ; Leukemia ; metabolism ; pathology ; Leukemia, Lymphocytic, Chronic, B-Cell ; metabolism ; Leukemia, Myeloid, Acute ; metabolism ; Myelodysplastic Syndromes ; metabolism ; Young Adult

This study was purposed to investigate the immunoregulatory effect of endothelial cells derived from mesenchymal stem cells (MSC). The human MSC was induced to differentiate into endothelial cells for one week. The phenotypes were evaluated by flow cytometry, the cell morphologic feature was observed by invert phase-contrast microscope and analysis of capillary formation was performed by using the in vitro angiogenesis kit. The immunoregulatory effect was detected by lymphocyte transformation test. The result indicated that during the differentiation cells grew fast and there was no significant change in the phenotypes, i.e. CD73, CD105, HLA-ABC were positive and CD34, CD80, CD86, HLA-DR, CD31 were negative. Immunofluorescence analysis showed typical expression of the von Willebrand factor. Differentiated MSCs formed capillary-like structure. Endothelial cells derived from MSC also revealed immunosuppressive effect on T cell proliferation in a dose-dependent manner. It is concluded that endothelial cells derived from MSC also harbor immunoregulatory effect on T lymphocytes.
5'-Nucleotidase ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Child ; Endothelial Cells ; cytology ; immunology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; T-Lymphocytes ; immunology ; von Willebrand Factor ; metabolism

OBJECTIVETo discuss the effect of protein kinase C (PKC) on regulation of ecto-5'-nucleotidase activity by lysophosphatidylcholine(LPC) in human umbilical endothelial cells (HUVEC).

METHODSExperiments were conducted in HUVEC grown on dishes which were divided into 4 groups (n=15): (1) Control group in which only eAMP (5 micromol/L) was added; (2) LPC group in which HUVEC were incubated with LPC (10 micromol/L) before eAMP was added; (3) Chelerythrine group in which cells were pre-incubated with the PKC inhibitor chelerythrine (100 micromol/L) before LPC and eAMP were added; (4) alpha, beta-Methyladenosine-5'-Diphosphate (AOPCP) group in which cells were incubated with AOPCP (10 micromol/L) before eAMP was added. Etheno-adenosine production was detected at 15th, 30th, 45th min with high performance liquid chromatography(HPLC) respectively.

RESULTSComparing to the control group LPC significantly increased etheno-adenosine production at three time points respectively (P < 0.05). Furthermore, PKC inhibitor chelerythrine abolished this effect of LPC and the ethenoadenosine production at three time points were at the same level of control group (P > 0.05). CD73 inhibitor AOPCP significantly decreased the etheno-adenosine production compared to the other three groups (P < 0.01).

CONCLUSIONEcto-5'-nucleotidase can be modulated within minutes following exposure of HUVEC to LPC and this response may be mediated by PKC in HUVEC.

5'-Nucleotidase ; metabolism ; Cells, Cultured ; GPI-Linked Proteins ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Lysophosphatidylcholines ; pharmacology ; Protein Kinase C ; physiology ; Up-Regulation ; drug effects

5' nucleotides (5'NT), a purine degradative enzyme, is capable of hydrolyzing nucleotide and acting as a phosphotransferase simultaneously. It has critical role in maintaining nucleotide metabolism balance. The present study was aimed to investigate the expression of 5'NT in bone marrow granulocytes (BMGs) from patients with acute myeloid leukemia (AML) and healthy donors comparatively. The BMGs were isolated from bone marrow of 33 patients with AML and 6 healthy donors by using lymphocyte isolating solution. The reactivity of 5'NT was detected by electron microscope and cytochemistry of cytidine monophosphate (CMP). The positive BMG ratio and their index were calculated on the base of ultrastructural observation semiquantitatively. The results indicated that electron microscopy revealed plasma membrane reacting pattern of CMP. Most BMGs from normal donors were CMP negative or exhibited lower active degree. All cases of M(0), M(1), M(2) and t (8; 21) showed high positive percentages and high indexes of BMGs, but no statistic differences between them. APL of t (15; 17) shared lower percentages and indexes than other subtypes. There was no significant difference between APL and normal donors statistically. In conclusions, the results suggested the expression of 5'NT may be associated with BMG differentiation in AML, and APL of t (15; 17) may be a highly differentiated leukemia subtype.
5'-Nucleotidase ; metabolism ; ultrastructure ; Adolescent ; Adult ; Aged ; Bone Marrow Cells ; cytology ; enzymology ; Child ; Female ; Granulocytes ; enzymology ; Humans ; Leukemia, Myeloid, Acute ; classification ; enzymology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo investigate the expression level and analyze the clinical significance of NT5C2, which is an nucleoside analogues metabolism related gene, in children with acute leukemia (AL).

METHODSReal-time PCR and immunohistochemistry were presented to detect the level of NT5C2 mRNA and its protein product cN- Ⅱ in bone marrow samples of 63 patients initially diagnosed with AL, 15 patients who achieved complete remission, 7 patients who relapsed and 16 non- hematologic malignancie controls. The expression of NT5C2 mRNA in different groups of AL and its relevance with clinical indicators were analyzed.

RESULTS①The expression of NT5C2 mRNA in newly diagnosed B-ALL, TALL, AML and controls were 1.16 (0.89-2.25, 0.96 (0.74-1.25, 1.66 (0.84-3.15) and 0.88 (0.61-1.21), respectively. NT5C2 mRNA expression in AML (P<0.01) and B-ALL (P<0.05) cases were higher than that in controls; NT5C2 mRNA expression in T- ALL and in controls showed no significant difference (P>0.05). Changes of NT5C2 mRNA level were observed between preliminary diagnosis and complete remission in 15 patients. NT5C2 mRNA levels were significantly decreased in complete remission stage than that in newly diagnosis AL (P<0.01). NT5C2 mRNA levels of relapsed-refractory group were higher than that of complete remission group and controls (P<0.01). ② Immunohistochemical staining results revealed that NT5C2 protein levels were consistent with the trend of mRNA levels. ③NT5C2 mRNA levels in AML (r=0.434) and T-ALL (r=0.389) were positively correlated with risk classification (P<0.05). ④ During chemotherapy of patients with AML, the NR rate of bone marrow in NT5C2 high expression group was higher than that of low expression group after 9 days induction chemotherapy (35.2% vs 0) and before consolidation therapy (25.0% vs 0); The positive rate of minimal-residual disease (36.4% vs 14.3%) and relapse rate of AL (38.5% vs 28.6%) were increased in NT5C2 high expressed patients than that in low expressed patients, but all the differences were insignificant (P>0.05).

CONCLUSIONHigh expression of NT5C2 was found to be a related risk factor of AL children with unfavourable prognosis. NT5C2 promises a new target for guiding individualized chemotherapy and evaluating the prognosis of childhood acute leukemia and monitoring recurrence.

5'-Nucleotidase ; metabolism ; Bone Marrow ; metabolism ; Child ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; Neoplasm, Residual ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; Prognosis ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Recurrence ; Remission Induction

The study was aimed to establish a protocol of isolating and culturing adult mesenchymal stem cells (MSC) from human bone marrow aspirate and identify them by surface antigen analysis and committed differentiation in order to provide an experimental foundation for achieving a therapeutic benefit in applying MSC in hematopoietic stem cell transplantation. MSCs were obtained from fresh human bone marrow aspirate by gradient centrifugation with Percoll (1.073 g/ml) and anchoring culture in L-DMEM with 10% fetal bovine serum by a full medium exchange every 3 days. The MSC surface antigens, including CD34, CD45, CD73, CD105, CD166, were analyzed on FACScan flow cytometer. Under culture in conditioned medium for osteogenesis (the hormone cocktail containing 0.1 micromol/L dexamethasone, 10 mmol/L glycerol-2-phosphate and 50 micromol/L ascorbic acid) and adipogenesis (the cocktail containing 1 micromol/L dexamethasone, 5 mg/L insulin, 0.5 mmol/L 1-methyl-3-isobutylxanthine and 60 micromol/L indomethacin), MSCs committedly differentiated into osteoblasts and adipocytes. The differentiated mesenchymal stem cells were identified by morphological observation and immunohistochemical staining. The results showed that by gradient centrifugation and adhesion culture, MSCs could be isolated and culture-expanded from human bone marrow aspirate. These cells were uniformly negative for CD34, CD45 and positive for CD73, CD105 and CD166. The osteogenic differentiated cells were positive for alkaline phosphatase (ALP) and the adipogenic differentiated cells displayed accumulation of lipid vacuoles, as detected by oil red O. It is concluded that MSC can be isolated and expand-cultured from adult human bone marrow aspirate and committedly differentiate into osteoblasts and adipocytes. MSC primary identification can be accomplished by flow cytometry and induced differentiation. The set of methods in current experiment shows somewhat practical value for basic research and clinical application.
5'-Nucleotidase ; metabolism ; Antigens, CD ; metabolism ; Bone Marrow Cells ; cytology ; Cell Adhesion Molecules, Neuronal ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Separation ; methods ; Endoglin ; Fetal Proteins ; metabolism ; Humans ; Mesenchymal Stromal Cells ; cytology ; Receptors, Cell Surface ; metabolism

AIMTo identify possible spermicidal agents through screening a number of edible medicinal plants with antimicrobial activity.

METHODSInitial screening was made on the basis of ram cauda epididymal sperm immobilization immediately after addition of extracts. The most potent extract was selected and was evaluated on both ram and human spermatozoa. To unravel its mode of action several sperm functional tests were carried out, namely viability of cells, hypo-osmotic swelling test for membrane integrity and assays of membrane-bound enzyme 5'-nucleotidase and acrosomal marker enzyme acrosin.

RESULTSThe crude aqueous extract of the bulb of Allium sativum L. showed the most promising results by instant immobilization of the ram epididymal sperm at 0.25 g/mL and human ejaculated sperm at 0.5 g/mL. Sperm immobilizing effects were irreversible and the factor of the extract responsible for immobilization was thermostable up to 90 deg. On boiling at 100 deg for 10 minutes, this activity was markedly reduced. Moreover, this extract was able to cause aggregation of ram sperms into small clusters after 30 minutes of incubation at 37 deg. However this property was not found in human spermatozoa. More than 50 % reduction in sperm viability and hypo-osmotic swelling occurred in treated sperm as compared with the controls, indicating the possibility of plasma membrane disintegration which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase and acrosomal acrosin.

CONCLUSIONThe crude aqueous extract of A. sativum bulb possesses spermicidal activity in vitro.

5'-Nucleotidase ; metabolism ; Animals ; Cell Aggregation ; drug effects ; Cell Membrane ; drug effects ; enzymology ; physiology ; Drug Stability ; Garlic ; chemistry ; Humans ; Male ; Plant Extracts ; chemistry ; pharmacology ; Sheep ; Sperm Motility ; drug effects ; Spermatozoa ; drug effects ; physiology

PURPOSE: This study examined a rapid isolation method decreasing the time and cost of the clinical application of adipose tissue-derived stem cells (ASCs). MATERIALS AND METHODS: Aliquots (10 g) of the lipoaspirates were stored at 4degrees C without supplying oxygen or nutrients. At the indicated time points, the yield of mononuclear cells was evaluated and the stem cell population was counted by colony forming unit-fibroblast assays. Cell surface markers, stem cell-related transcription factors, and differentiation potentials of ASCs were analyzed. RESULTS: When the lipoaspirates were stored at 4degrees C, the total yield of mononuclear cells decreased, but the stem cell population was enriched. These ASCs expressed CD44, CD73, CD90, CD105, and HLA-ABC but not CD14, CD31, CD34, CD45, CD117, CD133, and HLA-DR. The number of ASCs increased 1x1014 fold for 120 days. ASCs differentiated into osteoblasts, adipocytes, muscle cells, or neuronal cells. CONCLUSION: ASCs isolated from lipoaspirates and stored for 24 hours at 4degrees C have similar properties to ASCs isolated from fresh lipoaspirates. Our results suggest that ASCs can be isolated with high frequency by optimal storage at 4degrees C for 24 hours, and those ASCs are highly proliferative and multipotent, similar to ASCs isolated from fresh lipoaspirates. These ASCs can be useful for clinical application because they are time- and cost-efficient, and these cells maintain their stemness for a long time, like ASCs isolated from fresh lipoaspirates.
5'-Nucleotidase/metabolism ; Adipose Tissue/*cytology ; Adult ; Antigens, CD/metabolism ; Antigens, CD44/metabolism ; Antigens, Thy-1/metabolism ; Cell Differentiation/physiology ; Cells, Cultured ; Female ; Humans ; Immunoblotting ; Immunohistochemistry ; Immunophenotyping ; Mesenchymal Stem Cells/metabolism ; Muscle Development/genetics/physiology ; Osteogenesis/genetics/physiology ; Receptors, Cell Surface/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells/*cytology/metabolism ; Young Adult