Methylation of p15INK4B gene and its mechanism in patients with myelodysplastic syndrome and leukemia

AIM: To explore the relationship between hypermethylation of p15INK4B gene and the pathogenesis of hematopoietic malignances. METHODS: The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in bone marrow cells from 54 cases with hematopoietic malignances were detected by RT-PCR, Western blot, and methylation-specific PCR. RESULTS: The p15INK4B gene was methylated more often in high-risk myelodysplastic syndrome (MDS) patients, patients at blast phase of chronic myeloid leukemia (CML-BP) and acute leukemia patients than that in low-risk MDS patients (P<0.01). The expression levels of DNMT_3A and DNMT_3B in acute leukemia patients, high-risk MDS patients, and CML-BP patients were higher than that in low-risk MDS patients (P<0.05). CONCLUSION: The hypermethylation of p15INK4B gene may be one of the most common genetic event in pathogenesis of acute leukemia, high-risk MDS, and blast phase of chronic myeloid leukemia. Furthermore, DNMT_3A and DNMT_3B are substantially over-expressed in the bone marrow cells of these patients.     

Relationship between environmental factor and maximum daily stem shrinkage in apple tree in arid region of northwest China

After measuring maximum daily stem shrinkage (MDS) of irrigated apple tree using dendrometer during the year 2007–2010 in arid region of northwest China, we analyzed the respective relationships between MDS and single plant physiological index and environmental factors to investigate whether MDS can indicate the water status of apple tree and to establish empirical multiple regression equation among MDS and environmental factors. Results show that MDS increased at the beginning and then decreased gradually during whole growing stage. The close relationships between MDS and stem water potential, predawn water potential, stomatal conductance were found, showing that MDS can indicate the water status of mature apple tree. The relationships between MDS and single meteorological variable were significant at the other growth stages except at the bud development and flowering stage, because the canopy structure was not developed, and the order of determination coefficient (r²) over the whole growing stage was maximum vapour pressure deficit > maximum air temperature > net radiation. There were also significant correlations between MDS and soil volumetric water content and reference crop evapotranspiration over the whole growing stage. However, the determination coefficient among MDS and meteorological variables and soil volumetric water content at 0–120 cm depth was higher than those between MDS and single variable. Thus the multiple regression equation among MDS and meteorological variables and soil volumetric water content at 0–120 cm depth can be used to estimate MDS under fully irrigated apple orchard.     

Study on methylation of p15 INK4B in myelodysplastic syndrome

AIM: To investigate the variation and distribution of abnormaly methylated p15 INK4B in myelodysplastic syndrome (MDS) and its subgroups. METHODS: The abnormal methylation of p15 INK4B in 32 cases with MDS was studied using methylation-specific PCR (MSP). RESULTS:The positive rate of abnormal methylation of p15 INK4B was about 50% in MDS. The ratios in subtypes were: 0% (0/6) in RA,20% (1/5) in RA-S,57.1% (4/7) in RAEB,74.1% (5/7) in CMML,85.7% (6/7) in RAEB-t, respectively.It was worth noticing that 4 cases represented abnormal methylation of p15INK4B during their transformation and progression into AML. CONCLUSION:The abnormal methylation in p15 INK4B might be one of the causes of MDS, which was related to pathologial process of MDS.Every subtype was not solitary classification completely. Abnormal methylation of p15 INK4B was apt to occur accompanying the progression and transformation of the subtypes.     

Interaction of Maximum Daily Trunk Shrinkage and Fruit Quality in European Plum

Maximum daily trunk shrinkage (MDS) has been suggested as an appropriate indicator of plant water status because it is closely related to stem water potential. Interaction of MDS and fruit quality was studied in plum (Prunus domestica L. ‘Jojo’/Wavit and ‘Tophit plus’/Wavit) in temperate climate. According to the MDS data, trees were grouped as low MDS (LMDS) and high MDS (HMDS). Fruit quality was analysed during fruit development (95, 103, 117 DAFB for ‘Jojo’ and 99, 112, 121 DAFB for ‘Tophit plus’) before commercial harvest. Fruit picked at commercial harvest (137 DAFB and 140 DAFB for ‘Jojo’ and ‘Tophit plus’, respectively) were stored at 2 ± 0.5?°C (90 ± 2% RH) for 28 days, and 2 days shelf life at 20?°C providing 6 measuring dates postharvest. Results confirmed that MDS was positively correlated with water vapour pressure deficit also in the apparent temperate, semi-humid climate. Transpiration of fruit from high crop load and resulting HMDS trees, which can be assessed as physiologically drought, was low compared to that of fruit from LMDS trees. Furthermore, HMDS tree grown plums had enhanced soluble solids and dry matter contents with a tendency of reduced fruit size.     

The application of atomic force microscope in clinic diagnosis of erythrocytes

AIM: To investigate the application of atomic force microscope (AFM) in clinic diagnosis. METHODS: Topographic images and some parameters of large range field and microstructures of erythrocytes in the blood of normal subjects, lung cancer and myelodisplastic syndrome (MDS) patients were examined by atomic force microscope. RESULTS: Many clear topographic images of many erythrocytes, single erythrocyte, and microstructure of erythrocyte membrane surface were obtained. Many erythrocytes in lung cancer patients were found to change into echinocytes. One erythrocyte had 10-20 protuberances, most of which, with a mean width of 589. 0 nm and a length of 646. 7 nm, were on the edge of cells. The protuberances on the center of echinocytes are lodged and embedded. The erythrocytes of MDS patients were biconcave in shape. Many apertures with different diameters of tens to hundreds nanometer appeared on the surface of cell membrane. CONCLUSIONS: AFM can be widely applied in clinic pathological inspection, including quantification of cells, obtainment and comparison of many parameters (such as diameter, thickness, volume, surface, surface area/volume ratio), observation of topograph of single cell, and observation and comparison of membrane surface microstructure of cells, and so on.     

植物器官体积变化连续测微法指导果树自动化灌溉合理指标的研究

对盆栽葡萄（VitisviniferaL．）、苹果（MaluspumilaMill．）、山楂（CrataeguspinnatifidsBunge）和枣（ZiziphusjnyubaMill．）4种果树在不同水分营养状况下茎于微变化过程中的日最大收缩量（MDS）、日净增长量（DG）、当日完全复原所需时间（RT）等3个重要指标进行研究，结果表明：3个指标都比叶片日出前水势对土壤水分营养状况的反应灵敏得多；随着土壤可利用水分的不断减少，山楂和枣的MDS急剧增加，而葡萄和苹果的MDS减小，不同类型果树之间的反应不一致；在土壤水分状况较好时，4种果树的DG的变化平缓，变化幅度较小；在良好的土壤水分条件下，4种果树的RT随着土壤相对持水量的下降而迅速延长，并在土壤水分含量降低到一临界值以下时维持在24h。因此，在应用植物器官体积变化连续测微法指导果树灌溉时，MDS和DG并不适于单独作为所有果树的自动化灌溉指标，而RT作为果树自动化灌溉指标较为理想。     

Morphology and growth capacity of bone marrow stromal cells in vitro from APBSCT patients post pretreatment

AIM: To investigate the morphology and capacity of bone marrow colony forming unity-fibroblast (CFU-F) from APBSCT patients before and after pretreatment. METHODS: 21 case peripheral blood stem cell transplantation (PBSCT) patients were treated with pretreatment. The changes of morphology of the bone marrow stromal cells were assayed by light microscope and electron microscope, respectively. The numbers of CFU-F were assayed by Dexter type. RESULTS: The bone marrow stromal cells occured different types of morphology from PBSCT patients treated with chemotherapy or chemotherapy-TBI pretreatment, respectively, compared with controls. The transmission electron microscope showed that the endoplasmic reticula was dilated, the matrix of mitochondria appeared pale and the cristae of mitochondria became shorter in stromal cells from chemotherapy-TBI patients compared with those of controls. The structure of mitochondria from combined chemotherapy-TBI pretreatment appeared severe degeneration and disorder. The numbers of CFU-F from combined radiation-chemotherapy injury were significantly decreased compared with that before pretreatment and the chemotherapy injury (P<0.01), respectively. CONCLUSION: The change of cell morphology and capacity of CFU-F for bone marrow stromal cells is one of impairment injury mechanism of bone marrow hematopoietic inductive microenvironment from PBSCT patients post pretreatment.     

Phenology and drought affects the relationship between daily trunk shrinkage and midday stem water potential of peach trees

Summary

The relationship between maximum daily shrinkage in trunks (MDS), daily trunk growth (DTG), predawn water potential (Ψ_pd) and midday stem water potential (Ψ_stem) were studied in an irrigation experiment in peach trees. Control trees were irrigated to replace evapotranspiration, with trees receiving regulated deficit irrigation (RDI) watered at 35% of this rate during Stage II of fruit development and after harvest. The RDI trees were watered as controls during Stage III of fruit development. Minimum (Ψ_pd and Ψ_stem fell to –0.6.MPa and –1.2 MPa, respectively in RDI plots compared with –0.2 and –0.6 MPa in the controls. Trunk growth was less in the RDI plots than in the controls during drought. In contrast, MDS was higher when deficit irrigation was applied in the RDI trees. When site differences were considered the correlation between (Ψ_pd and accumulated trunk growth over an ample period was loose, while maximum daily shrinkage and midday stem water potential remarkably improved such a correlation. However, pooling all available data, the correlation between Ψ_stem and MDS was very poor (R²=0.44) and it substantially improved only when using data from specific phenological periods (i.e. R²=0.75). A seasonal drift in MDS values was observed and it was related to the seasonal changes in trunk growth rates, (i.e. highest shrinkage was found when growth rates were lowest). We concluded that phenology in combination with drought reduce the reliability of the water status information obtained from MDS.     

Bone marrow hematopoietic microenvironment in patients with systemic lupus erythmatosus

AIM: To investigate bone marrow hematopoietic microenvironment in patients with systemic lupus erythmatosus (SLE), and to explore the pathogenesis of SLE.METHODS: Bone marrow stroma cells were collected from SLE patients.Colony forming units of stromal cells, and expression of fibronectin, laminin and type IV collagen, adhesive molecules, and some cytokines were detected by cell culture, immunohistochemistry, flow-cytometry, ELISA, and RT-PCR assay, respectively.RESULTS: The number of the colony forming units of stromal cells and their morphology in SLE group were the same as the control group (P>0.05).We did not find any difference of the expression of fibronectin, laminin and type IV collagen in them.Expression of ICAM and VCAM were (56.4±14.8)% and (55.6±12.2)%, respectively, obviously higher than those in control group (P<0.01).IL-6 in bone marrow stromal cell culture suspension of SLE was higher than that in control group (P<0.01).SCF and SDF-1 were not different between two groups (P<0.05).MIP-1 and IFN-γ in SLE patients were obviously higher than those in control group (P<0.01).TGF-β was on the opposite (P<0.01).The later three cytokines were correlated to SLEDAI score (P<0.01).CONCLUSION: Bone marrow microenvironment of SLE patients was deficient in the expression of cell surface adhesion molecules and cytokine secretion, which contributed to the pathogenesis of SLE.     

Relation between telomerase activity and chronic myelocytic leukemia course

AIM: Studying the relation between telomerase activity and chronic myelocytic leukemia(CML) courses by detect telomerase activity(TMA) of mononuclear cells of bone marrow samples in different CML phases. METHOD: Using telomerase PCR-ELISA method. RESULTS: Telomerase activity of normal bone marrow is 9.85±0.68, CML chronic phase is 24.48±12.73 which is significantly higher than that of normal bone marrow(P＜0.05). CML accelerated phase TMA is 76.76±21.84 and blast crisis is 90.62±25.41, both are significantly higher than that of chronic phase(P＜0.001, P＜0.001)but there is no significant difference between accelerated phase and blast crisis(P＞0.05), at remisson TMA is 18.12±6.27 which is still higher than normal group(P＜0.05).CONCLUTIONS: Telomerase activity in different phases of CML is significantly higher than normal bone marrow group. When telomerase is extremely activated , it means that CML patients were entering the accelerated phase. Telomerase activity might be used as an useful new marker in detecting and curing CML.     

Inhibitory effects of Ginkgolide A on stress ulceration in rats

AIM: Studying the relation between telomerase activity and chronic myelocytic leukemia(CML) courses by detect telomerase activity(TMA) of mononuclear cells of bone marrow samples in different CML phases. METHOD: Using telomerase PCR-ELISA method. RESULTS: Telomerase activity of normal bone marrow is 9.85±0.68, CML chronic phase is 24.48±12.73 which is significantly higher than that of normal bone marrow(P＜0.05). CML accelerated phase TMA is 76.76±21.84 and blast crisis is 90.62±25.41, both are significantly higher than that of chronic phase(P＜0.001, P＜0.001)but there is no significant difference between accelerated phase and blast crisis(P＞0.05), at remisson TMA is 18.12±6.27 which is still higher than normal group(P＜0.05).CONCLUTIONS: Telomerase activity in different phases of CML is significantly higher than normal bone marrow group. When telomerase is extremely activated , it means that CML patients were entering the accelerated phase. Telomerase activity might be used as an useful new marker in detecting and curing CML.     

水杨酸和高温锻炼与葡萄抗热性及抗氧化的关系

 对‘京秀’葡萄(Vitis vinifera CV．Jingxiu)高温锻炼和外施水杨酸(salicylic acid，SA)都能显著提高其幼苗的抗热性。高温锻炼1 h，叶片内自由态sA含量急剧升高，其后又迅速下降。在抗热性诱导过程中(高温锻炼12 h或水杨酸喷施后6 h)，抗氧化酶系统中的过氧化物酶(POD)、超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GR)、抗坏血酸过氧化物酶(APX)的活性都明显升高，但过氧化氢酶(CAT)活性下降；高温锻炼或外施SA 1 h，过氧化氢(H2o2)含量急剧升高，之后又迅速下降，可能起着一种信号分子的作用。以上结果说明内源sA可能通过提高抗氧化酶活性参与了高温锻炼过程，外施sA和高温锻炼有相似的提高抗热性机制。     

Establishment of a human chronic myeloid leukemia-like mouse model

AIM：To establish a new model of chronic myeloid leukemia (CML) for further studying the pathogenesis mechanisms and discovering new therapeutic targets of this disease. METHODS：The retroviral packaging technique was improved by increasing retroviral titer with higher-quality plasmid, better cell state and appropriate cell seeding density for further studying. Donor BALB/c mice were pretreated with 5-fluorouracil (5-FU), and their bone marrow cells were infected with p210 BCR/ABL retroviral supernatant (BCR/ABL group) or empty retroviral vector (MigR1) supernatant (MigR1 group). Infected bone marrow cells were transplanted into lethally irradiated recipient BALB/c mice via tail vein. The activities of the mice were observed after bone marrow transplantation (BMT). The morphology of peripheral blood cells and bone marrow cells, and the pathological changes of the liver and spleen in dying mice were also determined. RESULTS：Retroviral packaging efficiency was improved by optimizing the experimental conditions with higher-quality plasmid, better cell state and appropriate cell seeding density. All mice in BCR/ABL group died at 19 to 25 d after BMT. Their myelocytes, metamyelocytes and mature granulocytes in peripheral blood and bone marrow were abundant. Hepatosplenomegaly and granulocyte infiltration in the liver and spleen were also observed. All mice in MigR1 group were normal. CONCLUSION: We have successfully set up a mouse model of CML by increasing retroviral titer with improved retroviral packaging technique, transfecting BCR/ABL into mouse bone marrow cells in vitro and transplanting the cells to the recipients of the same lineage.     

Differentiation of embryonic-like stem cells derived from human bone marrow into multinucleated fibers in vitro

AIM: To compare the capacity of in vitro differentiation into multinucleated fibers between embryonic-like stem cells (ELSCs) and mesenchymal stem cells (MSCs) derived from human bone marrow. METHODS: To isolate ELSCs, human bone marrow mononuclear cells were cultured in gelatin-coated flask with serum-free Knockout-DMEM medium designed for the expansion of human embryonic stem cells. MSCs were isolated from the same bone marrow by the traditional method. The morphological characters of both ELSCs and MSCs were observed under inverted phase-contrast microscope, and the expression of their multipotent antigen markers was identified by immunofluorescent staining. ELSCs and MSCs were cultured in myogenic differentiation medium. The protein levels of muscle-specific antigen markers myosin heavy chain (MHC), myogenin and MyoD were detected by the method of immunostaining. The mRNA expression of MHC, myogenin and MyoD was detected by RT-PCR. The capacity of in vitro differentiation into multinucleated fibers was compared between ELSCs and MSCs by calculating the proportion of MHC-positive multinucleated fibers. RESULTS: ELSCs, which weakly expressed the multipotential markers Oct-4, Nanog-3 and Sox-2, were isolated from bone marrow by the method of serum-free medium. ELSCs appeared smaller, slenderer and more homogeneous, and were morphologically different from MSCs derived from the same marrow. No multipotential marker in MSCs was expressed. ELSCs and MSCs were induced into long multinucleated fibers expressing MHC and myogenin at mRNA and protein levels by culturing in the myogenic differentiation medium. However, on the 10th day after induction, the proportion of the MHC-positive fibers in ELSCs was (25.7?4.1)%, and the proportion in MSCs was (15.8?7.6)%.The capacity for differentiation into muscle in ELSCs was significantly higher than that in MSCs (P<0.05). CONCLUSION: Bone marrow ELSCs are induced into multinucleated fibers and have the stronger myogenic differentiation capacity than MSCs derived from the same marrow. ELSCs are a more ideal candidate for muscular disease therapy.     

Study on bone marrow angiogenic mediators and inhibitors in aplastic anemia

AIM: Through detecting bone marrow angiogenic mediators and inhibitors in aplastic anemia (AA) patients,the value of angionesis in AA pathogenesis was elucidated.METHODS: The patients were divided into severe AA group (SAA,8 patients),non severe AA group (NSAA,10 patients),and normal control group (7 persons),5 patients were observed before treating (group beginning) and getting improvement (group improving).The angiogenic mediators vascular endothelial growth factor (VEGF) and bFGF were detected by ELISA,angiogenic inhibitors IFN-γ and TSP were detected by ELISA and flow cytometry,respectively.RESULTS: The levels of VEGF were lower in SAA group and NSAA group than those in control group significantly (P<0.05),the levels of IFN-γ and TSP were higher than those in control group (P<0.05),especially in SAA group (P<0.01).Compared with group beginning,the level of VEGF was higher in group improving (P<0.05),the levels of IFN-γ,TSP were lower (P<0.05),there was no obviously difference between group beginning and group improving except IFN-γ.CONCLUSION: The dropping of angiogenic mediators and the rising of angiogenic inhibitors may be one reason of reducing the number of microvessel,which result in deficiency in supporting hemapoietic stem cells by bone marrow microenvironment.     

Protein expression of TIMP3 and RUNX3 in bone marrow mononuclear cells from acute leukemia patients

AIM: To detect the protein expression of TIMP3 and RUNX3 in bone marrow mononuclear cells (BMMCs) from acute leukemia (AL) patients and to investigate the relationship between the methylation status of genes and their expressional levels. METHODS: Protein expression of TIMP3 and RUNX3 in 50 samples of BMMCs and 10 samples of peripheral blood mononuclear cells (PBMCs) from healthy volunteers was detected by Western blotting. The prognostic factors related to AL and data from methylation specific polymerase chain reaction were also analyzed. RESULTS: The expression level of RUNX3 with methylation was less than that without methylation in BMMCs from AL patients. The complete remission (CR) rate was related to RUNX3 expression and blasts in bone marrow (BM). BMMCs from patients with silencing of RUNX3 and higher blasts in BM had a lower CR rate. CONCLUSION: Absence of RUNX3 protein expression resulting from methylation of RUNX3 promoter probably plays a role in the pathogenesis of AL and is of value in prognosis. No relationship between methylation of TIMP3 promoter and the pathogenesis of AL is observed.     

Effects of cytomegalovirus on the adhesion of bone marrow stromal cells

AIM:To investigate the effect of cytomegalovirus (CMV) on the expression of ICAM-1 and VCAM-1 in bone marrow stromal cells and on the adhesion of bone marrow stromal cells to hematopoietic cells.METHODS:Flow cytometry was used to detect the expression of adhesion molecules ICAM-1 and VCAM-1 in bone marrow stromal cells, MTT method was used to perform the adhesion assay of bone marrow stromal cells to normal hematopoietic cells. RESULTS:Bone marrow stromal cells could be infected by the CMV used in this experiment; CMV below the dose of 100TCID₅₀ could not destroy bone marrow stromal cells apparently; The expression of ICAM-1 increased at the early stage(18 h) of CMV infection, the expression of ICAM-1 decreased at the late stage (120 h) of CMV infection. Inactived CMV could also increase the expression of ICAM-1 as alive CMV; The adhesion rate of bone marrow stromal cells to hematopoietic cells increased at the early stage of CMV infection. CMV had no significant effects on the expression of VCAM-1 in bone marrow stromal cells. CONCLUSION:The adhesion capacity of bone marrow stromal cells to hematopoietic cells increased at the early stage of CMV infection, while the adhesion capacity of bone marrow stromal cells to hematopoietic cells decreased at the late stage of CMV infection.     

Expression of SCL gene in bone marrow stromal cells from normal individuals and patients with aplastic anemia

AIM: To investigate the expression of SCL (stem cell leukemia) gene in bone marrow stromal cells (BMSCs) and bone marrow hematopoietic cells from patients with aplastic anemia （AA） and normal individuals. METHODS: Bone marrow stromal cells from AA (9 cases) and normal individuals (33 cases) were amplified by long-term in vitro culture. The adherent and nonadherent cells were collected respectively. RT-PCR-ELISA assay was then performed to detect the expression of SCL gene and the housekeeping gene β2 microglobulin (β2M). The expression ratio of SCL gene were analyzed and its expression level was normalized by β2M gene acting as an internal calibration for the purpose of semi-quantitative analysis. RESULTS: The expression ratio of SCL gene was lower in BMSCs from AA (22.2%) than that in normal controls (69.7%, P<0.05) and in the nonadherent cells from AA than that in their corresponding BMSCs (P<0.05). Semi-quantitative analysis by PCR-ELISA showed that SCL gene expression level in nonadherent cells from normal control was significantly higher than that in their corresponding BMSCs (P<0.05). CONCLUSION: The state of low expression of SCL gene in BMSCs from AA suggests that it may be involved in the abnormal regulation of hematopoiesis in AA.