The mechanism of apoptosis induced by homoharringtonine in HL-60 cells

AIM: To study the signal transduction pathway of apoptosis initiation induced by homoharringtonine in HL-60 cells. METHODS: After establishing the model of apoptosis initiation induced by homoharringtonine in HL-60 cells, at the point of apoptosis initiation, molecular caspase-3, Bcl-2, Bax and Fas/FasL were measured with flow cytometry and transmission electron microscope. ERK2 and P38 expression in HL-60 cells were detected by using immunohistochemistry. RESULTS: The model of apoptosis initiation induced by homoharringtonine was established in HL-60 cells. At the point of apoptosis initiation, upregulation of caspase-3 and decrease in Bcl-2/Bax were observed. However, the expression of Fas/FasL did not significantly change. ERK2 expression decreased and P38 expression increased. CONCLUSIONS: Caspase-3, Bcl-2, Bax and mitogen activated protein kinase pathways were involved in signal transduction of apoptosis initiation induced by homoharringtonine in HL-60 cells.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of HL60 and K562 cells to daunorubicin

AIM:To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin.METHODS:IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation.RESULTS:It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly (P＜0.05). Compared to the antisense oligonucleotide directed against the translation initiation of bcl-2 mRNA, the antisense oligonucleotide directed against the coding region showed stronger effects in the aspects of increasing the sensitivity of HL60 cells to daunorubicin (P＜0.05).CONCLUSIONS:These two antisense sequences in the translation initiation and the coding region of bcl-2 mRNA increased the sensitivity of HL60 and K562 cell lines to daunorubicin in a sequence-specific manner.     

Effects of Tribulus terrestris L. saponin on apoptosis and changes in cytosolic calcium induced by hypoxia/reoxygenation in rat cortical neurons

AIM: To study the effect of Tribulus terrestris L. saponin (TTLS) on apoptosis and changes in cytosolic calcium concentration induced by hypoxia/re-oxygenation in rat cortical neurons. METHODS: Rat cortical neurons in primary culture were used, and a apoptosis model was induced by hypoxia/reoxygenation. LDH releasing rate was detected by spectrophotometry. The apoptosis rate of cortical neurons was analyzed quantitatively by flow cytometry with Annexin V-FITC and PI staining. Intracellular free Ca2+(［Ca2+］i) was observed with a confocal laser-scanning microscope and determined by mean fluorescent value with Fluo-3 fluometry. RESULTS: Compared to control group, three hours of hypoxia and twelve hours of reoxygenation group induced cortical neuronal apoptosis and significantly increased the intracellular free Ca2+ concentration（P<0.01). Compared with model group, TTLS decreased the percentage of neuronal apoptosis and reduced neuronal ［Ca2+］i（P<0.01).CONCLUSION: TTLS could obviously reduce hypoxia/reoxygenation-induced apoptosis and alleviate the damage degree of rat cortical neurons.The mechanism might be related to inhibiting the calcium overload induced by hypoxia/reoxygenation in rat cortical neurons.     

Changes in the expression of Bcl-2 and Fas in HL-60 cells treated with cyclosporine A

AIM: To investigate the changes of the expression of Bcl-2 and Fas protein and the apoptosis in HL-60 cells induced by cyclosporine A. METHODS: The expression of Bcl-2 and Fas protein and apoptosis in HL-60 cells were measured by immunohistochemistry analysis and flow cytometric analysis. RESULTS: There was strong expression of Bcl-2 in HL-60 cells, treatment with cyclosporine A (CsA) for 8-10 h down-regulated the expression of Bcl-2. Fas protein expression in HL-60 cells was very low, CsA induced apoptosis of HL-60 cells, but didn't induce Fas protein expression. CONCLUSION: CsA induces apoptosis in HL-60 cells by down-regulating Bcl-2 expression.     

洋葱染色体核型的FISH分析

 挑选洋葱(Allium cepa L．) (2n=2x=16，CC)有丝分裂中期细胞，以洋葱卫星DNA序列(AC—SAT-DNA)为探针，DIG-1 1-dUTP为标记物，用荧光原位杂交(FISH)方法观察其在染色体上的定位，并进行洋葱的核型分析。6号染色体为近端部着丝点染色体，在其长臂末端有一个卫星DNA序列的主要位点。其余每条染色体在其长、短臂的末端都各具有一个主要位点。差异表现在各染色体上卫星DNA序列的次要位点分布不同。根据洋葱8对染色体各自具有的独特的卫星DNA分布位点，可将洋葱8对染色体全部分开。以此为基础，构建了洋葱卫星DNA在染色体上分布的模式图。     

大车前胚乳与胚发育的细胞胚胎学研究

应用石蜡制片技术研究了大车前胚乳与胚发育过程的细胞胚胎学特征,为车前科胚胎学及分类地位的确立提供相关资料。结果表明：大车前成熟雌配子体为七细胞七核的蓼型。经双受精后形成合子与初生胚乳核。胚乳发育类型为细胞型,初生胚乳核第1次横分裂产生珠孔室与合点室胚乳细胞。珠孔室胚乳细胞经1次纵分裂或2次连续相交的纵分裂形成2个或4个胚乳细胞;进而同步进行横分裂形成2排,即4个或8个胚乳细胞;靠近珠孔的2个或4个胚乳细胞分化并发育成为单核的管状胚乳珠孔吸器;靠近合点室胚乳细胞的2个或4个细胞进行各个方向的分裂形成胚乳本体,并将合子分裂所形成的胚包围其中;与此同时,合点室胚乳细胞的细胞核进行1次核分裂但不伴随胞质分裂,在合子期便形成具有两个细胞核的胚乳合点吸器;胚乳吸器为胚乳本体的发育从珠被与合点吸收并转送营养物质。胚胎发生属于柳叶菜型,经原胚期、心胚期、鱼雷胚、子叶胚形成成熟种子。珠被绒毡层发达。对胚乳本体以及球胚的流式细胞仪细胞核DNA分析表明,胚的DNA含量为二倍体,而胚乳为三倍体。     

Molecular polymorphism of cytoplasmic DNA in Ficus carica L.: Insights from non-coding regions of chloroplast DNA

The trnL (UAA) intron and the intergenic spacer between the 3′ exon of trnL (UAA) and trnF (GAA) sequences were used as genetic markers for differentiating Ficus carica cultivars and establishing refined genetic relationships. The study was based on 20 fig cultivars, collected from south and centre of Tunisia. Since, the intron was thought to be more variable among close relatives than is the chloroplast spacer. The size of these non-coding regions varied from 554 to 589 and from 989 to 1022 bases pairs for the intron and the combined sequences correspondingly. The average of GC content was 33.9% and 34.6% in the intron and the combined intron and spacer respectively. High values of A + T contents were detected in both data sets and may explain the high proportions of transversions founded. The observed variation pattern of plastid DNA provides evidence of an important genetic diversity. The overall transition/transversion bias (R) was 0.202 in the intron and 0.27 in the combined regions. The RI index of 0.592 indicates that these combined sequences have clearly more homoplasy then the intron (RI = 0.705) and spacer (RI = 0.777) sequences separately. Phylogenetic trees were generated based on maximum parsimony (MP) and neighbor-joining (NJ) analysis of the chloroplast sequences data. Results proved that a typically continuous genetic diversity characterizes the local fig germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical or tree sex correspondence. Although the level of apparent diversity is considerable, we may conclude that non-coding regions of chloroplast genome provide a new and practical opportunity to evaluate genetic diversity and to discriminate fig cultivars. Revealed cytoplasmic DNA markers are reliable to elaborate a molecular data base to conduct management and breeding programs on local fig germplasm.     

正交试验优化银杏胚愈伤组织继代培养及黄酮积累

采用正交试验研究了基本培养基、NAA、6-BA、植酸(PA)4因素各3水平对银杏胚愈伤组织继代培养及黄酮积累的影响,并测定了愈伤组织生长曲线.结果表明:银杏胚愈伤组织继代培养的最佳组合是N6+NAA 3.0 mg/L+6-BA 2.0 mg/L+0.1%PA,各因子影响程度为基本培养基＞NAA＞6-BA＞PA;愈伤组织黄酮积累量最佳组合是B5+NAA 0.1 mg/L+6-BA 0.5mg/L+0.05%PA,各因子影响程度为NAA＞PA＞6-BA＞基本培养基.愈伤组织生长曲线基本呈"S"型,黄酮积累的适宜培养周期为40～50 d.     

碱胁迫对蚕豆幼苗叶片质膜和光合性能的影响

研究不同浓度(51、01、52、5 mmol/L)Na2CO3胁迫对蚕豆幼苗质膜和光合性能的影响及作用机理。结果表明:较低浓度Na2CO3胁迫对蚕豆幼苗的质膜影响不大,而较高浓度的Na2CO3胁迫对其产生了明显的伤害。随Na2CO3浓度增加,MDA含量先降低后增加,在高浓度Na2CO3胁迫下蚕豆幼苗叶片发生了较严重的膜脂过氧化作用;蚕豆幼苗叶片净光合速率和叶绿素a、b、a+b的含量先升高后降低,25 mmol/L Na2CO3较严重抑制了其光合作用和叶绿素的合成。     

Silicon supplementation ameliorated the inhibition of photosynthesis and nitrate metabolism by cadmium (Cd) toxicity in Cucumis sativus L.

The effects of silicon (Si) application on plant growth, pigments, photosynthetic parameters, chlorophyll a (Chl a) fluorescence parameters and nitrogen metabolism were studied in Cucumis sativus L. under cadmium (Cd) toxicity. Compared with the control, 100 μM CdCl₂ treatment caused dramatic accumulation of Cd in cucumber leaves, greatly induced chlorosis, and the transmission electron microscope (TEM) analysis indicated that Cd treatment cucumber chloroplast showed obvious swollen, thylakoids and chloroplast membrane were seriously damaged, and could not be observed clearly. Application of Si reversed the chlorosis, protected the chloroplast from disorganization, and significantly increased the pigments contents, which might be mainly responsible for the higher photosynthetic rate and accumulation of biomass under Cd stress. Further investigation of chlorophyll a fluorescence indicated that Cd treatment decreasing photosynthesis was not due to stomatal restriction, while was closely related integrity damage or function lost of the photosynthetic machinery which can be concluded from the higher intercellular CO₂ concentration (Ci) and lower F_v/F_m and ΦPSII. Application of Si alleviated the inhibited level of photosynthesis and F_v/F_m and ΦPSII by Cd, which might imply that Si plays important roles in protecting photosynthetic machinery from damaging. The Cd treatment also greatly inhibited the enzymes of nitrogen metabolism including nitrogen reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH), and Si supply decreased the inhibiting effects of Cd.     

Artesunate negatively controls expression of cyclin D1 and activity of AP-1 by inhibiting the activation of ERK1/2 protein in HSC-T6 cells

AIM: To investigate the effects of artesunate(Art) on the expression of ERK1/2, AP-1 and cyclin D1 in rat hepatic stellate cells (HSCs), and to elucidate the molecular mechanism of Art against hepatic fibrosis. METHODS: HSC-T6 cells were treated with platelet-derived growth factor BB(PDGF-BB) to induce cell proliferation. The cells were divided into control group, PDGF-BB group, PDGF-BB+Art groups (with 6.25 mg稬^-1, 25 mg稬^-1or 50 mg稬^-1 of Art) and PDGF-BB+PD98059 group. The level of collagen type I in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of ERK1/2 and cyclin D1 were measured by RT-PCR. The protein levels of p-ERK1/2 and cyclin D1 in HSC-T6 cells were detected by Western blotting. The activity of AP-1 was analyzed by electrophoretic mobility shift assay. RESULTS: The concentration of collagen type I was significantly higher in PDGF-BB group than that in control group (P<0.05), and decreased in PDGF-BB+Art group and PDGF-BB+PD98059 group in comparison with that in PDGF-BB group (P<0.05, P<0.01). The protein level of ERK1/2 in PDGF-BB+Art group (50 mg稬^-1) was lower than that in PDGF-BB group (P<0.05), and was even lower in PDGF-BB+PD98059 group (P<0.01). The mRNA expression of cyclin D1 in PDGF-BB+Art groups (25 mg稬^-1and 50 mg稬^-1) and PDGF-BB+PD98059 group were significantly lower than that in PDGF-BB group (P<0.05). The protein levels of p-ERK1/2 and cyclinD1 were the highest in PDGF-BB group, and significantly lower in PDGF-BB+Art groups (6.25 mg稬^-1, 25 mg稬^-1 and 50 mg稬^-1) and PDGF-BB+PD98059 group (P<0.05, P<0.01). The AP-1 binding activity in HSC-T6 cells was down-regulated by Art. CONCLUSION: Artesunate inhibits the proliferation of HSC-T6 cells in vitro by inhibiting the activation of ERK1/2, thus down-regulating the activity of AP-1 and expression of cyclin D1.