红花桑寄生总黄酮提取物诱导淋巴瘤细胞株CA46凋亡及其分子机制研究

研究了一种寄主为夹竹桃的红花桑寄生总黄酮提取物(Nispex)对人Burkitt淋巴瘤细胞株CA46的抗肿瘤作用,探讨了其抗肿瘤作用的分子机制.应用MTT法研究Nispex对CA46细胞增殖的抑制效果,细胞集落培养法观察Nispex对CA46中增殖细胞群的影响.采用AO/EB荧光染色、TUNEL分析、DNA凝胶电泳分析以及AnnexinV流式细胞术检测细胞凋亡,Western blot检测Nispex对CA46细胞中NF-κBp65、Bcl-2、Bax、Caspase-3和PARP等蛋白表达的影响.结果表明,Nispex显著抑制CA46细胞增殖和诱导CA46细胞凋亡,作用48 h的IC50值为1.72μg/mL,细胞凋亡率与药物浓度正相关.Nispex能有效上调CA46细胞Bax、Caspase-3蛋白表达,下调NF-κBp65、Bcl-2、PARP蛋白表达.Nispex诱导CA46细胞凋亡可能是通过对NF-κB信号通路的抑制来实现的.     

5-烯丙基-7-二氟亚甲基白杨素（C19H14O4F2）诱导人卵巢癌CoC1细胞凋亡

目的 观察5-烯丙基-7-二氟亚甲基白杨素（ADFMChR）诱导人卵巢癌（CoCl）细胞凋亡的作用。方法 以体外培养的人卵巢癌CoCl为研究对象。采用软琼脂克隆测定ADFMChR对细胞集落的影响;流式细胞术（FCM）检测ADFMChR诱导细胞凋亡率;凝胶电泳观察ADFMChR诱导基因DNA梯形条带。Westernblot分析ADFMChR对CoCl细胞PPARγ,NF-κB,Bcl-2,Bax蛋白表达的影响。结果软琼脂克隆显示ADFMChR呈剂量依赖性抑制细胞集落形成;FCM分析发现ADFMChR呈剂量依赖性诱导细胞凋亡;ADFMChR（30μmol／L）孵育CoCl细胞48h后,DNA琼脂糖凝胶电泳呈现典型梯形条带。Westernblot分析结果表明ADFMChR以剂量依赖方式上调CoCl细胞PPARγ和Bax蛋白表达,下调NF-κB和Bcl-2蛋白表达。结论 ADFMChR诱导人卵巢癌CoCl细胞凋亡与其活化PPARγ,抑制NF-κB表达和提高Bax／Bcl-2比值有关。     

藁本内酯对H2O2诱导的B16黑素瘤细胞氧化损伤的保护作用

目的:观察藁本内酯对H_2O_2诱导的B16黑素瘤细胞氧化损伤的保护作用并探讨其可能机制。方法:以H_2O_2诱导B16黑素瘤细胞氧化损伤为模型,并以藁本内酯进行干预,采用MTT法测细胞活力,酶标仪检测乳酸脱氢酶(LDH)漏出量,流式细胞术测细胞凋亡率、线粒体膜电位(△Ψm)和细胞内游离钙离子浓度。结果:与H_2O_2诱导的B16黑素瘤细胞比较,应用藁本内酯(5、10、20μmol·L~(-1))处理的B16黑素瘤细胞活力和△Ψm明显提高,LDH漏出量明显减少,细胞凋亡率和细胞内游离钙离子浓度明显降低,差异均具有统计学意义(P0.05)。结论:藁本内酯对H_2O_2诱导的B16黑素瘤细胞氧化损伤具有保护作用,其作用机制可能通过恢复线粒体功能、抑制细胞凋亡有关。     

重楼皂苷Ⅶ抑制肺癌H460细胞增殖和迁移能力研究

为研究重楼皂苷Ⅶ(polyphyllin Ⅶ)抑制人肺癌H460细胞增殖、迁移能力和诱导凋亡的作用和机制。本实验采用MTT法检测重楼皂苷Ⅶ处理后H460细胞生长抑制率,Hoechst 33258染色观察细胞形态,细胞集落形成实验考察细胞的增殖能力,划痕实验和Transwell小室实验研究H460细胞迁移和侵袭能力的改变,并通过western blot法检测在重楼皂苷Ⅶ处理前后细胞蛋白表达变化情况。结果发现,重楼皂苷Ⅶ可显著抑制H460细胞增殖,影响其集落形成,并使细胞形态发生变化,抑制细胞体外的迁移和侵袭能力,并可诱导凋亡的发生。重楼皂苷Ⅶ处理后,H460细胞中基质金属蛋白酶MMP-2和MMP-9显著降低,凋亡相关蛋白剪切型caspase-3、Bax表达增加,ICAD、Bcl-2表达降低,提示重楼皂苷Ⅶ体外可能通过调节基质金属蛋白酶抑制H460细胞迁移和侵袭,同时诱导凋亡的发生。     

氧化苦参碱对K562肿瘤细胞增殖的影响

目的:研究氧化苦参碱(OM)对人白血痛细胞系K562生长增殖的影响.方法:运用MTT比色法、活细胞计数法、集落形成法以及透射电镜观察检测OM对人白血病细胞系K562增殖抑制作用.结果:MTT实验、生长曲线及集落形成实验显示OM能明显抑制K562细胞的增殖.随着OM浓度的增加,K562细胞存活细胞显著降低,呈现明显的刺量依赖性,经相关分析,细胞抑制率与OM浓度呈正相关(r=0.9010),其半数抑制浓度(IC50)为0.33 mg/ml.透射电镜下显示在低浓度即有明显的诱导细胞凋亡的作用,出现核固缩、核碎裂、凋亡小体等典型的凋亡形态.结论:OM具有抑制K562白血病细胞增殖诱导肿瘤细胞凋亡的作用.     

黄泥螺提取物对小鼠黑色素瘤细胞B16生长的影响

为研究黄泥螺提取物对小鼠黑色素瘤细胞B16生长的影响,以及初步研究其作用机制,采用磺酰罗丹明B(SRB)法测黄泥螺提取物对B16细胞的生长抑制作用,得到48 h后半数抑制浓度(IC50)为68.56 ug/ml.当黄泥螺提取物浓度为70ug/ml时,台盼蓝排斥试验显示有部分细胞死亡.经Hoechest 33258染色并用荧光显微镜观察,发现培养的细胞中出现凋亡小体,细胞核发生固缩;DNA琼脂糖凝胶电泳呈现出DNA大片段;用流式细胞仪进一步检测表明,细胞生长周期发生变化并检测到凋亡峰.结果表明,体外培养的B16细胞经过黄泥螺提取物处理后,B16细胞增殖受到抑制,细胞周期被阻滞,B16细胞受到诱导后存在凋亡.因此,黄泥螺提取物对小鼠黑色素瘤细胞B16生长有明显的影响.     

多胺类似物BENS和TBP对胰腺癌PANC-1细胞增殖、凋亡和迁移能力的影响

为探讨多胺类似物BENS和TBP对胰腺癌PANC-1细胞生长的影响、促进细胞凋亡的机制以及迁移能力的影响,运用MTT法检测药物对细胞生长的影响;亚凋亡峰流式细胞分析法及TUNEL法检测细胞凋亡;Western blotting法测定Bax蛋白、细胞色素C蛋白的表达变化;Ranswell技术检测细胞迁移能力的改变。分析发现,两种多胺类似物均可显著抑制PANC1癌细胞增殖,抑制作用随药物浓度加大而增加,其细胞毒性大小依次为BENSTBP。细胞凋亡分析发现,BENS和TBP均可诱导PANC1发生细胞程序性凋亡,导致亚凋亡峰出现和明显的DNA断裂现象。胞浆中Bax蛋白和细胞色素C含量显著增加,同时显著性抑制PANC1细胞的迁移能力。表明BENS和TBP能够抑制人胰腺癌PANC1细胞增殖;阻止PANC1细胞迁移的能力;同时诱导细胞发生凋亡,其作用机制可能与改变肿瘤细胞正常的细胞周期,活化线粒体参与的细胞凋亡途径相关。     

Ap5A延迟TRAIL诱导Novikoff细胞的凋亡

通过荧光分子标记和CONFOCAL技术检测方法,建立了TRAIL（TNF-related apoptosis inducing ligand, 一种类肿瘤坏死因子）诱导Novikoff细胞凋亡的模型,并探讨了TRAIL诱导凋亡的机制和Ap5A（P¹, P⁵-Di（adenosine-5′）pentaphosphate）在其中的作用.结果显示：TRAIL可诱导Novikoff细胞凋亡,且具有剂量和时间依赖性,同时胞内钙离子浓度显著上调.Ap5A能延迟TRAIL诱导的Novikoff细胞凋亡,同时下调胞内钙离子浓度.TRAIL和Ap5A分别上调和下调胞内钙离子浓度的作用可能是其诱发和延迟Novikoff细胞凋亡的一个机制.     

Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex.

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of Bacillus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The predicted molecular weights for the Fp and Ip subunits, 65,186 (585 amino acids) and 28,285 (252 amino acids), agreed with the values determined independently for the labeled Fp and Ip antigens, although it appeared that the B. subtilis Fp was not functional after expression of the sdhA gene in Escherichia coli. Both subunits closely resembled the corresponding Fp and Ip subunits of the succinate dehydrogenase (SDH) and fumarate reductase of E. coli in size, composition, and amino acid sequence. The sequence homologies further indicated that the B. subtilis SDH subunits are equally related to the SDH and fumarate reductase subunits of E. coli but are less closely related than are the corresponding pairs of E. coli subunits. The regions of highest sequence conservation were identifiable as the catalytically significant flavin adenine dinucleotide-binding sites and cysteine clusters of the iron-sulfur centers.     

Binding to cisplatin-modified DNA by the Saccharomyces cerevisiae HMGB protein Nhp6A

Nhp6A is an abundant non-histone chromatin-associated protein in Saccharomyces cerevisiae that contains a minor groove DNA binding motif called the HMG box. In this report, we show that Nhp6Ap binds to cisplatin intrastrand cross-links on duplex DNA with a 40-fold greater affinity than to unmodified DNA with the same sequence. Nevertheless, Nhp6Ap bound to cisplatinated DNA readily exchanges onto unmodified DNA. Phenanthroline-copper footprinting and two-dimensional NMR on complexes of wild-type and mutant Nhp6Ap with DNA were employed to probe the mode of binding to the cisplatin lesion. Recognition of the cisplatin adduct requires a surface-exposed phenylalanine on Nhp6Ap that promotes bending of DNA by inserting into the helix from the minor groove. We propose that Nhp6Ap targets the cisplatin adduct by means of intercalation by the phenylalanine and that it can bind in either orientation with respect to the DNA lesion. A methionine, which also inserts between base pairs and functions in target selection on unmodified DNA, plays no apparent role in recognition of the cisplatin lesion. Basic amino acids within the N-terminal arm of Nhp6Ap are required for high-affinity binding to the cisplatin adduct as well as to unmodified DNA. Cisplatin mediates its cytotoxicity by forming covalent adducts on DNA, and we find that Deltanhp6a/b mutants are hypersensitive to cisplatin in comparison with the wild-type strain. In contrast, Deltanhp6a/b mutants are slightly more resistant to hydrogen peroxide and ultraviolet irradiation. Therefore, Nhp6A/Bp appears to directly or indirectly function in yeast to enhance cellular resistance to cisplatin.     

Succinate dehydrogenase mutants of Bacillus subtilis lacking covalently bound flavin in the flavoprotein subunit

Succinate dehydrogenase consists of two unequal subunits; Fp and Ip. An FAD group is covalently linked to a histidyl residue in the Fp subunit. The mechanism by which flavin is attached to protein is not known. Covalently bound flavin was studied in wild-type and succinate-dehydrogenase-negative Bacillus subtilis. The Fp subunit of succinate dehydrogenase was found to be the only (major) flavinylated protein in the cell. Mutants lacking covalently bound flavin and still containing the Fp polypeptide are described. It is shown that the flavin is not essential for assembly and membrane binding of succinate dehydrogenase in B. subtilis.     

Experimental epizootiology of Zoophthora anhuiensis (Entomophthorales) against Myzus persicae (Homoptera: Aphididae) with a description of a modified Gompertz model for aphid epizootics

Epizootiological features of Zoophthora anhuiensis, a fungal pathogen specific to aphids in southern China, were studied in six aptera colonies of Myzus persicae at 16 regimes of temperature (T = 10, 15, 20 and 25 degrees C) and relative humidity (H = 90%, 95%, 98% and 100% RH) with initially infected proportion (Ip) of 0.5 in experiment (Expt) 1 or at a fixed regime of 15 degrees C and 100% RH with a variable Ip of 0.17-1.00 in Expt 2. Mycosis-caused mortalities (Mp) varied with aphid densities (D) over time after colony initiation (t) were well fitted to a Gompertz growth model modified to include the variables T, H, Ip and D in the form of Mp = 91.72exp[-5.282exp[-(0.0095T + 0.0128H/T-0.5407D2/H)t]] for Expt 1 (r2 = 0.94) or Mp = 95.49exp[-4.314exp[-(0.1479Ip + 0.1636/D)t]] for Expt 2 (r2 = 0.97). These variables were effective in determining the rate of epizootic development in the M. persicae colonies. Based on the observed and fitted results, Z. anhuiensis was most adaptive to cool climate or season and epizootiologically was subject to a minimal effect of RH at an optimum of approximately 15 degrees C. However, the humidity became determinant for epizootic progress at higher temperatures. Interactive effects of the variables and application of the fitted models for predicting epizootic development in field situations are discussed.     

Probing Aplysia californica adenosine 5'-diphosphate ribosyl cyclase for substrate binding requirements: design of potent inhibitors.

Readily synthesized nicotinamide adenine dinucleotide (NAD(+)) analogues have been used to investigate aspects of the cyclization of NAD(+) to cyclic adenosine 5'-O-diphosphate ribose (cADPR) catalyzed by the enzyme adenosine 5'-O-diphosphate (ADP) ribosyl cyclase and to produce the first potent inhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrate analogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behavior characteristics. Nicotinamide hypoxanthine dinucleotide (NHD(+)), nicotinamide guanine dinucleotide (NGD(+)), C1'-m-benzamide adenine dinucleotide (Bp(2)A), and C1'-m-benzamide nicotinamide dinucleotide (Bp(2)N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201 nM, respectively. However, NHD(+) and NGD(+) are also known substrates and are slowly converted to cyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap(2)A), bis-hypoxanthine dinucleotide (Hp(2)H), and bis-nicotinamide dinucleotide (Np(2)N), exhibited micromolar competitive inhibition, with Ap(2)A displaying the greatest affinity for the enzyme. 2',3'-Di-O-acetyl nicotinamide adenine dinucleotide (AcONAD(+)) was not a substrate for the A. californica cyclase but also displayed some inhibition at a micromolar level. Finally, inhibition of the cyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) was observed at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motif required for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamide ribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inability of the enzyme to release unprocessed material are both considered to explain nanomolar inhibition. Recognition of inhibitors by other ADP ribosyl cyclases has also been investigated, and this study now provides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitor Bp(2)A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamide derivatives did not inhibit CD38 cyclase or hydrolase activity when NGD(+) was used as substrate. These results emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activity predominates.     

Ligands to the 2Fe iron-sulfur center in succinate dehydrogenase

Membrane-bound succinate oxidoreductases are flavoenzymes containing one each of a 2Fe, a 3Fe and a 4Fe iron-sulfur center. Amino acid sequence homologies indicate that all three centers are located in the Ip (B) subunit. From polypeptide and gene analysis of Bacillus subtilis succinate dehydrogenase-defective mutants combined with earlier EPR spectroscopic data, we show that four conserved cysteine residues in the first half of Ip are the ligands to the [2Fe-2S] center. These four residues have previously been predicted to be the ligands. Our results also suggest that the N-terminal part of B. subtilis Ip constitutes a domain which can incorporate separately the 2Fe center and interact with Fp, the flavin-containing subunit of the dehydrogenase.     

Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos

Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (P < 0.05). The use of donor cells of any type in later passages decreased the rate of blastocyst formation. Treatment with trichostatin-A did not improve the rate of blastocyst formation from cleaved dewclaw cell-derived embryos but did so in the embryos derived from the tail-tip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.     

Cell-mediated immunity induced by recombinant Mycobacterium bovis Bacille Calmette-Guérin strains against an intracellular bacterial pathogen: importance of antigen secretion or membrane-targeted antigen display as lipoprotein for vaccine efficacy

Live recombinant vaccines expressing defined pathogen-derived Ags represent powerful candidates for future vaccination strategies. In this study, we report on the differential induction of protective cell-mediated immunity elicited by different recombinant Mycobacterium bovis Bacille Calmette-Guérin (BCG) strains displaying p60 Ag of Listeria monocytogenes in secreted, cytosolic, or membrane-attached form for T cell recognition. Anti-listerial protection evoked by the membrane-linked p60 lipoprotein of rBCG Mp60 and that of the p60 derivative secreted by rBCG Sp60-40 were nearly equal, whereas cytosolic p60 displayed by rBCG Np60 failed to protect mice from listeriosis. In vivo depletion of CD4 or CD8 T cell subpopulations in rBCG Mp60-vaccinated mice before listerial challenge revealed interactions of both T cell subsets in anti-listerial protection. In rBCG Sp60-40-vaccinated animals, CD4 T cells predominantly contributed to anti-listerial control as shown by the failure of anti-CD8 mAb treatment to impair the outcome of listeriosis in rBCG Sp60-40-vaccinated mice after L. monocytogenes challenge. Hence, differential Ag display by rBCG influences cell-mediated immunity, which in turn may impact vaccine efficacy due to the different requirements of CD4 or CD8 T cells for pathogen elimination.     

Presence of functional ATP and dinucleotide receptors in glutamatergic synaptic terminals from rat midbrain

Glutamatergic terminals from rat midbrain were characterized by immunolocalization of synaptophysin and the vesicular glutamate transporters, either VGLUT1 or VGLUT2. Terminals containing these markers represent about 31% (VGLUT1) and 16% (VGLUT2) of the total synaptosomal population. VGLUT1-positive glutamatergic terminals responded to ATP or P1,P 5-di(adenosine-5') pentaphosphate (Ap5A) with an increase in the intrasynaptosomal calcium concentration as measured by a microfluorimetric technique in single synaptosomes. Roughly 20% of the VGLUT1-positive terminals responded to ATP, 13% to Ap5A and 11% to both agonists. Finally 56% of the terminals labeled with the anti-VGLUT1 antibody did not show any calcium increase in response to ATP or Ap5A. A similar response distribution was also observed in the VGLUT2-positive terminals. The Ca2+ responses induced by ATP and Ap5A in the glutamatergic terminals could be selectively inhibited by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 80 micro m) and P1,P 5-di(inosine-5') pentaphosphate (Ip5I, 100 nm), respectively. Both ATP and Ap5A, once assayed in the presence of extrasynaptosomal calcium, were able to induce a concentration-dependent glutamate release from synaptosomal populations, EC50 values being 21 micro m and 38 micro m for ATP and Ap5A, respectively. Specific inhibition of glutamate release was obtained with PPADS on the ATP effect and with Ip5I on the dinucleotide response, indicating that separate receptors mediate the secretory effects of both compounds.