喜树果中喜树碱和10-羟基喜树碱的匀浆提取工艺

以喜树果为原料,对匀浆法提取喜树碱和10-羟基喜树碱的工艺进行了研究,确定了最佳的工艺条件：提取溶剂为体积分数55％的乙醇,匀浆时间为8min,料液比为1：15（g／mL）。在此工艺条件下,喜树碱和10-羟基喜树碱的提取率分别为0．801‰和0．546‰。将该法与超声提取、回流提取、常温冷浸提取、水浴振荡提取进行了比较,结果表明,匀浆提取具有得率高、省时间等方面的优势,是一种高效提取喜树碱和10．羟基喜树碱的方法。     

Establishment of persistent infection with HIV-1 abrogates the caspase-3-dependent apoptotic signaling pathway in U937 cells

Treatment of 26L cells, a subclone obtained from U937 cells, with TNF-alpha or DNA-damaging agents such as teniposide (VM26) and camptothecin (CPT) induced morphologically and biochemically typical apoptotic changes, including the activation of procaspase-3. The cells persistently infected with HIV-1 (26L/HIV), however, showed a marked resistance to VM26 and CPT, whereas they hardly lost the sensitivity to TNF-alpha. TNF-alpha-induced apoptosis of 26L/HIV cells proceeded without the increase in caspase-3 activity, indicating that signaling for apoptosis in the infected cells proceeded through an alternative caspase-3-independent pathway which could respond to TNF-alpha but not to VM26 and CPT. The evidence that p-toluenesulfonyl-l-lysine chloromethyl ketone (a trypsin-like serine protease inhibitor) blocked VM26- and CPT-induced apoptotic changes but not TNF-alpha-induced apoptosis also supported the existence of the alternative TNF-alpha-inducible pathway. The results also suggest that a TLCK-sensitive protease is involved upstream of the procaspase-3 activation process and that the protease is essential for the progress of VM26- and CPT-induced apoptosis. The similar effect of HIV-1-productive infection on the apoptosis induced by the DNA-damaging agents was also confirmed by utilizing U1 cells, which are latently HIV-1-infected U937 cells. The cells became resistant to these agents after induction of the viral production by pretreatment with PMA. These results suggest that persistent HIV-1 infection blocks an apoptotic pathway triggered by DNA damaging agents through the inhibition of the procaspase-3 activation process.     

A method to determine the incorporation capacity of camptothecin in liposomes

The purpose of this study was to establish a new experimental approach to determine the maximum amount of campothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultra-centrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubili ty of CPT in the buffer was improved by approximately a factor of 10 (from ∼2.7 to 15–50 μg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a maximum CPT-solubilization of ∼100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at ∼20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrisic water solubility of the drug.     

伤害及诱导子对喜树幼苗中喜树碱含量的影响

通过伤害处理和外施诱导子——水杨酸(SA)、茉莉酸甲酯(MeJa)和乙烯利(Ethrel)以及伤害和诱导子复合处理,研究不同处理对喜树中喜树碱(camptothein,CPT)含量的影响。结果表明:(1)4种单独诱导处理均能在一定程度上增强喜树碱的合成,其中水杨酸处理和机械伤害诱导效果最明显,较对照组的喜树碱含量分别提高了47.7%和27.2%,而机械伤害和诱导子复合处理均产生拮抗作用,喜树碱含量降低;(2)诱导作用受诱导剂浓度的影响,SA、MeJa和Ethrel的适宜质量浓度分别为50~100 mg.L-1、5~10 mg.L-1和5~10 mg.L-1。     

Regions within the N-terminal domain of human topoisomerase I exert important functions during strand rotation and DNA binding

The human topoisomerase I N-terminal domain is the only part of the enzyme still not crystallized and the function of this domain remains enigmatical. In the present study, we have addressed the specific functions of individual N-terminal regions of topoisomerase I by characterizing mutants lacking amino acid residues 1-202 or 191-206 or having tryptophane-205 substituted by glycine in a broad variety of in vitro activity assays. As a result of these investigations we find that mutants altered in the region 191-206 distinguished themselves from the wild-type enzyme by a faster strand rotation step, insensitivity towards the anti-cancer drug camptothecin in relaxation and the inability to ligate blunt end DNA fragments. The mutant lacking amino acid residues 1-202 was impaired in blunt end DNA ligation and showed wild-type sensitivity towards camptothecin in relaxation. Taken together, the presented data support a model according to which tryptophane-205 and possibly other residues located between position 191-206 coordinates the restriction of free strand rotation during the topoisomerization step of catalysis. Moreover, tryptophane-205 appears important for the function of the bulk part of the N-terminal domain in direct DNA interaction.     

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 M), kinetin (2.32–18.58 M), or thidiazuron (0.1–10 M) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 M 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 M). The lowest concentration of indole-3-butyric acid (4.9 M) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.Abbreviations BA 6-benzyladenine - B5 Gamborg's B5 medium (Gamborg et al., 1968) - CPT camptothecin - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - LS Linsmaier & Skoog medium (Linsmaier & Skoog, 1965) - MS Murashige & Skoog (Murashige & Skoog, 1962) - NAA I-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - WPM woody plant medium (Lloyd & McCown, 1981)     

Contribution of topoisomerase I to conversion of single-strand into double-strand DNA breaks

An in vitro system composed of nicked pBR322 DNA and purified topoisomerase I was employed to study the efficiency of the topoisomerase I-driven single-strand to double-strand DNA breaks conversion. At 1.4 × 10⁵ topoisomerase I activity units per mg DNA about 20% single-strand nicks were converted into double-strand breaks during 30 min due to topoisomerase I action. Camptothecin inhibited the conversion. The conversion was also inhibited when the relaxing activity of the used topoisomerase I was increased by phosphorylation of the enzyme with casein kinase 2. The presented data suggest that topoisomerase I may be involved in production of double-stranded breaks in irradiated cells and that this process positively depends on the amount of topoisomerase I but not on its phosphorylation state.     

Oncogene overexpression and de novo drug-resistance in human prostate cancer cells

We have isolated a variant [PC3(R)] of the human prostate PC3 tumor cell line which showed resistance to several anticancer drugs. Studies to evaluate the mechanisms of resistance to anticancer drugs in the PC3(R) cell line indicated that mdrl was not overexpressed. Studies also indicated that activities of topo I and topo II were not different in these cell lines, nor was there any difference in the formation of drug-induced KCl-SDS precipitable complexes, including that topoisomerases were not involved in the development of resistance in PC3(R) cells. While the activity of glutathione S-transferase and total glutathione levels were also similar in these cell lines, the glutathione peroxidase activity in PC3(R) cells was 5-fold lower than in PC3 cells. ] Furthermore, proto-oncogene expression for c-myc, and H-ras was significantly higher in resistant cell than in sensitive cells, indicating that the amplication of early response genes may play a role in the emergence of de novo resistance in PC3(R) cells.     

增强UV B辐射对喜树生理指标及喜树碱含量的影响

喜树(Camptotheca acuminata Decne.)隶属于蓝果树科(Nyssaceae)喜树属(Camptotheca),为抗癌药物喜树碱的主要资源,提高喜树碱的积累以满足临床需求是喜树碱开发的重要途径。该研究运用UV B辐射对2年生喜树进行每天8 h辐射处理,对1年生喜树分别设置每天2 h、4 h、6 h和8 h的辐射处理,连续处理12 d后分别测定各处理喜树叶的叶绿素、MDA、游离脯氨酸 (Fpro)含量和SOD活性,以及幼叶、幼枝和根中喜树碱含量,分析UV B辐射对喜树生理指标和次生代谢物的影响,以揭示喜树碱为喜树适应UV B辐射逆境的防御产物。结果显示：（1）2年生喜树经UV B每天8 h辐射处理12 d后,叶绿素含量较对照显著降低,而MDA、Fpro和喜树碱含量均增加,说明每天8 h UV B辐射对2年生喜树产生了较强的胁迫伤害。（2）1年生喜树经UV B辐射处理12 d后,随着每天UV B辐射时间的增加,叶绿素含量不断降低,Fpro含量显著增加;每天2~6 h处理的MDA含量与对照无显著差异,但总体随处理时间增加呈上升趋势;每天8 h UV B辐射的MDA含量较对照显著增加;SOD活性随每天处理时间的延长呈先下降、后上升、再下降的变化趋势,说明每天8 h的UV B辐射对一年生喜树也产生了胁迫伤害。（3）1年生喜树幼叶、幼枝和根中喜树碱含量随着每天UV B辐射时间的延长均呈递增趋势,而且每天8 h辐射处理的喜树碱含量均最高,其中幼叶和幼枝中喜树碱含量显著高于根中含量。实验结果表明,增强UV B辐射对喜树造成了一定的伤害,而喜树通过改变生理以及次生代谢机制,以进一步产生喜树碱来响应增强UV B的胁迫。     

喜树幼苗中喜树碱和10-羟基喜树碱对热激的响应特点和意义

植物在长期的生态环境适应过程中,产生了包括生物碱在内的大量次生代谢物.本文以我国特有树种--喜树(Camptotheca acuminata Decaisne)为材料,研究其不同器官中喜树碱(camptothecin,CPT)和10-羟基喜树碱(10-hydroxycamptothecin,HCPT)在不同热激温度和时间情况下的含量变化.CPT和HCPT变化呈现出较好的相互消长关系,并且分别在38℃和40℃达到各自的峰值,比以丙二醛和叶绿素为指标的致死温度低了2~4℃;HCPT在热激过程中的变化较CPT活跃;极易受到攻击和伤害的嫩叶中的生物碱含量变化最大.由此推断,CPT和HCPT遵循"幼嫩和生殖器官优先保护"的原则,从而有效地缓解了高温胁迫,并且HCPT和CPT代表了不同的防御策略.