Cytotoxic Mechanisms of FK317, a New Class of Bioreductive Agent with Potent Antitumor Activity

FK317 is a member of a new class of bioreductive agents that exhibit strong cytotoxicity against various human cancer cells. The effect of FK317 was found to be stronger than that of mitomycin C (MMC), adriamycin (ADR) or cisplatin (CDDP). Alkaline elution analysis indicated that FK317 formed interstrand DNA-DNA and DNA-protein cross-links in cells. On the other hand, no DNA single-strand breaks were observed in the cells treated with FK317. In a cell-free system the deacetylated metabolites produced cross-linked DNA under reductive conditions, though FK317 itself did not form DNA-DNA cross-links. In order to elucidate the metabolic activation mechanisms, we established an FK317-resistant subline from human non-small cell lung cancer cells (Lu99) by stepwise and brief exposure (1 h) to FK317. The resistant subline (Lu99/317) showed cross-resistance to MMC and carboquone (CQ), but not to ADR or CDDP. DT-diaphorase, which is one of the activation enzymes of MMC and CQ, was deficient in Lu99/317 cells as determined by enzyme activity assay. However, the levels of NADPH:cytochrome P450 reductase, which is another activation enzyme for MMC and CQ, were comparable in resistant and parent cell lines. Treatment of the cells with dicumarol, an inhibitor of DT-diaphorase, reduced the cytotoxicity of FK317 to Lu99 cells, but not to Lu99/317 cells. These results indicate that deacetylation of FK317 is necessary for its reductive activation, and deacetylated FK317 is reduced by DT-diaphorase to form an active metabolite, which produces DNA-DNA interstrand and DNA-protein cross-links that lead to cell death.     

Halichondria属软海绵次生代谢产物及其生物活性

Halichondria属软海绵分布广泛,含多种生物活性成分,引起人们的深入研究。近30年来,已从Halichon-dria属软海绵中分离得到了许多结构新颖,有抗菌、抗肿瘤和抗病毒等生物活性的次生代谢产物,如大环内酯、聚醚、生物碱、内酰胺、萜类、甾醇、鞘类脂糖苷等。在调研国内外相关资料的基础上,按化合物的结构类型简要介绍了1978—2003年Halichondria属软海绵次生代谢产物及其生物活性的研究概况,为进一步的研究工作提供参考。     

结扎橡皮圈应力松弛的研究

目的通过定期测量结扎橡皮圈负载后应力的变化，研究它应力松弛的过程及在不同负载下应力松弛率的异同。方法选取结扎橡皮圈30个，随机分为3组，每组10个，3组试样分别负载100g、150g和200g。通过定期测量结扎橡皮圈在不同负载下的应力，了解应力的变化过程。对3组试样的应力变化进行统计分析。结果结扎橡皮圈在最初的48h内应力衰减较快，负载48h后残余应力为初始应力的80％左右（78．4％-83．5％），之后应力松弛的速率逐渐减小，至第32天时残余应力为初始应力的73．1％-85．8％。结论结扎橡皮圈在不同负载下均存存应力松弛的现象，应力松弛率相同。     

高脂血症患者氧化修饰低密度脂蛋白对内皮依赖性血管舒张的影响

目的：探讨氧化修饰低密度脂蛋白（Ox-LDL）对血管舒张的影响及高脂血症男性患者易患动脉粥样硬化（AS）的机制。方法：采用Cu^2＊ 对13例健康自愿者及29例高脂血症患者的低密度脂蛋白(LDL)进行氧化修饰，硫代巴比妥酸法测定丙二醛（MDA）含量，Bartlett法测定溶血卵磷脂(LPC)含量，乙酰胆碱诱发血管舒张。结果：健康人和高脂血症患者血浆LDL被氧化修饰后与修饰前比较MDA含量显著增高（P<0.01）；两组Ox-LDL中的LPC水平均 明显高于各自天然低密度脂蛋白(N-LDL)中的LPC水平（P<0.05）；且健康人及高脂血症男性患者N- LDL中的LPC含量与女性基本相同，但两组男性Ox-LDL中的LPC含量高于女性（P<0.05）,男性高脂血症患 者Ox-LDL的LPC含量最高；两组Ox-LDL均明显抑制血管内皮依赖性舒张，与女性相比，健康男性Ox-LDL抑制血管舒张程度略强，但高脂血症男性患者Ox-LDL抑制血管舒张程度明显增强。高脂血症男性及女性患者Ox-LDL的抑制血管舒张效应与LPC量呈正相关（r ＝0.592，P<0.05；r＝0.816，P<0.05)。结论：男性高脂血症患者易患AS可能与Ox-LDL增加LPC含量及抑制内皮依赖性血管舒张有关。     

ATP通过P2受体调节大鼠近端结肠纵行肌的舒张与收缩反应(英文)

目的腺苷三磷酸(ATP)对大鼠离体远端结肠纵行肌运动的影响已明确,对近端结肠纵行肌的影响可能不同,但未有报告,为此对此进行观察并探讨其受体机制。方法观察静息张力时或预收缩时0.1μmol·L-1～1mmol·L-1ATP和1～100μmol·L-1腺苷对大鼠近端结肠纵行肌的抑制和兴奋作用。结果在静息张力下,1μmol·L-1～1mmol·L-1ATP对大鼠近端结肠纵行肌产生3种效应,即抑制自发性收缩反应,一过性轻度降低基础张力(0.05～0.08g),随后产生浓度依赖性收缩反应(0.04～0.44g)。0.1μmol·L-1河豚毒素不影响ATP的上述作用。在静息张力下,1～100μmol·L-1腺苷对近端结肠纵行肌未产生明显的收缩反应。应用5羟色胺(5HT)或乙酰胆碱(ACh)预收缩标本时,1μmol·L-1～1mmol·L-1ATP产生明显的浓度依赖性舒张反应(23.2%～94.6%,5HT预收缩;24.8%～92.4%,ACh预收缩),而腺苷引起的舒张反应明显小于ATP。结论在大鼠离体近端结肠纵行肌,ATP主要通过嘌呤嘧啶(P)2受体介导收缩反应,部分通过P1受体介导舒张反应。     

分次切除联合眼睑松弛矫正术在眼睑黄色瘤治疗中的应用

目的：探讨分次切除联合眼睑松弛矫正术对眼睑黄色瘤治疗的临床疗效。方法分次切除联合眼睑松弛矫正法治疗8例眼睑黄色瘤患者，观察随访疗效。结果8例病人中2例女性患者术后2次手术切除残留黄色瘤组织，其余6例患者外观满意放弃再次手术治疗；术后2年所有8例患者均无黄色瘤增生加重现象。结论分次切除联合眼睑松弛矫正对于眼睑黄色瘤治疗是一种有效的手术方法。     

Quantification of glutamate and glutamine using constant‐time point‐resolved spectroscopy at 3 T

Separate quantification of glutamate (Glu) and glutamine (Gln) using conventional MRS on clinical scanners is challenging. In previous work, constant‐time point‐resolved spectroscopy (CT‐PRESS) was optimized at 3 T to detect Glu, but did not resolve Gln. To quantify Glu and Gln, a time‐domain basis set was constructed taking into account metabolite T₂ relaxation times and dephasing from B₀ inhomogeneity. Metabolite concentrations were estimated by fitting the basis one‐dimensional CT‐PRESS diagonal magnitude spectra to the measured spectrum. This method was first validated using seven custom‐built phantoms containing variable metabolite concentrations, and then applied to in vivo data acquired in rats exposed to vaporized ethanol and controls. Separate metabolite quantification revealed increased Gln after 16 weeks and increased Glu after 24 weeks of vaporized ethanol exposure in ethanol‐treated compared with control rats. Without separate quantification, the signal from the combined resonances of Glu and Gln (Glx) showed an increase at both 16 and 24 weeks in ethanol‐exposed rats, precluding the determination of the independent and differential contribution of each metabolite at each time. Copyright © 2012 John Wiley & Sons, Ltd.     

Inhibition of human catechol-O-methyltransferase-mediated dopamine O-methylation by daphnetin and its Phase II metabolites

1.?Finding and developing inhibitors of catechol-O-methyltransferase (COMT) from natural products is highly recommended. Daphnetin, a naturally occurring catechol from the family thymelaeaceae, has a chemical structure similar to several potent COMT inhibitors reported previously. Here the potential of daphnetin and its Phase II metabolites as inhibitors of COMT was investigated with human liver cytosol (HLC).

2.?Daphnetin and its methylated metabolite (8-O-methyldaphnetin) were found to inhibit COMT-mediated dopamine O-methylation in a dose-dependent manner. The IC₅₀ values for daphnetin (0.51～0.53?μM) and 8-O-methyldaphnetin (22.5～24.3?μM) were little affected by changes in HLC concentrations. Further kinetic analysis showed the differences in inhibition type and parameters (K_i) between daphnetin (competitive, 0.37?μM) and 8-O-methyldaphnetin (noncompetitive, 25.7?μM). Other metabolites, including glucuronidated and sulfated species, showed negligible inhibition against COMT. By using in vitro–in vivo extrapolation (IV-IVE), a 24.3-fold increase in the exposure of the COMT substrates was predicted when they are co-administrated with daphnetin.

3.?With high COMT-inhibiting activity, daphnetin could serve as a lead compound for the design and development of new COMT inhibitors. Also, much attention should be paid to the clinical impact of combination of daphnetin and herbal preparations containing daphnetin with the drugs primarily cleared by COMT.     

Rapid multi‐echo measurement of brain metabolite T2 values at 7 T using a single‐shot spectroscopic Carr–Purcell–Meiboom–Gill sequence and prior information

We present a method for the robust and accurate estimation of brain metabolite transverse relaxation times (T₂) from multiple spin‐echo data acquired with a single‐shot Carr–Purcell–Meiboom–Gill (CPMG) spectroscopic sequence. Each acquired echo consists of a small number of complex time‐domain data points. The amplitudes of the spectral components in each echo are calculated by solving a set of linear equations in which previously estimated frequencies and linewidths serve as prior information. These priors are obtained from a short MRS experiment in which a large number of time‐domain data points are acquired, and are subsequently estimated using linear prediction with singular value decomposition (LPSVD) processing. We show that this process can be used to accurately and rapidly measure the T₂ values for the main singlet resonances in single‐volume MRS measurements in the brain. The proposed method can be generalized to any set of MRS experiments comprising repeated measurements of amplitude changes, e.g. as a function of an experimental parameter, such as TE, inversion time or diffusion weighting. Copyright © 2013 John Wiley & Sons, Ltd.     

UV-C radiation induced conformational relaxation of pMTa4 DNA in Escherichia coli may be the cause of single strand breaks

Purpose: The biological consequences of initial physicochemical events following exposure of DNA to germicidal (254 nm) ultraviolet C (UV-C) radiation are not fully understood despite progress that has been made. In particular the cause of UV-C induced single strand breaks is not known. This question has been addressed in the present investigation.

Materials and methods: A plasmid construct, pMTa4, was exposed to UV-C in vitro as well as in vivo after transforming the plasmid into a repair proficient wild type and repair deficient, recF, mutant of E. coli. Following UV exposure in vivo, the plasmid was isolated under repair non-permissive and permissive conditions. The plasmid isolate and the pure super-coiled closed circular (CC) topological form of the plasmid were analyzed by agarose gel electrophoresis. The dependence of UV-C induced damage and conformational changes on the dose of radiation as well as on the duration of post-irradiation repair incubations was observed. The influence of UV-C on hyperchromic change and intercalation of ethidium bromide into plasmid DNA were also recorded.

Results: UV-C exposure of pMTa4 DNA in vitro and in vivo induced dose dependent, but sparsely placed, single strand breaks (SSB). While the wild type (AB1157) E. coli was able to repair SSB nearly completely under repair permissive condition, the recF (JC9239) mutant failed to do so. A dose-dependent relaxation of super-structure of CC form of pMTa4 DNA concomitant with enhanced ethidium bromide intercalation into the plasmid DNA was observed.

Conclusion: It is proposed that the conformational relaxation generated negative super-coiling strain on the DNA backbone of CC form of plasmid as well as exposed chemical bonds for hydrolytic cleavage. This might be the cause of the production of sparsely placed single strand breaks in pMTa4 upon exposure to low doses of UV-C.