BRCA1 and its phosphorylation involved in caffeine-inhibitable event upstream of G2 checkpoint

Caffeine, which specifically inhibits ATM/ATR kinases, efficiently abrogates the ionizing radiation (IR)-induced G2 arrest and increases the sensitivity of various tumor cells to IR. Mechanisms for the effect of caffeine remain to be elucidated. As a target of ATM/ATR kinases, BRCA1 becomes activated and phosphorylated in response to IR. Thus, in this work, we investigated the possible role of BRCA1 in the effect of caffeine on G2 checkpoint and observed how BRCA1 phosphorylation was regulated in this process. For these purposes, the BRCA1 protein level and the phosphorylation states were analyzed by Western blotting by using an antibody against BRCA1 and phospho-specific antibodies against Ser-1423 and Ser-1524 residues in cells exposed to a combination of IR and caffeine. The results showed that caffeine down-regulated IR-induced BRCA1 expression and specifically abolished BRCA1 phosphorylation of Ser-1524, which was followed by an override of G2 arrest by caffeine. In addition, the ability of BRCA1 to transactivate p21 may be required for MCF-7 but not necessary for Hela response to caffeine. These data suggest that BRCA1 may be a potential target of caffeine. BRCA1 and its phosphorylation are most likely to be involved in the caffeine-inhibitable event upstream of G2 arrest.

Synthesis, crystal structure, and thermal property of a novel supramolecular assembly: (NH42810424)(CHNO) O-[H(V)O(]2H)4(10)28(2), constructed from decavanadate and caffeine

A novel 3D supramolecular assembly constructed from decavanadate and caffeine building blocks, (NH4)(2)(C8H10N4O2)(4)[H4V10O28].2H(2)O (1), has been synthesized in aqueous solution and characterized by elemental analysis, IR, H-1 NMR, V-51 NMR, TG-DTA, and single crystal X-Ray diffraction. The compound 1 crystallizes in monoclinic system, space group P2(1)/n, a = 15.801(1) Angstrom, b = 12.914(1) Angstrom, c = 15.913(2) Angstrom, beta = 113.55degrees, V = 2976.4 (5) Angstrom(3), Z = 2, R = 0.0498 with 6818 reflections. Water molecules, ammonium ions, and caffeine act as "cement" linking the polyanions into 1D chain along the c-axis by hydrogen bonding. In compound 1, extensive hydrogen-bond contacts and strong pi-pi interactions lead to an ordered 3D supramolecular framework. TG-DTA curves indicate that the weight loss of the complex can be divided into three stages.

12C6+束和Mitomycin C 诱导的DNA损伤效应

本文旨在分别研究重离子束及MMC在诱导细胞的DNA损伤效应中一些具体的分子机制，为治疗的进行以及相关辅助药物的开发提供理论依据。本文探索的重点有两个，第一个是重离子束辐射诱导的DNA损伤效应及p53在其中的激活，第二个是MMC诱导的DNA损伤效应及p53和BRCA1、H2AX等分子在其中的角色。 1. 12C6+离子束诱导HeLa细胞DNA损伤效应为了研究HeLa细胞经过12C6+ 束辐照之后的DNA损伤效应，及这个过程中p53激活的分子机制。我们运用中性单细胞电泳技术，检测了HeLa细胞经过4Gy 12C6+ 束辐照0h、3h、6h和12h之后DNA的损伤情况，以及0.5Gy、1Gy、2Gy和4Gy 12C6+ 束辐照0h后的DNA损伤情况。同时运用细胞生长实时监测仪监测了HeLa细胞在经过0Gy、0.5Gy和1Gy 12C6+ 束辐照之后的生长变化，并运用AO/EB双染检测了辐照24小时后的凋亡情况。另外，利用8mmol/L的caffeine（抑制ATM和ATR）和20μmol/L的wortmannin（抑制ATM和DNA-PK）处理HeLa细胞后再进行1Gy 12C6+ 束辐照，通过western blot检测p53的表达。结果显示，12C6+ 束辐照可造成HeLa细胞的DNA损伤，损伤随剂量升高而升高但随时间降低；并诱导HeLa细胞发生凋亡；而且辐照后p53表达升高，但经过caffeine或者wortmannin预先处理的细胞p53均没有显著升高。我们的结论是：12C6+ 束辐照可造成HeLa细胞的DNA损伤并诱导损伤修复及凋亡等效应，损伤效应相关的分子p53被激活，并且激活依赖于ATM。 2. MMC诱导的DNA损伤效应在这一部分研究中，首先，我们利用与上面相同的研究方法，探讨了p53在MMC诱导的DNA损伤效应中的激活情况，结果显示，MMC诱导的DNA损伤效应并不依赖于p53。另外，我们还探讨了， BRCA1在FANCD2的γ-H2AX依赖性转移中的作用。MMC可造成DNA的ICL（interstrand cross-link）损伤，ICL可通过FA(Fanconi Anemia)通路进行修复。FANCD2是FA通路的核心分子，在DNA产生ICL时被各种分子修饰然后转移到损伤部分，这个过程的涉及到ATR、γ-H2AX及BRCA1等，本文试图探讨BRCA1在其中的作用方式。研究中，我们监测了不同处理（包括对照、caffeine（可抑制ATR）、MMC及MMC ＋caffeine）的HCC1937(BRCA1缺陷型)和MCF7（BRCA1野生型）细胞的生长；并用Western blot检测MMC处理之后HCC1937细胞γ-H2AX的表达情况。结果表明，MMC和caffeine均可以抑制HCC1937的生长，但caffeine和MMC+caffeine的抑制效果是一样的；MMC和caffeine均可以抑制MCF7的生长，且MMC+caffeine处理比仅进行caffeine处理的抑制作用强；MMC处理之后，HCC1937的γ-H2AX表达显著升高。我们的结论是，在FANCD2的γ-H2AX依赖性转移中，H2AX的磷酸化并不依赖于BRCA1，不过，BRCA1和ATR应该参与一个相同的分子事件，可能是FANCD2的磷酸化。这个有待进一步的实验验证

On-line concentration of neutral and charged species in capillary electrochromatography with a methacrylate-based monolithic stationary phase

A method involving self-concentration, on-column enrichment and field-amplified sample stacking for on-line concentration in capillary electrochromatography with a polymer monolithic column is presented. Since monolithic columns eliminate the frit fabrication and the problems associated with frits, the experimental conditions could be more flexibly adjusted to obtain higher concentration factor in comparison with conventional particulate packed columns. With self-concentration effect, the detection sensitivity of benzene and hexylbenzene is improved by a factor of 4 and 8, respectively. With on-column enrichment and ultralong injection, improvement as high as 22 000 times in detection sensitivity of benzoin is achieved. Furthermore, a combination of the three above-mentioned methods yields up to a 24000-fold improvement in detection sensitivity for caffeine, a charged compound. Parameters affecting the efficiency of on-line concentration are investigated systematically. In addition, equations describing on-line concentration process are deduced.

Evaluation of nonlinear chromatographic performance by frontal analysis using a simple multi-plate mathematical model

A multi-plate (NIP) mathematical model was proposed by frontal analysis to evaluate nonlinear chromatographic performance. One of its advantages is that the parameters may be easily calculated from experimental data. Moreover, there is a good correlation between it and the equilibrium-dispersive (E-D) or Thomas models. This shows that it can well accommodate both types of band broadening that is comprised of either diffusion-dominated processes or kinetic sorption processes. The MP model can well describe experimental breakthrough curves that were obtained from membrane affinity chromatography and column reversed-phase liquid chromatography. Furthermore, the coefficients of mass transfer may be calculated according to the relationship between the MP model and the E-D or Thomas models. (C) 2004 Elsevier B.V. All rights reserved.