Dampening DNA damage checkpoint signalling via coordinated BRCT domain interactions

In response to DNA damage, checkpoint signalling protects genome integrity at the cost of repressing cell cycle progression and DNA replication. Mechanisms for checkpoint down‐regulation are therefore necessary for proper cellular proliferation. We recently uncovered a phosphatase‐independent mechanism for dampening checkpoint signalling, where the checkpoint adaptor Rad9 is counteracted by the repair scaffolds Slx4‐Rtt107. Here, we establish the molecular requirements for this new mode of checkpoint regulation. We engineered a minimal multi‐BRCT‐domain (MBD) module that recapitulates the action of Slx4‐Rtt107 in checkpoint down‐regulation. MBD mimics the damage‐induced Dpb11‐Slx4‐Rtt107 complex by synergistically interacting with lesion‐specific phospho‐sites in Ddc1 and H2A. We propose that efficient recruitment of Dpb11‐Slx4‐Rtt107 or MBD via a cooperative ‘two‐site‐docking’ mechanism displaces Rad9. MBD also interacts with the Mus81 nuclease following checkpoint dampening, suggesting a spatio‐temporal coordination of checkpoint signalling and DNA repair via a combinatorial mode of BRCT‐domains interactions.     

Assembly of Slx4 signaling complexes behind DNA replication forks

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.     

钙离子振荡对肝糖磷酸化酶激活的随机效应研究

在复杂生化系统的研究过程中,仿真与建模变得越来越重要.对于参与分子数量比较大的生化系统,通常可以采用常微分方程来解决这一问题.对于分子数量比较小的系统,离散粒子基础上的随机模拟方法更精确.然而目前还没有明确的理论方法来确定,对于实际问题用哪种方法能得到更合理的结果.因此需要在一个普遍研究的体系中,通过Ca~(2+)振荡传导信号来研究从随机行为到确定行为的过渡过程.本文以肝细胞中Ca~(2+)振荡对肝糖磷酸化酶激活随机效应为例,讨论了利用随机微分方程来解决分子数量比较小的生化系统的仿真与建模问题,利用细胞内Ca~(2+)有关的Li-Rinzel随机模型,研究了在磷酸化酶降解肝糖的磷酸化-去磷酸化作用循环过程中,三磷酸肌醇受体通道(IP_3R)释放Ca~(2+)的调控作用.结果表明,肝糖磷酸化酶的激活率随受体通道IP_3R的总数增大而减弱,而且三磷酸肌醇浓度比较小时出现相干共振.     

Electrospray Ionization Mass Spectrometric Study of the Multiple Intracellular Monomeric and Polymeric Hemoglobins of Glycera dibranchiata

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500–15,200 Da) and an approximately equal number of polymer Hb peaks (15,500–16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; 2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2–4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.     

Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4‐chloro‐7‐‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl)

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the anti‐epileptic drug carbamazepine (CBZ) in its dosage forms. The method was based on a nucleophilic substitution reaction of CBZ with 4‐chloro‐7‐nitrobenzo‐2‐ oxa‐1,3‐diazole (NBD‐Cl) in borate buffer (pH 9) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. Factors affecting the formation of the reaction product were studied and optimized, and the reaction mechanism was postulated. The fluorescence–concentration plot is rectilinear over the range of 0.6–8 µg/mL with limit of detection of 0.06 µg/mL and limit of quantitation of 0.19 µg/mL. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. Validation of the analytical procedures was evaluated according to ICH guidelines. Copyright © 2015 John Wiley & Sons, Ltd.     

Human embryonic and fetal mesenchymal stem cells differentiate toward three different cardiac lineages in contrast to their adult counterparts

Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.