Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2

The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC

Andrographolide Inhibits PI3K/AKT-Dependent NOX2 and iNOS Expression Protecting Mice against Hypoxia/Ischemia-Induced Oxidative Brain Injury

This study aimed to explore the mechanisms by which andrographolide protects against hypoxia-induced oxidative/nitrosative brain injury provoked by cerebral ischemic/reperfusion (CI/R) injury in mice. Hypoxia in vitro was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone CI/R injury with andrographolide (10-100 mu g/kg, i.v.) at 1 h after hypoxia ameliorated CI/R-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. CI/R induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (NOX2), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b cells due to activation of nuclear factor-kappa B (NF-kappa B) and hypoxia-inducible factor 1-alpha (HIF-1 alpha). All these changes were significantly diminished by andrographolide. In BV-2 cells, OGD induced ROS and nitric oxide production by upregulating NOX2 and iNOS via the phosphatidylinositol-3-kinase (PI3K)/AKT-dependent NF-kappa B and HIF-1 alpha pathways, and these changes were suppressed by andrographolide and LY294002. Our results indicate that andrographolide reduces NOX2 and iNOS expression possibly by impairing PI3K/AKT-dependent NF-kappa B and HIF-1 alpha activation. This compromises microglial activation, which then, in turn, mediates andrographolide's protective effect in the CI/R mice

A Human BRCA2 Complex Containing a Structural DNA Binding Component Influences Cell Cycle Progression

AbstractGermline mutations of the human BRCA2 gene confer susceptibility to breast cancer. Although the function of the BRCA2 protein remains to be determined, murine cells homozygous for BRCA2 inactivation display chromosomal aberrations. We have isolated a 2 MDa BRCA2-containing complex and identified a structural DNA binding component, designated as BR CA2-A ssociated F actor 35 (BRAF35). BRAF35 contains a nonspecific DNA binding HMG domain and a kinesin-like coiled coil domain. Similar to BRCA2, BRAF35 mRNA expression levels in mouse embryos are highest in proliferating tissues with high mitotic index. Strikingly, nuclear staining revealed a close association of BRAF35/BRCA2 complex with condensed chromatin coincident with histone H3 phosphorylation. Importantly, antibody microinjection experiments suggest a role for BRCA2/BRAF35 complex in modulation of cell cycle progression

Reversal of cardiac damage in patients with symptomatic severe aortic stenosis following transcatheter aortic valve implantation: An echocardiographic study

Background: Severe aortic stenosis (AS) results in cardiac damages, such as left ventricular hypertrophy, left atrial enlargement, pulmonary pressure elevation and in advanced stage, right ventricular damage. Généreux and colleagues proposed a staging classification based on these extra-valvular damages in 2017, with increasing stage representing more cardiac damage. While regression of these cardiac damages is expected following aortic valve replacement, the reversal of cardiac damage based on this staging system has not been described. Purpose: This study aimed to describe and stage the changes in cardiac structure and function at 6 months and 1 year after transcatheter aortic valve implantation (TAVI) in patients with symptomatic severe AS. Methods: This was a retrospective, single center, longitudinal observational study. Echocardiographic data of patients who underwent TAVI were retrieved and analysed. Results: From May 2018 to Feb 2021, 31 patients underwent TAVI. 5 patients were excluded due to death <6 months post-procedure (n=2) and incomplete echocardiographic data (n=3). The mean age of the remaining 26 patients was 70.9±9.4 years, 57.7% were male, and 34.6% bicuspid aortic valve. After TAVI, transvalvular aortic mean pressure gradient reduced from 45.2±14.5 mmHg to 8.0±5.4 mmHg (p<0.001), and aortic valve area increased from 0.57±0.21 cm2 to 1.75±0.68 cm2 (p<0.001). At baseline, 6-month and 1-year, the left ventricular mass index (LVMi) were 183.4±60.7g/m2, 150.8±55.3 g/m2 and 126.8±42.1 g/m2 (p<0.001) respectively; left-atrial volume index (LAVI) were 60.4±22.8 ml/m2 , 51.7±23.8ml/m2, and 48.1±23.6ml/m2 (p=0.009) respectively; left ventricular ejection fraction (LVEF) were 52.3±25.4%, 64.2±29.3%, and 62.4±12.1% (p=0.005) respectively. Based on the proposed cardiac damage staging for AS, at baseline 38% of patients were stage 1, 65.4% stage 2, 7.7% stage 3 and 23.1% stage 4. At 1 year, 8.3% were stage 0, 29.2% stage 1, 58.3% stage 2, and 4.2% stage 4. 12 patients (46%) showed improvement in cardiac damage staging, and the other 14 (54%) remained in the same stage. Conclusion: In patients with symptomatic severe AS, there were overall significant regression in LVMi and LAVI, and improvement in LVEF at 1 year after TAVI. However, improvement in cardiac damage staging was observed in only 46% of patients

Use of the normalized impact-echo spectrum to monitor the setting process of mortar

The objective of this paper is to introduce a newly developed nondestructive test technique called the normalized impact-echo spectrum and its application to monitoring the stiffening process of mortar. The impact-echo method is an effective and nondestructive technique for locating flaws in concrete. The depth of an air-concrete interface is determined by identifying the large amplitude peak in the spectrum at the dominant frequency corresponding to multiple wave reflections from the interface. However, the measure of the peak amplitude does not make sense because it changes with different impact forces. To make the peak amplitude meaningful, there is a need to normalize the spectrum on impact force. After normalization, the amplitudes in impact-echo spectra can provide useful information on the intensity of wave reflections at the interface. In this paper, the normalized impact-echo spectra were used to monitor the setting process of mortar. Experimental results demonstrate that the normalization of the impact-echo spectra can make the peak amplitude steady for various impacts and the coefficient of variation is less than 1.5%. The variation in the amplitude of the normalized impact-echo spectrum with the mortar age can indeed reflect the stiffening process of mortar. The results show that the proposed technique is suitable to monitor the setting process of mortar. (C) 2010 Elsevier Ltd. All rights reserved

Hyperhomocysteinemia causes ER stress and impaired autophagy that is reversed by vitamin B supplementation

10.1038/cddis.2016.374Cell Death and Disease712e251

oriC Region and replication termination site, dif, of the Xanthomonas campestris pv. campestris 17 chromosome

A 13-kb DNA fragment containing oriC and the flanking genes thdF, orf900, yidC, rnpA, rpmH, oriC, dnaA, dnaN, recF, and gyrB was cloned from the gram-negative plant pathogen Xanthomonas campestris pv. campestris 17. These genes are conserved in order with other eubacterial oriC genes and code for proteins that share high degrees of identity with their homologues, except for orf900, which has a homologue only in Xylella fastidiosa. The dnaA/dnaN intergenic region (273 bp) identified to be the minimal oriC region responsible for autonomous replication has 10 pure AT clusters of four to seven bases and only three consensus DnaA boxes. These findings are in disagreement with the notion that typical oriCs contain four or more DnaA boxes located upstream of the dnaA gene. The X campestris pv. campestris 17 attB site required for site-specific integration of cloned fragments from filamentous phage phiLf replicative form DNA was identified to be a dif site on the basis of similarities in nucleotide sequence and function with the Escherichia coli dif site required for chromosome dimer resolution and whose deletion causes filamentation of the cells. The oriC and dif sites were located at 12:00 and 6:00, respectively, on the circular X. campestris pv. campestris 17 chromosome map, similar to the locations found for E. coli sites. Computer searches revealed the presence of both the dif site and XerC/XerD recombinase homologues in 16 of the 42 fully sequenced eubacterial genomes, but eight of the dif sites are located far away from the 6:00 point instead of being placed opposite the cognate oriC. The differences in the relative position suggest that mechanisms different from that of E. coli may participate in the control of chromosome replication