RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly

DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 A. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.     

Pullout strength of suture anchors: Effect of mechanical properties of trabecular bone

This study investigated the relationships between trabecular microstructure and elastic modulus, compressive strength, and suture anchor pullout strength. Twelve fresh-frozen humeri underwent mechanical testing followed by micro-computed tomography (μCT). Either compression testing of cylindrical bone samples or pullout testing using an Arthrex 5 mm Corkscrew was performed in synthetic sawbone or at specific locations in the humerus such as the greater tuberosity, lesser tuberosity, and humeral head. Synthetic sawbone underwent identical mechanical testing and μCT analysis. Bone volume fraction (BVF), structural model index (SMI), trabecular thickness (TbTh), trabecular spacing (TbSp), trabecular number (TbN), and connectivity density were compared against modulus, compressive strength, and pullout strength in both materials. In cadaveric bone, modulus showed correlations to all of the microstructural properties, while compressive and pullout strength were only correlated to BVF, SMI, and TbSp. The microstructure of synthetic bone differed from cadaveric bone as SMI and TbTh showed little variation across the densities tested. Therefore, SMI and TbTh were the only microstructural properties that did not show correlations to the mechanical properties tested in synthetic bone. This study helps identify key microstructure–property relationships in cadaveric and synthetic bone as well as illustrate the similarities and differences between cadaveric and synthetic bone as biomechanical test materials.     

The tandem BRCT domain of 53BP1 is not required for its repair function

53BP1 plays an important role in cellular response to DNA damage. It is thought to be the mammalian homologue of budding yeast Rad9 and/or fission yeast Crb2. Rad9/Crb2 are bona fide checkpoint proteins whose activation requires their corresponding C-terminal tandem BRCT (BRCA1 C-terminal) motifs, which mediate their oligomerization and phosphorylation at multiple sites following DNA damage. Here we show that the function of human 53BP1 similarly depends on its oligomerization and phosphorylation at multiple sites but in a BRCT domain-independent manner. Moreover, unlike its proposed yeast counterparts, human 53BP1 only has limited checkpoint functions but rather acts as an adaptor in the repair of DNA double strand breaks. This difference in function may reflect the higher complexity of the DNA damage response network in metazoa including the evolution of other BRCT domain-containing proteins that may have functions redundant or overlapping with those of 53BP1.     

Reconstruction of rat femoral veins with microvascular prostheses

Synthetic conduits have not been suitable for microvascular reconstruction owing primarily to their high thrombogenicity. Vein replacements are the most vulnerable to thrombosis because of their low shear rates and low pressure. Experimental replacement of microvenous segments with prosthetic segments has shown little success. Recent technological advances in biomaterials and control of thrombogenesis provide the potential for success in the development of venous prostheses. The purpose of this study was to assess the use of nonbiodegradable composite polyurethane microvascular prostheses for reconstruction of rat femoral veins. Rat femoral venous defects of 10 mm were reconstructed with autogenous vein (n = 12), unprocessed plain polyurethane (n = 5), and nonbiodegradable composite polyurethane (n = 31). Patency was evaluated by direct observation and proximal venous milking tests. The patency rate of composite grafts was not significantly different from that of isotopic vein (p = 0.5, Fisher's exact test), and both had higher patency than unprocessed polyurethane (p less than 0.01). Composite grafts were examined sequentially using light and scanning electron microscopy. Grafts were fully endothelialized between the first and third months. The neointimal, neomedial, and neoadventitial layers could be seen more distinctly over time. New opportunities in reconstructive microsurgery may be opened by microvascular prostheses that are complaint and thromboresistant.     

A new aspect of the protective functions of the blood-brain barrier: activities of four drug-metabolizing enzymes in isolated rat brain microvessels

The multiple functions of the blood-brain barrier (BBB) consist mostly in membrane properties controlling the bidirectional exchange of molecules between the general circulation and the central nervous system. As lipophilic molecules are able to easily penetrate the membrane of brain microvessels endothelial cells, an Achilles' heel exists in the brain's protective system. We measured the activities of some enzymes involved in the metabolism of lipophilic xenobiotics, i.e. cytochrome P-450-linked monooxygenases, epoxide hydrolase, NADPH:cytochrome P-450 reductase and 1-naphthol UDP-glucuronosyl transferase in isolated rat brain microvessels. The relatively high activities observed indicate the capacity of endothelial cells to metabolize xenobiotics, and, thus, to give an additional protection to the brain.     

Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms.

A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Mutants were constructed which did not bind to Bax but retained anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain with the corresponding amino acids from Bax. The resulting chimera showed a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retained the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was introduced that rendered the chimera incapable of heterodimerization, the resulting protein failed to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial properties that were inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel.     

秦岭龙胆开花前后多糖含量的比较及其提取工艺优化与抗氧化活性

目的 对秦岭龙胆开花前后多糖含量进行比较并利用响应面法优化其多糖的提取工艺,并对多糖抗氧化活性进行测定。方法 利用单因素试验比较秦岭龙胆开花前后多糖的含量,对含量高者进行工艺优化。在单因素基础上选取因素水平,根据中心组合设计原理采用响应面法优化提取时间、料液比、醇沉比、离心转速对秦岭龙胆多糖提取率的影响,利用DPPH和ABTS自由基对多糖的抗氧化活性进行研究。结果 秦岭龙胆开花后多糖含量高于未开花药材。在分析各个因素的显著性和交互作用后,得到秦岭龙胆开花药材多糖最佳提取工艺为提取时间3h,料液比1:16,醇沉比为1:10,离心转速3250 r/min时,多糖的提取率为5.13%,与预测值5.42%接近。多糖对DPPH和ABTS均有一定的抗氧化能力,且其抗氧化能力高于对照品VC与BHT。结论 本研究建立的模型可对秦岭龙胆多糖提取率进行分析和预测,可为秦岭龙胆的开发和利用提供参考依据。     

Noggin Over-Expressing Mouse Embryonic Fibroblasts and MS5 Stromal Cells Enhance Directed Differentiation of Dopaminergic Neurons from Human Embryonic Stem Cells

Directed methods for differentiating human embryonic stem cells (hESCs) into dopaminergic (DA) precursor cells using stromal cells co-culture systems are already well established. However, not all of the hESCs differentiate into DA precursors using these methods. HSF6, H1, H7, and H9 cells differentiate well into DA precursors, but CHA13 and CHA15 cells hardly differentiate. To overcome this problem, we modified the differentiation system to include a co-culturing step that exposes the cells to noggin early in the differentiation process. This was done using γ-irradiated noggin-overexpressing CF1-mouse embryonic fibroblasts (MEF-noggin) and MS5 stromal cells (MS5-noggin and MS5-sonic hedgehog). After directed differentiation, RT-PCR analyses revealed that engrailed-1 (En-1), Lmx1b, and Nurr1, which are midbrain DA markers, were expressed regardless of differentiation stage. Moreover, tyrosine hydroxylase (Th) and an A9 midbrain-specific DA marker (Girk2) were expressed during differentiation, whereas levels of Oct3/4, an undifferentiated marker, decreased. Immunocytochemical analyses revealed that protein levels of the neuronal markers TH and TuJ1 increased during the final differentiation stage. These results demonstrate that early noggin exposure may play a specific role in the directed differentiation of DA cells from human embryonic stem cells.