小鼠动物实验方法系列专题(二) 肝脏大部分切除实验在小鼠肝再生研究中的应用

小鼠肝大部分切除(partial hepatectomy,PH)实验是研究肝再生的一个重要的实验。本文以C57小鼠为例,对肝大部分切除实验做了较为详细的介绍。实验结果显示,在术后的1～8天,小鼠的肝脏体重比值逐渐增加,在术后的7～10天里可以达到原来肝重的90%以上,10天以后肝细胞停止分裂。正常情况下,实施肝大部分切除后,小鼠的存活率可以达到90%以上。该模型的建立为研究肝脏再生的细胞和分子生物学机制奠定了基础。     

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed di...     

Hypoxia induces beta-amyloid in association with death of RGC-5 cells in culture

Beta-amyloid (Aβ) derived from amyloid precursor protein (APP) has been associated with retinal degeneration in Alzheimer’s disease (AD) and glaucoma. This study examined whether hypoxia exposure induces Aβ accumulation in RGC-5 cells. While levels of APP mRNA and protein significantly increased in the cells, elevated abundance of Aβ was also observed in cells and culture medium between 12 or 24 and 48 h after 5% O₂ hypoxia treatment. Additionally, there is a close relationship between induction of APP and Aβ and intracellular accumulation of ROS along with loss of mitochondrial membrane potential followed by the death of RGC-5 cells in culture under hypoxia. These results suggest a possible involvement of APP and Aβ in the death of RGCs challenged by hypoxia.     

Improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization

Most protein structural prediction algorithms assemble structures as reduced models that represent amino acids by a reduced number of atoms to speed up the conformational search. Building accurate full-atom models from these reduced models is a necessary step toward a detailed function analysis. However, it is difficult to ensure that the atomic models retain the desired global topology while maintaining a sound local atomic geometry because the reduced models often have unphysical local distortions. To address this issue, we developed a new program, called ModRefiner, to construct and refine protein structures from Cα traces based on a two-step, atomic-level energy minimization. The main-chain structures are first constructed from initial Cα traces and the side-chain rotamers are then refined together with the backbone atoms with the use of a composite physics- and knowledge-based force field. We tested the method by performing an atomic structure refinement of 261 proteins with the initial models constructed from both ab initio and template-based structure assemblies. Compared with other state-of-art programs, ModRefiner shows improvements in both global and local structures, which have more accurate side-chain positions, better hydrogen-bonding networks, and fewer atomic overlaps. ModRefiner is freely available at http://zhanglab.ccmb.med.umich.edu/ModRefiner.     

Cellular repressor of E1A-stimulated genes regulates vascular endothelial cell migration by the ILK/AKT/mTOR/VEGF(165) signaling pathway

The migration of vascular endothelial cells plays a critical role in a variety of vascular physiological and pathological processes, such as embryonic development, angiogenesis, wound healing, re-endothelialization, and vascular remodeling. This study clarified the role and mechanism of a new vascular homeostasis regulator, Cellular repressor of E1A-stimulated genes (CREG), in the migration of primary human umbilical vein endothelial cells (HUVECs). A wound healing assay and transwell migration model showed that upregulation of CREG expression induced HUVEC migration and it was positively correlated with the expression of vascular endothelial growth factor. Furthermore, wild type integrin-linked kinase reversed the poor mobility of CREG knock-down HUVECs; in contrast, kinase-dead integrin-linked kinase weakened the migration of HUVECs. We also studied the effect of CREG on HUVEC migration by the addition of an mTOR inhibitor, recombinant vascular endothelial growth factor₁₆₅, neutralizing antibody of vascular endothelial growth factor₁₆₅ and AKT siRNA, and we concluded that CREG induces endothelial cell migration by activating the integrin-linked kinase/AKT/mTOR/VEGF₁₆₅ signaling pathway.     

Biotransformation of p-, m-, and o-hydroxybenzoic acids by Panax ginseng hairy root cultures

The regioselective glycosylation of three isomers of hydroxybenzoic acids was observed in Panax ginseng hairy root cultures. p-Hydroxybenzoic acid (1) and m-hydroxybenzoic acid (2) were converted into their corresponding glycosides (1a and 2a) and glycosyl esters (1b and 2b) while no metabolite of o-hydroxybenzoic acid (3) was detected. A new compound, m-hydroxybenzoic acid β-d-xylopyranosyl (1 → 6)-β-d-glycopyranosyl ester (2c) was identified as a biotransformation product of 2. Further time-course studies of the biotransformation reactions showed that the glycosides were major products in the latter stage. The addition of carbohydrates or antioxidants increased glycosyl esters formation.     

High yield expression of non-phosphorylated protein tyrosine kinases in insect cells

The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.     

分子标记育种在我国的研究进展

现代生物技术的发展为利用外源基因培育优良作物品种提供了新的途径。简要综述了分子标记育种在我国研究的总体情况以及我国在分子标记育种领域取得的成就,并对分子标记育种的前景进行了展望。