回收琼脂糖凝胶中DNA的简便方法

多年来国内分子生物学实验室所采用的几种从琼脂糖凝胶中回收DNA的方法不同程度地存在着各种问题。现介绍一种新的从琼脂糖凝胶上分离和提取DNA的简易方法,采用实验室常用的吸附柱中的吸附膜对DNA进行拦截、纯化和回收。实验证明这种方法回收DNA具有简便快捷、成本低和效率高等优点,是一种切实可行的方法。     

舌骨肌切开悬吊术结合腭咽成形术治疗重度OSAHS

目的：应用联合应用舌骨肌切开悬吊术和腭咽成形术治疗重度阻塞性睡眠呼吸暂停低通气综合征。方法：用多导睡眠监测确定52例重度阻塞性睡眠呼吸暂停低通气综合征,不同方法确定均为以舌根平面阻塞为主的上气道多平面狭窄。采用舌骨肌切开悬吊术联合腭咽成形术进行治疗。结果：所有接受手术的患者术后鼾声均有不同程度的减轻,手术总有效率94．2％。无1例出现不良并发症。结论：舌骨肌切开悬吊术安全、易行,结合腭咽成形术治疗重度阻塞性睡眠呼吸暂停低通气综合征效果良好。     

The profile of MMP and TIMP in injured rat ACL

Human anterior cruciate ligament (ACL) has poor healing ability after injury. The devastating effects of matrix metalloproteinases (MMPs) excess expression are regarded as the main reason. Tissue inhibitor metalloproteinases (TIMPs) may be independent of MMPs inhibition. In this paper, a rat ACL rotating injury apparatus was designed to produce ACL injury. After inducing injury, joint fluids and ACL tissue total proteins were immediately extracted. In addition, ACL tissue was isolated in a culture plate with 1%FBS medium for the ex vivo study. We found MMP-2 in joint fluids increased significantly by 4 folds after ACL injury as a function of time. Ex vivo study showed MMP-2 in the medium and ACL cultured tissue increased significantly respectively to 3 folds and to 6 folds. The joint fluids global MMP increased to 3.5 folds with non-treatment and APMA-treatment in day three. On the gene expression level, the changes in MMP-1 and CD147 have the similar tendency. The ratio of MMP-1/TIMP-1 increased with time after ACL injury. We conclude that MMP-2 increased significantly in the early phase in the joint cavity after ACL injury. The ex vivo study demonstrated the same tendency. Generic MMP Activity Assay (global MMP assay) an dzymography also showed significant increase in MMP activity in joint fluids. These results showed ACL having poor healing ability after injury may not be only due to ACL release of large quantities of MMPs. The important factor may be the alterations in the whole joint cavity's internal environment.     

An Electrostatically Preferred Lateral Orientation of SNARE Complex Suggests Novel Mechanisms for Driving Membrane Fusion

Biological membrane fusion is a basic cellular process catalyzed by SNARE proteins and additional auxiliary factors. Yet, the critical mechanistic details of SNARE-catalyzed membrane fusion are poorly understood, especially during rapid synaptic transmission. Here, we systematically assessed the electrostatic forces between SNARE complex, auxiliary proteins and fusing membranes by the nonlinear Poisson-Boltzmann equation using explicit models of membranes and proteins. We found that a previously unrecognized, structurally preferred and energetically highly favorable lateral orientation exists for the SNARE complex between fusing membranes. This preferred orientation immediately suggests a novel and simple synaptotagmin-dependent mechanistic trigger of membrane fusion. Moreover, electrostatic interactions between membranes, SNARE complex, and auxiliary proteins appear to orchestrate a series of membrane curvature events that set the stage for rapid synaptic vesicle fusion. Together, our electrostatic analyses of SNAREs and their regulatory factors suggest unexpected and potentially novel mechanisms for eukaryotic membrane fusion proteins.     

Sequence variations of mitochondrial DNA D-loop region are highly frequent events in familial breast cancer

Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations. The non-coding displacement (D)-loop, especially a mononucleotide repeat (poly-C) between 303 and 315 nucleotides (D310), has been recently identified as a frequent hotspot of mutations in human neoplasia, including breast cancer. To further explore the sequence variations of mitochondrial D-loop region in familial breast cancer and their possible associations with breast cancer risk, PCR-SSCP and direct DNA sequencing methods were used to detect the variants of the mtDNA D-Loop in 23 familial breast cancer patients as well as three high-risk cancer families. Compared to that in sporadic breast tumors (53.3%, 16/30) and healthy blood donors (6.7%, 2/30), we identified a total of 126 sequence alterations in 23/23 (100%) of familial breast cancer patients, including eight novel nucleotide variants. Among these changes, A to G at nt.263, T to C at nt.489, T to C at nt.310, TC insertion at nt.311, CA deletion at nt.522, and C to G at nt.527 were highly frequent ones. In addition, among three high-risk cancer families, we found that individuals affected with breast cancer harbored more mtDNA sequence variants in mtDNA D310 area than other affected family members. Together, our data indicate that sequence variants within the mtDNA D-Loop region are frequent events in Chinese familial breast cancer patients. Some of these nucleotide abnormalities, particularly those in D310 segment, might be involved in the breast carcinogenesis and could be included in a panel of molecular biomarkers for cancer susceptibility early-detection strategy.     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.