Steady-state,Pre-steady-state,and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, k_off). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (k_off). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (<1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E>S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.     

阔筋膜与涤纶布修补术对胸壁肿瘤切除后胸壁缺损的临床疗效

目的:探讨阔筋膜修补术与涤纶布修补术对胸壁肿瘤切除后胸壁缺损的临床疗效观察。方法:选取我院胸外科收治的早期胸壁肿瘤患者81例,随机分为阔筋膜组40例行阔筋膜修补术,涤纶布组41例行涤纶布修补术。比较两组患者术后镇痛药用量、肺部体征、引流量、CRP、白细胞及临床康复情况。结果:治疗后与阔筋膜组比较,涤纶布组患者术后镇痛药量、呼吸受限时间及胸膜粘连发生率均较低,(P0.05)。与治疗前比较,两组患者白细胞水平均升高(P0.05),C反应蛋白水平均降低,(P0.05)。与阔筋膜组相比较,涤纶组白细胞水平,C反应蛋白水平均较低(P0.05),涤纶布组治疗后SPO2水平恢复正常的天数缩短(P0.05)。与阔筋膜组比较,涤纶布组患者术后发热天数,引流天数及住院天数均较少,(P0.05)。结论:涤纶布修补术较传统阔筋膜修补术临床疗效优越,且患者痛苦小,术后恢复快,对临床具有指导意义。     

离子注入对微生物细胞的刻蚀与对DNA的损伤及修复

以耐辐射异常球菌为试材,以E. coli 为对照,用显微扫描电镜和3H-TdR标记,研究了离子注入对微生物细胞的刻蚀与对DNA的损伤及其修复。结果表明,注入离子对细胞存在着刻蚀损伤;中性蔗糖梯度密度离心沉降分析证明, 大剂量下离子注入可直接导致DNA损伤,并观察到在对应的存活率峰值注入剂量下,D. radiodurans修复损伤DNA的能力比E. coli 强,还证明了细胞经不同时间温育后,损伤的DNA分子得到了部分修复。 Abstract：　The direct action of N+implantationin on D. radioduransand E. coliwas investigated by SEM, and their cells were labeled with 3H-TdR, which were implanted by 20keV N+after incubation 18hours, then the DNA of lysed cells was subjected to the neutral sucrose gradient(5%～20%) ultra-centrifugation sedimentation analysis. The results showed that N+implantation exerted direct action on two kinds of microorganisms; the momentum transfer and energy deposition of implantation ions produced the direct etching damage on cells, and repair DNA efficiency of D.radiodurans was higher than that of E. coli. Meanwhile, the damaged DNA incomplete repairing was observed. When incubation was continued up to 6 hours, the rejoined DNA molecules broke again. The repair of damaged DNA could be inhibited by 200μg/ml chloramphenicol. This suggested that DNA damage was serious by ion implantation and damaged DNA repair of cells need continuously synthesizing repair enzyme.     

生长激素-海藻酸钠-壳聚糖微胶囊促进骨折愈合的组织学研究

目的:观察生长激素-海藻酸钠-壳聚糖微胶囊促进兔挠骨骨折愈合的作用。方法:实验将新西兰兔80只,在制备新西兰兔右桡骨中段3mm骨缺损模型的基础上,随机分成四组:口服生长激素-海藻酸钠-壳聚糖微胶囊组、皮下注射生长激素组、口服空微胶囊组和生理盐水对照组。实验组口服生长激素-海藻酸钠-壳聚糖微胶囊和皮下注射生长激素,对照组口服空微胶囊。并于术后9、17、30、42d定期HE染色和地衣红染色观察各组的骨折愈合情况。结果:本实验HE染色结果表明,由于在骨缺损部位成纤维细胞产生的大量胶原纤维为基质,形成透明软骨及成骨细胞,骨小梁生长的基础,连接骨痂形成和骨髓腔贯通。而观察到生长激素微胶囊组各期提前生长及改建提前的形态。地衣红染色图像结果分析及直方图的分析表明:生长激素微胶囊组胶原纤维产生促进骨小梁提前形成,进而骨折处骨性骨痂的提前愈合和髓腔的提前贯通。结论:生长激素-海藻酸钠-壳聚糖微胶囊口服能促进骨折修复愈合。     

Pulsed electromagnetic fields promote repair of focal articular cartilage defects with engineered osteochondral constructs

Articular cartilage injuries are a common source of joint pain and dysfunction. We hypothesized that pulsed electromagnetic fields (PEMFs) would improve growth and healing of tissue-engineered cartilage grafts in a direction-dependent manner. PEMF stimulation of engineered cartilage constructs was first evaluated in vitro using passaged adult canine chondrocytes embedded in an agarose hydrogel scaffold. PEMF coils oriented parallel to the articular surface induced superior repair stiffness compared to both perpendicular PEMF (p = .026) and control (p = .012). This was correlated with increased glycosaminoglycan deposition in both parallel and perpendicular PEMF orientations compared to control (p = .010 and .028, respectively). Following in vitro optimization, the potential clinical translation of PEMF was evaluated in a preliminary in vivo preclinical adult canine model. Engineered osteochondral constructs (∅ 6 mm × 6 mm thick, devitalized bone base) were cultured to maturity and implanted into focal defects created in the stifle (knee) joint. To assess expedited early repair, animals were assessed after a 3-month recovery period, with microfracture repairs serving as an additional clinical control. In vivo, PEMF led to a greater likelihood of normal chondrocyte (odds ratio [OR]: 2.5, p = .051) and proteoglycan (OR: 5.0, p = .013) histological scores in engineered constructs. Interestingly, engineered constructs outperformed microfracture in clinical scoring, regardless of PEMF treatment (p < .05). Overall, the studies provided evidence that PEMF stimulation enhanced engineered cartilage growth and repair, demonstrating a potential low-cost, low-risk, noninvasive treatment modality for expediting early cartilage repair.     

植入了骨修复材料的小型猪腰椎椎体不脱钙病理组织切片制备方法

摘要 目的：建立植入了骨修复材料小型猪腰椎椎体骨组织标本的不脱钙病理组织切片制备方法。方法：将含骨修复材料的腰椎椎体骨组织标本进行分割暴露组织切面,梯度浓度乙醇脱水后经Technovit 7200 VLC光聚树脂浸润,经黄蓝光共同辐照进行光聚合包埋,借助硬组织病理切磨系统制备含骨修复材料不脱钙病理组织切片。结果：结果显示通过上述方法制备的病理组织切片,经苏木精-伊红（HE）染色及甲苯胺蓝染色后光学显微镜下观察能较好地显示骨的各种组织细胞结构,可清晰的观察到骨小梁的走向及连接情况。结论：研究建立了含骨修复材料骨组织标本病理组织切片制备方法,实现了含骨修复材料不脱钙骨组织病理切片的制备,经病理染色后实现了带植入物的组织学观察,为生物材料及医疗器械动物试验研究提供了新的病理检测手段及组织学评价途径。     

DNA损伤修复机制——解读2015年诺贝尔化学奖

Tomas Lindahl, Paul Modrich和Aziz Sancar三位科学家因发现“DNA损伤修复机制”获得了2015年诺贝尔化学奖．Lindahl首次发现Escherichia Coli中参与碱基切除修复的第一个蛋白质--尿嘧啶 DNA糖基化酶（UNG）; Modrich重建了错配修复的体外系统,从大肠杆菌到哺乳动物深入探究了错配修复的机制; Sancar利用纯化的UvrA、UvrB、UvrC重建了核苷酸切除修复的关键步骤,阐述了核苷酸切除修复的分子机制．DNA损伤是由生物所处体外环境和体内因素共同导致的,面对不同种类的损伤,机体启动多种不同的修复机制修复损伤,保护基因组稳定性．这些修复机制包括：光修复(light repairing);核苷酸切除修复（nucleotide excision repair, NER）;碱基切除修复（base excision repair, BER）;错配修复（mismatch repair, MMR）;以及DNA双链断裂修复（DNA double strand breaks repair, DSBR）．其中DNA双链断裂修复又分同源重组（homologous recombination, HR）和非同源末端连接（non homologous end joining, NHEJ）两种方式．本文将对上述几种修复的机制进行总结与讨论．     

sgf73+在裂殖酵母中的大规模遗传筛选

遗传相互作用(Genetic interaction, GI)直接提示了生物体内各个基因在功能上的关联性, 为研究一个基因的潜在功能提供了线索。遗传筛选是研究基因遗传相互作用的重要方法。文章以SAGA(Spt-Ada-Gcn5 acetyltransferase)复合物去泛素化模块亚基基因sgf73⁺为查询基因, 在裂殖酵母(Schizosaccharomyces pombe)中进行了大规模遗传筛选。结果显示, 164个基因与sgf73⁺具有负遗传相互作用, 42个基因具有正遗传相互作用。GO(Gene ontology)分析结果表明, 这些基因富集于染色质修饰、DNA损伤修复、压力应答、RNA转录等生物过程。通过组蛋白修饰检测实验首次发现, sgf73⁺的缺失导致组蛋白H3K9、H4K16位点乙酰化水平下降, H3K4位点甲基化修饰水平上升。此外, 系列稀释实验显示sgf73∆菌株对DNA损伤试剂HU和CPT的敏感性提高, 并且Sgf73参与高氧胁迫应答。这些结果显示sgf73⁺参与了染色质修饰、DNA损伤修复和高氧压力应答过程。     

Chlamydomonas raudensis (UWO 241), Chlorophyceae,exhibits the capacity for rapid D1 repair in response to chronic photoinhibition at low temperature1

Maximum photosynthetic capacity indicates that the Antarctic psychrophile Chlamydomonas raudensis H. Ettl UWO 241 is photosynthetically adapted to low temperature. Despite this finding, C. raudensis UWO 241 exhibited greater sensitivity to low‐temperature photoinhibition of PSII than the mesophile Chlamydomonas reinhardtii P. A. Dang. However, in contrast with results for C. reinhardtii, the quantum requirement to induce 50% photoinhibition of PSII in C. raudensis UWO 241 (50 μmol photons) was comparable at either 8°C or 29°C. To our knowledge, this is the first report of a photoautotroph whose susceptibility to photoinhibition is temperature independent. In contrast, the capacity of the psychrophile to recover from photoinhibition of PSII was sensitive to temperature and inhibited at 29°C. The maximum rate of recovery from photoinhibition of the psychrophile at 8°C was comparable to the maximum rate of recovery of the mesophile at 29°C. We provide evidence that photoinhibition in C. raudensis UWO 241 is chronic rather than dynamic. The photoinhibition‐induced decrease in the D1 content in C. raudensis recovered within 30 min at 8°C. Both the recovery of the D1 content as well as the initial fast phase of the recovery of F_v/F_m at 8°C were inhibited by lincomycin, a chloroplast protein synthesis inhibitor. We conclude that the susceptibility of C. raudensis UWO 241 to low‐temperature photoinhibition reflects its adaptation to low growth irradiance, whereas the unusually rapid rate of recovery at low temperature exhibited by this psychrophile is due to a novel D1 repair cycle that is adapted to and is maximally operative at low temperature.