鼻咽癌组织中氧化性DNA损伤标志物8-硝基鸟嘌呤和8-羟基脱氧鸟苷的检测及与iNOS的关系

8-硝基鸟嘌呤（8-nitroguanine, 8-NitroG）和8-羟基脱氧鸟苷（8-hydroxy-2′-deoxyguanosine, 8-OHdG）是2个氧化性DNA损伤生物标志物,而诱导型一氧化氮合酶(iNOS)在病理状态下催化细胞合成与氧化性DNA损伤有关的 氧自由基NO.本研究通过检测鼻咽癌组织中8-NitroG、8-OHdG和iNOS的免疫反应强度,初步探究鼻咽癌的发生和发展是否与氧化性DNA损伤有关以及8-NitroG、8-OHdG与iNOS表达的关系.利用多克隆抗体8-NitroG和单克隆抗体8-OHdG、iNOS,采用双色荧光免疫组织化学方法检测鼻咽癌组织中8-NitroG、8-OHdG和iNOS的免疫反应,秩和检验统计学方法分析鼻咽癌和慢性咽炎鼻咽组织之间8-NitroG、8-OHdG和iNOS免疫反应强度的差异.结果显示,19例鼻咽癌组织细胞中,8-NitroG、8-OHdG和iNOS均为强免疫反应,8-NitroG和8-OHdG阳性率100%,iNOS阳性率94.7 %,与13例慢性咽炎组织比较差异显著(P.<0.05).结果提示,鼻咽癌的发生和发展与氧化性DNA损伤有关,其原因与炎症等病理刺激下鼻咽组织高表达的iNOS催化细胞合成氧自由基NO引起的8-NitroG和8-OHdG DNA损伤密切相关.另外,8-NitroG和8-OHdG有望成为辅助鼻咽癌诊断的生物标志物.     

DNA氧化损伤的免疫组织化学研究

采用目前公认的DNA氧化损伤标志物8-OHdG的单抗,利用LSAB法研究不同年龄组BALB/C小鼠胸腺和脾脏8-OHdG的生成水平,对免疫组织在衰老过程中DNA氧化损伤的水平进行免疫组织化学研究。以探讨免疫系统的自由基损伤对衰老的影响。结果表明,在胸腺,8-OHdG^ 细胞密度随增龄而增多,并主要位于髓质区;脾脏中的阳性细胞则无明显的增龄性变化,但胸染形态却存在明显差异。本研究结果显示,衰老过程中,免疫细胞内8-OHdG含量发生改变,免疫系统受到了氧自由基的损伤。8-OHdG可作为免疫老化过程中的一种生物标志。     

化疗损伤性卵巢功能早衰小鼠动物模型的研究

目的探索建立化疗损伤性卵巢功能早衰动物模型的适宜方法及最佳时间。方法以70只ICR雌性小鼠为研究对象,腹腔连续注射顺铂7~14 d,观察不同剂量和时间条件下顺铂对小鼠的体重、卵巢功能、肝肾毒性及卵巢组织抗氧化系统各指标的变化。结果顺铂可引起小鼠体重明显下降,卵巢功能减退,出现肝肾毒性,并可引起卵巢抗氧化及氧化损伤指标的异常,与对照组比较均有明显差异。结论3.0~4.0 mg/(kg.bw)的顺铂作用7 d后即可引起小鼠出现明显的卵巢功能衰退、肝肾损伤,氧化损伤是顺铂产生其毒性作用的可能机制之一。该模型的生殖内分泌和病理组织学变化,与人类化疗损伤性卵巢功能早衰病变过程相似。     

急性缺氧对小鼠大脑皮质和海马的时间依赖性损伤

本文旨在研究急性缺氧对小鼠大脑皮质和海马组织的细胞损伤作用及其机制。利用密闭玻璃罐构建急性缺氧小鼠模型,采用激光散斑成像仪检测小鼠不同缺氧时间窗的脑血流变化,用总超氧化物歧化酶(total superoxide dismutase, T-SOD)和丙二醛(malondialdehyde, MDA)试剂盒检测大脑皮质和海马组织的氧化应激,用组织免疫荧光染色法检测小鼠大脑皮质和海马组织神经炎性反应,用一步法TUNEL细胞凋亡试剂盒检测大脑皮质和海马组织中神经元的凋亡。结果显示,与未缺氧(0min)小鼠相比,缺氧30 min小鼠脑血流显著降低,大脑皮质和海马组织CD68+/Iba1+小胶质细胞百分比显著增加,且神经元凋亡显著增多。与缺氧30 min小鼠相比,缺氧60 min小鼠脑血流相对值显著降低,大脑皮质MDA含量显著增加,大脑皮质和海马中CD68~+/Iba1~+小胶质细胞百分比显著增加,神经元凋亡显著增多。上述结果提示,急性缺氧诱发小鼠脑组织损伤程度具有时间依赖性,而氧化应激和神经炎性作用是重要的损伤机制。     

反复注射顺铂诱导小鼠慢性肾功能不全模型的建立

目的 构建慢性肾脏病(chronic kidney disease,CKD)C57BL/6小鼠模型,探索其小管损伤指标和间质纤维化程度随顺铂造模剂量的改变,为研究从AKI到CKD进展过程提供动物实验依据。方法 将24只8周龄雄性C57BL/6小鼠随机平均分为对照组和低、中、高剂量顺铂模型组。模型组小鼠按5、7、10 mg/kg顺铂腹腔注射给药,每周1次,连续注射4周构建模型。处死小鼠后,留取标本进行相关检测。检测血浆肌酐和24 h尿蛋白排泌量来评估小鼠肾功能;PAS染色观察肾脏病理学变化;免疫组化检测肾损伤分子1(KIM-1)和尿液检测N-乙酰-β-D氨基葡萄糖苷酶(NAG)水平以评估肾小管损伤情况;免疫组化法检测肾脏CD3阳性T细胞和免疫荧光法检测F4/80阳性巨噬细胞浸润情况;天狼星红染色、免疫组化法检测胶原I和α-平滑肌肌动蛋白(α-SMA)表达以评估肾脏纤维化情况。结果 与正常对照组相比,随着注射顺铂浓度的升高,小鼠肾脏损伤越明显,其中10 mg/kg顺铂高剂量组最为显著。与对照组相比,顺铂高剂量组小鼠肾功能下降,表现为血浆肌酐浓度和24 h尿蛋白排泌量显著升高(P0.05和P0.001);肾小管上皮细胞坏死、空泡变性等病理学改变显著,肾组织KIM-1表达显著上升(P0.05),尿NAG水平升高;肾组织浸润的CD3阳性T细胞和F4/80阳性巨噬细胞增多;肾组织天狼星红染色阳性胶原纤维区域显著增多(P0.001),胶原I和α-SMA表达也明显增多(P0.01),肾小管-间质发生纤维化。结论反复注射4周10 mg/kg顺铂可诱导小鼠慢性肾功能不全模型,可为研究AKI向CKD的转化机制提供了新的实验模型。     

8-OHdG在医学领域的应用与研究进展

氧化应激带来的氧化损伤是造成人体多种损伤和病变的重要因素。8-羟基脱氧鸟苷(8-hydroxy-2’-deoxyguanosine,8-OHdG)作为DNA氧化损伤产物是广泛用于研究疾病中氧化损伤机制的关键标志物。国内外大量研究已普遍应用8-OHdG作为分析指标,该文着眼于近年来研究动向,就8-OHdG的作用机理与检测方法,以及职业与环境暴露的危害评价、辅助疾病早期诊断、治疗和新药研发等方面的应用作一综述。     

不同天数姜黄素灌胃对顺铂所致小鼠胃排空障碍的作用

目的:探讨不同天数姜黄素灌胃对顺铂所致小鼠胃排空障碍的作用及其是否通过减轻氧化损伤发挥作用。方法:健康成年昆明种小鼠,随机分为对照组、顺铂组、姜黄素组和顺铂+姜黄素灌胃1天、5天、10天、15天、20天和30天组,每组8只。用药结束后24小时测量胃残留率并取胃组织检测MDA和SOD含量。结果:注射顺铂后,各组小鼠体重明显减轻,姜黄素灌胃10~30天可明显减轻顺铂导致的体重减少(P0.05);注射顺铂后各组胃残留率升高(P0.05),而姜黄素预先灌胃10~30天可明显减轻顺铂导致的胃残留率增高(P0.05),各组间治疗效应无明显差异。姜黄素灌胃1~5天组小鼠体重和胃残留率都无显著改善。各组胃组织中MDA和SOD含量差异无统计学意义(P0.05)。结论:姜黄素预先灌胃10天以上可改善顺铂所致的小鼠胃排空障碍,但延长天数并不会有进一步改善;其作用可能不主要通过抗氧化途径。     

高氟低碘对Wistar大鼠大脑组织病理学及DNA损伤的影响

目的研究高氟与低碘对子代大鼠大脑组织病理学及脑细胞DNA损伤的影响。方法断乳Wistar大鼠（雌∶雄=3∶1）32只,随机分为四组：对照组,饲喂大鼠标准饲料,去离子水;高氟组,饲喂大鼠标准饲料,饮100mg/L氟化钠（NaF）去离子水;低碘组,饲喂低碘饲料（定做）,去离子水;高氟低碘组,饲喂低碘饲料,饮100mg/L氟化钠（NaF）去离子水。雌雄大鼠自然交配,待子代大鼠出生后,观察其在10、20天时脑皮质、海马组织病理学变化,及30、60和90天时脑DNA的损伤。结果与对照组相比,高氟组和低碘组大鼠大脑皮质及海马内有较多神经元核固缩、核溶解及细胞轮廓不清晰。高氟低碘组,有大量神经元出现核固缩、核溶解、锥体突明显拉长等现象,并且在大鼠发育期内尤其明显。随着日龄的增加,各实验组子代大鼠脑细胞DNA损伤程度显著增加,以高氟低碘组90日龄大鼠DNA损伤最严重。结论高氟与低碘影响了大鼠大脑皮质及海马组织形态结构与引起了脑细胞的DNA损伤。     

NDV7793对肺腺癌皮下移植瘤小鼠肿瘤组织CEACAM1表达的影响

为研究NDV7793对肺腺癌皮下移植瘤小鼠肿瘤组织CEACAM1蛋白表达的影响,我们取72只健康C57BL/6小鼠,建模后随机分为3组(每组24只):PBS阴性对照组、顺铂阳性对照组和NDV7793给药组。观察各组小鼠的生存期及肿瘤生长情况;通过流式及免疫组化检测各组小鼠肿瘤组织CEACAM1表达水平。发现NDV7793给药组生存期显著长于PBS阴性对照组及顺铂阳性对照组,NDV7793给药组较顺铂阳性对照组抑瘤率更高,顺铂阳性对照组较PBS阴性对照组及NDV7793给药组小鼠肿瘤组织CEACAM1的表达水平更强,且NDV7793给药组较PBS阴性对照组小鼠肿瘤组织CEACAM1的表达水平更弱。提示NDV7793对肺腺癌皮下移植瘤小鼠的疗效比顺铂更佳,不仅延长小鼠生存期及抑制肿瘤生长,而且抑制肿瘤组织CEACAM1蛋白表达。     

姜黄素对顺铂所致胃组织ICC损伤的保护作用及机制

目的:探索顺铂对胃组织Cajal间质细胞(Cajal interstitial cells,ICCs)结构和功能的损伤以及姜黄素的保护作用。方法:选用成年雄性昆明种小鼠,随机分为对照组、顺铂组和顺铂+姜黄素组,每组各10只。姜黄素(200 mg/kg/d)混悬液连续灌胃15天。顺铂于实验结束前5天开始腹腔注射(2 mg/kg/d)共5天。计算每只小鼠最后5天的体重增减值,停药24 h后测量小鼠的胃排空率。电镜检测胃组织ICC超微结构,并测定特异性反映ICC功能变化的Ano1蛋白和m RNA的表达情况。结果:注射顺铂后各组小鼠的体重和胃排空率均显著降低(P0.01);与顺铂组相比,姜黄素预先灌胃组小鼠体重下降较少(P0.01),胃排空率有所回升(P0.05)。注射顺铂后,胃组织中ICCs受损,尤其与周围神经和肌肉间的缝隙连接增大甚至断裂,而姜黄素可以减轻这种损伤。同时,顺铂组胃组织中Ano1 m RNA和蛋白表达均下降(P0.01),加姜黄素组有所改善(P0.05)。结论:而姜黄素可通过减轻顺铂所致胃组织ICC结构损伤以及增强Ano1表达进而增强ICC慢波起博功能。     

Organelle pH in the Arabidopsis Endomembrane System

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein （GFP） （smGFP） with the pH-sensing capabil- ity of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin （plant-solubility-modified ecliptic pHluorin） gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin （plant- solubility-modified ratiomatric pHluorin）, a dual-excitation sensor, allowing for precise measurements. Compartment- specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar （pH 7.3 and 7.2）, while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum （ER） to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network （TGN） and mul- tivesicular body （MVB） is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H＋-ATPase （V-ATPase） with concanamycin A （ConcA） caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.     

Arabidopsis tic62 trol Mutant Lacking Thylakoid- Bound Ferredoxin-NADP＋ Oxidoreductase Shows Distinct Metabolic Phenotype

Ferredoxin-NADP＋ oxidoreductase （FNR）, functioning in the last step of the photosynthetic electron transfer chain, exists both as a soluble protein in the chloroplast stroma and tightly attached to chloroplast membranes. Surface plasmon resonance assays showed that the two FNR isoforms, LFNR1 and LFNR2, are bound to the thylakoid membrane via the C-terminal domains of Tic62 and TROL proteins in a pH-dependent manner. The tic62 trol double mutants contained a reduced level of FNR, exclusively found in the soluble stroma. Although the mutant plants showed no visual phenotype or defects in the function of photosystems under any conditions studied, a low ratio of NADPH/NADP~ was detected. Since the CO2 fixation capacity did not differ between the tic62 trol plants and wild-type, it seems that the plants are able to funnel reducing power to most crucial reactions to ensure survival and fitness of the plants. However, the activity of malate dehydrogenase was down-regulated in the mutant plants. Apparently, the plastid metabolism is able to cope with substantial changes in directing the electrons from the light reactions to stromal metabolism and thus only few differences are visible in steady-state metabolite pool sizes of the tic62 trol plants.     

Emerging roles of miR-210 and other non-coding RNAs in the hypoxic response

Hypoxia is a key component of the tumor microenviron- merit and represents a well-documented source of thera- peutic failure in clinical oncology. Recent work has provided support for the idea that non-coding RNAs, and in particular, microRNAs, may play important roles in the adaptive response to low oxygen in tumors. Specifically, all published studies agree that the induction of microRNA- 210 （miR-210） is a consistent feature of the hypoxic re- sponse in both normal and malignant cells, miR-210 is a robust target of hypoxia-inducible factors, and its overex- pression has been detected in a variety of diseases with a hypoxic component, including most solid tumors. High levels of miR-210 have been linked to an in vivo hypoxic sig- nature and to adverse prognosis in breast and pancreatic cancer patients. A wide variety of miR-210 targets have been identified, pointing to roles in mitochondrial metabol- ism, angiogenesis, DNA damage response, apoptosis, and cell survival. Such targets are suspected to affect the devel- opment of tumors in multiple ways; therefore, an increased knowledge about miR-210＇s functions may lead to novel diagnostic and therapeutic approaches in cancer.     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Inositol Polyphosphate Phosphatidylinositol 5-Phosphatase9 （At5PTase9） Controls Plant Salt Tolerance by Regulating Endocytosis

Phosphatidylinositol 5-phosphatases （5PTases） components of membrane trafficking system. Recently, we that hydrolyze the 5＇ position of the inositol ring are key reported that mutation in AtSPTase7 gene reduced produc- tion of reactive oxygen species （ROS） and decreased expression of stress-responsive genes, resulting in increased salt sensitivity. Here, we describe an even more salt-sensitive 5ptase mutant, At5ptase9, which also hydrolyzes the 5＇ phos- phate groups specifically from membrane-bound phosphatidylinositides. Interestingly, the mutants were more tolerant to osmotic stress. We analyzed the main cellular processes that may be affected by the mutation, such as production of ROS, influx of calcium, and induction of salt-response genes. The At5ptase9 mutants showed reduced ROS produc- tion and Ca2~ influx, as well as decreased fluid-phase endocytosis. Inhibition of endocytosis by phenylarsine oxide or Tyrphostin A23 in wild-type plants blocked these responses. Induction of salt-responsive genes in wild-type plants was also suppressed by the endocytosis inhibitors. Thus, inhibition of endocytosis in wild-type plants mimicked the salt stress responses, observed in the AtSptase9 mutants. In summary, our results show a key non-redundant role of At5PTase7 and 9 isozymes, and underscore the localization of membrane-bound Ptdlns in regulating plant salt tolerance by coordinating the endocytosis, ROS production, Ca2＋ influx, and induction of stress-responsive genes.     

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.     

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.     

Cybertaxonomy to accomplish big things in aphid systematics

Biodiversity sciences have progressed at such a pace that the taxonomic community has been unable to grow concomitantly to keep up with the influx of biologicaldata. This ＂taxonomic impediment＂ has led some to suggest that taxonomy is no longer pertinent and to the development of methodologies that circumvent the taxonomic process.This article does not seek to argue for the importance of taxonomy but rather is a call to the aphid taxonomy community to rise to the challenge by dramatically increasing the volume and comprehensiveness of its output without sacrificing quality. Recent informatics technology allows us to mobilize the 2 most important aphid taxonomy resources： expertsand specimens, both distributed globally. ＂Cyberspecimens,＂ museum specimens digitally rendered at a resolution sufficient for remote identification, and open ＂cybertaxonomic＂tools will allow the international aphid taxonomic community to carry out large, ambitious, projects. The global aphid cybertaxonomy proposed here will serve not only the ends ofresearch aphidologists, but also provide a model for other taxonomic communities to adapt and adopt as we confront both the taxonomic impediment and the taxonomic naysayers.     

Regulation of Cytokinesis by Exocyst Subunit SEC6 and KEULE in Arabidopsis thaliana

Proper vesicle tethering and membrane fusion at the cell plate are essential for cytokinesis. Both the vesicle tethering complex exocyst and membrane fusion regulator KEULE were shown to function in cell plate formation, but the exact mechanisms still remain to be explored. In this study, using yeast two-hybrid （Y-2-H） assay, we found that SEC6 interacted with KEULE, and that a small portion of C-terminal region of KEULE was required for the interaction. The direct SEC6-KEULE interaction was supported by further studies using in vitro pull-down assay, immunoprecipitation, and in vivo bimolecular florescence complementation （BIFC） microscopy, sec6 mutants were male gametophytic lethal as reported; however, pollen-rescued sec6 mutants （PRsec6） displayed cytokinesis defects in the embryonic cells and later in the leaf pavement cells and the guard cells. SEC6 and KEULE proteins were co-localized to the cell plate during cytokine- sis in transgenic Arabidopsis. Furthermore, only SEC6 but not other exocyst subunits located in the cell plate interacted with KEULE in vitro. These results demonstrated that, like KEULE, SEC6 plays a physiological role in cytokinesis, and the SEC6-KEULE interaction may serve as a novel molecular linkage between arriving vesicles and membrane fusion machin- ery or directly regulate membrane fusion during cell plate formation in plants.