Thermodynamic and EPR characteristics of two ferredoxin-type iron-sulfur centers in the succinate-ubiquinone reductase segment of the respiratory chain.

Two distinct ferredosin-type iron-sulfur centers (designated as Centers S-1 and S-2) are present in the soulble succinate dehydrogenase in approximately equivalent concentrations to that of bound flavin. Both Centers S-1 and S-2 exhibit electron paramagnetic resonance absorbance in the reduced state at the same magnetic field (gz = 2.03, gy = 1.93, and gx = 1.91) with similar line shape. Center S-2 is reducible only chemically with dithionite and remains oxidized under physiological conditions. Thus, its functional role is unknown; however, thermodynamic and EPR characterization of this iron-sulfur center has revealed important molecular events related to this dehydrogenase. The midpoint potentials of Centers S-1 and S-2 determined in the soluble succinate dehydrogenase preparations are -5 +/- 15 mV and -400 +/- 15 mV, respectively, while corresponding midpoint potentials determined in particulate preparations, such as succinate-cytochrome c reductase or succinate-ubiquinone reductase, are 0 +/- 15 mV and -260 +/- 15 mV. Reconstitution of soluble succinate dehydrogenase with the cytochrome b-c1 complex is accompanied by a reversion of the Center S-I midpoint from -400 +/- 15 mV to -250 +/- 15 mV with a concomitant restoration of antimycin A-sensitive succinate-cytochrome c reductase activity. There observations indicate that, during the reconstitution process, Center S-I is restored to its original molecular environment. In the reconstitutively active succinate dehydrogenase, the relaxation time of Center S-2 is much shorter than that of S-1, thus Center S-2 spectra are well discernible only below 20 K (at 1 milliwatt of power), while the resonance absorbance of Center S-1 is detectable at higher temperatures and readily saturates below 15 K. Over a wide temperature range the power saturation of Center S-1 resonance absorbance is relieved by Center S-2 in the paramagnetic state, and the Center S-2 central resonance absorbance is broadened by Center S-1 spins, due to a spin-spin interaction between these centers. These observations indicate an adjacent location of these centers in the enzyme molecule. In reconstitutively inactive enzymes, subtle modification of the enzyme structure appears to shift the temperature dependence of Center S-2 relaxation to the higher temperature. Thus the EPR signals of Center S-2 are also detectable at higher temperature. In this system a splitting of the central peak of the Center S-2 spectrum due to spin-spin interaction was observed at extremely low temperatures, while this was not observed in reconstitutively active enzymes or in paritculate preparations. This spin-spin interaction phenomena of inactive enzymes disappeared upon chemical reactivation with concomitant appearance of the reconstitutive activity. These observations provide a close correlation between the molecular integrity of the enzyme and its physiological function.     

利用长距离PCR扩增国内肠道病毒流行株基因组方法的建立和评价

目的:通过调查近年来我国肠道病毒EV-71型和柯萨奇病毒A16型流行株的全基因组序列,建立一种能够获得我国肠道病毒序列的通用扩增方法,为今后的手足口病流行病学分析、致病机理研究等打下基础。方法:收集我国近5年各地报道的肠道病毒流行株全基因组序列作为参考序列进行比对分析,在保守区设计通用引物,利用3'RACE、长距离PCR扩增及简并引物扩增肠道病毒全基因组序列,采用IonTorrentPGM二代测序仪对扩增产物进行深度测序,以对扩增方法进行验证和评价。结果:通过比对肠道病毒流行株序列设计了通用扩增引物,经二代测序实验获得了肠道病毒全基因组序列;以系列比例模拟混合病毒感染,该扩增方法能够同时获得2株肠道病毒的全基因组序列;能够完整地揭示肠道病毒重组情况。结论:建立了针对我国近年来肠道病毒流行株的通用全基因组扩增方法,在病毒培养液中肠道病毒的提取与扩增中显示了较高的灵敏度,能够反映混合病毒感染、重组病毒的情况。     

Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart.

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 degrees K) and potentiometric titration. Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix "N") resolves into two single electron titratins with EM7.2 values of minus 380 plus or minus 20 mV and minus 240 plus or minus 20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g parellel = 2.03, g = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with gz = 2.03, gy = 1.94 and gx = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g parallel = 2.05 and g = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high EM7.2 value (minus 20 plus or minus 20 mV). Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with EM7.2 = minus 240 plus or minus 20 mV is defined as Center N-3 (gz = 2.10, (gz = 2.10, (gy = 1.93?), GX = 1.87); the minus 405 plus or minus 20 mV component as Center N-4 (gz = 2.11, (gy = 1.93?), gx = 1.88). At temperatures close to 4.2 degrees K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with EM7.2 = minus 260 plus or minus 20 mV. The multiplicity of peaks suggests the presence of at least two different iron-sulfur centers having similar EM7.2 values (minus 260 plus or minus 20 mV); HENCE, tentatively assigned as N-5 and N-6. Consistent with the individual EM7.2 values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the EM7.2 of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. EM7.2 values and EPR lineshapes for the other iron-sulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show EM7.2 values around minus 250 mV; Center N-2 exhibits a high EM7.2 (minus 20 plus or minus 20 mV), while Center N-1a shows a very low EM7.2 (minus 380 plus or minus 20 mV).     

Thermodynamic and EPR characteristics of a HiPIP-type iron-sulfur center in the succinate dehydrogenase of the respiratory chain.

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a third iron-sulfur center (Center S-3), which is paramagnetic in the oxidezed state analogous to the bacterial high potential iron-sulfur protein, has bwen detected in the reconstitutively active soluble succinate dehydrogenase preparation. Midpoint potential (at pH 7.4) of Center S-3 determined in a particulate succinate-cytochrome c reductase is +60 +/- 15 mV. In soluble form, Center S-3 becomes extremely labile towards oxygen or ferricyanide plus phenazine methosulfate similar to reconstitutive activity of the dehydrogenase. Thus, even freshly prepared reconstitutively active enzyme preparations show EPR spectra of Center S-3 which correspond approximately to 0.5 eq per flavin; in particulate preparations this component was found in a 1:1 ratio to flavin. All reconstitutively inactive dehydrogenase preparations that Center S-3 is an innate constituent of succinate dehydrogenase and plays an important role in mediating electrons from the flavoprotein subunit to most probably ubiquinone and then to the cytochrome chain.     

Who Owns the Past?

Who Owns the Past? 2000. 56 minutes, color (and black and white). video by N. Jed Riffe. For more information, contact University of California Extension, Center for Media and Independent Learning, 2000 Center Street, Fourth Floor, Berkeley, CA 94704; cmil@uclink.berkeley.edu .     

EPR studies on a Hipip type iron-sulfur center in the succinate dehydrogenase segment of the respiratory chain

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a Hipip-type iron-sulfur center (Center S-3) has been detected in the reconstitutively active soluble succinate dehydrogenases. E_m7,4 determined in a particulate, antimycin A sensitive succinate-cytochrome $\text{c}$ reductase is +60 ± 15 mV. This center is extremely labile towards oxygen in a manner similar to the reconstitutive activity of the dehydrogenase. Even freshly prepared reconstitutively active enzyme shows a considerably diminished content of Center S-3 relative to flavin and displays a partly modified spectra. All reconstitutively inactive dehydrogenases give rise to a highly modified or no Center S-3 spectra at all. These observations indicate that Center S-3 is a constituent of succinate dehydrogenase and plays a role in the physiological function of the enzyme, $\text{i.e.}$ transferring electrons most probably to ubiquinone.     

5-HT和DA对虾蟹打斗行为影响的研究进展

5-羟色胺(5-HT)和多巴胺(DA)是两种神经递质,可与众多不同类型的受体结合发挥多种重要的生理功能.现已证明其广泛分布于多种动物的不同组织中,在动物的打斗行为活动中起着重要的调节作用.目前,在虾蟹中已被报道的5-HT受体主要有5种,分别是5-HT1A、5-HT1B、5HT2A、5HT2B和5-HT7;DA受体主要为DA1A、DA1B、DA2和DA4.5-HT和DA及其受体分布存在明显的种属和组织特异性.5-HT和DA参于了虾蟹打斗行为的调节过程并有不同的调节机理.5-HT可以调节环磷酸腺苷(cAMP)或高血糖激素(CHH)的释放,促进或抑制虾蟹打斗行为;而DA同样能够通过调节cAMP及COMT等物质的释放来调节虾蟹打斗行为.     

孕烷X受体配体结合结构域蛋白晶体的X线衍射结构解析

目的:利用X线衍射技术解析孕烷X受体(PXR)配体结合结构域(LBD)蛋白晶体的3维结构。方法:对PXR蛋白LBD(130~434氨基酸残基)序列进行密码子优化并化学合成后克隆至pRSFDuet-1表达载体,再将载体导入大肠杆菌BL21(DE3),对PXR-LBD蛋白进行原核表达与分离纯化;采用晶体筛选试剂盒筛选蛋白结晶条件,采用悬滴法获得目标蛋白的晶体;对获得的蛋白晶体进行X线晶体衍射检测,并收集相关数据建立PXR-LBD的三维结构。结果:获得了PXR-LBD的高质量晶体并利用X线衍射解析了该蛋白质晶体的结构数据,使用Phenix.refine软件和COOT软件等对结构进行修正,最终获得了高分辨率的3维结构数据。结论:完成了孕烷X受体配体结合结构域蛋白晶体的X线衍射结构解析,为研究和开发PXR相关药物奠定了基础。     

Diazonium modification of Photosystem I. A specific effect on iron-sulfur Center B

Modification of chloroplast membranes with diazonium benzene sulfonate (DABS) leads to a loss of Photosystem I-dependent ferredoxin reduction but not methyl viologen reduction. EPR studies of DABS-modified membranes show no inhibition of P-700⁺ formation at cryogenic temperatures, but iron-sulfur Center A photoreduction is markedly inhibited. Iron-sulfur Center B photoreduction at physiological temperatures in DABS-modified membranes is also markedly inhibited and little Center B can be detected after dark chemical reduction. These results indicate DABS specifically modifies iron-sulfur Center B of the spinach chloroplast Photosystem I electron acceptor complex and that Center B is obligately required for the reduction of Center A at cryogenic temperatures. Possible electron transport pathways at physiological temperatures are also considered.     

瑞戈非尼抑制三阴性乳腺癌MDA-MB-231细胞增殖的研究

目的:利用裸鼠皮下成瘤动物模型及裸鼠肝脏原位多发与弥散模型,探索新型分子靶向药物瑞戈非尼对三阴性乳腺癌MDA-MB-231增殖的抑制作用,确定其分子机制。方法:培养获得人高侵袭性三阴性乳腺癌细胞MDA-MB-231,经BALB/c裸鼠皮下注射形成皮下肿瘤,或经肝门静脉注射形成肝脏多发、弥散的肿瘤模型。给予瑞戈非尼灌胃给药治疗,3周后解剖,测量肿瘤体积并称重;或进行PET/CT检测,对核素强度、散发肿瘤占肝脏面积进行定量。通过定量PCR(q PCR)实验验证上皮-间质转化标志物与转移、侵袭相关标志物的表达水平。结果:在裸鼠皮下成瘤模型以及裸鼠肝脏原位多发与弥散肿瘤模型中,瑞戈非尼隔日灌胃给药持续3周能够显著抑制MDA-MB-231的增殖;皮下肿瘤体积、重量,以及裸鼠肝脏PET/CT检测显示瑞戈非尼持续给药组的相对核素强度均明显低于对照组;q PCR实验反映出瑞戈非尼能够抑制MDA-MB-231的上皮-间质转化与转移、侵袭作用。结论:揭示了瑞戈非尼对三阴性乳腺癌细胞系MDA-MB-231的增殖抑制作用,初步探索了其分子机制,为瑞戈非尼应用于三阴性乳腺癌的治疗提供参考。     

Three-iron-sulfur centers of pea mitochondria

EPR signals of three distinct types of three-iron-sulfur center were observed in pea mitochondria: the signal of Center S-3 (low-field peak at g = 2.016), the signal of Center ISP-1 (low-field peak at g = 2.024) and the signal of the axial Center ISP-2 with two maxima, at g = 2.027 and 2.016. Succinate increases the signal amplitude of Center ISP-1 and diminishes that of Center ISP-2; malate has an opposite effect. Membrane damage enhances the effect of malate and decreases that of succinate.     

芍药苷对结肠癌SW480细胞增殖、侵袭、迁移的影响

目的观察芍药苷对人结肠癌SW480细胞株增殖、侵袭、迁移的影响,探究其干预机制。方法含10%胎牛血清的DMEM/F12培养基常规培养人结肠癌SW480细胞株,CCK-8以及EdU-488法检测芍药苷对SW480细胞增殖的影响,Transwell小室检测芍药苷对SW480细胞侵袭、迁移的影响,Westernblot法检测beclin1、Bcl-2蛋白的表达。结果不同浓度芍药苷分别处理24h、48h、72h的结肠癌SW480细胞增殖活性受到显著抑制:相比较对照组,160μg/ml芍药苷处理48h后,SW480细胞内黄绿色荧光减弱,细胞增殖率显著下降,为(58.91±4.99)%;SW480细胞的侵袭细胞数、迁移细胞数显著下降:侵袭抑制率为26.50%,迁移抑制率为24.67%;beclin1蛋白表达高于对照组,Bcl-2蛋白表达低于对照组,beclin1与Bcl-2蛋白表达呈负相关。结论芍药苷能够抑制结肠癌SW480细胞增殖、侵袭和迁移,其机制可能通过抑制Bcl-2蛋白表达,上调beclin1蛋白的表达。     

The Reincarnation of Khensur Rinpoche: The Trials of Telo Rinpoche: A Stranger in My Native Land

The Reincarnation of Khensur Rinpoche. 1991. 55 minutes. color. video by Tenzing Sonam and Ritu Sarin. For more information, contact University of California Extension. Center Media and Independent Learning, 2000 Center Street. Suite 400. Berkeley, CA 94704.
The Trials of Telo Rinpoche. 1994. 50 minutes, color. video by Tenzing Sonam and Ritu Sarin. For more information, contact University of California Extension. Center Media and Independent Learning, 2000 Center Street. Suite 400. Berkeley, CA 94704.
Stranger in My Native Land. 1998. 30 minutes, color. video by Tenzing Sonam and Ritu Sarin. For more information, contact University of California Extension. Center Media and Independent Learning, 2000 Center Street. Suite 400. Berkeley, CA 94704.     

Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 °K) and potentiometric titration.Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix “N”) resolves into two single electron titrations with E_{m 7.2} values of ?380±20 mV and ?240±20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g_// = 2.03, g_⊥ = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with g_z = 2.03, g_y = 1.94 and g_x = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g_// = 2.05 and g_⊥ = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high E_{m 7.2} value (?20±20 mV).Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with E_{m 7.2} = ? 240±20 mV is defined as Center N-3 (g_z = 2.10, (g_y = 1.93?), g_x = 1.87); the ?405±20 mV component as Center N-4 (g_z = 2.11, (g_y = 1.93?), g_x = 1.88). At temperatures close to 4.2 °K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with E_{m 7.2} = ?260±20 mV. The multiplicity of peaks suggests the presence of at least two different ironsulfur centers having similar E_{m 7.2} values (?260±20 mV); hence, tentatively assigned as N-5 and N-6.Consistent with the individual E_{m 7.2} values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the E_{m 7.2} of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. E_{m 7.2} values and EPR lineshapes for the other ironsulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show E_{m 7.2} values around -250 mV; Center N-2 exhibits a high E_{m 7.2} (?20±20 mV), while Center N-1a shows a very low E_{m 7.2} (?380±20 mV).     

Ktaadn: Thoreau the anthropologist

Timothy Troy is a Librarian at the Center for Creative Photography, University of Arizona.     

The Japanese Tsukuba Primate Center for Medical Science (TPC): an outline

The facilities and activities of the Japanese Primate Center at Tsukuba, Japan are described. The Center became partially functional in 1978 and was completed in 1979. The three main aims of the Primate Center are: to quarantine newly imported primate animals, to breed, and to study them.     

广州首起输入性基孔肯雅热的调查分析

广州网络报告的疑似"登革热"患者经广州市疾病预防控制中心流行病学调查、血清学检测、病原学分离培养等做出的诊断为首例输入性基孔肯雅热。又经中国疾病预防控制中心和广东省疾病预防控制中心对疑似基孔肯雅热患者作进一步基孔肯雅病毒抗体和核酸等检测,并对扩增出的基孔肯雅病毒特异性片段进行了序列测定和分析。核苷酸同源性比较显示,E2区(336nt)和NS1区(314nt)的核苷酸序列与意大利基孔肯雅病毒分离株ITA07-RA1及多株印度的基孔肯雅病毒分离株的序列高度同源(98%以上)。根据现场流行病学和实验流行病学的调查结果,可以确认这例疑似境外感染的输入性登革热实为基孔肯雅热病例,也是中国首例输入性基孔肯雅热病例。     

Kinetic identification of component X as P430: a primary electron acceptor of Photosystem I

Kinetics of dark recoveries of Component X, Center A, and Center B at 20 and 0 °C after a 30-s illumination were studied in membrane fragments from a blue-green alga by using low temperature electron paramagnetic resonance spectroscopy in combination with a quick-freeze method. These kinetics were compared with those obtained by spectrophotometry under the same conditions. Contrary to the currently popular view, the result strongly suggests that Component X, rather than Center A or Center B, is P430.     

Issues on creating an autonomous region for the Cordillera,Northern Philippines

One of the most novel aspects of the 1987 Philippine Constitution is the provision of autonomous regions in the Cordillera and in Muslim Mindanao. Since late 1986, the Cordillera Studies Center has been interested in studying the institution of Cordillera autonomy. The Center was desirous of starting serious work on conceptualizing the research on Cordillera autonomy, in view of the constitutional timetable for implementing autonomy in the Cordillera (as well as in Muslim Mindanao). Thus, even before the research began in July 1987, the Center received a small grant to hold a conference, ‘Issues on Cordillera Autonomy’, in May 1987 (Rood 1987). In the first three sections of this report, I provide a background to autonomy, up to the time of consideration by Congress of the Draft Organic Act (January 1989). I then discuss some of the Center's early research results, by way of explicating some of the issues involved in setting up an autonomous region in the Cordillera.