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.     

EPR signals of NADH: Q oxidoreductase. Shape and intensity.

(1) The EPR spectrum of Center 1 of NADH dehydrogenase in isolated Complex I or submitochondrial particles from beef heart consists of two overlapping nearly axial signals of the same intensity. They are defined as Center 1a (gll = 0.021, gl = 1.938) and Center 1b (gll = 2.021, gl = 1.928). (2) The line shape of the EPR spectrum of the Center 3+4 can be interpreted as an overlap of two rhombic signals of the same intensity. We define Center 3 by the g-values: gz=2.103, gy = 1.93-1.94, gx=1.884, and Center 4 by the values gz=2.04, gy=1.92-1.93, gx=1.863. (3) Direct quantitation of the individuals signals as well as computer stimulation suggests that the amount of the Centers 1a and 1b is only 25% of that of the other individuals centers and FMN. As EPR spectra of beef-heart submitochondrial particles at 10-20 K are nearly identical to those of Complex I, the same relative concentrations of the Fe-S centers are also present in the particles. (4) The signals either observed by us in EPR spectra of Complex I and submitochondrial particles at 4.2 K and high microwave powers can now be explained without assuming more than 5 paramagnetic centers in NADH dehydrogenase.     

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.     

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。     

The Drum and the Mask: Time of the Tubuan.

The Drum and the Mask: Time of the Tubuan. 1999. 29 minutes. video by Caroline Yacoe. Charles Chess. and David Yacoe. For more information, contact University of California Extension Center for Media and Independent Learning, 2000 Center Street, Fourth Floor, Berkeley, CA 94704.     

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.     

Successful preservation of Daphnia for chemical and physical analysis

Daphnia may be preserved without loss of phosphorus or carbon by filtering them onto screens and quickly freezing them in the absence of excess water. Length is unaffected by the process and eggs and young may be counted accurately.Financial support provided by NSF Grant # BSR-8407431.Contribution # 362 from the Limnological Research Center.Contribution # 362 from the Limnological Research Center.     

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).     

Shifting Narratives and Mutable Meanings

In and Out of Africa . 1993. 59 minutes, color. A film by Ilisa Barbash and Lucien Taylor. For information contact University of California Extension Center for Media and Independent Learning, 2000 Center Street, Fourth Floor, Berkeley, CA 94704, 510–642–0460.     

The succinate dehydrogenase of Escherichia coli. Immunochemical resolution and biophysical characterization of a 4-subunit enzyme complex

Using EPR spectroscopy to monitor the integrity of the enzyme, conditions have been established which allow specific immunoprecipitation of the succinate dehydrogenase complex of Escherichia coli. The enzyme complex precipitated from Lubrol PX-solubilized membranes by monospecific antiserum in the presence of a cocktail of protease inhibitors contains four polypeptides of apparent MrS 71,000, 26,000, 17,000, and 15,000. The 71-kDa flavopeptide is readily susceptible to proteolysis, and the enzyme complex shows unusual facile dissociation. Spectroscopic measurements indicate the presence of a [2Fe-2S] cluster (Center 1), a [3Fe-xS] cluster (Center 3), and a b-type cytochrome. In addition, a change in relaxation of Center 1 at low potentials is indicative of Center 2. Midpoint redox potentials of Centers 1-3 for both the membrane-bound and detergent-solubilized enzyme were estimated to be +10 mV, -175 mV, and +65 mV, respectively.     

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。     

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.     

The spatial relationships and structure of the binuclear iron-sulfur clusters in succinate dehydrogenase.

Two binuclear iron-sulfur clusters (designated S-1 and S-2) are present in succinate dehydrogenase in approximately equal concentration to that of flavin. The large difference in their midpoint potentials (0 and -400 mV, respectively, in the soluble enzyme) permits the acquisition of individual electron paramagnetic resonance spectra characterized by nearly identical rhombic g tensors (gz = 2.025, gy = 1.93, gx = 1.905). Spin-coupling between the two centers is manifested by broadening and splitting of spectra of reconstitutively active and inactive succinate dehydrogenase, respectively, as the temperature is lowered; relief of power saturation of Center S-1 spectra on reduction of Center S 2; and observation of half-field ("delta ms = 2") signals in the dithionite-reduced enzyme. Saturation behavior of fully reduced dehydrogenase is consistent with the presence of S-1 and S-2 at equivalent concentrations/molecule. Simulation of the spin-coupled spectra, assuming dipolar interaction, provides information on molecular structure. Electron paramagnetic resonance spectra of the enzyme in 80% dimethylsulfoxide are nearly identical to the characteristic binuclear spectra obtained with adrenodoxin. These data provide additional evidence for binuclear structure of both Center S-1 and S-2. The extremely fast relaxation of Center S-2 at low temperatures would imply either an anomalously small value of J or an alternative relaxation mechanism, possibly due to the coupling between S-1 and S-2.     

A new EPR signal associated with photosystem 1 of algal and plant photosynthesis

A new low temperature electron paramagnetic resonance (EPR) signal with a g-value of 1.97 was found in Photosystem-1 particles from a blue-green alga, Anacystis nidulans, illuminated at room temperature. A similar signal was also found in spinach Photosystem-1 particles treated with thiophenol to decrease interference from a signal due to Center A. In the dark, the signal appeared only when the Anacystis particles were at redox potentials lower than -0.5 volts where Centers A and B were also reduced. The signal is most likely due to another iron-sulfur cluster, tentatively designated as Center C. Center C could be photoreduced at low temperatures like Center A when Centers A and B were partially reduced prior to illumination, indicating possible close association of these centers in Photosystem 1 of green plant and algal photosynthesis.     

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。     

Technology advancement for studying gene expression and gene function: a workshop report

Society News

Technology advancement for studying gene expression and gene function: a workshop reportSponsored by National Institute of Child Health and Human Development, National Institute of General Medical Sciences, National Center for Human Genome Research, National Center for Research Resources, National Institutes of Health, Bethesda, Maryland 20892, USA     

Complex biomedical systems: from basic science to translation

The Department of Biomedical Engineering (BME) of the University of Southern California (BME@USC) has a longstanding tradition of advancing biomedicine through the development and application of novel engineering ideas. More than 80 primary and affiliated faculty members conduct cutting-edge research in a wide variety of areas, such as neuroengineering, biosystems and biosignal analysis, medical devices (including biomicroelectromechanical systems (bioMEMS) and bionanotechnology), biomechanics, bioimaging, and imaging informatics. Currently, the department hosts six internationally recognized research centers: the Biomimetic MicroElectronic Systems Engineering Research Center (funded by the National Science Foundation), the Biomedical Simulations Resource [funded by the National Institutes of Health (NIH)], the Medical Ultrasonic Transducer Center (funded by NIH), the Center for Neural Engineering, the Center for Vision Science and Technology (funded by an NIH Bioengineering Research Partnership Grant), and the Center for Genomic and Phenomic Studies in Autism (funded by NIH). BME@USC ranks in the top tier of all U.S. BME departments in terms of research funding per faculty.     

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.     

Telling a Story about the Past: Fact and Fiction in Two Recent Films on the History of Anthropology

Ishi, The. Last Yahi. 1992. 57 minutes, color. Produced and directed by Jed Riffe and Pamela Roberts. Distributed by the Center for Media and Independent Learning, University of California Extension, 2000 Center Street, Fourth Floor, Berkeley, CA 94704; 510/642-0460. Fax 510/643-9271. Available in video and 16mm.
Margaret Mead: An Observer Observed. 1995.85 minutes, color. Produced by Virginia Yans-McLaughlin. Directed by Alan Berliner. Distributed by Filmakers Library, 124 East 40th Street, New York, NY l0016; 212/808-4980.     

EPR studies of higher plant mitochondria. II. Center S-3 of succinate dehydrogenase and its relation to alternative respiratory oxidations.

1. An electron paramagnetic resonance study of the high potential iron sulfur (HiPIP-type) Center S-3 of higher plant mitochondria is described. This center is the major HiPIP-type center associated with plant mitochondria and it displays physical properties which are similar to its mammalian counterpart. It has a pH-independent midpoint potential of +65 +/- 10 mV between pH 6.0 and 8.5. 2. The behavior of Center S-3 in a variety of steady-state conditions suggests that it is of physiological significance in electron transport. Furthermore, it can be shown that the alternative oxidase, which is present in many higher plant mitochondria, tends to keep this center oxidized in the presence of succinate and cyanide. This indicates that the alternative oxidation site is on the electron-donating side of the Center S-3. 3. Salicylhydroxamic acid, an inhibitor of the alternative pathway, does not affect the midpoint potential, signal size or shape, or temperature and power saturation profiles of Center S-3, suggesting that direct autoxidation of this center cannot account for alternative oxidase activity. This is further confirmed by the finding that the presence of succinate dehydrogenase is not necessary for alternative oxidase activity with NADH as respiratory substrate in submitochondrial particles.