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Electron transfer to 5-bromouracil (5-BrU) from nucleobase (N) electron adducts (and their protonated forms) has been studied by product analysis and pulse radiolysis. When an electron is transferred to 5-BrU, the ensuing 5-BrU radical anion rapidly loses a bromide ion; the uracilyl radical thus formed reacts with added t-butanol, yielding uracil. From the uracil yields measured as the function of [N]/[5-BrU] after gamma-radiolysis of Ar-saturated solutions it is concluded that thymine and adenine electron adducts and their heteroatomprotonated forms transfer electrons quantitatively to 5-BrU. Like the electron adduct of adenine, those of cytosine and guanine are rapidly protonated by water. The (protonated) electron adduct of guanine does not transfer an electron to 5-BrU, and in the case of the (protonated) cytosine electron adduct only partial electron transfer is observed. The results can be modelled if the protonated electron adduct (protonated at N(3) or at the amino group) of cytosine, CH., which can transfer its electron to 5-BrU (k approximately 2 x 10(7) dm3 mol-1 s-1) is transformed in a slow tautomerization reaction (k approximately 2.5 x +/- 10(3) s-1) into another form C'H. (possibly protonated at C(6) or C(5)) which does not transfer an electron to 5-BrU. There is also electron transfer from the electron adduct of thymine to cytosine and guanine which serve as electron sinks. The rate constant of electron transfer from the thymine electron adduct to cytosine is about 250 times greater than that of the reverse reaction. The heteroatom-protonated electron-adduct of thymidine transfers an electron to 5-BrU more slowly (k = 2.3 x 10(7) dm3 mol-1 s-1) than the electron-adduct itself (k = 7.2 x 10(8) dm3 mol-1 s-1). Phosphate buffer-induced protonation of the electron-adduct of thymine at carbon (C(6)) prevents electron transfer to 5-BrU. Such phosphate catalysis is also observed as an intramolecular process (k approximately 2 x 10(4) s-1) with thymidine-5'-phosphate but not with the 3'-phosphate. Phosphate-induced protonation at carbon also reduces transfer efficiency for the electron adducts of dinucleoside phosphates such as dTpdT and dTpdA. The data raise the question whether in DNA the guanine moiety may act as the ultimate sink of the electron in competition with other processes such as protonation at C(6) of the thymine electron adduct.
Both the United States Environmental Protection Agency (EPA) and the U.S. Food and Drug Administration (FDA) have issued regulatory documents that address the issues and requirements concerning electronic reporting to the Agencies. EPA has published two comprehensive and useful electronic data interchange (EDI) guidelines: 1) the EPA Electronic Data Interchange (EDI) Implementation Guideline, Draft of September 23, 1994 and October 18, 1994 that is available at the following EPA web site address: www.epa.gov/oppeedi1/guidelines/general.pdf and 2) the Interim Final Notice, Filing of Electronic Reports via Electronic Data Interchange, September 4, 1996, Federal Register Notice [FRL-5601-4, Volume 61, Number 172, page 46684], also available at: www.epa.gov/oppeedi1/edipoli.htm. The FDA has published a guidance document titled, "Guidance for Industry, Computerized Systems Used in Clinical Trials, April 1999" that is available at FDA's web site: www.fda.gov/ora/compliance_ref/bimo/ffinalcct.++ +htm. FDA's guidance document addresses a number of issues for electronic records that are applicable to all areas of GLP compliance. Another FDA document presently under development is titled, "Electronic Standards for the Transmission of Regulatory Information (ESTRI) Gateway." The ESTRI document defines strategic plans for electronic submissions to FDA. FDA has published a guidance document in this area titled, "Guidance for Industry: Providing Regulatory Submissions in Electronic Format--General Considerations, January 1999." This guidance document is available at: www.fda.gov/cder/guidance/index.htm. FDA has also published an important final rule applicable to all electronic records and signatures that is part of the U.S. Title 21 Code of Federal Regulations (CFR), Part 11, titled, "FDA's Final Rule, Electronic Records; Electronic Signatures, effective August 20, 1997." This FDA ruling is discussed below and is available at: www.fda.gov/cder/esig/index.htm.
The kinetics of electron transfer for rubredoxins are examined using density functional methods to determine the electronic structure characteristics that influence and allow for fast electron self-exchange in these electron-transport proteins. Potential energy surfaces for [FeX(4)](2-,1-) models confirm that the inner-sphere reorganization energy is inherently small for tetrathiolates ( approximately 0.1 eV), as evidenced by the only small changes in the equilibrium Fe-S bond distance during redox (Deltar(redox) approximately 0.05 A). It is concluded that electronic relaxation and covalency in the reduced state allow for this small in this case relative to other redox couples, such as the tetrachloride. Using a large computational model to include the protein medium surrounding the [Fe(SCys)(4)](2-,1-) active site in Desulfovibrio vulgaris Rubredoxin, the electronic coupling matrix element for electron self-exchange is defined for direct active-site contact (H0(DA)). Simple Beratan-Onuchic model is used to extend coupling over the complete surface of the protein to provide an understanding of probable electron-transfer pathways. Regions of similar coupling properties are grouped together to define a surface coupling map, which reveals that very efficient self-exchange occurs only within 4 sigma-bonds of the active site. Longer-range electron transfer cannot support the fast rates of electron self-exchange observed experimentally. Pathways directly through the two surface cysteinate ligands dominate, but surface-accessible amides hydrogen-bonded to the cysteinates also contribute significantly to the rate of electron self-exchange.
Electronic relaxation, the change in molecular electronic structure as a response to oxidation, is investigated in [FeX(4)](2)(-)(,1)(-) (X = Cl, SR) model complexes. Photoelectron spectroscopy, in conjunction with density functional methods, is used to define and evaluate the core and valence electronic relaxation upon ionization of [FeX(4)](2)(-). The presence of intense yet formally forbidden charge-transfer satellite peaks in the PES data is a direct reflection of electronic relaxation. The phenomenon is evaluated as a function of charge redistribution at the metal center (Deltaq(rlx)) resulting from changes in the electronic structure. This charge redistribution is calculated from experimental core and valence PES data using a valence bond configuration interaction (VBCI) model. It is found that electronic relaxation is very large for both core (Fe 2p) and valence (Fe 3d) ionization processes and that it is greater in [Fe(SR)(4)](2)(-) than in [FeCl(4)](2)(-). Similar results are obtained from DFT calculations. The results suggest that, although the lowest-energy valence ionization (from the redox-active molecular orbital) is metal-based, electronic relaxation causes a dramatic redistribution of electron density ( approximately 0.7ē) from the ligands to the metal center corresponding to a generalized increase in covalency over all M-L bonds. The more covalent tetrathiolate achieves a larger Deltaq(rlx) because the LMCT states responsible for relaxation are significantly lower in energy than those in the tetrachloride. The large observed electronic relaxation can make significant contributions to the thermodynamics and kinetics of electron transfer in inorganic systems.
We report a simple method to study the elemental content in cultured human adherent cells by electron probe X-ray microanalysis with scanning electron microscopy. Cells were adapted to grow on polycarbonate tissue culture cell inserts, washed with distilled water, plunge-frozen with liquid nitrogen and freeze-dried. Unstained, freeze-dried cultured cells were visualized in the secondary and backscattered electron imaging modes of scanning electron microscopy. With backscattered electron imaging it was possible to identify unequivocally major subcellular compartments, i.e. the nucleus, nucleoli and cytoplasm. X-ray microanalysis was used simultaneously to determine the elemental content in cultured cells at the cellular level. In addition, we propose some improvements to optimize backscattered electron and X-ray signal collection. Our findings demonstrate that backscattered electron imaging offers a powerful method to examine whole, freeze-dried cultured cells for scanning electron probe X-ray microanalysis.
The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.
This paper reports on the influence of the channeling effect on the energy distribution of electrons backscattered from crystals with different atomic numbers Z. These results can be used for the optimization of the contrast of electron backscattering and electron channeling patterns. Energy and angular resolved electron scattering distributions are obtained using a 4-axial experimental setup with a moveable high-resolution spherical spectrometer. Special care is taken to suppress undesired reflections of electrons inside the spectrometer. This experimental setup allows the direct observation of the excitation of different Bloch waves (anomalous absorption and transmission) within the crystal for different electron incidence angles and the observation of angular distributions of elastically scattered electrons. Results are presented for Si and Au monocrystals, showing that the influence of the channeling effect is more distinct for low atomic numbers.
An e.p.r. spectrum of the reduced form of the electron-transport component (X), thought to be the primary electron acceptor of Photosystem I, was obtained. By using line-shape simulations of this component and the free-radical e.p.r. signal I of the oxidized reaction-centre chlorophyll (P700), it was possible to determine the ratio of the number of electron spins to which these signals correspond in Photosystem-I particles under a variety of conditions. On illumination at cryogenic temperatures of Photosystem-I preparations, in which both bound iron-sulphur centres A and B were reduced, the measured ratio of free radical to component X varied between 1.04 and 2.23, with an average value of 1.54 +/- 0.18 where a Gaussian line-shape is assumed for the component-X signal in the simulation. The error in this measurement is estimated to be up to 50%. In a similar way component X and centre A of the bound iron-sulphur protein were quantified, the ratio between these two components varying between 1.26 and 0.61 with an average value of 0.75 +/- 0.06. These results indicate that the quantitative relationship, in terms of net electron spins, between centre A, component X and P700 is of the order to be expected if component X is indeed the primary electron acceptor in Photosystem I and a component of the photosynthetic electron-transport chain.
The kinetics of light-induced P700 redox transients in bean chloroplast was studied. It has been shown that the rate of electron transport decreased during few seconds of illumination of coupled chloroplasts without addition of ADP and inorganic phosphate. The evidence were obtained that there is a feedback inhibition of electron transport governed by the internal pH of thylakoid. This results in the overshoot in the kinetics of P700 redox transients induced by continuous actinic light. Under the phosphorylation condition (addition of Mg-ADP and inorganic phosphate) the effect of decreasing of the rate of electron transport between two photosystems was not observed. Addition of uncouplers (FCCP or gramicidine) also increased the steady-state rate of noncyclic electron transport. After adding only Mg-ADP (without phosphate) or Mg-ATP to coupled chloroplasts the effect of the light-driven inhibition of electron transport was observed as in the case of chloroplasts without any additions. We showed that the regulation for the electron transport rate was realized at the step of the plastoquinol oxidation by photosystem 1. Light-driven energization of the thylakoid membrane also leads to the the slowing of the reduction of spin label TEMPO. Evidences were obtained that TEMPO interacts with the semiquinone localized in the acceptor side of photosystem 2. From the comparative study of P700+ and TEMPO reduction by photosystem 2 we have concluded that there are two points of inhibitory action of DCMU localized at the acceptor and donor sides of photosystem 2. The mechanisms of photosynthetic control and the role of transmembrane proton gradient for energy transmission in chloroplasts are discussed.
A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP+). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, I1/2, is defined as the actinic light intensity that, at a given concentration of electron donro, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, I1/2 is compared with another parameter, equivalent to I1/2, that is obtained independently of the reaciton-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow. The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP+ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.
A major challenge to the development of a fundamental understanding of transport and retardation mechanisms of trace metal contaminants (<10 ppm) is their identification and characterization at the nanoscale. Atomic-scale techniques, such as conventional transmission electron microscopy, although powerful, are limited by the extremely small amounts of material that are examined. However, recent advances in electron microscopy provide a number of new analytical techniques that expand its application in environmental studies, particularly those concerning heavy metals on airborne particulates or water-borne colloids. High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM), STEM-energy-dispersive X-ray spectrometry (EDX), and energy-filtered TEM (EFTEM) can be effectively used to identify and characterize nanoparticles. The image contrast in HAADF-STEM is strongly correlated to the atomic mass: heavier elements contribute to brighter contrast. Gold nanocrystals in pyrite and uranium nanocrystals in atmospheric aerosols have been identified by HAADF-STEM and STEM-EDX mapping and subsequently characterized by high-resolution TEM (HRTEM). EFTEM was used to identify U and Fe nanocrystals embedded in an aluminosilicate. A rare, As-bearing nanophase, westerveldite (FeAs), was identified by STEM-EDX and HRTEM. The combined use of these techniques greatly expands the effective application of electron microscopy in environmental studies, especially when applied to metals of very low concentrations. This paper describes examples of how these electron microbeam techniques can be used in combination to characterize a low concentration of heavy metals (a few ppm) on nanoscale particles.
The energy component associated with the cyclotron motion of the beam electrons in the Livermore EBIT-II electron beam ion trap was inferred from measurements of the linear polarization of the K-shell x-ray lines emitted from helium-like Mg(10+) ions. The average line polarization was found to be reduced by about 20% from its nominal value. From this it was inferred that the electron cyclotron motion accounted for 190+/-30 eV of the total electron beam energy. The measured value is in good agreement with the predictions of optical electron beam propagation. It does not agree with the estimates derived from other model assumptions, such as the rigid-rotator model, for determining the size of the energy stored in the electron motion perpendicular to the beam propagation direction.
In order to describe initial events in enamel mineralization and to help characterize inorganic-organic interactions in this tissue, the earliest rod and interrod enamel in mandibular incisors from normal young adult (100 gm) rats, perfused with 100% ethylene glycol, has been studied by transmission electron microscopy, selected area electron diffraction, and high-spatial-resolution electron probe microanalysis. Diffraction and probe data were correlated precisely from the same extracellular regions of the tissue. Sites were examined progressively as a function of location a) from the most recently deposited enamel adjacent to ameloblasts toward the dentin-enamel junction and b) from the apical portion of the tooth longitudinally toward its incisal end. Electron diffraction patterns consistent with that of a poorly crystalline hydroxyapatite were generated at all locations. Diffraction characteristics changed only slightly toward that of more crystalline hydroxyapatite at different locations. Earliest apical enamel generated molar Ca/P ratios in a range of 0.99-1.46 (average 1.24 +/- 0.15). Molar Ca/P ratios of the first enamel interrod elements increased from approximately 1.24 at ameloblast-enamel boundaries to approximately 1.40 at the dentin-enamel junction, small changes corresponding to those observed in electron diffraction characteristics.
The electron transport chain of Escherichia coli grown anaerobically on glycerol with fumarate as terminal electron acceptor, has been studied using electron paramagnetic resonance (EPR) spectroscopy. The analysis did not include cytochromes, but was confined to the lower potential (dehydrogenase) section of the electron transport chain. Several ferredoxin-type centres were detected and partially characterized, and the possible presence of iron-sulphur centres paramagnetic in their oxidized form ('HiPIP-type') was also noted. An EPR-detectable signal that may have been due to the presence of molybdenum in the electron transport chain is described and assessed. Most of the centres detected were reducible by substrate in the absence of oxidant and some were found to be wholly or partly reduced during steady state oxidation of substrates in the presence of oxygen.
The effect of Mg2+-ions on the physical state of thylakoid membrane and kinetics of electron transport between two photosystems were studied. The rate of electron transport from photosystem 2 to P700+ and the activity of photosystem 2 were obtained from the kinetics of P700 redox transients induced by flashes of white light (t1/2 = 7 musec or 0.75 msec) fired simultaneously with the background continuous far-red light (707 nm). The spin-labeled stearic acids (I1.14 and I12.3) were used as indicators of Mg2+-induced structural changes. Addition of MgCl2 stimulates incorporation of spin-labels into the lipid region of the thylakoid membrane. It was found that Mg2+-ions modify the ESR spectrum of I12.3. The results evidence that the screening of charged groups on the thylakoid membrane surface induces structural changes in the lipid region of the membrane. We have concluded that these structural changes result in reorientation of lipid molecules in the thylakoid membrane. There is a correlation between Mg2+-induced structural changes and electron transport in chloroplasts. Addition of Mg2+-ions stimulates the photochemical activity of photosystem 2 by increasing the amount of active reaction centres and modifies the rate constant of electron transport from photosystem 2 to P700+. It has been demonstrated that ion regulation of electron transport in more effective in the oxidising side than in the reducing side of plastoquinone shuttle.
A measurement technique for incident electron current in secondary electron (SE) detectors, especially the Everhart-Thornley (ET) detector, based on signal-to-noise ratio (SNR), which uses the histogram of a digital scanning electron microscope (SEM) image, is described. In this technique, primary electrons are directly incident on the ET detector. This technique for measuring the correlation between incident electron current and SNR is applicable to the other SE detectors. This correlation was applied to estimate the efficiency of the ET detector itself, to evaluate SEM image quality, and to measure the geometric SE collection efficiency and the SE yield. It was found that the geometric SE collection efficiency at each of the upper and lower detectors of a Hitachi S-4500 SEM was greater than 0.78 at all working distances.