The membrane-induced proton motive force influences the metal binding ability of Bacillus subtilis cell walls.

Bacillus subtilis 168 is a gram-positive bacterium whose cell wall contains the highly electronegative polymers peptidoglycan (chemotype A1 gamma) and glycerol-based teichoic acid to produce a surface with a net negative charge with high metal binding capacity. During metabolism, a membrane-induced proton motive force continuously pumps protons into the wall fabric. As a result, a competition between protons and metal ions for anionic wall sites occurs, and less metal is bound in living cells than in nonliving cells or those in which the plasma membrane has been uncoupled. This was shown by using two metallic ions, UO2(2+) and Sc3+, on control cells, cells uncoupled with either carbonyl cyanide m-chlorophenylhydrazone or NaN3, or cells killed by gamma radiation. Transmission electron microscopy, energy-dispersive X-ray spectroscopy, and inductively coupled plasma atomic-emission spectroscopy showed that more metal was retained in the walls of nonliving cells and those with deenergized membranes than in their living counterparts.     

Selective Electrodiffusion of Zinc Ions in a Zrt-, Irt-like Protein,ZIPB

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn²⁺ and Cd²⁺, whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.     

Response of the carotenoidless mutant Rhodobacter sphaeroides growing cells to cobalt and nickel exposure

The interaction of cobalt (Co²⁺) and nickel (Ni²⁺) ions with whole cells of the photosynthetic purple bacterium Rhodobacter sphaeroides strain R26 was investigated. Active and passive uptakes were examined in cells grown in the presence of increasing amounts of Co²⁺ and Ni²⁺. Inductively coupled plasma atomic emission spectroscopy (ICP-AES), pH titration, and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to assess the role of cell envelope and metabolism in accumulating the two heavy metals. The chosen microorganism was able to uptake cobalt and nickel up to 2.2 and 0.25 mg per gram of dried cells respectively, with the largest part found bound to the cell surface. Carboxylate groups lying on the cell wall of this Gram-negative bacterium proved to be the major candidates for binding protons and metal cations. Co²⁺ was found to interfere with Mg²⁺ extracellular immobilization and transport across the membrane, indicating that these ions share binding sites on the cell envelope and ion transport systems. According to the presence of a competition mechanism, bacterial growth experiments showed that high Mg²⁺ concentrations are able to rescue R. sphaeroides from Co²⁺ toxicity.     

Effects of 28Si Ions, 56Fe Ions,and Protons on the Induction of Murine Acute Myeloid Leukemia and Hepatocellular Carcinoma

Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the carcinogenic effects of 300 MeV/n ²⁸Si or 600 MeV/n ⁵⁶Fe ions in a mouse model for radiation-induced acute myeloid leukemia and hepatocellular carcinoma. C3H/HeNCrl mice were irradiated with 0.1, 0.2, 0.4, or 1 Gy of 300 MeV/n ²⁸Si ions, 600 MeV/n ⁵⁶Fe ions or 1 or 2 Gy of protons simulating the 1972 solar particle event (1972SPE) at the NASA Space Radiation Laboratory. Additional mice were irradiated with ¹³⁷Cs gamma rays at doses of 1, 2, or 3 Gy. All groups were followed until they were moribund or reached 800 days of age. We found that ²⁸Si or ⁵⁶Fe ions do not appear to be substantially more effective than gamma rays for the induction of acute myeloid leukemia. However, ²⁸Si or ⁵⁶Fe ion irradiated mice had a much higher incidence of hepatocellular carcinoma than gamma ray irradiated or proton irradiated mice. These data demonstrate a clear difference in the effects of these HZE ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis. Also seen in this study was an increase in metastatic hepatocellular carcinoma in the ²⁸Si and ⁵⁶Fe ion irradiated mice compared with those exposed to gamma rays or 1972SPE protons, a finding with important implications for setting radiation exposure limits for space-flight crew members.     

Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent.

The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.     

Yeast SMF1 mediates H(+)-coupled iron uptake with concomitant uncoupled cation currents

Yeast membrane proteins SMF1, SMF2, and SMF3 are homologues of the DCT1 metal ion transporter family. Their functional characteristics and the implications of these characteristics in vivo have not yet been reported. Here we show that SMF1 expressed in Xenopus oocytes mediates H(+)-dependent Fe(2+) transport and uncoupled Na(+) flux. SMF1-mediated Fe(2+) transport exhibited saturation kinetics (K(m) = 2.2 microM), whereas the Na(+) flux did not, although both processes were electrogenic. SMF1 is also permeable to Li(+), Rb(+), K(+), and Ca(2+), which likely share the same uncoupled pathway. SMF2 (but not SMF3) mediated significant increases in both Fe(2+) and Na(+) transport compared with control oocytes. These data are consistent with the concept that uptake of divalent metal ions by SMF1 and SMF2 is essential to yeast cell growth. Na(+) inhibited metal ion uptake mediated by SMF1 and SMF2 expressed in oocytes. Consistent with this, we found that increased sensitivity of yeast to EGTA in the high Na(+) medium is due to inhibition of SMF1- and SMF2-mediated metal ion transport by uncoupled Na(+) pathway. Interestingly, DCT1 also mediates Fe(2+)-activated uncoupled currents. We propose that uncoupled ion permeabilities in metal ion transporters protect cells from metal ion overload.     

Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis.

Bacterial metabolism excretes protons during normal metabolic processes. The protons may be recycled by chemiosmosis, diffuse through the wall into the medium, or bind to cell surface constituents. Calculations by Koch (J. Theor. Biol. 120:73-84, 1986) have suggested that the cell wall of gram-positive bacteria may serve as a reservoir of protons during growth and metabolism, causing the wall to have a relatively low pH. That the cell wall may possess a pH lower than the surrounding medium has now been tested in Bacillus subtilis by several independent experiments. When cultures of B. subtilis were treated with the proton conductors azide and carbonylcyanide m-chlorophenylhydrazone, the cells bound larger amounts of positively charged probes, including the chromium (Cr3+) and uranyl (UO2(2+) ions and were readily agglutinated by cationized ferritin. In contrast, the same proton conductors caused a decrease in the binding of the negatively charged probe chromate (CrO4(2-)). Finally, when levansucrase was induced in cultures by the addition of sucrose, the enzyme was inactive as it traversed the wall during the first 0.7 to 1.0 generation of growth. The composite interpretation of the foregoing observations suggests that the wall is positively charged during metabolism, thereby decreasing its ability to complex with cations while increasing its ability to bind with anions. This may be one reason why some enzymes, such as autolysins, are unable to hydrolyze their substrata until they reach the wall periphery or are in the medium.     

31P and 23Na nuclear magnetic resonance studies of resting and stimulated mast cells

Exocytosis induced by crosslinking the type I receptor for Fc epsilon domains present on rat mucosal mast cells (RBL-2H3-line) requires the influx of Ca2+ ions and is markedly influenced by the concentration of monovalent cations (K+, Na+ and protons) in their medium. We investigated the role of these ions in coupling the immunological stimulus to secretion using NMR spectroscopy to monitor simultaneously intracellular pH, ATP and Na+ concentrations and the secretory response of living adherent mast cells. Using this methodology we observed that: (i) ATP concentration and intracellular pH are highly regulated and no changes could be resolved in them upon stimulation and during exocytosis. (ii) In the absence of potassium ions in the cells' medium, a decrease is observed in the intracellular pH and ATP concentration and an increase in the Na+ concentration. (iii) From the influx of extracellular Na+ following inhibition of the Na+, K(+)-ATPase by ouabain, we estimated the inward Na+ current of resting cells to 5 x 10(7) ions/(cell.s). This value does not vary by more than 10% during exocytosis.     

Energy coupling to nitrite respiration in the sulfate-reducing bacterium Desulfovibrio gigas.

By use of a membrane fraction prepared from Desulfovibrio gigas grown in a lactate-sulfate medium, synthesis of ATP was demonstrated to be coupled to the oxidation of molecular hydrogen and reduction of either nitrite or hydroxylamine. This phosphorylation was uncoupled from electron transport by pentachlorophenol, methyl viologen, and gramicidin, but not by oligomycin. The extrusion of protons from the cells was shown to be coupled to the hydrogen-nitrite respiratory system, and, assuming the localization of nitrite reductase on the outer side of the plasma membrane, H+/2e- values of 2.0 +/- 0.3 were obtained. Energy coupling observed with this system appears to be due to electron transfer-coupled proton translocation rather than vectorial electron transfer associated with hydrogen oxidation.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na+/H+ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

Effects of dietary supplements on space radiation-induced oxidative stress in Sprague-Dawley rats

Of particular concern for the health of astronauts during space travel is radiation from protons and high-mass, high-atomic-number (Z), and high-energy particles (HZE particles). Space radiation is known to induce oxidative stress in astronauts after extended space flight. In the present study, the total antioxidant status was used as a biomarker to evaluate oxidative stress induced by gamma rays, protons and HZE-particle radiation. The results demonstrate that the plasma level of total antioxidants in Sprague-Dawley rats was significantly decreased (P < 0.01) in a dose-dependent manner within 4 h after exposure to gamma rays. Exposure to protons and HZE-particle radiation also significantly decreased the serum or plasma level of total antioxidants in the irradiated animals. Diet supplementation with L-selenomethionine alone or a combination of selected antioxidant agents was shown to partially or completely prevent the decrease in the serum or plasma levels of total antioxidants in animals exposed to gamma rays, protons or HZE particles. These findings suggest that exposure to space radiation may compromise the capacity of the host antioxidant defense and that this adverse biological effect can be prevented at least partially by dietary supplementation with L-selenomethionine and antioxidants.     

NMR study of a lymphocyte differentiating thymic factor. An investigation of the Zn(II)-nonapeptide complexes (thymulin)

Detailed investigations of a serum peptide (less than Glu1-Ala2-Lys3-Ser4-Gln5-Gly6-Gly7-Ser8-++ +Asn9) were carried out by 1H and 13C NMR spectroscopy to elucidate the structure of the complex formed with Zn(II), thymulin, which has been found to be active in vivo. These experiments were performed in dimethyl sulfoxide-d6 solution at different metal:peptide ratios. The results suggest the following conclusions. (i) The Zn(II) complexation corresponds to a fast exchange on the NMR time scale. (ii) The evolution of 1H and 13C NMR chemical shifts indicates the existence of two types of complexes: a 1:2 species associating two peptide molecules and one Zn(II) ion and a complex with 1:1 stoichiometry. The former is predominant for metal:peptide ratios below unity. (iii) In the 1:2 complex, Zn(II) is coordinated by the Ser4-O gamma H and Asn9-CO2- sites, while in the 1:1 complex, Ser8-O gamma H is the third ligand to the Zn(II) ion. The results are compared with those for the [Ala4] and [Ala8] analogues, and those for the complexes of thymulin with other metal ions (Cu2+ and Al3+) in terms of its biological activity. These comparative studies suggested that the 1:1 complex is the only conformation recognized by the antibodies.     

Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme

A modified hammerhead ribozyme (R32S) with a phosphorothioate linkage between G(8) and A(9), a site that is considered to play a crucial role in catalysis, was examined by high-resolution 1H and (31)P nuclear magnetic resonance (NMR) spectroscopy. Signals due to imino protons that corresponded to stems were observed, but the anticipated signals due to imino protons adjacent to the phosphorothioate linkage were not detected and the (31)P signal due to the phosphorothioate linkage was also absent irrespective of the presence or absence of the substrate. (31)P NMR is known to reflect backbone mobility, and thus the absence of signals indicated that the introduction of sulfur at P9 had increased the mobility of the backbone near the phosphorothioate linkage. The addition of metal ions did not regenerate the signals that had disappeared, a result that implied that the structure of the core region of the hammerhead ribozyme had fluctuated even in the presence of metal ions. Furthermore, kinetic analysis suggested that most of the R32S-substrate complexes generated in the absence of Mg(2+) ions were still in an inactive form and that Mg(2+) ions induced a further conformational change that converted such complexes to an activated state. Finally, according to available NMR studies, signals due to the imino protons of the central core region that includes the P9 metal binding site were broadened or not observed, suggesting that this catalytically important region might be intrinsically flexible. Our present analysis revealed a significant change in the structure of the ribozyme upon the introduction of the single phosphorothioate linkage at P9 that is in general considered to be a conservative modification.     

Ion-exchange aspects of toxic metal uptake by Indian mustard

Uptake of lead (Pb), copper (Cu), zinc (Zn), and cadmium (Cd) as +2 ions by excised roots of Indian mustard was demonstrated to be an ion-exchange process with existing Ca or protons released to the solution. This initial reaction at the root-aqueous interface is important in the uptake of these toxic metals from contaminated soil. Ethylene diamine tetraacetic acid (EDTA)-amended soil for phytoremediation has Pb in anionic form as [Pb-EDTA]2-, which was not taken up by excised roots. In nonliving B. juncea, Pb2+ was translocated from a solution through a cut stem to petiole and leaves much more quickly than anionic [Pb-EDTA]2-. However, in living plants [Pb-EDTA]2- was more quickly translocated from a solution through roots and petiole to leaves than Pb2+. The final amount of uptake on roots of the living plants was the same for both forms of Pb. The present results are important toward understanding the mechanism of phytoremediation of toxic metal-contaminated soil for two reasons: 1) the initial process, uptake of metal ions by roots, was shown to occur by cation exchange and 2) since [Pb-EDTA]2- was not sorbed by excised roots, other factors such as transpiration and active transport are important in applications using EDTA-amended soils contaminated by Pb.     

Proton-HZE-particle sequential dual-beam exposures increase anchorage-independent growth frequencies in primary human fibroblasts

The radiation field in deep space contains high levels of high-energy protons and substantially lower levels of high-atomic-number, high-energy (HZE) particles. Calculations indicate that cellular nuclei of human space travelers will be hit during a 3-year Mars mission by approximately 400 protons and approximately 0.4 HZE particles. Thus most cells in astronauts will be hit by a proton(s) before being hit by an HZE particle. To investigate effects of dual ion irradiations on human cells, we irradiated primary human neonatal fibroblasts with protons (1 GeV/nucleon, 20 cGy) followed from 2.5 min to 48 h later by iron or titanium ions (1 GeV/nucleon, 20 cGy) and then measured clonogenic survival and frequency of anchorage-independent growth. This frequency depends on the interval between hydrogen- and iron-ion irradiation, with a critical window between 2.5 min and 1 h producing about three times more anchorage-independent colonies per survivor than expected from simple addition of the two ions separately. The hydrogen-titanium-ion dual-beam irradiation produced similar increases that persisted to approximately 6 h. At longer intervals, anchorage-independent growth frequencies were similar to those expected for additivity. However, irradiation of cells with either an iron or a titanium particle first followed by protons produced only additive levels.     

Determination of copper binding in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CZ1 by chemical modifications and X-ray absorption spectroscopy

Previously performed studies have shown that Pseudomonas putida CZ1 biomass can bind an appreciable amount of Cu(II) and Zn(II) ions from aqueous solutions. The mechanisms of Cu- and Zn-binding by P. putida CZ1 were ascertained by chemical modifications of the biomass followed by Fourier transform infrared and X-ray absorption spectroscopic analyses of the living or nonliving cells. A dramatic decrease in Cu(II)- and Zn(II)-binding resulted after acidic methanol esterification of the nonliving cells, indicating that carboxyl functional groups play an important role in the binding of metal to the biomaterial. X-ray absorption spectroscopy was used to determine the speciation of Cu ions bound by living and nonliving cells, as well as to elucidate which functional groups were involved in binding of the Cu ions. The X-ray absorption near-edge structure spectra analysis showed that the majority of the Cu was bound in both samples as Cu(II). The fitting results of Cu K-edge extended X-ray absorption fine structure spectra showed that N/O ligands dominated in living and nonliving cells. Therefore, by combining different techniques, our results indicate that carboxyl functional groups are the major ligands responsible for the metal binding in P. putida CZ1.     

Covalent incorporation of 3'-O-(4-benzoyl)benzoyl-ATP into a P2 purinoceptor in transformed mouse fibroblasts.

ATP, 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, and several other ATP analogs induced an increase in plasma membrane permeability to monovalent ions and normally impermeant metabolites, including nucleotides, in transformed 3T6 mouse fibroblasts. The rank order of agonist potency for induction of nucleotide channels was BzATP (EC50 = 15 microM) greater than ATP (EC50 = 50 microM) approximately adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) greater than 2-methylthio-ATP (EC50 = 75 microM) approximately 3'-amino-3'-deoxy-ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) (EC50 = 175 microM). Long wavelength UV illumination of 3T6 cells in the presence of greater than or equal to 20 microM BzATP at 4 degrees C, a nonpermeabilizing temperature, followed by removal of unbound BzATP, resulted in the efflux of 86Rb+ and the release of a prelabeled pool of cytoplasmic nucleotides when the temperature was shifted to 37 degrees C. Photoincorporation of BzATP was inhibited by ATP, ATP alpha S, ATP gamma S, and other ATP analogs that induced an increase in plasma membrane permeability to nucleotides in 3T6 cells under nonphotoactivating conditions. GTP, ITP, UTP, adenosine, and ATP analogs that did not alter plasma membrane permeability to nucleotides under nonphotoactivating conditions also had no effect on BzATP photoincorporation. Photoincorporation of BzATP occurred optimally between pH 6.6 and pH 8.2 but was inhibited at pH 6.0. Photoincorporation of BzATP was also modulated by the osmolarity and the divalent cation concentration of the assay medium. The increase in plasma membrane permeability to nucleotides induced by photoincorporated BzATP occurred at the same rate and had the same temperature, pH, ionic strength, and divalent cation requirements as the increase in plasma membrane permeability to nucleotides induced by ATP and BzATP under nonphotoactivating conditions. These findings support the hypothesis that BzATP can be covalently incorporated into a P2 purinoceptor in 3T6 cells that is coupled to plasma membrane channels for ions and other metabolites.     

Characterization of human DNase I family endonucleases and activation of DNase gamma during apoptosis

We describe here the characterization of the so far identified human DNase I family DNases, DNase I, DNase X, DNase gamma, and DNAS1L2. The DNase I family genes are found to be expressed with different tissue specificities and suggested to play unique physiological roles. All the recombinant DNases are shown to be Ca(2+)/Mg(2+)-dependent endonucleases and catalyze DNA hydrolysis to produce 3'-OH/5'-P ends. High activities for DNase I, DNase X, and DNase gamma are observed under neutral conditions, whereas DNAS1L2 shows its maximum activity at acidic pH. These enzymes have also some other peculiarities: different sensitivities to G-actin, aurintricarboxylic acid, and metal ions are observed. Using a transient expression system in HeLa S3 cells, the possible involvement of the DNases in apoptosis was examined. The ectopic expression of each DNase has no toxic effect on the host cells; however, extensive DNA fragmentation is observed only in DNase gamma-transfected cells after the induction of apoptosis. Furthermore, DNase gamma is revealed to be located at the perinuclear region in living cells, and to translocate into the nucleus during apoptosis. Our results demonstrate that DNase I, DNase X, DNase gamma, and DNAS1L2 have similar but unique endonuclease activities, and that among DNase I family DNases, DNase gamma is capable of producing apoptotic DNA fragmentation in mammalian cells.     

Solution structure and thermodynamics of a divalent metal ion binding site in an RNA pseudoknot.

Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies. The pseudoknot causes efficient -1 ribosomal frameshifting in mouse mammary tumor virus. Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+preferentially stabilizes the pseudoknot relative to its constituent hairpins. The use of Co(NH3)63+as a substitute for Mg2+was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+or Co (NH3)63+. The structure of the pseudoknot-Co(NH3)63+complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem. Co(NH3)63+stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands. Hydrogen bonds are identified between the Co(NH3)63+protons and non-bridging phosphate oxygen atoms, 2' hydroxyl groups, and nitrogen and oxygen acceptors on the bases. The binding site is significantly different from that previously characterized in the major groove surface of tandem G.U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron. Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+as on addition of Co(NH3)63+, indicating that both ions bind at the same site. Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+and a temperature of 15 degrees C) were obtained for Co(NH3)63+and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration. An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot. These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots.     

Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to gamma rays at 77 K. I. Radical formation as studied by electron spin resonance

Formation of free radicals in golden hamster embryo (GHE) cells in the frozen living state by gamma irradiation has been studied by electron spin resonance spectroscopy at 4.2 and 77 K. The relative yields of H atoms, OH radicals, and organic radicals trapped in the irradiated GHE cells are 12, 72, and 16%, respectively, of total radical yields. When dimethylsulfoxide (DMSO) is added to GHE cells at 77 K, a large quantity of CH2SOCH3 radicals (DMSO radicals) are formed after gamma irradiation. The yields of OH radicals are not affected by the addition of DMSO. When the GHE cell-DMSO mixtures are irradiated with gamma rays at 77 K and then warmed to 111 K, the OH radicals decay, whereas the DMSO radicals do not increase complementarily. Moreover, the decay rates of the OH radicals at 111 K do not depend upon the concentration of DMSO. Thus OH radicals do not react with DMSO during warming of the irradiated sample. When H atoms are produced by gamma irradiation of acid ice at 60 K, the decay rates of the H atoms at 77 K increase with increasing DMSO concentration, indicating that DMSO reacts with H atoms (CH3SOCH3 + H----.CH2SOCH3 + H2) at 77 K by quantum-mechanical tunneling. When the GHE cell-DMSO mixture is irradiated with gamma rays at 77 or 4.2 K in the dark, DMSO ions are produced in addition to DMSO radicals. Therefore it is concluded that DMSO does not scavenge OH radicals, but does capture H atoms, holes and/or electrons in the gamma-irradiated cells, resulting in the remarkable formation of DMSO radicals. This scavenger effect of DMSO may be related to the radioprotection of DMSO against cell killing described in the companion paper (Watanabe et al., Radiat. Res., this issue).