Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

Summary The fluorescent intercalation complex of ethidium bromide (ETB) with DNA was used as a probe to compare the effects of various radicals with respect to impairment of the DNA base-pair region.OH radicals inhibit up to 0.7 dye intercalations perOH at low salt concentration, and for various oxidizing species the effect decreases in the orderOH > Br ₂ ^– > N ₃ > $$ " align="middle" border="0"> (SCN) ₂ ^–. DNA impairment by theOH product of Met-Gly is comparable to that of N ₃ , but no effect was found due to the interaction between DNA and Lys-Tyr-Lys phenoxyl radicals. The reducing speciese _aq ^– , H, O ₂ ^–, and CO ₂ ^– hardly affect the DNA-ETB intercalation.     

Contrasting oxygen-effects in the inactivation of ribonuclease A by N 3 ⋅ , (SCN) 2 ⋅ − and⋅OH radicals

Summary N ₃ exhibits higher efficiency thanOH in the inactivation of RNase in de-acerated (neutral) aqueous solution. In O₂-saturated solution theOH-induced inactivation is enhanced, but N ₃ and (SCN) ₂ ^– become remarkably inefficient. Our results suggest that semi-oxidized tyrosine, the predominant initial defect induced by N ₃ and (SCN) ₂ ^– but not byOH, can be re-reduced upon reaction with O ₂ ^– or cysteine.     

Polysaccharide-enriched fraction isolated from Duchesnea chrysantha protects against oxidative damage

Reactive oxygen species (ROS)-related oxidative damages have been implicated in a wide variety of the pathological processes such as atherosclerosis, inflammation and carcinogenesis. A polysaccharide-enriched fraction (PEF) was isolated from Duchesnea chrysantha, a herbaceous plant, and its antioxidant activity was demonstrated using several assay systems in vitro. The PEF effectively inhibited Cu²⁺-stimulated low density lipoprotein oxidation in a concentration-dependent way and retarded the conjugated diene formation with the enhancement of the lag phase during oxidation. An assay for DNA strand breaks showed that PEF strongly protected DNA against damage caused by UV or by OH or O₂ ^– generated in the metal-catalyzed oxidation system. PEF effectively inhibited Nitroblue Tetrazolium reduction mediated by O₂ ^– in a dose-dependent manner. It also competed with 2-deoxy-d-ribose in absorbing OH generated by -irradiation (600 Gy) and thus inhibited the formation malondialdehyde. In conclusion, these evidences indicate that PEF may act as an antioxidant to scavenge O₂ ^–, OH or LO₂ directly. Our findings suggest the possibility that it may play a role as a potential therapeutic antioxidant in treatment of oxidative damage-derived diseases.     

Secondary H/D isotope effect on hydrogen-bonded hydroxyl groups as a tool for recognizing distance constraints in conformational analysis of oligosaccharides

An isotopomer-selected NOE (ISNOE) method for the unequivocal identification of mutually hydrogen-bond-linked hydroxyl groups is described. It relies on the fact that the OH group's signal patterns obtained for a partially deuterated sample originate from both isotopomers of the partner hydroxyl, whereas a NOE for this group can originate from cross-relaxation with the protio isotopomer of this hydroxyl only. Hence, the isotopically shifted component of this group's signal does not appear in a ROE difference spectrum obtained with selective excitation of the partner hydroxyl. This method is also applicable in those cases when only one of two mutually hydrogen-bonded groups exhibits resolvable isotope shifts. Furthermore, it is shown that isotope shifts may occur even for pairs of OH groups that are not mutually hydrogen-bonded, if these participate in hydrogen bonds with other hydroxyls and thereby affect conformational equilibria. The ISNOE experiment enables one to distinguish between these two sources of isotope shifts. Since the OO distance for hydrogen-bonded hydroxyls in sugars is known to lie between 2.7 and 3.0 Å , the hydrogen bonds established by ISNOE can be used in conformational analysis as reliable, motionally non-averaged distance constraints for the conformations containing these bonds.     

Dual Role of Superoxide Radicals in the Chilling-Induced photoinhibition in Maize Seedlings

Maize (Zea mays) seedlings were exposed for 6 h to strong irradiance (1 000 mol m^–1 s^–1 of PPFD) at 5, 12, 17, or 25 °C, followed by an exposure to the darkness for 6 h at 22 °C. Leaf chlorophyll fluorescence, net photosynthetic rate (P _N), and the amount of superoxide radicals (O₂ ^–) in relation to chilling-induced photoinhibition were investigated. During the photophase, a good correlation (r=–0.879) was observed between _PS2 (relative quantum efficiency of PS2 electron transport) and the amount of O₂ ^–. Treatment with exogenous O₂ ^– reduced the P _N and _PS2 as the chilling stress did, that was inhibited by specific scavenger of O₂ ^–. Hence chilling-induced photoinhibition might be due to the production of O₂ ^–. In contrast, in the dark period, P _N and _PS2 of the seedlings treated with the exogenous O₂ ^– were enhanced, but they were inhibited by the specific scavenger of O₂ ^–, showing the photoprotective role of O₂ ^– in the recovery phase. Furthermore, in terms of the effect of exogenous O₂ ^– on the xanthophyll cycle, the O₂ ^– production suggested a promotion effect for the de-epoxidation of violaxanthin during the photophase, the epoxidation of zeaxanthin at the dark stage, and the increase of the xanthophyll pool both in the photophase and dark phase, resulting in an enhancement of the ability of non-photochemical quenching to avoid or alleviate the damage to photosynthetic apparatus.     

Increased erythritol production in Torula sp. by Mn2+and Cu2+

The production of erythritol and the erythritol yield from glucose by Torula sp. were improved, in increasing order, by supplementing with 10 mg MnSO₄4H₂O l^–1, 2 mg CuSO₄5H₂O l^–1, and both 10 mg MnSO₄4H₂O l^–1 and 2 mg CuSO₄5H₂O l^–1. Mn²⁺ decreased the intracellular concentration of erythritol, whereas Cu²⁺ increased the activity of erythrose reductase in cells. These results suggest that Mn²⁺ altered the permeability of cells, whereas Cu²⁺ increased the activity of erythrose reductase in cells.     

Myeloperoxidase from Neutrophil Peroxisomes

Peroxisomal myeloperoxidase plays a key role in synthesis of oxidants by neutrophilic leukocytes. This heme protein consists of two subunits connected by a disulfide bond. The enzyme uses ₂₂ and l^– for synthesizing HOCl, the major oxidant produced by neutrophils. In addition to the chlorination reaction, myeloperoxidase exhibits some other properties depending on its oxidation state. The enzyme significantly affects synthesis of oxidants in the cells depending on the competing substrate concentrations and other factors. ^¯ ₂ is also a physiological substrate of myeloperoxidase. Its reaction with the enzyme determines how the cells utilize ^¯ ₂ for pathogen elimination. ^¯ ₂ affects the chlorinating and peroxidase activities of myeloperoxidase. In addition, ^¯ ₂ reacts with the enzyme yielding the catalytically active compound III that hydroxylates phenols.     

IGF-I activates the eIF4F system in cardiac muscle in vivo

IGF-I acutely stimulates protein synthesis in cardiac muscle through acceleration of mRNA translation. In the present study, we examined the regulatory signaling pathways and translation protein factors that potentially contribute to the myocardial responsiveness of protein synthesis to IGF-I in vivo. IGF-I was injected IV into rats and 20 min later the hearts were excised and homogenized for assay of regulatory proteins. IGF-I increased assembly of the translationally active eukaryotic initiation factor (eIF)4GeIF4E complex. The increased assembly of eIF4GeIF4E was associated with an enhanced eIF4G phosphorylation and increased availability of eIF4E. Increased availability of eIF4E occurred as a consequence of diminished abundance of the inactive 4E-BP1eIF4E complex following IGF-I. The assembly of the 4E-BP1eIF4E complex appeared to be decreased through an IGF-I-induced phosphorylation of 4E-BP1. IGF-I also caused an increase in the phosphorylation of S6K1. Activation of the potential upstream regulators of 4E-BP1 and S6K1 phosphorylation via PKB and mTOR was also observed. In contrast, there was no effect of IGF-I on phosphorylation of elongation factor (eFE)2. The results suggest the major impact of IGF-I in cardiac muscle occurred via stimulation of translation initiation rather than elongation. Furthermore, the results are consistent with a role for assembly of active eIF4GeIF4E complex and activation of S6K1 in mediating the stimulation of mRNA translation initiation by IGF-I through a PKB/mTOR signaling pathway.     

The effect of specific chromosome and cytoplasm substitutions on the tissue culture response of wheat (Triticum aestivum) callus

Summary Calli were initiated from immature embryos of four lines of hexaploid wheat (Triticum aestivum L. em. Thell), the euplasmic nuclear donor Chinese Spring, Chinese Spring in which both 4B chromosomes were substituted by those of the variety Cappelle-Desprez and two alloplasmic lines in which these nuclei were substituted into the cytoplasm of Aegilops ovata. The calli were found to differ in their initia growth rates and their ability to organise shoot primordia and regenerate shoots. The Cappelle 4B chromosomes had a very significant effect on all these characters. The potential for modelling genotypes for improved tissue culture characteristics is discussed.     

Expression of Pyruvate Carboxylase Enhances Succinate Production in Escherichia coli without Affecting Glucose Uptake

Plasmids carrying the pyc gene from Rhizobium etli were used to express pyruvate carboxylase in Escherichia coli. Results of batch fermentations of a wild-type E. coli (MG1655), this wild-type with the pUC18 cloning/expression vector (MG1655/pUC18) and this wild-type carrying the pyc gene (MG1655/pUC18-pyc) were compared in glucose-limited medium. The results indicate that the final succinate concentration upon complete glucose utilization was increased from 1.18 g/L to 1.77 g/L by the expression of pyc, while the final succinate concentration in MG1655/pUC18 was slightly lower than in the parent strain. This increased succinate concentration came at the expense of lactate synthesis, whose final concentration decreased from 2.33 g/L to 1.88 g/L. The expression of pyc did not affect the maximum glucose uptake (2.17 g/Lh for MG1655 versus 2.47 g/Lh for MG1655/pUC18-pyc), but did decrease the maximum rate of cell mass production (0.213 g/Lh for MG1655, 0.169 g/Lh for MG1655/pUC18 and 0.199 g/Lh for MG1655/pUC18-pyc).     

Interactions of growth inhibitory factor with hydroxyl and superoxide radicals

To show the effects of growth inhibitory factor (Cu₄Zn₃MT-III) involved in the scavenging of reactive oxygen species (ROS), a pulse radiolytic study was employed using N₂O-saturated Cu₄Zn₃MT-III aqueous solutions. It was demonstrated that the oxidizing OH radical efficiently reacted with Cu₄Zn₃MT-III by forming a thiyl radical RS with a second-order constant of 1.46×10¹¹ mol l^–1s^–1, which was determined by competition kinetics against KSCN. The thiyl radical RS reacted rapidly and reversibly with a thiolate in Cu₄Zn₃MT-III to form radical anion RSSR ^– with a constant of 1.65×10⁹ mol lL^–1s^–1 per thiolate, while the constant of the decay of this radical anion was 2.72×10⁵ s^–1, and the equilibrium constant of the formation for RSSR ^– was 6.08×10³ mol^–1 l. These values were close to those of Cd₅Zn₂MT-II. The SOD activity of Cu₄Zn₃MT-III to quench O₂ ^–was assayed by the riboflavine-methionine-nitrobluetetrazolium (NBT) method which catalyzed the dismutation of superoxide (O₂ ^–) at pH 7.8 with an IC₅₀ value of 1.50×10^–6 M for Cu₄Zn₃MT-III and 1.62×10^–6 M for Cd₅Zn₂MT-II. Additionally, the down-regulation of GIF may be a main factor in the decrease of the scavenging ability for the free OH and O₂ ^–radicals, which is possibly associated with the pathogenesis of neurodegenerative disease.     

Improved production of ginseng polysaccharide by adding conditioned medium to Panax notoginseng cell cultures

By adding 50% (v/v) filtered culture broth to fresh MS medium, the specific growth rate of Panax notoginseng was increased from 0.046 d–1 to 0.068 d–1, and the polysaccharide production and productivity reached 1.21 g l–1 and 61 mg/(ld), respectively, which were 1.3- and 2.3-fold of the control. Further supplementation of the conditioned medium with sucrose, ammonium, nitrate and phosphate gave a cell density of 13.7 g l–1 and a specific growth rate of 0.086 d–1. Polysaccharide production was 1.65 g l–1 and the productivity was 78 mg/(ld).     

Interaction of Phomopsin A with Normal and Subtilisin-Treated Bovine Brain Tubulin

Tubulin, the major component of microtubules, has a tendency to lose its ability to assemble or to bind to ligands in a time-dependent process known as decay. The decay process also causes tubulin to expose sulfhydryl groups and hydrophobic areas. The antimitotic drug phomopsin A strongly protects the tubulin molecule from decay. Here we have studied the interaction of phomopsin A with tubulin and tubulin which has been treated with subtilisin to remove selectively the C-termini of the and chains (_ss). The binding of phomopsin A to tubulin decreases the sulfhydryl titer by approximately 1.0 mol/mol. Selective removal of the peptides from the C-terminal ends does not affect phomopsin A's interaction with tubulin. Moreover, the _ss tubulin–phomopsin A complex appears to be more stable than the tubulin–phomopsin A complex as determined by the time-dependent increase in exposure of sulfhydryl groups and hydrophobic areas on tubulin. In fact, phomopsin A inhibits the decay process of _ss tubulin completely. This observation raises the possibility of determining the conformtion of this configuration of tubulin.     

Sorbitol as a non-repressing carbon source for fed-batch fermentation of recombinant Pichia pastoris

Glycerol/methanol and sorbitol/methanol mixed-feed fermentation strategies for the production of recombinant proteins by Pichia pastoris were compared in order to examine sorbitol's potential as a carbon source. Although P. pastoris does have a lower cell yield on sorbitol than on glycerol, the specific rate of product formation is higher (60 g protein g^–1 dry wth for sorbitol/methanol, vs 45 g protein g^–1 dry wth for glycerol/methanol), resulting in comparable final recombinant expression levels. Importantly, the presence of residual sorbitol in the growth medium appears to be less repressive to the alcohol oxidase promoter in this organism, providing a more forgiving means of operating mixed-feed fed-batch recombinant P. pastoris fermentations.     

A comparative study of conserved protein interactions of the primary electron donor in photosynthetic purple bacterial reaction centers

The pigment–protein interactions within the binding site of the bacteriochlorophyll (BChl) dimer constituting the primary electron donor (P) in several, native, photosynthetic bacterial reaction centers have been determined using Fourier transform Raman spectroscopy. For reaction centers whose primary sequence data are available, and assuming a structural analogy with the Rb. sphaeroides RC whose high-resolution three-dimensional structure is known, amino acid residues donating hydrogen bonds to P are proposed. Consequently, one may propose the microenvironment structure of the primary donors studied and correlate this deduced structure with the known absorption and redox properties of the primary donors. In this mini-review of our past work, we group and classify the primary donors with respect to their specific H-bonding interactions with the protein. This classification reveals trends in the H-bonding and certain physicochemical properties such as the P°/P+ redox midpoint potential, the positive charge distribution over the dimeric primary donors in their oxidized radical cation state P+, and the absorption maxima of the lower exciton Q_y absorption band of P.     

Removal of239Pu from mice with 3,4,3 LICAM(C) or N,N′, N″, N‴-tetra-(2,3-dihydroxybenzoyl)-spermine

Summary Male mice SAS/4 were injected i.v. with²³⁹Pu citr(IV) 0.27 µCikg^–1–9.99 kBqkg^–1. After 1 h 30 µmol kg^–1 of 3,4,3 LICAM(C), N, N, N, N-tetra-(2,3-dihydroxybenzoyl)-spermine or Na₃CaDTPA as a reference compound was given intraperitoneally. After 4 days the animals were sacrified and the Pu content in livers, kidneys, femurs and carcasses was determined by the liquid scintillation method. It was found that, as compared with the control, 3,4,3 LICAM(C) removed 83% of the Pu activity deposited in the liver, 71% of that in the femur and 79% of the Pu in the whole body. The Pu content in the kidneys exceeded the control value by about 50%. Na₃CaDTPA removed 96, 86, 40 and 72% of plutonium from the liver, kidneys, femurs and carcasses respectively.Tetra-DHB-spermine caused the excretion of 50, 57 and 39% of Pu from liver, bone and whole body respectively. The retention of Pu in the kidneys was increased to 400% of the control value.     

Synthesis of RGD amphiphilic cyclic peptide as fibrinogen or fibronectin antagonist

This paper investigates the effect of the incorporation of adiazaethylene glycol derivative (Deg, 2) into a cyclic peptide containingthe tripeptide sequence Arg-Gly-Asp (RGD). This motif is a common structuralelement of many integrin ligands. The synthesis of cyclo-(Arg-Gly-Asp-Deg)(7) has been accomplished in solution using standard peptide chemistry. Theintent was to improve the bioavailability of this new RGD cyclic peptide,which is shown to interact with _IIb33or _{5 1} receptors. A preliminary stepfor the conformational study of peptide 7 was done in DMSO-d₆using nuclear magnetic resonance spectroscopy techniques.     

Oxygen-centred free radicals can efficiently degrade the polypeptide of proteoglycans in whole cartilage

Bovine nasal cartilage slices, biosynthetically labelled in their proteoglycan with³⁵SO₄, were used as substrate for the attack of free radicals generated on exposure to a Co⁶⁰ source (which allows study of single radical species), and by chemical and enzymatic means. Systems generating hydroxyl (OH) and superoxide (0₂ -) radicals degraded the proteoglycan efficiently, while the hydroperoxy radical (HO₂ ) was less efficient ; addition of appropriate radical scavengers inhibited degradation. The radioactive products were heterogeneous in molecular size, but with doses up to 3600 Gy were the same size range as intact chondroitin sulphate. They contained free amino groups, and more were liberated by aminopeptidase M digestion, implying that at least a small peptide was present. Thus a major site of radical attack may be the polypeptide chain. We suggest that free-radical fragmentation of polypeptides may be important both in extracellular catabolism and in intracellular proteolysis.     

Induction of ELF transmembrane potentials in relation to power-frequency electric field bioeffects in a plant root model system

Summary Seminal roots ofCucumis sativus andCucurbita maxima were exposed to 60 Hz electric fields of 100–500 Vm^–1 in a conducting aqueous inorganic growth medium. Root growth rates were measured to produce a dose-response relationship for each species. The species were selected for study because of their familial relationship, reported sensitivity to 60 Hz, 360 Vm^–1 electric fields, and differing average root cell sizes. The latter characteristic influences the magnitude of ELF membrane potentials induced by constant-strength applied electric fields, but does not affect the magnitude of the electric field strength tangent to the cell surface. The difference in average root cell size betweenC. sativus (smaller cells) andC. maxima (larger cells) was used to evaluate two alternate hypotheses that the observed effect on root growth is stimulated by [1] the electric field tangent to the cell surface, or (2) a field-induced perturbation in the normal transmembrane potential of the cells.The results of the dose-response relationship studies are qualitatively consistent with the hypothesis that the effect is elicited by induced transmembrane potentials. The smaller-celled roots showed a substantially higher response threshold [C. sativus; E ₀ ^TH 330 Vm^–1] than did the larger-celled species [C. maxima; E ₀ ^TH 200 Vm^–1]. At field strengths above the response thresholds in both species, the growth rate ofC. sativus roots was less affected than that ofC. maxima roots exposed to the same field strength.