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.     

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.     

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.     

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.     

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.     

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.     

Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture

Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483, when grown on lactose in continuous culture, showed increasing specific yields and volumetric productivities of exopolysaccharide (EPS) with increasing dilution rate. Specific and volumetric productivities of lactate and galactose, as extracellular metabolites, increased in response to the incremental changes in the dilution rate up to 0.4 h^–1. Elevated Y_p/s values determined for EPS (0.025 g EPSg lactose^–1) at the dilution rates of 0.3 h^–1–0.4 h^–1, relative to those determined at lower dilution rates, suggest a diversion of carbon flux towards EPS being associated with the higher rates of growth.     

Ca2+ and Cu2+ supplementation augments vancomycin production by Amycolatopsis orientalis

Supplementation with CaCl₂2H₂O (40 mg l^–1) or CuSO₄5H₂O (10 mg l^–1) improved vancomycin production by Amycolatopsis orientalis by 12 and 11%, respectively; used in combination, they acted synergistically. Ca²⁺ decreased the intracellular concentration of vancomycin 36%, whereas Cu²⁺ increased the intracellular activity of TDP-glucose:aglycosylvancomycin glucosyltransferase 3 times more than control. Ca²⁺ probably works by altering the permeability of cells to vancomycin, whereas Cu²⁺ increases the activity of an enzyme responsible for vancomycin biosynthesis.     

Aluminum Enhances Melanin-Induced Lipid Peroxidation

The present study was conducted to characterize the possible interaction of Al³⁺ and Fe²⁺ with synthetic melanin in the potentiation of lipid peroxidation in liposomes and rat caudate-putamen homogenates. Al³⁺ stimulated melanin-initiated lipid peroxidation as measured by the production of 2-thiobarbituric acid-reactive substances (TBARS) and conjugated dienes. The effect of Al³⁺ was dependent on melanin (10–100 g/ml) and Al³⁺ (2.5–250 M) concentrations and no synergism between Fe²⁺ and Al³⁺ was observed. The prooxidant effect of Al³⁺ was partially inhibited by superoxide dismutase indicating the involvement of O ₂ ^- . Ga³⁺ and Be²⁺ which can increase NADH oxidation in the presence of O ₂ ^- , also were shown to stimulate melanin-initiated TBARS production. Based on the effect of Al³⁺ and other non redox metals, we suggest that Al³⁺ does not act through either the induction of melanin free radicals, or the induction of changes in membrane physical properties. Results show that Al³⁺ enhances melanin-initiated lipid peroxidation in part through an interaction with O ₂ ^- generated from the autoxidation of melanin. We speculate that Al³⁺ contributes to neuromelanin-mediated oxidative damage in dopaminergic neurons and subsequent neuronal degeneration and death in Parkinson's disease.     

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.     

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.     

Carbonate bioformations around underwater freshwater springs in the north-eastern Adriatic Sea

Large carbonate, bryozoan-serpulid constructions, made by Pentapora fascialis and Salmacina dysteri respectively, were found around karstic freshwater springs, called vruljas, in the Senj Archipelago (Velebit Channel, Croatia). In June 2002, several sites were investigated by SCUBA divers on the rocky cliffs of Grmac and dralova at depths ranging from 19 to 32 m. Mean colony diameter decreased with increasing distance from the vruljas: in the vicinity the mean diameter was 65.8±21 cm, at 2-m distance it was 40.4±8.2. Carbonate contribution was to a great extent due to the bryozoan (5,784±1,186 gm^–2 CaCO₃) rather than to the serpulid (383±218 gm^–2 CaCO₃). P. fascialis carbonate standing stock was remarkably high if compared with data from literature for shallow carbonate producers. The bryozoan-serpulid constructions can be indicated as important, even if localised, contributions to the carbonate budget in the Adriatic Sea.     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Antibiotic purification from fermentation broths by counter-current chromatography: analysis of product purity and yield trade-offs

Counter-current chromatography (CCC) is a low pressure, liquid–liquid chromatographic technique which has proven to be a powerful purification tool for the high-resolution fractionation of a variety of active pharmaceutical compounds. The successful integration of CCC into either existing or new manufacturing processes requires the predictable purification of target compounds from crude, fermentation-derived, feed streams. This work examines the feasibility of CCC for the purification of fermentation-derived erythromycin A (EA) from its structurally and chemically similar analogues. At the laboratory scale, the effect of feed pre-treatment using either clarified, forward extracted (butyl acetate) or back extracted broth on EA separation was investigated. This defined the degree of impurity removal required, i.e. back extracted broth, to ensure a reproducible elution profile of EA during CCC. Optimisation and scale-up of the separation studied the effects of mobile phase flow (2–40 mlmin^–1) and solute loading (0.1–10 g) on the attainable EA purity and yield. The results in all cases demonstrated a high attainable EA purity (>97% w/w) with throughputs up to 0.33 kgday^–1. Secondly, a predictive scale-up model was applied demonstrating, that from knowledge of the solute distribution ratio of EA (K_EA) at the laboratory scale, the EA elution time at the pilot scale could be predicted to within 3–10%, depending upon the solute injection volume. In addition, this study has evaluated a fractionation diagram approach to visually determine the effects of key operational variables on separation performance. This resulted in accurate fraction cut-point determination for a required degree of product purity and yield. Overall, the results show CCC to be a predictable and scaleable separation technique capable of handling real feed streams.     

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).     

The Effects of Hypoxia on Three Sympatric Shark Species: Physiological and Behavioral Responses

Behavioral and physiological responses to hypoxia were examined in three sympatric species of sharks: bonnethead shark Sphyrna tiburo, blacknose shark, Carcharhinus acronotus, and Florida smoothhound shark, Mustelus norrisi, using closed system respirometry. Sharks were exposed to normoxic and three levels of hypoxic conditions. Under normoxic conditions (5.5–6.4mg l^–1), shark routine swimming speed averaged 25.5 and 31.0cm s^–1 for obligate ram-ventilating S. tiburo and C. acronotus respectively, and 25.0cm s^–1 for buccal-ventilating M. norrisi. Routine oxygen consumption averaged about 234.6 mg O₂kg^–1h^–1 for S. tiburo, 437.2mg O₂kg^–1h^–1 for C. acronotus, and 161.4mg O₂ kg^–1 h^–1 for M. norrisi. For ram-ventilating sharks, mouth gape averaged 1.0cm whereas M. norrisi gillbeats averaged 56.0 beats min^–1. Swimming speeds, mouth gape, and oxygen consumption rate of S. tiburo and C. acronotus increased to a maximum of 37–39cm s^–1, 2.5–3.0cm and 496 and 599mg O₂ kg^–1 h^–1 under hypoxic conditions (2.5–3.4mg l^–1), respectively. M. norrisi decreased swimming speeds to 16cm s^–1 and oxygen consumption rate remained similar. Results support the hypothesis that obligate ram-ventilating sharks respond to hypoxia by increasing swimming speed and mouth gape while buccal-ventilating smoothhound sharks reduce activity.     

Contribution of methanol to the production of methane and its <Superscript>13</Superscript>C-isotopic signature in anoxic rice field soil

Conversion of methanol to CH₄ has a large isotope effect so that a small contribution of methanol-dependent CH₄ production may decrease the ¹³CH₄ of total CH₄ production. Therefore, we investigated the role of methanol for CH₄ production. Methanol was not detectable above 10 M in anoxic methanogenic rice field soil. Nevertheless, addition of ¹³C-labeled methanol (99% enriched) resulted in immediate accumulation of ¹³CH₄. Addition of 0.1 M ¹³C-methanol resulted in increase of the ¹³CH₄ from –47 to –6 within 2 h, followed by a slow decrease. Addition of 1 M ¹³C-methanol increased ¹³CH₄ to +500 within 4 h, whereas 10 M increased ¹³CH₄ to +2500 and continued to increase. These results indicate that the methanol concentrations in situ, which diluted the ¹³C-methanol added, were 0.1 M and that the turnover of methanol contributed only about 2% to total CH₄ production at 0.1 M. However, contribution increased up to 5 and 17% when 1 and 10 M methanol were added, respectively. Anoxic rice soil that was incubated at different temperatures between 10 and 37 °C exhibited maximally 2–6% methanol-dependent methanogenesis about 1–2 h after addition of 1 M ¹³C-methanol. Only at 50 °C, contribution of methanol to CH₄ production reached a maximum of 10%. After longer (7–10 h) incubation, however, contribution generally was only 2–4%. Methanol accumulated in the soil when CH₄ production was inhibited by chloroform. However, the accumulated methanol accounted for only up to 0.7 and 1.2% of total CH₄ production at 37 and 50 °C, respectively. Collectively, our results show that methanol-dependent methanogenesis was operating in anoxic rice field soil but contributed only marginally to total CH₄ production and the isotope effect observed at both low and high temperature.     

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Role of disulfide and sulfhydryl groups

The triated adrenergic antagonists Prazosin ([³H]PRZ) and Idazoxan ([³H]IDA, or RX-781094) bind specifically and with high affinity to ₁ and ₂-adrenoceptors respectively, in membrane preparations from cerebral cortex. Saturation experiments performed to determine the density of receptors and the dissociation constant (K _d) were analyzed by the methods of Eadie Hofstee, iterative modelling, and the procedure of Hill, while the specificity of the labelling was verified by displacement experiments. Since receptors are proteins, we examined the role of disulfide (–SS–) bridges and sulfhydryl (–SH) groups in the specific combination of [³H]PRZ and [³H]IDA to the ₁ and ₂-adrenoceptors. Pretreatment of the membranes with the –SS– reactive DL-dithiothreitol (DTT) or the alkylating agent N-ethylmaleimide (NEM), alone or in combination, decreased specific binding of both ligands, with only minor changes in the non-specific counts. The [³H]IDA binding (₂-sites) was more sensitive to both DTT and NEM than the [³H]PRZ sites (₂-adrenoceptors), and the initial changes induced by alkylation of the ₂-site were due to an important decrease in the affinity for [³H]IDA, as judged by the increase in theK _d. This modulation in the affinity caused by alkylation of a thiol group could explain the higher potency of the blocking agent tetramine disulfide benextramine at the ₂-site. The results provide evidence for the participation of –SS– and –SH groups in the binding site of ₁ and ₂-adrenoceptors in the cerebral cortex.