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.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on ⋅OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as β-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against ^⋅OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for ^⋅OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of ^⋅OH from H₂O₂. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H₂O₂. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on ^⋅OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

Weak coherent radiation of OH and ortho- H2O space masers as a carrier in biocommunication: the ortho/para-conversion of H2O spin isomers?

A conception of biocommunication based on the principle of radiophysics stating that the carrier modulation takes place at the resonance frequency in the transmission-receiver system has been substantiated and proved. The coherent radiation of space OH-masers (1.6-1.7 GHz) and ortho-H2O-maser (22.3 GHz) is proposed as a source of the carrier frequency. The narrow lines of rotational transition of H2O and OH molecules in liquid water were proposed to be considered as an analog of selective resonances of transmitter and receiver in radiocommunication. The possibility of the ortho-para conversion of H2O spin isomers, induced by weak electromagnetic fields, is discussed.     

Biological Activation of Heteropoly Complex of Molybdotungstosilicate Containing Lanthanum K10H3La(SiMo6W5O39)2⋅26H2O: Spectroscopic Approach and Microcalorimetry

In this paper, the biological activation of heteropoly complex of molybdotungstosilicate containing lanthanum K₁₀H₃La(SiMo₆W₅O₃₉)₂?26H₂O (LaW₅) was investigated by spectroscopic approach and microcalorimetry under the human physiological conditions. Fluorescence spectroscopy in combination with UV–Vis absorption spectroscopy was employed to investigate the binding of LaW₅ to bovine serum albumin (BSA). In the mechanism discussion, it was proved that the fluorescence quenching of BSA by LaW₅ is a result of the formation of LaW₅–BSA complex. Binding parameters were determined using the Stern–Volmer equation. The results of thermodynamic parameters ?G, ?H, ?S at different temperatures indicate that van der Waals interactions and hydrogen bonds play a major role for LaW₅–BSA association. The distance r between donor (BSA) and acceptor (LaW₅) was obtained according to fluorescence resonance energy transfer. Furthermore, the calorimetric method was used to monitor the biological activity of LaW₅ in Escherichia coli.     

β-Arrestins facilitate ubiquitin-dependent degradation of apoptosis signal-regulating kinase 1 (ASK1) and attenuate H2O2-induced apoptosis

β-Arrestins are ubiquitously expressed proteins that play important roles in receptor desensitization, endocytosis, proteosomal degradation, apoptosis and signaling. It has been reported that β-Arrestin2 acts as a scaffold by directly interacting with the JNK3 isoform and recruiting MKK4 and the apoptosis-signaling kinase-1 (ASK1). Here, we report a novel function of β-Arrestins in regulating H₂O₂-induced apoptosis. Our study demonstrates that β-Arrestins physically associate with C-terminal domain of ASK1, and moreover, both over-expression and RNA interference (RNAi) experiments indicate that β-Arrestins down-regulate ASK1 protein. In detail, β-Arrestin-induced reduction of ASK1 protein is due to ubiquitination and proteasome-dependent degradation of ASK1 in response to association of β-Arrestins and ASK1. Upon H₂O₂ stimulation, the protein binding between β-Arrestins and ASK1 increases and ASK1 degradation is expedited. In consequence, β-Arrestins prevent ASK1-JNK signaling and as a result attenuate H₂O₂-induced apoptosis. Structurally, C-terminal domain of ASK1 is essential for β-Arrestins and ASK1 association. We also found that CHIP is required for β-Arrestins-induced ASK1 degradation, which suggested that β-Arrestins function as a scaffold of ASK1 and CHIP, leading to CHIP-mediated ASK1 degradation. All these findings indicate that β-Arrestins play a negative regulatory role in H₂O₂-induced apoptosis signaling through associating with ASK1 and CHIP and facilitating ASK1 degradation, which provides a new insight for analyzing the effects of β-Arrestins on protecting cells from oxidative stress-induced apoptosis.     

Microsolvation and hydration enthalpies of CaC2O4(H2O) n (n = 0-16) and C2O4 2-(H2O) n (n = 0-14): an ab initio study

We studied hydrated calcium oxalate and its ions at the restricted Hartree–Fock RHF/6-31G* level of theory. Performing a configurational search seems to improve the fit of the HF/6-31G* level to experimental data. The first solvation shell of calcium oxalate contains 13 water molecules, while the first solvation shell of oxalate ion is formed by 14 water molecules. The first solvation shell of Ca(II) is formed by six water molecules, while the second shell contains five. At 298.15 K, we estimate the asymptotic limits (infinite dilution) of the total standard enthalpies of hydration for Ca(II), oxalate ion and calcium oxalate as ?480.78, –302.78 and –312.73 kcal mol^?1, resp. The dissociation of hydrated calcium oxalate is an endothermic process with an asymptotic limit of +470.84 kcal mol^?1.

Lack of interaction between α2 and dopamine D2-receptors in mediating their inhibitory effects on [3H]dopamine release from rat nucleus accumbens slices

The ₂-adrenoceptor agonist, UK14304, dose-dependently inhibited the electrically stimulated release of dopamine (DA) from rat nucleus accumbens slices. This effect was antagonized by idazoxan, confirming that it was an ₂-adrenoceptor mediated effect. There was no evidence of endogenous activation of noradrenergic receptors suggesting that the ₂-adrenoceptor agonist was not acting presynaptically to inhibit noradrenaline release. An in vitro superfusion technique was used to investigate wheher there was any interaction between ₂-adrenoceptors and DA D₂-receptors in mediating their inhibitory effects on [³H]DA release from rat nucleus accumbens slices. ₂-Adrenoceptor and DA D₂-receptors interact with similar second messenger systems and it was considered that they may compete for a common pool of G-proteins. The inhibitory effects of the ₂-adrenoceptor agonist, UK14304, and the DA receptor agonists, quinpirole, apomorphine and pergolide were not independent. However, there was no evidence of any interaction between UK14304 and the DA D₂-receptor antagonists, sulpiride or haloperidol, suggesting that the two receptors do not compete for a common pool of G-proteins in mediating their inhibitory effects on DA release.     

Correlated behavior of the EPR signal of cytochrome b-559 heme Fe(III) ligated by OH− and the multiline signal of the Mn cluster in PS-II membrane fragments

EPR signals of Cyt b-559 heme Fe(III) ligated by OH⁻ and the multiline signal of the Mn cluster in PS-II membrane fragments have been investigated. In 2,3-dicyano-5,6-dichloro-p-benzoquinone-oxidized PS-II membrane fragments the light-induced decrease of the EPR signal of the heme Fe(III)-OH⁻ is accompanied by the appearance of the EPR multiline signal of the Mn cluster. Addition of F⁻ ions, which act as a stronger ligand for heme Fe(III) than OH⁻, decreases to the same extent the dark- and light-induced signal of the heme Fe(III)-OH⁻ and the light-induced multiline signal of the Mn cluster. These results are discussed in terms of the light-induced formation of a bound OH′ radical shared between the Cyt b-559 heme Fe and the Mn cluster as a first step of water oxidation.     

Relationship between copper biosorption and microbial inhibition of hydroxyl radical formation in a copper(II)–hydrogen peroxide system

The microbial retardation of the spin adduct, DMPO-OH, formed in a copper(II)–hydrogen peroxide–DMPO (5,5-dimethyl-1-pyrroline N-oxide) solution was examined in relation to copper biosorption. A hydroxyl radical is formed in the solution through two steps, the reduction of Cu(II) to Cu(I) by H₂O₂ and the Fenton-type reaction of Cu(I) with H₂O₂. The resultant radical is trapped by DMPO to form DMPO-OH. Microbial cells retarded the DMPO-OH in the Cu(II)–H₂O₂–DMPO far more significantly than in the UV-irradiated H₂O₂–DMPO solution. Egg albumin showed a higher DMPO-OH retardation than microbial cells both in the Cu(II)–H₂O₂–DMPO and the UV-irradiated H₂O₂–DMPO solutions. These results indicated that the retardation effect is related to organic matter and not to microbial activity. Microorganisms having higher affinities for copper ion retarded DMPO-OH more significantly. The linear relationship between the amounts of copper biosorption and the inverse of the median inhibitory doses for DMPO-OH indicated that the microbial cells inhibited the reduction of Cu(II) to Cu(I) by H₂O₂, followed by the decrease of hydroxyl radical formation and the retardation of DMPO-OH. These results also suggest that the coupling between microbial cells and Cu(II) ion can be estimated from their ability to retard DMPO-OH.     

Unimolecular study of the interaction between the outer membrane protein OmpF from E. coli and an analogue of the HP(2–20) antimicrobial peptide

Little is known on antimicrobial peptide permeation through outer membrane channels in Gram-negative bacteria. Herein, we probed at a single-molecule level the interaction of two different peptides, magainin 2 and HPA3P with OmpF from E. coli. HPA3P is an analogue of the antimicrobial peptide HP(2–20) isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Our data show that the shorter and more charged HPA3P peptide is more accessible to the inner volume of the OmpF than magainin 2. We demonstrate the ability of HPA3P peptides to interact with OmpF in a voltage- and concentration-dependent manner, which does not rule out a novel mechanism by which such peptides could reach the periplasmic space of Gram-negative bacteria. Unexpectedly, we found that increasing the applied voltage led to an increase of the residence time of HPA3P peptide inside the pore, possibly reflecting electric field-induced changes in pore and peptide geometry.     

Crystal structure,magnetic and thermal properties of the one-dimensional complex [Nd(pzam)3(H2O)Mo(CN)8] · H2O

The new d–f cyanido-bridged 1D assembly [Nd(pzam)₃(H₂O)Mo(CN)₈] · H₂O was prepared by self-assembly of pyrazine-2-carboxamide (pzam), Nd(NO₃) · nH₂O and (Bu₃NH)₃[Mo(CN)₈] · 4H₂O in acetonitrile. X-ray crystallographic studies indicate that the complex comprises chains of alternating, cyanido-bridged [Nd(pzam)₃(H₂O)]³⁺ and Mo(CN)₈]^3? fragments. The magneto-structural properties have been studied by field-dependent magnetization and specific heat measurements at low temperatures (?0.3 K). Below ≈10 K the Nd(III) moment is well approximated by an effective spin S = 1/2, with anisotropic g-tensor. The exchange coupling between the Nd(III) and the Mo(V) spins S = 1/2 along the structural chains is found to be ferromagnetic, with J/k_B = 1.8 ± 0.2 K and approximately XY (planar) anisotropy. No evidence for 3D interchain magnetic ordering is found. A comparison with magneto-structural data of other cyanido-bridged complexes involving the Nd(III) ion is presented.     

Hydrothermal synthesis and structural characterization of an organic–inorganic hybrid material: (H2tptz)2[δ-Mo8O26]·2H2O (tptz=2,4,6-tripyridyltriazine)

The reaction of MoO₃ and 2,4,6-tripyridyltriazine (tptz) in water at 180°C for 48 h and pH 5.5 produces (H₂tptz)₂[Mo₈O₂₆]·2H₂O in 70% yield. The structure is constructed from δ-Mo₈O₂₆ ⁴⁻ clusters, H₂tptz²⁺ and H₃O⁺ cations linked through hydrogen bonding into a network. Crystal data: C₁₈H₁₆Mo₄N₆O₁₄; monoclinic P2₁/n; a=10.2225(5) Å, b=14.0072(6) Å, c=18.1154(8) Å, β=93.896(1)°, V=2587.9(2) Å³, Z=4, D_calc=2.372 g cm⁻³; R₁=0.0271 based on 3212 reflections.     

Potential role of Thermus thermophilus and T. oshimai in high rates of nitrous oxide (N2O) production in ∼80 °C hot springs in the US Great Basin

Ambient nitrous oxide (N(2)O) emissions from Great Boiling Spring (GBS) in the US Great Basin depended on temperature, with the highest flux, 67.8 ± 2.6 μmol N(2)O-N m(-2) day(-1) , occurring in the large source pool at 82 °C. This rate of N(2)O production contrasted with negligible production from nearby soils and was similar to rates from soils and sediments impacted with agricultural fertilizers. To investigate the source of N(2)O, a variety of approaches were used to enrich and isolate heterotrophic micro-organisms, and isolates were screened for nitrate reduction ability. Nitrate-respiring isolates were identified by 16S rRNA gene sequencing as Thermus thermophilus (31 isolates) and T. oshimai (three isolates). All isolates reduced nitrate to N(2)O but not to dinitrogen and were unable to grow with N(2)O as a terminal electron acceptor. Representative T. thermophilus and T. oshimai strains contained genes with 96-98% and 93% DNA identity, respectively, to the nitrate reductase catalytic subunit gene (narG) of T. thermophilus HB8. These data implicate T. thermophilus and T. oshimai in high flux of N(2)O in GBS and raise questions about the genetic basis of the incomplete denitrification pathway in these organisms and on the fate of biogenic N(2)O in geothermal environments.     

Cu(Nor)2·5H2O, a complex of Cu(II) with Norfloxacin: theoretic approach and biological studies. Cytotoxicity and genotoxicity in cell cultures

Norfloxacin is a fluoroquinolone antibiotic used in the treatment of bacterial infections. In this article, we studied the potential antitumoral action of a complex of Norfloxacin with Cu(II), Cu(Nor)₂·5H₂O on osteosarcoma cells (UMR106) and calvaria-derived cells (MC3T3-E1), evaluating its cytotoxicity and genitoxicity. We have also elucidated the more stable conformation of this complex under physiologic conditions by Molecular Dynamic simulations based on the model of the canonical ensemble and PM6 force field. When solvent effect was taken into account, the complex conformation with both carbonyl groups in opposite sides displayed lower energy. Cu(Nor)₂·5H₂O caused an inhibitory effect on the proliferation on both cell lines from 300 μM (P < 0.01). Nevertheless, the decline on cell proliferation of UMR106 cells was more pronounced (45 % vs basal) than in MC3T3-E1 cells (20 % vs basal) at 300 μM (P < 0.01). Cu(Nor)₂·5H₂O altered lysosomal metabolism (Neutral Red assay) in a dose-dependent manner from 300 μM (P < 0.001). Morphological studies showed important transformations that correlated with a decrease in the number of cells in a dose-dependent manner. Moreover, Cu(Nor)₂·5H₂O caused statistically significant genotoxic effects on both osteoblast cell lines in a lower range of concentrations (Micronucleus assay) (P < 0.05 at 10 μM, P < 0.001 from 25 to 50 μM). UMR106 cells displayed a dose-related genotoxic effect between 5 and 25 μM while the MC3T3-E1 cells showed a narrower concentration dependent range. Altogether, these results suggest that Cu(Nor)₂·5H₂O is a good candidate to be further evaluated for alternative therapeutics in cancer treatment.     

Is the tissue persistence of O(6)-methyl-2'-deoxyguanosine an indicator of tumour formation in the gastrointestinal tract?

Azoxymethane (AOM) is a methylating agent capable of inducing mutations in DNA by forming adducts with DNA bases. It has been used to understand the mechanisms involved in colon carcinogenesis. Of the adducts formed in response to AOM, O(6)-methyl-2'-deoxy-guanosine (O(6)-mdGua) is the most mutagenic. Based on studies in rodents of the abundance and persistence of DNA adducts in various tissues after treatment with alkylating agents, previous results suggest, as a generalization, that the longer O(6)-mdGua adducts remain unrepaired in the cells of a tissue, the greater the risk for tumorigenesis. To test this hypothesis, we have built on these studies, expanding the number of tissues in which O(6)-mdGua abundance and persistence were examined and correlating these data with tumour distribution and abundance in rats maintained for 26 weeks after the treatment with AOM. Our study revealed firstly the existence of groups of tissues that developed relatively large amounts (proximal and distal colon, proximal small intestine (SI), liver and kidney) and relatively low levels (stomach, distal SI, bladder, spleen, blood and lung) of O(6)-mdGua after AOM exposure. Secondly, while all tissues showed an increase in adduct levels at 6h after mutagen treatment and most showed a significant drop in adduct levels between 6h and 48h (stomach, proximal and distal SI, liver, spleen, blood and lung), one group of tissues displayed O(6)-mdGua levels that did not decrease at 48h (proximal and distal colon, kidney and bladder). Predictably, the colon displayed tumours 26 weeks after treatment. Interestingly, however, the proximal SI also displayed significant tumour formation at that time. Our findings demonstrate (1) a direct association between exposure to O(6)-mdGua and tumours of the distal colon and (2) a dissociation of the relationship between adduct clearance and tumorigenesis in the SI. This diversity of response in the gastrointestinal tract warrants further analysis.