Inhibition of Mitochondrial Permeability Transition by Low pH is Associated with Less Extensive Membrane Protein Thiol Oxidation

Ca²⁺ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H₂O₂ production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca²⁺ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues.     

Sulfur isotope values in a forested catchment over four years: Evidence for oxidation and reduction processes

A small catchment on the Swedish West Coast has been studied over four years to determine S dynamics by using S isotope ratios. A Norway spruce dominated forest covers the catchment, and small peat areas occur in the lower parts of the catchment. The runoff values varied both during the year, and from year to year. Over the period from February 1990 to December 1993, the values ranged from — 1%. to +11%. Over the same period, the throughfall values ranged from +1%. to +15%. There was no correlation (r ²= 0.01; Pr(F)=0.57) between values in throughfall and runoff. Since the only input of S to the catchment is atmospheric deposition, the long-term runoff S mass flux is controlled by the deposition. Therefore, processes in the catchment are responsible for the variation in the runoff values. During periods with enriched runoff, bacterial dissimilatory SO ₄ ^2– reduction occurs in the catchment. After very dry periods, oxidation of this reduced S, which is³²S-enriched, can be traced in runoff. Previous studies of the catchment have not been able to distinguish between: 1) oxidation of reduced S and dry deposition, and 2) reduction and adsorption. From the current study, it can be concluded that adsorption and dry deposition cannot cause the observed variation in runoff .     

Release of fossil methane from mineral soil particles, and its implication for estimation of methane oxidation in a mineral subsoil

In a preliminary experiment we found that methane evolved from a sandy subsoil during aerobic incubation of shaken soil slurries. In the study presented here the methane was found to be released from the sand particles by mechanical weathering, caused by the grinding effect of the shaking. Large amounts of gas (about 0.5 ml gas g^–1 soil) were extracted by intense grinding of the soil in gas tight serum vials. Methane was the main hydrocarbon in the emitted gas, but also a considerable amount of ethane was present, as well as minor amounts of heavier hydrocarbons (up to C₆). The ¹³C-values of the emitted methane and ethane were –33 and –29 , respectively. Together these results demonstrate a thermogenic origin of the gas. This paper also reports the results of an incubation experiment where possible methane oxidation was looked for. If a possible release of methane is not accounted for, methane oxidation may be overlooked, as illustrated in this paper. Methane consumption was detected only in soil from 40 cm, in contrast to soil sampled at 100 cm and deeper where a slight production was measured. When methane oxidation was inhibited by dimethyl-ether, a significant release of methane was seen. The release was probably caused by chemical weathering. When this methane release was taken into account, methane oxidation was found to be present at all measured depths (40 to 200 cm). Fertilization with urea inhibited the methane oxidation at 40 cm but not at deeper layers. It is hypothesized that ammonia oxidizing bacteria were the main methane oxidizers in this mineral subsoil (deeper than 1 m), and that oxidation of methane might be a survival mechanism for ammonia oxidizers in ammonia limited environments.     

Metabolism of Anxiolytics and Hypnotics: Benzodiazepines, Buspirone, Zoplicone, and Zolpidem

1. The benzodiazepines are among the most frequently prescribed of all drugs and have been used for their anxiolytic, anticonvulsant, and sedative/hypnotic properties. Since absorption rates, volumes of distribution, and elimination rates differ greatly among the benzodiazepine derivatives, each benzodiazepine has a unique plasma concentration curve. Although the time to peak plasma levels provides a rough guide, it is not equivalent to the time to clinical onset of effect. The importance of and half-lives in the actions of benzodiazepines is discussed.2. The role of cytochrome P450 isozymes in the metabolism of benzodiazepines and in potential pharmacokinetic interactions between the benzodiazepines and other coadministered drugs is discussed.3. Buspirone, an anxiolytic with minimal sedative effects, undergoes extensive metabolism, with hydroxylation and dealkylation being the major pathways. Pharmacokinetic interactions of buspirone with other coadministered drugs seem to be minimal.4. Zopiclone and zolpidem are used primarily as hypnotics. Both are extensively metabolized; N-demethylation, N-oxidation, and decarboxylation of zopiclone occur, and zolpidem undergoes oxidation of methyl groups and hydroxylation of a position on the imidazolepyridine ring system. Zopiclone has a chiral centre, and demonstrates stereoselective pharmacokinetics. Metabolic drug–drug interactions have been reported with zopiclone and erythromycin, trimipramine, and carbamazepine. Reports to date indicate minimal interactions of zolpidem with coadministered drugs; however, it has been reported to affect the C _max and clearance of chlorpromazepine and to decrease metabolism of the antiviral agent ritonavin. Since CYP3A4 has been reported to play an important role in metabolism of zolpidem, possible interactions with drugs which are substrates and/or inhibitors of that CYP isozyme should be considered.     

An in vitro study on the role of metal catalyzed oxidation in glyeation and crosslinking of collagen

The present investigation was carried out to understand the effect of metal catalyzed oxidation on glycation and crosslinking of collagen. Tail tendons obtained from rats weighing 200-225 g were incubated with glucose (250 mM) and increasing concentrations of copper ions (5, 25, 50 and 100 M) under physiological conditions of temperature and pH. Early glycation, crosslinking and late glycation (fluorescence) of collagen samples were analyzed periodically. Early glycation was estimated by phenol sulfuric acid method, and the crosslinking was assessed by pepsin and cyanogen bromide digestion. A concentrationdependent effect of metal ions on the rate of glycation and crosslinking of collagen was observed. Tendon collagen incubated with glucose and 100 M copper ions showed 80% reduction in pepsin digestion within seven days, indicating extensive crosslinking, whereas collagen incubated with glucose alone for the same period showed only 7% reduction. The presence of metal ions in the incubation medium accelerated the development of Maillard reaction fluorescence on collagen, and the increase was dependent on the concentration of metal ions used. The metal chelator Diethylene triamine penta-acetate significantly prevented the increase in collagen crosslinking by glucose and copper ions. Free radical scavengers benzoate and mannitol effectively prevented the increased crosslinking and browning of collagen by glucose. The results indicate that the metal catalyzed oxidation reactions play a major role in the crosslinking of collagen by glucose. It is also suggested that the prevention of increased oxidative stress in diabetes may prevent the accelerated advanced glycation and crosslinking of collagen.     

Stable markers of oxidant damage to proteins and their application in the study of human disease

The mechanisms of formation and the nature of the altered amino acid side chains formed on proteins subjected to oxidant attack are reviewed. The use of stable products of protein side chain oxidation as potential markers for assessing oxidative damage in vivo in humans is discussed. The methods developed in the authors laboratories are outlined, and the advantages and disadvantages of these techniques compared with other methodologies for assessing oxidative damage to proteins and other macromolecules. Evidence is presented to show that protein oxidation products are sensitive markers of oxidative damage, that the pattern of products detected may yield information as to the nature of the original oxidative insult, and that the levels of oxidized side-chains can, in certain circumstances, be much higher than those of other markers of oxidation such as lipid hydroperoxides.     

Electron transfer of dissolved organic matter and its potential significance for anaerobic respiration in a northern bog

We investigated electron transfer processes of dissolved organic matter (DOM) and their potential importance for anaerobic heterotrophic respiration in a northern peatland. Electron accepting and donating capacities (EAC, EDC) of DOM were quantified using dissolved H₂S and ferric iron as reactants. Carbon turnover rates were obtained from porewater profiles (CO₂, CH₄) and inverse modeling. Carbon dioxide was released at rates of 0.2–5.9 mmol m⁻² day⁻¹ below the water table. Methane (CH₄) formation contributed <10%, and oxygen consumption 2% to 40%, leaving a major fraction of CO₂ production unexplained. DOM oxidized H₂S to thiosulfate and was reduced by dissolved ferric iron. Reduction with H₂S increased the subsequently determined EDC compared to untreated controls, indicating a reversibility of the electron transfer. In situ redox capacities of DOM ranged from 0.2 to 6.1 mEq g⁻¹ C (EAC) and from 0.0 to 1.4 mEq g⁻¹ C (EDC), respectively. EAC generally decreased with depth and changed after a water table drawdown and rebound by 20 and −45 mEq m⁻², respectively. The change in EAC during the water table fluctuation was similar to CH₄ formation rates. In peatlands, electron transfer of DOM may thus significantly contribute to the oxidation of reduced organic substrates by anaerobic heterotrophic respiration, or by maintaining the respiratory activity of sulfate reducers via provision of thiosulfate. Part of the anaerobic electron flow in peat soils is thus potentially diverted from methanogenesis, decreasing its contribution to the total carbon emitted to the atmosphere.     

Ultrasensitive peroxynitrite-based luminescence with L-012 as a screening system for antioxidative/antinitrating substances, e.g. Tylenol (acetaminophen), 4-OH tempol, quercetin and carboxy-PTIO.

Previously our group developed a water-soluble antioxidant screening system using the luminescence of the reaction of peroxynitrite and luminol. In the present study we replaced luminol with the luminol-like compound L-012. This increases the production of luminescence approximately 100-fold and therefore, with a higher signal:noise ratio, this new system can detect antioxidation and antinitration effects at lower doses of the inhibitor. We studied acetaminophen (Tylenol) and its metabolite 3-nitroacetaminophen, tyrosine and nitrotyrosine and all these substances were inhibitory in a dose-responsive manner and below micromolar amounts. In addition quercetin, a polyphenol, was highly active (below micromolar amounts) as an antioxidant and antinitrating compound. 4-OH tempol, the stable free radical, superoxide dismutase (SOD) mimetic, was inhibitory in a dose-responsive manner and below micromolar amounts. Carboxy-PTIO was inhibitory at 10 times micromolar amount but not below that dose, which may be related to colour quenching, since the drug is deeply blue, or possibly it is an inhibitor with a slow kinetic profile. Finally, the amino acid tyrosine has been found to be inhibitory in micromolar amounts, similar to acetaminophen. This indicates that tyrosine can act as an antioxidant and antinitration target alone or conjugated in protein, e.g. insulin.     

Generating oxidation-resistant variants of <Emphasis Type="Italic">Bacillus kaustophilus</Emphasis> leucine aminopeptidase by substitution of the critical methionine residues with leucine

Bacillus kaustophilus leucine aminopeptidase (bkLAP) was sensitive to oxidative damage by hydrogen peroxide. To improve its oxidative stability, the oxidation-sensitive methionine residues in the enzyme were replaced with leucine by site-directed mutagenesis. The variants, each with an apparent molecular mass of approximately 54 kDa, were overexpressed in recombinant Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. The specific activity for M282L, M285L, M289L and M321L decreased by more than 43%, while M400L, M426L, M445L, and M485L showed 191, 79, 313, and 103%, respectively, higher activity than the wild-type enzyme. Although the mutations did not cause significant changes in the K _m value, more than 67.8% increase in the value of k _cat/K _m was observed in the M400L, M426L, M445L and M485L. In the presence of 50 mM H₂O₂, most variants were more stable with respect to the wild-type enzyme, indicating that the oxidative stability of the enzyme can be improved by engineering the methionine residues. This revised version was published online in June 2006 with corrections to the Cover Date.