EXAFS study on arsenic species and transformation in arsenic hyperaccumulator

Synchrotron radiation extended X-ray absorption fine structure (SR EXAFS) was employed to study the transformation of coordination environment and the redox speciation of arsenic in a newly discovered arsenic hyperaccumulator, Cretan brake (Pteris cretica L. var nervosa Thunb). It showed that the arsenic in the plant mainly coordinated with oxygen, except that some arsenic coordinated with S as As-GSH in root. The complexation of arsenic with GSH might not be the predominant detoxification mechanism in Cretan brake. Although some arsenic in root presented as As(V) in Na₂HAsO₄ treatments, most of arsenic in plant presented as As(III)-O in both treatments, indicating that As(V) tended to be reduced to As(III) after it was taken up into the root, and arsenic was kept as As(III) when it was transported to the above-ground tissues. The reduction of As(V) primarily proceeded in the root.     

Intracellular distribution and chemical forms of arsenic in rabbits exposed to arsenate

Inhibition of the methylation of arsenic in rabbits by ip injection of periodate-oxidized adenosine (PAD) prior to an iv injection of⁷⁴As-arsenate (AsV; 0.4 mg As/kg body wt) caused a marked increase in the retention of⁷⁴As in both the cellular organelles and the soluble fractions of liver and kidney. One day after exposure, almost 30% of the arsenic in the liver and about 40% of the arsenic in the kidney was recovered in the nuclear fraction. In the liver nuclei, the inhibition of the methylation increased the⁷⁴As content of the insoluble fraction and most of this arsenic was protein-bound. The major part of the soluble intranuclear⁷⁴As was in the form of AsIII, formed by reduction of the administered AsV. In the liver, PAD also caused a pronounced increase in the⁷⁴As content of the microsomal fraction. In the kidneys, where most of the arsenic was present as AsV, there was a marked accumulation of arsenic in the mitochondria.     

The degradation of arsenobetaine to inorganic arsenic by sedimentary microorganisms

Two growth media containing arsenobetaine [(CH₃)₃ As⁺ CH₂COO^–] were mixed with coastal marine sediments, the latter providing a source of microorganisms. The mixtures were kept at 25 °C in the dark and shaken for several weeks under an atmosphere of air. The disappearance of arsenobetaine and the appearance of two metabolites were followed by HPLC. The HPLC-retention time of the first metabolite agreed with that of trimethylarsine oxide [(CH₃)₃AsO]. The second metabolite was identified as arsenate (As(V)) using hydride generation/cold trap/GC MS analysis and thin layer chromatography. This is the first scientific evidence showing that arsenobetaine is degraded by microorganisms to inorganic arsenic via trimethylarsine oxide. The degradation of arsenobetaine to inorganic arsenic completes the marine arsenic cycle that begins with the methylation of inorganic arsenic on the way to arsenobetaine.     

Properties of rat 6-N-trimethyl-l-lysine hydroxylases: Similarities among the kidney,liver, heart,and skeletal muscle activities

Hydroxylation of 6-N-trimethyl-l-lysine(lys(Me₃)) to 3-hydroxy-6-N-trimethyl-l-lysine(3-HO-lys(Me₃)) by several rat tissues has been examined and compared. The kidney enzyme, which previously was shown to require molecular oxygen and α-ketoglutarate as cosubstrates, ferrous iron and ascorbate as cofactors, and to be stimulated by catalase, has a broad pH optimum ranging between 6.5 to 7.5 at 37 °C. As determined with crude tissue extracts from kidney, liver, heart, and skeletal muscle, similar apparent K_m values were obtained for substrate, cosubstrates, and cofactors. In view of similar kinetic parameters among the several lys(Me₃) hydroxylases examined in rat tissues, and the fact that the level of skeletal muscle lys(Me₃) hydroxylase activity is comparable to that of heart, liver, and kidney, because of its large total mass, skeletal muscle may contribute significantly to the biosynthesis of l-carnitine from lys(Me₃). The most effective inhibitors found, competitive with lys(Me₃), were 2-N-acetyl-6-N-trimethyl-l-lysine, 6-N-monomethyl-l-lysine, and 6-N-dimethyl-l-lysine. l-2-Amino-6-N-trimethylammonium-4-hexynoate, d-2-amino-6-N-trimethylammonium-4-hexynoate, and dl2-amino-6-N-trimethylammonium-cis-4-hexenoate, also inhibited hydroxylase activity but by a yet undetermined mechanism. Oxalacetate, succinate, and citrate inhibited the hydroxylation reaction by competing with α-ketoglutarate. The binding of ferrous iron to the enzyme was competitively inhibited by ions of “soft metals” (e.g., Cd²⁺, Zn²⁺) but not by those of “hard metals” (e.g., Ca²⁺, Mg²⁺). Preincubation of the crude kidney enzyme for 15 min at 37 °C with mercuriphenylsulfonate, N-ethylmaleimide, iodoacetate, or iodoacetamide resulted in considerable inhibition of 3-HO-lys(Me₃) formation. The degree of inhibition by N-ethylmaleimide could be reduced by including Zn (II) during preincubation of the enzyme. The effects of “soft” metals and sulfhydryl reagents on the enzyme suggest that sulfhydryl groups are required for ferrous iron binding in the active site.     

Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids

The reactions of copper(II)-ahphatic polyamine complexes with cysteine, cysteine methyl ester, penicillamine. and glutathione have been investigated, with the goal of understanding the relationship between RS^?-Cu(II) adduct structure and preferred redox decay pathway. Considerable mechanistic flexibility exists within this class of mercapto ammo acid oxidations, as changes in the rate law could be induced by modest variations in reductant concentration (at fixed [Cu(II)]_o), pH, and the structure of the redox partners. With excess cysteine present at 25°C, pH 5 0, I = 0 2 M (NaOAc), decay of 1:1 cys-S^?-Cu(II) transient adducts was found to be first order in both cys-SH and transient. Second-order rate constants characteristic of Cu(dien)²⁺ (6 1 × 10³M^?1sec^?1), Cu(Me₅dien)²⁺ (2.7 × 10³M^?1 sec^?1), Cu(en)₂²⁺ (2.1 × 10³M^?1 sec^?1), and Cu(dien)₂²⁺ (4.7 × 10³ M^?1 sec ^?1) are remarkably similar, considering substantial differences in the composition and geometry of the oxidant first coordination sphere. A mechanism involving attack of cysteine on the coordinated sulfur atom of the transient, giving a disulfide anion radical intermediate, is proposed to account for these results Moderate reactivity decreases in the cysteine-Cu(dien)²⁺, Cu(Me₅dien)²⁺ reactions with increasing [H⁺] (pH 4–6) reflect partial protonation of the polyamine ligands. A very different rate law, second order in the RS^?-Cu(II) transient and approximately zeroth order in mercaptan, applies in the pH 5.0 oxidations of cysteine methyl ester, penicillamine, and glutathione by Cu(dien)²⁺ and Cu(Me₅dien)²⁺. This behavior suggests the mtermediacy of di-μ-mercapto-bridged binuclear Cu(II) species, in which a concerted two-electron change yields the disulfide and Cu(I) products. Similar hydroxo-bridged intermediates are proposed to account for the transition from first- to second-order transient dependence in cysteine oxidations by Cu(dien)²⁺ and Cu(Me₅dien)²⁺ as the pH is increased from 5 to 7. Yet another rate law, second order in transient and first order in cysteine, applies in the pH 5.0 oxidation of cysteine by Cu(Me₆tren)²⁺ (k(25°C) 7.5 × 10⁷ M^?2 sec^?1, I = 0.2 M). Steric rigidity of this trigonal bipyramidal oxidant evidently protects the coordinated sulfur atom from attack in a RSSR^?-forming pathway. Formation of a coordinated disulfide in the rate-determining step is purposed, coupled with attack of a noncoordinated cysteine molecule on a vacated coordination position to stabilize the (Me₆(tren)Cu(I) product.     

Measurement of bacterial sulfate reduction in sediments: Evaluation of a single-step chromium reduction method

A procedure which includes the Total Reduced Inorganic Sulfur (TRIS) in a single distillation step is described for the radiotracer measurement of sulfate reduction in sediments. The TRIS includes both Acid Volatile Sulfide (AVS: H₂S + FeS) and the remaining Chromium Reducible Sulfur (CRS: S⁰, FeS₂). The single-step distillation was simpler and faster than the consecutive distillations of AVS and CRS. It also resulted in higher (4–50%) sulfate reduction rates than those obtained from the sum of³⁵S in AVS and CRS. The difference was largest when the sediment had been dried after AVS but before CRS distillation. Relative to the³⁵S-AVS distillation alone, the³⁵S-TRIS single-step distallation yielded 8–87% higher reduction rates. The separation and recovery of FeS, S⁰ and FeS₂ was studied under three distillation conditions: 1) cold acid, 2) cold acid with Cr²⁺, and 3) hot acid with Cr²⁺. The FeS was recovered by cold acid alone while pyrite was recovered by cold acid with Cr²⁺. A smaller S⁰ fraction, presumably of the finer crystal sizes, was recovered also in the cold acid with Cr²⁺ while most of the S⁰ required hot acid with Cr²⁺ for reduction to H₂S.     

Radical ions derived from hydrides and methyls of aluminum,silicon and phosphorus: A semi-empirical SCFMO study

Semi-empirical SCFMO calculations were made of the energies, and geometric and electronics structures of a range of radical ions of type MR₃^± and M₂R₆^± where M = Al, Si or P, and R = H or CH₃. In each of the MH₃ radicals, methylation effects an increase in the HMH angle: the structure of Al₂Me₆^?, formed by γ-irradiation of Al₂Me₆, is found to have C_s symmetry and to resemble a weak complex of AlMe₂ and AlMe₄^?. Possible identities for the radical, other than AlH₃^?, formed on γ-irradiation of LiAlH₄ are suggested, and a considerable number of plausible identities are firmly ruled out.     

The Regulation of the Plasma Membrane Redox System and H+-transport in Adaptation of Reed Ecotypes to Their Habitats

The redox system and H⁺-transport activities in the plasma membranes from two ecotypes of reed (Phragmites communis Trin.), named swamp reed (SR) and dune reed (DR) according to their habitats, were investigated. Compared to the SR, the DR possessed the very high rates of NADH oxidation and Fe(CN)₆ ^3– and EDTA-Fe³⁺ reduction when NADH was taken as the electron donor. As NADPH was an electron donor, the rate of NADPH oxidation was also significantly higher in the DR than that in the SR. In addition, the H⁺-transport activity in the plasma membranes was also significantly higher in the DR than in the SR.     

Rapid Equilibrium Kinetic Analysis of Arsenite Methylation Catalyzed by Recombinant Human Arsenic (+3 Oxidation State) Methyltransferase (hAS3MT)

In the human body, arsenic is metabolized by methylation. Understanding this process is important and provides insight into the relationship between arsenic and its related diseases. We used the rapid equilibrium kinetic model to study the reaction sequence of arsenite methylation. The results suggest that the mechanism for arsenite methylation is a completely ordered mechanism that is also of general interest in reaction systems with different reductants, such as tris(2-carboxyethyl)phosphine, cysteine, and glutathione. In the reaction, cysteine residues of recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) coordinate with arsenicals and involve the methyl transfer step. S-Adenosyl-l-methionine (AdoMet) is the first-order reactant, which modulates the conformation of hAS3MT to a best matched state by hydrophobic interaction. As the second-order reactant, reductant reduces the disulfide bond, most likely between Cys-250 and another cysteine residue of hAS3MT, and exposes the active site cysteine residues for binding trivalent inorganic arsenic (iAs³⁺) to give monomethylarsonic dicysteine (MADC³⁺). In addition, the reaction can be extended to further methylate MADC³⁺ to dimethylarsinic cysteine (DAMC³⁺). In the methylation reaction, the β-pleated sheet content of hAS3MT is increased, and the hydrophobicity of the microenvironment around the active sites is decreased. Similarly, we confirm that both the high β-pleated sheet content of hAS3MT and the high dissociation ability of the enzyme-AdoMet-reductant improve the yield of dimethylated arsenicals.     

Biogeochemical transformations of arsenic in circumneutral freshwater sediments

Arsenic is a wide-spread contaminant of soils and sediments, andmany watersheds worldwide regularly experience severe arsenic loading. While the toxicityof arsenic to plants and animals is well recognized, the geochemical and biological transformationsthat alter its bioavailability in the environment are multifaceted and remain poorly understood.This communication provides a brief overview of our current understanding of the biogeochemistryof arsenic in circumneutral freshwater sediments, placing special emphasis on microbialtransformations. Arsenic can reside in a number of oxidation states and complex ions. The commoninorganic aqueous species at circumneutral pH are the negatively charged arsenates(H₂As^VO₄ ^- and Has^VO₄ ^2-) and zero-charged arsenite(H₃As^IIIO₃ ⁰). Arsenic undergoes diagenesis in response to both physicaland biogeochemical processes. It accumulates in oxic sediments by adsorption on and/orco-precipitation with hydrous iron and manganese oxides. Burial of such sediments in anoxic/suboxicenvironments favors their reduction, releasing Fe(II), Mn(II) and associatedadsorbed/coprecipitated As. Upward advection can translocate these cations and As into theoverlying oxic zone where they may reprecipitate. Alternatively, As may be repartitioned tothe sulfidic phase, forming precipitates such as arsenopyrite and orpiment. Soluble and adsorbedAs species undergo biotic transformations. As(V) can serve as the terminal electronacceptor in the biological oxidation of organic matter, and the limited number of microbes capableof this transformations are diverse in their phylogeny and physiology. Fe(III)-respiring bacteriacan mobilize both As(V) and As(III) bound to ferric oxides by the reductive dissolution ofiron-arsenate minerals. SO₄ ^2--reducing bacteria canpromote deposition of As(III) as sulfide minerals via their production of sulfide. A limited number of As(III)-oxidizing bacteriahave been identified, some of which couple this reaction to growth. Lastly, prokaryotic andeukaryotic microbes can alter arsenic toxicity either by coupling cellular export to its reductionor by converting inorganic As to organo-arsenical compounds. The degree to which each ofthese metabolic transformations influences As mobilization or sequestration in differentsedimentary matrices remains to be established.     

Comparison of Indolidan Analog Binding Sites of Drug Antibody and Sarcoplasmic Reticulum with Inhibition of Cyclic AMP Phosphodiesterase

Abstract

Dihydropyridazinone(DHP) derivatives such as indolidan are positive inotropic agents that show inhibition of cyclic AMP phosphodiesterase(PDE) activity. Indolidan inhibition is selective for PDE3 among the seven PDE gene families. DHP derivatives and related analogs have been used to define critical regions of the active site of PDE3 isoforms and radiolabeled analogs have been used to define indolidan sarcoplasmic reticulum (SR) receptor sites. We report here studies comparing the structure-activity relationships (SAR) for PDE3 inhibition with indolidan binding to two types of sites: canine SR and a monoclonal antibody derived against indolidan conjugated to a hemocyanin. SR and monoclonal antibody binding both fit single-site, high affinity models (IC₅₀ = 1.2 and 62 nM) that were near 52 and 360 times that of SR PDE3. Indolidan and thirteen analogs showed similar competition with either SR ³H-LY186126 binding or SR PDE3 inhibition. Antibody binding maintained selectivity but showed a different rank order potency for SR binding. Indole ring C3 methylation increased and DHP ring C4′ methylation decreased indolidan monoclonal antibody binding while both substitutions increased SR binding. These studies support the hypothesis that SR PDE3 is a cardiotonic receptor site in myocardial membranes and indicate that models of the structural features of binding sites derived from inhibitor data alone could produce models with limited topography relative to the natural ligand.     

The Functions of Crucial Cysteine Residues in the Arsenite Methylation Catalyzed by Recombinant Human Arsenic (III) Methyltransferase

Arsenic (III) methyltransferase (AS3MT) is a cysteine (Cys)-rich enzyme that catalyzes the biomethylation of arsenic. To investigate how these crucial Cys residues promote catalysis, we used matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to analyze Cys residues in recombinant human arsenic (III) methyltransferase (hAS3MT). We detected two disulfide bonds, Cys250-Cys32 and Cys368-Cys369, in hAS3MT. The Cys250-Cys32 disulfide bond was reduced by glutathione (GSH) or other disulfide bond reductants before the enzymatic methylation of arsenite (iAs³⁺). In addition to exposing residues around the active sites, cleavage of the Cys250-Cys32 pair modulated the conformation of hAS3MT. This adjustment may stabilize the binding of S-Adenosyl-L-methionine (AdoMet) and favor iAs³⁺ binding to hAS3MT. Additionally, we observed the intermediate of Cys250-S-adenosylhomocysteine (AdoHcy), suggesting that Cys250 is involved in the transmethylation. In recovery experiments, we confirmed that trivalent arsenicals were substrates for hAS3MT, methylation of arsenic occurred on the enzyme, and an intramolecular disulfide bond might be formed after iAs³⁺ was methylated to dimethylarsinous acid (DMA³⁺). In this work, we clarified both the functional roles of GSH and the crucial Cys residues in iAs³⁺ methylation catalyzed by hAS3MT.     

Characterization of polycyclic aromatic hydrocarbons degradation and arsenate reduction by a versatile Pseudomonas isolate

A Pseudomonas isolate, designated PAHAs-1, was found capable of reducing arsenate and degrading polycyclic aromatic hydrocarbons (PAHs) independently and simultaneously. This isolate completely reduced 1.5 mM arsenate within 48 h and removed approximately 100% and 50% of 60 mg l⁻¹ phenanthrene and 20 mg l⁻¹ pyrene within 60 h, respectively. Using PAHs as the sole carbon source, however, this isolate showed a slow arsenate reduction rate (4.62 μM h⁻¹). The presence of arsenic affected cell growth and concurrent PAHs removal, depending on PAH species and arsenic concentration. Adding sodium lactate to the medium greatly enhanced the arsenate reduction and pyrene metabolism. The presence of the alpha subunit of the aromatic ring-hydroxylating dioxygenase (ARHD) gene, arsenate reductase (arsC) and arsenite transporter (ACR3(2)) genes supported the dual function of the isolate. The finding of latter two genes indicated that PAHAs-1 possibly reduced arsenate via the known detoxification mechanism. Preliminary data from hydroponic experiment showed that PAHAs-1 degraded the majority of phenanthrene (>60%) and enhanced arsenic uptake by Pteris vittata L. (from 246.7 to 1187.4 mg kg⁻¹ As in the fronds). The versatile isolate PAHAs-1 may have potentials in improving the bioremediation of PAHs and arsenic co-contamination using the plant-microbe integrated strategy.     

Volatile Flavor Components of ZINGIBERIS RHIZOMA (Zingiber officinale Roscoe)

The toxicity of dimethyl sulfoxide (Me₂SO) was examined in HeLa cells cultured at 37°C for up to 72 hr. The growth of the cells was measured by a colorimetric method with the use of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which gave good correlation between the cell number and the color development from the reduction of MTT under suitable conditions. When the initial number of cells was 3 × 10⁴/ml, Me₂SO at 1% or less had no apparent effect on prolifiration for up to 48 hr of incubation, but in longer incubations, cell growth was repressed. When the initial number of cells was 3 × 10⁵/ml, the effect of Me₂SO was similar.     

8-methyl-2'-deoxyguanosine incorporation into parallel DNA quadruplex structures

This paper concerns the Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) structural studies of the quadruple helix arrangements adopted by three tailored oligodeoxyribonucleotide analogues, namely d(TG^MeGGT), d(TGG^MeGT) and d(TGGG^MeT), where dG^Me represents a 8-methyl-2′-deoxyguanosine residue. The results of this study clearly demonstrate that the effects of the incorporation of dG^Me instead of a dG residue are strongly dependant upon the positioning of a single base replacement along the sequence. As such, d(TG^MeGGT), d(TGG^MeGT) have been found to form 4-fold symmetric quadruplexes with all strands parallel and equivalent to each other, each more stable than their natural counterpart. NMR experiments clearly indicate that [d(TG^MeGGT)]₄ possesses a G^Me-tetrad with all dG^Me residues in a syn-glycosidic conformation while an anti-arrangement is apparent for the four dG^Me of [d(TGG^MeGT)]₄. As the two complexes show a quite different CD behaviour, a possible relationship between the presence of residues adopting syn-glycosidic conformations and CD profiles is briefly discussed. As far as d(TGGG^MeT) is concerned, NMR data indicate that at 25°C it exists primarily as a single-strand conformation in equilibrium with minor amounts of a quadruplex structure.     

Biological Responses to Arsenic Compounds

Arsenic is a metalloid that generates various biological effects on cells and tissues. Depending on the specific tissue exposed and the time and degree of exposure, diverse responses can be observed. In humans, prolonged and/or high dose exposure to arsenic can have a variety of outcomes, including the development of malignancies, severe gastrointestinal toxicities, diabetes, cardiac arrhythmias, and death. On the other hand, one arsenic derivative, arsenic trioxide (As₂O₃), has important antitumor properties. This agent is a potent inducer of antileukemic responses, and it is now approved by the Food and Drug Administration for the treatment of acute promyelocytic leukemia in humans. The promise and therapeutic potential of arsenic and its various derivatives have been exploited for hundreds of years. Remarkably, research focused on the potential use of arsenic compounds in the treatment of human diseases remains highly promising, and it is an area of active investigation. An emerging approach of interest and therapeutic potential involves efforts to target and block cellular pathways activated in a negative feedback manner during treatment of cells with As₂O₃. Such an approach may ultimately provide the means to selectively enhance the suppressive effects of this agent on malignant cells and render normally resistant tumors sensitive to its antineoplastic properties.Arsenic forms complexes with other elements, and it exists in inorganic and organic forms (1–3). The three major inorganic forms of arsenic are arsenic trisulfide (As₂S₃, yellow arsenic), arsenic disulfide (As₂S₂, red arsenic), and arsenic trioxide (As₂O₃, white arsenic) (1–3). There are two different oxidative states of arsenic that correlate with its cytotoxic potential, As(III) and As(V). Among them, As(III) is the most potent form and primarily accounts for its pro-apoptotic and inhibitory effects on target cells and tissues (3). The various forms of arsenic exist in nature primarily in a complex with pyrite (4, 5), although under certain circumstances, arsenic can dissociate from soil and enter natural waters (6), providing a contamination source for humans or animals who ingest such waters. In fact, most associations between long term exposure to arsenic and development of malignancies or other health disorders result from drinking contaminated water, especially in developing countries. Interestingly, pollution of the air with arsenic can also occur under certain circumstances, such as in the case of emissions from coal burning in China (7), providing an additional source of human exposure.The metabolism of arsenic in humans includes reduction to the trivalent state and oxidative methylation to the pentavalent state (reviewed in Ref. 2). There is also reduction of arsenic acid to the arsenous form and subsequent methylation (2). The generation of inorganic or organic trivalent arsenic forms has important implications with regard to the toxicity of this agent, as such compounds are more toxic to the cells and exhibit more carcinogenic properties (2, 3). Thus, many of the consequences of exposure to arsenic as discussed below are the result of the activities and toxicities of the various metabolic products of arsenic compounds. It should be also noted that arsenic has the ability to bind to reduced thiols, including sulfhydryl groups in some proteins (2). Depending on the cellular context, such protein targeting may explain some of its cellular effects and generation of its toxicities and/or therapeutic effects.     

The effect of inoculation and the type of carrier material used on the biofiltration of methyl sulphides

 Low elimination capacities (less than 10 g m^-3 day^-1) were observed for the odorant dimethyl sulphide (Me₂S) when either wood bark or compost was used as the carrier material in a laboratory-scale biofilter. Enrichment experiments were set up by incubation of garden soil samples during 4 weeks with 100 ppm (v/v) headspace concentrations of both Me₂S and dimethyl disulphide (Me₂S₂). After transfer to a mineral medium, Me₂S- and Me₂S₂-degrading enrichment cultures were obtained for all five soil samples tested, both compounds being converted stoichiometrically to sulphuric acid. Upon inoculation of the laboratory-scale biofilter with one of these enrichment cultures (±120 g cell dry weight m^-3 reactor), the elimination capacity for Me₂S increased in a 3-week period to 35 g m^-3 day^-1 and 680 g m^-3 day^-1 when wood bark and compost were used as the respective carrier materials. Both inoculated biofilters were able to degrade Me₂S₂, however the elimination capacities obtained for Me₂S₂ were lower (e.g. 24 g m^-3 day^-1 for the wood bark filter) compared to those for Me₂S. For both inoculated biofilters, a gradual decrease of the elimination capacity for the methyl sulphides was observed as a result of acidification of the carrier material, suggesting that pH regulation is necessary if long-term biofiltration experiments are to be performed. Received: 6 June 1995/Received revision: 10 August 1995/Accepted: 22 August 1995     

Mutations in human lipoyltransferase gene <Emphasis Type="Italic">LIPT1</Emphasis>cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase

Background

Synthesis and apoenzyme attachment of lipoic acid have emerged as a new complex metabolic pathway. Mutations in several genes involved in the lipoic acid de novo pathway have recently been described (i.e., LIAS, NFU1, BOLA3, IBA57), but no mutation was found so far in genes involved in the specific process of attachment of lipoic acid to apoenzymes pyruvate dehydrogenase (PDHc), α-ketoglutarate dehydrogenase (α-KGDHc) and branched chain α-keto acid dehydrogenase (BCKDHc) complexes.

Methods

Exome capture was performed in a boy who developed Leigh disease following a gastroenteritis and had combined PDH and α-KGDH deficiency with a unique amino acid profile that partly ressembled E3 subunit (dihydrolipoamide dehydrogenase / DLD) deficiency. Functional studies on patient fibroblasts were performed. Lipoic acid administration was tested on the LIPT1 ortholog lip3 deletion strain yeast and on patient fibroblasts.

Results

Exome sequencing identified two heterozygous mutations (c.875C?>?G and c.535A?>?G) in the LIPT1 gene that encodes a mitochondrial lipoyltransferase which is thought to catalyze the attachment of lipoic acid on PDHc, α-KGDHc, and BCKDHc. Anti-lipoic acid antibodies revealed absent expression of PDH E2, BCKDH E2 and α-KGDH E2 subunits. Accordingly, the production of ¹⁴CO₂ by patient fibroblasts after incubation with ^14Cglucose, ^14Cbutyrate or ^14C3OHbutyrate was very low compared to controls. cDNA transfection experiments on patient fibroblasts rescued PDH and α-KGDH activities and normalized the levels of pyruvate and 3OHbutyrate in cell supernatants. The yeast lip3 deletion strain showed improved growth on ethanol medium after lipoic acid supplementation and incubation of the patient fibroblasts with lipoic acid decreased lactate level in cell supernatants.

Conclusion

We report here a putative case of impaired free or H protein-derived lipoic acid attachment due to LIPT1 mutations as a cause of PDH and α-KGDH deficiencies. Our study calls for renewed efforts to understand the mechanisms of pathology of lipoic acid-related defects and their heterogeneous biochemical expression, in order to devise efficient diagnostic procedures and possible therapies.

     

Sulfate reduction and trichloroethylene biodegradation by a marine microbial community from hydrothermal vents sediments

Sulfate reduction (SR) and trichloroethylene (TCE) biodegradation at two different temperatures (37 and 70 °C) were investigated in enrichment cultures prepared with two different samples of sediments collected from hydrothermal vents. The unadapted sediments were incubated with sulfate (4 g L⁻¹) as the electron acceptor before TCE addition to enrich them in biomass and to establish a constant sulfate reduction (SR, 87% sulfate conversion and specific H₂S concentration of 90.81 ± 8.19 mg H₂S g VSS⁻¹), afterwards TCE was added at an initial concentration of 300 ??mol L⁻¹. The best results for TCE biodegradation were obtained at 37 °C. At this temperature, SR was up to 92%, whereas TCE biodegradation reached 75% and ethane was detected as the main degradation product. Under thermophilic conditions (70 °C) TCE biodegradation reached up to approximately 60% and the SR was 30% in 30 days of incubation with the chlorinated solvent. Along with these results, the 16S rDNA analysis from samples at 37 °C showed the presence of bacteria belonging to the genera: Clostridium, Bacillus and Desulfuromonas. The overall results on TCE degradation and SR suggest that cometabolic TCE degradation is carried out by sulfate or sulfur reducers and fermentative bacteria at mesophilic conditions.     

Hg2+-stimulated NADH-oxidase activity of Ascaris muscle microsomal lipoamide dehydrogenase.

In the presence of Hg²⁺$\text{Ascaris}$ lipoamide dehydrogenase stimulated the reduction of oxygen, ferricyanide, and 2,6-dichlorophenolindophenol with NADH, which was inhibited by lipoic acid. On the other hand, Cu²⁺ stimulated the reduction of the artificial dyes, but only a little the reduction of oxygen. Hg²⁺ changed the visible absorption spectrum of the lipoamide dehydrogenase, but did not change the fluorescence curve. Lipoic acid decreased the fluorescence, but did not change the visible absorption spectrum. The $\text{Ascaris}$ lipoamide dehydrogenase have two SH groups per one subunit and 5–6 moles of HgCl₂ and 3–4 moles of CuSO₄ per one subunit were required for the maximal activity.