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Lactoperoxidase, a dithionite ion dismutase.

The dithionite ion is catalytically disproportionated by lactoperoxidase with Km = 0.36 mM in 100 mM glycine HCl pH 3.0. The products formed are thiosulfate and hydrogensulfite ions. The rate of reaction is considerably increased at low pH with a pKa at 3-3.5 possibly indicating the involvement of a carboxyl group. The reaction is competitively inhibited by hydrogensulfite, Ki = 5.5 mM in 100 mM glycine HCl pH 3.50. Four different spectral forms of reduced lactoperoxidase appear during the reaction. The first two forms are found during the lag phase of the reaction. The third form, which is interpreted as a ternary complex, exists under the dismutation phase. After exhaustion of the substrate a visible spectrum similar to that of lactoperoxidase H2O2 compound III appears. A mechanistic model for the lactoperoxidase dismutation of the dithionite ion is proposed and discussed.

Animals↗

Enzymic synthesis of the iron-sulfur cluster of spinach ferredoxin.

A biologically active spinach ferredoxin was reconstituted from the apoprotein by incubation with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, reduced lipoate and ferric ammonium citrate. Analytical and spectroscopical features of the reconstituted ferredoxin were identical to those of the native one; yield of the reconstitution reaction was 80%. Yields and kinetic parameters of the enzymic and chemical reconstitution were also compared. The higher efficiency of the enzymic system is ascribed to a productive interaction between rhodanese and apoferredoxin favouring the process of cluster build-up and insertion. The physiological relevance of this synthetic activity is discussed.

Animals↗

Enzymic synthesis of the 4Fe-4S clusters of Clostridium pasteurianum ferredoxin.

Ex novo enzymic synthesis of the two 4Fe-4S clusters of Clostridium pasteurianum ferredoxin has been achieved by incubation of the apoprotein with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, DL-dihydrolipoate and ferric ammonium citrate. This enzymic reconstitution procedure was compared to a chemical one, in which the enzyme was replaced by sodium sulfide. A further comparison was made with the results previously obtained in the enzymic synthesis of the 2Fe-2S cluster of spinach ferredoxin, allowing the following conclusions to be drawn. The nature of the cluster to be inserted into the reconstituted iron-sulfur protein is determined by the apoprotein itself. The refolding of the structure of the iron-sulfur proteins around the newly inserted cluster is the rate-limiting step in both chemical and enzymic reconstitution. Rhodanese appears to play a role in the recovery of the native architecture of the reconstituted iron-sulfur protein(s). The extension to the 4Fe-4S centers of the rhodanese-based biosynthetic system allows this enzymic route to be proposed as a general way to the in vivo synthesis of iron-sulfur structures.

Apoproteins↗

Mitochondrial sulfide oxidation in Arenicola marina. Evidence for alternative electron pathways.

Sulfide is oxidized in the mitochondria of the lugworm Arenicola marina. Mitochondrial sulfide oxidation is coupled with oxygen consumption and with an equimolar production of thiosulfate [Völkel, S. & Grieshaber, M. K. (1994) Mar. Biol. 118, 137-147]. Mitochondrial respiration in the presence of malate (or succinate) and ADP but without sulfide could be completely inhibited by rotenone, antimycin, cyanide, and sulfide. Only 40% inhibition was achieved by salicylhydroxamic acid. Sulfide oxidation (with sulfide as the only substrate) was fully inhibited by antimycin and by salicylhydroxamic acid but not by rotenone or sulfide. Moreover, sulfide oxidation was 3-4-fold less sensitive to cyanide as compared to normal respiration. The data indicate that sulfide oxidation in A. marina is linked to the respiratory electron transport chain. We suggest that electrons from sulfide enter the respiratory chain via ubiquinone or at the ubiquinol-cytochrome-c oxidoreductase. At sulfide concentrations higher than 10 microM, the cytochrome-c oxidase is blocked and electrons from sulfide are transferred to oxygen via an alternative terminal oxidase.

Animals↗

AF64A: an active site directed irreversible inhibitor of choline acetyltransferase.

Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuron-specific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma X glioma cell line, NG-108-15, with 100 microM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60-85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration- and time-dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl-coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64A's capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.

Animals↗

Oral lichenoid lesions, mercury hypersensitivity and combined hypersensitivity to mercury and other metals: histologically-proven reproduction of the reaction by patch testing with metal salts.

We report 11 patients seen between 1991 and 1994 with oral lichenoid lesions (OLL). In 10 cases, there was contact with dental amalgam fillings, and in patient no. 10 with both amalgam restorations and a gold crown. The last patient had, in addition to her OLL, lichen planus of the skin and genital mucosa. In 5 cases, combined sensitization to mercury and other metal salts, particularly gold sodium thiosulfate (GST) and palladium chloride (PDC), was observed. In 10 patients, the lesions considerably improved or totally cleared within 1 to 9 months of replacement of restoration materials. Histological examination of biopsies from the test sites of amalgam, mercuric chloride, GST and PDC, taken 10 or 17 days after application of patch tests, showed lichenoid changes in 7 patients with at least 1 of the allergens. As at least 2 patients had inflammatory lesions of the oral mucosa related to both amalgam and gold restorations, combined sensitization to inorganic and organic mercury derivatives, GST and, in 1 case, PDC, a "dental restoration metal intolerance syndrome" is proposed.

Adult↗

Prevalence of gold contact hypersensitivity in the west of Scotland.

373 patients attending for routine patch testing were tested with 0.5% and 0.05% gold sodium thiosulfate (GST). 8 (2.1%) patients had a positive patch test, and a further 4 (1.0%) patient reactions which were interpreted as irritant 2 out of 8 patients with a positive patch test to GST suspected gold allergy prior to testing, and both of these patients reported that their eczema resolved if they avoided gold jewellery. This is the lowest prevalence of hypersensitivity to GST reported and suggests that gold contact allergy may not be as widespread as has been recently proposed.

Adolescent↗

A controlled study of gold contact hypersensitivity.

1203 patients attending for routine patch testing at 3 hospitals and 105 volunteers were tested with 0.5% and 0.05% gold sodium thiosulfate (GST). 38 patients (3.2%) and 5 volunteers (4.8%) had positive patch tests to GST. There were no significant differences between volunteers and patients with respect to age, sex, atopy or exposure to gold in dental restorations, jewellery or through occupation. There were no significant differences in prevalence of GST hypersensitivity in the 3 hospitals, or between patients and controls. This is the 1st controlled study of hypersensitivity to GST, and suggests that routine patch testing to gold is of limited clinical benefit.

Adolescent↗

Laser Doppler perfusion imaging for the documentation of flare-up in contact allergy to gold.

A healed or healing allergic contact dermatitis is easily reactivated to a flare-up if the patient is provoked systemically with his particular allergen. 5 patients with contact allergy to gold were patch tested with a serial dilution of gold sodium thiosulfate. 1 to 2 months later, when positive test reactions were healed, the patients were given 1 intramuscular injection of gold sodium thiomalate. This resulted in a flare-up of patch tests as well as a transient fever and toxicoderma-like reactions. The flaring tests were registered by digital photographs and laser Doppler imaging (LDI). An increased cutaneous blood flow in the formerly positive patch tests was discernible within 1 h after provocation, increased to a vigorous maximum after 4-6 h, and subsided during the following few days. The earliest increase and the highest peak of blood flow were observed in the test areas of the strongest original test concentration. LDI provides a most suitable technique for studying the dynamics of this rapidly developing inflammation of a mainly dermal character.

Adult↗

In vitro lymphocyte reactivity to gold compounds in the diagnosis of contact hypersensitivity.

The use of the lymphocyte transformation test (LTT) in the diagnosis of contact hypersensitivity to gold was studied in 8 patients who had positive patch tests to gold salts, and in 8 control subjects who were negative to such patch tests. Gold sodium thiosulfate and gold chloride were added to cultures of lymphocytes, which were labeled by 3H-thymidine after 96 h. The lymphocyte stimulation index was calculated as the beta-counts in stimulated cultures divided by those in control cultures. The index was statistically significantly higher for the patient group (p=0.005-0.04) than for the control group. Levels of interferon-gamma (IFN-gamma) were determined for the supernatants of the lymphocyte cultures. An index IFN-gamma, which is defined as the level of IFN-gamma in stimulated cultures divided by that in control cultures, was statistically significantly higher for the patient group (p=0.01-0.006). The LTT stimulation index showed specificity and sensitivity between 67 and 80%, the respective values for Index IFN-gamma being between 73 and 100% when the patch test was used as a reference method. Evaluation of lymphocyte reactivity might be of future interest in the diagnosis of allergic reactions to gold if the sensitivity and specificity can be improved.

Adult↗

Calciphylaxis: no therapeutic concepts for a poorly understood syndrome?

Calciphylaxis is a very uncommon and severe disease which mainly appears in patients with chronic renal insufficiency. It presents with ischemia and necrosis of the skin, subcutaneous adipose tissue, muscles and rarely viscera. The pathogenetic mechanisms inducing calciphylaxis are for the most part unknown. The mortality rate of 80% in the first year is very high. Patients experience marked pain, recurrent infections and the constant risk of secondary sepsis. Even multidisciplinary therapeutic strategies are limited, although there are recent case reports providing promising new therapeutic options including sodium thiosulfate and cinacalcet. This review summarizes the important aspects of diagnosis, pathogenesis, prevention and the possible therapeutic strategies of this intriguing, rare and often fatal disease.

Age Factors↗

Sex-specific ethylene responses drive floral sexual plasticity in Cannabis sativa.

Cannabis sativa L. exhibits pronounced sexual plasticity in which both XX and XY plants can undergo floral phenotypic sex reversal in response to ethylene modulation, yet the underlying molecular mechanisms remain poorly defined. Here, we present the most extensive multi-omic analysis of ethylene-induced sex change in C. sativa to date, integrating over 130 RNA-seq libraries, ethylene pathway metabolite quantification, and whole-genome sequencing across three XX and XY genotypes. Treatments with silver thiosulfate and ethephon induced more than 80% phenotypic conversion, but transcriptomic responses diverged sharply between XX and XY plants. Profiling 47 ERGs revealed 14 high-confidence candidates, including CsACS1, CsACO5, CsERF1, and CsMTN, with sex-specific and temporal expression patterns that show dynamic ethylene mediation of plasticity. Early transcriptional activation occurred prior to the emergence of flowers, within 18 h of sex-change treatments and the photoperiod-induced transition to flowering. As opposite-sex floral tissues emerged, ethylene-related gene expression shifted accordingly within developing floral organs, with distinct sets of genes stabilizing the opposite-sex phenotype in XX and XY plants. Several candidates were located in non-recombining regions of the X chromosome or were absent from the Y chromosome, and most exhibited low nucleotide diversity, consistent with functional constraint. These results provide a high-resolution view of ethylene-responsive sexual plasticity in cannabis and show that the shared capacity for sex reversal in XX and XY plants is implemented through distinct regulatory trajectories that produce opposite-sex floral phenotypes. This work expands the mechanistic understanding of sex expression in dioecious species and identifies candidate genes relevant to the development of sex-stable cultivars.

Ethylenes↗

Sulfite oxidase deficiency in man: demonstration of the enzymatic defect.

An infant who died with neurological abnormalities, mental retardation, and dislocated ocular lenses excreted in his urine abnormally large amounts of S-sulfo-L-cysteine, sulfite, and thiosulfate and virtulally no inorganic sutlfate. The present report establishes the occurrence of an ezymatic defect in this infant. His liver, brain, and kidney specifically lacked sulfite oxidase activity. Deficiency of sulfite oxidase, which has not apparently been described in man, provides a reasonable explanation for the abnormalities in this infant.

Brain↗

Involvement of Pseudomonas aeruginosa rhodanese in protection from cyanide toxicity.

Cyanide is a serious environmental pollutant and a biocontrol metabolite in plant growth-promoting Pseudomonas species. Here we report on the presence of multiple sulfurtransferases in the cyanogenic bacterium Pseudomonas aeruginosa PAO1 and investigate in detail RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) which converts cyanide to less toxic thiocyanate. RhdA is a cytoplasmic enzyme acting as the principal rhodanese in P. aeruginosa. The rhdA gene forms a transcriptional unit with the PA4955 and psd genes and is controlled by two promoters located upstream of PA4955 and rhdA. Both promoters direct constitutive RhdA expression and show similar patterns of activity, involving moderate down-regulation at the stationary phase or in the presence of exogenous cyanide. We previously observed that RhdA overproduction protects Escherichia coli against cyanide toxicity, and here we show that physiological RhdA levels contribute to P. aeruginosa survival under cyanogenic conditions. The growth of a DeltarhdA mutant is impaired under cyanogenic conditions and fully restored upon complementation with rhdA. Wild-type P. aeruginosa outcompetes the DeltarhdA mutant in cyanogenic coculture assays. Hence, RhdA could be regarded as an effector of P. aeruginosa intrinsic resistance to cyanide, insofar as it provides the bacterium with a defense mechanism against endogenous cyanide toxicity, in addition to cyanide-resistant respiration.

Amino Acid Sequence↗

Carbon dioxide fixation and mixotrophic metabolism by strain DCB-1, a dehalogenating anaerobic bacterium.

Fixation by strain DCB-1 of CO2 carbon into cell material and organic acids occurred during growth on pyruvate both with and without thiosulfate. By using sodium [14C]bicarbonate and sodium [2-14C]pyruvate, the isotopic composition of products and cells was investigated. Up to 70% of cell carbon was derived from CO2. CO2 carbon was also incorporated into succinate, formate, and acetate. Both carbons of acetate underwent exchange reactions with CO2, although the carboxyl-group exchange was twice as fast. Because strain DCB-1 uses CO2 as its major but not sole carbon source while deriving energy from pyruvate metabolism, we describe its metabolism as mixotrophic. Other mixotrophic conditions also supported growth. Lactate or butyrate, which could not support growth in mineral medium, could replace pyruvate as the oxidizable substrate only when acetate was added to the medium.

Bicarbonates↗

Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU.

Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.

Acetates↗

Impact of different in vitro electron donor/acceptor conditions on potential chemolithoautotrophic communities from marine pelagic redoxclines.

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO2 dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO2 dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO2 dark fixation rates, ranging from 0.7 to 1.4 micromol liter(-1) day(-1), were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO2 dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 micromol liter(-1) CO2 day(-1). Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 micromol liter(-1) NO3(-) day(-1). Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.

Carbon Dioxide↗