Utilization of cystine by dermatophytes on glucose-peptone media

All the 16 strains of dermatophytes tested here metabolized cystine (3 mmol/L) in two glucose-peptone media with a different C: N ratio. Cystine was utilized as a sulfur source and, in addition, as a carbon and nitrogen source, in parallel with growth. Excess sulfur was excreted to the medium after its oxidation as inorganic sulfate and sulfite. In a physiologically alkaline medium the growth was fast and was accompanied by a pH increase and cystine was utilized intensively. Eleven species used up all cystine available. Sulfate was the main oxidation product, sulfite was produced at a low concentration, at the beginning of growth in particular. Only traces of thiol compounds (cysteine) were present in the medium. In a physiologically acid medium growth was soon limited by a decreased pH (below 5.0) but cystine continued to be utilized at an identical rate. All cystine was used up by 5 species. The tendency to produce sulfite in addition to sulfate further increased and sulfite was often the predominant product. Concentrations of thiol compounds were also substantially higher. Thus, dermatophytes can utilize cystine even under conditions that do not support good growth and increase the sulfite production.     

The interrelation between high- and low-molecular-weight forms of GM1-beta-galactosidase purified from porcine spleen

1) Two forms of acid beta-galactosidase [EC 3.1.23] with different molecular weights catalyzing the hydrolysis of GM1-ganglioside and p-nitrophenyl-beta-D-galactoside were separated and purified from porcine spleen. 2) The apparent molecular weights were 400,000-600,000 and 70,000-74,000 for the high (termed Am form) and low (termed A1 form) molecular weight forms, respectively. 3) On examination by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis, both forms of the enzyme had a common protein band of molecular weight 63,000, and the Am form showed three additional protein bands with molecular weights of 31,000, 21,000, and 20,000. 4) Both forms of the enzyme had similar catalytic functions with regard to pH-optimum, Km, substrate specificity and sensitivity to substrate analogues and other substances such as detergents, bovine serum albumin (BSA) and NaCl. 5) Both forms of the enzyme were fairly stable upon preincubation at 45 degrees C at acidic pH (pH 4.5), but lost their activities at neutral pH (pH 7.0). 6) The A1 form was a monomer at neutral pH (pH 7.0) and formed a dimer at acidic pH (pH 4.5). However, most of the Am form could not be converted to a dimeric form on gel filtration at acidic pH.     

Sulfite oxidase controls sulfur metabolism under SO2 exposure in Arabidopsis thaliana

In the present study, the significance of sulfite oxidase (SO) for sulfite detoxification and sulfur assimilation was investigated. In response to sulfur dioxide (SO(2)) exposure, a remarkable expansion of sulfate and a significant increase of GSH pool were observed in wild-type and SO-overexpressing Arabidopsis. These metabolic changes were connected with a negative feedback inhibition of adenosine 5'-phosphosulfate reductase (APR), but no alterations in gas exchange parameters or visible symptoms of injury. However, Arabidopsis SO-KO mutants were consistently negatively affected upon 600 nL L(-1) SO(2) exposure for 60 h and showed phenotypical symptoms of injury with small necrotic spots on the leaves. The mean g(H2O) was reduced by about 60% over the fumigation period, accompanied by a reduction of net CO(2) assimilation and SO(2) uptake of about 50 and 35%. Moreover, sulfur metabolism was completely distorted. Whereas sulfate pool was kept constant, thiol-levels strongly increased. This demonstrates that SO should be the only protagonist for back-oxidizing and detoxification of sulfite. Based on these results, it is suggested that co-regulation of SO and APR controls sulfate assimilation pathway and stabilizes sulfite distribution into organic sulfur compounds. In conclusion, a sulfate-sulfite cycle driven by APR and SO can be postulated for fine-tuning of sulfur distribution that is additionally used for sulfite detoxification, when plants are exposed to atmospheric SO(2).     

Purification and characterization of a sulfite:cytochrome c oxidoreductase from Thiobacillus acidophilus

Cell-free extracts of Thiobacillus acidophilus prepared at neutral pH showed oxidation of sulfite to sulfate with ferricyanide as electron acceptor. Horse heart cytochrome c could be used as alternative electron acceptor; however, the observed activity was only 0.1% of that found for ferricyanide. The enzyme responsible for the oxidation of sulfite was purified to homogeneity. The purified enzyme was a monomer of 42 kDa and contained one haem c per monomer. Electron paramagnetic resonance (EPR) spectroscopical analysis of the sulfite:cytochrome c oxidoreductase showed the presence of molybdenum (V), only after reduction of the enzyme with sulfite. The pH optimum for the enzymatic reaction was 7.5 and the temperature optimum 40°C. Enzymatic activity was strongly reduced in the presence of the anions: chloride, phosphate and nitrate. In contrast to other enzymes involved in sulfur metabolism and previously isolated from T. acidophilus, sulfite:cytochrome c oxidoreductase activity is not stimulated by the presence of sulfate ions.     

Human sulfide:quinone oxidoreductase catalyzes the first step in hydrogen sulfide metabolism and produces a sulfane sulfur metabolite

Sulfide:quinone oxidoreductase (SQOR) is a membrane-bound enzyme that catalyzes the first step in the mitochondrial metabolism of H(2)S. Human SQOR is successfully expressed at low temperature in Escherichia coli by using an optimized synthetic gene and cold-adapted chaperonins. Recombinant SQOR contains noncovalently bound FAD and catalyzes the two-electron oxidation of H(2)S to S(0) (sulfane sulfur) using CoQ(1) as an electron acceptor. The prosthetic group is reduced upon anaerobic addition of H(2)S in a reaction that proceeds via a long-wavelength-absorbing intermediate (λ(max) = 673 nm). Cyanide, sulfite, or sulfide can act as the sulfane sulfur acceptor in reactions that (i) exhibit pH optima at 8.5, 7.5, or 7.0, respectively, and (ii) produce thiocyanate, thiosulfate, or a putative sulfur analogue of hydrogen peroxide (H(2)S(2)), respectively. Importantly, thiosulfate is a known intermediate in the oxidation of H(2)S by intact animals and the major product formed in glutathione-depleted cells or mitochondria. Oxidation of H(2)S by SQOR with sulfite as the sulfane sulfur acceptor is rapid and highly efficient at physiological pH (k(cat)/K(m,H(2)S) = 2.9 × 10(7) M(-1) s(-1)). A similar efficiency is observed with cyanide, a clearly artificial acceptor, at pH 8.5, whereas a 100-fold lower value is seen with sulfide as the acceptor at pH 7.0. The latter reaction is unlikely to occur in healthy individuals but may become significant under certain pathological conditions. We propose that sulfite is the physiological acceptor of the sulfane sulfur and that the SQOR reaction is the predominant source of the thiosulfate produced during H(2)S oxidation by mammalian tissues.     

Characteristics of type IV collagen unfolding under various pH conditions as a model of pathological disorder in tissue

The overall structure of type IV collagen is the same at neutral and acidic pH, as determined by circular dichroism spectra. The heating rate dependence of denaturation midpoint temperature (T(m)) shows that type IV collagen is unstable at body temperature, similarly to type I collagen. The heating rate dependence of T(m) at neutral pH has two phases, but that at acidic pH apparently has a single phase. The T(m) of the first phase (lower T(m)) at neutral pH is consistent with that at acidic pH, and the activation energy of these phases is consistent, within experimental error. The triple helix region of type IV collagen corresponding to the second phase (higher T(m)) at neutral pH is thermally stable when compared to the triple helical structure at acidic pH. At acidic pH, as the loosely packed and unstable region has spread throughout the whole molecule, the thermal transition is thought to be cooperative and is observed as a single phase. Structural flexibility is related to protein function and assembly; therefore, the unstable structure and increased flexibility of type IV collagen induced at acidic pH may affect diseases accompanied by type IV collagen disorder.     

Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis

Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD.     

Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors

Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10 mM dithionite to medium containing 2.5 mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5 mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15 mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5).     

Accumulation of Sulfite by a Sulfate-Using Revertant of Salmonella pullorum

Sulfate-utilizing revertants of a cysteine auxotroph of Salmonella pullorum have been found that cause an accumulation in growth medium of a sulfur compound more oxidized than sulfide. The sulfur compound was presumptively identified as sulfite by the formation of a colored complex in the presence of basic fuchsin and formaldehyde, oxidation to sulfate by peroxide, and acid-volatility. The acid-volatile material was identified as sulfite by formation of an S-sulfonyl derivative of 5,5'-dithio-bis(2-nitrobenzoic acid) which was chromatographically and electrophoretically identical to an authentic S-sulfonyl derivative. The presence of sulfate in minimal medium is required for sulfite accumulation, and both cysteine and selenate inhibit the accumulation. No evidence was obtained to indicate that a reduced sulfur compound was the precursor of the accumulated sulfite.     

Mechanism of oxidation of inorganic sulfur compounds by thiosulfate-grown Thiobacillus thiooxidans

Thiobacillus thiooxidans was grown at pH 5 on thiosulfate as an energy source, and the mechanism of oxidation of inorganic sulfur compounds was studied by the effect of inhibitors, stoichiometries of oxygen consumption and sulfur, sulfite, or tetrathionate accumulation, and cytochrome reduction by substrates. Both intact cells and cell-free extracts were used in the study. The results are consistent with the pathway with sulfur and sulfite as the key intermediates. Thiosulfate was oxidized after cleavage to sulfur and sulfite as intermediates at pH 5, the optimal growth pH on thiosulfate, but after initial condensation to tetrathionate at pH 2.3 where the organism failed to grow. N-Ethylmaleimide (NEM) inhibited sulfur oxidation directly and the oxidation of thiosulfate or tetrathionate indirectly. It did not inhibit the sulfite oxidation by cells, but inhibited any reduction of cell cytochromes by sulfur, thiosulfate, tetrathionate, and sulfite. NEM probably binds sulfhydryl groups, which are possibly essential in supplying electrons to initiate sulfur oxidation. 2-Heptyl-4-hydroxy-quinoline N-oxide (HQNO) inhibited the oxidation of sulfite directly and that of sulfur, thiosulfate, and tetrathionate indirectly. Uncouplers, carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), inhibited sulfite oxidation by cells, but not the oxidation by extracts, while HQNO inhibited both. It is proposed that HQNO inhibits the oxidation of sulfite at the cytochrome b site both in cells and extracts, but uncouplers inhibit the oxidation in cells only by collapsing the energized state of cells, delta muH+, required either for electron transfer from cytochrome c to b or for sulfite binding.     

Cellular regulation of cytosolic group IV phospholipase A2 by phosphatidylinositol bisphosphate levels

Cytosolic group IV phospholipase A2 (cPLA2) is a ubiquitously expressed enzyme with key roles in intracellular signaling. The current paradigm for activation of cPLA2 by stimuli proposes that both an increase in intracellular calcium and mitogen-activated protein kinase-mediated phosphorylation occur together to fully activate the enzyme. Calcium is currently thought to be needed for translocation of the cPLA2 to the membrane via a C2 domain, whereas the role of cPLA2 phosphorylation is less clearly defined. Herein, we report that brief exposure of P388D1 macrophages to UV radiation results in a rapid, cPLA2-mediated arachidonic acid mobilization, without increases in intracellular calcium. Thus, increased Ca2+ availability is a dispensable signal for cPLA2 activation, which suggests the existence of alternative mechanisms for the enzyme to efficiently interact with membranes. Our previous in vitro data suggested the importance of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in the association of cPLA2 to model membranes and hence in the regulation of cPLA2 activity. Experiments described herein show that PtdInsP2 also serves a similar role in vivo. Moreover, inhibition of PtdInsP2 formation during activation conditions leads to inhibition of the cPLA2-mediated arachidonic acid mobilization. These results suggest that cellular PtdInsP2 levels are involved in the regulation of group IV cPLA2 activation.     

Acid tolerance response of biofilm cells of Streptococcus mutans

Streptococcus mutans, a major etiological agent of dental caries, is a component of the dental plaque biofilm and functions during caries progression in acidic lesions that may be at or below pH 4. In this study, we were interested in determining the acid tolerance of 1-7-day chemostat-grown biofilm cells of S. mutans BM71 growing in a semi-defined medium at a rate consistent with that of cells in dental plaque (dilution rate=0.1 h(-1)), as well as, assessing the capacity of 2- and 5-day biofilms to induce an acid tolerance response that would enhance survival at a killing pH (3.5). As expected, biofilm cell growth increased (2.5-fold) from day 1 to day 7 (10.6-25.7 x 10(6) cells cm(-)(2)) with the percentage live cells over that period averaging 79.4%, slightly higher than that of planktonic cells (77.4%). Biofilms were highly resistant to acid killing at pH 3.5 for 2 h with survival ranging from 41.8 (1 day) to 63.9% (7 day), while the percentage of live cells averaged 43.4%. Planktonic and dispersed biofilm cells were very acid-sensitive with only 0.0009%- and 0.0002-0.2% survivors, respectively. Unlike the planktonic cells, the incubation of 2- and 5-day biofilms at pH 5.5 for periods of up to 6 h induced strong acid tolerance responses that enhanced survival during a subsequent exposure to acid killing at pH 3.5.     

Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles

In ambient aerosols, ultrafine particles (UFP) and their agglomerates are considered to be major factors contributing to adverse health effects. Reactivity of agglomerated UFP of elemental carbon (EC), Printex 90, Printex G, and diesel exhaust particles (DEP) was evaluated by the capacity of particles to oxidize methionine in a cell-free in vitro system for determination of their innate oxidative potential and by alveolar macrophages (AMs) to determine production of arachidonic acid (AA), including formation of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), reactive oxygen species (ROS), and oxidative stress marker 8-isoprostane. EC exhibiting high oxidative potential induced generation of AA, PGE2, LTB4, and 8-isoprostane in canine and human AMs. Printex 90, Printex G, and DEP, showing low oxidative capacity, still induced formation of AA and PGE2, but not that of LTB4 or 8-isoprostane. Aging of EC lowered oxidative potential while still inducing production of AA and PGE2 but not that of LTB4 and 8-isoprostane. Cellular ROS production was stimulated by all particles independent of oxidative potential. Particle-induced formation of AA metabolites and ROS was dependent on mitogen-activated protein kinase kinase 1 activation of cytosolic phospholipase A2 (cPLA2) as shown by inhibitor studies. In conclusion, cPLA2, PGE2, and ROS formation was activated by all particle types, whereas LTB4 production and 8-isoprostane were strongly dependent on particles' oxidative potential. Physical and chemical parameters of particle surface correlated with oxidative potential and stimulation of AM PGE2 and 8-isoprostane production.     

Vesicular stomatitis virus-mediated cell fusion subsequent to virus adsorption at different pH values.

Vesicular stomatitis virus (VSV)-mediated cell fusion from without can be induced by transient exposure to low pH, subsequent to adsorption of VSV at neutral pH. To study the mechanism of VSV-induced cell fusion, we examined the effect of pH condition at virus adsorption on acid-inducible VSV-mediated cell fusion. Although the binding of VSV to BHK-21 cells was most efficient under acidic condition (pH 5.7-6.3), extensive cell fusion was not observed under this condition. A temporary exposure to low pH after binding at neutral pH also decreased fusion activity. However, return to neutral pH for 2 min just after the acid binding restored the fusion activity. These results indicate the requirement of neutral pH condition for VSV-mediated cell fusion prior to the acid stimulation which induces conformational change of the virus glycoprotein into a fusogenic form.     

Enzymatic properties of human cytosolic phospholipase A(2)gamma

The enzymatic properties of cytosolic phospholipase A(2)gamma (cPLA(2)gamma), an isoform of 85-kDa group IV cPLA(2)alpha (cPLA(2)alpha) were studied in vitro and when the enzyme was expressed in cells. cPLA(2)gamma expressed in Sf9 cells is associated with membrane. Membranes isolated from [(3)H]arachidonic acid-labeled Sf9 cells expressing cPLA(2)gamma, constitutively release [(3)H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA(2) inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA(2) inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA(2)gamma has higher lysophospholipase activity than PLA(2) activity. Purified His-cPLA(2)gamma does not exhibit phospholipase A(1) activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA(2)gamma overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA(2)gamma overexpressed in lung fibroblasts from cPLA(2)alpha-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA(2)alpha results primarily in arachidonic acid release.     

Linkage of high rates of sulfate reduction in Yellowstone hot springs to unique sequence types in the dissimilatory sulfate respiration pathway

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89 degrees C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.     

Effect of pH on sulfite oxidation by Thiobacillus thiooxidans cells with sulfurous acid or sulfur dioxide as a possible substrate.

The oxidation of sulfite by Thiobacillus thiooxidans was studied at various pH values with changing concentrations of potassium sulfite. The optimal pH for sulfite oxidation by cells was a function of sulfite concentrations, rising with increasing substrate concentrations, while that by the cell extracts was unaffected. The sulfite oxidation by cells was inhibited at high sulfite concentrations, particularly at low pH values. The results from kinetic studies show that the fully protonated form of sulfite, sulfurous acid or sulfur dioxide, is the form which penetrates the cells for the oxidation.     

Effect of sulfite on the energy metabolism of mammalian tissues in correlation to sulfite oxidase activity

Mammalian tissues show significant differences in the activity of sulfite oxidase (EC 1.8.3.1) which detoxifies sulfite by oxidation to sulfate. Lung tissue and phagocytic cells such as alveolar macrophages, peritoneal macrophages, Kupffer cells and granulocytes show very low activities of sulfite oxidase. Liver tissue and hepatocytes, however, exhibit high activities of sulfite oxidase. Lung tissue and macrophages show an almost 100% decrease of the intracellular ATP levels when incubated with 1 mM sulfite at pH 6 for 30 min. In addition, the O₂ consumption of lung tissue is inhibited by 1 mM sulfite at pH 6 by more than 80%. This sulfite-induced decrease of the ATP level and of the O₂ consumption of lung tissue is enhanced between pH 6.0 and pH 7.4 with decreasing pH value of the incubation medium. In contrast, the ATP levels in liver tissue and hepatocytes are not affected by 1 mM sulfite at pH 6. The O₂ consumption of liver tissue and hepatocytes is significantly increased by sulfite due to the high activities of sulfite oxidase. Therefore, the activity of the ‘sulfite-detoxifying enzyme’ sulfite oxidase and the sensitivity of the energy metabolism to sulfite show a reciprocal relationship in the tissues and cells studied.