Factors affecting iron sulfide-enhanced bacteriophage plaque assays in Salmonella

Reaction of ferric ions with hydrogen sulfide (H(2)S) enhances contrast of phage plaques in H(2)S+ Salmonella, but contrast diminishes in weak H(2)S+ strains. H(2)S was affected by concentrations of peptones, glucose, ferric ammonium citrate (FAC) and sodium thiosulfate (ST), and by FAC:ST ratio, temperature, pH, air, and host strain. Increasing peptone levels was most important for improving contrast in weak H(2)S+ strains.     

Direct measurements of ferric reductase activity of human 101F6 and its enhancement upon reconstitution into phospholipid bilayer nanodisc

We studied human 101F6 protein to clarify its physiological function as a ferric reductase and its relationship to tumor suppression activity. We found for the first time that purified 101F6 both in detergent micelle state and in phospholipid bilayer nanodisc state has an authentic ferric reductase activity by single turnover kinetic analyses. The kinetic analysis on the ferrous heme oxidation of reduced 101F6 upon the addition of a ferric substrate, ferric ammonium citrate (FAC), showed concentration-dependent accelerations of its reaction with reasonable values of K_M and V_max. We further verified the authenticity of the ferric reductase activity of 101F6 using nitroso-PSAP as a Fe²⁺-specific colorimetric chelator. 101F6 in nanodisc state showed higher efficiency for FAC than in detergent micelle state.     

Inhibitory effect of ursolic acid derivatives on hydrogen peroxide- and glutathione-mediated degradation of hemin: A possible additional mechanism of action for antimalarial activity

Compounds obtained by the condensation of ursolic acid (UA) with 1,4-bis(3-aminopropyl)piperazines have previously been shown as cytocidal to Plasmodium falciparum strains. Preliminary results indicated that the inhibition of β-hematin formation (one of the possible mechanisms of action of antimalarial drugs) was achieved by a few of these molecules with varying efficiencies. To gain further insight in the antimalarial action of UA derivatives, we report here the results of additional pathways that may explain their in vitro cytocidal activity such as inhibition of hemin degradation by H₂O₂ or glutathione (GSH). H₂O₂-mediated hemin degradation was drastically reduced by hydroxybenzyl-substituted UA derivatives while UA and intermediate compounds displayed weaker inhibitory actions. The results of GSH-mediated hemin degradation inhibition did not parallel those of H₂O₂ degradation as hydroxybenzyl-substituted UA only proved to be a weak inhibitor. As H₂O₂ interaction with the iron moiety of hemin is the first step towards its degradation, we assume that the interaction of our products with the ferric ion in the hemin structure is of upmost importance in inhibiting its peroxidative degradation. A two-step mechanism of action implying (1) stacking of the acetylursolic acid structure to hemin and (2) additive protection of hemin ferric iron from H₂O₂ by hydroxyphenyl groups through steric hindrance and/or trapping of oxygen reactive species in the direct neighborhood of ferric iron can be put forward. For GSH degradation pathway, grafting of UA structure with a piperazine structure gave the best inhibition, pleading for the implication of this latter moiety in the inhibitory process.     

The hemoglobin of the crossopterygian fish, Latimeria chalumnae (Smith). Subunit structure and oxygen equilibria

There are five oxidation-reduction states of horseradish peroxidase which are interconvertible. These states are ferrous, ferric, Compound II (ferryl), Compound I (primary compound of peroxidase and H₂O₂), and Compound III (oxy-ferrous). The presence of heme-linked ionization groups was confirmed in the ferrous enzyme by spectrophotometric and pH stat titration experiments. The values of pK were 5.87 for isoenzyme A and 7.17 for isoenzymes (B + C). The proton was released when the ferrous enzyme was oxidized to the ferric enzyme while the uptake of the proton occurred when the ferrous enzyme reacted with oxygen to form Compound III. The results could be explained by assuming that the heme-linked ionization group is in the vicinity of the sixth ligand and forms a stable hydrogen bond with the ligand.The measurements of uptake and release of protons in various reactions also yielded the following stoichiometries: Ferric peroxidase + H₂O₂ → Compound I, Compound I + e^? + H⁺ → Compound II, Compound II + e^? + H⁺ → ferric peroxidase, Compound II + H₂O₂ → Compound III, Compound III + 3e^? + 3H⁺ → ferric peroxidase.Based on the above stoichiometries and assuming the interaction between the sixth ligand and heme-linked ionization group of the protein, it was possible to picture simple models showing structural relations between five oxidation-reduction states of peroxidase. Tentative formulae are as follows: [Pr·Po·Fe-(II) $\text{\$}\text{?}$PrH⁺·Po·Fe(II)] is for the ferrous enzyme, Pr·Po·Fe(III)OH₂ for the ferric one, Pr·Po·Fe(IV)OH^? for Compound II, Pr(OH^?)·Po⁺·Fe(IV)OH^? for Compound I, and PrH⁺·Po·Fe(III)O₂^? for Compound III, in which Pr stands for protein and Po for porphyrin. And by Fe(IV)OH^?, for instance, is meant that OH^? is coordinated at the sixth position of the heme iron and the formal oxidation state of the iron is four.     

Effects of pH and Lactate on Hydrogen Sulfide Production by Oral Veillonella spp.

Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H₂S), one of the major components of oral malodor. However, there is little information on the physiological properties of H₂S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H₂S production. Thus, in the present study, the H₂S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica, Veillonella dispar, and Veillonella parvula were used. These Veillonella species produced H₂S during growth in the presence of l-cysteine. Resting cells of these bacteria produced H₂S from l-cysteine, and the cell extracts showed enzymatic activity to convert l-cysteine to H₂S. H₂S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H₂S production by resting cells (4.5- to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H₂S, ammonia was produced in cell extracts of all the strains, indicating that H₂S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine β-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H₂S from l-cysteine and that their H₂S production can be regulated by oral environmental factors, namely, pH and lactate.     

Immune Response Elicited by DNA Vaccination Using Lactococcus lactis Is Modified by the Production of Surface Exposed Pathogenic Protein

In this study, we compared immune responses elicited by DNA immunization using Lactococcus lactis or L. lactis expressing the Staphylococcus aureus invasin Fibronectin Binding Protein A (FnBPA) at its surface. Both strains carried pValac:BLG, a plasmid containing the cDNA of Beta-Lactoglobulin (BLG), and were designated LL-BLG and LL-FnBPA+ BLG respectively. A T_H2 immune response characterized by the secretion of IL-4 and IL-5 in medium of BLG reactivated splenocytes was detected after either oral or intranasal administration of LL-FnBPA+ BLG. In contrast, intranasal administration of LL-BLG elicited a T_H1 immune response. After BLG sensitization, mice previously intranasally administered with LL-BLG showed a significantly lower concentration of BLG-specific IgE than the mice non-administered. Altenatively administration of LL-FnBPA+ BLG didn''t modify the BLG-specific IgE concentration obtained after sensitization, thus confirming the T_H2 orientation of the immune response. To determine if the T_H2-skewed immune response obtained with LL-FnBpA+ BLG was FnBPA-specific or not, mice received another L. lactis strain producing a mutated form of the Listeria monocytogenes invasin Internalin A intranasally, allowing thus the binding to murine E-cadherin, and containing pValac:BLG (LL-mInlA+ BLG). As with LL-FnBPA+ BLG, LL-mInlA+ BLG was not able to elicit a T_H1 immune response. Furthermore, we observed that these difference were not due to the peptidoglycan composition of the cell wall as LL-FnBPA+ BLG, LL-mInlA+ BLG and LL-BLG strains shared a similar composition. DNA vaccination using LL-BLG elicited a pro-inflammatory T_H1 immune response while using LL-FnBPA+ BLG or LL-mInlA+ BLG elicited an anti-inflammatory T_H2 immune response.     

Hydrogen sulphide production by Zymomonas and its elimination in a mutant strain

Strains of Zymomonas mobilis grown in media containing either glucose or sucrose were assessed for the production of hydrogen sulphide (H₂S). In a liquid medium with low glucose concentration (20 g l^?1) only a proportion of the strains tested formed H₂S, but in medium containing a higher glucose concentration (100 g l^?1) all the strains tested produced H₂S. Four Z. mobilis strains were assayed quantitatively for H₂S production and strain ZM4 was found to produce the most H₂S in glucose medium. The amount of yeast extract and glucose, and the type of sugar used in the medium affected the amount of H₂S formed by strain ZM4. A mutant, designated ZM4701, of strain ZM4 was isolated which did not produce any detectable H₂S in liquid medium containing yeast extract plus either glucose or sucrose. The nutritional requirements of ZM4701 were investigated.     

MET17 and Hydrogen Sulfide Formation in Saccharomyces cerevisiae

Commercial isolates of Saccharomyces cerevisiae differ in the production of hydrogen sulfide (H₂S) during fermentation, which has been attributed to variation in the ability to incorporate reduced sulfur into organic compounds. We transformed two commercial strains (UCD522 and UCD713) with a plasmid overexpressing the MET17 gene, which encodes the bifunctional O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase), to test the hypothesis that the level of activity of this enzyme limits reduced sulfur incorporation, leading to H₂S release. Overexpression of MET17 resulted in a 10- to 70-fold increase in OAS/OAH SHLase activity in UCD522 but had no impact on the level of H₂S produced. In contrast, OAS/OAH SHLase activity was not as highly expressed in transformants of UCD713 (0.5- to 10-fold) but resulted in greatly reduced H₂S formation. Overexpression of OAS/OAH SHLase activity was greater in UCD713 when grown under low-nitrogen conditions, but the impact on reduction of H₂S was greater under high-nitrogen conditions. Thus, there was not a good correlation between the level of enzyme activity and H₂S production. We measured cellular levels of cysteine to determine the impact of overexpression of OAS/OAH SHLase activity on sulfur incorporation. While Met17p activity was not correlated with increased cysteine production, conditions that led to elevated cytoplasmic levels of cysteine also reduced H₂S formation. Our data do not support the simple hypothesis that variation in OAS/OAH SHLase activity is correlated with H₂S production and release.     

The cycloauration of pyridine-2-thiocarboxamide ligands

Reactions of H[AuCl₄] with N-substituted 2-pyridine thiocarboxamide ligands 2-(C₅H₄N)C(S)NHR (R = p-C₆H₄Me, CH₂Ph, Me, p-C₆H₄OMe) gave cycloaurated derivatives {(C₅H₄N)C(S)NR}AuCl₂, with the ligand bonded as the thiol tautomer through the deprotonated SH group and the pyridine N atom to give a five-membered metallacyclic ring. The X-ray structure determination of the R = CH₂Ph derivative shows a square-planar gold(III) complex that dimerises in the solid state by weak Au···S intermolecular interactions. In contrast, in the reaction of H[AuCl₄] with 2-(C₅H₄N)C(S)NHR where R = 2-pyridyl, the ligand was oxidised to give a 1,2,4-thiadiazolo[2,3-a]pyridinium heterocyclic ring that was crystallographically characterised.     

Diversity of Capsular Polysaccharide Gene Clusters in Kpc-Producing Klebsiella pneumoniae Clinical Isolates of Sequence Type 258 Involved in the Italian Epidemic

Strains of Klebsiella pneumoniae producing KPC-type beta-lactamases (KPC-Kp) are broadly disseminating worldwide and constitute a major healthcare threat given their extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In this work we report on the characterization of two different capsular polysaccharide (CPS) gene clusters, named cps _BO-4 and cps _207-2, from two KPC-Kp clinical strains from Italy belonging in sequence type (ST) 258, which is one of the most successful ST of KPC-Kp spreading worldwide. While cps _BO-4 was different from known 78 K-types according to the recently proposed typing schemes based on the wzi or wzc gene sequences, cps _207-2 was classified as K41 by one of these methods. Bioinformatic analysis revealed that they were represented in the genomic sequences of KPC-Kp from strains of ST258 from different countries, and cps _BO-4 was also detected in a KPC-Kp strain of ST442 from Brazil. Investigation of a collection of 46 ST258 and ST512 (a single locus variant of ST258) clinical strains representative of the recent Italian epidemic of KPC-Kp by means of a multiplex PCR typing approach revealed that cps _BO-4 was the most prevalent type, being detected both in ST258 and ST512 strains with a countrywide distribution, while cps _207-2 was only detected in ST258 strains with a more restricted distribution.     

Hydrogen sulfide interacts with calcium signaling to enhance the chromium tolerance in Setaria italica

The oscillation of intracellular calcium (Ca²⁺) concentration is a primary event in numerous biological processes in plants, including stress response. Hydrogen sulfide (H₂S), an emerging gasotransmitter, was found to have positive effects in plants responding to chromium (Cr⁶⁺) stress through interacting with Ca²⁺ signaling. While Ca²⁺ resemblances H₂S in mediating biotic and abiotic stresses, crosstalk between the two pathways remains unclear. In this study, Ca²⁺ signaling interacted with H₂S to produce a complex physiological response, which enhanced the Cr⁶⁺ tolerance in foxtail millet (Setaria italica). Results indicate that Cr⁶⁺ stress activated endogenous H₂S synthesis as well as Ca²⁺ signaling. Moreover, toxic symptoms caused by Cr⁶⁺ stress were strongly moderated by 50 μM H₂S and 20 mM Ca²⁺. Conversely, treatments with H₂S synthesis inhibitor and Ca²⁺ chelators prior to Cr⁶⁺-exposure aggravated these toxic symptoms. Interestingly, Ca²⁺ upregulated expression of two important factors in metal metabolism, MT3A and PCS, which participated in the biosynthesis of heavy metal chelators, in a H₂S-dependent manner to cope with Cr⁶⁺ stress. These findings also suggest that the H₂S dependent pathway is a component of the Ca²⁺ activating antioxidant system and H₂S partially contributes Ca²⁺-activating antioxidant system.     

PerR-Regulated Manganese Ion Uptake Contributes to Oxidative Stress Defense in an Oral Streptococcus

Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H₂O₂, whereas Dpr (an Fe²⁺-chelating protein)-dependent H₂O₂ protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H₂O₂-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl₂, S. oligofermentans increased survival after H₂O₂ stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn²⁺-mediated H₂O₂ resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ∼0.4 μM Mn²⁺) unless it was supplemented with ≥2.5 μM MnCl₂ and caused 82 to 95% reduction of the cellular Mn²⁺ level, while mntABC overexpression increased cellular Mn²⁺ 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn²⁺ transporter in S. oligofermentans. mntA mutation reduced the survival of H₂O₂-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H₂O₂-challenged survival 12-fold, indicating that MntABC-mediated Mn²⁺ uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H₂O₂ pulsing upregulated mntABC, while H₂O₂-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H₂O₂ can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H₂O₂ stress defenses and may be distributed across all oral streptococci lacking catalase.     

Competitive Inhibition of Ferrous Iron Oxidation by Thiobacillus ferrooxidans by Increasing Concentrations of Cells

The oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺) with dioxygen (O₂) by various strains of Thiobacillus ferrooxidans was studied by measuring the rate of O₂ consumption at various Fe²⁺ concentrations and cell concentrations. The apparent K_m values for Fe²⁺ remained constant at different cell concentrations of laboratory strains ATCC 13661 and ATCC 19859 but increased with increasing cell concentrations of mine isolates SM-4 and SM-5. The latter results are explained by the competitive inhibition of the Fe²⁺-binding site of a cell by other cells in the reaction mixture. Possible mechanisms involving cell surface properties are discussed.     

Detection of Methicillin-Susceptible Staphylococcus aureus ST398 and ST133 Strains in Gut Microbiota of Healthy Humans in Spain

Fecal samples of 100 healthy humans were tested for Staphylococcus aureus recovery. Fifteen samples (15 %) contained S. aureus, all methicillin-susceptible (MSSA), being one isolate/sample further studied. These 15 isolates were characterized by spa and agr typing as well as multi-locus sequence typing. High diversity of spa types (n?=?11) and sequences types (n?=?8) was detected. Two S. aureus of lineages ST398 or ST133 were detected, and six isolates were ascribed to clonal complex 30 (CC30). Strains were susceptible to most of the 17 antimicrobial agents tested with exceptions: erythromycin/clindamycin (three strains, containing erm(C) and/or erm(A) + mph(C) genes) and tobramycin and mupirocin (one strain containing ant(4′)-Ia + mup(A) genes). The presence of 18 staphylococcal enterotoxin genes was studied by PCR, and isolates were negative for lukF/lukS-PV genes, although strain ST133 harbored the lukD-lukE + lukM genes. Other virulence genes detected were (number of strains): tsst-1 (6), hla (15), hlb (9), hld (15), hlg (6), hlgv (9), cna (2), aur (14), and egc-like cluster (3). Analysis of immune evasion cluster genes showed six types, highlighting their absence in two strains of lineages ST133 and ST5. A high clonal diversity of MSSA strains was identified in the intestinal microbiota of healthy humans, being CC30 the most frequent one. This is the first report of MSSA ST133 and ST398 isolates in gut microbiota of healthy humans.     

THE KINETICS OF PENETRATION : XII. HYDROGEN SULFIDE

The rate of entrance of H₂S into cells of Valonia macrophysa has been studied and it has been shown that at any given time up to 5 minutes the rate of entrance of total sulfide (H₂S + S^-) into the sap is proportional to the concentration of molecular H₂S in the external solution. This is in marked contrast with the entrance of ammonia, where Osterhout has shown that the rate of entrance of total ammonia (NH₃ + NR₄ ⁺) does not increase in a linear way with the increase in the external concentration of NH₃, but falls off. The strong base guanidine also acts thus. It has been shown that the rate of entrance of H₂S is best explained by assuming that it enters by diffusion of molecular H₂S through the non-aqueous protoplasmic surface. It has been pointed out that the simple diffusion requires that the rate of entrance might be expected to be monomolecular. Possible causes of the failure of H₂S to follow this relationship have been discussed.     

Copper sulfide precipitation by yeasts from Acid mine-waters

Two strains of Rhodotorula and one of Trichosporon precipitated dissolved copper with H₂S formed by reducing elemental sulfur with glucose. Iron stimulated this activity under certain conditions. In the case of Rhodotorula strain L, iron stimulated copper precipitation aerobically at a copper concentration of 18 but not 180 μg/ml. Anaerobically, the L strain required iron for precipitation of copper from a medium with 180 μg of copper per ml. Rhodotorula strain L was able to precipitate about five times as much copper anaerobically as aerobically. The precipitated copper was identified as copper sulfide, but its exact composition could not be ascertained. Iron was not precipitated by the H₂S formed by any of the yeasts. Added as ferric iron, it was able to redissolve copper sulfide formed aerobically by Rhodotorula strain L from 18 but not 180 μg of copper per ml of medium. Since the yeasts were derived from acid mine-waters, their ability to precipitate copper may be of geomicrobial importance.     

Overlapped Sequence Types (STs) and Serogroups of Avian Pathogenic (APEC) and Human Extra-Intestinal Pathogenic (ExPEC) Escherichia coli Isolated in Brazil

Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitD_chrom, fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpV _Sakai and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some “zoonotic-related” STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential.     

MET2 affects production of hydrogen sulfide during wine fermentation

The production of hydrogen sulfide (H₂S) during yeast fermentation contributes negatively to wine aroma. We have mapped naturally occurring mutations in commercial wine strains that affect production of H₂S. A dominant R₃₁₀G mutant allele of MET2, which encodes homoserine O-acetyltransferase, is present in several wine yeast strains as well as in the main lab strain S288c. Reciprocal hemizygosity and allele swap experiments demonstrated that the MET2 _R310G allele confers reduced H₂S production. Mutations were also identified in genes encoding the two subunits of sulfite reductase, MET5 and MET10, which were associated with reduced H₂S production. The most severe of these, an allele of MET10, showed five additional phenotypes: reduced growth rate on sulfate, elevated secretion of sulfite, and reduced production in wine of three volatile sulfur compounds: methionol, carbon disulfide and methylthioacetate. Alleles of MET5 and MET10, but not MET2, affected H₂S production measured by colour assays on BiGGY indicator agar, but MET2 effects were seen when bismuth was added to agar plates made with Sauvignon blanc grape juice. Collectively, the data are consistent with the hypothesis that H₂S production during wine fermentation results predominantly from enzyme activity in the sulfur assimilation pathway. Lower H₂S production results from mutations that reduce the activity of sulfite reductase, the enzyme that produces H₂S, or that increase the activity of l-homoserine-O-acetyltransferase, which produces substrate for the next step in the sulfur assimilation pathway.     

Hydrogen peroxide production in wheat leaves infected with the fungus Septoria nodorum Berk. Strains with different virulence

The effect of two strains of the phytopathogenic fungus Septoria nodorum Berk. of different virulence on the intensity of local generation of hydrogen peroxide in common wheat leaves and the role of oxidoreductases in this process was studied. Differences in the pattern of hydrogen peroxide production in wheat plants infected with high- and low-virulence pathogen strains have been found. The low-virulent S. nodorum strain caused a long-term hydrogen peroxide (H₂O₂) generation in the infection zone, whereas the inoculation of leaves with the highly virulent strain resulted in a transient short-term increase in the H₂O₂ concentration at the initial moment of contact between the plant and the fungus. It was shown that the low level of H₂O₂ production by plant cells at the initial stages of pathogenesis facilitates S. nodorum growth and development. The decrease in the H₂O₂ concentration induced by the highly virulent S. nodorum strain is determined by inhibition of the oxalate oxidase activity in plant tissues and by the ability of the fungus to actively synthesize an extracellular catalase. The pattern of hydrogen peroxide generation at the initial stages of septoriosis may serve as an index of virulence of S. nodorum population.     

Ketomethylthiobutyric acid formation from methylthioadenosine: A diffusion assay

Typical enzyme kinetics were observed when 5′-methylthioadenosine was used as substrate with extracts of malignant murine cells in a diffusion assay. The volatile product was measured after diffusion into a solution of the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoic acid), which it reduced to a yellow chromophore. Cysteine was required in the system. The volatile product was identified as H₂S derived from the cysteine. The yield of H₂S was similar to the amount of 2-keto-4-methylthiobutyric acid (KMTB) formed from methylthioadenosine when the KMTB was measured simultaneously in an ether extraction assay. KMTB could replace methylthioadenosine as a substrate capable of causing the formation of the diffusible product from cysteine. It is concluded that the following sequence of reactions takes place in the diffusion assay system: (1) 5′-methylthioadenosine + P_i → adenine + 5-methylthioribose-1-P, (2) 5-methylthioribose-1-P → KMTB, (3) KMTB + cysteine → methionine + 3-mercaptopyruvate, (4) 3-mercaptopyruvate + excess R-SH → pyruvate + H₂S, (5) H₂S + 5,5′-dithiobis(2-nitrobenzoic acid) → 5-mercapto-2-nitrobenzoic acid. Thus, the diffusion assay measures the amount of KMTB formed. The key enzyme, cysteine aminotransferase, EC 2.6.1.3, was partially purified from malignant cells and from liver and several of its characteristics are described. The diffusion assay using this enzyme is useful in measuring de novo synthesis of α-keto acids and it is applicable to crude enzyme preparations. The sensitivity is about 5 nmol of keto acid and the accurate range is 5 to 100 nmol.