Clinical and laboratory features associated with myeloperoxidase expression in pediatric B-lymphoblastic leukemia.

BACKGROUND: B-lymphoblastic leukemia (B-ALL) is the most common childhood malignancy, and its diagnosis requires immunophenotypically demonstrating blast B cell lineage differentiation. Expression of myeloperoxidase (MPO) in B-ALL is well-described and it has been recognized that a diagnosis of mixed phenotype acute leukemia should be made cautiously if MPO expression is the sole myeloid feature in these cases. We sought to determine whether MPO expression in pediatric B-ALL was associated with differences in laboratory, immunophenotypic, or clinical features. METHODS: We reviewed clinical, diagnostic bone marrow flow cytometry, and laboratory data for all new B-ALL diagnoses at our pediatric institution in 5 years. Cases were categorized as MPO positive (MPO+) or negative (MPO-) using a threshold of ≥20% blasts expressing MPO at intensity greater than the upper limit of normal lymphocytes on diagnostic bone marrow flow cytometry. RESULTS: A total of 148 cases were reviewed, 32 of which (22%) were MPO+. MPO+ B-ALL was more frequently hyperdiploid and less frequently harbored ETV6-RUNX1; no MPO+ cases had KMT2A rearrangements or BCR-ABL1. Although not significantly so, MPO+ B-ALL was less likely than MPO- B-ALL to have positive end-of-induction minimal residual disease studies (9.4 and 24%, respectively), but relapse rates and stem cell transplantation rates were similar between groups. Aberrant expression of other more typically myeloid markers was similar between these groups. CONCLUSION: In our study cohort, MPO+ B-ALL showed minimal residual disease persistence less often after induction chemotherapy but otherwise had similar clinical outcomes to MPO- B-ALL, with similar rates of additional myeloid antigen aberrancy.

Production and characterisation of a novel actinobacterial DyP-type peroxidase and its application in coupling of phenolic monomers.

The extracellular peroxidase from Streptomyces albidoflavus BSII#1 was purified to near homogeneity using sequential steps of acid and acetone precipitation, followed by ultrafiltration. The purified peroxidase was characterised and tested for the ability to catalyse coupling reactions between selected phenolic monomer pairs. A 46-fold purification of the peroxidase was achieved, and it was shown to be a 46 kDa haem peroxidase. Unlike other actinobacteria-derived peroxidases, it was only inhibited (27 % inhibition) by relatively high concentrations of sodium azide (5 mM) and was capable of oxidising eleven (2,4-dichlorophenol, 2,6-dimethoxyphenol, 4-tert-butylcatechol, ABTS, caffeic acid, catechol, guaiacol, l-DOPA, o-aminophenol, phenol, pyrogallol) of the seventeen substrates tested. The peroxidase remained stable at temperatures of up to 80 °C for 60 min and retained >50 % activity after 24 h between pH 5.0-9.0, but was most sensitive to incubation with hydrogen peroxide (H2O2; 0.01 mM), l-cysteine (0.02 mM) and ascorbate (0.05 mM) for one hour. It was significantly inhibited by all organic solvents tested (p ≤ 0.05). The Km and Vmax values of the partially purified peroxidase with the substrate 2,4-DCP were 0.95 mM and 0.12 mmol min-1, respectively. The dyes reactive blue 4, reactive black 5, and Azure B, were all decolourised to a certain extent: approximately 30 % decolourisation was observed after 24 h (1 µM dye). The peroxidase successfully catalysed coupling reactions between several phenolic monomer pairs including catechin-caffeic acid, catechin-catechol, catechin-guaiacol and guaiacol-syringaldazine under the non-optimised conditions used in this study. Genome sequencing confirmed the identity of strain BSII#1 as a S. albidoflavus strain. In addition, the genome sequence revealed the presence of one peroxidase gene that includes the twin arginine translocation signal sequence of extracellular proteins. Functional studies confirmed that the peroxidase produced by S. albidoflavus BSII#1 is part of the dye-decolourising peroxidase (DyP-type) family.

Peroxidase zymograms obtained by agarose native gel electrophoresis have unmet resolution and completeness.

A recently published method to separate protein isoforms by means of a flat-bed agarose native gel was adapted for identifying simultaneously both acid and basic isoforms of plant peroxidases. These were evidenced by in situ activity staining using alternative substrates for which the isoforms showed specific preference. Such approach allowed the detection of a significantly higher number of horseradish peroxidases than the conventional methods based on sample separation by acrylamide gel and the single bands were clearer to observe. Samples recovered from different plant species and with variable level of purity were successfully analyzed for their peroxidase (POX) isoform pattern. We expect that the innovative electrophoretic methodology illustrated in this work will strongly improve the output of experiments that aim at relating the activity and expression variation of specific enzyme biomarkers with physiological and pathological conditions.

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity.

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of ß-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

Function of hesperidin alleviating inflammation and oxidative stress responses in COPD mice might be related to SIRT1/PGC-1α/NF-κB signaling axis.

Purpose: Hesperidin has anti-inflammatory and anti-oxidant stress effects, but its functions in chronic obstructive pulmonary disease (COPD) remains unknown. This study analyzed the role of hesperidin in COPD mice, aiming to provide a basis for the hesperidin application.Materials and methods: Mice were injected with cigarette smoke extract (CSE) to construct COPD models and then treated with budesonide or hesperidin. Hematoxylin-eosin (HE) and TUNEL assays were used to observe the pathological changes and cell death of lung tissue. The levels of interleukin (IL)-6, IL-8, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) in bronchoalveolar lavage fluid (BLAF), as well as myeloperoxidase (MPO) content in lung tissues were confirmed. The expression levels of SIRT1, PGC-1α, and p65 proteins were measured by western blotting (WB) analysis.Results: CSE induced inflammatory cell infiltration and cell death in the lung tissues of mice, whereas budesonide and hesperidin effectively alleviated these pathological changes. The levels of IL-6, IL-8, and MDA in BLAF and pulmonary MPO content in the COPD mice were effectively increased, while the levels of SOD and CAT in BLAF were decreased, which could be reversed by budesonide and hesperidin. Moreover, the addition of budesonide or hesperidin reliably accelerated the expression levels of PGC-1α and SIRT1 but suppressed the phosphorylation of p65 in COPD mice. In general, high-dose hesperidin had a stronger regulatory effect on COPD mice.Conclusions: Hesperidin alleviated inflammation and oxidative stress responses in CES-induced COPD mice, associated with SIRT1/PGC-1α/NF-κB signaling axis, which might become a new direction for COPD treatment.

The ant Lasius niger is a new source of bacterial enzymes with biotechnological potential for bleaching dye.

Industrial synthetic dyes cause health and environmental problems. This work describes the isolation of 84 bacterial strains from the midgut of the Lasius niger ant and the evaluation of their potential application in dye bioremediation. Strains were identified and classified as judged by rRNA 16S. The most abundant isolates were found to belong to Actinobacteria (49%) and Firmicutes (47.2%). We analyzed the content in laccase, azoreductase and peroxidase activities and their ability to degrade three known dyes (azo, thiazine and anthraquinone) with different chemical structures. Strain Ln26 (identified as Brevibacterium permense) strongly decolorized the three dyes tested at different conditions. Strain Ln78 (Streptomyces ambofaciens) exhibited a high level of activity in the presence of Toluidine Blue (TB). It was determined that 8.5 was the optimal pH for these two strains, the optimal temperature conditions ranged between 22 and 37 °C, and acidic pHs and temperatures around 50 °C caused enzyme inactivation. Finally, the genome of the most promising candidate (Ln26, approximately 4.2 Mb in size) was sequenced. Genes coding for two DyP-type peroxidases, one laccase and one azoreductase were identified and account for the ability of this strain to effectively oxidize a variety of dyes with different chemical structures.

Purification of peroxidase enzyme from radish species in fast and high yield with affinity chromatography technique.

In this study, an effective single step affinity method is presented for purifying plant peroxidase (POD) enzymes from radish species. This method make possible to purify the enzymes in high yield and purity. Briefly, 10 different 4-amino benzohydrazide derivatives were synthesized and identified as new competitive POD inhibitors. Then, these derivatives were coupled to Sepharose 4B-L-Tyrosine support matrix by diazotization to form the affinity gels. Purification factors were recorded as 54.8% yield - 665-fold, 33.8% yield - 613-fold, 22.7% yield - 595-fold, 34.4% yield - 781-fold, 40.9% yield - 282-fold for turnip (T-POD), black radish (BR-POD), daikon (D-POD), sweet radish (SR-POD) and kohlrabi radish, (KR-POD), respectively. It has also been shown that the affinity gels, which prepared using the 4-amino 3-bromo benzohydrazide and 4-amino 2-nitro benzohydrazide molecules, capable to purify all radish species POD enzymes in high purity and yield.

A novel peroxidase from Arabian balsam (Commiphora gileadensis) stems: Its purification, characterization and immobilization on a carboxymethylcellulose/Fe3O4 magnetic hybrid material.

A novel plant peroxidase was isolated from the stem of Arabian balsam (Commiphora gileadensis) and purified using ammonium sulfate, followed by ion exchange chromatography (DEAE-Sepharose) and gel filtration (Sephcryl S-200). The newly isolated peroxidase was characterized as having a specific activity of 9503.3 unit/mg of protein after 20.3-fold purification, which yielded a recovery of 18.5%. Based on the subunit size, the purified peroxidase was a 40 kDa monomeric structure and presented high thermostability, as it was entirely stable at 55 °C for 30 min and retained approximately 13.6% of its activity at 85 °C. The optimal pH exhibited a broad value range (pH 7.0- 7.5). The kinetic parameters for the purified peroxidase were obtained. To increase the enzyme durability, efficiency and reusability, the peroxidase was entrapped onto a carboxymethyl cellulose/Fe3O4 magnetic hybrid material. The immobilized enzyme was characterized by scanning electron microscopy (SEM) and FT-IR spectroscopy. It was tested at different pH values, storage times and temperatures, and its kinetic behavior was assessed. The immobilized enzyme maintained its activity upon storage at 4 and 25 °C for 8 weeks, and upon recycling for up to 15 uses. Arabian balsam peroxidase appears to be candidate for industrial applications.

Antigen-Specific Detection of Autoantibodies Against Myeloperoxidase (MPO) and Proteinase 3 (PR3).

ANCA testing was introduced in many laboratories throughout the world when it was recognized that a significant subset of patients with small vessel vasculopathies presented with such antibodies. Many laboratories developed and introduced in-house testing methods for antigen-specific ANCA detection complementary to indirect immune fluorescence screening. Such in-house tests have proven their merit in diagnosing vasculitis and were important to identify critical steps in the development of antigen-specific assays with high sensitivity and specificity. In the meantime various commercial assays became available for antigen-specific ANCA testing. Because of the high diagnostic accuracy of such assays it can be anticipated that commercial, antigen-specific tests will completely replace in-house testing for MPO- and PR3-ANCA. Furthermore, such tests will replace the need for IIF in the diagnostic workup of AAV. In this light it can be foreseen that the knowledge that underlies the development of in-house ANCA testing will gradually disseminate over time. Therefore we describe the current antigen-specific ANCA ELISAs (direct and capture) with the intention to maintain the knowledge and the identification of the critical steps in the development of robust assays.

Monocyte phagocytosis of malaria ß-haematin in the presence of artemisinin, amodiaquine, chloroquine, doxycycline, primaquine, pyrimethamine and quinine.

The intraerythrocytic malaria parasite digests haemoglobin to provide amino acids for metabolism and releases toxic haem that is sequestered into haemozoin, a non-toxic, insoluble, crystalline pigment. Following erythrocyte rupture, haemozoin is released into circulation and phagocytosed by monocytes. Phagocytosed haemozoin and antimalarial drugs have both been reported to modulate monocyte functions. This study determined the effects of therapeutic concentrations of seven antimalarial drugs; amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of ß-haematin (synthetic haemozoin) by two monocytic cell lines, J774A.1 and U937, and human peripheral blood mononuclear cells. A novel spectrophotometric method based on the absorbance (O.D 400 nm) of alkali/SDS treated monocytes containing ß-haematin was developed to complement counting phagocytosis with microscopy. The method has potential use for the large scale screening of monocyte phagocytic activity. Artemisinin, quinine, primaquine and pyrimethamine activated ß-haematin phagocytosis by 12% or more, whereas amodiaquine, chloroquine and doxycyline inhibited ß-haematin phagocytosis. In contrast, antimalarial drugs had minimal inhibitory effects on the phagocytosis of latex beads with only quinine resulting in more than 20% inhibition. Antimalarial drugs appear to alter monocyte phagocytic activity which has implications for the treatment, pathogenicity and adjunct therapies for malaria.

Improvement of kinetic properties and thermostability of recombinant Lepidium draba peroxidase (LDP) upon exposed to osmolytes.

In this study, effects of different concentrations of glycine and D-sorbitol were analyzed on the activity and thermostability of recombinant Lepidium draba peroxidase (LDP). Based on the results, activity of the enzyme increased in the presence of various concentrations of these osmolytes. Maximum activity was detected for the enzyme in the presence of 300 mM glycine and 600 mM sorbitol. In presence of the aforementioned doses of osmolytes, enzyme affinity for substrate (3,3',5,5'-tetramethylbenzidine and H2O2) and Vmax increased. According to the results, enzyme stability improved against temperature and H2O2. Furthermore, structural changes of the enzyme upon exposure to the osmolytes were revealed by the use of far-UV circular dichroism and fluorescence methods. The results showed, whereas the secondary structure of the enzyme was not significantly changed upon exposed to the osmolytes, the fluorescence studies revealed microenvironment of the aromatic residues dramatically affected by them. Overall, it may be speculated, structural changes of the enzyme upon exposed to the osmolytes, lead to the improvement of its kinetic properties and stability that can be benefit for using of it in in vitro applications.

Cationic peroxidase from proso millet induces human colon cancer cell necroptosis by regulating autocrine TNF-α and RIPK3 demethylation.

A cationic peroxidase (POD) was purified from proso millet seeds (PmPOD) using ammonium sulfate fractionation, cation exchange, and size exclusion chromatography. The purified PmPOD showed toxicity to normal cells and tumor cells, but was more sensitive in HT29 cells. Furthermore, the mechanism driving HCT116 and HT29 cell death by PmPOD was the induction of receptor interacting protein kinase 1 (RIPK1)- and RIPK3-dependent necroptosis, independent of apoptosis. More importantly, PmPOD could induce tumor necrosis factor-α (TNF-α) production through transcriptional upregulation. In addition, PmPOD could restore RIPK3 expression in HCT116 cells via the demethylation of the RIPK3 genomic sequence. Taken together, these results suggest that two distinct mechanisms are involved in PmPOD-induced necroptosis: the autocrine production of TNF-α and the restoration of RIPK3 expression.

High isostatic pressure and thermal processing of açaí fruit (Euterpe oleracea Martius): Effect on pulp color and inactivation of peroxidase and polyphenol oxidase.

The present study evaluated the effect of high isostatic pressure (HIP) on the activity of peroxidase (POD) and polyphenol oxidase (PPO) from açaí. Açaí pulp was submitted to several combinations of pressure (400, 500, 600MPa), temperature (25 and 65°C) for 5 and 15min. The combined effect of HIP technology and high temperatures (690MPa by 2 and 5min at 80°C) was also investigated and compared to the conventional thermal treatment (85°C/1min). POD and PPO enzyme activity and instrumental color were examined after processing and after 24h of refrigerated storage. Results showed stability of POD for all pressures at 25°C, which proved to be heat-resistant and baro-resistant at 65°C. For PPO, the inactivation at 65°C was 71.7% for 600MPa after 15min. In general, the increase in temperature from 25°C to 65°C reduced the PPO relative activity with no changes in color. Although the thermal treatment and the HIP (690MPa) along with high temperature (80°C) reduced the PPO relative activity, and relevant darkening was observed in the processed samples. Thus, it can be concluded that POD is more baro-resistant than PPO in açaí pulp subjected to the same HIP processing conditions and processing at 600MPa/65°C for 5min may be an effective alternative for thermal pasteurization treatments.

Neutrophil granule proteins generate bactericidal ammonia chloramine on reaction with hydrogen peroxide.

The neutrophil enzyme, myeloperoxidase, by converting hydrogen peroxide (H2O2) and chloride to hypochlorous acid (HOCl), provides important defense against ingested micro-organisms. However, there is debate about how efficiently HOCl is produced within the phagosome and whether its reactions with phagosomal constituents influence the killing mechanism. The phagosome is a small space surrounding the ingested organism, into which superoxide, H2O2 and high concentrations of proteins from cytoplasmic granules are released. Previous studies imply that HOCl is produced in the phagosome, but a large proportion should react with proteins before reaching the microbe. To mimic these conditions, we subjected neutrophil granule extract to sequential doses of H2O2. Myeloperoxidase in the extract converted all the H2O2 to HOCl, which reacted with the granule proteins. 3-Chlorotyrosine, protein carbonyls and large amounts of chloramines were produced. At higher doses of H2O2, the extract developed potent bactericidal activity against Staphylococcus aureus. This activity was due to ammonia monochloramine, formed as a secondary product from protein chloramines and dichloramines. Isolated myeloperoxidase and elastase also became bactericidal when modified with HOCl and antibacterial activity was seen with a range of species. Comparison of levels of protein modification in the extract and in phagosomes implies that a relatively low proportion of phagosomal H2O2 would be converted to HOCl, but there should be sufficient for substantial protein chloramine formation and some breakdown to ammonia monochloramine. It is possible that HOCl could kill ingested bacteria by an indirect mechanism involving protein oxidation and monochloramine formation.

Heterologous Expression, Purification and Characterization of a Peroxidase Isolated from Lepidium draba.

Peroxidase is one of the most widely used enzymes in biotechnology and medicine. In the current study, cDNA encoding peroxidase from Lepidium draba (LDP) was cloned and expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies (IBs). To achieve purified active enzyme, IBs were solubilized before being purified and refolded. The deduced amino acid sequence (308) of the LDP gene (924 bp) revealed 88.96% identity to horseradish peroxidase C1A (HRP C1A). The results of basic local alignment search tool (BLAST) and phylogenetic analysis of the protein sequence showed that this enzyme belongs to the neutral group of class III plant peroxidases. According to sequence analysis and structural modeling, critical amino acids in heme and calcium binding domain as well as cysteine residues were conserved as HRP C1A except for calcium binding domain where valine228 was replaced with isoleucine. The far-UV circular dichroism (CD) results were confirmed by homology modeling data showing the enzyme consists mainly of α-helices as other plant peroxidases. Overall, according to the results of catalytic activity and refolding yield, LDP can be introduced as a novel peroxidase for medical and biotechnology applications.

Characterization and application of the enzyme peroxidase to the degradation of the mycotoxin DON.

Deoxynivalenol (DON), one of the main mycotoxins found in food matrices, has high level of toxicity. This study aimed to characterize the peroxidase enzyme extracted from rice bran to be applied to the biodegradation of DON in order to evaluate the potential peroxidase (PO) from rice bran (RB) has to degrade DON in optimal conditions. Purification and recovery factors of PO extracted from RB and purified by three-phase partitioning were 5.7% and 50%, respectively. PO had the highest level of activity in the phosphate buffer 5 mM pH 5.5 in both crude and purified forms, whose reaction temperatures were 25°C and 10°C. At the end of production, purification and characterization steps, specific activities of the bran were 115.79 U mg-1 and 4363 U g-1. Reduction in the mycotoxin DON in optimal conditions determined for PO from RB was 20.3%, a promising result when the aim is to adequate mycotoxicological levels to foods.

Kinetic and amperometric study of the MtPerII peroxidase isolated from the ascomycete fungus Myceliophthora thermophila.

The enzyme MtPerII is a new peroxidase which has been isolated only recently from fungus Myceliophthora thermophila and has significant thermostability and stability at high H2O2 concentrations. In the present work, an electrochemical kinetic study, based on cyclic voltammetry, is performed for the first time for the catalytic decomposition of H2O2 by MtPerII, at 18°C. Leuco methylene blue (LMB) is used as a mediator and the catalytic and Michaelis constants are determined, assuming a Michaelis-Menten mechanism. Experimental evidence suggest the absence of inhibition by H2O2, for concentrations up to 16mM, and increasing catalytic activity for temperatures up to 50°C. Moreover, a modified electrode is constructed, by attempting the entrapment of MtPerII on a dodecanothiol self-assembled monolayer on gold. The modified electrode is studied chronoamperometrically in solutions containing methylene blue mediator and different concentrations of H2O2. It is shown that adsorbed MtPerII retains its activity and the modified electrode exhibits a considerably high linear region for the detection of H2O2. The experimental findings indicate that MtPerII is a new candidate for analytical and industrial applications.

CodY Regulates Thiol Peroxidase Expression as Part of the Pneumococcal Defense Mechanism against H2O2 Stress.

Streptococcus pneumoniae is a facultative anaerobic pathogen. Although it maintains fermentative metabolism, during aerobic growth pneumococci produce high levels of H2O2, which can have adverse effects on cell viability and DNA, and influence pneumococcal interaction with its host. The pneumococcus is unusual in its dealing with toxic reactive oxygen species (ROS) in that it neither has catalase nor the global regulators of peroxide stress resistance. Previously, we identified pneumococcal thiol peroxidase (TpxD) as the key enzyme for enzymatic removal of H2O2, and showed that TpxD synthesis is up-regulated upon exposure to H2O2. This study aimed to reveal the mechanism controlling TpxD expression under H2O2 stress. We hypothesize that H2O2 activates a transcription factor which in turn up-regulates tpxD expression. Microarray analysis revealed a pneumococcal global transcriptional response to H2O2. Mutation of tpxD abolished H2O2-mediated response to high H2O2 levels, signifying the need for an active TpxD under oxidative stress conditions. Bioinformatic tools, applied to search for a transcription factor modulating tpxD expression, pointed toward CodY as a potential candidate. Indeed, a putative 15-bp consensus CodY binding site was found in the proximal region of tpxD-coding sequence. Binding of CodY to this site was confirmed by EMSA, and genetic engineering techniques demonstrated that this site is essential for TpxD up-regulation under H2O2 stress. Furthermore, tpxD expression was reduced in a ΔcodY mutant. These data indicate that CodY is an activator of tpxD expression, triggering its up-regulation under H2O2 stress. In addition we show that H2O2 specifically oxidizes the 2 CodY cysteines. This oxidation may trigger a conformational change in CodY, resulting in enhanced binding to DNA. A schematic model illustrating the contribution of TpxD and CodY to pneumococcal global transcriptional response to H2O2 is proposed.

Mesna (2-mercaptoethane sodium sulfonate) functions as a regulator of myeloperoxidase.

Myeloperoxidase (MPO), an abundant protein in neutrophils, monocytes, and macrophages, is thought to play a critical role in the pathogenesis of various disorders ranging from cardiovascular diseases to cancer. We show that mesna (2-mercaptoethanesulfonic acid sodium salt), a detoxifying agent, which inhibits side effects of oxazaphosphorine chemotherapy, functions as a potent inhibitor of MPO; modulating its catalytic activity and function. Using rapid kinetic methods, we examined the interactions of mesna with MPO compounds I and II and ferric forms in the presence and absence of chloride (Cl-), the preferred substrate of MPO. Our results suggest that low mesna concentrations dramatically influenced the build-up, duration, and decay of steady-state levels of Compound I and Compound II, which is the rate-limiting intermediate in the classic peroxidase cycle. Whereas, higher mesna concentrations facilitate the porphyrin-to-adjacent amino acid electron transfer allowing the formation of an unstable transient intermediate, Compound I*, that displays a characteristic spectrum similar to Compound I. In the absence of plasma level of chloride, mesna not only accelerated the formation and decay of Compound II but also reduced its stability in a dose depend manner. Mesna competes with Cl-, inhibiting MPO's chlorinating activity with an IC50 of 5µM, and switches the reaction from a 2e- to a 1e- pathway allowing the enzyme to function only with catalase-like activity. A kinetic model which shows the dual regulation through which mesna interacts with MPO and regulates its downstream inflammatory pathways is presented further validating the repurposing of mesna as an anti-inflammatory drug.

A novel peroxidase purified from Marsdenia megalantha latex inhibits phytopathogenic fungi mediated by cell membrane permeabilization.

An antifungal class III peroxidase was purified from Marsdenia megalantha latex (named Mo-POX) using DEAE-cellulose and gel filtration chromatography on a Superose 12 HR 10/30 column. Mm-POX has an apparent molecular mass of 67.0kDa and a pI of 5.2, shares identity with other peroxidases, and follows Michaelis-Menten kinetics. It has a high affinity for guaiacol and hydrogen peroxide. The pH and temperature optima for Mm-POX were 5.0-7.0 and 60°C, respectively. The catalytic activity of Mm-POX was decreased in the presence of classic peroxidase inhibitors including azide, dithiothreitol, ethylenediamine tetraacetic acid, and sodium metabisulfite and high concentrations of Na+, Mn+, and salicylic acid. In contrast, Ca+ and Mg+, even at low concentrations, enhanced the Mm-POX enzymatic activity. This protein inhibited the germination of the conidia of the phytopathogenic fungi Fusarium oxysporum and Fusarium solani by acting through a membrane permeabilization mechanism. Mm-POX also induced oxidative stress in F. solani. Mm-POX is the first enzyme to be isolated from the M. megalantha species and it has potential use in the control of plant disease caused by important phytopathogenic fungi. This adds biotechnological value to this enzyme.