Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

Degradation of salicylic acid to catechol in Solanaceae by SA 1-hydroxylase.

The hormone salicylic acid (SA) plays crucial roles in plant defense, stress responses, and in the regulation of plant growth and development. Whereas the biosynthetic pathways and biological functions of SA have been extensively studied, SA catabolism is less well understood. In this study, we report the identification and functional characterization of an FAD/NADH-dependent SA 1-hydroxylase from tomato (Solanum lycopersicum; SlSA1H), which catalyzes the oxidative decarboxylation of SA to catechol. Transcript levels of SlSA1H were highest in stems and its expression was correlated with the formation of the methylated catechol derivatives guaiacol and veratrole. Consistent with a role in SA catabolism, SlSA1H RNAi plants accumulated lower amounts of guaiacol and failed to produce any veratrole. Two O-methyltransferases involved in the conversion of catechol to guaiacol and guaiacol to veratrole were also functionally characterized. Subcellular localization analyses revealed the cytosolic localization of this degradation pathway. Phylogenetic analysis and functional characterization of SA1H homologs from other species indicated that this type of FAD/NADH-dependent SA 1-hydroxylases evolved recently within the Solanaceae family.

Protective effects of zingerone on cisplatin-induced nephrotoxicity in female rats.

Zingerone (ZO), one of the active components of ginger (Zingiber officinale), is a phenolic alkanone with antioxidant, antiapoptotic, and anti-inflammatory properties. Cisplatin (CP) is a widely used chemotherapeutic drug for solid tumors, but its therapeutic use is limited due to dose-dependent nephrotoxicity. In the present study, we investigated the ameliorative effect of ZO against CP-induced nephrotoxicity. Intraperitoneal administration of single-dose CP (7 mg/kg body weight) on the first day enhanced kidney lipid peroxidation and reduced antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GSH). CP increased serum urea and creatinine levels and disrupted histological integrity while causing a decrease aquaporin 1 (AQP1) level in the kidney tissues. CP induced inflammatory responses by elevating the levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-33 (IL-33) and nuclear factor kappa B (NF-κB), and activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, it also caused oxidative DNA damage and activation of apoptotic pathway by increasing of 8-hydroxy-2'-deoxyguanosine (8-OHdG), p53, cysteine aspartate-specific protease-3 (caspase-3), and Bcl-2-associated x protein (bax) while decreasing B cell lymphoma-2 (Bcl-2). However, treatment with ZO at a dose of 25 and 50 mg/kg b.wt. for 7 days significantly decreased oxidative stress, apoptosis, inflammation, and histopathological alterations while increased AQP1 levels in the kidney tissue. The results of the current study suggested that ZO as an effective natural product attenuates CP-induced nephrotoxicity.

Contribution of aorta glycosaminoglycans and PCSK9 to hyperlipidemia in experimental rabbits: the role of 10-dehdrogingerdione as effective modulator.

10-Dehydrogingerdione (10-DHGD) was previously reported to possess a hypolipidemic, anti-inflammatory and anti-oxidant properties in hyperlipidemic rabbit model. In this study, we investigated a possible new role for 10-DHGD in modulating atherogenic lipid profile by targeting proprotein convertase subtilisin kexin-9 (PCSK-9). Cholesterol (0.2% w/w)-fed rabbits received either atorvastatin (20 mg/kg) or 10-DHGD (10 mg/kg) for 12 weeks along with cholesterol feeding (HCD). Lipid profile, serum PCSK-9 and macrophage migration inhibitory factor (MIF), and aorta level of tumor necrosis factor-alpha (TNF-α) and glycosaminoglycans (GAGs) were measured. HCD-fed rabbits revealed an atherogenic lipid profile along with increased serum level of PCSK-9 (p < 0.001) and increased serum MIF and aortic TNF-α and GAGs (p < 0.001). 10-DHGD administration to HCD-fed rabbits prevented this atheogenicity by modulating the release of PCSK-9, inflammation extent (serum MIF and aortic TNF-α) and GAGs. These results provide new insights on the hypolipidemic potential of 10-DHGD. The effects of 10-DHGD was superior to that of atorvastatin in most studied parameters modulating atherogenicity. 10-DHGD is found to be able to suppress the release of PCSK-9, decrease aortic expression of GAGs in cholesterol-fed rabbits and halt the inflammation extent. These effects may provide new insights on the hypolipidemic potential of 10-DHGD.

Renal protective effects of zingerone in a mouse model of sepsis.

Zingerone (ZGR), a phenolic alkanone isolated from ginger, has been reported to possess pharmacological activities such as anti-inflammatory and anti-apoptotic effects. This study was initiated to determine whether ZGR could modulate renal functional damage in a mouse model of sepsis and to elucidate the underlying mechanisms. The potential of ZGR treatment to reduce renal damage induced by cecal ligation and puncture (CLP) surgery in mice was measured by assessment of serum creatinine, blood urea nitrogen (BUN), lipid peroxidation, total glutathione, glutathione peroxidase activity, catalase activity, and superoxide dismutase activity. Treatment with ZGR resulted in elevated plasma levels of BUN and creatinine, and of protein in urine in mice with CLP-induced renal damage. Moreover, ZGR inhibited nuclear factor-κB activation and reduced the induction of nitric oxide synthase and excessive production of nitric acid. ZGR treatment also reduced the plasma levels of interleukin-6 and tumor necrosis factor-α, reduced lethality due to CLP-induced sepsis, increased lipid peroxidation, and markedly enhanced the antioxidant defense system by restoring the levels of superoxide dismutase, glutathione peroxidase, and catalase in kidney tissues. Our study showed renal suppressive effects of zingerone in a mouse model of sepsis, suggesting that ZGR protects mice against sepsis-triggered renal injury. [BMB Reports 2019; 52(4): 271-276].

Cell-assisted synthesis of conducting polymer - polypyrrole - for the improvement of electric charge transfer through fungal cell wall.

In this research we report the biological synthesis of electrically conducting polymer - Polypyrrole (Ppy). Cell-assisted enzymatic polymerization/oligomerization of Ppy was achieved using whole cell culture and cell-free crude enzyme extract from two white-rot fungal cultures. The selected fungal strains belong to Trametes spp., known laccase producers, commonly applied in bioremediation and bioelectrochemical fields. The biocatalytic reaction was initiated in situ through the copper-containing enzymes biosynthesized within the cell cultures under submerged aerobe cultivation conditions. The procedure was inspired by successful reports of laccase-catalyzed pyrrole polymerization. The usage of whole culture and/or crude enzyme extract has the advantage of overcoming enzyme purification and minimizing the liability of enzyme inactivation through improved stability of enzymes in their natural environment. Spectral and electrochemical techniques (UV-vis spectroscopy, infrared spectroscopy; cyclic voltammetry (CV)) and pH measurements provided insight into the evolution of pyrrole polymerization/oligomerization and the electrochemical features of the final product. Microscopy techniques (optical microscopy and scanning electron microscopy (SEM)) were primary tools for visualization of the formed Ppy particles. The relevance of our research is twofold: Ppy prepared in crude enzyme extract results in enzyme encapsulated within Ppy and/or Ppy-modified fungal cells can be formed when polymerization occurs in whole cell culture. The route of biocatalysis can be chosen according to the desired bioelectrochemical application. The reported study focuses on the improvement of charge transfer through the fungal cell membrane and/or cell wall by modification of the fungal cells with conducting polymer - polypyrrole.

Bacillus amyloliquefaciens CotA degradation of the lignin model compound guaiacylglycerol-ß-guaiacyl ether.

The cotA gene from Bacillus amyloliquefaciens MN-13 was cloned and expressed in Escherichia coli Transetta. Nucleotide sequence analysis showed an open reading frame of 1542 bp encoding a polypeptide comprised of 513 amino acids. The degradation of lignin model compounds by recombinant CotA was investigated by HPLC-MS with guaiacylglycerol-ß-guaiacyl ether as the substrate. The compounds including guaiacol, 3-(4-hydro-3-methoxyphenyl)-3-oxo-propanol and 4-hydro-3-methoxy acetophenone detected by HPLC-MS verified the rupture of ß-O-4 bond and oxidation Cα bond of guaiacylglycerol-ß-guaiacyl ether by CotA. 4-vinylguaiacol and 1-(4-hydroxy-3-methoxyl phenyl)-1-(2-methoxyl) phenoxyl ethylene were first time found in the degradation products of guaiacylglycerol-ß-guaiacyl ether. The appearance of 4-vinylguaiacol and 4-hydro-3-methoxy acetophenone confirmed the cleavage of Cß-Cγ bond. 1-(4-hydroxy-3-methoxyl phenyl)-2-(2-methoxyl) phenoxyl ethylene was coupled by the radical reaction of 4-vinylguaiacol with guaiacol. Otherwise, no corresponding degradation product was found to give a proof of cleavage of Cα-Cß bond in guaiacylglycerol-ß-guaiacyl ether by CotA.

Biosynthesis of 4-vinylguaiacol from crude ferulic acid by Bacillus licheniformis DLF-17056.

4-vinylguaiacol, a kind of volatile phenolic compound with tobacco flavor, is widely used as a component of edible flavor and intermediate of medicine. Ferulic acid is usually used as substrate for the biosynthesis of 4-vinylguaiacol. However, the price of ferulic acid is high, leading to high production cost. In this study, a feasible low-cost process for the production of 4-vinylguaiacol was developed. The ultrasonic assisted weak alkali was used to extract protein from rice bran, and waste liquid and residue were then mixed to extract crude ferulic acid by alkaline hydrolysis. Subsequently crude ferulic acid without further purification was directly converted into 4-vinylguaiacol via alginate-immo cells of the strain Bacillus licheniformis DLF-17056, which was newly isolated and highly active with ferulic acid conversion to 4-vinylguaiacol. 4-Vinylguaiacol could be produced up to 0.76 g/L from 1.0 g/L ferulic acid within 24 h biotransformation. Furthermore, the immobilized biocatalysts retained above 60% initial activity even after 8 times biotransformations. Thereby, it was assumed that our study would contribute to the industrial production of 4-vinylguaiacol from ferulic acid.

Inhibition of epidermal growth factor receptor by ferulic acid and 4-vinylguaiacol in human breast cancer cells.

OBJECTIVES: To examine the potential of ferulic acid and 4-vinylguaiacol for inhibiting epidermal growth factor receptor (EGFR) in human breast cancer cells in vitro. RESULTS: Ferulic acid and 4-vinylguaiacol limit the EGF (epidermal growth factor)-induced breast cancer proliferation and new DNA synthesis. Western blot analysis revealed both ferulic acid and 4-vinylguaiacol exhibit sustained inhibition of EGFR activation through down-regulation of Tyr 1068 autophosphorylation. Molecular docking analysis shows ferulic acid forming hydrogen bond interaction with Lys 745 and Met 793 whereas, 4-vinylguaiacol forms two hydrogen bonds with Phe 856 and exhibits stronger hydrophobic interactions with multiple amino acid residues at the EGFR kinase domain. CONCLUSIONS: Ferulic acid and 4-vinylguaiacol could serve as a potential structure for the development of new small molecule therapeutics against EGFR.

Thermal stability, antioxidant, and anti-inflammatory activity of curcumin and its degradation product 4-vinyl guaiacol.

Curcumin is a secondary plant metabolite present in Curcuma longa L. Since curcumin is widely used as a food colorant in thermally processed food it may undergo substantial chemical changes which in turn could affect its biological activity. In the current study, curcumin was roasted at 180 °C up to 70 minutes and its kinetic of degradation was analyzed by means of HPLC-PDA and LC-MS, respectively. Roasting of curcumin resulted in the formation of the degradation products vanillin, ferulic acid, and 4-vinyl guaiacol. In cultured hepatocytes roasted curcumin as well as 4-vinyl guaiacol enhanced the transactivation of the redox-regulated transcription factor Nrf2, known to be centrally involved in cellular stress response and antioxidant defense mechanisms. The antioxidant enzyme paraoxonase 1 was induced by roasted curcumin and 4-vinyl guaiacol. Furthermore, roasted curcumin and 4-vinyl guaiacol decreased interleukin-6 gene expression in lipopolysaccharide stimulated murine macrophages. Current data suggest that curcumin undergoes degradation due to roasting and its degradation product exhibit significant biological activity in cultured cells.

Identification of the metabolites of gigantol in rat urine by ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Gigantol is a typical bibenzyl compound isolated from Dendrobii Caulis that has been widely used as a medicinal herb in China for the treatment of diabetic cataract, cancer and arteriosclerosis obliterans and as a tonic for stomach nourishment, saliva secretion promotion and fever reduction. However, few studies have been carried out on its in vivo metabolism. In the present study, a rapid and sensitive method based on ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) in positive ion mode was developed and applied to identify the metabolites of gigantol in rat urine after a single oral dose (100 mg/kg). Chromatographic separation was performed on an Acquity UPLC HSS T3 column (100 × 2.1 mm i. d., 1.8 µm) using acetonitrile and 0.1% aqueous formic acid as mobile phases. A total of 11 metabolites were detected and identified as all phase II metabolites. The structures of the metabolites were identified based on the characteristics of their MS, MS(2) data and chromatographic retention times. The results showed that glucuronidation is the principal metabolic pathway of gigantol in rats. The newly identified metabolites are useful to understand the mechanism of elimination of gigantol and, in turn, its effectiveness and toxicity. As far as we know, this is the first attempt to investigate the metabolic fate of gigantol in vivo.

Curcumin analogue identified as hyaluronan export inhibitor by virtual docking to the ABC transporter MRP5.

Hyaluronan is overproduced in many diseases including metastasis, inflammation or ischemia, but there is no drug to attenuate hyaluronan production. Hyaluronan is exported from fibroblasts by the multidrug resistance associated protein 5 (MRP5) which is inhibited by the plant phenols curcumin or xanthohumol. We performed virtual docking and chemical synthesis of analogues to optimize the inhibitors. The AutoDock software was used to identify the binding cavity within the open conformation of MRP5. Inhibitory plant phenols bound to the ATP binding site between the two nucleotide binding domains NBD1 and NBD2. This binding cavity was chosen to screen about 120 derivatives and analogues. The superior hyaluronan export inhibitor was 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one (hylin). It inhibited hyaluronan export from fibroblasts with an IC50 of 4.9 µM. Hylin is a minor component in natural curcumin preparations and has previously been described as anti-metastatic and anti-inflammatory. Since curcumin itself is unstable under physiological conditions, the active component for many cell biological and pharmaceutical effects of natural curcumin preparations could be hylin that acts by hyaluronan export inhibition.

An iron(III) tetradentate monoamido complex as a nonheme iron-based peroxidase mimetic.

A mononuclear iron(III) complex of a noncyclic tetradentate monoamido ligand, Fe(III)mpaq, catalyses the oxidation of Orange II, guaiacol, ABTS and Amplex Red with H2O2 in aqueous solutions at neutral pH. Under identical conditions, other structurally related nonheme iron complexes showed only negligible activities.

Small molecules interacting with α-synuclein: antiaggregating and cytoprotective properties.

Curcumin, a dietary polyphenol, has shown a potential to act on the symptoms of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, as a consequence of its antioxidant, anti-inflammatory and anti-protein aggregation properties. Unfortunately, curcumin undergoes rapid degradation at physiological pH into ferulic acid, vanillin and dehydrozingerone, making it an unlikely drug candidate. Here, we evaluated the ability of some curcumin by-products: dehydrozingerone (1), its O-methyl derivative (2), zingerone (3), and their biphenyl analogues (4-6) to interact with α-synuclein (AS), using CD and fluorescence spectroscopy. In addition, the antioxidant properties and the cytoprotective effects in rat pheochromocytoma (PC12) cells prior to intoxication with H2O2, MPP+ and MnCl2 were examined while the Congo red assay was used to evaluate the ability of these compounds to prevent aggregation of AS. We found that the biphenyl zingerone analogue (6) interacts with high affinity with AS and also displays the best antioxidant properties while the biphenyl analogues of dehydrozingerone (4) and of O-methyl-dehydrozingerone (5) are able to partially inhibit the aggregation process of AS, suggesting the potential role of a hydroxylated biphenyl scaffold in the design of AS aggregation inhibitors.

[10]-Gingerdiols as the major metabolites of [10]-gingerol in zebrafish embryos and in humans and their hematopoietic effects in zebrafish embryos.

Gingerols are a series of major constituents in fresh ginger with the most abundant being [6]-, [8]-, and [10]-gingerols (6G, 8G, and 10G). We previously found that ginger extract and its purified components, especially 10G, potentially stimulate both the primitive and definitive waves of hematopoiesis (blood cell formation) in zebrafish embryos. However, it is still unclear if the metabolites of 10G retain the efficacy of the parent compound toward pathological anemia treatment. In the present study, we first investigated the metabolism of 10G in zebrafish embryos and then explored the biotransformation of 10G in humans. Our results show that 10G was extensively metabolized in both zebrafish embryos and humans, in which two major metabolites, (3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol, were identified by analysis of the MS(n) spectra and comparison to authentic standards that we synthesized. After 24 h of treatment of zebrafish embryos, 10G was mostly converted to its metabolites. Our results clearly indicate that the reductive pathway is a major metabolic route for 10G in both zebrafish embryos and humans. Furthermore, we investigated the hematopoietic effect of 10G and its two metabolites, which show similar hematopoietic effects as 10G in zebrafish embryos.

Combretazet-3 a novel synthetic cis-stable combretastatin A-4-azetidinone hybrid with enhanced stability and therapeutic efficacy in colon cancer.

In recent years an extensive series of synthetic combretastatin A-4 (CA-4)-azetidinone (ß-lactam) hybrids were designed and synthesised with a view to improve the stability, therapeutic efficacy and aqueous solubility of CA-4. Lead compounds containing a 3,4,5-trimethoxy aromatic ring at position 1 and a variety of substitution patterns at positions 3 and 4 of the ß-lactam ring were screened in three adenocarcinoma-derived colon cancer cell lines (CT-26, Caco-2 and the CA-4 resistant cell line, HT-29). In both CT-26 and Caco-2 cells all ß-lactam analogues analysed displayed potent therapeutic efficacy within the nanomolar range. Substitution of the ethylene bridge of CA-4 with the ß-lactam ring together with the aforementioned aryl substitutions improved the therapeutic efficacy of CA-4 up to 300­fold in the combretastatin refractory HT-29 cells. The lead compound combretazet-3 (CAZ-3); chemical name [4-(3-hydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one] demonstrated improved chemical stability together with enhanced therapeutic efficacy as compared with CA-4 whilst maintaining the natural biological properties of CA-4. Furthermore, CAZ-3 demonstrated significant tumour inhibition in a murine model of colon cancer. Our results suggest that combretastatin-azetidinone hybrids represent an effective novel therapy for the treatment of combretastatin resistant carcinomas.

A Solanum lycopersicum catechol-O-methyltransferase involved in synthesis of the flavor molecule guaiacol.

O-methyltransferases (OMT) are important enzymes that are responsible for the synthesis of many small molecules, which include lignin monomers, flavonoids, alkaloids, and aroma compounds. One such compound is guaiacol, a small volatile molecule with a smoky aroma that contributes to tomato flavor. Little information is known about the pathway and regulation of synthesis of guaiacol. One possible route for synthesis is via catechol methylation. We identified a tomato O-methyltransferase (CTOMT1) with homology to a Nicotiana tabacum catechol OMT. CTOMT1 was cloned from Solanum lycopersicum cv. M82 and expressed in Escherichia coli. Recombinant CTOMT1 enzyme preferentially methylated catechol, producing guaiacol. To validate the in vivo function of CTOMT1, gene expression was either decreased or increased in transgenic S. lycopersicum plants. Knockdown of CTOMT1 resulted in significantly reduced fruit guaiacol emissions. CTOMT1 overexpression resulted in slightly increased fruit guaiacol emission, which suggested that catechol availability might limit guaiacol production. To test this hypothesis, wild type (WT) and CTOMT1 that overexpress tomato pericarp discs were supplied with exogenously applied catechol. Guaiacol production increased in both WT and transgenic fruit discs, although to a much greater extent in CTOMT1 overexpressing discs. Finally, we identified S. pennellii introgression lines with increased guaiacol content and higher expression of CTOMT1. These lines also showed a trend toward lower catechol levels. Taken together, we concluded that CTOMT1 is a catechol-O-methyltransferase that produces guaiacol in tomato fruit.

4-fluoro-2-methoxyphenol, an apocynin analog with enhanced inhibitory effect on leukocyte oxidant production and phagocytosis.

Apocynin, a methoxy-substituted catechol (4-hydroxy-3-methoxyacetophenone), originally extracted from the roots of Picrorhiza kurroa, has been extensively used as a non-toxic inhibitor of the multienzymatic complex NADPH oxidase. We discovered that the analogous methoxy-substituted catechol, 4-Fluoro-2-methoxyphenol (F-apocynin), in which the acetyl group present in apocynin was changed to a fluorine atom, was significantly more potent as an inhibitor of NADPH oxidase activity, myeloperoxidase (MPO) chlorinating activity and phagocytosis of microorganisms by neutrophils; it was also as potent as apocynin in inhibiting tumor necrosis factor-alpha (TNFα) release by peripheral blood mononuclear cells. We attribute the increased potency of F-apocynin to its increased lipophilicity, which could facilitate the passage of the drug through the cell membrane. The inhibition of MPO chlorination activity, phagocytosis and TNFα release shows that apocynin and F-apocynin actions are not restricted to reactive oxygen species inhibition, but further studies are needed to clarify if these mechanisms are related. Like apocynin, F-apocynin did not show cell toxicity, and is a strong candidate for use in the treatment of inflammatory diseases.

Cloning, sequencing, and overexpression in Escherichia coli of the Enterobacter sp. Px6-4 gene for ferulic acid decarboxylase.

Ferulic acid decarboxylase (FADase) can catalyze the transformation of ferulic acid into 4-vinyl guaiacol via decarboxylation in microorganisms. In this study, a gene encoding FADase was first isolated from the bacterium Enterobacter sp. Px6-4 using degenerate primers and a genome walking technique. The putative encoding gene (fad) of FADase consists of 507-bp nucleotides, coding a polypeptide of 168 amino acid residues. In addition, a putative gene encoding the transcriptional regulator was identified from the upstream of the fad gene. The deduced peptide sequence of the FADase from Enterobacter sp. Px6-4 showed a 51.2-53.3% sequence identity to decarboxylases from other bacteria. The gene fad was successfully expressed in Escherichia coli BL21, and the recombinant FADase was purified as a protein of ca. 23 kDa with an optimal activity at pH 4.0 and 28 °C. The purified FADase could convert ferulic acid to 4-vinyl guaiacol effectively, and its hydrolytic activity could be inhibited by Cu(2+) (99%) and Hg(2+) (99.5%). A phylogenetic analysis of the FADase protein from bacteria revealed several different clades. Our result provided a basis for further studies of the ferulic acid transformation pathway and for enhanced production of vanillin in the future.

Myeloperoxidase-dependent oxidation of etoposide in human myeloid progenitor CD34+ cells.

Etoposide is a widely used anticancer drug successfully used for the treatment of many types of cancer in children and adults. Its use, however, is associated with an increased risk of development of secondary acute myelogenous leukemia involving the mixed-lineage leukemia (MLL) gene (11q23) translocations. Previous studies demonstrated that the phenoxyl radical of etoposide can be produced by action of myeloperoxidase (MPO), an enzyme found in developing myeloid progenitor cells, the likely origin for myeloid leukemias. We hypothesized, therefore, that one-electron oxidation of etoposide by MPO to its phenoxyl radical is important for converting this anticancer drug to genotoxic and carcinogenic species in human CD34(+) myeloid progenitor cells. In the present study, using electron paramagnetic resonance spectroscopy, we provide conclusive evidence for MPO-dependent formation of etoposide phenoxyl radicals in growth factor-mobilized CD34(+) cells isolated from human umbilical cord blood and demonstrate that MPO-induced oxidation of etoposide is amplified in the presence of phenol. Formation of etoposide radicals resulted in the oxidation of endogenous thiols, thus providing evidence for etoposide-mediated MPO-catalyzed redox cycling that may play a role in enhanced etoposide genotoxicity. In separate studies, etoposide-induced DNA damage and MLL gene rearrangements were demonstrated to be dependent in part on MPO activity in CD34(+) cells. Together, our results are consistent with the idea that MPO-dependent oxidation of etoposide in human hematopoietic CD34(+) cells makes these cells especially prone to the induction of etoposide-related acute myeloid leukemia.