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Cyanobacteria produce high levels of ergothioneine
Abstract
Ergothioneine (ET) is a unique natural antioxidant. We have examined the origin of ET in zebrafish. There was virtually no ET, measured by LC–MS, in most tank vegetation (plant, green and red alga). However, ET was detected in a Phormidium sample, a cyanobacterium. In commercial fish feed preparations, ET content increased with the content of cyanobacteria Arthrospira platensis or Arthrospira maxima (Spirulina). High levels of ET (up to 0.8mg per g dry mass) were measured in cyanobacteria preparations sold as dietary supplements for humans and in fresh Scytonema and Oscillatoria cultures. Cyanobacteria contained as much ET as King Oyster mushrooms (Pleurotus eryngii). All samples with substantial ET content also contained the biosynthesis intermediate hercynine; this strongly suggests that cyanobacteria synthesise ET de novo. In conclusion, our data establish that cyanobacteria can produce high levels of ergothioneine. Spirulina is a novel, safe, accessible, and affordable source of ergothioneine for humans.
... Ergothioneine (EGT) is a naturally occurring hydrophilic amino acid that is only synthesized by selected bacteria and fungi (Genghof, 1970;Genghof and Damme, 1964;Pfeiffer et al., 2011). It is a derivative of hercynine in thiol-thione tautomerism form ( Fig. 1), that in solution and at physiological state exists predominantly in the thione form , providing greater stability. ...
... EGT can only be obtained through diet, therefore prompting us to explore the dietary sources of EGT. Since EGT is only synthesized by certain bacteria and fungi (Genghof, 1970;Genghof and Damme, 1964;Pfeiffer et al., 2011), it is predictable that in mushrooms, the fruiting body is the supreme source of EGT. A systematic analysis of EGT occurrences in different types of mushrooms has been reported (Kalaras et al., 2017). ...
Healthy ageing is a crucial process that needs to be highlighted as it affects the quality of lifespan. An increase in oxidative stress along with ageing is the major factor related to the age-associated diseases, especially neurodegenerative disorders. An antioxidant-rich diet has been proven to play a significant role in the ageing process. Targeting ageing mechanisms could be a worthwhile approach to improving health standards. Ergothioneine (EGT), a hydrophilic compound with specific transporter known as OCTN1, has been shown to exert anti-ageing properties. In addition to its antioxidant effect, EGT has been reported to have anti-senescence, anti-inflammatory and anti-neurodegenerative properties. This review aims to define the pivotal role of EGT in major signalling pathways in ageing such as insulin/insulin-like growth factor (IGF) signalling (IIS), sirtuin 6 (SIRT6) and mammalian target of rapamycin complex (mTOR) pathways. The review further discusses evidence of EGT on neurodegeneration in its therapeutic context in various model organisms, providing new insights into improving health. In conclusion, an ergothioneine-rich diet may be beneficial in preventing age-related diseases, resulting in a healthy ageing population.
... The levels of 10 acquired by G. elata correlated with those in A. mellea [79]. In order to examine the organ-specific accumulation and the physiological effects of 10 in zebrafish, a reliable dietary source was needed [80]. Analysis of various options revealed oyster mushrooms, the alga Arthrospira platensis (spirulina), and the cyanobacterium Oscillatoria sp. to be leading sources, with other cyanobacteria also producing 10 and its precursor 13. ...
... Analysis of various options revealed oyster mushrooms, the alga Arthrospira platensis (spirulina), and the cyanobacterium Oscillatoria sp. to be leading sources, with other cyanobacteria also producing 10 and its precursor 13. Indeed, cyanobacteria, such as spirulina, are regarded as a promising source of 10 for the future [80,81]. ...
The history, chemistry, biology, and biosynthesis of the globally occurring histidine-derived alkaloids ergothioneine (10), ovothiol A (11), and selenoneine (12) are reviewed comparatively and their significance to human well-being is discussed.
... This is partly supported by the finding that glutathione (gshB)-deficient Synechocystis sp. PCC 6803 mutants were unable to produce ergothioneine (Table 2; Narainsamy et al., 2015;Pfeiffer et al., 2011;Pluskal et al., 2014). ...
Many of the world’s freshwater ecosystems suffer from cyanobacteria-mediated blooms and their toxins. However, a mechanistic understanding of why and how Microcystis aeruginosa dominates over other freshwater cyanobacteria during warmer summers is lacking. This paper utilizes comparative genomics with other cyanobacteria and literature reviews to predict the gene functions and genomic architectures of M. aeruginosa based on complete genomes. The primary aim is to understand this species' survival and competitive strategies in warmer freshwater environments. M. aeruginosa strains exhibiting a high proportion of insertion sequences (~ 11%) possess genomic structures with low synteny across different strains. This indicates the occurrence of extensive genomic rearrangements and the presence of many possible diverse genotypes that result in greater population heterogeneities than those in other cyanobacteria in order to increase survivability during rapidly changing and threatening environmental challenges. Catalase-less M. aeruginosa strains are even vulnerable to low light intensity in freshwater environments with strong ultraviolet radiation. However, they can continuously grow with the help of various defense genes (e.g., egtBD, cruA, and mysABCD) and associated bacteria. The strong defense strategies against biological threats (e.g., antagonistic bacteria, protozoa, and cyanophages) are attributed to dense exopolysaccharide (EPS)-mediated aggregate formation with efficient buoyancy and the secondary metabolites of M. aeruginosa cells. Our review with extensive genome analysis suggests that the ecological vulnerability of M. aeruginosa cells can be overcome by diverse genotypes, secondary defense metabolites, reinforced EPS, and associated bacteria.
... Ergothioneine, a thiohistidine derivative, is ubiquitously present in most plants and mammals and provides multiple health benefits, such as antioxidative, antiaging and anti-inflammatory effects [5,6]. Few bacteria and non-yeast fungi can synthesize ergothioneine [7,8]. A sulfur-to-selenium substitution in ergothioneine results in the analog selenoneine. ...
Selenoneine, an ergothioneine analog, is important for antioxidation and detoxification. SenB and SenA are two crucial enzymes that form carbon–selenium bonds in the selenoneine biosynthetic pathway. To investigate their underlying catalytic mechanisms, we obtained complex structures of SenB with its substrate UDP-N-acetylglucosamine (UDP-GlcNAc) and SenA with N-α-trimethyl histidine (TMH). SenB adopts a type-B glycosyltransferase fold. Structural and functional analysis of the interaction network at the active center provide key information on substrate recognition and suggest a metal-ion-independent, inverting mechanism is utilized for SenB-mediated selenoglycoside formation. Moreover, the complex structure of SenA with TMH and enzymatic activity assays highlight vital residues that control substrate binding and specificity. Based on the conserved structure and substrate-binding pocket of the type I sulfoxide synthase EgtB in the ergothioneine biosynthetic pathway, a similar reaction mechanism was proposed for the formation of C–Se bonds by SenA. The structures provide knowledge on selenoneine synthesis and lay groundwork for further applications of this pathway.
... By contrast, production of EGT in microbial cell factories would provide a sustainable low-cost alternative to its current manufacturing processes. According to available reports, a variety of bacteria and fungi are able to act as producers of EGT (Supporting Information Table S1), including cyanobacteria with a yield of 0.8 mg g −1 dry cell weight (DCW), 26 Methylobacterium aquaticum with a yield of 10 mg L −1 , 7 and Rhodotorula mucilaginosa with a yield of 24 mg L−1. 27 In addition, model strains such as Escherichia coli and Saccharomyces cerevisiae have also been successfully constructed as engineered strains for EGT production. ...
BACKGROUND
Ergothioneine (EGT) is a high‐value food functional factor that cannot be synthesized by humans and other vertebrates, and the low yield limits its application.
RESULTS
In this study, the optimal fermentation temperature, fermentation time, initial pH, inoculum age, and inoculation ratio on EGT biosynthesis of Rhodotorula mucilaginosa DL‐X01 were optimized. In addition, the effects of three key precursor substances – histidine, methionine, and cysteine – on fungal EGT synthesis were verified. The optimal conditions were further obtained by response surface optimization. The EGT yield of R. mucilaginosa DL‐X01 under optimal fermentation conditions reached 64.48 ± 2.30 mg L⁻¹ at shake flask fermentation level. Finally, the yield was increased to 339.08 ± 3.31 mg L⁻¹ (intracellular) by fed‐batch fermentation in a 5 L bioreactor.
CONCLUSION
To the best of our knowledge, this is the highest EGT yield ever reported in non‐recombinant strains. The fermentation strategy described in this study will promote the efficient biosynthesis of EGT in red yeast and its sustainable production in the food industry. © 2024 Society of Chemical Industry.
... Moreover, ergothioneine present in SP is known as a potent peroxynitrite, hypochlorous acid, and hydroxyl radical scavenger [47]. It can scavenge nonfree radical oxidizing species [48], decrease lipid peroxidation, and suppress IL-8, TNF-α, and IL-6 expression in epithelial cells [47]. Some studies have suggested that ergothioneine can maintain the proper ratio of GSH to oxidized glutathione and in vivo thiol defenses [47].Reduction of the MPO level of colonic tissue in our study showed the ability of honey, SP, and concurrent administration of honey with SP to inhibit neutrophil infiltration in inflamed colonic tissue similar to mesalazine and SSZ. ...
 Background: Antioxidant therapy has gained attention for the treatment of ulcerative coliÂtis (UC). The excessive generation of reactive oxygen/nitrogen species in the gastrointestinal tract increases oxidative stress, thereby leading to antioxidant defense depletion, lipid peroxÂidation, inflammation, tissue damage, and ulceration. Spirulina platensis (SP) and honey are excellent sources of potent antioxidants such as polyphenols and other bioactive compounds. We aimed to investigate antioxidant and anti-inflammatory effects of honey and SP in comÂparison with sulfasalazine (SSZ) and mesalazine on acetic acid-induced colitis (AA-colitis) in rats. Materials and Methods: Fifty-six Sprague Dawley male rats were allocated to sevÂen groups, with each group comprising eight rats. UC was induced, except in normal conÂtrols (NC). All groups received oral treatments for seven days. The normal saline solution of 2 mL was intrarectally administered to the NC group. The AA-colitis and NC groups received 2 mL acetic acid intrarectally as a single dose and 2 mL normal saline for seven consecutive days orally. The mesalazine group received 100 mg/kg mesalazine, the SSZ group 360 mg/kg SSZ, the honey or H group 1 mL honey diluted with 1 mL distilled water, the SH group 1g/kg SP and 1 mL honey, and the SP group 1g/kg SP. After clinical activity score assessment, the rats were sacrificed. Colonic weight/length ratio, prostaglandin E2 (PGE2), myeloperoxidase (MPO), nitric oxide (NO), malondialdehyde (MDA), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), glutathione peroxidase (GPx), total antioxidant capacity (TAC), reduced glutathione (GSH), and superoxide dismutase (SOD) were measured. Colonic histopathological changes were observed microscopically. Results: Treatment of UC with SP, honey, and combination regimen significantly reduced TNF-α, IL-1β, IL-6, MDA, MPO, NO, and PGE2, and increased TAC, GSH, GPx, and SOD in interventional groups compared to the AA-colitis group (P<0.05). Conclusion: Honey and SP might be beneficial food suppleÂments for medical nutrition therapy in UC. [GMJ.2019;8:e1095]
... Hence it is noteworthy to study the role of mushrooms as a leading source of antioxidant-rich food. EGT can only be obtained through dietary sources as it is only produced by certain fungi and bacteria (Pfeiffer et al., 2011). Extra attention has also been diverted to EGT as a potential antioxidant due to the presence of specific cation transporter type 1 (OCTN1) also known as ergothioneine transporter (ETT) (Gründemann, 2012). ...
... Among the potential bioactive compounds contained in foods, Lergothioneine (ERT, 2-mercaptohistidine trimethylbetaine), an unusual water-soluble thiol-imidazole amino acid, has been gaining increased interest over the last decade [11,12]. This thiourea derivative of histidine is not biosynthesized by plants and animals but only by a few organisms, such as mycobacteria [13], cyanobacteria [14], and non-yeast-like fungi, including edible mushrooms [15,16]. In humans, a specific transporter (ETT, human gene SLC22A4) facilitates the cellular uptake and accumulation of dietary ERT [17], particularly in organs and tissues that are exposed to oxidative stress and inflammatory processes [18]. ...
Among plasma metabolites linked with a health-conscious food pattern (HCFP) identified in the Malmö Diet and Cancer epidemiological study, circulating ergothioneine (ERT) concentrations exhibited the strongest independent association with reduced risk of cardiometabolic disease and all-cause mortality and were also related to alcohol consumption. Thus, we first assessed whether alcohol intake and ERT were similarly associated in participants of the Hunter Community Study (HCS) that did not follow an HCFP-based diet. Then, we sought to identify the presence of associations with some biomarkers associated with cardiovascular disease. In a multivariable adjusted, robust regression analysis, compared to non-drinkers, safe drinkers had, on average, a serum ERT concentration 0.112 (95% CI: 0.0–0.225; P = 0.051) units higher and moderate-hazardous drinkers had a serum ERT concentration 0.240 (95% CI: 0.093–0.387; P = 0.001) units higher. Moreover, stepwise multiple linear regression shows that age (P = 0.025), and asymmetric dimethyl-l-arginine (ADMA) (P = 0.001) were independently associated with serum ERT concentrations, independently of age, sex, education, household income, marital status, and health status of participants, or possible alcohol-induced organ damage. The relationship between ERT and ADMA offers a potential explanation for the interplay between ERT, and decreased risk of cardiometabolic disease and all-cause mortality. Also, it provides new mechanistic insights into the association between alcohol consumption and cardiovascular diseases, possibly mediated by ADMA metabolic pathways.
... L-Erg is an intracellular antioxidant molecule with cytoprotective and anti-inflammatory effects which role associated with pathologies has been described in several organs [27][28][29][30], including the kidney [31][32][33]. It is a natural thiol molecule synthesized by cyanobacteria [34], mycobacteria [35] and non-yeast-fungi [36], that animals must absorb from diet. However, although animals cannot synthesize L-Erg, the existence of a specific transporter (OCTN1-SLC22A4) in most tissues that ensures its absorption and retention in the body [37], L-Erg long half-life [38] and its ability to accumulate in injured tissues [39], suggest that L-Erg has an important physiological role. ...
The high recurrence rate of cystine lithiasis observed in cystinuria patients highlights the need for new therapeutic options to address this chronic disease. There is growing evidence of an antioxidant defect in cystinuria, which has led to test antioxidant molecules as new therapeutic approaches. In this study, the antioxidant l-Ergothioneine was evaluated, at two different doses, as a preventive and long-term treatment for cystinuria in the Slc7a9-/- mouse model. l-Ergothioneine treatments decreased the rate of stone formation by more than 60% and delayed its onset in those mice that still developed calculi. Although there were no differences in metabolic parameters or urinary cystine concentration between control and treated mice, cystine solubility was increased by 50% in the urines of treated mice. We also demonstrate that l-Ergothioneine needs to be internalized by its transporter OCTN1 (Slc22a4) to be effective, as when administrated to the double mutant Slc7a9-/-Slc22a4-/- mouse model, no effect on the lithiasis phenotype was observed. In kidneys, we detected a decrease in GSH levels and an impairment of maximal mitochondrial respiratory capacity in cystinuric mice that l-Ergothioneine treatment was able to restore. Thus, l-Ergothioneine administration prevented cystine lithiasis in the Slc7a9-/- mouse model by increasing urinary cystine solubility and recovered renal GSH metabolism and mitochondrial function. These results support the need for clinical trials to test l-Ergothioneine as a new treatment for cystinuria.
... Many cyanobacteria synthesize large amounts of the EGT antioxidant [61, [168][169][170][171], and fishes that feed, to some extent, on cyanobacteria are thus provided with plenty of EGT [137]. This finding is interesting because there is an increasing demand for EGT [135,140], and extracting it and chemically synthesizing it from edible fungi has a high cost and low yield [137,139]. ...
From bacteria to plants and humans, the glutathione system plays a pleiotropic role in cell defense against metabolic, oxidative and metal stresses. Glutathione (GSH), the γ-L-glutamyl-L-cysteinyl-glycine nucleophile tri-peptide, is the central player of this system that acts in redox ho-meostasis, detoxification and iron metabolism in most living organisms. GSH directly scavenges diverse reactive oxygen species (ROS), such as singlet oxygen, superoxide anion, hydrogen perox-ide, hydroxyl radical, nitric oxide and carbon radicals. It also serves as a cofactor for various enzymes , such as glutaredoxins (Grxs), glutathione peroxidases (Gpxs), glutathione reductase (GR) and glutathione-S-transferases (GSTs), which play crucial roles in cell detoxication. This review summarizes what is known concerning the GSH-system (GSH, GSH-derived metabolites and GSH-dependent enzymes) in selected model organisms (Escherichia coli, Saccharomyces cerevisiae, Arabidop-sis thaliana and human), emphasizing cyanobacteria for the following reasons. Cyanobacteria are environmentally crucial and biotechnologically important organisms that are regarded as having evolved photosynthesis and the GSH system to protect themselves against the ROS produced by their active photoautotrophic metabolism. Furthermore, cyanobacteria synthesize the GSH-derived metabolites, ergothioneine and phytochelatin, that play crucial roles in cell detoxication in humans and plants, respectively. Cyanobacteria also synthesize the thiol-less GSH homologs ophthalmate and norophthalmate that serve as biomarkers of various diseases in humans. Hence, cyanobacteria are well-suited to thoroughly analyze the role/specificity/redundancy of the players of the GSH-system using a genetic approach (deletion/overproduction) that is hardly feasible with other model organisms (E. coli and S. cerevisiae do not synthesize ergothioneine, while plants and humans acquire it from their soil and their diet, respectively).
... Compounds derived from amino acids, like γ-glutamylcysteine (γ-GC), PCs and ergothioneine, may represent more recent additions to the metal chelating repertoire of cells. Ergothioneine, derived from histidine, is a competent Cu chelator found in select filamentous fungi (Genghof, 1970;Zhang et al., 2022) and some cyanobacteria (Genghof, 1970;Pfeiffer et al., 2011;Zhang et al., 2022). In yeast, the dipeptide of γ-glutamylcysteine (γ-GC), formerly also known as cadystins (Kondo et al., 1985;Hayashi et al., 1991) which also accumulates in Zn limited Chlamydomonas (likely driven by the high Cys content), but to a much lower level than (which was not certified by peer review) is the author/funder. ...
Growth of Chlamydomonas reinhardtii in zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. We show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding “first responder” proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, γ-glutamylcysteine and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ~80-fold, corresponding to ~ 2.8 × 10 ⁹ molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is an in vivo Cu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.
... Fermented foods can also be significant sources of ergothioneine (Table 1), with the concentration of ergothioneine dependent on the different species of bacteria used in fermentation (20) . In addition, spirulina, the dried biomass of cyanobacteria (Arthrospira platensis) sold commonly as a dietary supplement, contains relatively high amounts of ergothioneine (21) . It is interesting to observe that ergothioneine is found in high amounts in certain plants, mushrooms and spirulina, that have been used and investigated for their medicinal properties (14,22,23) . ...
Ergothioneine is a naturally occurring amino acid and thiol antioxidant found in high amounts in mushrooms and fermented foods. Humans and animals acquire ergothioneine from the diet through the pH-dependent activity of a membrane transporter, the large solute carrier 22A member 4 (SLC22A4), expressed on the apical membrane of the small intestine. The SLC22A4 transporter also functions in the renal reabsorption of ergothioneine in the kidney, with avid absorption and retention of ergothioneine from the diet observed in both animals and humans. Ergothioneine is capable of scavenging a diverse range of reactive oxygen and nitrogen species, has metal chelation properties, and is predicted to directly regulate nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Although not lethal, the genetic knockout of the SLC22A4 gene in multiple organisms increases susceptibility to oxidative stress, damage and inflammation; in agreement with a large body of preclinical data suggesting the physiological function of ergothioneine is as a cellular antioxidant and cytoprotectant agent. In humans, blood levels of ergothioneine decline after the age of 60 years, and lower levels of ergothioneine are associated with more rapid cognitive decline. Conversely, high plasma ergothioneine levels have been associated with significantly reduced cardiovascular mortality and overall mortality risks. In this horizon’s manuscript, we review evidence suggesting critical roles for dietary ergothioneine in healthy ageing and the prevention of cardiometabolic disease. We comment on some of the outstanding research questions in the field and consider the question of whether or not ergothioneine should be considered a conditionally essential micronutrient.
... In nature, many organisms such as bacteria, fungi, yeast, cyanobacteria, actinomycetes and plants can synthesize EGT [15,16], but only mushrooms can accumulate high levels of EGT, such as Pleurotus eryngii, Lentinus edodes, Pleurotus ostreatus, Agaricus bisporus, Pleutotus citrinopileatus, Cantharellus cibarius and Boletus edulis [17][18][19][20]. In most of the fungi, only two enzymes, Egt1 and Egt2, are required for ergothioneine synthesis [21]. ...
Ergothioneine is a natural and safe antioxidant that plays an important role in anti-aging and the prevention of various diseases. This study aimed to report on a kind of medicinal mushroom of Panus conchatus with great potential for the bioproduction of ergothioneine. The effect of different nutritional and environmental conditions on the growth of Panus conchatus and ergothioneine production were investigated. Molasses and soy peptone were found to promote cell growth of Panus conchatus and enhance ergothioneine accumulation. Adding precursors of histidine, methionine and cysteine could increase ergothioneine production and the highest ergothioneine concentration of 148.79 mg/L was obtained. Finally, the extraction and purification processes were also established to obtain the crude ergothioneine extract for further antioxidant property evaluation. The ergothioneine from Panus conchatus showed high antioxidant activity with good stability in a lower pH environment. This study provided a new strain and process for the bioproduction of ergothioneine.
... Mycobacterium tuberculosis/smegmatis [31,32]), Cyanobacteria (e.g. Arthrospira platensis/maxima [33]), Proteobacteria (e.g. Burkholderia pseudomallei/thailandensis [34] and Methylobacterium [35]) and Clorobiota (namely the obligate anaerobe Chlorobium limicola [36]). ...
The dietary thione‐thiol, ergothioneine (ET), accumulates in human and animal tissues and may play important roles in disease prevention. ET biosynthesis has only been described in fungi and certain bacteria, and humans and animals are widely assumed to accumulate ET solely from diet. However, a recent study suggested that Lactobacillus/Limosilactobacillus reuteri, a commensal gut bacterium, may produce ET, thereby protecting the host against social defeat stress and sleep disturbances. Upon our further investigation, no evidence of ET biosynthesis was observed in L. reuteri when a heavy‐labelled histidine precursor was administered. Instead, we discovered that L. reuteri avidly accumulates ET. This observation may indicate a possible mechanism by which the gut microbiota could influence tissue levels of ET in the host. The commensal bacterium Lactobacillus reuteri does not appear to synthesise ergothioneine, nor does it possess the necessary ergothioneine biosynthesis enzymes. However, this study demonstrates the bacterium can accumulate ergothioneine from its surroundings (e.g., dietary ergothioneine in the host gut) and may demonstrate how L. reuteri, and other gut microbiota, may influence the uptake and accumulation of ergothioneine in the host.
... Ergothioneine (EGT) is a thiohistidine derivative that ubiquitously exists in live cells and participates in various physiological and biochemical processes (Beliaeva et al., 2021;Borodina et al., 2020;Han et al., 2021;Qiu et al., 2021;Zhao et al., 2015). It can be endogenously synthesized by mushrooms (Kalaras et al., 2017), cyanobacteria (Pfeiffer et al., 2011), Mycolicibacterium smegmatis (Seebeck, 2010), and Neurospora crassa , but it does not exist in plants or animals (Borodina et al., 2020). A significant source of EGT was found even in strictly anaerobic green sulfur bacteria (Burn et al., 2017), indicating that this simple metabolite might be crucial to life. ...
Ergothioneine (EGT) represents valuable protective functions for humans, but EGT from the diet cannot meet daily requirements. Although the heterologous synthesis of EGT had been achieved, it is still a challenge to obtain stable and high‐yield EGT‐producing cell factories. Here, after the co‐overexpression of the EGT synthetic gene cluster and hisG, hisC, and allB1 in Mycolicibacterium neoaurum, the natural EGT titer was increased by 7.2‐folds. However, the degradation problem of EGT in large‐scale fermentation needs to be urgently solved. A putative lyase gene Mn_3042 was inactivated, thus inhibiting the product degradation and increasing the EGT titer by 21%. Moreover, the enhancement of S‐adenosyl‐ l‐methionine regeneration further increased EGT titer by 28%. After optimization of fed‐batch fermentation, the yield of EGT was boosted to 1.56 g/L with a productivity of 7.2 mg/L/h. This study provides a systematic engineering strategy for developing EGT‐producing cell factories. M. neoaurum was systematically engineered to produce ergothioneine. Regulating of a lyase and SAM regeneration boosted the production of ergothioneine. The engineered M. neoaurum realized the production of 1.56 g/L ergothioneine. M. neoaurum was systematically engineered to produce ergothioneine. Regulating of a lyase and SAM regeneration boosted the production of ergothioneine. The engineered M. neoaurum realized the production of 1.56 g/L ergothioneine.
... Ergothioneine (ERG), discovered in 1909, is an unusual amino acid derived from histidine and contains a sulfur atom on the imidazole ring (Tanret, 1909). While ERG is universally present in high organisms, such as plants and mammals (Halliwell et al., 2018;Melville, 1959), it is only biosynthesized by bacteria and fungi, including cyanobacteria, actinobacteria, and basidiomycete mushrooms (Genghof, 1970;Genghof et al., 1956;Kalaras et al., 2017;Pfeiffer et al., 2011). For humans, the main dietary source of ERG is mushrooms. ...
Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%–51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.
... Ergothioneine (ERG) is a water-soluble amino acid derived from dietary sources usually synthesized by fungi and bacteria [85]. The antioxidant potential of ERG has been evaluated in several disease models [86,87]. ...
Despite considerable research efforts over the past few decades, the pathology of preeclampsia (PE) remains poorly understood with no new FDA-approved treatments. There is a substantial amount of work being conducted by investigators around the world to identify targets to develop therapies for PE. Oxidative stress has been identified as one of the crucial players in pathogenesis of PE and has garnered a great deal of attention by several research groups including ours. While antioxidants have shown therapeutic benefit in preclinical models of PE, the clinical trials evaluating antioxidants (vitamin E and vitamin C) were found to be disappointing. Although the idea behind contribution of mitochondrial oxidative stress in PE is not new, recent years have seen an enormous interest in exploring mitochondrial oxidative stress as an important pathological mediator in PE. We and others using animals, cell models, and preeclamptic patient samples have shown the evidence for placental, renal, and endothelial cell mitochondrial oxidative stress, and its significance in PE. These studies offer promising results; however, the important and relevant question is can we translate these results into clinical efficacy in treating PE. Hence, the purpose of this review is to review the existing literature and offer our insights on the potential of mitochondrial antioxidants in treating PE.
... Unfortunately, the American diet has been shown to be quite low in ERGO compared to four European countries, and this lack was associated with a higher incidence of chronic diseases of aging and reduced life expectancy [1]. ERGO is produced in nature primarily by fungi and some bacteria [3][4][5][6]. Cultivated mushrooms have been shown to be, by far, the leading dietary source of ERGO. However, it is also detected in small quantities in plants, even though plants are not known to produce ERGO themselves. ...
Ergothioneine (ERGO) is a potent antioxidant and anti-inflammatory amino acid that is produced in nature mainly by non-yeast fungi, cyanobacteria, and mycobacteria. Mounting evidence suggests that ERGO can be considered a longevity vitamin that can mitigate chronic diseases of aging and thereby increase life expectancy. Humans must obtain ERGO from their diet, and it is therefore important to know which foods contain it. Although ERGO is not produced by plants it is found in plant products such as grain, apparently because detrital or symbiotic soil fungi pass on ERGO to plants through their roots. Besides differences between plant species in their ability to accumulate ERGO, how they are managed might also affect its concentration. Soil tillage has been shown to reduce soil fungal biomass, and therefore ERGO contents in maize, soybeans, and oats grown in soil managed with annual moldboard plowing (most intensive), chisel/disking (less intensive), or no-tillage (least intensive) in crop rotation were compared. ERGO concentrations declined in all three crops as tillage intensity increased, with reductions from no till to moldboard plow of approximately 30% in all three crops. Because crop yield was also negatively impacted by intensive tillage, ERGO yield per hectare was reduced even more due to increasing tillage intensity. This study is one of the first to show that soil health improving practices that minimize soil disturbance can directly enhance a key dietary factor associated with long-term human health.
... Several species of cyanobacteria e.g., Phormidium, Arthrospira maxima, Scytonema, Oscillatoria sp. (Pfeiffer et al., 2011;Baran et al., 2013) produce large amounts of ergothioneine (EGT) that functions as a stable antioxidant and cytoprotectant (Fahey, 2013;Cheah and Halliwell, 2012). Ergothioneine is a histidine betaine thiol metabolite that scavenges oxidizing species other than free radicals (Baran et al., 2013;Liao and Seebeck, 2017). ...
23 24 25 J o u r n a l P r e-p r o o f 2 Regulation of antioxidant defense and glyoxalase systems in cyanobacteria 26 27 Abstract 28 Oxidative stress is common consequence of abiotic stress in plants as well as cyanobacteria 29 caused by generation of reactive oxygen species (ROS), an inevitable product of respiration 30 and photosynthetic electron transport. ROS act as signalling molecule at low concentration 31 however, when its production exceeds the endurance capacity of antioxidative defence 32 system, the organisms suffer oxidative stress. A highly toxic metabolite, methylglyoxal (MG) 33 is also produced in cyanobacteria in response to various abiotic stresses which consequently 34 augment the ensuing oxidative damage. Taking recourse to the common lineage of eukaryotic 35 plants and cyanobacteria, it would be worthwhile to explore the regulatory role of glyoxalase 36 system and antioxidative defense mechanism in combating abiotic stress in cyanobacteria. 37 This review provides comprehensive information on the complete glyoxalase system (GlyI, 38 GlyII and GlyIII) in cyanobacteria. Furthermore, it elucidates the recent understanding 39 regarding the production of ROS and MG, noteworthy link between intracellular MG and 40 ROS and its detoxification via synchronization of antioxidants (enzymatic and non-41 enzymatic) and glyoxalase systems using glutathione (GSH) as common co-factor. 42
... Several species of cyanobacteria e.g., Phormidium, Arthrospira maxima, Scytonema, Oscillatoria sp. (Pfeiffer et al., 2011;Baran et al., 2013) produce large amounts of ergothioneine (EGT) that functions as a stable antioxidant and cytoprotectant (Fahey, 2013;Cheah and Halliwell, 2012). Ergothioneine is a histidine betaine thiol metabolite that scavenges oxidizing species other than free radicals (Baran et al., 2013;Liao and Seebeck, 2017). ...
Oxidative stress is common consequence of abiotic stress in plants as well as cyanobacteria caused by generation of reactive oxygen species (ROS), an inevitable product of respiration and photosynthetic electron transport. ROS act as signalling molecule at low concentration however, when its production exceeds the endurance capacity of antioxidative defence system, the organisms suffer oxidative stress. A highly toxic metabolite, methylglyoxal (MG) is also produced in cyanobacteria in response to various abiotic stresses which consequently augment the ensuing oxidative damage. Taking recourse to the common lineage of eukaryotic plants and cyanobacteria, it would be worthwhile to explore the regulatory role of glyoxalase system and antioxidative defense mechanism in combating abiotic stress in cyanobacteria. This review provides comprehensive information on the complete glyoxalase system (GlyI, GlyII and GlyIII) in cyanobacteria. Furthermore, it elucidates the recent understanding regarding the production of ROS and MG, noteworthy link between intracellular MG and ROS and its detoxification via synchronization of antioxidants (enzymatic and non-enzymatic) and glyoxalase systems using glutathione (GSH) as common co-factor.
... Mycobacteria, members of the genus Actinomycetales, and fungi were the first organisms reported to produce ET (37,38). Later, ET was also discovered in cyanobacteria, methylobacteria, and other microbes (3,75,105). Unlike eukaryotes and Gram-negative bacteria, where glutathione (GSH) is the primary thiol, in mycobacteria, GSH is absent and small molecules such as ET and mycothiol (MSH) constitute the major thiol reserve. ...
Significance:
Ergothioneine (ET) is an unusual sulfur containing amino acid derived from histidine, acquired predominantly from the diet. Its depletion is associated with deleterious consequences in response to stress stimuli in cell culture models, prompting us to classify it as a vitamin in 2010, which was later supported by in vivo studies. ET is obtained from a variety of foods and is taken up by a selective transporter. ET possesses antioxidant and anti-inflammatory properties, which confers cytoprotection. ET crosses the blood brain barrier and has been reported to have beneficial effects in the brain. Here we discuss the cytoprotective and neuroprotective properties of ET, which may be harnessed for combating neurodegeneration and decline during aging. Recent Advances: The designation of ET as a stress vitamin is gaining momentum, opening a new field of investigation involving small molecules that are essential for optimal physiological functioning and maintenance of healthspan.
Critical issues:
Although ET was discovered more than a century ago, its physiological functions are still being elucidated, especially in the brain. As ET is present in most foods, toxicity associated with its deprivation has been difficult to assess.
Future directions:
Using genetically engineered cells and mice, it may now be possible to elucidate roles of ET. This coupled with advances in genomics and metabolomics may lead to identification of ET function. As ET is a stable antioxidant with anti-inflammatory properties, whose levels decline during aging, supplementing ET in the diet or consuming an ET-rich diet may prove beneficial.
... Mushrooms are also rich sources of powerful antioxidants ergothioneine and glutathione, which play a significant role in preventing chronic diseases and mortality [19][20][21][22][23]. Ergothioneine is an amino acid with a unique chemical structure produced by certain fungi and a few mycobacteria but not by animals or higher plants [24][25][26]. Consequently, ergothioneine is obtained exclusively through dietary sources, with mushrooms having the highest levels compared to other foods [4,7,20,23,27,28]. ...
Background
Whether mushroom consumption, which is a rich source of potent antioxidants ergothioneine and glutathione, vitamins, and minerals (e.g., selenium & copper), is associated with a lower mortality risk is not well understood. This study aimed to examine the association between mushroom consumption and risk of mortality in a prospective cohort study and a meta-analysis of prospective cohort studies.
Methods
We followed 30,378 participants from the continuous National Health and Nutrition Examination Survey (NHANES) extant data (2003-2014). Dietary mushroom intake was assessed using up to two 24-h recalls. Mortality was evaluated in all participants linked to the National Death Index mortality data through December 31, 2015. We used Cox proportional hazards regression models to calculate multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (95% CIs). We also conducted a meta-analysis, including results from our present study and 4 other cohort studies.
Results
During a mean (SD) of 6.7 (3.4) years of follow-up, a total of 2855 death cases were documented among NHANES participants. In our analysis of continuous NHANES, we found a non-significant association between mushroom consumption and all-cause mortality (adjusted hazard ratio (HR) = 0.84; 95% CI: 0.67-1.06) after adjusting for demographic, major lifestyle factors, overall diet quality, and other dietary factors, including total energy. The meta-analysis of prospective cohort studies, including 601,893 individuals, showed that mushroom consumption was associated with a lower risk of all-cause mortality (pooled risk ratio: 0.94; 95% CI: 0.91, 0.98).
Conclusion
In a meta-analysis of prospective cohort studies, mushroom consumption was associated with a lower risk of all-cause mortality.
... L-Ergothioneine (L-Erg) is a natural thiohistidine [1] absorbed from the diet in mammals and synthesized by non-yeast-like fungi, actinobacteria [2], methylotrophs [3] and cyanobacteria [4]. L-Erg is widely distributed among tissues, being more abundant in erythrocytes, liver, seminal fluid, bone marrow, eye lens, cornea and kidneys [5][6][7]. ...
Cystinuria, a rare inherited aminoaciduria condition, is characterized by the hyperexcretion of cystine, ornithine, lysine, and arginine. Its main clinical manifestation is cystine stone formation in the urinary tract, being responsible for 1–2% total and 6–8% pediatric lithiasis. Cystinuria patients suffer from recurrent lithiasic episodes that might end in surgical interventions, progressive renal functional deterioration, and kidney loss. Cystinuria is monitored for the presence of urinary cystine stones by crystalluria, imaging techniques or urinary cystine capacity; all with limited predicting capabilities. We analyzed blood and urine levels of the natural antioxidant L-ergothioneine in a Type B cystinuria mouse model, and urine levels of its metabolic product S-methyl-L-ergothioneine, in both male and female mice at two different ages and with different lithiasic phenotype. Urinary levels of S-methyl-L-ergothioneine showed differences related to age, gender and lithiasic phenotype. Once normalized by L-ergothioneine to account for interindividual differences, the S-methyl-L-ergothioneine to L-ergothioneine urinary ratio discriminated between cystine lithiasic phenotypes. Urine S-methyl-L-ergothioneine to L-ergothioneine ratio could be easily determined in urine and, as being capable of discriminating between cystine lithiasis phenotypes, it could be used as a lithiasis biomarker in cystinuria patient management.
... EGT is a unique thiol derivative of histidine and was first discovered by Charles Tanret in 1909 in the ergot fungus, Claviceps purpurea [5]. Although EGT is widely distributed within tissues of both plants and animals, it is exclusively synthesized in non-yeast fungi, actinomycete bacteria, lactobacillus bacteria, and some cyanobacteria [6][7][8][9]. Plants absorb EGT produced by microorganisms in the soil via their roots, whereas animals and humans acquire EGT only through their diet [10]. Edible mushrooms are the food source most enriched in EGT, while other dietary sources include some meat products, oat bran and beans [10]. ...
Medicinal use of mushrooms has been documented since ancient times, and in the modern world, mushrooms have a longstanding history of use in Eastern medicine. Recent interest in plant-based diets in Westernized countries has brought increasing attention to the use of mushrooms and mushroom-derived compounds in the prevention and treatment of chronic diseases. Edible mushrooms are the most abundant food sources of the modified amino acid, ergothioneine. This compound has been shown to accumulate in almost all cells and tissues, but preferentially in those exposed to oxidative stress and injury. The demonstrated cytoprotectant effect of ergothioneine has led many to suggest a potential therapeutic role for this compound in chronic conditions that involve ongoing oxidative stress and inflammation, including cardiovascular and metabolic diseases. However, the in vivo effects of ergothioneine and its underlying therapeutic mechanisms in the whole organism are not as clear. Moreover, there are no well-defined, clinical prevention and intervention trials of ergothioneine in chronic disease. This review highlights the cellular and molecular mechanisms of action of ergothioneine and its potential as a Traditional, Complementary and Alternative Medicine for the promotion of cardiometabolic health and the management of the most common manifestations of cardiometabolic disease.
... Ergothioneine is a strong antioxidant of hydroxyl radicals, hypochlorous acid, and peroxynitrite (Motohashi and Mori 1986;Franzoni et al. 2006), and has been shown to be effective in preventing oxidative damage to cells. Ergothioneine is found in many living organisms (Melville et al. 1956;Melville et al. 1957;Mayumi et al. 1982;Hartman 1990;Shires et al. 1997;Dubost et al. 2007;Ey et al. 2007;Kato et al. 2010;Seebeck 2010), but is synthesized by only a few organisms, such as certain microbes and mushrooms (Genghof and Vandamme 1964;Pfeiffer et al. 2011;Woldegiorgis et al. 2014;Alamgir et al. 2015;Burn et al. 2017). The microbial ergothioneine biosynthetic pathway, which is the source of ergothioneine for higher organisms, has been extensively investigated (Seebeck 2010;Burn et al. 2017). ...
Here, we report the identification of the gene encoding a novel enzyme, 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase, in Burkholderia sp. HME13. The enzyme converts 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid and H2O to 3-(2,5-dioxoimidazolidin-4-yl) propionic acid and H2S. Amino acid sequence analysis of the enzyme indicates that it belongs to the DUF917 protein family, which consists of proteins of unknown function.
... Ergothioneine is a thio-histidine amino acid that is synthesized by several fungi and bacteria including mushroom fungus, actinobacteria as well as cyanobacteria. [18][19][20] In contrast, it is not synthesized by higher organisms including humans. [20] Structurally, ergothioneine has a thiol group on its imidazole ring that tautomerizes to thione. ...
The liver is highly susceptible to iron overload-evoked oxidative injury. Ergothioneine is a thio-histidine amino acid that has exhibited strong antioxidant and metal chelating activities. This study aimed at exploring the potential modulating effects of ergothioneine on iron-triggered liver injury. The results showed that ergothioneine inhibited iron-evoked inflammation and apoptosis as demonstrated by a significant reduction in tumor necrosis factor-α and interleukin-6 levels and in caspase-3 activity. Ergothioneine significantly improved liver cell survival as indicated by modulating phosphatidylinositol 3-kinase/protein kinase B signaling. Consistent with reduced necrotic cell death, ergothioneine diminished the iron-evoked histopathological changes and decreased serum activity of the liver enzymes. Mechanistically, ergothioneine reduced nuclear translocation of nuclear factor kappa B p65 and modulated p38 mitogen-activated protein kinase/c-Fos signaling. In addition, it enhanced the liver tissue antioxidant potential and curbed hepatic iron load. Together, these results point out the modulatory effects of ergothioneine on iron-evoked liver cell injury that are possibly mediated via anti-inflammatory, antioxidant, and possible iron chelation capabilities.
... ET is present in a wide array of foods, but the highest dietary levels of ET are found in certain mushrooms [29,30,47,130] and Spirulina [131], which produce their own ET [132]. Several studies have investigated the putative health benefits of mushroom consumption. ...
There has been a recent surge of interest in the unique low molecular weight dietary thiol/thione, ergothioneine. This compound can accumulate at high levels in the body from diet and may play important physiological roles in human health and development, and possibly in prevention and treatment of disease. Blood levels of ergothioneine decline with age and onset of various diseases. Here we highlight recent advances in our knowledge of ergothioneine.
Background
Ergothioneine (ERT) and asymmetric dimethyl-L-arginine (ADMA) have been associated with cognitive decline and dementia in older adults, but their interplay with psychological distress remains unexplored. This study aimed to measure the serum concentrations of ERT and ADMA in a representative sample of older community-dwelling adults and to determine their association with psychological distress.
Methods
Data on clinical, lifestyle, demographic characteristics, and serum concentrations of ERT and ADMA were collected from a population-based sample of older Australian adults (mean age 65.5 ± 7.5 years) from the Hunter Community Study. Psychological distress was assessed using the self-reported Kessler Psychological Distress Scale (K10).
Results
In individuals with psychological distress, serum ERT concentrations decreased by 24%, while ADMA concentrations increased by 6%. In adjusted analysis, accounting for age and sex, only ERT remained independently associated with psychological distress. For each unit increase in ERT, the odds of experiencing psychological distress decreased by approximately 68%.
Conclusion
The trend of decreasing serum ERT concentrations observed in older adults with increasing psychological distress suggests a potential link between this compound and mental health. Given the dietary origin of ERT, its integration offers therapeutic opportunities that warrant investigation in intervention studies.
Sixteen strains of basidiomycetous yeasts were evaluated for their capability to produce ergothioneine (EGT), an amino acid derivative with strong antioxidant activity. The cells were cultured in either two synthetic media or yeast mold (YM) medium for 72 h, after which cytosolic constituents were extracted from the cells with hot water. After analyzing the extracts via liquid chromatography-mass spectrometry (LC-MS), we found that all strains produced varying amounts of EGT. The EGT-producing strains, including Ustilago siamensis, Anthracocystis floculossa, Tridiomyces crassus, Ustilago shanxiensis, and Moesziomyces antarcticus, were subjected to flask cultivation in YM medium. U. siamensis CBS9960 produced the highest amount of EGT at 49.5 ± 7.0 mg/L after 120 h, followed by T. crassus at 30.9 ± 1.8 mg/L. U. siamensis was also cultured in a jar fermenter and produced slightly higher amounts of EGT than under flask cultivation. The effects of culture conditions, particularly the addition of precursor amino acids, on EGT production by the selected strains were also evaluated. U. siamensis showed a 1.5-fold increase in EGT production with the addition of histidine, while U. shanxiensis experienced a 1.8-fold increase in EGT production with the addition of methionine. These results suggest that basidiomycetous yeasts could serve an abundant source for natural EGT producers.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13568-024-01672-w.
Growth of Chlamydomonas reinhardtii in zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. We show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding "first responder" proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, γ-glutamylcysteine and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ∼80-fold, corresponding to ∼ 2.8×109 molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is an in vivo Cu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.
The chemical diversification of biomolecules set forth a significant area of research that constitutes an important intersection between chemistry and biology. Amino acids and peptides are the fundamental building blocks of proteins and play essential roles in all living organisms. While significant efforts have been geared toward the chemical modification of amino acid residues, particularly the functionalization of reactive functional groups such as lysine NH2 and cysteine SH, the exploration of the aromatic amino acid residues of tryptophan, tyrosine, phenylalanine, and histidine has been relatively limited. Therefore, this review highlights strategies for the side-chain functionalization of these four aromatic amino acids in peptides, with a focus on elucidating the underlying mechanisms. We have also illustrated the use of these modifications in the chemical and biological realm.
1 Introduction
2 Tryptophan Modifications
3 Tyrosine Modifications
4 Phenylalanine Modifications
5 Histidine Modifications
6 Perspectives and Future Outlook
Ergothioneine (EGT) is a high-value natural sulfur-containing amino acid and has been shown to possess extremely potent antioxidant and cytoprotective activities. At present, EGT has been widely used in food, functional food, cosmetics, medicine, and other industries, but its low yield is still an urgent problem to overcome. This review briefly introduced the biological activities and functions of EGT, and expounded its specific applications in food, functional food, cosmetic, and medical industries, introduced and compared the main production methods of EGT and respective biosynthetic pathways in different microorganisms. Furthermore, the use of genetic and metabolic engineering methods to improve EGT production was discussed. In addition, the incorporation of some food-derived EGT-producing strains into fermentation process will allow the EGT to act as a new functional factor in the fermented foods.
Ergothioneine (ERG) is an unusual sulfur-containing amino acid with antioxidant activity that can be synthesized by certain bacteria and fungi. Microbial fermentation is a promising method for ERG production. In this study, the bifunctional enzyme methyltransferase-sulfoxide synthase NcEgt1 from Neurospora crassa was truncated to obtain sulfoxide synthase TNcEgt1, which showed a higher expression level in Escherichia coli BL21(DE3). Then, the genes egtD encoding methyltransferase EgtD and egtE encoding C-S lyase EgtE from Mycobacterium smegmatis were cloned with TncEgt1 into E. coli BL21(DE3) to produce 70 mg/L ERG. To improve ERG production, TNcEgt1 and EgtD were modified, and the resulting mutants were screened with an established high-throughput method which could directly analyze the ERG content in culture broths. After several rounds of mutation and screening, the optimal mutant MD4 was obtained and produced 290 mg/L ERG. Furthermore, a fed-batch culture was conducted in a 5 L bioreactor. After optimizing the fermentation process, the ERG yield reached 5.4 g/L after 94 h of cultivation supplemented with amino acids and glycerol, which is the highest ERG yield reported to date. The results showed that ERG production was significantly improved by modifying the key enzymes, and the engineered strains constructed in this study have potential industrial application prospects.
L -Ergothioneine (ET), the 2-thioimidazole derivative of trimethylhistidine, is biosynthesized by select fungi and bacteria, notably Mycobacterium tuberculosis , and functions as a scavenger of reactive oxygen species. The extent to which ET broadly functions in bacterial cells unable to synthesize it is unknown. Here we show that spd _1642-1643 in Streptococcus pneumoniae , a Gram-positive respiratory pathogen, encodes an ET uptake ATP-binding cassette (ABC) transporter, designated EgtU. The solute binding domain (SBD) of EgtU, EgtUC, binds ET with high affinity and exquisite specificity in a cleft between the two subdomains, with cation-π interactions engaging the betaine moiety and a network of water molecules that surround the thioimidazole ring. EgtU is highly conserved among known quaternary amine compound-specific transporters and widely distributed in Firmicutes, including the human pathogens Listeria monocytogenes , as BilEB, Enterococcus faecalis and Staphylococcus aureus . ET increases the chemical diversity of the low molecular weight thiol pool in Gram-positive human pathogens and may contribute to antioxidant defenses in the infected host.
In this study, Corynebacterium glutamicum was engineered to produce ergothioneine, an amino acid derivative with high antioxidant activity. The ergothioneine biosynthesis genes, egtABCDE, from Mycolicibacterium smegmatis were introduced into wild-type and l-cysteine-producing strains of C. glutamicum to evaluate their ergothioneine production. In the l-cysteine-producing strain, ergothioneine production reached approximately 40 mg L⁻¹ after 2 weeks, and the amount was higher than that in the wild-type strain. As C. glutamicum possesses an ortholog of M. smegmatis egtA, which encodes an enzyme responsible for γ-glutamyl-l-cysteine synthesis, the effect of introducing egtBCDE genes on ergothioneine production in the l-cysteine-producing strain was evaluated, revealing that a further increase to more than 70 mg L⁻¹ was achieved. As EgtBs from Methylobacterium bacteria are reported to use l-cysteine as a sulfur donor in ergothioneine biosynthesis, egtB from Methylobacterium was expressed with M. smegmatis egtDE in the l-cysteine-producing strain. As a result, ergothioneine production was further improved to approximately 100 mg L⁻¹. These results indicate that utilization of the l-cysteine-producing strain and introduction of heterologous biosynthesis pathways from M. smegmatis and Methylobacterium bacteria are effective for improved ergothioneine production by C. glutamicum.
Ergothioneine is a histidine derivative with a 2-mercaptoimidazole side chain and a trimethylated α-amino group. Although the physiological function of this natural product is not yet understood, the facts that many bacteria, some archaea, and most fungi produce ergothioneine and that plants and animals have specific mechanisms to absorb and distribute ergothioneine in specific tissues suggest a fundamental role in cellular life. The observation that ergothioneine biosynthesis has emerged multiple times in molecular evolution points to the same conclusion. Aerobic bacteria and fungi attach sulfur to the imidazole ring of trimethylhistidine via an O2-dependent reaction that is catalyzed by a mononuclear non-heme iron enzyme. Green sulfur bacteria and archaea use a rhodanese-like sulfur transferase to attach sulfur via oxidative polar substitution. In this report, we describe a third unrelated class of enzymes that catalyze sulfur transfer in ergothioneine production. The metallopterin-dependent ergothioneine synthase from Caldithrix abyssi contains an N-terminal module that is related to the tungsten-dependent acetylene hydratase and a C-terminal domain that is a functional cysteine desulfurase. The two modules cooperate to transfer sulfur from cysteine onto trimethylhistidine. Inactivation of the C-terminal desulfurase blocks ergothioneine production but maintains the ability of the metallopterin to exchange sulfur between ergothioneine and trimethylhistidine. Homologous bifunctional enzymes are encoded exclusively in anaerobic bacterial and archaeal species.
The effect of dried and UV-C-irradiated mushroom powder on lipid oxidation and vitamin D content in fish meat was investigated. To this end, Flammulina velutipes, Grifola frondosa, Hypsizygus marmoreus, Lentinula edodes, Pleurotus eryngii, Pleurotus ostreatus were dried by hot air and irradiated by UV-C and evaluated the effect of these treatments on the components. In general, the ergothioneine content did not change substantially, the total phenolic compound content decreased by hot-air drying, and the ergocalciferol content increased by UV-C irradiation. To the evaluate effect of mushroom powder on lipid oxidation and vitamin D content in fish meat, 5% of the hot air dried and UV-C-irradiated mushroom powder was added to fish meat and oxidized. Consequently, all six mushrooms prevented lipid oxidation, and ergocalciferol content in each mushroom powder remained between 58.2% and 69.7%. Overall, P. eryngii, L. edodes, and P. ostreatus strongly prevented the generation of lipid peroxide and aldehyde.
Background
Ergothioneine (ERG) is a potent histidine-derived antioxidant that confers health-promoting effects. Only certain bacteria and fungi can biosynthesize ERG, but the ERG productivity in natural producers is low. ERG overproduction through genetic engineering represents an efficient and cost-effective manufacturing strategy.
Results
Here, we showed that Trichoderma reesei can synthesize ERG during conidiogenesis and hyphal growth. Co-expression of two ERG biosynthesis genes (tregt1 and tregt2) from T. reesei enabled E. coli to generate 70.59 mg/L ERG at the shaking flask level after 48 h of whole-cell biocatalysis, whereas minor amounts of ERG were synthesized by the recombinant E. coli strain bearing only the tregt1 gene. By fed-batch fermentation, the extracellular ERG production reached 4.34 g/L after 143 h of cultivation in a 2-L jar fermenter, which is the highest level of ERG production reported thus far. Similarly, ERG synthesis also occurred in the E. coli strain engineered with the two well-characterized genes from N. crassa and the ERG productivity was up to 4.22 g/L after 143 h of cultivation under the above-mentioned conditions.
Conclusions
Our results showed that the overproduction of ERG in E. coli could be achieved through two-enzymatic steps, demonstrating high efficiency of the fungal ERG biosynthetic pathway. Meanwhile, this work offers a more promising approach for the industrial production of ERG.
Ergothioneine (ET) is the 2-thiourea derivative of trimethylhistidine that is biosynthesized only by select fungi and bacteria, notably Mycobacterium tuberculosis, and functions as a potent scavenger of reactive oxygen species. Although ET is obtained in the diet and accumulates in vertebrate cells via an ET-specific transporter, the extent to which ET broadly functions in bacterial cells unable to synthesize it is unknown. Here we show that spd_1642-1643 in Streptococcus pneumoniae D39, a Gram-positive respiratory pathogen, encodes a novel ergothioneine uptake ATP-binding cassette (ABC) transporter, which we designate EgtUV. EgtU is a permease-solute binding domain (SBD) fusion protein, and the SBD binds ET with high affinity and exquisite specificity in the cleft between the two subdomains, with cation-π interactions engaging the betaine moiety and a water-mediated hydrogen bonding network surrounding the C2-sulfur-containing imidazole ring. Bioinformatics studies reveal that EgtUV is uniquely strongly conserved among known quaternary amine-specific transporters and widely distributed in firmicutes, including the human pathogens Listeria monocytogenes, as BilEB, Enterococcus faecalis and Staphylococcus aureus. This discovery significantly diversifies the LMW thiol pool in Gram-positive human pathogens that may contribute to antioxidant defenses in the infected host.
L-Ergothioneine (EGT) is a natural antioxidant derived from microorganisms, especially in edible mushrooms. EGT is found to be highly accumulated in tissues that are susceptible to oxidative damage, and it has attracted extensive attention due to its powerful antioxidant activity and the tight relationships of this natural product with various oxidative stress-related diseases. Herein, we 1) introduce the biological source and in vivo distribution of EGT; 2) review the currently available evidence concerning the relationships of EGT with diabetes, ischemia-reperfusion injury-related diseases like cardiovascular diseases and liver diseases, neurodegenerative diseases, and other diseases pathogenically associated with oxidative stress; 3) summarize the potential action mechanisms of EGT against these diseases; 4) discuss the advantages of EGT over other antioxidants; and 5) also propose several future research perspectives for EGT. These may help to promote the future application of this attractive natural antioxidant.
In all vertebrates including mammals, the ergothioneine transporter ETT (obsolete name OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). ETT is not expressed ubiquitously and only cells with high ETT cell-surface levels can accumulate ET to high concentration. Without ETT, there is no uptake because the plasma membrane is essentially impermeable to this hydrophilic zwitterion. Here, we review the substrate specificity and localization of ETT, which is prominently expressed in neutrophils, monocytes/macrophages, and developing erythrocytes. Most sites of strong expression are conserved across species, but there are also major differences. In particular, we critically analyze the evidence for the expression of ETT in the brain as well as recent data suggesting that the transporter SLC22A15 may transport also ET. We conclude that, to date, ETT remains the only well-defined biomarker for intracellular ET activity. In humans, the ability to take up, distribute, and retain ET depends principally on this transporter.
The global population increase has increased the demand for food products. However, post-harvest deterioration because of oxidation and discoloration results in a drastic loss of food quality and supply. Thus, research has focused on developing strategies to minimize such losses. One of those strategies includes the application of ergothioneine (ET), a potent hydrophilic antioxidant, to several food products to overcome their short shelf-life. ET can be synthetic or derived from several species of edible mushrooms and their extracts, which are known sources of natural ET. Given the reported potential of ET in food quality preservation, this review compiles the recent applications of ET as a preservative for maintaining the quality of food commodities.
L-ergothioneine is a low-molecular weight natural product, the chemical structure of which comprises oxygen-, nitrogen- and sulfur-containing functional groups. This gives L-ergothioneine specific physicochemical properties and allows to better understanding its chemical reactivity, which is primarily due to the 2-thio-imidazole group. Here, I review how different modes of chemical reactivity account for the reported molecular biological activities of L-ergothioneine. By matching the physicochemical properties to the biological properties of L-ergothioneine, a new perspective of the function and the mode of action of this enigmatic molecule emerges into the limelight.
Ergothioneine is a natural and safe antioxidant playing an important role in anti-aging and the prevention of various diseases. Mushrooms are the main dietary source of ergothioneine. This study aims to report a kind of medicinal mushroom Panus conchatus with great potential for the bioproduction of ergothioneine. Molasses and soy peptone could promote cell growth of Panus conchatus and enhance the accumulation of ergothioneine. Meanwhile, three precursors of cysteine, histidine and methionine were added to the broth, and the highest ergothioneine concentration of 148.79mg/L was obtained when adding 0.4 g/L cysteine. Finally, the crude ergothioneine extract was purified and further evaluated. The ergothioneine from Panus conchatus showed higher antioxidant activity than vitamin C and glutathione with good stability at lower pH environment. This research would provide a new method for the bioproduction of ergothioneine and lay foundation for the in-depth study about the exploration of natural products from Panus conchatus .
Aim
Cisplatin is a potent chemotherapeutic agent whose therapeutic application is hindered by the associated nephrotoxicity. Cisplatin-evoked nephrotoxicity has been largely attributed to the induction of oxidative stress and inflammatory responses. The current study aimed at investigating the ability of ergothioneine to mitigate cisplatin-evoked nephrotoxicity and to elucidate the underlining molecular mechanisms.
Main methods
Wistar rats were treated with a daily dose of ergothioneine (70 mg/kg, po) for fourteen days and a single dose of cisplatin (5 mg/kg, ip) on day ten. On day fifteen, kidneys and blood specimens were collected and subjected to Western blotting, ELISA, histopathological, and spectrophotometric analysis.
Key findings
Ergothioneine significantly enhanced renal function in cisplatin-treated rats as manifested by increased GFR and decreased serum creatinine and blood urea nitrogen. Ergothioneine effectively reduced the cisplatin-induced oxidative stress and mitigated apoptosis and the histopathological changes. Mechanistically, ergothioneine induced the expression of the antioxidant transcription factor Nrf2 and up-regulated its downstream targets NQO1 and HO-1. Equally important, ergothioneine inhibited γ-glutamyl transpeptidase that plays crucial roles in biotransformation of cisplatin into a toxic metabolite. Additionally, it reduced the pro-apoptotic protein p53 and the inflammatory transcription factor NF-κB along with its downstream pro-inflammatory cytokines TNF-α and IL-1β.
Significance
The results of the current work shed the light on the ameliorating effect of ergothioneine on cisplatin-evoked nephrotoxicity that is potentially mediated through modulation of Nrf2, p53, and NF-κB signaling and inhibition of γ-glutamyl transpeptidase. This findings support the potential application of ergothioneine in controlling cisplatin-associated nephrotoxicity although clinical investigations are warranted.
Ergothioneine is a sulfur-containing histidine derivative, that possessesexcellent antioxidant activity and has been used in the food and cosmetics industries. It plays a significant role in anti-aging and the prevention of various diseases. This review will briefly introduce the functions and applications of ergothioneine, elaborate the biosynthetic pathways of ergothioneine and describe several strategies to increase the production of ergothioneine. Then the efficient extraction and detection methods of ergothioneine will be presented. Finally, several proposals are put forward to increase the yield of ergothioneine, and the development prospects of ergothioneine will be discussed.
Ergothioneine is a histidine-derived sulfur metabolite that is biosynthesized by bacteria and fungi. Plants and animals absorb ergothioneine as a micronutrient from their environment or nutrition. Several different mechanisms of microbial ergothioneine production have been described in the past ten years. Much less is known about the genetic and structural basis for ergothioneine catabolism. In this report, we describe the in vitro reconstitution of a five-step pathway that degrades ergothioneine to l-glutamate, trimethylamine, hydrogen sulfide, carbon dioxide, and ammonia. The first two steps are catalyzed by the two enzymes ergothionase and thiourocanate hydratase. These enzymes are closely related to the first two enzymes in histidine catabolism. However, the crystal structure of thiourocanate hydratase from the firmicute Paenibacillus sp. reveals specific structural features that strictly differentiate the activity of this enzyme from that of urocanate hydratases. The final two steps are catalyzed by metal-dependent hydrolases that share most homology with the last two enzymes in uracil catabolism. The early and late part of this pathway are connected by an entirely new enzyme type that catalyzes desulfurization of a thiohydantoin intermediate. Homologous enzymes are encoded in many soil-dwelling firmicutes and proteobacteria, suggesting that bacterial activity may have a significant impact on the environmental availability of ergothioneine.
Oxidative damage has been proposed as a possible mechanism involved in lead toxicity, specially affecting the liver and kidney. Previous studies have shown the antioxidant effect of Spirulina maxima in several experimental models of oxidative stress. The current study was carried out to evaluate the antioxidant activity of Spirulina maxima against lead acetate-induced hyperlipidemia and oxidative damage in the liver and kidney of male rats. Control animals were fed on a standard diet and did not receive lead acetate (Control group). Experimental animals were fed on a standard laboratory diet with or without Spirulina maxima 5% in the standard laboratory diet and treated with three doses of lead acetate (25 mg each/weekly, intraperitoneal injection) (lead acetate with Spirulina, and lead acetate without Spirulina groups).
The results showed that Spirulina maxima prevented the lead acetate-induced significant changes on plasma and liver lipid levels and on the antioxidant status of the liver and kidney. On the other hand, Spirulina maxima succeeded to improve the biochemical parameters of the liver and kidney towards the normal values of the Control group.
It was concluded that Spirulina maxima has protective effects on lead acetate-induced damage, and that the effects are associated with the antioxidant effect of Spirulina.
In the present study, we focused on the protective effect of Spirulina against 4-nitroquinoline-1-oxide (4NQO) induced hepato and nephrotoxicity in the experimental rats. The 4NQO administration resulted in increased levels of hepatic and renal markers [Alanine Transaminase (ALT), Aspartate Transaminase (AST), Lactate Dehydrogenase (LDH), urea, creatinine and uric acid] in the serum of experimental animals. It also increased the oxidative stress resulting in increased levels of the lipid peroxidation with a concomitant decline in the levels of non enzymic [reduced glutathione (GSH)] and enzymic antioxidants [(Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), and Glutathione-S-transferase (GST)] in both liver and kidney. Oral pretreatment with aqueous extract of Spirulina prevented 4NQO induced changes in the levels of hepatic and kidney diagnostic marker enzymes in the serum of experimental rats. It counteracted the 4NQO induced lipid peroxidation and maintained the hepatic and kidney antioxidant defense system at near normal in both liver and kidney. The antioxidant responsiveness mediated by Spirulina may be anticipated to have biological significance in eliminating reactive free radicals that may otherwise affect normal cell functioning and provide a scientific rationale for the use of Spirulina.
Spirulina is a popular nutritional supplement that is accompanied by claiMSS for antioxidant and performance-enhancing effects. Therefore, the aim of the present study was to examine the effect of spirulina supplementation on (i) exercise performance, (ii) substrate metabolism, and (iii) blood redox status both at rest and after exercise.
Nine moderately trained males took part in a double-blind, placebo-controlled, counterbalanced crossover study. Each subject received either spirulina (6 g x d(-1)) or placebo for 4 wk. Each subject ran on a treadmill at an intensity corresponding to 70%-75% of their VO2max for 2 h and then at 95% VO2max to exhaustion. Exercise performance and respiratory quotient during exercise were measured after both placebo and spirulina supplementation. Blood samples were drawn before, immediately after, and at 1, 24, and 48 h after exercise. Reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG, thiobarbituric acid-reactive substances (TBARS), protein carbonyls, catalase activity, and total antioxidant capacity (TAC) were determined.
Time to fatigue after the 2-h run was significantly longer after spirulina supplementation (2.05 +/- 0.68 vs 2.70 +/- 0.79 min). Ingestion of spirulina significantly decreased carbohydrate oxidation rate by 10.3% and increased fat oxidation rate by 10.9% during the 2-h run compared with the placebo trial. GSH levels were higher after the spirulina supplementation compared with placebo at rest and 24 h after exercise. TBARS levels increased after exercise after placebo but not after spirulina supplementation. Protein carbonyls, catalase, and TAC levels increased similarly immediately after and 1 h after exercise in both groups.
Spirulina supplementation induced a significant increase in exercise performance, fat oxidation, and GSH concentration and attenuated the exercise-induced increase in lipid peroxidation.
Spirulina maxima, which is used as a food additive, is a microalga rich in protein and other essential nutrients. Spirulina contains phenolic acids, tocopherols and beta-carotene which are known to exhibit antioxidant properties. The aim of the present study was to evaluate the antioxidant capacity of a Spirulina extract. The antioxidant activity of a methanolic extract of Spirulina was determined in vitro and in vivo. The in vitro antioxidant capacity was tested on a brain homogenate incubated with and without the extract at 37 degrees C. The IC50 (concentration which causes a 50% reduction of oxidation) of the extract in this system was 0.18 mg/ml. The in vivo antioxidant capacity was evaluated in plasma and liver of animals receiving a daily dose of 5 mg for 2 and 7 weeks. Plasma antioxidant capacity was measured in brain homogenate incubated for 1 h at 37 degrees C. The production of oxidized compounds in liver after 2 h of incubation at 37 degrees C was measured in terms of thiobarbituric acid reactant substances (TBARS) in control and experimental groups. Upon treatment, the antioxidant capacity of plasma was 71% for the experimental group and 54% for the control group. Data from liver spontaneous peroxidation studies were not significantly different between groups. The amounts of phenolic acids, alpha-tocopherol and beta-carotene were determined in Spirulina extracts. The results obtained indicate that Spirulina provides some antioxidant protection for both in vitro and in vivo systems.
Crohn disease is a chronic, inflammatory disease of the gastrointestinal tract. A locus of approximately 250 kb at 5q31 (IBD5) was previously associated with susceptibility to Crohn disease, as indicated by increased prevalence of a risk haplotype of 11 single-nucleotide polymorphisms among individuals with Crohn disease, but the pathogenic lesion in the region has not yet been identified. We report here that two variants in the organic cation transporter cluster at 5q31 (a missense substitution in SLC22A4 and a G-->C transversion in the SLC22A5 promoter) form a haplotype associated with susceptibility to Crohn disease. These variants alter transcription and transporter functions of the organic cation transporters and interact with variants in another gene associated with Crohn disease, CARD15, to increase risk of Crohn disease. These results suggest that SLC22A4, SLC22A5 and CARD15 act in a common pathogenic pathway to cause Crohn disease.
Variants of the SLC22A4 gene are associated with susceptibility to rheumatoid arthritis and Crohn's disease. SLC22A4 codes for an integral membrane protein, OCTN1, that has been presumed to carry organic cations like tetraethylammonium across the plasma membrane. Here, we show that the key substrate of this transporter is in fact ergothioneine (ET). Human OCTN1 was expressed in 293 cells. A substrate lead, stachydrine (alias proline betaine), was identified by liquid chromatography MS difference shading, a new substrate search strategy. Analysis of transport efficiency of stachydrine-related solutes, affinity, and Na⁺ dependence indicates that the physiological substrate is ET. Efficiency of transport of ET was as high as 195 μl per min per mg of protein. By contrast, the carnitine transporter OCTN2 from rat did not transport ET at all. Because ET is transported >100 times more efficiently than tetraethylammonium and carnitine, we propose the functional name ETT (ET transporter) instead of OCTN1. ET, all of which is absorbed from food, is an intracellular antioxidant with metal ion affinity. Its particular purpose is unresolved. Cells with expression of ETT accumulate ET to high levels and avidly retain it. By contrast, cells lacking ETT do not accumulate ET, because their plasma membrane is virtually impermeable for this compound. The real-time PCR expression profile of human ETT, with strong expression in CD71⁺ cells, is consistent with a pivotal function of ET in erythrocytes. Moreover, prominent expression of ETT in monocytes and SLC22A4 polymorphism associations suggest a protective role of ET in chronic inflammatory disorders.
• erythrocyte
• inflammation
Spirulina, a filamentous cyanobacterium, possesses diverse biological activities and nutritional significance due to high concentration of natural nutrients, having bio-modulatory and immuno-modulatory functions. Different Spirulina preparations influence immune system viz. increase phagocytic activity of macrophages, stimulating the production of antibodies and cytokines, increase accumulation of NK cells into tissue and activation and mobilization of T and B cells. Spirulina have also shown to perform regulatory role on lipid and carbohydrate metabolism by exhibiting glucose and lipid profile correcting activity in experimental animals and in diabetic patients. Preparations have been found to be active against several enveloped viruses including herpes virus, cytomegalovirus, influenza virus and HIV. They are capable to inhibit carcinogenesis due to anti-oxidant properties that protect tissues and also reduce toxicity of liver, kidney and testes.
The SLC22A4 and SLC22A5 genes within the IBD5 risk locus encode the organic cation transporters OCTN1 and OCTN2. Two variants, 1672C>T in SLC22A4 and -207G>C in SLC22A5, were shown to alter these genes' functions and were identified as genetic susceptibility factors for Crohn's disease (CD). We pursued to check both putative etiologic variants in an independent population through a case-control study with 309 Spanish CD patients and 408 ethnically matched healthy subjects. Both polymorphisms were found in partial linkage disequilibrium (D'=0.86). The separate analysis of each OCTN variant evidenced no association. However, when the simultaneous presence of mutant variants in both genes was analyzed, an effect on CD susceptibility was observed (P=0.026, odds ratio (OR) (95% confidence interval (CI))=1.59 (1.03-2.45)). The previously described predisposition conferred by the 5q31-risk haplotype increased in the absence of the etiologic 1672T and -207C alleles (P=0.0006, OR (95% CI)=10.14 (1.97-98.04)). Moreover, the risk contributed by these polymorphisms was higher in the IBD5 wild-type population (P=0.003, OR (95% CI)=2.65 (1.32-5.35)), arguing against the exclusive etiological role of the OCTN variants. The haplotype pattern inferred led to the consideration of these variants as susceptibility markers only in a defined genetic context. Our data support the interpretation of the 1672C>T SLC22A4 and -207G>C SLC22A5 polymorphisms as genetic markers of susceptibility/protection haplotypes.
Genetic association between Crohn's disease (CD) and OCTN1 (SLC22A4) C1672T/OCTN2 (SLC22A5) G-207C variants in IBD5 has recently been reported. These genes encode solute carriers and the association was suggested to be distinct from the background IBD5 risk haplotype. There have been conflicting reports of the association between markers in the IBD5 region and ulcerative colitis (UC) and interaction (epistasis) between this locus and CARD15. Our aim was to ascertain the contribution of OCTN variants to UC and CD in a large independent UK dataset, to seek genetic evidence that the OCTN association is distinct from the IBD5 risk haplotype and to identify interactions between the IBD5 and CARD15 loci.
A total of 1104 unrelated Caucasian subjects with inflammatory bowel disease (IBD) (496 CD, 512 UC, 96 indeterminate) and 750 ethnically matched controls were genotyped for three single nucleotide polymorphisms (SNPs) in the CD associated genes (OCTN1+1672, OCTN2-207, and IGR2230), and two flanking IBD5 tagging SNPs, IGR2096 and IGR3096. Data were analysed by logistic regression methods within STATA.
OCTN variants were as strongly associated with UC and IBD overall as they were with CD (p = 0.0001; OR 1.3 (95% confidence interval 1.1-1.5)). OCTN variants were in tight linkage disequilibrium with the extended IBD5 risk haplotype D' 0.79 and 0.88, and r2 = 0.62 and 0.72 for IGR2096 and 3096, respectively. There was no deviation from a multiplicative model of interaction between CARD15 and IBD5 on the penetrance scale.
The OCTN variants were associated with susceptibility to IBD overall. The effect was equally strong in UC and CD. Although OCTN variants may account for the increased risk of IBD associated with IBD5, a role for other candidate genes within this extended haplotype was not excluded. There was no statistical evidence of interaction between CARD15 and either OCTN or IBD5 variants in susceptibility to IBD.
Polymorphisms in the organic cation transporter (OCTN) genes SLC22A4 (OCTN1; polymorphism 1672C/T) and SLC22A5 (OCTN2; polymorphism -207G/C) at the inflammatory bowel disease (IBD) 5 locus comprise a two-allele haplotype (SLC22A-TC) associated with increased risk for Crohn's disease (CD). In this study, we examined the contribution of the disease susceptibility haplotype SLC22A-TC to CD in a New Zealand Caucasian population. The frequencies of the gene polymorphisms 1672C/T and -207G/C were examined in 182 patients with CD and 188 ethnically matched controls by PCR-RFLP analysis. There was a significant difference in the allele frequency (0.444 vs 0.519; P = 0.041) of the 1672T polymorphism in the SLC22A4 gene between controls and patients with CD. In contrast, there was no significant difference (0.497 vs 0.552; P = 0.135) for the -207C polymorphism in the SLC22A5 gene. The homozygote SLC22A-TC diplotype was significantly associated with an increased risk for CD (odds ratio 2.19), and the SLC22A-TC haplotype was associated with increased risk (P = 0.0007) of ileocolonic involvement. The population-attributable risk for the SLC22A-TC haplotype is 15.1%. Thus, SLC22A-TC is associated with an increased risk of CD and disease phenotype in our New Zealand CD cohort.
Type 1 diabetes (T1D) is a chronic, autoimmune and multifactorial disease characterized by abnormal metabolism of carbohydrate and fat. Diminished carnitine plasma levels have been previously reported in T1D patients and carnitine increases the sensitivity of the cells to insulin. Polymorphisms in the carnitine transporters, encoded by the SLC22A4 and SLC22A5 genes, have been involved in susceptibility to two other autoimmune diseases, rheumatoid arthritis and Crohn's disease. For these reasons, we investigated for the first time the association with T1D of six single nucleotide polymorphisms (SNPs) mapping to these candidate genes: slc2F2, slc2F11, T306I, L503F, OCTN2-promoter and OCTN2-intron.
A case-control study was performed in the Spanish population with 295 T1D patients and 508 healthy control subjects. Maximum-likelihood haplotype frequencies were estimated by applying the Expectation-Maximization (EM) algorithm implemented by the Arlequin software.
When independently analyzed, one of the tested polymorphisms in the SLC22A4 gene at 1672 showed significant association with T1D in our Spanish cohort. The overall comparison of the inferred haplotypes was significantly different between patients and controls (chi2 = 10.43; p = 0.034) with one of the haplotypes showing a protective effect for T1D (rs3792876/rs1050152/rs2631367/rs274559, CCGA: OR = 0.62 (0.41-0.93); p = 0.02).
The haplotype distribution in the carnitine transporter locus seems to be significantly different between T1D patients and controls; however, additional studies in independent populations would allow to confirm the role of these genes in T1D risk.
The effects of spirulina supplementation on preventing skeletal muscle damage on untrained human beings were examined. Sixteen students volunteered to take Spirulina platensis in addition to their normal diet for 3-weeks. Blood samples were taken after finishing the Bruce incremental treadmill exercise before and after treatment. The results showed that plasma concentrations of malondialdehyde (MDA) were significantly decreased after supplementation with spirulina (P < 0.05). The activity of blood superoxide dismutase (SOD) was significantly raised after supplementation with spirulina or soy protein (P < 0.05). Both of the blood glutathione peroxidaes (GPx
) and lactate dehydrogenase (LDH) levels were significantly different between spirulina and soy protein supplementation by an ANCOVA analysis (P < 0.05). In addition, the lactate (LA) concentration was higher and the time to exhaustion (TE) was significantly extended in the spirulina trail (P < 0.05). These results suggest that ingestion of S. platensis showed preventive effect of the skeletal muscle damage and that probably led to postponement of the time of exhaustion during the all-out exercise.
The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.
This chapter presents a brief review of the accumulated knowledge concerning ergothioneine. Although several biological activities have been suggested for ergothioneine in the past few years, the full meaning of these activities is not yet clear. It is quite likely that its chief function or functions have not yet been uncovered. It is obviously not yet possible to classify ergothioneine in the realm of hiologically active compounds such as vitamins or hormones. It now appears fairly certain that ergothioneine is not synthesized by higher animals. On the other hand, experiments so far have failed to demonstrate that animals need the compound for survival under normal laboratory conditions. At the present state of knowledge, it would appear that ergothioneine cannot be classified as a vitamin in the usual sense of the term. On the other hand, recent developments have suggested that ergothioneine may have regulatory effects on enzyme activity. In addition, a possibility which has not yet been explored is the effect of ergothioneine on animals under various stress conditions. It is within the realm of possibility that the substance cannot be neatly categorized in any of the recognized classes of biologically active compounds; indeed, it is perhaps for this reason that its function or functions have defied elucidation for so long.
L-ergothioneine is a naturally occurring antioxidant that is available from dietary sources. There is a lack of an adequate assay applicable to identify and quantify this antioxidant in plant material. Thus, the objective was to identify and quantify the ergothioneine content of mushrooms including Agaricus bisporus (white and brown strains), Lentinus edodes, Pleurotus ostreatus, P. eryngii, and Grifola frondosa by an analytical method utilizing a high-performance liquid chromatography and liquid chromatography-mass spectroscopy. Freeze dried mushroom powder was analyzed with two C18 columns in tandem utilizing an isocratic mobile phase consisting of an aqueous sodium phosphate buffer with 3% acetonitrile and 0.1% triethylamine. Ergothioneine was identified by matching the retention time and mass spectra of the authentic compound with the mushroom samples, while quantification was completed via absorbance at 254 nm. The ergothioneine content of the mushrooms ranged from 0.4-2.0 mg/g (dry wt). The white Agaricus bisporus contained the least ergothioneine and portabellas (brown) contained the highest within the varieties of A. bisporus. The specialty mushrooms tested (Lentinus edodes, Pleurotus ostreatus, P. eryngii, Grifola frondosa) all contained a statistically significant greater amount of ergothioneine compared to the A. bisporus; however, no significant difference was found between these specialty mushrooms.
Spirulina maxima is an alga, rich in organic nitrogenous constituents, used for food in the Chad Republic. Amino acids, vitamins, and nutritive value were determined for a strain of the algae grown in an open-air pilot production unit. The dried alga, more than 60% of which was proteinaceous material, contained all the essential amino acids in fairly good concentration except that the amount of the sulphur amino acids was low. Nutrition experiments on rats showed that the biological value of the alga was acceptable and was correlated with the chemical score (protein index). Dried Spirulina maxima also contained several vitamins, particularly β-carotene (pro-vitamin A).
Conditions for growing spirulina (Spirulina platensis) were developed. The amino-acid and carbohydrate compositions were determined. Lipids and vitamins of the culture biomass were studied.
Agaricus bisporus, Lentinula edodes, Pleurotus ostreatus, Pleurotus eryngii and Grifola frondosa were analyzed for antioxidant capacity, as measured by oxygen radical absorbance capacity (lipophilic and hydrophilic) (ORACtotal), hydroxyl radical averting capacity (HORAC), peroxynitrite radical averting capacity (NORAC), superoxide radical averting capacity (SORAC) assays, Folin-Ciocalteu reagent and ergothioneine (ERG) content. ORACtotalvalues ranged from 39 to 138 μmol of Trolox equivalents (TE)/g dry weight (dw). HORAC values ranged from 3.0 to 13.6 μmol of caffeic acid equivalents/g dw. NORAC values ranged from 2.0 to 9.0 μmol TE/g dw. SORAC values ranged from 0.37 to 2.6 kunit superoxide dismutate equivalents/g dw. Polyphenols ranged from 4.2 to 10.6 mg gallic acid equivalents/g dw. A. bisporus mushrooms, especially portabellas, had higher antioxidant capacity relative to the specialty mushrooms tested. ERG ranged from 0.21–2.6 mg/g dw with L. edodes, P. ostreatus, G. frondosa containing a statistically significant greater amount compared to A. bisporus. A good correlation was found between ORACtotal and polyphenols (R2 = 0.86).
Spirulina is free-floating filamentous microalgae growing in alkaline water bodies. With its high nutritional value, Spirulina has been consumed as food for centuries in Central Africa. It is now widely used as nutraceutical food supplement worldwide. Recently, great attention and extensive studies have been devoted to evaluate its therapeutic benefits on an array of diseased conditions including hypercholesterolemia, hyperglycerolemia, cardiovascular diseases, inflammatory diseases, cancer, and viral infections. The cardiovascular benefits of Spirulina are primarily resulted from its hypolipidemic, antioxidant, and antiinflammatory activities. Data from preclinical studies with various animal models consistently demonstrate the hypolipidemic activity of Spirulina. Although differences in study design, sample size, and patient conditions resulting in minor inconsistency in response to Spirulina supplementation, the findings from human clinical trials are largely consistent with the hypolipidemic effects of Spirulina observed in the preclinical studies. However, most of the human clinical trials are suffered with limited sample size and some with poor experimental design. The antioxidant and/or antiinflammatory activities of Spirulina were demonstrated in a large number of preclinical studies. However, a limited number of clinical trials have been carried out so far to confirm such activities in human. Currently, our understanding on the underlying mechanisms for Spirulina's activities, especially the hypolipidemic effect, is limited. Spirulina is generally considered safe for human consumption supported by its long history of use as food source and its favorable safety profile in animal studies. However, rare cases of side-effects in human have been reported. Quality control in the growth and process of Spirulina to avoid contamination is mandatory to guarantee the safety of Spirulina products.
Ergothioneine is a histidine-derived thiol of bacterial and fungal origin that has also been isolated from animal and human tissue. Recent findings point to critical functions of ergothioneine in human physiology, but its role in microbial life is poorly understood. This report describes the identification of the ergothioneine biosynthetic gene cluster from mycobacteria and in vitro reconstitution of this process using recombinant proteins from Mycobacterium smegmatis. The key reactions are catalyzed by a methyltransferase that transfers three methyl groups to the alpha-amino moiety of histidine and an iron(II)-dependent enzyme that catalyzes oxidative sulfurization of trimethylhistidine. A search for homologous genes indicated that ergothioneine production is a frequent trait among fungi, actinobacteria, and cyanobacteria but also occurs in numerous bacteroidetes and proteobacteria.
ETT (originally designated as OCTN1; human gene symbol SLC22A4) and CTT (OCTN2; SLC22A5) are highly specific transporters of ergothioneine and carnitine, respectively. Despite a high degree of sequence homology, both carriers discriminate precisely between substrates: ETT does not transport carnitine, and CTT does not transport ergothioneine. Our aim was to turn ETT into a transporter for carnitine and CTT into a transporter for ergothioneine by a limited number of point mutations. From a multiple alignment of several mammalian amino acid sequences, those positions were selected for conversion that were momentously different between ETT and CTT from human but conserved among all orthologues. Mutants were expressed in 293 cells and assayed for transport of ergothioneine and carnitine. Several ETT mutants clearly catalyzed transport of carnitine, up to 35% relative to wild-type CTT. Amazingly, complementary substitutions in CTT did not provoke transport activity for ergothioneine. In similar contrast, carnitine transport by CTT mutants was abolished by very few substitutions, whereas ergothioneine transport by ETT mutants was maintained even with the construct most active in carnitine transport. To explain these results, we propose that ETT and CTT use dissimilar pathways for conformational change, in addition to incongruent substrate binding sites. In other words, carnitine is excluded from ETT by binding, and ergothioneine is excluded from CTT by turnover movement. Our data indicate amino acids critical for substrate discrimination not only in transmembrane segments 5, 7, 8, and 10, but also in segments 9 and 12 which were hitherto considered as unimportant.
This study was conducted to determine the antioxidant capacity, immunomodulatory and lipid-lowering effects of spirulina in healthy elderly subjects and to document the effectiveness of spirulina as a functional food for the elderly.
A randomized double-blind, placebo-controlled study was performed. The subjects were 78 individuals aged 60-87 years and were randomly assigned in a blinded fashion to receive either spirulina or placebo. The elderly were instructed to consume the spirulina or placebo at home, 8 g/day, for 16 consecutive weeks.
In male subjects, a significant plasma cholesterol-lowering effect was observed after the spirulina intervention (p < 0.05). Spirulina supplementation resulted in a significant rise in plasma interleukin (IL)-2 concentration, and a significant reduction in IL-6 concentration. A significant time-by-treatment intervention for total antioxidant status was observed between spirulina and placebo groups (p < 0.05). In female subjects, significant increases in IL-2 level and superoxide dismutase activity were observed (p < 0.05) after spirulina supplementation. There were significant reductions in total cholesterol in female subjects.
The results demonstrate that spirulina has favorable effects on lipid profiles, immune variables, and antioxidant capacity in healthy, elderly male and female subjects and is suitable as a functional food.
Intracellular antioxidants include low molecular weight scavengers of oxidizing species, and enzymes which degrade superoxide and hydroperoxides. Such antioxidants systems prevent the uncontrolled formation of free radicals and activated oxygen species, or inhibit their reactions with biological structures. Hydrophilic scavengers are found in cytosolic, mitochondrial and nuclear compartments. Ascorbate and glutathione scavenge oxidizing free radicals in water by means of one-electron or hydrogen atom transfer. Similarly, ergothioneine scavenges hydroxyl radicals at very high rates, but it acts more specifically as a chemical scavenger of hypervalent ferryl complexes, halogenated oxidants and peroxynitrite-derived nitrating species, and as a physical quencher of singlet oxygen. Hydrophobic scavengers are found in cell membranes where they inhibit or interrupt chain reactions of lipid peroxidation. In animal cells, they include alpha-tocopherol (vitamin E) which is a primary scavenger of lipid peroxyl radicals, and carotenoids which are secondary scavengers of free radicals as well as physical quenchers of singlet oxygen. The main antioxidant enzymes include dismutases such as superoxide dismutases (SOD) and catalases, which do not consume cofactors, and peroxidases such as selenium-dependent glutathione peroxidases (GPx) in animals or ascorbate peroxidases (APx) in plants. The reducing coenzymes of peroxidases, and as a rule all reducing components of the antioxidant network, are regenerated at the expense of NAD(P)H produced in specific metabolic pathways. Synergistic and co-operative interactions of antioxidants rely on the sequential degradation of peroxides and free radicals as well as on mutual protections of enzymes. This antioxidant network can induce metabolic deviations and plays an important role in the regulation of protein expression and/or activity at the transcriptional or post-translational levels. Its biological significance is discussed in terms of environmental adaptations and functional regulations of aerobic cells.
The presence of blue-green algae (BGA) toxins in surface waters used for drinking water sources and recreation is receiving increasing attention around the world as a public health concern. However, potential risks from exposure to these toxins in contaminated health food products that contain BGA have been largely ignored. BGA products are commonly consumed in the United States, Canada, and Europe for their putative beneficial effects, including increased energy and elevated mood. Many of these products contain Aphanizomenon flos-aquae, a BGA that is harvested from Upper Klamath Lake (UKL) in southern Oregon, where the growth of a toxic BGA, Microcystis aeruginosa, is a regular occurrence. M. aeruginosa produces compounds called microcystins, which are potent hepatotoxins and probable tumor promoters. Because M. aeruginosa coexists with A. flos-aquae, it can be collected inadvertently during the harvesting process, resulting in microcystin contamination of BGA products. In fall 1996, the Oregon Health Division learned that UKL was experiencing an extensive M. aeruginosa bloom, and an advisory was issued recommending against water contact. The advisory prompted calls from consumers of BGA products, who expressed concern about possible contamination of these products with microcystins. In response, the Oregon Health Division and the Oregon Department of Agriculture established a regulatory limit of 1 microg/g for microcystins in BGA-containing products and tested BGA products for the presence of microcystins. Microcystins were detected in 85 of 87 samples tested, with 63 samples (72%) containing concentrations > 1 microg/g. HPLC and ELISA tentatively identified microcystin-LR, the most toxic microcystin variant, as the predominant congener.
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The IBD5 locus on chromosome 5q31 is a confirmed Crohn's disease (CD) susceptibility locus in adults. Recently, two polymorphisms in the organic cation transporter (OCTN) gene cluster within the IBD5 locus have been found to be associated with CD. Although the original report of significant linkage to IBD5 was in families with at least one case of early age at onset CD, there are no published reports on the role of OCTN genes in pediatric onset CD. We performed a comprehensive analysis of OCTN variants in an independent, exclusively pediatric onset CD cohort and examined the genotype/phenotype correlations.
264 Caucasian CD children (172 of them were trios) were genotyped along with 527 controls for OCTN1 (SLC22A4 C1672T), OCTN2 (SLC22A5 G-207C), and two haplotype-tagging SNPs (IGR2230 and IGR2198).
TDT confirmed the association of SLC22A4 and SLC22A5. Case-control analysis of the SLC22A4 1672T, SLC22A5-207C diplotype showed significant association (p=0.04) with CD susceptibility compared with controls. Little correlation was seen with regard to clinical phenotype and the SLC22A4/SLC22A5 diplotype. There was no significant interaction between the SLC22A4/SLC22A5 diplotype and the three CD-associated CARD15 SNPs.
We confirm the association of the OCTN variants (SLC22A4 and SLC22A5) in pediatric onset CD as seen in adult CD cohorts. However, when an extended IBD5 haplotype was examined, no independent association between OCTN variants and pediatric onset CD can be demonstrated. Compared with adults, a relatively weak association of the OCTN variants was observed in our CD cohort. No definitive genotype-phenotype correlation or gene-gene interactions with CARD15 were observed. Although the IBD5 locus is associated with pediatric onset CD, no definitive conclusions can be drawn about OCTN variants as causative genes in pediatric CD at this point.
A common haplotype spanning 250 kb on chromosome 5q31 is strongly associated with Crohn disease (CD). Recently, two functional variants within the SLC22A4 and SLC22A5 genes at this locus (IBD5), L503F (c.1507C > T) and G-207C (c.-207G > C), have been proposed to contribute directly to susceptibility to CD. However, extensive linkage disequilibrium at the IBD5 locus has complicated efforts to distinguish causal variants from association of the general risk haplotype. We genotyped the SLC22A4 and SLC22A5 variants and other polymorphisms across the risk haplotype in four populations of European origin, and applied regression-based haplotype analysis to over 1,200 fully genotyped case-control pairs, modeling case/control status on the presence of one or more SNPs to test for conditional association and to identify risk haplotypes. We found highly significant association of SNPs at the IBD5 locus with Crohn disease in all populations tested. However, the frequencies of L503F and G-207C in individuals who did not carry the general IBD5 risk haplotype were not significantly different in cases and controls, with associated disease odds ratios (ORs) of 0.90 (95% CI, 0.57-1.40) and 0.90 (95% CI, 0.65-1.23), respectively. Haplotype analysis showed that addition of the SLC22A4 and SLC22A5 variants to a null model that included the background risk haplotype did not significantly improve the model fit. In addition to the common risk haplotype, several rare haplotypes had an increased frequency in cases compared to controls. This study suggests that the molecular basis for Crohn disease susceptibility at the IBD5 locus remains to be defined, and highlights the challenge of the identification of causal variants in a complex disease in regions of extensive linkage disequilibrium.
Recent data have controversially suggested that variants of the organic cation transport genes SLC22A4 (OCTN1) and SLC22A5 (OCTN2) are responsible for the contribution of IBD5 to disease susceptibility in Crohn's disease (CD). The objective of this study was to assess the contribution of the SLC22A4 variant (1672T) and SLC22A5 variant (-207C) together with three IBD5 haplotype markers in the previously uninvestigated Swedish CD population.
The study comprised 178 CD patients and 143 healthy controls (HC). Genotyping for IBD5 single nucleotide polymorphisms (SNPs) IGR2096a_1, IGR2198a_1, IGR2230a_1, SLC22A4 1672T and SLC22A5 -207C was carried out using the TaqMan system. Associations with disease susceptibility and disease phenotype were investigated.
Strong linkage disequilibrium was observed between the investigated SNPs (D prime >0.92). IGR2096a_1 allelic frequency and homozygosity rates were associated with CD (44% CD versus 33.8% HC, p=0.008, OR=1.55 and 20% CD versus 12% HC, p=0.04, OR=1.93, respectively). Variant allelic frequency of SLC22A4, 1672T (44% versus 36%, p=0.03, OR=1.4) and homozygosity for the SLC22A4, SLC22A5 TC haplotype (1672T, -207C) (21.3% versus 12%, p=0.03, OR=1.78, population attributable risk (PAR)=11%) were associated with CD. There was no association between the allelic frequency of SLC22A5 and CD (46.6% CD versus 41.5% HC, p=0.82). The association of the TC haplotype with CD was not independent of the SNPs representing the extended IBD5 linkage interval.
The IBD5 locus is associated with CD in the Swedish population. The strongest association is with the marker SNP IGR2096a_1, lying p-telomeric to SLC22A4 and SLC22A5. The effect of the TC haplotype was not an independent determinant in this population.
Recently, we have identified the ergothioneine (ET) transporter ETT (gene symbol SLC22A4). Much interest in human ETT has been generated by case-control studies that suggest an association of polymorphisms in the SLC22A4 gene with susceptibility to chronic inflammatory diseases. ETT was originally designated a multispecific novel organic cation transporter (OCTN1). Here we reinvestigated, based on stably transfected 293 cells and with ET as reference substrate, uptake of quinidine, verapamil, and pyrilamine. ETT from human robustly catalyzed transport of ET (68micfrol/(minmgprotein)), but no transport of organic cations was discernible. With ET as substrate, ETT was relatively resistant to inhibition by selected drugs; the most potent inhibitor was verapamil (K(i)=11micromol/l). The natural compound hercynine and antithyroid drug methimazole are related in structure to ET. However, efficiency of ETT-mediated transport of methimazole (K(i)=7.5mmol/l) was 130-fold lower, and transport of hercynine (K(i)=1.4mmol/l) was 25-fold lower than transport of ET. ETT from mouse, upon expression in 293 cells, catalyzed high affinity, sodium-driven uptake of ET very similar to ETT from human. Additional real-time PCR experiments based on 16 human tissues revealed ETT mRNA levels considerably lower than in bone marrow. Our experiments establish that ETT is highly specific for its physiological substrate ergothioneine. ETT is not a cationic drug transporter, and it does not have high affinity for organic cation inhibitors. Detection of ETT mRNA or protein can therefore be utilized as a specific molecular marker of intracellular ET activity.
Considering drug-induced lipid peroxidation as a possible mediator of drug-induced toxicity and exploiting the free radical scavenging action of antioxidants, the present study was designed to evaluate the protective effects of ascorbic acid (AA) and water extract of Spirulina plantesis (SP) to minimize 5-fluorouracil (5-FU)-induced lipid peroxidation. The study has been performed in vitro using goat liver as an experimental model. This evaluation was done by measuring the malondialdehyde (MDA), reduced glutathione (GSH), 4-hydroxy-2-nonenal (4-HNE) and nitric oxide (NO) content of the tissue as markers of lipid peroxidation. The results suggest that ascorbic acid and water extract of Spirulina plantesis could suppress the 5-FU-induced lipid peroxidation to a significant extent.