Search

In this study, we measured conjugated and unconjugated free bile acids (BAs) in plasma from patients with schizophrenia and healthy subjects to examine the possibility of BA as a biomarker for schizophrenia. Although the levels of each BA conjugate showed no significant differences, significant differences for three unconjugated bile acids were observed in the plasma between patients with schizophrenia and healthy subjects. Additionally, a more than three times difference between patients and healthy subjects was observed in the mean value of the total concentrations of primary BAs. These results indicate that cholic acid and chenodeoxycholic acid levels in plasma may be novel diagnostic markers for a sub-population of patients with schizophrenia. Thus, future studies should elucidate the relationship between this increase in BA levels and the pathology of schizophrenia and verify the potential of unconjugated BA in plasma as biomarkers for schizophrenia.

Water transport in the brain is tightly controlled by blood-brain-barrier (BBB) composed of capillary endothelial cells expressing AQP1/AQP11 and glial foot processes expressing AQP4. Here we examined each AQP mRNA expression in acute hyponatremic and hypernatremic mouse models of wild type (WT) and AQP11 KO mice (KO). The expressions of AQP1, AQP4 and AQP11 mRNAs were quantified by real-time qRT-PCR analysis of whole brain RNA. Acute hyponatremia enhanced AQP4 expression without changing AQP1 expression in KO, whereas it did not change the expression of all AQPs in WT. On the other hand, acute hypernatremia increased AQP4 but decreased AQP1 expression by half in KO, whereas it decreased AQP1 and AQP11 by half without changing AQP4 expression in WT. Enhanced AQP4 expression by osmotic challenges with sodium in KO seems to be a compensation for the loss of AQP11. A stronger hypertonic stimulation with mannitol decreased all AQPs by 30-80% in WT. Since AQP4 plays an important role in the regulation of brain edema at BBB, the results suggest that AQP11 may also be involved in the osmotic regulation of the brain.

Our previous studies have shown that glycerin, which is present at high concentrations in moisturizers and skin lotions, gradually oxidizes to produce methylglyoxal (MGO). In this study, we observed that MGO-treated porcine dermis type-I collagen was carbonylated in an MGO concentration- and time-dependent manner. Furthermore, we examined the structure of advanced glycation end products (AGEs) induced by MGO reacting with type-I collagen. Our findings demonstrate that the α chains of collagen reacted with MGO and easily transformed into a modified protein containing a methylglyoxal-derived hydroimidazolone (MG-H1) moiety in a concentration- and time-dependent manner. Moreover, porcine skin proteins underwent carbonylation when the skin section was treated with MGO for four weeks. Analysis of the structure of AGEs on the carbonylated proteins extracted from MGO-treated skin sections revealed that skin collagen had been converted to MG-H1-modified protein. These novel findings suggest that continuous application of MGO to the skin leads to carbonylation of proteins, which may cause prompt accumulation of MG-H1-modified dermis collagen, thereby resulting in morphological and functional changes of collagen in the skin.

In this study, we developed an analytical method using LC-MS/MS for the simultaneous determination of five bile acids (BAs) that have been recently reported as candidate diagnostic biomarkers for Alzheimer's disease (AD) or AD related factors in the brain. The measurement of BAs in the brains of healthy mice led to the determination of candidate diagnostic markers for AD, such as cholic acid and deoxycholic acid, and other bile acids, such as chenodeoxycholic acid noted for the ameliorating effect on the symptoms of AD. Significant positive correlations were observed between the brain and plasma concentrations of four BAs in healthy young mice. These results indicate that the BA level in the brain may be estimated by the corresponding BA level in the plasma. Thus, our study suggested that the proposed method for the analysis of the five bile acids would aid in the diagnosis of AD or in studies that use AD model mice.

Large adrenocortical adenomas have rarely been reported. We describe a case of a 26-year-old man who underwent an adrenalectomy for a large adrenocortical adenoma (8.6 × 7.7 cm). Although the lesion had typical malignant features on imaging, histopathological examination revealed an adrenocortical adenoma. This highlights that imaging alone may not be able to distinguish adrenocortical carcinomas from adrenal masses. In most cases, a resection should be performed for early diagnosis and management of large adrenal masses with malignant features on imaging. To our knowledge, this is the first report of a large adrenocortical adenoma diagnosed with multiple imaging investigations., Highlights • A patient presented with abdominal and left flank pain. • We observed an ≥8-cm lesion that appeared to be malignant on imaging. • Histologically, the lesion was diagnosed as an adrenocortical adenoma. • Adrenocortical adenomas with malignant imaging features must be resected. • This will allow their early diagnosis and treatment.

Background and Aims As the brain edema influences the outcomes of many edematous brain diseases due to the limited intracranial space, the role of aquaporins (AQPs) in brain edema needs to be clarified to advance its treatment especially in increasing ischemic brain diseases. Although the importance of AQP4 at the Blood Brain Barrier (BBB) has been well characterized, the roles of other AQPs is still unknown, especially a relatively new AQP11. As AQP11 is expressed in the brain capillary (Koike S et al. Int J Mol Sci. 17:861, 2016), AQP11 may also be important for the regulation of brain edema. The aim of the present study is to clarify the role of AQP11 in cerebral ischemic model to identify new treatment of cerebral edema. Method AQP expression in the brain was examined by RT-PCR. Common cervical artery was ligated for 15 min to produce ischemic-reperfusion model of brain infarction to induce brain edema. As the first step to study the involvement of AQPs in this process, the mRNA expression of AQP1, AQP4, AQP11 and GFAP were monitored after reperfusion which are expressed at BBB. As mannitol is often employed for the treatment of brain edema, the effects of single or twice doses of 1.1M mannitol 0.1 mL/g body weight i.p. on the expression of AQP1, AQP4 and AQP11 mRNA were examined after 6 h in control mice. Furthermore, hypertonic 2M NaCl was also challenged to simulate the osmotic effect: a single or twice doses of 2M NaCl 0.16 mL/g body weight were administered intraperitoneally, i.p. Results The expression of AQP1 mRNA increased by 20% one and two hours after 15-min ligation, while the expression of AQP4 mRNA decreased transiently just after the ligation but increased at 24 h by 10%. On the other hand, the expression of AQP11 mRNA started to decrease from 30 min after the ligation to continue decreasing up to 24h by 40% in wild mice, while the decrease of AQP11 mRNA only observed at 24h by 20% in AQP11 heterogenous KO mice. A single mannitol i.p. did not change serum osmolality from 320 to 317 mOsm, while twice i.p. in 6 h interval increased serum osmolality to 328 mOsm. Both procedures similarly decreased the expression of all AQPs: AQP1 by 80%, AQP4 by 40%, and AQP11 by 50%. Similar studies were conducted with 2M NaCl. A single dose of 2M NaCl increased serum Na/Osm from 151/319 to 154/322 mEq/mOsm, which decreased AQP1 by 50%, AQP11 by 35% and no change of AQP4. On the other hand, twice doses in 3 h interval increased serum Na/Osm to 208/435 mEq/Osm, which did not change all AQP expressions. Conclusion As AQP4 KO mice survive longer in brain edema models, decrease of AQP4 by mannitol will be beneficial as brain infarction increased AQP4 expression in our study. However, further decrease of AQP11 by mannitol will be detrimental as AQP11 was already decreased in brain infarction. As AQP11 KO mice die within a month due to polycystic kidneys, the studies on brain infarction in brain capillary specific AQP11 conditionaly KO mice are currently underway.

Recent studies have shown that carbonyl stress is a causative factor of schizophrenia, categorized as carbonyl stress-related schizophrenia (CS-SCZ). However, the correlation between carbonyl stress and the pathogenesis of this disease is not well established. In this study, glyoxalase 1(Glo1)-knockout and vitamin B6-deficient mice (KO/VB6 (-) mice), which are susceptible to methylglyoxal (MGO)-induced oxidative damages, were used as a CS-SCZ model to analyze MGO-modified protein and the carbonyl stress status in the brain. A comparison between Wild/VB6(+) mice and KO/VB6(−) mice for accumulated carbonyl proteins levels, with several advanced glycation end products (AGEs) in the brain, revealed that carbonyl protein levels with the Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl) ornithine (MG-H1) moiety were significantly increased in the hippocampus, prefrontal cortex, striatum, cerebral cortex, and brainstem regions of the brain in KO/VB6(−) mice. Moreover, two-dimensional electrophoresis and Liquid chromatography-tandem mass spectrometry analysis showed MG-H1-modified arginine residues in mitochondrial creatine kinase, beta-adrenergic receptor kinase 1, and T-complex protein in the hippocampus region of KO/VB6(−) mice, but not in Wild/VB6(+) mice. In particular, MG-H1 modification of mitochondrial creatine kinase was quite notable. These results suggest that further studies focusing on MG-H1-modified and accumulated proteins in the hippocampus may reveal the onset mechanism of CS-SCZ induced by MGO-induced oxidative damages.

Methylglyoxal (MG) is a cytotoxicα-dicarbonyl byproduct of glycolysis. Our bodies have several bio-defense systems to detoxify MG, including an enzymatic system by glyoxalase (GLO) 1 and a scavenge system by vitamin B6 (VB6). We know a population of patients with schizophrenia impaired MG detoxification systems. However, the molecular mechanism connecting them remains poorly understood. We created a novel mouse model for MG detoxification deficits by feedingGlo1knockout mice VB6-lacking diets (KO/VB6(-)) and evaluated the effects of impaired MG detoxification systems on brain function. KO/VB6(-) mice accumulated MG in the prefrontal cortex (PFC), hippocampus, and striatum, and displayed schizophrenia-like behavioral deficits. Furthermore, we found aberrant gene expression related to mitochondria function in the PFC of the KO/VB6(-) mice. We demonstrated respiratory deficits in mitochondria isolated from the PFC of KO/VB6(-) mice. These findings suggest that MG detoxification deficits might cause schizophrenia-like behavioral deficits via mitochondrial dysfunction in the PFC.

Methylglyoxal (MGO) is a reactive α-dicarbonyl compound that causes carbonylation of protein and DNA through the pathways of the Maillard reaction. It is known that MGO is physiologically involved in renal dysfunction, vascular disorders, and the acceleration of aging. In this study, we showed for the first time, that a trace amount of MGO was present as an impurity in glycerol preparations used as external medicines and intravenous infusions, when kept unused. The concentration of MGO in the glycerol solutions, diluted to a concentration of 20%, significantly increased after storage for one month when compared to the MGO concentration immediately after opening. Following storage for 6 months at 25°C, MGO concentration increased by about 300 times (approx. 170 µM), and at 40°C, it increased by about 600 times (approx. 350 µM). In the case of intravenous infusion preparations containing 10% glycerol, the MGO concentration increased by 4-15 times (approx. 70 µM) after 2 months of storage at 40°C, and reached over 200 µM after 6 months. Results from the present study showed that glycerol in pharmaceutical preparations is gradually oxidized to form MGO via autoxidation, depending on the temperature and dissolved oxygen content. Thus, we suggest that precautions should be taken when storing glycerol preparations in bottles or plastic containers, with respect to the storage temperature and sealability to prevent MGO formation due to oxidation of glycerol.

Methylglyoxal (MG) is an α-dicarbonyl compound that is naturally produced in vivo through glucose metabolism. In general, MG is metabolized by the glyoxalase 1(GLO1)/GLO2 system and aldose reductase (AR); however, excessive MG can react with proteins and nucleic acids to induce the accumulation of advanced glycation end products (AGEs). Recently, the accumulation of AGEs in the brain has been presumed to be related to neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease, respectively. Research investigating the role of AGEs in such diseases is ongoing. However, the changes in MG concentration that occur in the brain during healthy ageing remain unclear. Therefore, we performed fractionation of the brains of aged and young mice, measured the MG concentration in each part of the brain, and then examined the distribution. We also investigated the expression levels of GLO1 and AR, the main metabolizing enzymes of MG, in various brain regions, across age groups. We show that MG concentration varies among different regions of the brain, and that MG concentration in aged mice is significantly lower than that in young mice across all regions of the brain, except the brain stem. In addition, although the expression level of the GLO1 protein in the brain did not change with ageing, the expression level of AR was higher in aged than in young mice. Moreover, although a significant positive correlation was observed between GLO1 expression and MG concentration in the brains of young mice, no significant correlations were observed in the brains of aged mice. Meanwhile, the production of protein carbonyls and the accumulation of AGEs were not observed in the brains of aged mice. These results suggest that the accumulation of MG in the brain, along with the carbonyl stress are suppressed and regionally controlled during healthy ageing. This finding is useful as the foundation for further studies to investigate the role and toxicity of MG in various age-related disease conditions.

It has been known that hydrogen sulfide and/or polysulfides are produced from a (poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase (MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the donor substrate and the catalytic reaction was terminated on the formation of a persulfide or polysulfides. We can present alternative pathway of production of hydrogen sulfide and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometric analysis revealed that after prolonged incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPH = 1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric analysis for monobromobimane-derivatized H2Sn revealed that H2S2 first appeared, and then H2S and H2S3 did later. The results were confirmed by high-performance liquid chromatography-fluorescence analysis.

Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H2Sn), have various physiological functions in mammalian cells. Although H2Sn molecules have been considered as secondary metabolites derived from hydrogen sulfide (H2S) based on in vitro studies or predetermined reaction formula, the physiological form of BSS and their endogenous concentration remain unclear. In the present study, we aimed to improve the usual method using monobromobimane (mBB) followed by high performance liquid chromatographic (HPLC) analysis for HS－ for simultaneous determination of H2S, H2S2, H2S3 and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of H2S and H2Sn standards under alkaline conditions (pH 9.5) induced significant decreases in H2S2 and H2S3 levels and a significant increase in the H2S level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of H2S2 and H2S3 were stable under neutral conditions (pH 7.0), which is physiologically relevant. Therefore, we re-examined the method using mBB and applied an improved method for the evaluation of H2S, H2S2, and H2S3 in mouse brain under physiological pH conditions. The concentrations of H2S and H2S2 were 0.030 ± 0.004μmol/g protein and 0.026 ± 0.002μmol/g protein, respectively. Although the level of H2S3 was below the quantification limit of this method, H2S3 was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous H2S2 and H2S3 in mammalian brain tissues. H2S2 and H2S3 exert anti-oxidant activity and anti-carbonyl stress effects through the regulation of redox balance in neuronal cells. Thus, our observations provide novel insights into the physiological functions of BSS in the brain and into neuronal diseases involved in redox imbalance.

Argpyrimidine (ARP) is an advanced glycation end product thought to be generated from a reaction between methylglyoxal and arginine residues in proteins. In this study, we observed marked accumulation of an approximately 56 kD protein, reactive to anti-ARP antibodies, in the red blood cells (RBCs) of some patients with refractory schizophrenia. This ARP-modified protein was purified from the blood of schizophrenic patients and identified as selenium binding protein 1 (SBP1) by LC-MS/MS. This is the first report of ARP-modified proteins accumulating in RBCs of patients with diseases involving carbonyl stress. We also observed high accumulation of ARP-modified SBP1 in the RBCs of patients with chronic kidney disease. Therefore, this modified protein may be a novel marker of carbonyl stress.

Alzheimer's disease (AD) destroys brain function, especially in the hippocampus, and is a social problem worldwide. A major pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) peptides, resulting in neuronal cell death in the brain. Here, we isolated four saponins (1-4) and elucidated their structures from 1D and 2D NMR and HRFABMS spectral data. The structures of 1 and 2 were determined as new saponins which have cochalic acid as the aglycon, and 3 was determined as a new saponin with oleanolic acid as the aglycon. Compound 4 was confirmed as the known saponin chikusetsusaponin V (=ginsenoside R0). Isolated saponins (1-4) and six previously reported saponins (5-10) were tested for their inhibitory effects of Aβ aggregation and their protective effects on SH-SY5Y cells against Aβ-associated toxicity. As the results, compounds 3 and 4 showed inhibitory effect of Aβ aggregation and compounds 5-8 exerted the protective effects on SH-SY5Y cells against Aβ-associated toxicity.

Peroxiredoxins (Prxs) detoxify hydrogen peroxide (H2O2), peroxynitrite, and various organic hydroperoxides. However, the differential oxidative status of Prxs reacted with each peroxide remains unclear. In the present study, we focused on the oxidative alteration of Prxs and demonstrated that, in human red blood cells (RBCs), peroxiredoxin 2 (Prx2) is readily reactive with H2O2, forming disulfide dimers, but was not easily hyperoxidized. In contrast, Prx2 was highly sensitive to the relatively hydrophobic oxidants, such as tert-butyl hydroperoxide (t-BHP) and cumene hydroperoxide. These peroxides hyperoxidized Prx2 into oxidatively damaged forms in RBCs. The t-BHP treatment formed hyperoxidized Prx2 in a dose-dependent manner. When organic hydroperoxide-treated RBC lysates were subjected to reverse-phase high performance liquid chromatography, two peaks derived from hyperoxidized Prx2 appeared along with the decrease of that corresponding to native Prx2. Liquid chromatography-tandem mass spectrometry analysis clearly showed that hyperoxidation to sulfonic acid (-SO3H) at Cys-51 residue was more advanced in a newfound hyperoxidized Prx2 compared to another hydrophobic hyperoxidized form previously identified. These results indicate that irreversible hyperoxidation of the Prx2 monomer in RBCs was easily caused by organic hydroperoxide but not H2O2. Thus, it is important to detect the hyperoxidation of Prx2 into sulfinic or sulfonic acid derivates of Cys-51 because hyperoxidized Prx2 is a potential marker of oxidative injury caused by organic hydroperoxides in human RBCs.

The present study focused on the methylglyoxal (MG) detoxification mechanism in neuroblastoma cells. The involvement of nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway as a defense response against the formation of MG-modified proteins, which is well-known evidence of carbonyl stress, was also examined. We found that MG treatment resulted in accumulation of modified proteins bearing the structure of advanced glycation end products (AGEs) derived from MG in SH-SY5Y cells. This accumulation was suppressed by activation of the Nrf2 pathway prior to MG exposure via pre-treatment with an Nrf2 activator, carnosic acid and CDDO-Im, confirming the involvement of the Nrf2 pathway in MG detoxification. Although pre-treatment with the Nrf2 activator did not affect mRNA levels of GLO1, AKR1B1, and AKR7A2, the expressions of GCL and xCT mRNA, involved in GSH synthesis, were induced prior to increase in GSH levels. Furthermore, we demonstrated that a GSH synthesis inhibitor eliminated the MG detoxification effect derived from pretreatment with the Nrf2 activator. These results indicated that increase in GSH levels, induced by pre-treatment with carnosic acid, promoted the formation of the GLO1 substrate, hemithioacetal, thereby accelerating MG metabolism via the glyoxalase system and suppressing its toxicity. It was, therefore, determined that promotion of GSH synthesis via the Nrf2/Keap1pathway is important in the MG detoxification mechanism against neuronal MG-induced carbonyl stress, and Nrf2 activators contribute to reduction in the accumulation and toxic expression of carbonyl proteins.

Methylglyoxal (MG) is a highly reactive dicarbonyl compound that promotes the non-enzymatic glycation of proteins to yield irreversible advanced glycated end products, leading to the cross-linking or degradation of proteins. The physiological relevance of MG currently remains unclear because its metabolic behavior has not yet been elucidated in detail. Although several labeling methods that require a HPLC system have been developed and used to measure MG, a standard method to analyze the content of MG in biological samples has not been established. We herein present a practical method based on HPLC with fluorescence detection to measure low MG levels. MG concentrations were also measured in human blood plasma using the present method in order to demonstrate its utility. A calibration curve was produced using freshly purified MG at concentrations ranging between 0.05 and 1.0 μM. The intra-day and inter-day relative standard diviations of the method were 2.55% and 4.03%, respectively. The limit of detection and limit of quantification were 60 fmol and 200 fmol, respectively for MG with a 10-μl injection volume of the derivatized sample solution. When the optimized method was applied to human plasma, the resulting concentrations of MG in the plasma of healthy subjects (n = 23) ranged between 0.024 and 0.258 μM (mean ± SD = 0.098 ± 0.066). Thus, the method developed herein is simple, sensitive, and easy to operate for the measurement of MG in biological samples.

Although it's well known that protein carbonyl (PCO) and advanced glycation end-products (AGEs) levels are elevated in plasma from patients with renal dysfunction, we recently identified patients who had no renal dysfunction but possessed high levels of plasma pentosidine (PEN), which is an AGEs, and low vitamin B6 levels in serum. In this study, we investigated the status of carbonyl stress to characterize the subtype of schizophrenia. When plasma samples were subjected to Western blot analysis for various AGEs, clear differences were only observed with the anti-PEN antibody in the plasma from schizophrenic patients. Moreover, we determined the formation of protein carbonyl (PCO), a typical indicator of carbonyl stress, occurred prior to the accumulation of PEN in the plasma of schizophrenic patients. PCO levels in the plasma from schizophrenic patients were significantly higher than that from healthy subjects. Western blots analysis clearly showed that albumin and IgG were markedly carbonylated in the plasma of some patients. Thus, PCOs may be a novel marker of carbonyl stress-type schizophrenia in addition to albumin containing PEN structure.

Polysulfides are a typical type of bound sulfur, which is physiologically stable form of sulfur species, derived from the hydrogen sulfide (H2S) that is generated endogenously in cells. We previously reported that bound sulfur protects neuronal cells from oxidative injury. In the present study, we demonstrated that polysulfides inhibited cell growth and promoted neurite outgrowth in mouse neuroblastoma Neuro2A (N2A) cells. However, Na2S showed no effect on neurite outgrowth in N2A cells. Furthermore, 2-APB and SKF96365, which are typical transient receptor potential (TRP) channel inhibitors, suppressed the neurite outgrowth induced by Na2S4. These new findings suggest that bound sulfur could induce neurite outgrowth and cell differentiation of N2A cells by accelerating calcium influx.

It has been known that hydrogen sulfide and/or polysulfides are produced from a (poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase (MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the donor substrate and the catalytic reaction was terminated on the formation of a persulfide or polysulfides. We can present alternative pathway of production of hydrogen sulfide and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometric analysis revealed that after prolonged incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPH = 1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric analysis for monobromobimane-derivatized H

Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H

Hydrogen sulfide (H2S) exerts synergistic effects with another gaseous signaling molecule nitric oxide (NO) on ion channels and vasculature. However, the mechanism of the synergy is not well understood. Here, we show that the interaction between H2S and NO generates polysulfides (H2Sn), which activate transient receptor potential ankyrin 1 (TRPA1) channels. High performance liquid chromatography with tandem mass spectrometry analysis, along with the imaging of intracellular Ca2+ and H2Sn, showed that H2Sn and their effects were abolished by cyanolysis and by reducing substances such as dithiothreitol (DTT), cysteine, and glutathione (GSH). However, the effects of nitroxyl or nitrosopersulfide, other potential products of H2S and NO interaction, are not affected by cyanolysis or reducing substances. This study demonstrates that H2Sn are products of synergy between H2S and NO and provides a new insight into the signaling mechanisms.

Polysulfide is a bound sulfur species derived from endogenous H2S. When mouse neuroblastoma, Neuro2A cells were exposed to tert-butyl hydroperoxide after treatment with polysulfide, a significant decline in cell toxicity was observed. Rapid uptake of polysulfides induced translocation of Nrf2 into the nucleus, resulting in acceleration of GSH synthesis and HO-1 expression. We demonstrated that polysulfide reversibly modified Keap1 to form oxidized dimers and induced the translocation of Nrf2. Moreover, polysulfide treatment accelerated Akt phosphorylation, which is a known pathway of Nrf2 phosphorylation. Thus, polysulfide may mediate the activation of Nrf2 signaling, thereby exerting protective effects against oxidative damage in Neuro2A cells.

3-Mercaptopyruvate (3-MP) is a metabolite of cysteine present in mammalian tissues and is known to be a substrate of 3-mercaptopyruvate sulfurtransferase (3MST, EC.3.4.1.2). The physiological relevance of the 3-MP pathway has not been fully recognized because the metabolic behavior of 3-MP remains unclear. Here, we describe a novel method using high-performance liquid chromatography with fluorescence detection to measure 3-MP formation from cysteine. To demonstrate the practical value of the present method, we applied it to analyze the 3-MP produced in biological samples from mouse tissue.

Hydrogen sulfide (H2S) exerts synergistic effects with another gaseous signaling molecule nitric oxide (NO) on ion channels and vasculature. However, the mechanism of the synergy is not well understood. Here, we show that the interaction between H2S and NO generates polysulfides (H2Sn), which activate transient receptor potential ankyrin 1 (TRPA1) channels. High performance liquid chromatography with tandem mass spectrometry analysis, along with the imaging of intracellular Ca2+ and H2Sn, showed that H2Sn and their effects were abolished by cyanolysis and by reducing substances such as dithiothreitol (DTT), cysteine, and glutathione (GSH). However, the effects of nitroxyl or nitrosopersulfide, other potential products of H2S and NO interaction, are not affected by cyanolysis or reducing substances. This study demonstrates that H2Sn are products of synergy between H2S and NO and provides a new insight into the signaling mechanisms.

The formation of advanced glycation end products (AGEs) is associated with various neurological disorders, such as Alzheimer's disease, Parkinson's disease and schizophrenia. Methylglyoxal (MG), a highly reactive dicarbonyl compound, is known to be a major precursor for AGEs in modified proteins. Thus, a scavenger of MG might provide beneficial effects by suppressing the accumulation of AGEs and the occurrence of diseases induced by carbonyl stress. Meanwhile, polysulfides, one of the typical bound sulfur species, are oxidized forms of hydrogen sulfide (H2S) and may play a variety of roles in the brain. Herein, we assessed the scavenging ability of polysulfides against neuronal carbonyl stress induced by MG. First, we showed that polysulfides could protect differentiated (df)-SH-SY5Y cells from MG-induced cytotoxicity. When cells were pretreated with polysulfides, MG-induced cytotoxicity was attenuated with a rapid decrease in intracellular MG levels. Moreover, we found that polysulfides significantly suppressed the formation of MG-modified proteins in df-SH-SY5Y cells. Although polysulfide treatment increased endogenous GSH levels in the neuronal cells, its effects on MG-induced cytotoxicity were not affected by GSH concentration. Our results demonstrated that polysulfides had the direct potentials to protect neuronal cells against MG separate to the enzymatic detoxification system that required GSH.

Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles. We recently found H2Sn in the brain. H2Sn induced some responses previously attributed to H2S but with much greater potency than H2S. However, the number of sulfur atoms in H2Sn and its producing enzyme were unknown. Here, we detected H2S3 and H2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain. High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H2S3 and H2S were produced from 3 MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not. H2S3 was localized in the cytosol of cells. H2S3 was also produced from H2S by 3MST and rhodanese. H2S2 was identified as a minor H2Sn and 3 MP did not affect the H2S5 level. The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

Peroxiredoxins (Prxs) are a family of thiol peroxidases, which have been suggested to serve as biomarkers for diseases caused by oxidative stress. In this study, we established a high performance liquid chromatography (HPLC) method for quantifying the amount of Prx2 in red blood cells (RBCs). RBC proteins were separated using HPLC, and a single peak was detected that matched that produced by recombinant Prx2. Under improved conditions, the calibration curve for Prx2 (reduced form) was linear over the range of 0.5-20.0μg with a correlation coefficient of 0.999. The minimum detectable level of the recombinant Prx2 was 0.2μg, with a signal-to-noise ratio of 3 per 20μl of injection volume. SDS-PAGE and mass spectrometric analysis showed that the proteins comprising the peak were almost exclusively Prx2. Further high-resolution analysis using nanoLC-MS/MS demonstrated that the oxidation sensitive, Cys-51 was carbamidomethylated by iodoacetamide-alkylation during in-gel digestion but was not modified with sulfinic acid (-SO2H) or sulfonic acid (-SO3H). These results indicated that the separated Prx2 was the reduced form and not the hyperoxidized form. These basic experiments allowed us to determine the relative amounts of native Prx2 in RBCs taken from healthy subjects. The average levels of Prx2 in male and female subjects were 7.28ng/mg and 8.29ng/mg, respectively, and no significant difference was observed between the sexes. Therefore, the HPLC method with UV detection described herein offers a convenient method to quantitatively determine the levels of reduced form of Prx2 and its oxidative decrease in human RBCs.

Cysteine-persulfide (Cys-SSH) is a cysteine whose sulfhydryl group is covalently bound to sulfur (sulfane sulfur). Cys-SSH and its glutathione (GSH) counterpart (GSSH) have been recognized as redox regulators, some of which were previously ascribed to cysteine and GSH. However, the production of Cys-SSH and GSSH is not well understood. Here, we show that 3-mercaptopyruvate sulfurtransferase (3MST) produces Cys-SSH and GSSH together with the potential signaling molecules hydrogen per- and tri-sulfide (H2S2 and H2S3). Cys-SSH and GSSH are produced in the brain of wild-type mice but not in those of 3MST-KO mice. The levels of total persulfurated species in the brain of 3MST-KO mice are less than 50% of that in the brain of wild-type mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST showed that Cys-SSH and GSSH were produced in the presence of physiological concentrations of cysteine and glutathione, while those with longer sulfur chains, Cys-SSnH and GSSnH, were produced in the presence of lower than physiological concentrations of cysteine and glutathione. The present study provides new insights into the production and physiological roles of these persulfurated species as well as the therapeutic targets for diseases in which these molecules are involved.

The excessive intake of dietary salt is a global issue in health. Attempts have been made to address this issue, including the development of salt substitutes. Yet, none of these substances are currently in wide use, because of their weak saltiness. The purpose of this study was to assess the effects of sodium aspartate (Asp-Na) on salty taste perception using the bullfrog glossopharyngeal nerve response and human sensory tests. When added to the mixture of NaCl and KCl, Asp-Na significantly enhanced the glossopharyngeal nerve response to the mixture by 1.6-fold compared to control. Asp-Na did not enhance the response to NaCl, nor did Asp-Na enhance the response to sour, bitter, or umami stimuli. The optimal concentration for Asp-Na to enhance the salt mixture was 1.7 mM. The largest enhancement was induced when NaCl and KCl were mixed at equimolar concentrations. Asp-Na significantly suppressed the glossopharyngeal nerve response to quinine hydrochloride, which suggests that bitterness of KCl is suppressed by Asp-Na. The salty taste enhancing effect of Asp-Na was also confirmed with human sensory tests. The present results suggested that the mixture of NaCl and KCl containing Asp-Na can be used as a salt substitute. In addition to demonstrating that Asp-Na enhanced salt taste responses in an experimental animal and human, our findings provide clues to identify the elusive salty taste receptors.

In eukaryotes, hydrogen sulphide acts as a signalling molecule and cytoprotectant. Hydrogen sulphide is known to be produced from L-cysteine by cystathionine β-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase coupled with cysteine aminotransferase. Here we report an additional biosynthetic pathway for the production of hydrogen sulphide from D-cysteine involving 3-mercaptopyruvate sulfurtransferase and D-amino acid oxidase. Unlike the L-cysteine pathway, this D-cysteine-dependent pathway operates predominantly in the cerebellum and the kidney. Our study reveals that administration of D-cysteine protects primary cultures of cerebellar neurons from oxidative stress induced by hydrogen peroxide and attenuates ischaemia-reperfusion injury in the kidney more than L-cysteine. This study presents a novel pathway of hydrogen sulphide production and provides a new therapeutic approach to deliver hydrogen sulphide to specific tissues.