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PURPOSE: Music therapy is widely used to enhance well-being, reduce pain, and distract patients from unpleasant symptoms in the clinical setting. However, the degree to which music modulates pain perception is unknown. The medial pain pathway including the limbic system is associated with emotion, but how music alters pathway activity is unclear. The aim of the study was to investigate pain thresholds and pain-related responses in the anterior cingulate cortex (ACC) and whether they were modulated when subjects listened to their favorite music genre. SUBJECTS AND METHODS: First, 30 subjects were examined for left forearm pain threshold using electrical stimulation with Pain Vision PS-2011N. The pain thresholds with and without music were compared. Second, when an 80-µA current from Pain Vision was applied to the left ankle of eight women, the pain-related responses of the ACC with and without music were observed with functional magnetic resonance device (fMRI). The changes in the pain-related activity in both parameters were discussed. RESULTS: The median pain threshold with favorite music was 38.9 µA, compared to 29.0 µA without, which was significantly different (p<0.0001). The men's thresholds were significantly higher than women's both with music (p<0.05) and without music (p<0.01). The pain threshold in women was more strongly affected by music than in men. The fMRI results showed that the pain-related response in the ACC in five of eight subjects was attenuated while they listened to their favorite music. No change was observed in the other three subjects. CONCLUSION: The present findings suggest that pain perception might be strongly affected by listening to favorite music, possibly through modulation of pain-related responses in the ACC.

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Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and ß-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

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We previously reported that shokyo and kankyo, which are water-extracted fractions of ginger, reduced LPS-induced PGE2 production in human gingival fibroblasts. In this study, we examined the effects of these herbs on LPS-treated mouse macrophage RAW264.7 cells. Both shokyo and kankyo reduced LPS-induced PGE2 production in a concentration-dependent manner. Shokyo and kankyo did not inhibit cyclooxygenase (COX) activity, nor did they alter the expression of molecules in the arachidonic acid cascade. In addition, these herbs did not alter NF-κB p65 translocation into nucleus, or phosphorylation of p65 or ERK. These results suggest that shokyo and kankyo inhibit cPLA2 activity. Although 6-shogaol produced similar results to those of shokyo and kankyo, the concentration of 6-shogaol required for the reduction of PGE2 production were higher than those of 6-shogaol in shokyo and kankyo. Therefore, several gingerols and shogaols other than 6-shogaol may play a role in the reduction of LPS-induced PGE2 production. Thus, 6-shogaol, and other gingerols and shogaols inhibit cPLA2 activity and reduce LPS-induced PGE2 production via a different mechanism from traditional anti-inflammatory drugs. Moreover, kampo medicines that contain shokyo or kankyo are considered to be effective for inflammatory diseases.

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Cancer invasion, metastasis, and therapy resistance are the crucial phenomena in cancer malignancy. The high expression of matrix metalloproteinase 9 (MMP9) is a biomarker as well as a causal factor of cancer invasiveness and metastatic activity. However, a regulatory mechanism underlying MMP9 expression in cancer is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming an important clue. In the present study, we aimed (i) to develop a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional tumoroid model and Mmp9 promoter and (ii) to examine pharmacological actions of anticancer medications using this reporter system. High expression and genetic amplification of MMP9 were found in colon cancer cases. We found that proximal promoter sequences of MMP9 in murine and human contained conserved binding sites for transcription factors ß-catenin/TCF/LEF, glucocorticoid receptor (GR), and nuclear factor kappa-B (NF-κB). The murine Mmp9 promoter (-569 to +19) was markedly activated in metastatic colon cancer cells and additionally activated by tumoroid formation and by ß-catenin signaling stimulator lithium chloride. The Mmp9 promoter-driven fluorescent reporter cells enabled the monitoring of activities of MMP9/gelatinase, tumorigenesis, invasion, and metastasis in syngeneic transplantation experiments. We also demonstrated pharmacological actions as follows: dexamethasone and hydrocortisone, steroidal medications binding to GR, inhibited the Mmp9 promoter but did not inhibit tumorigenesis. On the contrary, antimetabolite 5-fluorouracil, a gold standard for colon cancer chemotherapy, inhibited tumoroid formation but did not inhibit Mmp9 promoter activity. Notably, antimalaria medication artesunate inhibited both tumorigenesis and the Mmp9 promoter in vitro, potentially through inhibition of ß-catenin/TCF/LEF signaling. Thus, this novel reporter system enabled monitoring tumorigenesis, invasiveness, metastasis, key regulatory signalings such as ß-catenin/MMP9 axis, and druggability. Impact Statement Cancer invasion and metastasis have been shown to be driven by matrix metalloproteinase 9 (MMP9), whose expression mechanism is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming important. We established a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional (3D) tumoroid model and Mmp9 promoter. Using this reporter system, we demonstrated pharmacological actions of anticancer medications such as antimetabolite 5-fluorouracil (5-FU) and antimalaria medication artesunate (ART), which inhibited both tumorigenesis and ß-catenin/MMP regulatory signaling. Our study impacts the translational fields of oncology, drug discovery, and organoid model.

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The oral inflammatory diseases are divided into two types: acute and chronic inflammatory diseases. In this review, we summarize the biological efficacy of herbal medicine, natural products, and their active ingredients against acute and chronic inflammatory diseases in the oral region, especially stomatitis and periodontitis. We review the effects of herbal medicines and a biscoclaurin alkaloid preparation, cepharamthin, as a therapy against stomatitis, an acute inflammatory disease. We also summarize the effects of herbal medicines and natural products against periodontitis, a chronic inflammatory disease, and one of its clinical conditions, alveolar bone resorption. Recent studies show that several herbal medicines such as kakkonto and ninjinto reduce LPS-induced PGE 2 production by human gingival fibroblasts. Among herbs constituting these herbal medicines, shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) strongly reduce PGE 2 production. Moreover, anti-osteoclast activity has been observed in some natural products with anti-inflammatory effects used against rheumatoid arthritis such as carotenoids, flavonoids, limonoids, and polyphenols. These herbal medicines and natural products could be useful for treating oral inflammatory diseases.

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Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel that is expressed in the sensory neurons and responds to various noxious stimuli including heat and capsaicin. The molecular properties of TRPV1 have been clearly examined; however, there are obvious individual differences in human sensitivity to thermal stimuli and capsaicin. Here, we examined the possibility that different genome sequence of human TRPV1 caused the different sensitivity to heat or capsaicin. The sensitivities to burning pain and capsaicin of Japanese adult subjects were compared with their TRPV1 genome sequence, and we detected 6 single-nucleotide polymorphisms and 11 single-nucleotide polymorphisms related to burning pain and capsaicin sensitivity, respectively. In particular, homozygous I585V, a single-nucleotide polymorphism with amino acid substitution, significantly related to higher capsaicin sensitivity.

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Previously, we revealed that several kampo medicines used for patients with excess and/or medium patterns (kakkonto (TJ-1), shosaikoto (TJ-9), hangeshashinto (TJ-14), and orento (TJ-120)) reduced prostaglandin (PG)E2 levels using LPS-treated human gingival fibroblasts (HGFs). Recently, we examined other kampo medicines used for patients with the deficiency pattern [bakumondoto (TJ-29), shinbuto (TJ-30), ninjinto (TJ-32), and hochuekkito (TJ-41)] and the herbs comprising shinbuto and ninjinto using the same experimental model. Shinbuto and ninjinto concentration-dependently reduced LPS-induced PGE2 production by HGFs, whereas hochuekkito weakly reduced and bakumondoto did not reduce PGE2 production. Shinbuto and ninjinto did not alter cyclooxygenase (COX) activity or the expression of molecules involved in the arachidonic acid cascade. Therefore, we next examined which herbs compromising shinbuto and ninjinto reduce LPS-induced PGE2 production. Among these herbs, shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) strongly and concentration-dependently decreased LPS-induced PGE2 production. However, both shokyo and kankyo increased the expression of cytosolic phospholipase (cPL)A2 but did not affect annexin1 or COX-2 expression. These results suggest that shokyo and kankyo suppress cPLA2 activity. We demonstrated that kampo medicines suppress inflammatory responses in patients with the deficiency pattern, and in those with excess or medium patterns. Moreover, kampo medicines that contain shokyo or kankyo are considered to be effective for the treatment of inflammatory diseases.

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We previously demonstrated that a kampo medicine, kakkonto, decreases lipopolysaccharide- (LPS-) induced prostaglandin E2 (PGE2) production by human gingival fibroblasts. In this study, we examined the herbs constituting kakkonto that exhibit this effect. Shokyo strongly and concentration dependently and kanzo and keihi moderately decreased LPS-induced PGE2 production. Shokyo did not alter cyclooxygenase-2 (COX-2) activity, cytosolic phospholipase A2 (cPLA2), annexin 1 and COX-2 expression, and LPS-induced extracellular signal-regulated kinase (ERK) phosphorylation. Kanzo inhibited COX-2 activity but increased annexin 1 and COX-2 expression and did not alter LPS-induced ERK phosphorylation. Keihi inhibited COX-2 activity and LPS-induced ERK phosphorylation but slightly increased COX-2 expression and did not alter cPLA2 and annexin 1 expression. These results suggest that the mechanism of shokyo is through the inhibition of cPLA2 activity, and that of kanzo and keihi is through the inhibition of COX-2 activity and indirect inhibition of cPLA2 activity. Therefore, it is possible that shokyo and kakkonto are clinically useful for the improvement of inflammatory responses.

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Histatins are salivary proteins with antimicrobial activities. We previously reported that histatin 3 binds to heat shock cognate protein 70 (HSC70), which is constitutively expressed, and induces DNA synthesis stimulation and promotes human gingival fibroblast (HGF) survival. However, the underlying mechanisms of histatin 3 remain largely unknown. Here, we found that the KRHH sequence of histatin 3 at the amino acid positions 5-8 was essential for enhancing p27(Kip1) (a cyclin-dependent kinase inhibitor) binding to HSC70 that occurred in a dose-dependent manner; histatin 3 enhanced the binding between p27(Kip1) and HSC70 during the G1/S transition of HGFs as opposed to histatin 3-M(5-8) (substitution of KRHH for EEDD in histatin 3). Histatin 3, but not histatin 3-M(5-8), stimulated DNA synthesis and promoted HGF survival. Histatin 3 dose-dependently enhanced both p27(Kip1) and HSC70 ubiquitination, whereas histatin 3-M(5-8) did not. These findings provide further evidence that histatin 3 may be involved in the regulation of cell proliferation, particularly during G1/S transition, via the ubiquitin-proteasome system of p27(Kip1) and HSC70.

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PURPOSE: To evaluate correlations between serotonin transporter (SERT) uptake ability in human peripheral platelets and sleep bruxism (SB) frequency. METHODS: Subjects were consecutively recruited from sixth-year students at Okayama University Dental School. Subjects were excluded if they (1) were receiving orthodontic treatment, (2) had a dermatological disease, (3) had taken an antidepressant within 6 months, or (4) had used an oral appliance within 6 months. SB frequency was determined as the summary score of three consecutive night assessments using a self-contained electromyography detector/analyzer in their home. Fasting peripheral venous blood samples were collected in the morning following the final SB assessment. SERT amount and platelet number were quantified via an ELISA assay and flow cytometry, respectively. Functional SERT characterization, 5-hydroxytryptamine (5-HT) uptake, maximum velocity (V max), and an affinity constant (K m ) were assessed with a [(3)H] 5-HT uptake assay. The correlations between these variables and SB level were evaluated. RESULTS: Among 50 eligible subjects (26 males, mean age 25.4 ± 2.41 years), 7 were excluded because of venipuncture failure, smoking, and alcohol intake during the experimental period. A small but significant negative correlation between SB level and [(3)H] 5-HT uptake was observed (Spearman's correlation R (2) = 0.063, p = 0.04). However, there were no significant correlations between SB level and total platelet amount, SERT, V max, and K m values (p = 0.08, 0.12, 0.71, and 0.68, respectively). CONCLUSIONS: Platelet serotonin uptake is significantly associated with SB frequency, yet only explains a small amount of SB variability.

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PURPOSE: The aim of this study was to evaluate the correlation between sleep bruxism (SB) frequency and serotonin transporter (SERT)-driven serotonin (5-HT)-uptake in platelets. METHODS: Subjects were dental trainee residents and faculty members of Okayama University Hospital who were aware of having severe or no SB. SB frequency was assessed for 3-consecutive nights by a self-contained electromyographic detector/analyzer, which indicated individual SB levels as one of four grades (score 0, 1, 2 and 3). Subjects were classified as normal control (NC) when SB scores indicated only 0 or 1 during the 3 nights, or as severe SB for scores 2 or 3. Those subjects whose scores fluctuated from 0 to 3 during the 3 nights were omitted from further analysis. Fasting peripheral venous blood samples were collected in the morning following the final SB assessment. Amounts of SERTs proteins collected from peripheral platelets were quantified using ELISA, and SERTs transport activity was assessed by uptake assay using [3H]-5-HT. RESULTS: Thirteen severe SB subjects and 7 NC subjects were eligible. Gender distribution, mean age, 5-HT concentration and total amounts of SERT protein in platelets showed no significant differences between NC and severe SB (p=0.85: Chi-squared test; p=0.64, 0.26, 0.46: t-test). However, [3H]-5-HT uptake by platelets was significantly greater in NC compared to severe SB subjects (12.79±1.97, 8.27±1.91 fmol/10(5) platelets/min, p<0.001, t-test). CONCLUSION: The results of this pilot study suggest a possible correlation between peripheral platelet serotonin transporter uptake ability and SB severity.

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Parkinson's disease (PD) is a neurodegenerative disease with motor symptoms as well as non-motor symptoms that precede the onset of motor symptoms. Mitochondrial complex I inhibitor, rotenone, has been widely used to reproduce PD pathology in the central nervous system (CNS) and enteric nervous system (ENS). We reported previously that metallothioneins (MTs) released from astrocytes can protect dopaminergic neurons against oxidative stress. The present study examined the changes in MT expression by chronic systemic rotenone administration in the striatum and colonic myenteric plexus of C57BL mice. In addition, we investigated the effects of MT depletion on rotenone-induced neurodegeneration in CNS and ENS using MT-1 and MT-2 knockout (MT KO) mice, or using primary cultured neurons from MT KO mice. In normal C57BL mice, subcutaneous administration of rotenone for 6 weeks caused neurodegeneration, increased MT expression with astrocytes activation in the striatum and myenteric plexus. MT KO mice showed more severe myenteric neuronal damage by rotenone administration after 4 weeks than wild-type mice, accompanied by reduced astroglial activation. In primary cultured mesencephalic neurons from MT KO mice, rotenone exposure induced neurotoxicity in dopaminergic neurons, which was complemented by addition of recombinant protein. The present results suggest that MT seems to provide protection against neurodegeneration in ENS of rotenone-induced PD model mice.

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The GABAergic system in the spinal cord has been shown to participate in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling the synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In the present study, we compared the betaine/GABA transporter (BGT-1) with other GAT subtypes to determine its participation in neuropathic pain using a mouse model of sciatic nerve ligation. 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), an inhibitor that displays moderate selectivity for BGT-1, had an antiallodynic action on model mice treated through both intrathecally and intravenous administration routes. On the other hand, SKF89976A, a selective GAT-1 inhibitor, had a weak antiallodynic action, and (S)-SNAP5114, an inhibitor that displays selectivity for GAT-3, had no antiallodynic action. Systemic analysis of these compounds on GABA uptake in CHO cells stably expressing BGT-1 revealed that NNC05-2090 not only inhibited BGT-1, but also serotonin, noradrenaline, and dopamine transporters, using a substrate uptake assay in CHO cells stably expressing each transporter, with IC50: 5.29, 7.91, and 4.08 µM, respectively. These values were similar to the IC50 value at BGT-1 (10.6 µM). These results suggest that the antiallodynic action of NNC05-2090 is due to the inhibition of both BGT-1 and monoamine transporters.

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Oxidative stress and inflammation play important roles in diabetic complications, including diabetic nephropathy. Metallothionein (MT) is induced in proximal tubular epithelial cells as an antioxidant in the diabetic kidney; however, the role of MT in renal function remains unclear. We therefore investigated whether MT deficiency accelerates diabetic nephropathy through oxidative stress and inflammation. Diabetes was induced by streptozotocin injection in MT-deficient (MT(-/-)) and MT(+/+) mice. Urinary albumin excretion, histological changes, markers for reactive oxygen species (ROS), and kidney inflammation were measured. Murine proximal tubular epithelial (mProx24) cells were used to further elucidate the role of MT under high-glucose conditions. Parameters of diabetic nephropathy and markers of ROS and inflammation were accelerated in diabetic MT(-/-) mice compared with diabetic MT(+/+) mice, despite equivalent levels of hyperglycemia. MT deficiency accelerated interstitial fibrosis and macrophage infiltration into the interstitium in the diabetic kidney. Electron microscopy revealed abnormal mitochondrial morphology in proximal tubular epithelial cells in diabetic MT(-/-) mice. In vitro studies demonstrated that knockdown of MT by small interfering RNA enhanced mitochondrial ROS generation and inflammation-related gene expression in mProx24 cells cultured under high-glucose conditions. The results of this study suggest that MT may play a key role in protecting the kidney against high glucose-induced ROS and subsequent inflammation in diabetic nephropathy.

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AIMS: Cisplatin (CDDP) is a potent anticancer agent, but severe renal toxicity can limit its use. We investigated the protective effect of cepharanthin (CE), a biscoclaurin alkaloid, on the renal toxicity of CDDP. MAIN METHODS: Mice were given CDDP along with CE. Effects of CE on CDDP toxicity were investigated by assaying markers of renal toxicity together with MT expression, and by histopathological examination of the kidney. MT-null mice were also examined. KEY FINDINGS: CE induced expression of metallothionein (MT). Pre-administration of CE attenuated an increase in blood urea nitrogen (BUN) concentrations after the CDDP injection. A histochemical analysis demonstrated protection against CDDP-induced necrocytosis of kidney tissues by CE. The protective effect of CE did not occur in the MT-null mice. SIGNIFICANCE: Pretreatment with CE may reduce the renal toxicity of CDDP through expression of MT.

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The potency of anesthetics changes during development, probably due not only to pharmacokinetic factors such as differential distribution and/or metabolism, but also to pharmacodynamic factors such as changes to the GABAergic system in the brain. To explore the latter mechanism, we focused on the GABA transporter (GAT), the uptake system for GABA, which participates in the synaptic clearance of GABA. Thiopental-induced anesthesia, as assessed by the onset and duration of loss of the righting reflex, was more pronounced in 3-week-old mice than in 7-week-old mice. Both NO-711 and SKF89976A, selective GAT-1 inhibitors, significantly enhanced the anesthesia in the 7-week-old but not in the 3-week-old mice. In synaptosomes prepared from the cerebral cortex, the kinetics of GABA transport was similar between the two age groups, as assessed by [(3)H]GABA uptake assay. In addition, expression of GAT mRNA was similar between the two age groups, as assessed by quantitative RT-PCR. Thiopental reduced [(3)H]GABA uptake only at high concentrations in a similar manner at both ages. Conversely, the ability of SKF89976A to inhibit [(3)H]GABA uptake was greater in the 7-week-old mice than in the 3-week-old mice. Based on these results, GAT seems unlikely to contribute to the greater susceptibility to thiopental anesthesia in 3-week-old mice, while the increased ability of GABA uptake inhibitors to enhance thiopental-induced anesthesia in 7-week-old mice is at least partly due to higher sensitivity of GAT to the inhibitors.

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Betaine/γ-aminobutyric acid (GABA) transporter (BGT1, SLC6A12) is a member of the Na(+)- and Cl(-)-dependent neurotransmitter transporter gene family with a homology to the GABA transporters (GATs), GAT1 (SLC6A1), GAT2 (SLC6A13) and GAT3 (SLC6A11) (HUGO nomenclature). Since antidepressants have been reported to inhibit GABA uptake, we examined those effects on mouse BGT1 (mBGT1) in comparison with other mouse GAT (mGAT) subtypes in the heterologously expressed cell cultures. All antidepressants tested here inhibited the [(3)H]GABA uptake through mBGT1 and mGATs in a rank order of potency with mBGT1 > mGAT1-3. Kinetic analyses for maprotilline, mianserine and trimipramine revealed that they inhibited mBGT1 and mGAT1 noncompetitively, except that mianserine competitively inhibited mBGT1. These results provided a clue to investigate the structure-function relationship of mBGT1 using antidepressants as a tool, leading to the identification of potential candidates for selective and specific inhibitors of mBGT1.

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Metallothionein (MT) is an intracellular metal-binding, cysteine-rich protein, and is a potent antioxidant that protects cells and tissues from oxidative stress. Although the major isoforms MT-1 and -2 (MT-1/-2) are highly inducible in many tissues, the distribution and role of MT-1/-2 in diabetic nephropathy are poorly understood. In this study, diabetes was induced in adult male rats by streptozotocin, and renal tissues were stained with antibodies for MT-1/-2. MT-1/-2 expression was also evaluated in mProx24 cells, a mouse renal proximal tubular epithelial cell line, stimulated with high glucose medium and pretreated with the antioxidant vitamin E. MT-1/-2 expression was gradually and dramatically increased, mainly in the proximal tubular epithelial cells and to a lesser extent in the podocytes in diabetic rats, but was hardly observed in control rats. MT-1/-2 expression was also increased by high glucose stimulation in mProx24 cells. Because the induction of MT was suppressed by pretreatment with vitamin E, the expression of MT-1/-2 is induced, at least in part, by high glucose-induced oxidative stress. These observations suggest that MT-1/-2 is induced in renal proximal tubular epithelial cells as an antioxidant to protect the kidney from oxidative stress, and may offer a novel therapeutic target against diabetic nephropathy.

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Methylone (2-methylamino-1-[3,4-methylenedioxyphenyl]propane-1-one) is a synthetic hallucinogenic amphetamine analog, like MDMA (3,4-methylenedioxy- methamphetamine), considered to act on monoaminergic systems. However, the psychopharmacological profile of its cytotoxicity as a consequence of monoaminergic deficits remains unclear. We examined here the effects of methylone on the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT), using a heterologous expression system in CHO cells, in association with its cytotoxicity. Methylone inhibited the activities of DAT, NET, and SERT, but not GABA transporter-1 (GAT1), in a concentration-dependent fashion with a rank order of NET > DAT > SERT. Methylone was less effective at inhibiting DAT and NET, but more effective against SERT, than was methamphetamine. Methylone alone was not toxic to cells except at high concentrations, but in combination with methamphetamine had a synergistic effect in CHO cells expressing the monoamine transporters but not in control CHO cells or cells expressing GAT1. The ability of methylone to inhibit monoamine transporter function, probably by acting as a transportable substrate, underlies the synergistic effect of methylone and methamphetamine.

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Our previous studies demonstrated the involvement of quinone formation in dopaminergic neuron dysfunction in the L-DOPA-treated parkinsonian model and in methamphetamine (METH) neurotoxicity. We further reported that the cysteine-rich metal-binding metallothionein (MT) family of proteins protects dopaminergic neurons against dopamine (DA) quinone neurotoxicity by its quinone-quenching property. The aim of this study was to examine MT induction in astrocytes in response to excess DA and the potential neuroprotective effects of astrocyte-derived MTs against DA quinone toxicity. DA exposure significantly upregulated MT-1/-2 in cultured striatal astrocytes, but not in mesencephalic neurons. This DA-induced MT upregulation in astrocytes was blocked by treatment with a DA-transporter (DAT) inhibitor, but not by DA-receptor antagonists. Expression of nuclear factor erythroid 2-related factor (Nrf2) and its binding activity to antioxidant response element of MT-1 gene were significantly increased in the astrocytes after DA exposure. Nuclear translocation of Nrf2 was suppressed by the DAT inhibitor. Quinone formation and reduction of mesencephalic DA neurons after DA exposure were ameliorated by preincubation with conditioned media from DA-treated astrocytes. These protective effects were abrogated by MT-1/-2-specific antibody. Adding exogenous MT-1 to glial conditioned media also showed similar neuroprotective effects. Furthermore, MT-1/-2 expression was markedly elevated specifically in reactive astrocytes in the striatum of L-DOPA-treated hemi-parkinsonian mice or METH-injected mice. These results suggested that excess DA taken up by astrocytes via DAT upregulates MT-1/-2 expression specifically in astrocytes, and that MTs or related molecules secreted specifically by astrocytes protect dopaminergic neurons from damage through quinone quenching and/or scavenging of free radicals.

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