Association of risk factors and bleeding complications in Asian patients taking edoxaban.

AIMS: Asian patients are known to be more prone to bleeding complications than patients of other ethnicities. Therefore, there are possibilities of other risk factors that should be given special consideration for dosage adjustment in this specific ethnic group. This study aimed to investigate the risk factors for bleeding complications in Asian patients under appropriate edoxaban dosage regimens. METHODS: Data on patients taking proper dosages, based on the Lixiana package insert, were analysed. Univariate and multivariable analyses were conducted to evaluate associations between risk factors and bleeding outcomes. Subgroup analysis was performed on high-risk patients for bleeding complications whose edoxaban dose was reduced according to the package insert. RESULTS: In total, 346 patients were included. Among them, 32 patients experienced bleeding complications. Patients with weight ≤60 kg and with cancer showed around 3.3- and 3.4-fold increased risk of bleeding complications compared to heavier patients (>60 kg) and those without cancer, respectively. In subgroup analysis with high-risk patients who took low-dose edoxaban (15 and 30 mg), weight ≤60 kg remained a significant factor for bleeding outcomes. CONCLUSION: This study showed that weight ≤60 kg and the presence of cancers could affect bleeding complications, which occurred despite proper edoxaban treatment in Asian patients. Therefore, more strict dosage guideline could be considered in populations with high proportions of Asian ethnicities.

Ethanol extract from Gynostemma pentaphyllum ameliorates dopaminergic neuronal cell death in transgenic mice expressing mutant A53T human alpha-synuclein.

Gynostemma (G.) pentaphyllum (Cucurbitaceae) contains various bioactive gypenosides. Ethanol extract from G. pentaphyllum (GP-EX) has been shown to have ameliorative effects on the death of dopaminergic neurons in animal models of Parkinson's disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- and 6-hydroxydopamine. PD patients exhibit multiple symptoms, so PD-related research should combine neurotoxin models with genetic models. In the present study, we investigated the ameliorative effects of GP-EX, including gypenosides, on the cell death of dopaminergic neurons in the midbrain of A53T α-synuclein transgenic mouse models of PD (A53T). Both GP-EX and gypenosides at 50 mg/kg per day were orally administered to the A53T mice for 20 weeks. α-Synuclein-immunopositive cells and α-synuclein phosphorylation were increased in the midbrain of A53T mice, which was reduced following treatment with GP-EX. Treatment with GP-EX modulated the reduced phosphorylation of tyrosine hydroxylase, extracellular signal-regulated kinase (ERK1/2), Bcl-2-associated death promoter (Bad) at Ser112, and c-Jun N-terminal kinase (JNK1/2) due to α-synuclein overexpression. In the A53T group, GP-EX treatment prolonged the latency of the step-through passive avoidance test and shortened the transfer latency of the elevated plus maze test. Gypenosides treatment exhibited the effects and efficacy similar to those of GP-EX. Taken together, GP-EX, including gypenosides, has ameliorative effects on dopaminergic neuronal cell death due to the overexpression of α-synuclein by modulating ERK1/2, Bad at Ser112, and JNK1/2 signaling in the midbrain of A53T mouse model of PD. Further studies are needed to investigate GP-EX as a treatment for neurodegenerative synucleinopathies, including PD. This study was approved by the Animal Ethics Committee of Chungbuk National University (approval No. CBNUA-956-16-01) on September 21, 2016.

Oral diabetes medication and risk of dementia in elderly patients with type 2 diabetes.

AIM: To examine the effect of oral diabetes medication on the risk of dementia in an elderly cohort with type 2 diabetes. METHODS: This was a population-based cohort study using the Korean National Health Insurance claims data from 2002 to 2013. Elderly subjects (60 years of age or older) with and without type 2 diabetes were included; patients with new-onset type 2 diabetes were further divided into the oral diabetes medication group and no-medication group. RESULTS: Among 278,290 patients with type 2 diabetes, 56,587 developed dementia (20.3%) over 11 years of follow-up. Type 2 diabetes was associated with a 1.69-fold increased risk of dementia (95% CI 1.66-1.72). Among patients with newly diagnosed type 2 diabetes, the risk of dementia was lower in the oral diabetes medication group than in the no-medication group (adjusted hazard ratio [aHR], 0.79; 95% CI 0.77-0.81). Lower risk of dementia was particularly noticeable in all of the combination therapy groups and especially lower in the combination therapy group treated with dipeptidyl peptidase 4 inhibitor (aHR 0.48, 95% CI 0.45-0.51). CONCLUSION: Overall, the use of oral diabetes medication in type 2 diabetes patients significantly decreased the risk of dementia.

Repeated treatments with the D1 dopamine receptor agonist SKF-38393 modulate cell viability via sustained ERK-Bad-Bax activation in dopaminergic neuronal cells.

The D1 dopamine receptor agonist, SKF-38393, induces cytotoxicity in striatal dopaminergic neurons via an extracellular signal-regulated kinase (ERK) signaling cascade. However, the underlying mechanism remains unclear. We hypothesized that repeated activation of dopaminergic receptors by agonists could lead to neuronal cell death. This study investigated the effects of SKF-38393 on dopaminergic neuronal cell death in a 6-hydroxydopamine-lesioned rat model of Parkinson's disease (PD) and PC12 cells. In the PD model, SKF-38393 administration (3 and 10 mg/kg per day, s.c.) for 8 weeks significantly increased the number of tyrosine hydroxylase-immunopositive neuronal cells in nigrostriatal regions. SKF-38393 administration for 8 weeks induced phosphorylation of sustained ERK1/2 and Bad (Bcl-2-associated death promoter) at Ser155 (BadSer155), and augmented Bax (Bcl-2-associated X protein) expression. However, SKF-38393 only increased Bad phosphorylation at Ser112 (BadSer112) when administered for 4 weeks. In PC12 cells, toxic levels of SKF-38393 (20 and 50 µM) rapidly induced formation of neurite-like processes, but not in the presence of an adenylyl cyclase inhibitor (MDL-12330 A). SKF-38393 (20 and 50 µM) induced sustained ERK1/2 and BadSer155 phosphorylation as well as caspase-3 activation. At a non-toxic level (5 µM), SKF-38393 produced only transient ERK1/2 and BadSer112 phosphorylation. Repeated treatments with SKF-38393 (5 µM) for 1-3 days activated BadSer112. Repeated treatments for 4-7 days induced sustained ERK1/2 and BadSer155 phosphorylation as well as Bax and caspase-3 activation. These results suggest that SKF-38393 induces neurotoxicity by activation of the sustained ERK-Bad-Bax system. These findings contribute to an understanding of the adverse effects of D1 dopamine receptor agonists in patients with PD.

1-O-Hexyl-2,3,5-Trimethylhydroquinone Ameliorates l-DOPA-Induced Cytotoxicity in PC12 Cells.

1-O-Hexyl-2,3,5-trimethylhydroquinone (HTHQ) has previously been found to have effective anti-oxidant and anti-lipid-peroxidative activity. We aimed to elucidate whether HTHQ can prevent dopaminergic neuronal cell death by investigating the effect on l-DOPA-induced cytotoxicity in PC12 cells. HTHQ protected from both l-DOPA-induced cell death and superoxide dismutase activity reduction. When assessing the effect of HTHQ on oxidative stress-related signaling pathways, HTHQ inhibited l-DOPA-induced phosphorylation of sustained extracellular signal-regulated kinases (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK1/2). HTHQ also normalized l-DOPA-reduced Bcl-2-associated death protein (Bad) phosphorylation and Bcl-2-associated X protein (Bax) expression, promoting cell survival. Taken together, HTHQ exhibits protective effects against l-DOPA-induced cell death through modulation of the ERK1/2-p38MAPK-JNK1/2-Bad-Bax signaling pathway in PC12 cells. These results suggest that HTHQ may show ameliorative effects against oxidative stress-induced dopaminergic neuronal cell death, although further studies in animal models of Parkinson's disease are required to confirm this.

Supplementation with extract of Gynostemma pentaphyllum leaves reduces anxiety in healthy subjects with chronic psychological stress: A randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: The ethanol extract of Gynostemma pentaphyllum Makino leaves (EGP) has been reported recently to have anxiolytic effects on chronically stressed mice models. PURPOSE: We aimed to investigate the efficacy and safety of EGP on anxiety level in healthy Korean subjects under chronic stressful conditions. STUDY DESIGN: Double-blind, placebo-controlled trial. METHODS: This study was conducted with 72 healthy adults who had perceived chronic stress and anxiety with a score on the State-Trait Anxiety Inventory (STAI) from 40 to 60. Participants were randomly assigned to receive either EGP (200 mg, twice a day, N = 36) or placebo (N = 36). All participants were exposed to repetitive loads of stress by performing the serial subtraction task for 5 min every second day during the 8-week intervention. Primary outcome of Trait-STAI and secondary outcomes of State-STAI, total score of STAI, Hamilton Anxiety Inventory (HAM-A), Beck Anxiety Inventory (BAI), blood norepinephrine and adrenocorticotropic hormone (ACTH), salivary cortisol and alpha-amylase, cardiovascular autonomic nervous system (ANS) functional test, and heart rate variability (HRV) test were measured before and after intervention. RESULTS: After the 8-week intervention, the EGP significantly lowered the score of the Trait Anxiety Scale of the STAI (T-STAI) by 16.8% compared to the placebo (p = 0.041). The total score on the STAI decreased by 17.8% in the EGP group and tended to improve compared with that of the placebo group (p = 0.067). There were no significant differences in the changes in score of S-STAI, HAM-A, BAI, and other parameters from baseline between the two groups. There was no causal relationship between the ingestion of EGP and adverse drug reactions. CONCLUSION: We found that supplementation with EGP reduced "anxiety proneness" in subjects under chronic psychological stress, as shown by a decrease in the score of T-STAI and the tendency for decrease in the total score of STAI. This result suggests that EGP supplementation can be used as a regimen to safely reduce stress and anxiety; however, more studies are needed to establish the long-term safety and effectiveness.

High-Intensity Statin Therapy Is "Too Much," Thus Not Indicated for Very Elderly Patients.

PURPOSE: Although moderate- to high-intensity statin therapy is increasingly recommended in cardiovascular disease patients, the efficacy and safety in elderly patients have not been proven clearly. Here, we compare the effect of various-intensity statins between elderly and very elderly patients. METHODS: 43,870 patients over 65 years old who were treated with statins were screened using electronic medical record data. RESULTS: We evaluated 451 patients in the elderly group aged 65-74 years and 159 patients in the very elderly group over 75 years old. Baseline cholesterol profiles were similar between the 2 groups, but the 10-year atherosclerotic cardiovascular disease (ASCVD) risk was significantly higher in the very elderly (20.9 ± 11.5$ vs. 37.2 ± 13.6$, p < 0.001). The reduction rate of low-density lipoprotein (LDL) (-40.2 ± 21.3$ vs. -39.3 ± 21.0$, p = 0.634) and the ratio of target LDL attainment (74.2 vs. 79.2$, p = 0.252) were similar between the 2 groups. Low-intensity statins showed comparable LDL cholesterol reduction with moderate-intensity statins both in the elderly and the very elderly groups. The 10-year ASCVD risk reduction was similar between the 2 groups (-3.5 ± 4.9$ vs. -3.0 ± 8.4$, p = 0.480), but in the very elderly group, no different ASCVD reduction rate was shown in low- to high-intensity statins (p = 0.784). Only the elderly group showed a significant correlation (r = 0.112, p = 0.017) with LDL reduction and 10-year ASCVD risk. Interestingly, the incidence of adverse drug reaction (ADR) was higher in the very elderly group (4.4$) than in the elderly group (2.7$) and was more frequent in high-intensity statin therapy. CONCLUSION: The efficacy of statins in LDL reduction was similar between the elderly and very elderly population. However, the benefit of moderate- to high-intensity statins is limited considering potential ADR. Therefore, the stepwise intensification of statin therapy might be necessary for the very elderly in spite of the higher cardiovascular risk.

Ombuoside from Gynostemma pentaphyllum Protects PC12 Cells from L-DOPA-Induced Neurotoxicity.

This study investigated the effects of ombuoside on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced neurotoxicity in PC12 cells. Ombuoside did not affect cell viability at concentrations of up to 50 µM for 24 h, and ombuoside (1, 5, and 10 µM) significantly inhibited L-DOPA-induced (100 and 200 µM) decreases in cell viability. L-DOPA (100 and 200 µM) induced sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) for 6 h, which were significantly decreased by cotreatments with ombuoside (1, 5, and 10 µM). L-DOPA (100 and 200 µM) alone significantly increased c-Jun N-terminal kinase (JNK1/2) phosphorylation for 6 h and cleaved-caspase-3 expression for 24 h, both of which were partially, but significantly, blocked by ombuoside (1, 5, and 10 µM). In addition, ombuoside (1, 5, and 10 µM) significantly restored the L-DOPA-induced (100 and 200 µM) decrease in superoxide dismutase (SOD) activity for 24 h. Taken together, these findings indicate that ombuoside protects against L-DOPA-induced neurotoxicity by inhibiting L-DOPA-induced increases in sustained ERK1/2 and JNK1/2 phosphorylation and caspase-3 expression and L-DOPA-induced decrease in SOD activity in PC12 cells. Thus, ombuoside might represent a novel neuroprotective agent that warrants further study.

Effects of gynosaponin TN-2 on L-DOPA-induced cytotoxicity in PC12 cells.

Gynosaponins have pharmacological effects on 3,4-L-dihydroxyphenylalanine (L-DOPA)-related or dopamine-related neurological diseases; however, the neuroprotective functions of single compound of gynosaponins remain undefined. This study investigated the cytotoxic effects of gynosaponin TN-2 on L-DOPA in pheochromocytoma 12 cells. Gynosaponin TN-2, at 0.5-3 µM, did not exhibit cytotoxicity and protected against L-DOPA (100 and 200 µM)-induced cell death. Gynosaponin TN-2 (0.5 and 1.0 µM) inhibited the L-DOPA (100 and 200 µM)-induced sustained extracellular signal-regulated protein kinases 1 and 2 phosphorylation. Gynosaponin TN-2 at 0.5 and 1.0 µM also reduced L-DOPA (100 and 200 µM)-induced JNK1/2 phosphorylation and cleaved caspase-3 expression. These results suggested that gynosaponin TN-2 exerts protective effects on L-DOPA (100 and 200 µM)-induced apoptotic cell death by modulating extracellular signal-regulated protein kinases 1 and 2 activation in pheochromocytoma 12 cells.

Gypenosides ameliorate memory deficits in MPTP-lesioned mouse model of Parkinson's disease treated with L-DOPA.

BACKGROUND: Previous studies have revealed that gypenosides (GPS) improve the symptoms of anxiety disorders in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned rat model of Parkinson's disease (PD). The present study aimed to investigate the effects of GPS on memory deficits in an MPTP-lesioned mouse model of PD treated with L-3,4-dihydroxyphenylalanine (L-DOPA). METHODS: MPTP (30 mg/kg/day, 5 days)-lesioned mice were treated with GPS (50 mg/kg) and/or L-DOPA (10 and 25 mg/kg) for 21 days. After the final treatments, behavioral changes were assessed in all mice using passive avoidance and elevated plus-maze tests. We then evaluated the biochemical influences of GPS treatment on levels of tyrosine hydroxylase (TH), dopamine, N-methyl-D-aspartate (NMDA) receptors, extracellular signal-regulated kinase (ERK1/2), and cyclic AMP-response element binding protein (CREB) phosphorylation. RESULTS: MPTP-lesioned mice exhibited deficits associated with habit learning and spatial memory, which were further aggravated by treatment with L-DOPA (25 mg/kg). However, treatment with GPS (50 mg/kg) ameliorated memory deficits. Treatment with GPS (50 mg/kg) also improved L-DOPA (25 mg/kg)-treated MPTP lesion-induced decreases in retention latency on the passive avoidance test, as well as levels of TH-immunopositive cells and dopamine in the substantia nigra and striatum. GPS treatment also attenuated increases in retention transfer latency on the elevated plus-maze test and in NMDA receptor expression, as well as decreases in the phosphorylation of ERK1/2 and CREB in the hippocampus. Treatment with L-DOPA (10 mg/kg) also ameliorated deficits in habit learning and spatial memory in MPTP-lesioned mice, and this effect was further enhanced by treatment with GPS (50 mg/kg). CONCLUSION: GPS ameliorate deficits in habit learning and spatial memory by modulating the dopaminergic neuronal and N-methyl-D-aspartate receptor-mediated signaling systems in MPTP-lesioned mice treated with L-DOPA. GPS may serve as an adjuvant therapeutic agent for memory deficits in patients with PD receiving L-DOPA.

Effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine-induced cytotoxicity in PC12 cells.

This study investigated the effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in rat adrenal pheochromocytoma (PC12) cells. Treatment with asarinin (25-50 µM) increased intracellular dopamine levels and enhanced L-DOPA-induced increases in dopamine levels. Asarinin (25 µM) induced cyclic AMP-dependent protein kinase A (PKA) signaling, leading to increased cyclic AMP-response element binding protein (CREB) and tyrosine hydroxylase (TH) phosphorylation, which in turn stimulated dopamine production. Asarinin (25 µM) also activated transient phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Bad phosphorylation at Ser 112, both of which have been shown to promote cell survival. In contrast, asarinin (25 µM) inhibited sustained ERK1/2, Bax, c-Jun N-terminal kinase (JNK1/2) and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and caspase-3 activity, which were induced by 6-OHDA (100 µM). These results suggest that asarinin induces dopamine biosynthesis via activation of the PKA-CREB-TH system and protects against 6-OHDA-induced cytotoxicity by inhibiting the sustained activation of the ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells.

Anti-Prion Screening for Acridine, Dextran, and Tannic Acid using Real Time-Quaking Induced Conversion: A Comparison with PrPSc-Infected Cell Screening.

Prion propagation is mediated by the structural alteration of normal prion protein (PrPC) to generate pathogenic prion protein (PrPSc). To date, compounds for the inhibition of prion propagation have mainly been screened using PrPSc-infected cells. Real time-quaking-induced conversion (RT-QuIC) is one alternative screening method. In this study, we assessed the propagation inhibition effects of known anti-prion compounds using RT-QuIC and compared the results with those from a PrPSc-infected cell assay. Compounds were applied to RT-QuIC reactions at 0 h or 22 h after prion propagation to determine whether they inhibited propagation or reduced amplified aggregates. RT-QuIC reactions in presence of acridine, dextran sulfate sodium (DSS), and tannic acid inhibited seeded aggregation with sporadic Creutzfeldt-Jakob disease at 0 h. After treatment at 22 h, amplified fluorescence was decreased in wells treated with either acridine or tannic acid. Compound activities were verified by western blot of RT-QuIC products and in a dye-independent conversion assay, the Multimer Detection System. Protease K-resistant PrPSc fragments (PrPres) were reduced by DSS and tannic acid in the PrPSc-infected cell assay. Importantly, these inhibitory effects were similar despite different treatment times (0 h versus 3 days). Consequentially, RT-QuIC enabled the more specific classification of compounds according to action (i.e., inhibition of prion propagation versus reduction of amplified aggregates). RT-QuIC addresses the limitations of cell-based screening methods and can be used to further aid our understanding of the mechanisms of action of anti-prion compounds.

Effects of (-)-Sesamin on Chronic Stress-Induced Anxiety Disorders in Mice.

This study investigated the effects of (-)-sesamin on chronic electric footshock (EF) stress-induced anxiety disorders in mice. Mice were treated with (-)-sesamin (25 and 50 mg/kg) orally once a day for 21 days prior to exposure to EF stress (0.6 mA, 1 s every 5 s, 3 min). Mice treated with (-)-sesamin (25 and 50 mg/kg) exhibited less severe decreases in the number of open arm entries and time spent on open arms in the elevated plus-maze test and the distance traveled in the open field test following exposure to chronic EF stress. Similarly, mice treated with (-)-sesamin exhibited significantly less severe decreases in brain levels of dopamine, norepinephrine, and serotonin following exposure to chronic EF stress. Increases in serum levels of corticosterone and expression of c-Fos were also less pronounced in mice treated with (-)-sesamin (25 and 50 mg/kg). These results suggest that (-)-sesamin may protect against the effects of chronic EF stress-induced anxiety disorders by modulating dopamine, norepinephrine, and serotonin levels, c-Fos expression, and corticosterone levels.

Gynostemma pentaphyllum Ethanolic Extract Protects Against Memory Deficits in an MPTP-Lesioned Mouse Model of Parkinson's Disease Treated with L-DOPA.

This study investigated the effects of ethanol extract from Gynostemma pentaphyllum (GP-EX) on memory deficits in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of Parkinson's disease (PD) (MPTP-lesioned mice). MPTP (30 mg/kg/day, 5 days)-lesioned mice showed deficits of habit learning memory and spatial memory, which were further aggravated by treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) (25 mg/kg, 21 days). However, treatment with GP-EX (50 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice treated with L-DOPA (25 mg/kg): GP-EX prevented the decreases in retention latency time in the passive avoidance test and tyrosine hydroxylase-immunopositive cells and dopamine levels in the nigrostriatum. GP-EX also reduced increases in retention transfer latency time of the elevated plus-maze test and expression of N-methyl-D-aspartate (NMDA) receptor and improved decreases in phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-response element binding protein (CREB) in the hippocampus in the same models. By contrast, L-DOPA treatment (10 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice, which were further improved by GP-EX treatment. These results suggest that GP-EX ameliorates habit learning memory deficits by activating dopaminergic neurons and spatial memory deficits by modulating NMDA receptor-ERK1/2-CREB system in MPTP-lesioned mice treated with L-DOPA. GP-EX may serve as an adjuvant phytonutrient for memory deficits in PD.

Effects of (-)-sesamin on chronic stress-induced memory deficits in mice.

This study investigated the effects of (-)-sesamin on memory deficits induced by chronic electric footshock (EF)-induced stress in mice. Mice were treated with (-)-sesamin (25 and 50mg/kg, p.o., daily for 21day) prior to chronic EF stress (0.6mA, 1s every 5s for 3min, daily for 21day). Transfer retention latencies in the elevated plus maze test and N-methyl-d-aspartate (NMDA) receptor (type 1) phosphorylation in the hippocampus increased with chronic EF stress, and they were reduced by treatment with (-)-sesamin at both doses. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-responsive element binding protein (CREB), which were reduced by chronic EF stress, were increased by treatment with (-)-sesamin. Retention latencies in the passive avoidance test and dopamine levels in the substantia nigra-striatum were also reduced by chronic EF stress, and similarly recovered with (-)-sesamin treatment. These results suggest that (-)-sesamin ameliorates the effects of chronic EF stress-induced spatial and habit learning memory deficits by modulating both NMDA receptor and dopaminergic neuronal systems.

Flavonol glycosides from the aerial parts of Gynostemma pentaphyllum and their antioxidant activity.

The phytochemical investigation of the aerial parts of Gynostemma pentaphyllum led to the isolation of a new flavonol glycoside, gynopentaphylloside (1), along with seven known compounds (2-8). The structure of the new compound was determined on the basis of 1D, 2D NMR and HRESIMS data as well as acid hydrolysis. The antioxidant activity of the isolates was evaluated by a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay.

L-DOPA modulates cell viability through the ERK-c-Jun system in PC12 and dopaminergic neuronal cells.

L-DOPA causes neurotoxicity by modulating the Epac-ERK system in PC12 cells. This study investigated the effects of a single treatment with L-DOPA and multiple treatments with L-DOPA (MT-LD) on ERK1/2 and JNK1/2-c-Jun systems. In PC12 cells, a toxic L-DOPA concentration (200 µM) induced sustained ERK1/2 and JNK1/2 phosphorylation that was inhibited by the Epac inhibitor brefeldin A, but not by the PKA inhibitor H89. This ERK1/2 and JNK1/2 phosphorylation was also inhibited by ERK1/2 (U0126) and JNK1/2 (SP600125) inhibitors, respectively, but sustained ERK1/2 phosphorylation was not affected by JNK1/2 phosphorylation. A non-toxic L-DOPA concentration (20 µM) induced c-Jun phosphorylation (Ser73) via transient ERK1/2 phosphorylation, whereas the toxic L-DOPA concentration induced c-Jun phosphorylation (Ser63) and c-Jun expression via Epac-sustained ERK1/2-JNK1/2 phosphorylation, which then enhanced cleaved caspase-3 expression. MT-LD (20 µM) initially enhanced c-Jun phosphorylation (Ser73) (for 1-4 days), but later (5-6 days) induced c-Jun phosphorylation (Ser63) and c-Jun expression. In the 6-hydroxydopamine-lesioned rat model of Parkinson's disease, L-DOPA administration (10 mg/kg) protected against neurotoxicity through c-Jun phosphorylation (Ser73) for 1-2 weeks. However, L-DOPA administration (10 or 30 mg/kg) showed neurotoxicity through c-Jun phosphorylation (Ser63) and c-Jun expression via ERK1/2 phosphorylation for 3-4 weeks. Thus, in PC12 cells, non-toxic L-DOPA treatment maintained cell survival through c-Jun phosphorylation (Ser73). By contrast, toxic L-DOPA treatment or MT-LD (20 µM) induced c-Jun phosphorylation (Ser63) and c-Jun expression via Epac-dependent sustained ERK1/2 and JNK1/2 phosphorylation, which subsequently led to cell death. These results were validated by those obtained after long-term L-DOPA administration in a rat model of Parkinson's disease. Our data indicate that L-DOPA causes neurotoxicity via the ERK1/2-c-Jun system in dopaminergic neuronal cells.

Gintonin enhances performance of mice in rotarod test: Involvement of lysophosphatidic acid receptors and catecholamine release.

Ginseng has a long history of use as a tonic for restoration of vigor. One example of ginseng-derived tonic effect is that it can improve physical stamina under conditions of stress. However, the active ingredient and the underlying molecular mechanism responsible for the ergogenic effect are unknown. Recent studies show that ginseng contains a novel ingredient, gintonin, which consists of a unique class of herbal-medicine lysophosphatidic acids (LPAs). Gintonin activates G protein-coupled LPA receptors to produce a transient [Ca(2+)]i signal, which is coupled to diverse intra- and inter-cellular signal transduction pathways that stimulate hormone or neurotransmitter release. However, relatively little is known about how gintonin-mediated cellular modulation is linked to physical endurance. In the present study, systemic administration of gintonin, but not ginsenosides, in fasted mice increased blood glucose concentrations in a dose-dependent manner. Gintonin treatment elevated blood glucose to a maximum level after 30min. This elevation in blood glucose level could be abrogated by the LPA1/3 receptor antagonist, Ki16425, or the ß-adrenergic receptor antagonist, propranolol. Furthermore, gintonin-dependent enhanced performance of fasted mice in rotarod test was likewise abrogated by Ki16425. Gintonin also elevated plasma epinephrine and norepinephrine concentrations. The present study shows that gintonin mediates catecholamine release through activation of the LPA receptor and that activation of the ß-adrenergic receptor is coupled to liver glycogenolysis, thereby increasing the supply of glucose and enhancing performance in the rotarod test. Thus, gintonin acts via the LPA-catecholamine-glycogenolysis axis, representing a candidate mechanism that can explain how ginseng treatment enhances physical stamina.

Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory.

The 14-3-3 protein has been used as a biomarker for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, weakly positive 14-3-3 leads to false positive results and an incorrect diagnosis. We attempted to use quantitative data for tau protein to provide an accurate diagnosis based on weak 14-3-3 protein. Sixty-two patients with sCJD, including pathologically confirmed, clinically definite, and probable cases, and 89 non-CJD patients were investigated based on a Korean population. Among them, 20 sCJD and 14 non-CJD showed weakly positive 14-3-3. The total tau (t-tau) and phosphorylated tau (p-tau) protein levels were measured by ELISA, and the p-tau to t-tau ratio (p/t ratio) was calculated. The combined use of the 14-3-3 protein assay, t-tau levels, and p/t ratio improved the specificity of diagnosis compared with the use of the 14-3-3 protein assay alone (47% for 14-3-3 alone; 85.94% for 14-3-3 combined with t-tau; 90.62% for 14-3-3 combined with the p/t ratio). In addition, 18 of 20 sCJD and 12 of 14 non-CJD who were weakly positive for 14-3-3 were positive for the p/t ratio and negative for the p/t ratio, respectively. When used in combination with the 14-3-3 protein, the tau protein is useful as a biomarker for the precise diagnosis of sCJD.

Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches.

Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrP(C)) to the pathogenic form, PrP(Sc). Compounds that inhibit this process by blocking conversion to the PrP(Sc) could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrP(C) interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrP(Sc) levels and one of these compounds also showed a high binding affinity for PrP(C). These results provide a promising starting point for the development of anti-prion compounds.