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Abstract: In the present study, we investigated the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) and which types of neuronal cells contain CaMK II and phosphorylated CaMK II (p-CaMK II) in the CA3 hippocampal region of mice using conforcal immunofluorescence study. KA increased the CaMK II, p-CaMK II, glial fibrillary acidic protein (GFAP) and complement receptor type 3 (OX-42) immunoreactivities (IR) at 30min after KA treatment in mouse hippocampal area. In studies, nevertheless KA-induced CaMK II is expressed in neurons or astrocytes or microglia, p-CaMK II is expressed only in neurons. Thus, our results suggest that the activated CaMK II in early time may be performed important roles only in neurons but not in the astrocytes and microglia. [Copyright &y& Elsevier]

In an immunohistochemical study, kainic acid (KA, 0.1 μg) administered intracerebroventricularly (i.c.v.) dramatically increased the expression of Ca2+/calmodulin-dependent protein kinase II (CaMK II) and the phosphorylation of CaMK II (p-CaMK II) in the CA3 hippocampal region of mice. Pre-treatment with cycloheximide (a protein synthesis inhibitor; 200 mg/kg) intraperitoneally prevented the expression of CaMK II and phosphorylation of CaMK II induced by KA. In addition, pre-treatment with MK-801 ((5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; an NMDA receptor blocker, 1 μg, i.c.v.) or CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; a non-NMDA receptor blocker, 0.5 μg, i.c.v.) attenuated the p-CaMK II, but not CaMK II, expression induced by KA. Our results suggest that KA administered supraspinally induces CaMK II and the phosphorylation of CaMK II expression in the CA3 hippocampal region, for which an on-going protein synthesis is needed. Furthermore, both NMDA and non-NMDA receptors appear to be involved in supraspinally administered KA-induced phosphorylation of CaMK II. [Copyright &y& Elsevier]

Background: Identifying genetic variants which impact the level of cell cycle reentry and establishing the degree of cell cycle progression in those variants could help guide development of therapeutic interventions aimed at effecting cardiac regeneration. We observed that C57Bl6/NCR (B6N) mice have a marked increase in cardiomyocyte S-phase activity following permanent coronary artery ligation as compared to infarcted DBA/2J (D2J) mice.Methods: Cardiomyocyte cell cycle activity post-infarction was monitored in D2J, (D2J x B6N)-F1 and [(D2J x B6N)-F1 x D2J] backcross mice via bromodeoxyuridine or 5-ethynyl-2' -deoxyuridine incorporation, using a nuclear-localized transgenic reporter to identify cardiomyocyte nuclei. Genome-wide quantitative trait locus (QTL) analysis, fine scale genetic mapping, whole exome sequencing and RNA-seq analyses of the backcross mice were performed to identify the gene responsible for the elevated cardiomyocyte S-phase phenotype.Results: (D2J x B6N)-F1 mice exhibited a 14-fold increase in cardiomyocyte S-phase activity in ventricular regions remote from infarct scar as compared to D2J mice (0.798 ± 0.09% vs. 0.056 ± 0.004%; p < 0.001). QTL analysis of [(D2J x B6N)-F1 x D2J] backcross mice revealed that the gene responsible for differential S-phase activity was located on the distal arm of Chromosome 3 (LOD score = 6.38; p < 0.001). Additional genetic and molecular analyses identified 3 potential candidates. Of these, troponin I-interacting kinase (Tnni3k) is expressed in B6N hearts but not in D2J hearts. Transgenic expression of Tnni3k in a D2J genetic background results in elevated cardiomyocyte S-phase activity post-injury. Cardiomyocyte S-phase activity in both TNNI3K-expressing and TNNI3K-nonexpressing mice results in the formation of polyploid nuclei.Conclusions: These data indicate that TNNI3K expression increases the level of cardiomyocyte S-phase activity following injury. [ABSTRACT FROM AUTHOR]

A large body of evidence support major roles of mitochondrial dysfunction and insulin action in the Alzheimer's disease (AD) brain. However, interaction between cellular expression of β-amyloid (Aβ) and insulin resistance on mitochondrial metabolism has not been explored in neuronal cells. We investigated the additive and synergistic effects of intracellular Aβ42 and ceramide-induced insulin resistance on mitochondrial metabolism in SH-SY5Y and Neuro-2a cells. In our model, mitochondria take-up Aβ42 expressed through viral-mediated transfection and exposure of the same cells to ceramide produces resistance to insulin signaling. Ceramide alone increased phosphorylated MAP kinases while decreasing phospho-Akt (Ser473). The combination of Aβ42 and ceramide synergistically decreased phospho-Thr308 on Akt. Aβ42 and ceramide synergistically also decreased mitochondrial complex III activity and ATP generation whereas Aβ alone was largely responsible for complex IV inhibition and increases in mitochondrial reactive oxygen species production (ROS). Proteomic analysis showed that a number of mitochondrial respiratory chain and tricarboxylic acid cycle enzymes were additively or synergistically decreased by ceramide in combination with Aβ42 expression. Mitochondrial fusion and fission proteins were notably dysregulated by Aβ42 (Mfn1) or Aβ42 plus ceramide (OPA1, Drp1). Antioxidant vitamins blocked the Aβ42 alone-induced ROS production, but did not reverse Aβ42-induced ATP reduction or complex IV inhibition. Aβ expression combined with ceramide exposure had additive effects to decrease cell viability. Taken together, our data demonstrate that Aβ42 expression and ceramide-induced insulin resistance synergistically interact to exacerbate mitochondrial damage and that therapeutic efforts to reduce insulin resistance could lessen failures of energy production and mitochondrial dynamics. [ABSTRACT FROM AUTHOR]

The Alzheimer's brain is affected by multiple pathophysiological processes, which include a unique, organ-specific form of insulin resistance that begins early in its course. An additional complexity arises from the four-fold risk of Alzheimer's Disease (AD) in type 2 diabetics, however there is no definitive proof of causation. Several strategies to improve brain insulin signaling have been proposed and some have been clinically tested. We report findings on a small allosteric molecule that reverses several indices of insulin insensitivity in both cell culture and in vitro models of AD that emphasize the intracellular accumulation of β-amyloid (Aβi). PS48, a chlorophenyl pentenoic acid, is an allosteric activator of PDK-1, which is an Akt-kinase in the insulin/PI3K pathway. PS48 was active at 10 nM to 1 μM in restoring normal insulin-dependent Akt activation and in mitigating Aβi peptide toxicity. Synaptic plasticity (LTP) in prefrontal cortical slices from normal rat exposed to Aβ oligomers also benefited from PS48. During these experiments, neither overstimulation of PI3K/Akt signaling nor toxic effects on cells was observed. Another neurotoxicity model producing insulin insensitivity, utilizing palmitic acid, also responded to PS48 treatment, thus validating the target and indicating that its therapeutic potential may extend outside of β-amyloid reliance. The described in vitro and cell based-in vitro coupled enzymatic assay systems proved suitable platforms to screen a preliminary library of new analogs. [ABSTRACT FROM AUTHOR]

There is an error in affiliation 3 for authors Keykavous Parang and Rakesh Tiwari. The correct affiliation 3 is: Center for Targeted Drug Delivery, Chapman University, School of Pharmacy, Irvine, CA, United States of America.By Henry Querfurth; John Marshall; Keykavous Parang; Mengia S. Rioult-Pedotti; Rakesh Tiwari; Bumsup Kwon; Steve Reisinger and Han-Kyu LeeReported by Author; Author; Author; Author; Author; Author; Author; Author [Extracted from the article]

Spin transfer torque magnetic random access memory (STT-MRAM) is a promising candidate for next generation memory as it is non-volatile, fast, and has unlimited endurance. Another important aspect of STT-MRAM is that its core component, the nanoscale magnetic tunneling junction (MTJ), is thought to be radiation hard, making it attractive for space and nuclear technology applications. However, studies on the effects of ionizing radiation on the STT-MRAM writing process are lacking for MTJs with perpendicular magnetic anisotropy (pMTJs) required for scalable applications. Particularly, the question of the impact of extreme total ionizing dose on perpendicular magnetic anisotropy, which plays a crucial role on thermal stability and critical writing current, remains open. Here we report measurements of the impact of high doses of gamma and neutron radiation on nanoscale pMTJs used in STT-MRAM. We characterize the tunneling magnetoresistance, the magnetic field switching, and the current-induced switching before and after irradiation. Our results demonstrate that all these key properties of nanoscale MTJs relevant to STT-MRAM applications are robust against ionizing radiation. Additionally, we perform experiments on thermally driven stochastic switching in the gamma ray environment. These results indicate that nanoscale MTJs are promising building blocks for radiation-hard non-von Neumann computing. [ABSTRACT FROM AUTHOR]

Abstract For over two decades, fast-scan cyclic voltammetry (FSCV) has served as a reliable analytical method for monitoring dopamine release in near real-time in vivo. However, contemporary FSCV techniques have been limited to measure only rapid (on the order of seconds, i.e. phasic) changes in dopamine release evoked by either electrical stimulation or elicited by presentation of behaviorally salient stimuli, and not slower changes in the tonic extracellular levels of dopamine (i.e. basal concentrations). This is because FSCV is inherently a differential method that requires subtraction of prestimulation tonic levels of dopamine to measure phasic changes relative to a zeroed baseline. Here, we describe the development and application of a novel voltammetric technique, multiple cyclic square wave voltammetry (M-CSWV), for analytical quantification of tonic dopamine concentrations in vivo with relatively high temporal resolution (10 s). M-CSWV enriches the electrochemical information by generating two dimensional voltammograms which enable high sensitivity (limit of detection, 0.17 nM) and selectivity against ascorbic acid, and 3,4-dihydroxyphenylacetic acid (DOPAC), including changes in pH. Using M-CSWV, a tonic dopamine concentration of 120 ± 18 nM (n = 7 rats, ± SEM) was determined in the striatum of urethane anethetized rats. Pharmacological treatments to elevate dopamine by selectively inhibiting dopamine reuptake and to reduce DOPAC by inhibition of monoamine oxidase supported the selective detection of dopamine in vivo. Overall, M-CSWV offers a novel voltammetric technique to quantify levels and monitor changes in tonic dopamine concentrations in the brain to further our understanding of the role of dopamine in normal behavior and neuropsychiatric disorders. Highlights • Highly sensitive and selective electrochemical method for detecting tonic dopamine level in vivo. • Enhanced visualization of the data by two-dimensional voltammogram of electrochemical responses. • High temporal resolution (10 s). [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM), peripheral insulin resistance (IR) and obesity are clear risk factors for Alzheimer's disease. Several anti-diabetic drugs and insulin have been tested in rodents and humans with MCI or AD, yielding promising but inconclusive results. The PDK-1/Akt axis, essential to the action of insulin, has not however been pharmacologically interrogated to a similar degree. Our previous cell culture and in vitro studies point to such an approach. Double transgenic APPsw/PSENdE9 mice, a model for Alzheimer's disease, were used to test the oral administration of PS48, a PDK-1 agonist, on preventing the expected decline in learning and memory in the Morris Water Maze (MWM). Mice were raised on either standard (SD) or high fat (HFD) diets, dosed beginning 10 months age and tested at an advanced age of 14 months. PS48 had positive effects on learning the spatial location of a hidden platform in the TG animals, on either SD or HFD, compared to vehicle diet and WT animals. On several measures of spatial memory following successful acquisition (probe trials), the drug also proved significantly beneficial to animals on either diet. The PS48 treatment-effect size was more pronounced in the TG animals on HFD compared to on SD in several of the probe measures. HFD produced some of the intended metabolic effects of weight gain and hyperglycemia, as well as accelerating cognitive impairment in the TG animals. PS48 was found to have added value in modestly reducing body weights and improving OGTT responses in TG groups although results were not definitive. PS48 was well tolerated without obvious clinical signs or symptoms and did not itself affect longevity. These results recommend a larger preclinical study before human trial. • Disturbed brain insulin signaling characterizes Alzheimer's Disease (AD). • Intracellular β-amyloid is toxic to neurons, in part by affecting the activation of Akt by PDK-1. • PDK-1 allosteric activators reversed insulin insensitivity in cell culture and in vitro models of AD • βAPP/PS-1 mice fed standard and high fat diets showed spatial learning and memory deficits. • Oral dosing of a lead compound improved Morris Water Maze indices. FC results were inconclusive. [ABSTRACT FROM AUTHOR]

Risk for ischemic stroke has a strong genetic basis, but heritable factors also contribute to the extent of damage after a stroke has occurred. We previously identified a locus on distal mouse chromosome 7 that contributes over 50% of the variation in postischemic cerebral infarct volume observed between inbred strains. Here, we used ancestral haplotype analysis to fine-map this locus to 12 candidate genes. The gene encoding the IL-21 receptor (Il21r) showed a marked difference in strain-specific transcription levels and coding variants in neonatal and adult cortical tissue. Collateral vessel connections were moderately reduced in Il21r-deficient mice, and cerebral infarct volume increased 2.3-fold, suggesting that Il21r modulates both collateral vessel anatomy and innate neuroprotection. In brain slice explants, oxygen deprivation (OD) activated apoptotic pathways and increased neuronal cell death in IL-21 receptor-deficient (IL-21R-deficient) mice compared with control animals. We determined that the neuroprotective effects of IL-21R arose from signaling through JAK/STAT pathways and upregulation of caspase 3. Thus, natural genetic variation in murine Il21r influences neuronal cell viability after ischemia by modulating receptor function and downstream signal transduction. The identification of neuroprotective genes based on naturally occurring allelic variations has the potential to inform the development of drug targets for ischemic stroke treatment. [ABSTRACT FROM AUTHOR]

Cyclooxygenase metabolizes dihomo-γ-linolenic acid and arachidonic acid to form prostaglandin (PG) E, including PGE1 and PGE2, respectively. Although PGE2 is well known to play an important role in the development and maintenance of hyperalgesia and allodynia, the role of PGE1 in pain is unknown. We confirm whether PGE1 induced pain using orofacial pain behavioral test in mice and determine the target molecule of PGE1 in TG neurons with whole-cell patch-clamp and immunohistochemistry. Intradermal injection of PGE1 to the whisker pads of mice induced a reduced threshold, enhancing the excitability of HCN channel-expressing trigeminal ganglion (TG) neurons. The HCN channel-generated inward current (Ih) was increased by 135.3 ± 4.8% at 100 nM of PGE1 in small- or medium-sized TG, and the action of PGE1 on Ih showed a concentration-dependent effect, with a median effective dose (ED50) of 29.3 nM. Adenylyl cyclase inhibitor (MDL12330A), 8-bromo-cAMP, and the EP2 receptor antagonist AH6809 inhibited PGE1-induced Ih. Additionally, PGE1-induced mechanical allodynia was blocked by CsCl and AH6809. PGE1 plays a role in mechanical allodynia through HCN2 channel facilitation via the EP2 receptor in nociceptive neurons, suggesting a potential therapeutic target in that PGE1 could be involved in pain as endogenous substances under inflammatory conditions. [ABSTRACT FROM AUTHOR]

Purpose: The aims of this study were 1) to investigate the effects of a subepithelial connective tissue graft (SCTG) and a volume-stable collagen matrix (VCMX) on soft-tissue volume gain in the immediate implant placement protocol, and 2) to determine whether polydeoxyribonucleotide (PDRN) can enhance the effects of a VCMX., Methods: Dental implants were placed in 4 mongrel dogs immediately after extracting the distal roots of their third and fourth mandibular premolars. The gap between the implant and the buccal bone plate was filled with synthetic bone substitute particles. The following soft-tissue augmentation modalities were applied buccally: 1) control (no augmentation), 2) SCTG, 3) VCMX, and 4) VCMX/PDRN. After 4 months, histomorphometric analysis was performed. Tissue changes were evaluated using superimposed standard tessellation language (STL) files., Results: Wound dehiscence was found in more than half of the test groups, but secondary wound healing was successfully achieved in all groups. Histomorphometrically, tissue thickness was favored in group SCTG at or above the implant platform level (IP), and group SCTG and the groups with VCMX presented similar tissue thickness below the IP. However, the differences in such thickness among the groups were minor. The keratinized tissue height was greater in group VCMX/PDRN than in groups SCTG and VCMX. Superimposing the STL files revealed a decrease in soft-tissue volume in all groups., Conclusions: Wound dehiscence after soft-tissue volume augmentation might be detrimental to obtaining the expected outcomes. PDRN appears not to have a positive effect on the soft-tissue volume gain., Competing Interests: No potential conflict of interest relevant to this article was reported., (Copyright © 2024. Korean Academy of Periodontology.)

Purpose: To investigate the effect of xenogeneic collagen matrix (XCM) with polydeoxyribonucleotide (PDRN) for gingival phenotype modification compared to autogenous connective tissue graft., Methods: Five mongrel dogs were used in this study. Box-type gingival defects were surgically created bilaterally on the maxillary canines 8 weeks before gingival augmentation. A coronally positioned flap was performed with either a subepithelial connective tissue graft (SCTG) or XCM with PDRN (2.0 mg/mL). The animals were sacrificed after 12 weeks. Intraoral scanning was performed for soft tissue analysis, and histologic and histomorphometric analyses were performed., Results: One animal exhibited wound dehiscence, leaving 4 for analysis. Superimposition of STL files revealed no significant difference in the amount of gingival thickness increase (ranging from 0.69±0.25 mm to 0.80±0.31 mm in group SCTG and from 0.48±0.25 mm to 0.85±0.44 mm in group PDRN; P >0.05). Histomorphometric analysis showed no significant differences between the groups in supracrestal gingival tissue height, keratinized tissue height, tissue thickness, and rete peg density ( P >0.05)., Conclusions: XCM soaked with PDRN yielded comparable gingival augmentation to SCTG., Competing Interests: No potential conflict of interest relevant to this article was reported., (Copyright © 2023. Korean Academy of Periodontology.)

Sturge-Weber Syndrome (SWS) is a sporadic (non-inherited) syndrome characterized by capillary vascular malformations in the facial skin, leptomeninges, or the choroid. A hallmark feature is the mosaic nature of the phenotype. SWS is caused by a somatic mosaic mutation in the GNAQ gene (p.R183Q), leading to activation of the G protein, Gαq. Decades ago, Rudolf Happle hypothesized SWS as an example of "paradominant inheritance", that is, a "lethal gene (mutation) surviving by mosaicism". He predicted that the "presence of the mutation in the zygote will lead to death of the embryo at an early stage of development". We have created a mouse model for SWS using gene targeting to conditionally express the GNAQ p.R183Q mutation. We have employed two different Cre-drivers to examine the phenotypic effects of expression of this mutation at different levels and stages of development. As predicted by Happle, global, ubiquitous expression of this mutation in the blastocyst stage results in 100% embryonic death. The majority of these developing embryos show vascular defects consistent with the human vascular phenotype. By contrast, global but mosaic expression of the mutation enables a fraction of the embryos to survive, but those that survive to birth and beyond do not exhibit obvious vascular defects. These data validate Happle's paradominant inheritance hypothesis for SWS and suggest the requirement of a tight temporal and developmental window of mutation expression for the generation of the vascular phenotype. Furthermore, these engineered murine alleles provide the template for the development of a mouse model of SWS that acquires the somatic mutation during embryonic development, but permits the embryo to progress to live birth and beyond, so that postnatal phenotypes can also be investigated. These mice could then also be employed in pre-clinical studies of novel therapies., Competing Interests: Conflicts of interest statement The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ischemic stroke, caused by vessel blockage, results in cerebral infarction, the death of brain tissue. Previously, quantitative trait locus (QTL) mapping of cerebral infarct volume and collateral vessel number identified a single, strong genetic locus regulating both phenotypes. Additional studies identified RAB GTPase-binding effector protein 2 (Rabep2) as the casual gene. However, there is yet no evidence that variation in the human ortholog of this gene plays any role in ischemic stroke outcomes. We established an in vivo evaluation platform in mice by using adeno-associated virus (AAV) gene replacement and verified that both mouse and human RABEP2 rescue the mouse Rabep2 knockout ischemic stroke volume and collateral vessel phenotypes. Importantly, this cross-species complementation enabled us to experimentally investigate the functional effects of coding sequence variation in human RABEP2. We chose four coding variants from the human population that are predicted by multiple in silico algorithms to be damaging to RABEP2 function. In vitro and in vivo analyses verify that all four led to decreased collateral vessel connections and increased infarct volume. Thus, there are naturally occurring loss-of-function alleles. This cross-species approach will expand the number of targets for therapeutics development for ischemic stroke., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Although studies with inbred strains of mice have shown that infarct size is largely determined by the extent of collateral vessel connections between arteries in the brain that enable reperfusion of the ischemic territory, we have identified strain pairs that do not vary in this vascular phenotype, but which nonetheless exhibit large differences in infarct size. In this study we performed quantitative trait locus (QTL) mapping in mice from an intercross between two such strains, WSB/EiJ (WSB) and C57BL/6J (B6). This QTL mapping revealed only one neuroprotective locus on Chromosome 8 (Chr 8) that co-localizes with a neuroprotective locus we mapped previously from F2 progeny between C3H/HeJ (C3H) and B6. The allele-specific phenotypic effect on infarct volume at the genetic region identified by these two independent mappings was in the opposite direction of the parental strain phenotype; namely, the B6 allele conferred increased susceptibility to ischemic infarction. Through two reciprocal congenic mouse lines with either the C3H or B6 background at the Chr 8 locus, we verified the neuroprotective effects of this genetic region that modulates infarct volume without any effect on the collateral vasculature. Additionally, we surveyed non-synonymous coding SNPs and performed RNA-sequencing analysis to identify potential candidate genes within the genetic interval. Through these approaches, we suggest new genes for future mechanistic studies of infarction following ischemic stroke, which may represent novel gene/protein targets for therapeutic development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lee, Wetzel-Strong, Aylor and Marchuk.)

Novel targets to arrest neurodegeneration in several dementing conditions involving misfolded protein accumulations may be found in the diverse signaling pathways of the Mammalian/mechanistic target of rapamycin (mTOR). As a nutrient sensor, mTOR has important homeostatic functions to regulate energy metabolism and support neuronal growth and plasticity. However, in Alzheimer's disease (AD), mTOR alternately plays important pathogenic roles by inhibiting both insulin signaling and autophagic removal of β-amyloid (Aβ) and phospho-tau (ptau) aggregates. It also plays a role in the cerebrovascular dysfunction of AD. mTOR is a serine/threonine kinase residing at the core in either of two multiprotein complexes termed mTORC1 and mTORC2. Recent data suggest that their balanced actions also have implications for Parkinson's disease (PD) and Huntington's disease (HD), Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Beyond rapamycin; an mTOR inhibitor, there are rapalogs having greater tolerability and micro delivery modes, that hold promise in arresting these age dependent conditions.

Riboflavin, also known as vitamin B 2 , isfound in foods and is used as a dietary supplement. Its deficiency (also called ariboflavinosis) results in some skin lesions and inflammations, such as stomatitis, cheilosis, oily scaly skin rashes, and itchy, watery eyes. Various therapeutic effects of riboflavin, such as anticancer, antioxidant, anti-inflammatory, and anti-nociceptive effects, are well known. Although some studies have identified the clinical effect of riboflavin on skin problems, including itch and inflammation, its underlying mechanism of action remains unknown. In this study, we investigated the molecular mechanism of the effects of riboflavin on histamine-dependent itch based on behavioral tests and electrophysiological experiments. Riboflavin significantly reduced histamine-induced scratching behaviors in mice and histamine-induced discharges in single-nerve fiber recordings, while it did not alter motor function in the rotarod test. In cultured dorsal root ganglion (DRG) neurons, riboflavin showed a dose-dependent inhibitory effect on the histamine- and capsaicin-induced inward current. Further tests wereconducted to determine whether two endogenous metabolites of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), have similar effects to those of riboflavin. Here, FMN, but not FAD, significantly inhibited capsaicin-induced currents and itching responses caused by histamine. In addition, in transient receptor potential vanilloid 1 (TRPV1)-transfected HEK293 cells, both riboflavin and FMN blocked capsaicin-induced currents, whereas FAD did not. These results revealed that riboflavin inhibits histamine-dependent itch by modulating TRPV1 activity. This study will be helpful in understanding how riboflavin exerts antipruritic effects and suggests that it might be a useful drug for the treatment of histamine-dependent itch., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lee, Choi, Im, Hwang, Lee, Im, Kim, Jung and Park.)

Understanding the dynamics of harmful algal blooms is important to protect the aquatic ecosystem in regulated rivers and secure human health. In this study, artificial neural network (ANN) and support vector machine (SVM) models were used to predict algae alert levels for the early warning of blooms in a freshwater reservoir. Intensive water-quality, hydrodynamic, and meteorological data were used to train and validate both ANN and SVM models. The Latin-hypercube one-factor-at-a-time (LH-OAT) method and a pattern search algorithm were applied to perform sensitivity analyses for the input variables and to optimize the parameters of the models, respectively. The results indicated that the two models well reproduced the algae alert level based on the time-lag input and output data. In particular, the ANN model showed a better performance than the SVM model, displaying a higher performance value in both training and validation steps. Furthermore, a sampling frequency of 6- and 7-day were determined as efficient early-warning intervals for the freshwater reservoir. Therefore, this study presents an effective early-warning prediction method for algae alert level, which can improve the eutrophication management schemes for freshwater reservoirs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Modulation of the function of somatosensory neurons is an important analgesic strategy, requiring the proposal of novel molecular targets. Many G-protein-coupled receptors (GPRs) have been deorphanized, but the receptor locations, outcomes due to their activations, and their signal transductions remain to be elucidated, regarding the somatosensory nociceptor function. Here we report that GPR171, expressed in a nociceptor subpopulation, attenuated pain signals via Gi/o-coupled modulation of the activities of nociceptive ion channels when activated by its newly found ligands. Administration of its natural peptide ligand and a synthetic chemical ligand alleviated nociceptor-mediated acute pain aggravations and also relieved pathologic pain at nanomolar and micromolar ranges. This study suggests that functional alteration of the nociceptor neurons by GPR171 signaling results in pain alleviation and indicates that GPR171 is a promising molecular target for peripheral pain modulation.

Major depressive disorder (MDD) is a devastating disease that arises in a background of environmental risk factors, such as chronic stress, that produce reactive oxygen species (ROS) in the brain. The chronic stress-induced ROS production involves Ca 2+ signals; however, the mechanism is poorly understood. Transient receptor potential melastatin type 2 (TRPM2) is a Ca 2+ -permeable cation channel that is highly expressed in the brain. Here we show that in animal models of chronic unpredictable stress (CUS), deletion of TRPM2 ( Trpm2 -/- ) produces antidepressant-like behaviors in mice. This phenotype correlates with reduced ROS, ROS-induced calpain activation, and enhanced phosphorylation of two Cdk5 targets including synapsin 1 and histone deacetylase 5 that are linked to synaptic function and gene expression, respectively. Moreover, TRPM2 mRNA expression is increased in hippocampal tissue samples from patients with MDD. Our findings suggest that TRPM2 is a key agent in stress-induced depression and a possible target for treating depression., Competing Interests: The authors declare no conflict of interest.

We established a unique platform for proteomic analysis of cultured three-dimensional (3D) neurons and brain tissue from Alzheimer's disease (AD) patients. We collected peripheral blood mononuclear cells (PBMC), converted PBMC to induced pluripotent stem cell (iPSC) lines, and differentiated the iPSC into human 3D neuro-spheroids. The postmortem brain tissue from the superior frontal cortex, inferior frontal cortex and cerebellum area of the AD patients was compared to the same regions from the control subjects. Proteomic analysis of 3D neuro-spheroids derived from AD subjects revealed the alteration of a number of proteins involved in axon growth, mitochondrial function, and antioxidant defense. Similar analysis of post-mortem AD brain tissue revealed significant alteration in proteins involved in oxidative stress, neuro-inflammation, along with proteins related to axonal injury. These results clearly indicate that the dysfunction of 3D neurons from AD patients in our in vitro environment is comparable to the post-mortem AD brain tissue in vivo. In conclusion, our study revealed a number of candidate proteins that have important implications in AD pathogenesis and supports the notion that the iPSC-derived 3D neuronal system functions as a model to examine novel aspects of AD pathology., Significance: In this study, we present a unique platform for proteomic analysis of induced pluripotent stem cell-derived three dimensional (3D) neurons and compare the results to those from three regions of post-mortem brain tissue from Alzheimer's disease patients and normal control subjects. Our results show that the dysfunction of 3D neurons from AD patients in our in vitro environment is comparable to the post-mortem AD brain tissue in vivo. Our results revealed several candidate proteins that have important implications in AD pathogenesis., (Copyright © 2018. Published by Elsevier B.V.)

In an aging society with an increasing threat to higher brain cognitive functions due to dementia, it becomes imperative to identify new molecular remedies for supporting adult neurogenesis. Interleukin-6 (IL-6) is a promising cytokine that can support neurogenesis under conditions of neurodegeneration, and neuron replacement is eventually possible due to its agonistic acting soluble receptor sIL-6R. Here, we report that activation of the IL-6-signal transducer and activator of transcription 3 (STAT3) axis is neurogenic and has potential therapeutic applications for the treatment of neurodegenerative diseases such as Parkinson's disease (PD).

The analgesic mechanism of opioids is known to decrease the excitability of substantia gelatinosa (SG) neurons receiving the synaptic inputs from primary nociceptive afferent fiber by increasing inwardly rectifying K(+) current. In this study, we examined whether a µ-opioid agonist, [D-Ala2,N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), affects the two-pore domain K(+) channel (K2P) current in rat SG neurons using a slice whole-cell patch clamp technique. Also we confirmed which subtypes of K2P channels were associated with DAMGO-induced currents, measuring the expression of K2P channel in whole spinal cord and SG region. DAMGO caused a robust hyperpolarization and outward current in the SG neurons, which developed almost instantaneously and did not show any time-dependent inactivation. Half of the SG neurons exhibited a linear I~V relationship of the DAMGO-induced current, whereas rest of the neurons displayed inward rectification. In SG neurons with a linear I~V relationship of DAMGO-induced current, the reversal potential was close to the K(+) equilibrium potentials. The mRNA expression of TWIK (tandem of pore domains in a weak inwardly rectifying K(+) channel) related acid-sensitive K(+) channel (TASK) 1 and 3 was found in the SG region and a low pH (6.4) significantly blocked the DAMGO-induced K(+) current. Taken together, the DAMGO-induced hyperpolarization at resting membrane potential and subsequent decrease in excitability of SG neurons can be carried by the two-pore domain K(+) channel (TASK1 and 3) in addition to inwardly rectifying K(+) channel., Competing Interests: The authors declare no conflict of interest.

Risk for ischemic stroke has a strong genetic basis, but heritable factors also contribute to the extent of damage after a stroke has occurred. We previously identified a locus on distal mouse chromosome 7 that contributes over 50% of the variation in postischemic cerebral infarct volume observed between inbred strains. Here, we used ancestral haplotype analysis to fine-map this locus to 12 candidate genes. The gene encoding the IL-21 receptor (Il21r) showed a marked difference in strain-specific transcription levels and coding variants in neonatal and adult cortical tissue. Collateral vessel connections were moderately reduced in Il21r-deficient mice, and cerebral infarct volume increased 2.3-fold, suggesting that Il21r modulates both collateral vessel anatomy and innate neuroprotection. In brain slice explants, oxygen deprivation (OD) activated apoptotic pathways and increased neuronal cell death in IL-21 receptor-deficient (IL-21R-deficient) mice compared with control animals. We determined that the neuroprotective effects of IL-21R arose from signaling through JAK/STAT pathways and upregulation of caspase 3. Thus, natural genetic variation in murine Il21r influences neuronal cell viability after ischemia by modulating receptor function and downstream signal transduction. The identification of neuroprotective genes based on naturally occurring allelic variations has the potential to inform the development of drug targets for ischemic stroke treatment.

Background: The role of increased insulin resistance in the pathogenesis of type 2 diabetes has been emphasized in Asian populations. Thus, we evaluated the proportion of insulin resistance and the insulin secretory capacity in patients with early phase type 2 diabetes in Korea., Methods: We performed a cross-sectional analysis of 1,314 drug-naive patients with newly diagnosed diabetes from primary care clinics nationwide. The homeostasis model assessment of insulin resistance (HOMA-IR) was used as an index to measure insulin resistance, which was defined as a HOMA-IR ≥2.5. Insulin secretory defects were classified based on fasting plasma C-peptide levels: severe (<1.1 ng/mL), moderate (1.1 to 1.7 ng/mL) and mild to non-insulin secretory defect (≥1.7 ng/mL)., Results: The mean body mass index (BMI) was 25.2 kg/m(2); 77% of patients had BMIs >23.0 kg/m(2). Up to 50% of patients had central obesity based on their waist circumference (≥90 cm in men and 85 cm in women), and 70.6% had metabolic syndrome. Overall, 59.5% of subjects had insulin resistance, and 20.2% demonstrated a moderate to severe insulin secretory defect. Among those with insulin resistance, a high proportion of subjects (79.0%) had a mild or no insulin secretory defect. Only 2.6% of the men and 1.9% of the women had both insulin resistance and a moderate to severe insulin secretory defect., Conclusion: In this study, patients with early phase type 2 diabetes demonstrated increased insulin resistance, but preserved insulin secretion, with a high prevalence of obesity and metabolic syndrome.

Inclusion body myositis (IBM), a degenerative and inflammatory disorder of skeletal muscle, and Alzheimer's disease share protein derangements and attrition of postmitotic cells. Overexpression of cyclins and proliferating cell nuclear antigen (PCNA) and evidence for DNA replication is reported in Alzheimer's disease brain, possibly contributing to neuronal death. It is unknown whether aberrant cell cycle reentry also occurs in IBM. We examined cell cycle markers in IBM compared with normal control, polymyositis (PM) and non-inflammatory dystrophy sample sets. Next, we tested for evidence of reentry and DNA synthesis in C2C12 myotubes induced to express β-amyloid (Aβ42). We observed increased levels of Ki-67, PCNA and cyclins E/D1 in IBM compared with normals and non-inflammatory conditions. Interestingly, PM samples displayed similar increases. Satellite cell markers did not correlate with Ki-67-affected myofiber nuclei. DNA synthesis and cell cycle markers were induced in Aβ-bearing myotubes. Cell cycle marker and cyclin protein expressions were also induced in an experimental allergic myositis-like model of PM in mice. Levels of p21 (Cip1/WAF1), a cyclin-dependent kinase inhibitor, were decreased in affected myotubes. However, overexpression of p21 did not rescue cells from Aβ-induced toxicity. This is the first report of cell cycle reentry in human myositis. The absence of rescue and evidence for reentry in separate models of myodegeneration and inflammation suggest that new DNA synthesis may be a reactive response to either or both stressors., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Elevated circulating levels of saturated free fatty acids (sFFAs; e.g. palmitate) are known to provoke inflammatory responses and cause insulin resistance in peripheral tissue. By contrast, mono- or poly-unsaturated FFAs are protective against sFFAs. An excess of sFFAs in the brain circulation may also trigger neuroinflammation and insulin resistance, however the underlying signaling changes have not been clarified in neuronal cells. In the present study, we examined the effects of palmitate on mitochondrial function and viability as well as on intracellular insulin and nuclear factor-κB (NF-κB) signaling pathways in Neuro-2a and primary rat cortical neurons. We next tested whether oleate preconditioning has a protective effect against palmitate-induced toxicity. Palmitate induced both mitochondrial dysfunction and insulin resistance while promoting the phosphorylation of mitogen-activated protein kinases and nuclear translocation of NF-κB p65. Oleate pre-exposure and then removal was sufficient to completely block subsequent palmitate-induced intracellular signaling and metabolic derangements. Oleate also prevented ceramide-induced insulin resistance. Moreover, oleate stimulated ATP while decreasing mitochondrial superoxide productions. The latter were associated with increased levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Inhibition of protein kinase A (PKA) attenuated the protective effect of oleate against palmitate, implicating PKA in the mechanism of oleate action. Oleate increased triglyceride and blocked palmitate-induced diacylglycerol accumulations. Oleate preconditioning was superior to docosahexaenoic acid (DHA) or linoleate in the protection of neuronal cells against palmitate- or ceramide-induced cytotoxicity. We conclude that oleate has beneficial properties against sFFA and ceramide models of insulin resistance-associated damage to neuronal cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The GluA2 subunit of AMPA-type glutamate receptors (AMPARs) regulates excitatory synaptic transmission in neurons. In addition, the transsynaptic cell adhesion molecule N-cadherin controls excitatory synapse function and stabilizes dendritic spine structures. At postsynaptic membranes, GluA2 physically binds N-cadherin, underlying spine growth and synaptic modulation. We report that N-cadherin binds to PSD-95/SAP90/DLG/ZO-1 (PDZ) domain 2 of the glutamate receptor interacting protein 1 (GRIP1) through its intracellular C terminus. N-cadherin and GluA2-containing AMPARs are presorted to identical transport vesicles for dendrite delivery, and live imaging reveals cotransport of both proteins. The kinesin KIF5 powers GluA2/N-cadherin codelivery by using GRIP1 as a multilink interface. Notably, GluA2 and N-cadherin use different PDZ domains on GRIP1 to simultaneously bind the transport complex, and interference with either binding motif impairs the turnover of both synaptic cargoes. Depolymerization of microtubules, deletion of the KIF5 motor domain, or specific blockade of AMPAR exocytosis affects delivery of GluA2/N-cadherin vesicles. At the functional level, interference with this cotransport reduces the number of spine protrusions and excitatory synapses. Our data suggest the concept that the multi-PDZ-domain adaptor protein GRIP1 can act as a scaffold at trafficking vesicles in the combined delivery of AMPARs and N-cadherin into dendrites.

Aim: To evaluate the anticancer efficacy of CKD-516, a novel vascular-disrupting agent, alone and in combination with doxorubicin in the treatment of hepatocellular carcinoma (HCC)., Materials and Methods: In mice bearing luciferized HCC cells, therapeutic efficacy was assessed for seven days after single administration of CKD-516, doxorubicin, or combination of CKD-516 and doxorubicin., Results: Bioluminescence-imaging (BLI) signals in the CKD-516 group abruptly decreased initially, but recovered at seven days after treatment. BLI signals in the doxorubicin group gradually decreased over the 7-day period. In the combination group, BLI signals were abruptly reduced and remained suppressed for the 7-day period. On histopathological examination, CKD-516-treated tumors showed extensive central necrosis, whereas the peripheral layers remained viable. Doxorubicin-treated tumors showed mild and scattered necrosis. Tumors from the combination group showed more extensive central and peripheral necrosis, with smaller viable peripheral layers than the CKD-516 group., Conclusion: Combination therapy can have additive effects for treatment of HCC compared with CKD-516 or doxorubicin monotherapy.

Skeletal muscle atrophy can occur rapidly in various fasting, cancerous, systemic inflammatory, deranged metabolic or neurogenic states. The ubiquitin ligase Atrogin-1 (MAFbx) is induced in animal models of these conditions, causing excessive myoprotein degradation. It is unknown if Atrogin upregulation also occurs in acquired human myositis. Intracellular β-amyloid (Aβi), phosphorylated neurofilaments, scattered infiltrates and atrophy involving selective muscle groups characterize human sporadic Inclusion Body Myositis (sIBM). In Polymyositis (PM), inflammation is more pronounced and atrophy is symmetric and proximal. IBM and PM share various inflammatory markers. We found that forkhead family transcription factor Foxo3A is directed to the nucleus and Atrogin-1 transcript is increased in both conditions. Expression of Aβ in transgenic mice and differentiated C2C12 myotubes was sufficient to upregulate Atrogin-1 mRNA and cause atrophy. Aβi reduces levels of p-Akt and downstream p-Foxo3A, resulting in Foxo3A translocation and Atrogin-1 induction. In a mouse model of autoimmune myositis, cellular inflammation alone was associated with similar Foxo3A and Atrogin changes. Thus, either Aβi accumulation or cellular immune stimulation may independently drive muscle atrophy in sIBM and PM, respectively, through pathways converging on Foxo and Atrogin-1. In sIBM it is additionally possible that both mechanisms synergize.

The significance of intracellular beta-amyloid (Abeta(42)) accumulation is increasingly recognized in Alzheimer's disease (AD) pathogenesis. Abeta removal mechanisms that have attracted attention include IDE/neprilysin degradation and antibody-mediated uptake by immune cells. However, the role of the ubiquitin-proteasome system (UPS) in the disposal of cellular Abeta has not been fully explored. The E3 ubiquitin ligase Parkin targets several proteins for UPS degradation, and Parkin mutations are the major cause of autosomal recessive Parkinson's disease. We tested whether Parkin has cross-function to target misfolded proteins in AD for proteasome-dependent clearance in SH-SY5Y and primary neuronal cells. Wild-type Parkin greatly decreased steady-state levels of intracellular Abeta(42), an action abrogated by proteasome inhibitors. Intracellular Abeta(42) accumulation decreased cell viability and proteasome activity. Accordingly, Parkin reversed both effects. Changes in mitochondrial ATP production from Abeta or Parkin did not account for their effects on the proteasome. Parkin knock-down led to accumulation of Abeta. In AD brain, Parkin was found to interact with Abeta and its levels were reduced. Thus, Parkin is cytoprotective, partially by increasing the removal of cellular Abeta through a proteasome-dependent pathway.

Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery.

Intraneuronal beta-amyloid (Abeta(i)) accumulates early in Alzheimer's disease (AD) and inclusion body myositis. Several organelles, receptor molecules, homeostatic processes, and signal transduction components have been identified as sensitive to Abeta. Although prior studies implicate the insulin-PI3K-Akt signaling cascade, a specific step within this or any essential metabolic or survival pathway has not emerged as a molecular target. We tested the effect of Abeta42 on each component of this cascade. In AD brain, the association between PDK and Akt, phospho-Akt levels and its activity were all decreased relative to control. In cell culture, Abeta(i) expression inhibited both insulin-induced Akt phosphorylation and activity. In vitro experiments identified that beta-amyloid (Abeta), especially oligomer preparations, specifically interrupted the PDK-dependent activation of Akt. Abeta(i) also blocked the association between PDK and Akt in cell-based and in vitro experiments. Importantly, Abeta did not interrupt Akt or PI3K activities (once stimulated) nor did it affect more proximal signal events. These results offer a novel therapeutic strategy to neutralize Abeta-induced energy failure and neuronal death.

Kainic acid (KA) is well-known as an excitatory, neurotoxic substance. In mice, KA administered intracerebroventricularly (i.c.v.) lead to morphological damage of hippocampus expecially concentrated on the CA3 pyramidal neurons. In the present study, the possible role of gamma-aminobutyric acid B (GABA(B)) receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. 5-Aminovaleric acid (5-AV; GABA(B) receptors antagonist, 20 mug) reduced KA-induced CA3 pyramidal cell death. KA increased the phosphorylated extracellular signal-regulated kinase (p-ERK) and Ca(2+)/calmodulin-dependent protein kinase II (p-CaMK II) immunoreactivities (IRs) 30 min after KA treatment, and c-Fos, c-Jun IR 2 h, and glial fibrillary acidic protein (GFAP), complement receptor type 3 (OX-42) IR 1 day in hippocampal area in KA-injected mice. 5-AV attenuated KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. These results suggest that p-CaMK II may play as an important regulator on hippocampal cell death induced by KA administered i.c.v. in mice. Activated astrocytes, which was presented by GFAP IR, and activated microglia, which was presented by the OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA excitotoxicity. Furthermore, it showed that GABA(B) receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

The present study was designed to characterize the possible roles of spinally located cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G-proteins in pro-inflammatory cytokine induced pain behaviors. Intrathecal injection of tumor necrosis factor-alpha (TNF-alpha; 100 pg), interleukin-1beta (IL-1beta; 100 pg) and interferon-gamma (INF-gamma; 100 pg) showed pain behavior. Intrathecal pretreatment with CTX (0.05, 0.1 and 0.5 mg) attenuated pain behavior induced by TNF-alpha and INF-gamma administered intrathecally. But intrathecal pretreatment with CTX (0.05, 0.1 and 0.5 microg) did not attenuate pain behavior induced by IL-1beta. On the other hand, intrathecal pretreatment with PTX further increased the pain behavior induced by TNF-alpha and IL-1beta administered intrathecally, especially at the dose of 0.5 microg. But intrathecal pretreatment with PTX did not affect pain behavior induced by INF-gamma. Our results suggest that, at the spinal cord level, CTX- and PTX-sensitive G-proteins appear to play important roles in modulating pain behavior induced by pro-inflammatory cytokines administered spinally. Furthermore, TNF-alpha, IL-1beta and INF-gamma administered spinally appear to produce pain behavior by different mechanisms.

We examined the effect of the subcutaneous (s.c.) pretreatment of formalin into both hind paws of mice on the antinociception induced by the intracerebroventricularly (i.c.v.) or intrathecally (i.t.) administration of beta-endorphin using the tail-flick test. Pretreatment with formalin (5%) for 5 h had no affect on the i.c.v. administered beta-endorphin-induced tail-flick response. However, pretreatment with formalin for 40 h attenuated the tail-flick inhibition induced by i.c.v. administered beta-endorphin. This antinociceptive tolerance to i.c.v. beta-endorphin continued up to 1 week, but to a lesser extent. Pretreatment with formalin for 5 and 40 h significantly reduced the i.t. beta-endorphin-induced inhibition of the tail-flick response, which continued up to 1 week. The s.c. formalin treatment increased the hypothalamic pro-opiomelanocortin (POMC) mRNA level at 2 h, but this returned to the basal level after 40 h. Our results suggest that the increase in the POMC mRNA level in the hypothalamus appears to be involved in the supraspinal or spinal beta-endorphin-induced antinociceptive tolerance in formalin-induced inflammatory pain.

The antinociceptive effect of nicotine administered intracereboventricularly (i.c.v.) or intrathecally (i.t) in several pain models was examined in the present study. We found that i.t. treatment with nicotine (from 5 to 20 g) dose-dependently blocked pain behavior revealed during the second phase, but not during the first phase in the formalin test. In addition, i.c.v. treatment with nicotine (from 0.1 to 10 microg) dose-dependently attenuated pain behavior revealed during both the first and second phases. In addition to the formalin test, nicotine administered i.c.v. or i.t. attenuated acetic acid-induced writhing response. Furthermore, i.c.v. or i.t. administration of nicotine did not cause licking, scratching and biting responses induced by substance P, glutamate, TNF-alpha (100 pg), IL-1beta (100 pg) and INF-gamma (100 pg) injectied i.t. The antinociception induced by supraspinally-administered nicotine appears to be more effective than that resulting from spinally administered nicotine. Our results suggest that nicotine administration induces antinociception by acting on the central nervous system and has differing antinociceptive profiles according to the various pain models.

Effects of ginsenosides on nitric oxide (NO) production induced by lipopolysaccharide plus TNF-alpha (LNT) were examined in C6 rat glioma cells. Among several ginsenosides, ginsenoside Rd showed a complete inhibition against LNT-induced NO production. Ginsenoside Rd attenuated LNT-induced increased phosphorylation of ERK. Among several immediate early gene products, only Jun B and Fra-1 protein levels were increased by LNT, and ginsenoside Rd attenuated Jun B and Fra-1 protein levels induced by LNT. Furthermore, LNT increased AP-1 DNA binding activities, which were partially inhibited by ginsenoside Rd. Our results suggest that ginsenoside Rd exerts an inhibitory action against NO production via blocking phosphorylation of ERK, in turn, suppressing immediate early gene products such as Jun B and Fra-1 in C6 glioma cells.