Down-regulation of RalBP1 expression reduces seizure threshold and synaptic inhibition in mice.

Idiopathic epilepsy is characterized by seizures without a clear etiology and is believed to have a strong genetic component but exhibits a complex inheritance pattern. Genetic factors seem to confer a low seizure threshold to susceptible individuals and thereby enhance epileptogenesis. However, the identity of susceptibility genes and the mechanisms regulating seizure threshold are still poorly understood. Here, we describe that reduced expression of RalBP1, a downstream effector of the small GTPases RalA and RalB, lowers the seizure threshold in mice. The intraperitoneal injection of the chemoconvulsant pentylenetetrazol induced more severe seizures in RalBP1 hypomorphic mice than in their wild-type littermates. The reduction of RalBP1 in the brain has no effect on neuronal excitability, but does decrease the inhibitory synaptic transmission onto CA1 pyramidal neurons. This impaired synaptic inhibition was associated with the loss of GABAergic interneurons in the CA1 subfield of the hippocampus. The present study identifies RalBP1 as a gene regulating the seizure threshold in mice and provides direct evidence for the role of RalBP1 in synaptic inhibition in vivo.

Developmental retardation, microcephaly, and peptiduria in mice without aminopeptidase P1.

Cytosolic aminopeptidase P1 (APP1) is one of the three known mammalian aminopeptidase Ps (APPs) that cleave the N-terminal amino acid residue of peptides in which the penultimate amino acid is proline. In mammals, many biologically active peptides have a highly conserved N-terminal penultimate proline. However, little is known about the physiological role of APP1. In addition, there is no direct evidence to associate a deficiency in APP1 with metabolic diseases. Although two human subjects with reduced APP activity exhibited peptiduria, it is unclear which of the three APP isoforms is responsible for this disorder. In this study, we generated APP1-deficient mice by knocking out Xpnpep1. Mouse APP1 deficiency causes severe growth retardation, microcephaly, and modest lethality. In addition, imino-oligopeptide excretion was observed in urine samples from APP1-deficient mice. These results suggest an essential role for APP1-mediated peptide metabolism in body and brain development, and indicate a strong causal link between APP1 deficiency and peptiduria.

Casuarinin suppresses TARC/CCL17 and MDC/CCL22 production via blockade of NF-κB and STAT1 activation in HaCaT cells.

Casuarinin is a naturally occurring tannin that is isolated from the leaves of Hippophae rhamnoides. It has been shown to have anti-oxidant, anti-cancer, anti-viral, and anti-inflammatory activities. The aim of this study was to investigate the possible mechanism by which casuarinin inhibits TNF-α/IFN-γ-induced Th2 chemokines expression in the human keratinocytes cell line HaCaT. We found that casuarinin suppressed TNF-α/IFN-γ-induced expression of TARC and MDC mRNA and protein in HaCaT cells. Casuarinin significantly inhibited TNF-α/IFN-γ-induced activation of NF-κB, STAT1, and p38 MAPK. Furthermore, we observed that p38 MAPK contributes to inhibition of TNF-α/IFN-γ-induced TARC and MDC production by blocking NF-κB and STAT1 activation in HaCaT cells. Taken together, these results suggest that casuarinin may exert anti-inflammatory responses by suppressing TNF-α/IFN-γ-induced expression of TARC and MDC via blockage of p38 MAPK activation and subsequent activation of NF-κB and STAT1. We propose that it could therefore be used as a therapeutic agent against inflammatory skin diseases.

Suppression of exaggerated NMDAR activity by memantine treatment ameliorates neurological and behavioral deficits in aminopeptidase P1-deficient mice.

Inborn errors of metabolism (IEMs) are common causes of neurodevelopmental disorders, including microcephaly, hyperactivity, and intellectual disability. However, the synaptic mechanisms of and pharmacological interventions for the neurological complications of most IEMs are unclear. Here, we report that metabolic dysfunction perturbs neuronal NMDA receptor (NMDAR) homeostasis and that the restoration of NMDAR signaling ameliorates neurodevelopmental and cognitive deficits in IEM model mice that lack aminopeptidase P1. Aminopeptidase P1-deficient (Xpnpep1-/-) mice, with a disruption of the proline-specific metalloprotease gene Xpnpep1, exhibit hippocampal neurodegeneration, behavioral hyperactivity, and impaired hippocampus-dependent learning. In this study, we found that GluN1 and GluN2A expression, NMDAR activity, and the NMDAR-dependent long-term potentiation (LTP) of excitatory synaptic transmission were markedly enhanced in the hippocampi of Xpnpep1-/- mice. The exaggerated NMDAR activity and NMDAR-dependent LTP were reversed by the NMDAR antagonist memantine. A single administration of memantine reversed hyperactivity in adult Xpnpep1-/- mice without improving learning and memory. Furthermore, chronic administration of memantine ameliorated hippocampal neurodegeneration, hyperactivity, and impaired learning and memory in Xpnpep1-/- mice. In addition, abnormally enhanced NMDAR-dependent LTP and NMDAR downstream signaling in the hippocampi of Xpnpep1-/- mice were reversed by chronic memantine treatment. These results suggest that the metabolic dysfunction caused by aminopeptidase P1 deficiency leads to synaptic dysfunction with excessive NMDAR activity, and the restoration of synaptic function may be a potential therapeutic strategy for the treatment of neurological complications related to IEMs.

Effects of taxifolin from enzymatic hydrolysis of Rhododendron mucrotulatum on hair growth promotion.

Flavonoid aglycones possess biological activities, such as antioxidant and antidiabetic activities compared to glycosides. Taxifolin, a flavonoid aglycones, is detected only in trace amounts in nature and is not easily observed. Therefore, in this study, to investigate the hair tonic and hair loss inhibitors effect of taxifolin, high content of taxifolin aglycone extract was prepared by enzymatic hydrolysis. Taxifolin effectively regulates the apoptosis of dermal papilla cells, which is associated with hair loss, based on its strong antioxidant activities. However, inhibition of dihydrotestosterone (DHT), which is a major cause of male pattern hair loss, was significantly reduced with taxifolin treatment compared with minoxidil, as a positive control. It was also confirmed that a representative factor for promoting hair growth, IGF-1, was significantly increased, and that TGF-ß1, a representative biomarker for hair loss, was significantly reduced with taxifolin treatment. These results suggest that taxifolin from enzymatic hydrolysis of RM is a potential treatment for hair loss and a hair growth enhancer.

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells.

Hippophae rhamnoides has been extensively used in oriental traditional medicines for treatment of asthma, skin diseases, gastric ulcers, and lung disorders. In this study, we isolated casuarinin from the leaves of H.rhamnoides and examined the effect of casuarinin on the TNF-α-induced ICAM-1 expression in a human keratinocytes cell line HaCaT. Pretreatment with casuarinin inhibited TNF-α-induced protein and mRNA expression of ICAM-1 and subsequent monocyte adhesiveness in HaCaT cells. Casuarinin significantly inhibited TNF-α-induced NF-κB activation. In addition, casuarinin inhibited activation of ERK and p38 MAPK in a dose-dependent manner. Furthermore, pretreatment with casuarinin decreased TNF-α-induced pro-inflammatory mediators, such as IL-1ß, IL-6, IL-8, and MCP-1. These results demonstrated that casuarinin exerts its anti-inflammatory activity by suppressing TNF-α-induced expression of ICAM-1 and pro-inflammatory cytokines/chemokines via blockage of activation of NF-κB and ERK/p38 MAPK and can be used as a therapeutic agent against inflammatory skin diseases.

Inhibition of aldose reductase by phenylethanoid glycoside isolated from the seeds of Paulownia coreana.

Aldose reductase (AR) inhibitors have considerable therapeutic potential against diabetic complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of the 70% acetone extract obtained from Paulownia coreana seeds, phenylpropanoid glycosides (compounds 1-4) and 5 phenolic compounds were isolated (compounds 5-9). Their structures were determined on the basis of spectroscopic analysis and comparison with reported data. All the isolates were subjected to in vitro bioassays to evaluate their inhibitory activities against recombinant human aldose reductase (rhAR) and sorbitol formation in human erythrocytes. Phenylethanoid glycosides showed more effective than the phenolic compounds in inhibiting rhAR. Among the compounds, isocampneoside II (3) was found to significantly inhibit rhAR with an IC(50) value of 9.72 µM. In kinetic analyses performed using Lineweaver-Burk plots of 1/velocity and 1/concentration of substrate, isocampneoside II (3) showed uncompetitive inhibition against rhAR. Furthermore, it inhibited sorbitol formation in a rat lens incubated with a high concentration of glucose; this finding indicated that isocampneoside II (3) may effectively prevent osmotic stress in hyperglycemia. Thus, the P. coreana-derived phenylethanoid glycoside isocampneoside II (3) may have a potential therapeutics against diabetic complications.

Altered hippocampal gene expression, glial cell population, and neuronal excitability in aminopeptidase P1 deficiency.

Inborn errors of metabolism are often associated with neurodevelopmental disorders and brain injury. A deficiency of aminopeptidase P1, a proline-specific endopeptidase encoded by the Xpnpep1 gene, causes neurological complications in both humans and mice. In addition, aminopeptidase P1-deficient mice exhibit hippocampal neurodegeneration and impaired hippocampus-dependent learning and memory. However, the molecular and cellular changes associated with hippocampal pathology in aminopeptidase P1 deficiency are unclear. We show here that a deficiency of aminopeptidase P1 modifies the glial population and neuronal excitability in the hippocampus. Microarray and real-time quantitative reverse transcription-polymerase chain reaction analyses identified 14 differentially expressed genes (Casp1, Ccnd1, Myoc, Opalin, Aldh1a2, Aspa, Spp1, Gstm6, Serpinb1a, Pdlim1, Dsp, Tnfaip6, Slc6a20a, Slc22a2) in the Xpnpep1-/- hippocampus. In the hippocampus, aminopeptidase P1-expression signals were mainly detected in neurons. However, deficiency of aminopeptidase P1 resulted in fewer hippocampal astrocytes and increased density of microglia in the hippocampal CA3 area. In addition, Xpnpep1-/- CA3b pyramidal neurons were more excitable than wild-type neurons. These results indicate that insufficient astrocytic neuroprotection and enhanced neuronal excitability may underlie neurodegeneration and hippocampal dysfunction in aminopeptidase P1 deficiency.

Application of Ethanol Extracts From Alnus sibirica Fisch. ex Turcz in Hair Growth Promotion.

Alnus sibirica Fisch. ex Turcz (ASFT), belonging to the family of Betulaceae, grows naturally in Asia, Europe, and America. The aims of this study are determining the efficacy of various biomarkers related to hair loss, evaluated by extracting the branch with 60% alcohol, and purely separating diarylheptanoid oregonin, an indicator and active substance, from 60% alcohol extract of the tree. To determine the preventive effects on hair loss, we investigated the anti-oxidative and anti-apoptotic effects on hydrogen peroxide-induced cytotoxicity on human hair dermal papilla cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Western blotting analysis for proving of apoptosis-related marker alteration, respectively. Moreover, we examined the ameliorative effects of 60% alcohol extract of the tree and oregonin against changes of oxidative stress-induced cytokine and testosterone-induced dihydrotestosterone production as crucial pathways of the hair loss mechanism. These results suggest that 60% alcohol extract of the tree and oregonin were available as novel natural materials for maintaining hair health in mammals.

Enhanced NMDA receptor-mediated synaptic transmission, enhanced long-term potentiation, and impaired learning and memory in mice lacking IRSp53.

IRSp53 is an adaptor protein that acts downstream of Rac and Cdc42 small GTPases and is implicated in the regulation of membrane deformation and actin filament assembly. In neurons, IRSp53 is an abundant postsynaptic protein and regulates actin-rich dendritic spines; however, its in vivo functions have not been explored. We characterized transgenic mice deficient of IRSp53 expression. Unexpectedly, IRSp53(-/-) neurons do not show significant changes in the density and ultrastructural morphologies of dendritic spines. Instead, IRSp53(-/-) neurons exhibit reduced AMPA/NMDA ratio of excitatory synaptic transmission and a selective increase in NMDA but not AMPA receptor-mediated transmission. IRSp53(-/-) hippocampal slices show a markedly enhanced long-term potentiation (LTP) with no changes in long-term depression. LTP-inducing theta burst stimulation enhances NMDA receptor-mediated transmission. Spatial learning and novel object recognition are impaired in IRSp53(-/-) mice. These results suggest that IRSp53 is involved in the regulation of NMDA receptor-mediated excitatory synaptic transmission, LTP, and learning and memory behaviors.

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-beta-D-glucose via blockade of NF-kappaB and STAT1 activation in the HaCaT cells.

Keratinocytes, one of major cell types in the skin, can be induced by TNF-alpha and IFN-gamma to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-alpha/IFN-gamma induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-alpha/IFN-gamma-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-alpha/IFN-gamma-induced NF-kappaB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-gamma. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-alpha and/or IFN-gamma-induced activation of NF-kappaB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.

Phenolic compounds in the leaves of Populus ussuriensis and their antioxidant activities.

Two new phenolic glucosides [isograndidentatin A (1) isograndidentatin B (2)], 5 known phenolic glucosides [grandidentatin (3), salireposide (4), populoside (5), populoside A (6), and salicortin (7)], and 2 known phenolic acids [P-coumaric acid (8) and caffeic acid (9)] were isolated from the leaves of Populus ussuriensis. Structure elucidation of 1 and 2 was achieved through extensive spectroscopic techniques. Compounds 1-6 and 9 showed significant antioxidant activities, which were evaluated by the DPPH radical-scavenging method (IC(50) values of 6.68, 6.61, 6.75, 6.84, 6.76, 6.79, and 5.92 microM, respectively) and the ABTS .+ radical-scavenging system (TEAC values of 1.21, 1.28, 1.26, 1.05, 1.69, 1.60, and 2.00 mM, respectively).

Induction of cell proliferation and neuroblasts in the subgranular zone of the dentate gyrus by aqueous extract from Platycodon grandiflorum in middle-aged mice.

In the present study, we observed the neurogenic effects of an aqueous extract from the root of Platycodon grandiflorum (EPG) in middle-aged (12-month-old) mice. For this, 100mg/kg EPG was administered orally to mice for 30 days before sacrifice and 5-bromodeoxyguanosine (BrdU) was injected intraperitoneally every 8h for 24h on the day prior to sacrifice. The increase of neurogenesis was estimated by immunohistochemical staining for cellular proliferation markers (BrdU and Ki67) and a marker for neuroblasts (Doublecortin, DCX). These markers were detected in the subgranular zone of the dentate gyrus in vehicle- and EPG-treated groups. The number of BrdU-, Ki67- and DCX-positive cells in the EPG-treated group was significantly increased compared to that in the vehicle-treated group. In addition, DCX-positive cells in the EPG-treated group showed well-developed processes. These results suggest that the number of neuroblasts is increased by the repeated treatment of EPG in middle-aged mice.

Antiinflammatory effect of the ethanol extract of Berberis koreana in a gerbil model of cerebral ischemia/reperfusion.

Berberis koreana extract (BE) has a strong neuroprotective effect after ischemic stroke in gerbils, which is associated with the inhibition of the N-methyl-d-aspartate receptor. The present study examined the antiinflammatory mechanism of BE after ischemic damage in vitro and in vivo. The BE used contained on average 7.39 +/- 0.78 mg/g of berberine. In PC12 cells with inflammation, prostaglandin E2 (PGE2) production was significantly reduced by BE. About 75% of pyramidal cells in the hippocampal CA1 region of gerbils exposed to 5 min of transient ischemia were protected from ischemic damage by BE. Cyclooxygenase-2 (COX-2) immunoreactivity and its protein level in the CA1 region of vehicle-treated animals exposed to an ischemic insult increased with time post-ischemia, whereas no such changes were observed in BE-treated animals exposed to ischemia. PGE2 production in BE-treated ischemic animals was significantly lower than that observed in vehicle-treated ischemic animals. Summarizing, the potent neuroprotective effect of BE was found to be due to the inhibitions of COX-2 expression and PGE2 production and its antiinflammatory activity.

Phenylpropanoid glycosides from the leaves of Paulownia coreana.

Study on the water soluble fraction from the leaves of Paulownia coreana led to the isolation of verbascoside (1), isoverbascoside (2), campneoside II (3), and a new phenylpropanoid glycoside, (R,S)-7-hydroxy-7-(3,4-dihydroxyphenyl)-ethyl-O-alpha-L-rhamnopyranosyl(1 --> 3)-beta-d-(6-O-caffeoyl)-glucopyranoside (4). The structures of these compounds were established on the basis of spectroscopic evidence.

Purification of Four Flavonoid Glycosides from Lotus (Nelumbo nucifera Gaertn) plumule by Macroporous Resin Combined with HSCCC.

High-speed counter-current chromatography (HSCCC) combined with macroporous resin (MR) column was successfully applied to the isolation and purification of four flavonoid glycosides from the medicinal herb Lotus plumule (LP). A polar two-phase solvent system composed of ethyl acetate-n-butanol-water (1:2:3, v/v/v) was selected by high-performance liquid chromatography (HPLC) and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. Quercetin-3-O-ß-D-glucopyranoside (15 mg), isorhamnetin-3-O-ß-D-glucopyranoside (13 mg), apigenin 6-C-ß-D-glucopyranosyl-8-C-α-L-arabinopyranoside (18 mg) and apigenin 6,8-di-C-ß-D-glucopyranoside (48 mg) were obtained in a one-step HSCCC separation from 240 mg of the sample. The purity of each compound was over 95% as determined by HPLC. Chemical structures of the isolated compounds were identified by electrospray ionization mass spectrometry (ESI-MS-MS) and nuclear magnetic resonance (NMR) methods. Moreover, the four compounds were isolated from LP for the first time.

The repopulating cancer cells in melanoma are characterized by increased mitochondrial membrane potential.

Although considerable effort has been expended in identifying definitive markers for cancer stem cells (CSCs) or cancer-initiating cells (CICs), the phenotypic plasticity of these cells obviates simple characterization using cell surface markers. We hypothesized that these cells could be characterized by their metabolic properties because they are in a quiescent state with low energy needs. We examined whether cancer cells differ in mitochondrial membrane potential (Δψm) when they are under stress. The Δψm of B16-F10 melanoma cells increased when they were exposed in vitro to serum starvation and chemotherapeutic agents, but not when exposed to hypoxia. Such TMREhigh cells were also present in tumor tissue. They primarily used glucose and/or lactate, and were superior to TMRElow B16-F10 cells in their ability to drive tumor growth. These findings suggest that CSCs or CICs could be identified in heterogeneous melanoma populations by measuring Δψm.

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages.

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1ß and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages.

Antioxidant phenolic glycosides from the bark of Populus ussuriensis Kom.

Study on the EtOAc soluble fraction from the bark of Populus ussuriensis Kom. resulted in the isolation of three phenolic glycosides, including populoside (1), 7-O-ρ-coumaroylsalirepin (2) and 7-O-caffeoylsalirepin (3), among which 3 is a new compound. The structures of these compounds were elucidated on the basis of spectroscopic evidence. Phenolic glycosides 1-3 exhibited excellent antioxidant activity, evaluated in the ABTS⁺ radical scavenging assay.

Inhibitory effect of glucodistylin from the bark of Quercus acutissima on human recombinant aldose reductase and sorbitol accumulation.

A flavanol glycoside, glucodistylin (1) and three polyphenol derivatives, gallate (2), (+)-catechin (3) and (+)-gallocatechin (4) were isolated from an aqueous acetone extract of the bark of Quercus acutissima. Of these compounds, glucodistylin exhibited uncompetitive inhibitory activity against recombinant human aldose reductase with an IC(50) value of 7.2 µM. Furthermore, glucodistylin inhibited sorbitol accumulation by 48.84% at 50 µM. This flavonoid showed therapeutic potential in the prevention and treatment of diabetes-related complications.