Neuroprotection and axon regeneration by novel low-molecular-weight compounds through the modification of DOCK3 conformation.

Dedicator of cytokinesis 3 (DOCK3) is an atypical member of the guanine nucleotide exchange factors (GEFs) and plays important roles in neurite outgrowth. DOCK3 forms a complex with Engulfment and cell motility protein 1 (Elmo1) and effectively activates Rac1 and actin dynamics. In this study, we screened 462,169 low-molecular-weight compounds and identified the hit compounds that stimulate the interaction between DOCK3 and Elmo1, and neurite outgrowth in vitro. Some of the derivatives from the hit compound stimulated neuroprotection and axon regeneration in a mouse model of optic nerve injury. Our findings suggest that the low-molecular-weight DOCK3 activators could be a potential therapeutic candidate for treating axonal injury and neurodegenerative diseases including glaucoma.

Vision protection and robust axon regeneration in glaucoma models by membrane-associated Trk receptors.

Activation of neurotrophic factor signaling is a promising therapy for neurodegeneration. However, the transient nature of ligand-dependent activation limits its effectiveness. In this study, we solved this problem by inventing a system that forces membrane localization of the intracellular domain of tropomyosin receptor kinase B (iTrkB), which results in constitutive activation without ligands. Our system overcomes the small size limitation of the genome packaging in adeno-associated virus (AAV) and allows high expression of the transgene. Using AAV-mediated gene therapy in the eyes, we demonstrate that iTrkB expression enhances neuroprotection in mouse models of glaucoma and stimulates robust axon regeneration after optic nerve injury. In addition, iTrkB expression in the retina was also effective in an optic tract transection model, in which the injury site is near the superior colliculus. Regenerating axons successfully formed pathways to their brain targets, resulting in partial recovery of visual behavior. Our system may also be applicable to other trophic factor signaling pathways and lead to a significant advance in the field of gene therapy for neurotrauma and neurodegenerative disorders, including glaucoma.

Carob pod polyphenols suppress the differentiation of adipocytes through posttranscriptional regulation of C/EBPß.

Obesity is a major risk factor for various chronic diseases such as diabetes, cardiovascular disease, and cancer; hence, there is an urgent need for an effective strategy to prevent this disorder. Currently, the anti-obesity effects of food ingredients are drawing attention. Therefore, we focused on carob, which has high antioxidant capacity and various physiological effects, and examined its anti-obesity effect. Carob is cultivated in the Mediterranean region, and its roasted powder is used as a substitute for cocoa powder. We investigated the effect of carob pod polyphenols (CPPs) on suppressing increases in adipose tissue weight and adipocyte hypertrophy in high fat diet-induced obesity model mice, and the mechanism by which CPPs inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes in vitro. In an in vivo experimental system, we revealed that CPPs significantly suppressed the increase in adipose tissue weight and adipocyte hypertrophy. Moreover, in an in vitro experimental system, CPPs acted at the early stage of differentiation of 3T3-L1 preadipocytes and suppressed cell proliferation because of differentiation induction. They also suppressed the expression of transcription factors involved in adipocyte differentiation, thereby reducing triacylglycerol synthesis ability and triglycerol (TG) accumulation. Notably, CPPs regulated CCAAT/enhancer binding protein (C/EBP)ß, which is expressed at the early stage of differentiation, at the posttranscriptional level. These results demonstrate that CPPs suppress the differentiation of adipocytes through the posttranscriptional regulation of C/EBPß and may serve as an effective anti-obesity compound.

Conflicting actions of 4-vinylcatechol in rat lymphocytes under oxidative stress induced by hydrogen peroxide.

4-Vinylcatechol (4VC) has been identified as an aroma compound in roasted foods, especially coffee. It is also a component in traditional herbal medicines. This compound may be subconsciously ingested through foods and herbs. Recent experimental evidence has shown that 4VC possesses an antioxidative action. However, the antioxidative action of 4VC at cellular levels is not well characterized. The effects of 4VC (0.1-100 µM) were examined on rat thymic lymphocytes without and with oxidative stress induced by 300 µM hydrogen peroxide (H2O2). Cell treatment with 100 µM 4VC alone for 4 h significantly increased the population of dead cells. Thus, 4VC at 100 µM or above elicits cytotoxicity. However, 4VC at sublethal concentrations (1-10 µM) significantly attenuated the H2O2-induced increase in cell lethality in a concentration-dependent manner. While application of 10 µM 4VC slowed the process of cell death induced by H2O2, 4VC did not antagonize the H2O2-induced reduction of cellular nonprotein thiols. Although 4VC at 10 µM did not affect intracellular Ca2+ and Zn2+ levels, the agent potentiated the H2O2-induced increases in these levels. These actions of 10 µM 4VC are adverse to the cells under the oxidative stress. However, 10 µM 4VC partly attenuated the cell death induced by 100 nM A23187, a calcium ionophore. There are conflicting actions of 4VC at 1-100 µM on the cells under oxidative stress although the agent is used for an antioxidant. Thus, caution is required when using 4VC as a therapeutic agent.

Survival of Alpha and Intrinsically Photosensitive Retinal Ganglion Cells in NMDA-Induced Neurotoxicity and a Mouse Model of Normal Tension Glaucoma.

Purpose: We assess if α retinal ganglion cells (αRGCs) and intrinsically photosensitive retinal ganglion cells (ipRGCs) survive in mouse models of glaucoma. Methods: Two microliters of N-methyl-D-aspartate (NMDA; 1 mM) or PBS were injected intraocularly 7 days before sacrifice. Immunohistochemical analyses of the retina were performed using antibodies against RNA-binding protein with multiple splicing (RBPMS), osteopontin, and melanopsin. Immunohistochemical analyses also were performed in adult mice with glutamate/aspartate transporter (GLAST) deletion (GLAST knockout [KO] mice), a mouse model of normal tension glaucoma. Results: NMDA-induced loss of RBPMS-positive total RGCs was 58.4% ± 0.4% compared to PBS-treated controls, whereas the loss of osteopontin-positive αRGCs was 5.0% ± 0.6% and that of melanopsin-positive ipRGCs was 7.6% ± 1.6%. In GLAST KO mice, the loss of total RGCs was 48.4% ± 0.9% compared to wild-type mice, whereas the loss of αRGCs and ipRGCs was 3.9% ± 0.4% and 9.3% ± 0.5%, respectively. The distribution of survived total RGCs, αRGCs, and ipRGCs was similar regardless of the location of the retina. Conclusions: These results suggest that αRGC and ipRGC are highly tolerant to NMDA-induced neurotoxicity and NTG-like neurodegeneration in GLAST KO mice.

Propolis Components from Stingless Bees Collected on South Sulawesi, Indonesia, and Their Xanthine Oxidase Inhibitory Activity.

Three new compounds, namely, 4-(4'-hydroxy-3'-methoxyphenyl)-3,5,7-trihydroxycoumarin (1) and sulawesins A (2) and B (3), were isolated from the propolis of stingless bees ( Tetragonula aff. biroi) collected on South Sulawesi, Indonesia. In addition, five known compounds, glyasperin A, broussoflavonol F, (2 S)-5,7-dihydroxy-4'-methoxy-8-prenylflavanone, (1' S)-2- trans,4- trans-abscisic acid, and (1' S)-2- cis,4- trans-abscisic acid, were identified. The structures of the new compounds were determined by a combination of methods that included mass spectrometry and NMR spectroscopy. The absolute configuration of sulawesin A (2), a new podophyllotoxin derivative, was determined by X-ray crystallography. The absolute configuration of sulawesin B (3) was also determined by the ECD calculation. 4-(4'-Hydroxy-3'-methoxyphenyl)-3,5,7-trihydroxycoumarin (1) and sulawesin A (2) were examined for xanthine oxidase (XO) inhibitory activity; 1 exhibited XO inhibitory activity, with an IC50 value of 3.9 µM.

Radical Scavenging Properties of Roasted Egoma (Perilla frutescens var. frutescens) Oils and Identification of Their Characteristic Scavengers.

The radical scavenging activity of commercially available roasted (deep colored) and unroasted (light colored) egoma (Perilla frutescens var. frutescens) oils was evaluated by the DPPH radical scavenging method. The antiradical activity of roasted oils was higher than that of unroasted oils, and the activity of methanol-water extracts from the roasted egoma oils was significantly higher than that of unroasted oils. The antiradical activity of the methanol-water fractions was strongly correlated to that of whole oils (r=0.72) and the color depth of oils (r=0.93), which was an index of roasting. Fractionation of the methanol-water extract of a roasted egoma oil according to molecular size using ultra membrane filters revealed that the fraction under 3 kDa had the strongest radical scavenging activity. Subsequent preparative HPLC separation using an ODS column also revealed that the second fraction was the most active. Our HPLC analytical method for DPPH radical scavengers in complex mixtures detected four strong radical scavenger peaks in the fraction. Among the detected peaks, two peaks were determined to be derived from rosmarinic acid and luteolin by comparison with the retention times and UV spectra of the authentic samples, and the other two compounds could not be identified because no characteristic UV spectra were observed. These identified polyphenols (rosmarinic acid and luteolin) have been reported to be present in the non-oily part of egoma seeds. They probably migrated to the oily part during the egoma oil roasting process.

Hydroxyhydroquinone, a by-product of coffee bean roasting, increases intracellular Ca2+ concentration in rat thymic lymphocytes.

Hydroxyhydroquinone (HHQ) is generated during coffee bean roasting. A cup of coffee contains 0.1-1.7 mg of HHQ. The actions of HHQ on mammalian DNA were examined because HHQ is a metabolite of benzene, which causes leukemia. Currently, information on the cellular actions of HHQ is limited. We examined the effects of sublethal levels of HHQ on the concentration of intracellular Ca2+ in rat thymic lymphocytes by using a flow cytometric technique with fluorescent probes. HHQ at 10 µM or more significantly elevated intracellular Ca2+ levels by increasing the membrane permeability of divalent cations, resulting in hyperpolarization via the activation of Ca2+-dependent K+ channels. HHQ-induced changes in the intracellular Ca2+ concentration and membrane potential may affect the cell functions of lymphocytes. HHQ-reduced coffee may be preferable in order to avoid the possible adverse effects of HHQ.

Conversion to purpurogallin, a key step in the mechanism of the potent xanthine oxidase inhibitory activity of pyrogallol.

In this study, the mechanism of the xanthine oxidase (XO) inhibitory activity of pyrogallol, the main inhibitor found in roasted coffee, was investigated. Pyrogallol was unstable and readily converted to purpurogallin in a pH 7.4 solution, a physiological model of human body fluids. The XO inhibitory activity of the produced purpurogallin was higher than that of pyrogallol, as evidenced by comparing their IC50 values (0.2µmolL-1 for purpurogallin, 1.6µmolL-1 for pyrogallol). The XO activity of pyrogallol was enhanced by pre-incubation in pH 7.4 solution. Although the initial XO inhibitory activity of 4-methylpyrogallol was weak (IC50 33.3µmolL-1), its XO inhibitory activity was also enhanced by pre-incubation in the pH 7.4 solution. In contrast, 5-methylpyrogallol, which could not be transformed into corresponding purpurogallin derivatives, did not show XO inhibitory activity before or after incubation in pH 7.4 solution. Molecular docking simulations clarified that purpurogallins have stronger affinities for XO than corresponding pyrogallols. These results revealed that the potent XO inhibitory activity seemingly observed in pyrogallol is actually derived from its chemical conversion, under alkaline conditions, into purpurogallin.

Identification of Pyrogallol in the Ethyl Acetate-Soluble Part of Coffee as the Main Contributor to Its Xanthine Oxidase Inhibitory Activity.

In this study, ethyl acetate-soluble parts of hot-water extracts from roasted coffee beans were found to demonstrate potent xanthine oxidase (XO) inhibition. The XO inhibitory activities and chlorogenic lactone contents (chlorogenic lactones have previously been identified as XO inhibitors in roast coffee) were measured for ethyl acetate-soluble parts prepared from coffee beans roasted to three different degrees. Although chlorogenic lactone contents decreased with higher degrees of roasting, the XO inhibitory activity did not decrease. These data led us to investigate new potent inhibitors present in these ethyl acetate-soluble extracts. Repeated assay-guided purifications afforded a highly potent XO inhibitor, which was eluted before chlorogenic lactones via medium-pressure chromatography using an octadecylsilica gel column. The obtained inhibitor was identified as pyrogallol (1,2,3-trihydroxybenzene), which had an IC50 of 0.73 µmol L-1, much stronger than that of other related polyphenolic compounds. Quantitative analysis of pyrogallol and chlorogenic lactones revealed that pyrogallol (at concentrations of 33.9 ± 4.2 nmol mL-1 in light roast coffee and 39.4 ± 3.9 nmol mL-1 in dark roast coffee) was the main XO inhibitor in hot-water extracts of roasted coffee beans (i.e., drinking coffee).

Effective Conversion of Metmyoglobin to Oxymyoglobin by Cysteine-Substituted Polyphenols.

Reaction products from the peroxidase-catalyzed oxidation of polyphenols in the presence of cysteine showed a potent activity for reducing metmyogolobin (MetMb) to bright-colored oxymyogolobin (MbO2). High-performance liquid chromatography (HPLC) purification of the reaction products from catechin, chlorogenic acid, dihydrocaffeic acid, hydroxytyrosol, nordihydroguaiaretic acid, and rosmarinic acid afforded corresponding S-cysteinyl compounds, the structures of which were determined by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The isolated cysteinyl polyphenols showed a concentration-dependent reducing activity for MetMb to MbO2 for the initial 1 h. However, after 1 h, some of them decreased the amount of MbO2 produced. The effect of the number of cysteinyl sulfur substitutions in polyphenols on both MetMb reduction and MbO2 maintenance was examined using hydroxytyrosols with different numbers of cysteine substitutions; these hydroxytyrosols were synthesized from hydroxytyrosol and an N-acetylcysteine methyl ester. The hydroxytyrosol derivative substituted with two N-acetylcysteine esters exhibited the most effective reducing activity without any effect on MbO2.

An oxidative coupling product of luteolin with cysteine ester and its enhanced inhibitory activity for xanthine oxidase.

Oxidative coupling reactions of several flavonoids with a cysteine ester (a radicalic and nucleophilic biochemical) were carried out and the abilities of the coupling products against xanthine oxidase (XO) were screened. One of the products, derived from luteolin, showed a notable inhibitory effect. A potent XO inhibitory compound was isolated from the complex mixture of the product of the coupling of luteolin and cysteine ethyl ester, and its structure was determined by NMR and MS analysis. The compound has a unique 1,4-thiazine ring unit on the luteolin B-ring and is inhibited XO 4.5 times more strongly than it did luteolin.

Reducing effects of polyphenols on metmyoglobin and the in vitro regeneration of bright meat color by polyphenols in the presence of cysteine.

The effect of polyphenols and related phenolic compounds on the reduction of metmyoglobin (MetMb) to oxymyoglobin (MbO2), in the presence of cysteine, was investigated. Caffeic acid, dihydrocaffeic acid, and hydroxtyrosol (600 µmol/L) did not show any reducing activity individually. However, their highly potent activity in the reduction of MetMb to MbO2 was observed in the presence of equimolar amounts of cysteine. On the basis of the analytical results for the redox reaction products generated during the MetMb-reducing reaction of caffeic acid, we proposed a mechanism for the polyphenol-mediated reduction of MetMb. As per the proposed mechanism, the antioxidant polyphenols having a catechol substructure can effectively reduce MetMb to MbO2 with chemical assistance from nucleophilic reactive thiol compounds such as cysteine. Moreover, cysteine-coupled polyphenols such as cysteinylcaffeic acids (which are coupling products of caffeic acid and cysteine) can be used as preserving agents for retaining the fresh meat color, because of their powerful reducing effect on MetMb. The reduction of MetMb to MbO2 changes the color of meat from brown to the more desirable bright red.

Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans.

Xanthine oxidase (XO) inhibitory activity has been found in boiling water extracts from roasted coffee beans. Therefore, assay-guided purification of the extracts was performed using size-exclusion column chromatography, and subsequently with reversed phase HPLC to afford lactone derivatives of chlorogenic acids. Among the tested lactones, crypto- and neochlorogenic lactones showed potent XO inhibitory activities compared with three major chlorogenic acids found in coffee beans. These XO inhibitory lactones may ameliorate gout and hyperuricemia in humans who drink coffee.

Antioxidant activities of cysteine derivatives against lipid oxidation in anhydrous media.

This study investigated antioxidant activities of cysteine derivatives of amino and carboxylic acid moieties against lipid oxidation in anhydrous acetonitrile. Only cysteine derivatives bearing free amino or carboxylate ion were found to exert potent antioxidant activities. Sequential proton loss and electron transfer-like proton shift and subsequent electron transfer (PS-ET) mechanism may facilitate the antioxidant activities of cysteine derivatives against lipid oxidation in anhydrous media.

Identification of a potent xanthine oxidase inhibitor from oxidation of caffeic acid.

Inhibitory activity of Fe-ion-catalyzed radical oxidation products from 22 types of phenolic compounds toward xanthine oxidase (XO) was investigated. Phenols are readily oxidizable compounds in nature and, thus, showed potent antioxidant activities. Among the phenols screened in this study, noticeable activity was observed in the oxidation product of caffeic acid, whereas almost no XO-inhibitory activity of caffeic acid was observed. Assay-guided purification of the oxidation product of caffeic acid afforded a highly potent XO inhibitor, with an IC50 value that was calculated to be 60 nmol L(-1), which indicated XO-inhibitory activity much stronger than that of allopurinol (IC50 = 1 µmol L(-1)), a potent XO inhibitor and excellent medicine for the treatment of gout. The chemical structure of this new XO inhibitor was investigated by one- and two-dimensional NMR and HR-ESI-MS analyses, and the unique tetracyclic structure was confirmed by synthesis starting from commercially available 1,2,4-trimethoxybenzene and 3,4-dimethoxylbenzoyl chloride.

Metmyoglobin reduction by polyphenols and mechanism of the conversion of metmyoglobin to oxymyoglobin by quercetin.

The effect of antioxidant polyphenols and related phenolic compounds from plants on the reduction of metmyoglobin (MetMb) was investigated. Potent activity in the reduction of MetMb to oxymyoglobin (MbO2), a bright red protein in meat, was observed for three flavonols, kaempferol, myricetin, and quercetin, at 300 µmol/L against 60 µmol/L MetMb. Sinapic acid, catechin, nordihydroguaiaretic acid, taxifolin, morin, and ferulic acid promoted reduction at 600 µmol/L. A mechanism for the reduction by one of the active flavonols, quercetin, was proposed on the basis of analytical results for redox reaction products derived from quercetin. This suggested the importance of a high propensity toward reduction of the flavonol structure and rapid convertibility of the quinone form to the phenol form for the MbO2 reduction and the maintenance of the level of MbO2 produced.