Total Synthesis, Structure Revision, and Neuroprotective Effect of Hericenones C-H and Their Derivatives.

The first total syntheses of hericenones C-H and "putative 3-hydroxyhericenone F" were achieved. Highlights of the synthesis include the straightforward construction of the resorcinol core and geranyl side chain, assembly of the natural product skeleton by sequential O-geranylation and a clay/zeolite-mediated O → C rearrangement reaction, and a biomimetic cyclization to form a variety of bicyclic natural hericenones and their congeners. The structure of the "putative 3-hydroxyhericenone F" was revised as the 5-exo cyclization product (named: hericenone Z) of epoxyhericenone C through in-depth analyses of the cyclization modes in addition to NMR spectroscopic studies. To gain insights into the biological functions of geranyl-resorcinols in Hericium erinaceus, potential neuroprotective effects against endoplasmic reticulum (ER) stress-dependent cell death were evaluated systematically to clarify a fundamental structure-activity relationship. Among the compounds assayed, the linoleate-containing hericenone analogue, i.e., the regioisomer of hericene D, was found to possess the most potent neuroprotective effect against tunicamycin and thapsigargin-induced ER stress-dependent cell death.

Endoplasmic reticulum stress suppressive compounds from the edible mushroom Mycoleptodonoides aitchisonii.

Two novel compounds, 1 and 7, along with six known compounds (2-6 and 8), were isolated from the edible mushroom Mycoleptodonoides aitchisonii (bunaharitake in Japanese). The structures of the new compounds were determined by the interpretation of spectroscopic data. Compounds 1-4 and 6-8 showed protective activity against endoplasmic reticulum stress-dependent cell death.

Biotransformation and detoxification of tetrabromobisphenol A by white-rot fungus Phanerochaete sordida YK-624.

The bulky phenolic compound tetrabromobisphenol A (TBBPA) is a brominated flame retardant used in a wide range of products; however, it diffuses into the environment, and has been reported to have toxic effects. Although it is well-known that white-rot fungi degrade TBBPA through ligninolytic enzymes, no other metabolic enzymes have yet been identified, and the toxicity of the reaction products and their risks have not yet been examined. We found that the white-rot fungus Phanerochaete sordida YK-624 converted TBBPA to TBBPA-O-ß-D-glucopyranoside when grown under non-ligninolytic-enzyme-producing conditions. The metabolite showed less cytotoxicity and mitochondrial toxicity than TBBPA in neuroblastoma cells. From molecular biological and genetic engineering experiments, two P. sordida glycosyltransferases (PsGT1c and PsGT1e) that catalyze the glycosylation of TBBPA were newly identified; these enzymes showed dramatically different glycosylation activities for TBBPA and bisphenol A. The results of computational analyses indicated that the difference in substrate specificity is likely due to differences in the structure of the substrate-binding pocket. It appears that P. sordida YK-624 takes up TBBPA, and reduces its cytotoxicity via these glycosyltransferases.

Association between laughter, frailty, and depression in rheumatoid arthritis patients.

OBJECTIVE: This study aimed to determine whether there are associations between laughter, disease activity, frailty, and depression in rheumatoid arthritis (RA) patients. METHODS: A total of 240 patients were included in this prospective cohort study on frailty in RA patients between March 2021 and June 2022. Patients were divided into the following four groups according to the frequency of laughter: "almost every day," "1-5 days per week," "1-3 days per month," and "never or almost never." Patient characteristics were compared among the four groups by analysis of variance. Factors associated with laughter were identified by multivariable logistic analysis. RESULTS: The mean 28-joint Disease Activity Score using CRP was 1.91, with 70.7% of patients in remission and 12.6% in low disease activity. For the "almost every day" (42.5% of patients), "1-5 days per week" (40.0%), "1-3 days per month" (11.3%), and "never or almost never" (6.3%) groups, scores of the Kihon Checklist (KCL) for assessing frailty status were 3.5, 4.6, 7.3, and 8.1 (p < .001), respectively, and scores of the Beck Depression Inventory (BDI-II) were 8.4, 10.7, 15.1, and 16.5 (p < .001), respectively. Multivariable analysis revealed that KCL (OR: 0.81, 95% CI: 0.73-0.90) and BDI-II (OR: 0.91, 95% CI: 0.86-0.95) scores were independently associated with the frequency of laughter. CONCLUSION: Frailty and depression were associated with laughter in RA patients with controlled disease activity. Interventions aimed at not only disease activity control but also frailty prevention may lead to a life filled with laughter.

Makomotindoline from Makomotake, Zizania latifolia infected with Ustilago esculenta.

Makomotindoline (1) was isolated from Makomotake, Zizania latifolia infected with Ustilago esculenta. The structure was determined by the interpretation of spectroscopic data and synthesis. Makomotindoline (1), its l-Glc isomer (2) and its aglycon (3) were synthesized and their effects on rat glioma cells showed adverse effects on the cell growth.

Functional-food constituents in the fruiting bodies of Stropharia rugosoannulata.

Nine compounds (1-9) were isolated from the fruiting bodies of Stropharia rugosoannulata. Compounds 1-5, 8, and 9 suppressed the formation of osteoclast. Compounds 2 and 5 showed anti-fungal activity, and their MIC were 250 µM and 500 µM respectively. Compounds 2-6 showed inhibitory effects on thapsigargin toxicity.

Novel etiological and therapeutic strategies for neurodiseases: epigenetic understanding of gene-environment interactions.

Epigenetics is a mechanism that regulates gene expression not depending on the underlying DNA sequence, but on the chemical modifications of DNA and histone proteins. Defects in the factors involved in epigenetic regulation cause congenital neurodevelopmental diseases, and thus, epigenetic regulation is essential for normal brain development. Besides these intrinsic defects, it is becoming increasingly apparent that extrinsic factors, such as insufficient nutrition, psychiatric drugs, and mental stress, also alter epigenetic regulation. Therefore, environmental factors may lead to "acquired" neurodevelopmental disorders through the failure of epigenetic regulation. Epigenetics is a biological key to understand the gene-environment interactions in neurodevelopmental disorders. As the mechanism is reversible, its comprehensive understanding will result in the development of new therapies for these disorders.

Inhibition of alpha-mannosidase attenuates endoplasmic reticulum stress-induced neuronal cell death.

N-glycosylation is crucial for proper folding of most of the proteins in the endoplasmic reticulum (ER). The N-glycans in the ER are mainly constructed of mannose. In this study, we examined whether inhibition of mannose trimming in the ER affects the susceptibility of PC-12 cells to ER stress. Pretreatment with 100 microM alpha-mannosidase inhibitor 1-deoxymannojirimycin (DMJ) in PC-12 cells significantly attenuated the cytotoxicity by ER stressors tunicamycin (TM), thapsigargin (TG), and amyloid beta1-42 (Abeta1-42), and reduced caspase-3 activation by TM and TG. Pretreatment with DMJ also protected primary cultured mouse cortical neurons from Abeta1-42 toxicity. With regard to the effect of DMJ pretreatment on ER stress signaling in PC-12 cells, DMJ attenuated TM- and TG-induced CHOP expression and TG stimulated JNK phosphorylation, which is associated with ER stress dependent cell death. Next, we examined the effect of mannose oligosaccharides, which have similar structures to N-glycans in the ER, on amyloidogenesis of Abeta1-42 that causes ER stress dependent neuronal cell death. Mannopentaose (M5) and Man9GlcNAc2 (M9) oligosaccharides significantly inhibited the amyloidogenesis of Abeta1-42. Our data suggests that inhibition of N-glycan processing in the ER attenuates ER stress-induced cell death by increasing high-mannose type oligosaccharides that reduce protein aggregation, such as amyloidogenesis.

Endoplasmic reticulum (ER) stress-suppressive compounds from scrap cultivation beds of the mushroom Hericium erinaceum.

Four compounds were isolated from scrap cultivation beds of the mushroom, Hericium erinaceum. Compounds 1-4 were identified as methyl 4-hydroxy-3-(3-methylbutanoyl) benzoate, 2-chloro-1,3-dimethoxy-5-methylbenzene, methyl 4-chloro-3,5-dimethoxybenzoate, and 4-chloro-3,5-dimethoxybenzaldehyde by an interpretation of the NMR and MS data, respectively. This is the first reported isolation of 1 from a natural source. All the compounds showed protective activity against endoplasmic reticulum stress-dependent cell death.

Influence of methotrexate on gastrointestinal symptoms in patients with rheumatoid arthritis.

AIM: This study aimed to determine the influence of methotrexate (MTX) on gastrointestinal (GI) symptoms in patients with rheumatoid arthritis (RA). METHODS: This cross-sectional study examined 529 consecutive patients with RA receiving oral MTX in our department between April 1 and September 30, 2017. GI symptoms were evaluated by the Gastrointestinal Symptom Rating Scale (GSRS); a score of ≥2 was considered "symptomatic." Prevalence of GI symptoms was compared between patients receiving ≤8 mg/wk (low-dose) vs >8 mg/wk (high-dose) of MTX. RESULTS: Of our study population, 313 (59%) received low-dose MTX at a median (interquartile range) dose of 6 (6-8) mg/wk, whereas 216 (41%) received high-dose MTX at a median dose of 12 (10-12) mg/wk. Relative to the low-dose MTX group, the high-dose MTX group exhibited a higher prevalence of reflux (32% vs 24%, P = 0.043) and abdominal pain (28% vs 18%, P = 0.007). There was no significant group-dependent difference in the prevalence of indigestion, diarrhea or constipation. Multivariate logistic regression analysis revealed that high-dose MTX (>8 mg/wk) was independently associated with reflux (odds ratio [OR]: 1.62, 95% confidence interval [CI]: 1.07-2.43) and abdominal pain (OR: 1.60, 95% CI: 1.04-2.43), and that the ORs for reflux and abdominal pain among those receiving high-dose MTX (>8 mg/wk) were similar to those using nonsteroidal anti-inflammatory drugs. CONCLUSION: High-dose MTX is independently associated with the prevalence of upper GI symptoms in Japanese patients with RA.

An endoplasmic reticulum (ER) stress-suppressive compound and its analogues from the mushroom Hericium erinaceum.

Three new compounds, 3-hydroxyhericenone F (1), hericenone I (2), and hericenone J (3), were isolated from the mushroom Hericium erinaceum. The structures of 1-3 were determined by the interpretation of spectral data. Compound 1 showed the protective activity against endoplasmic reticulum (ER) stress-dependent Neuro2a cell death, however, compounds 2 and 3 did not.

Augmentation of polysialic acid by valproic acid in early postnatal mouse hippocampus and primary cultured hippocampal neurons.

We examined the effects of valproic acid (VPA) on hippocampal neurons. Prenatal VPA exposure significantly increased polysialic acid (PSA) expression in the early postnatal mouse hippocampus. Moreover, VPA treatment significantly enhanced PSA expression in primary cultured hippocampal neurons and stimulated neurite growth. Our results suggest that VPA exposure in ovo affects hippocampal development.

Phosphorylation of methyl-CpG binding protein 2 (MeCP2) regulates the intracellular localization during neuronal cell differentiation.

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional repressor which recognizes methylated CpG dinucleotides. Mutations in the MeCP2 gene is known to cause human autistic disease Rett syndrome, but its molecular mechanisms remain to be elucidated. Since MeCP2 is a DNA-binding protein, it has been believed that MeCP2 functions only in the nucleus. We herein show that MeCP2 is localized in the cytosol as well as in the nucleus of neuronal cells. Through the use of immunofluorescence and Western blot analyses, MeCP2 was found to be localized both in the nucleus and cytosol of rat PC-12 and mouse Neuro2a cells before neuronal differentiation, and it was translocated into the nucleus during differentiation. In primary cultured neurons from mouse cortex, MeCP2 was expressed in whole cell bodies on the first day of culture while after 7 days of culture, MeCP2 was localized mainly in the nucleus. Furthermore, MeCP2 was re-localized in the nucleus and cytosol after 14 days of culture. To study the molecular mechanisms of translocation, we analyzed the post-translational modification of MeCP2. The cytosolic MeCP2 was Ser/Thr-phosphorylated, while the nuclear MeCP2 was not. Both the cytosolic and nuclear MeCP2 were SUMOylated, which has been reported to be a nuclear transport signal. Our data suggests that the nuclear translocation of neuronal MeCP2 was induced during differentiation and/or maturation, and that Ser/Thr-phosphorylation regulates its translocation.

Bioremediation of the neonicotinoid insecticide clothianidin by the white-rot fungus Phanerochaete sordida.

Clothianidin (CLO) is a member of the neonicotinoid pesticides, which have been widely used worldwide over the last two decades. However, its toxicity for bees and neurological toxicity for humans are urgent problems. Here, the degradation of CLO by the white-rot fungus Phanerochaete sordida was examined in nitrogen-limited liquid medium. After incubation for 20days at 30°C, 37% of CLO was degraded in the cultures. High-resolution ESI-MS and NMR analyses of the culture supernatant identified N-(2-chlorothiazol-5-yl-methyl)-N'-methylurea (TZMU) as a metabolite of CLO degradation. The addition of cytochrome P450 inhibitors to the culture medium markedly reduced the degradation of CLO by P. sordida. And manganese peroxidase, a major ligninolytic enzyme secreted by this fungus, were not carried out CLO degradation. The effects of CLO and TZMU on the viability of the neuronal cell line Neuro2a demonstrated that P. sordida effectively degrades CLO into a metabolite that lacks neurotoxicity.

Cordyceps militaris improves the survival of Dahl salt-sensitive hypertensive rats possibly via influences of mitochondria and autophagy functions.

The genus Cordyceps and its specific ingredient, cordycepin, have attracted much attention for multiple health benefits and expectations for lifespan extension. We analyzed whether Cordyceps militaris (CM), which contains large amounts of cordycepin, can extend the survival of Dahl salt-sensitive rats, whose survival was reduced to ∼3 months via a high-salt diet. The survival of these life-shortened rats was extended significantly when supplemented with CM, possibly due to a minimization of the effects of stroke. Next, we analyzed the effect of CM on hypertension-sensitive organs, the central nervous systems (CNS), heart, kidney and liver of these rats. We attempted to ascertain how the organs were improved by CM, and we paid particular attention to mitochondria and autophagy functions. The following results were from CM-treated rats in comparison with control rats. Microscopically, CNS neurons, cardiomyocytes, glomerular podocytes, renal epithelial cells, and hepatocytes all were improved. However, immunoblot and immunohistochemical analysis showed that the expressions of mitochondria-related proteins, ATP synthase ß subunit, SIRT3 and SOD2, and autophagy-related proteins, LC3-II/LC3-I ratio and cathepsin D all were reduced significantly in the CNS neurons, but increased significantly in the cells of the other three organs, although p62 was decreased in its expression in all the organs tested. Activity of Akt and mTOR was enhanced but that of AMPK was reduced in the CNS, while such kinase activity was completely the opposite in the other organs. Together, the influence of CM may differ between mitochondria and autophagy functioned between the two organ groups, as mitochondria and autophagy seemed to be repressed and promoted, respectively, in the CNS, while both mitochondria and autophagy were activated in the others. This could possibly be related to the steady or improved cellular activity in both the organs, which might result in the life extension of these rats.

Secreted protein-based reporter systems for monitoring inflammatory events: critical interference by endoplasmic reticulum stress.

A number of recent reports have used secreted protein-based reporter assays for monitoring intercellular and intracellular events involved in inflammation. However, we found that these assay systems are critically affected by endoplasmic reticulum (ER) stress. When reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of NF-kappaB were exposed to IL-1beta or TNF-alpha, induction of SEAP activity was markedly reduced under ER stress conditions. Downregulation of SEAP activity was observed regardless of cell types and type of regulatory elements; e.g., when reporter hepatocytes that express SEAP under the control of the dioxin responsive elements were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene, ER stress similarly suppressed the increase in SEAP activity despite its transcriptional upregulation. Activity of constitutively expressed SEAP in various cells was also reduced by ER stress in a magnitude-dependent manner, and it was associated with disturbed subcellular transport of SEAP to the Golgi. Furthermore, interference by ER stress was similarly observed in other reporter assay using secreted luciferase. These results evidenced critical interference by ER stress in secreted protein-based reporter systems. The suppression of reporter responses by ER stress should be considered carefully for experimental design and interpretation of data when secreted protein-based reporter systems are used for investigation.

Dilinoleoyl-phosphatidylethanolamine from Hericium erinaceum protects against ER stress-dependent Neuro2a cell death via protein kinase C pathway.

In many types of neurodegeneration, neuronal cell death is induced by endoplasmic reticulum (ER) stress. Hence, natural products able to reduce ER stress are candidates for use in the attenuation of neuronal cell death and, hence, in the reduction of the damage, which occurs in neurodegenerative disease. In this study, we investigated ER stress-reducing natural products from an edible mushroom, Hericium erinaceum. As a result of screening by cell viability assay on the protein glycosylation inhibitor tunicamycin-induced (i.e., ER stress-dependent) cell death, we found that dilinoleoyl-phosphatidylethanolamine (DLPE) was one of the molecules effective at reducing ER stress-dependent cell death in the mouse neuroblastoma cell line Neuro2a cells. A purified DLPE, commercially available, also exhibited a reducing effect on this ER stress-dependent cell death. Therefore, we concluded that DLPE has potential as a protective molecule in ER stress-induced cell death. From the structure of DLPE, it was hypothesized that it might activate protein kinase C (PKC). The activity of PKC-epsilon, a novel-type PKC, was increased by adding DLPE, and PKC-gamma, a conventional-type PKC, was activated on the coaddition of diolein and DLPE, as shown by in vitro enzyme activity analysis. The protecting activity of DLPE was attenuated in the presence of a PKC inhibitor GF109203X but not completely diminished. Therefore, DLPE can protect neuronal cells from ER stress-induced cell death, at least in part by the PKC pathway.

A transcriptional repressor MeCP2 causing Rett syndrome is expressed in embryonic non-neuronal cells and controls their growth.

An epigenetic key protein MeCP2 is thought to be expressed exclusively in mature neurons. Here, we show that MeCP2 is expressed in embryonic non-neuronal cells by immunocytochemistry and Western blotting, and that knockdown of MeCP2 levels using RNA interference reduces their proliferation. These findings suggest that MeCP2 is essential to non-neuronal cell growth during brain development, which may be associated with microcephaly of Rett syndrome patients with MeCP2 mutations.