Contributions of the N-terminal flanking residues of an antigenic peptide from the Japanese cedar pollen allergen Cry j 1 to the T-cell activation by HLA-DP5.

Cry j 1 is a major allergen present in Japanese cedar (Cryptomeria japonica) pollens. Peptides with the core sequence of KVTVAFNQF from Cry j 1 ('pCj1') bind to HLA-DP5 and activate Th2 cells. In this study, we noticed that Ser and Lys at positions -2 and -3, respectively, in the N-terminal flanking (NF) region to pCj1 are conserved well in HLA-DP5-binding allergen peptides. A competitive binding assay showed that the double mutation of Ser(-2) and Lys(-3) to Glu [S(P-2)E/K(P-3)E] in a 13-residue Cry j 1 peptide (NF-pCj1) decreased its affinity for HLA-DP5 by about 2-fold. Similarly, this double mutation reduced, by about 2-fold, the amount of NF-pCj1 presented on the surface of mouse antigen-presenting dendritic cell line 1 (mDC1) cells stably expressing HLA-DP5. We established NF-pCj1-specific and HLA-DP5-restricted CD4+ T-cell clones from HLA-DP5 positive cedar pollinosis (CP) patients, and analyzed their IL-2 production due to the activation of mouse TG40 cells expressing the cloned T-cell receptor by the NF-pCj1-presenting mDC1 cells. The T-cell activation was actually decreased by the S(P-2)E/K(P-3)E mutation, corresponding to the reduction in the peptide presentation by this mutation. In contrast, the affinity of NF-pCj1·HLA-DP5 for the T-cell receptor was not affected by the S(P-2)E/K(P-3)E mutation, as analyzed by surface plasmon resonance. Considering the positional and side-chain differences of these NF residues from previously reported T-cell activating sequences, the mechanisms of enhanced T-cell activation by Ser(-2) and Lys(-3) of NF-pCj1 may be novel.

Tolerogenic properties of CD206+ macrophages appeared in the sublingual mucosa after repeated antigen-painting.

The sublingual mucosa (SLM) in the oral cavity is utilized as the site for sublingual immunotherapy to induce tolerance against allergens. We previously reported that CD206+ round-type macrophage-like cells were induced in the SLM after repeated antigen (e.g. cedar pollen or fluorescein isothiocyanate (FITC))-painting. In this study, we examined the phenotypic and functional properties of CD206+ cells induced by repeated FITC-painting on the SLM. CD206+ cells after the repeated FITC-painting possessed a macrophage-like CD11b+Ly6C+ F4/80+CD64+ phenotype and expressed TIM-4, which was expressed in tolerogenic tissue-resident macrophages, at a high level. SLM CD206+ cells preferentially expressed molecules related to endocytosis and homeostatic processes, including the novel B7 family of immune checkpoint molecules, as assessed by microarray analyses. SLM CD206+ cells showed preferential expression of M2-related genes such as Fizz1, Aldh1a1 and Aldh1a2 but not Ym-1 and Arginase-1. A CD206+ cell-rich status inhibited OVA-specific CD4+ T-cell responses but reciprocally enhanced the proportion of both IL-10+CD4+ cells and Foxp3+ regulatory T-cells in regional lymph nodes. Co-culture of CD206+ cells with dendritic cells (DCs) showed that IL-12 production was suppressed in DCs concurrent with the decline of the MHC class IIhiCD86+ population, which was restored by neutralization of IL-10. These results demonstrate SLM CD206+ cells show the feature of tolerogenic macrophages and down-regulate the antigen-presenting cell function of mature DCs resulting in the inhibition of CD4+ T-cell responses.

Artificially lignified cell wall catalyzed by peroxidase selectively localized on a network of microfibrils from cultured cells.

MAIN CONCLUSION: An artificial lignified cell wall was synthesized in three steps: (1) isolation of microfibrillar network; (2) localization of peroxidase through immunoreaction; and (3) polymerization of DHP to lignify the cell wall. Artificial woody cell wall synthesis was performed following the three steps along with the actual formation in nature using cellulose microfibrils extracted from callus derived from Cryptomeria japonica. First, we constructed a polysaccharide network on a transmission electron microscopy (TEM) grid. The preparation method was optimized by chemical treatment, followed by mechanical fibrillation to create a microfibrillated network. Morphology was examined by TEM, and chemical characterization was by Fourier transform infrared (FTIR) spectroscopy. Second, we optimized the process to place peroxidase on the microfibrils via an immunoreaction technique. Using a xyloglucan antibody, we could ensure that gold particles attached to the secondary antibodies were widely and uniformly localized along with the microfibril network. Third, we applied the peroxidase attached to secondary antibodies and started to polymerize the lignin on the grid by simultaneously adding coniferyl alcohol and hydrogen peroxide. After 30 min of artificial lignification, TEM observation showed that lignin-like substances were deposited on the polysaccharide network. In addition, FTIR spectra revealed that the bands specific for lignin had increased, demonstrating the successful artificial formation of woody cell walls. This approach may be useful for studying woody cell wall formation and for producing made-to-order biomaterials.

Bioactive Dimeric Abietanoid Peroxides from the Bark of Cryptomeria japonica.

Three new dimeric abietane-type diterpenoids, abieta-6,8,11,13-tetraen-12-yl 12-hydroxyabieta-8,11,13-trien-7α-yl peroxide (1), abieta-6,8,11,13-tetraen-12-yl 12-hydroxyabieta-8,11,13-trien-7ß-yl peroxide (2), and 12-hydroxyabieta-8,11,13-trien-7ß-yl 7-oxoabieta-5,8,11,13-tetraen-12-yl peroxide (3), together with four known abietane-type diterpenoids (4-7) were isolated from the methanol extract of the bark of Cryptomeria japonica. Their structures were elucidated on the basis of spectroscopic analysis and comparison of NMR data with those of known analogues. At a concentration of 50 µM, compounds 1, 2, and 3 showed 26.2%, 23.6%, and 35.7% inhibition towards xanthine oxidase enzyme, respectively. In addition, compound 3 also showed 24.9% inhibition toward angiotensin-converting enzyme (ACE).

A Pair of Enantiomeric Bis- seco-abietane Diterpenoids from Cryptomeria fortunei.

(±)-Cryptomeriolide, a pair of racemic bis- seco-abietane diterpenoids, were isolated from the bark of Cryptomeria fortunei. The separation of enantiomers was achieved by using chiral stationary phase HPLC. Their structures including the absolute configuration and conformations in solution and solid state were determined by extensive analysis of spectroscopic data, single-crystal X-ray diffraction, and comparison of calculated and experimental electronic circular dichroism data. A bioinspired one-pot enantiomeric synthesis of 1a and 1b was accomplished via a readily made intermediate orthoquinone from sugiol. All compounds including the synthetic intermediates were assayed for their cytotoxic activities on human cancer cell lines HL-60, A549, and SGC7901.

Gender differences in the psychophysiological effects induced by VOCs emitted from Japanese cedar (Cryptomeria japonica).

BACKGROUND: Wood is a valuable material for interiors, and the psychophysiological relaxation effects of volatile organic compounds (VOCs) from wood chips and essential oils have been reported. However, few studies have identified the odors in full-scale wooden environment, and also, differences in gender have not been clarified. In this study, we aimed to confirm the effects of VOCs emitted from interior wood walls in both human male and female participants. METHODS: We used Japanese cedar timber and analyzed VOCs in the experimental rooms with and without Japanese cedar timber by gas chromatography-mass spectrometry (GC-MS). The physiological effects were measured using neuroendocrinological and immunological parameters in saliva. A questionnaire was used to evaluate the subjective responses to each odor in the experimental rooms. RESULTS: The main compound emitted from Japanese cedar timber was δ-cadinene, and the total volume of VOCs in the wood condition (presence of VOCs emitted from Japanese cedar) was 282.4 (µg/m3). Significant differences between genders in salivary parameters were shown that there were decreases of α-amylase in wood condition and increases of cortisol in the control (absence of VOCs) condition in female participants compared to male participants. The results demonstrated that VOCs in the experimental room with Japanese cedar timber tend to suppress the activation of the sympathetic nervous activity and non-VOCs of Japanese cedar in the control room increase cortisol in female participants. CONCLUSIONS: These results suggest that an indoor environment with wood interior materials has the potential to be useful for health management, especially women's health.

Effect of epicuticular wax crystals on the localization of artificially deposited sub-micron carbon-based aerosols on needles of Cryptomeria japonica.

Elucidation of the mechanism of adsorption of particles suspended in the gas-phase (aerosol) to the outer surfaces of leaves provides useful information for understanding the mechanisms of the effect of aerosol particles on the growth and physiological functions of trees. In the present study, we examined the localization of artificially deposited sub-micron-sized carbon-based particles on the surfaces of needles of Cryptomeria japonica, a typical Japanese coniferous tree species, by field-emission scanning electron microscopy. The clusters (aggregates) of carbon-based particles were deposited on the needle surface regions where epicuticular wax crystals were sparsely distributed. By contrast, no clusters of the particles were found on the needle surface regions with dense distribution of epicuticular wax crystals. Number of clusters of carbon-based particles per unit area showed statistically significant differences between regions with sparse epicuticular wax crystals and those with dense epicuticular wax crystals. These results suggest that epicuticular wax crystals affect distribution of carbon-based particles on needles. Therefore, densely distributed epicuticular wax crystals might prevent the deposition of sub-micron-sized carbon-based particles on the surfaces of needles of Cryptomeria japonica to retain the function of stomata.

Transcutaneous Peptide Immunotherapy of Japanese Cedar Pollinosis Using Solid-in-Oil Nanodispersion Technology.

Peptide immunotherapy is an attractive approach to relieve allergic symptoms such as rhinitis and asthma. Treatment of Japanese cedar pollinosis (Cryptomeria japonica; Cj), from which over one quarter of Japanese population suffer, is becoming a great concern. Recently, oral feeding of a peptide (7crp) consisting of seven immunodominant human T cell epitopes derived from two enzymes present in Cj pollen was demonstrated to have a benefit in treating Cj pollinosis. In this work, we aimed to apply a novel transcutaneous administration system as a simple and easy peptide delivery for an immunotherapy using a T cell epitope peptide. A modified 7crp peptide (7crpR) which contained triarginine linkers between each epitopes was designed to increase water solubility and was encapsulated in a unique solid-in-oil (S/O) nanodispersion. The S/O nanodispersion consists of a nano-sized peptide-surfactant complex dispersed in an oil vehicle. The S/O nanopartilces having an average diameter of 230 nm facilitated the permeation of the peptide 7crpR into the skin and suppressed serum total IgE and antigen-specific IgE levels in a Cj pollinosis mouse model. Transcutaneous administration of the T cell epitope peptide using the S/O nanodispersion system has potential for future simple and easy immunotherapy of Cj pollinosis.

Characterization of sulfur deposition over the period of industrialization in Japan using sulfur isotope ratio in Japanese cedar tree rings taken from stumps.

We characterized the sulfur deposition history over the period of industrialization in Japan based on the sulfur isotope ratio (δ(34)S) in tree rings of Japanese cedar (Cryptomeria japonica D. Don) stumps. We analyzed and compared δ(34)S values in the rings from two types of disk samples from 170-year-old stumps that had been cut 5 years earlier (older forest stand) and from 40-year-old living trees (younger forest stand) in order to confirm the validity of using stump disks for δ(34)S analysis. No differences in δ(34)S values by age were found between the sample types, indicating that stump disks can be used for δ(34)S analysis. The δ(34)S profile in tree rings was significantly correlated with anthropogenic SO2 emissions in Japan (r = -0.76, p < 0.05) and, thus, tree rings serve as a record of anthropogenic sulfur emissions. In addition, the values did not change largely from pre-industrialization to the 1940s (+4.2 to +6.1‰). The values before the 1940s are expected to reflect the background sulfur conditions in Japan and, thus, disks containing rings formed before the 1940s contain information about the natural environmental sulfur, which is useful for biogeochemical studies.

Anti-SARS-CoV-2 activity of microwave solvolysis lignin from woody biomass.

The global pandemic caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had profoundly detrimental effects on our society. To combat this highly pathogenic virus, we turned our attention to an abundant renewable natural aromatic polymer found in wood. Through a chemical modification of Eucalyptus and Japanese cedar wood via acidic microwave solvolysis in equivolume mixture of 2 % (w/w) aqueous H2SO4, ethylene glycol, and toluene at 190 °C. Subsequently, we separated the resulting solvolysis products through extractions with toluene, ethyl acetate, and ethanol. Among these products, the ethyl acetate extract from Eucalyptus wood (eEAE) demonstrated the highest inhibition effects against the novel SARS-CoV-2. We further divided eEAE into four fractions, and a hexane extract from the ethanol-soluble portion, termed eEAE3, exhibited the most substantial inhibitory rate at 93.0 % when tested at a concentration of 0.5 mg/mL. Analyzing eEAE3 using pyrolysis gas chromatography-mass spectrometry revealed that its primary components are derived from lignin. Additionally, 1H-13C edited-heteronuclear single quantum coherence nuclear magnetic resonance analysis showed that the solvolysis process cleaved major lignin interunit linkages. Considering the abundance and renewability of lignin, the lignin-derived anti-SARS-CoV-2 agent presents a promising potential for application in suppressing infections within our everyday environment.

Oral immunotherapy with transgenic rice seed containing destructed Japanese cedar pollen allergens, Cry j 1 and Cry j 2, against Japanese cedar pollinosis.

Transgenic rice accumulating the modified major Japanese cedar pollen allergens, Cryptomeria japonica 1 (Cry j 1) and Cryptomeria japonica 2 (Cry j 2), which were deconstructed by fragmentation and shuffling, respectively, in the edible part of the seed was generated by transformation of a good-tasting rice variety, 'Koshihikari'. These modified cedar pollen antigens were deposited in ER-derived protein bodies (PB-I), which are suitable for delivery to the mucosal immune system in gut-associated lymphoid tissue when orally administered because antigens bioencapsulated in PB-I are resistant against hydrolysis by intestinal enzymes and harsh environments. Mice fed transgenic seeds daily for three weeks and then challenged with crude cedar pollen allergen showed marked suppression of allergen-specific CD4(+) T-cell proliferation, IgE and IgG levels compared with mice fed nontransgenic rice seeds. As clinical symptoms of pollinosis, sneezing frequency and infiltration of inflammatory cells such as eosinophils and neutrophils were also significantly reduced in the nasal tissue. These results imply that oral administration of transgenic rice seeds containing the structurally disrupted Cry j 1 and Cry j 2 antigens, serving as universal antigens, is a promising approach for specific immunoprophylaxis against Japanese cedar pollinosis.

Bioethanol production from Lignocellulosic biomass by a novel Kluyveromyces marxianus strain.

The yeast Kluyveromyces marxianus is considered as a potential alternative to Saccharomyces cerevisiae in producing ethanol as a biofuel. In this study, we investigated the ethanol fermentation properties of novel K. marxianus strain DMB1, isolated from bagasse hydrolysates. This strain utilized sorbitol as well as various pentoses and hexoses as single carbon sources under aerobic conditions and produced ethanol from glucose in hydrolysates of the Japanese cedar at 42 °C. Reference strains K. marxianus NBRC1777 and S. cerevisiae BY4743 did not assimilate sorbitol or ferment lignocellulosic hydrolysates to ethanol at this temperature. Thus strain DMB1 appears to be optimal for producing bioethanol at high temperatures, and might provide a valuable means of increasing the efficiency of ethanol fermentation.

Whitening and antioxidant activities of bornyl acetate and nezukol fractionated from Cryptomeria japonica essential oil.

OBJECTIVES: The aim of this study was to investigate the whitening and antioxidant activities of essential oils from Cryptomeria japonica by determining their tyrosinase inhibition, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and superoxide dismutase (SOD)-like activities. METHODS: Essential oils of C. japonica leaves were extracted with distilled water, and after condensation of volatile constituents, the condensates were extracted with ethyl acetate. Crude essential oils of C. japonica were divided into six fractions by thin layer chromatography and open column chromatography, and their chemical analysis was performed by GC/MS. Major compounds of fractions were composed of kaurene, bornyl acetate, nezukol, (-)-4-terpineol, δ-cadinene, α-terpineol, γ-eudesmol, α-eudesmol and elemol. RESULTS: For tyrosinase inhibitory activity using two substrates, l-tyrosine and 3,4-dihydroxyphenylalanine (l-DOPA), kaurene, bornyl acetate and nezukol were highly effective. In antioxidant activity, (-)-4-terpinenol and δ-cadinene showed high DPPH radical scavenging activity, and bornyl acetate and nezukol indicated extremely high SOD-like activity. CONCLUSION: Therefore, bornyl acetate and nezukol fractionated from C. japonica essential oil, which showed highly active whitening and antioxidant activities, have potential applications in cosmeceutical materials.

Biological activities of organic extracts and specialized metabolites from different parts of Cryptomeria japonica (Cupressaceae) - A critical review.

Forest operations and wood industry generate large amounts of residues that are discarded in the field and cause environmental pollution. However, these biomass residues are still raw materials to obtain value-added products, such as essential oils, organic/aqueous extracts and resins that are among the great natural sources of bioactive metabolites. Thus, in recent years, the scientific community is giving special attention to their valorization. To date, different uses of biomass residues have been proposed, such as a source of renewable energy, fertilizers, animal feed and bioactive molecules. In this context, Cryptomeria japonica biomass residues (e.g., bark and its exudate, heartwood, sapwood, leaves, cones and roots) represent a source of diverse specialized metabolites (e.g., sesqui-, di-, tri- and sesquarterpenes, flavonoids, lignans and norlignans) with potential application in different fields, particularly in the agrochemical, food, cosmeceutical, pharmaceutical, phytomedicine and esthetic, due to their valuable multi-bioactivities determined over the last decades. Thus, this review provides an overview of the reported biological activities of organic extracts/fractions and their specialized metabolites obtained from different parts of C. japonica, in order to encourage the alternative uses of C. japonica wastes/byproducts, and implement a sustainable and circular bioeconomy.

Changes in the quality of chromophoric dissolved organic matter leached from senescent leaf litter during the early decomposition.

Chromophoric dissolved organic matter (CDOM) leached from leaf litter is a major source of humus in mineral soil of forest ecosystems. While their functions and refractoriness depend on the physicochemical structure, there is little information on the quality of CDOM, especially for that leached in the very early stages of litter decomposition when a large amount of dissolved organic matter (DOM) is leached. This study aimed to better understand the variations/changes in the composition of CDOM leached from senescent leaf litter from two tree species during the early stage of decomposition. Leaf litter from a conifer tree (Japanese cedar, D. Don) and a deciduous broad-leaved tree (Konara oak, Thunb.) were incubated in columns using simulated rainfall events periodically for a total of 300 d at 20°C. The quality of CDOM was investigated based on the fluorescence properties by using a combination of excitation-emission matrix fluorescence (EEM) and parallel factor analysis (PARAFAC). In addition, the phenolic composition of DOM was investigated at a molecular level by thermally assisted hydrolysis and methylation-gas chromatography-mass spectrometry (THM-GC-MS) in the presence of tetramethylammonium hydroxide (TMAH). The EEM was statistically decomposed into eight fluorescence components (two tannin/peptide-like peaks, one protein-like peak, and five humic-like peaks). A significant contribution of tannin/peptide-like peaks was observed at the beginning of incubation, but these peaks decreased quickly and humic-like peaks increased within 1 mo of incubation. The composition of humic-like peaks was different between tree species and changed over the incubation period. Since tannin-derived phenolic compounds were detected in the DOM collected after 254 d of incubation on THM-GC-MS, it was suggested that tannins partially changed its structure, forming various humic-like peaks during the early decomposition.

Effect of Ganoderma lucidum on pollen-induced biphasic nasal blockage in a guinea pig model of allergic rhinitis.

Ganoderma lucidum (GL), an oriental medical mushroom, has been used in Asia for the prevention and treatment of a variety of diseases. However, the effect of GL on allergic rhinitis has not been well defined. The current study describes the inhibitory effect of GL on the biphasic nasal blockage and nasal hyperresponsiveness induced by repeated antigen challenge in a guinea pig model of allergic rhinitis. Intranasally sensitized guinea pigs were repeatedly challenged by inhalation of Japanese cedar pollen once every week. Ganoderma lucidum was orally administered once daily for 8 weeks from the time before the first challenge. The treatment with GL dose-dependently inhibited the early and late phase nasal blockage at the fifth to ninth antigen challenges. Furthermore, nasal hyperresponsiveness to intranasally applied leukotriene D4 on 2 days after the eighth antigen challenge was also inhibited by the treatment with GL. However, Cry j 1-specific IgE antibody production was not affected by the treatment. In conclusion, we demonstrated that the pollen-induced biphasic nasal blockage and nasal hyperresponsiveness were suppressed by the daily treatment with GL in the guinea pig model of allergic rhinitis. These results suggest that GL may be a useful therapeutic drug for treating patients with allergic rhinitis.

Chemical composition and antitermitic activity against Coptotermes formosanus Shiraki of Cryptomeria japonica leaf essential oil.

Cryptomeria japonica D. Don, called 'sugi' in Japanese, is an abundant and renewable potential resource of valuable natural products that may serve as natural biocides in Taiwan. Hydrodistillation (HD) and steam distillation (SD) were used for extracting the essential oils from C. japonica leaves in this study. The chemical constituents of the two leaf essential oils were identified by GC/MS analysis, and their antitermitic activities were evaluated. The results from the antitermitic tests against Coptotermes formosanus Shiraki showed that the leaf essential oils extracted by HD and SD had excellent antitermitic activities, with LD(50) values of 1.57 and 1.72 mg/g after 7 d of testing, respectively. Comparison of the chemical composition of the two leaf essential oils revealed that oxygenated diterpenes (kaur-16-ene) and oxygenated sesquiterpenes (ß-elemol) were the dominant constituents. In addition, 13 constituents isolated from the C. japonica leaf essential oils were tested individually against C. formosanus. Among these constituents, ß-elemol and α-terpineol achieved 100% termite mortality at the dosage of 1 mg/g after 7 d of testing. These results demonstrated that both C. japonica leaf essential oils as well as ß-elemol and α-terpineol possessed commendable antitermitic activity.

Essential oil from Cryptomeria japonica induces apoptosis in human oral epidermoid carcinoma cells via mitochondrial stress and activation of caspases.

Cryptomeria japonica D. Don (C. japonica) has been used in traditional medicines from Asia for a variety of indications, including liver ailments, and an antitussive, and for its antiulcer activities. We examined the cell viability and apoptosis of KB cells treated with C. japonica essential oil at several concentrations for 12 h by MTT assay, Hoechst-33258 dye staining, DNA fragmentation, flow cytometry (cell cycle), and Western blotting for mitochondria stress, activation of caspases, and poly (ADP-ribose) polymerase. The essential oil induced the apoptosis of KB cells in a dose-dependent manner, which was verified by DNA fragmentation, appearance of apoptotic bodies, and the sub-G1 ratio. The essential oil also induced rapid and transient caspase-3 activity and cleavage of PARP of the KB cells. Treating the cells with the oil also caused changes in the mitochondrial level of the Bcl-2 family proteins such as Bcl-2 and Bax, thereby inducing the release of cytochrome c into the cytosol. The essential oil of C. japonica may have potential as a cancer chemopreventive and therapeutic agent.

Light-scattering analysis of native wood holocelluloses totally dissolved in LiCl-DMI solutions: high probability of branched structures in inherent cellulose.

Three holocelluloses (i.e., cellulose and hemicellulose fractions) are prepared from softwood and hardwood by the Wise method. These holocelluloses completely dissolve in 8% lithium chloride/1,3-dimethyl-2-imidazolidinone (LiCl/DMI) after an ethylenediamine (EDA) pretreatment. After diluting the holocellulose solutions to 1% LiCl/DMI, they are subjected to size-exclusion chromatography/multiangle laser-light scattering/photodiode array (SEC-MALLS-PDA) analysis. All holocelluloses exhibit bimodal molecular weight distributions primarily due to high-molecular-weight (HMW) cellulose and low-molecular-weight hemicellulose fractions. Plots of molecular weight vs root-mean-square radius obtained by SEC-MALLS analysis revealed that all the wood celluloses comprise dense conformations in 1% LiCl/DMI. In contrast, bacterial cellulose, which was used as a pure cellulose model, has a random coil conformation as a linear polymer. These results show that both softwood and hardwood HMW celluloses contain branched structures, which are probably present on crystalline cellulose microfibril surfaces. These results are consistent with those obtained by permethylation analysis of wood celluloses.