• Title/Summary/Keyword: methylation analysis

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Prognostic Significance of $O^6$-MGMT and Promotor Hypermethylation in Patients with Soft Tissue Sarcomas (연부조직육종 환자에서 $O^6$-MGMT 와 촉진자 과메틸화의 예후적 중요성)

  • Suh, Jeung-Tak;Kim, Jeung-Il;Oh, Jong-Seok;Choi, Kyung-Un
    • The Journal of the Korean bone and joint tumor society
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    • v.15 no.1
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    • pp.13-25
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    • 2009
  • Purpose: The DNA repair protein, $O^6$-methylguanine-DNA methyltransferase (MGMT), removes alkyl adducts from the $O^6$ position of guanine. Epigenetic inactivation of MGMT has been found in human neoplasia and considered one of the implicated factors in chemoresistance. Materials and Methods: Sixty-two patiensts with soft tissue sarcomas (STS) were analyzed for the status of MGMT protein expression by immunohistochemistry and the promoter hypermethylation of the MGMT gene using methylation-specific PCR. Result: The loss of MGMT expression was found in 20 cases (32.3%) of total 62 STS. MGMT promoter hypermethylation rate was 25.0% (11/44 cases). The loss of MGMT expression showed significant association with high AJCC stage, high FNCLCC grade, and aggressive behavior. However,when the group who received chemotherapy was analyzed (n=27), loss of MGMT expression was correlated with worse survival in multivariate analysis (p=0.024). MGMT promoter hypermethylation is associated with high FNCLCC grade. MGMT promoter hypermethylation status had a strong correlation with loss of MGMT expression (p=0.000). Conclusion: Our results suggest that MGMT promoter hypermethylation and loss of MGMT expression had a tendency to be associated with poor prognosis and that loss of MGMT protein expression is frequently occurs via MGMT promoter hypermethylation.

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Characterization of the Immunologically Active Components of Glycyrrhiza uralensis Prepared as Herbal Kimchi

  • Hwang, Jong-Hyun;Lee, Kyong-Haeng;Yu, Kwang-Won
    • Preventive Nutrition and Food Science
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    • v.8 no.1
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    • pp.29-35
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    • 2003
  • A crude polysaccharide fraction (GU-3) from the roots of Glycyrrhiza uralensis (licorice root), a screened herbal plant used in the preparation of herbal kimchi, enhanced Peyer's patch mediated bone marrow cell proliferation and NK cell-mediated tumor cytotoxicity against Yac-1 cells. GU-3 was further purified by DEAE-Sepharose CL-6B yielding fractions designated as GU-3I, and 3IIa∼3IIe. GU-3IIa is mainly composed of arabinose, galactose and galacturonic acid, and showed the highest bone marrow cell proliferation activity. In addition, GU-3IIb had arabinose, galactose, rhamnose and galacturonic acid as the component sugars with a small quantity of protein; GU-3IIb also enhanced activity of NK cell-mediated tumor cytotoxicity. After these fractions were further fractionated via gel filtration on Sepharose CL-6B or Sephacryl S-300, two immunological active polysaccharides, GU-3IIa-2 and 3IIb-1 were purified from the respective fractions. GU-3IIa-2 mostly contained neutral sugars (75%) such as arabinose and galactose (molar ratio; 1.0 : 0.7) in addition to a considerable amount of galacturonic acid (20%), whereas GU-3IIb-1 was composed of arabinose, galactose, rhamnose and galacturonic acid (molar ratio; 0.3 : 0.5 : 0.1 : 1.0). Methylation analysis indicated that GU-3IIa-2 was composed mainly of terminal, 4- or 5-linked and 3,4- or 3,5-branched arabinose, 3-linked, 4-linked and 3,6-branched galactose, and terminal and 4-linked galacturonic acid whereas GU-3IIb-1 contained various glycosidic linkages such as terminal and 4- or 5-linked arabinose, 2,4-branched rhamnose, terminal and 4-linked galactose, and terminal and 4-galacturonic arid. Single radial gel diffusion indicated that only GU-3IIa-2 strongly reacted with β-D-glucosyl-Yariv antigen. These results suggest that bone marrow cell proliferating activity and enhancement of NK cell-mediated tumor cytotoxicity of GU-3 are caused by polysaccharides containing a pectic arabinogalactan (GU-3IIa-2) and pectic polysaccharide (GU-3IIb-1).

Analysis of Binding Media in Dancheong Sample from Unhangak Hall of Hwaryeongjeon Shrine, Suwon (수원 화령전 운한각 단청시편의 교착제 분석)

  • Park, Jongseo;Lee, Sun Myung;Mun, Seong Woo;Kang, Yeong Seok
    • Journal of Conservation Science
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    • v.37 no.3
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    • pp.245-254
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    • 2021
  • In this study, organic materials in Dancheong specimens were analyzed to establish a Dancheong preparation method in Unhangak Hall of Hwaryeongjeon Shrine, Suwon. IR and pyrolysis/GC/MS were applied to a Zuhong specimen and a gilded one. The IR spectra showed absorption peaks corresponding to C-O, C=O, OH, although they were different from those of animal glue, Asian lacquer, and drying oil. The Pyrolysis/GC/MS after the on-line methylation revealed Asian lacquer-derived components, such as methyl 7-(2,3-dimethoxyphenyl)heptanoate, and drying oil-derived components, such as nonanedioic acid, dimethyl ester. Based on these results, we estimated that Asian lacquer and drying oil were used in the Dancheong preparation. Small amounts of 3-methyl-1H-pyrrole were also detected, which could possibly originate from animal glue. The radiocarbon age of the specimens appeared to be between the late 17th to the early 20th century. This study potentially reports the first case of Asian lacquer detection in a Dancheong specimen in Korea.

Structural Analysis of the Unusual Sugar-Containing Oligosaccharides Formed by the Selective Cleavage of Weakly Acidic Polysaccharide (약산성 다당의 선택적 분해 과정에서 얻어진 특이당 함유 Oligo당의 구조적 분석)

  • Shin, Kwang-Soon;Lee, Ho
    • Korean Journal of Food Science and Technology
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    • v.29 no.6
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    • pp.1105-1112
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    • 1997
  • By sequential degradation using partial acid hydrolysis of a weakly acidic polysaccharide (GL-4IIb2'), two acidic oligosaccharide fragments, PA-2' and PA-1-III were isolated and their structures were characterized. PA-2' consisted of almost equal proportion of a rhamnose (Rha) and an unusual sugar, 3-deoxy-D-manno-2-octurosonic acid (Kdo). When permethylated oligosaccharide-alditol derived from PA-2' was analyzed by GC-MS, the peak gave the fragment ions at m/z 189 $(bA_1,\;6-deoxyhexose)$ and at m/z 308 $(aJ_2,\;alditol\;from\;Kdo)$. The peak also gave the characteristic ion at m/z 162 but it did not give the fragment ion at m/z 177, suggesting that Kdo is substituted at C5 but not at C4. Methylation analysis also indicated that PA-2' was composed mainly of terminal Rhap and 5-substituted Kdo. When the reduced product from PA-2' was analyzed by $^1H-NMR$, it gave a signal at 5.09 ppm due to an anomeric proton of ${\alpha}-L-Rha$. These results indicated that PA-2' mainly contained ${\alpha}-L-Rhap-(1{\rightarrow}5)-Kdo$. On the other hand, PA-1-III mainly comprised Rha and Kdo in addition to small proportions of arabinose (Ara) and 3-deoxy-D-lyxo-2-heptulosaric acid (Dha). MS analysis of permethylated oligosaccharide-alditols from PA-1-III suggested that the major peak 1P was $Rhap-(1{\rightarrow}5)-Kdo$ whereas the minor peaks 2P and 3P possessed $Araf-(1{\rightarrow}5)-Dha$ unit and these peaks were produced as epimers during reduction of carbonyl groups in Dha.

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Studies on the Chemical Structure of the New Polysaccharide C - (The New Polysaccharides of Gum Tragacanth. II) - (Tragacanth gum 의 신다당류(新多糖類) C 의 화학구조(化學構造) - Tragacanth gum의 신다당류(新多糖類)에 관(關)한 연구(硏究) 제2보(第二報) -)

  • Lee, Sung-Hwan
    • Applied Biological Chemistry
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    • v.3
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    • pp.25-48
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    • 1962
  • The polysaccharide C prepared from gum tragacanth powder (U. S. P. grade) by the precipitation method with 85% ethanol was a neutral polysaccharide, $[{\alpha}]^{30}_D-72.2$. The polysaccharide C consisted of L-rhamnose, D-xylose, L-arabinose and D-galactose in the molar ratio 2:1:17:9 (Table 1, 2, 3, ). The polysaccharide C was methylated with dimethylsulphate and 40% NaOH, and Purdies regent. The hydrolyzate of fully methlated product ($[{\alpha}]^{22}_D-102$ in chloroform, the methoxy content 40.6%) was composed of 2, 3, 5-tri-O-methyl-L-arabofuranose (I), 3,4-di-O-methyl-L-rhamnopyranose (II), 2,3-di-O-methyl-D-xylose (III), 2,3,4-tri-O-methyl-D-galactopyranose (IV), 2,4-di-O-methyl-L-arabopyranose (?), 2,4-di-O-methyl-D-galactose(VI), 2-O-methyl-D-arabinose (VII), and L-arabopyranose(VIII) (Table 4, 5, and Fig. 4). The first partial hydrolysis (A) of the polysaccharide C with 0.05N-HCl for 4.5 hours at $80-85^{\circ}C$ released only L-arabinose: the second hydrolysis (B) with 0.1N-HCl for 5 hours at $80-85^{\circ}C$, L-arabinose and D-galactose; and the third hydrolysis (C) with 0.3N-HCl at $90-95^{\circ}C$ in sealed tube, L-rhamnose, D-xylose, L-arabinose and D-galactose. From the unhydrolyzate A' were found L-rhamnose, D-xylose, L-arabinose, and D-galactose; from B' L-rhamnose, d-xylose, L-arabinose and D-galactose; and from C' D-xylose and D-galactose respectively (Table 6). The periodate consumption and formic acid production of the polysaccharide C were measured at various time intervals. After 120 hours periodat was consumed by 1.23 mole per $C_5H_8O_4$ and formic acid was produced 0.78 mole per $C_5H_8O_4$ (Table 7). Although a definite chemical structure for this polysaccharide C may not be formulated, experimental data, especially, from methylation, partial hydrolysie and determination of its molar ratio, and periodate analysis showed that the polysaccharide C is a highly branched polysaccharide and would be constructed of galactoaraban as a main chain residue and L-arabofuranose, D-galactopyranosyl $(1{\rightarrow}1)$-L-arabofuranose, D-xylopyranosyl $(1{\rightarrow}2)$-L-rhamnopyranosyl $(1{\rightarrow}1)$-L-arabofuranose, and L-rhamnopyranosyl $(1{\rightarrow}1)$-arabofuranose, and D-galactopyranosyl-$(1{\rightarrow}2)$-L-arabopyranosyl-$(1{\rightarrow}1)$-I-arabofuranose as a branch chain or end group (page 21).

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Structural Characterization of the Anti-Complementary and Macrophage Activating Polysaccharides Isolated from Agaricus bisporus (양송이에서 분리한 보체계와 대식세포 활성화 다당류의 구조적 특성)

  • Kim, Byung-Hee;Kweon, Mee-Hyang;Lim, Wang-Jin;Sung, Ha-Chin;Yang, Han-Chul
    • Korean Journal of Food Science and Technology
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    • v.30 no.3
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    • pp.709-716
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    • 1998
  • Three kinds of anti-complementary system and macrophage activating polysaccharides, AB-20-Ia, AB-20-IIa-2a and AB-20-IVa-2 were isolated from the fruit body of Agaricus bisporus and their structures were characterized. The proteoglycan, AB-20-IVa-2 showing the most potent anti-complementary and macrophage activity was composed of glucose, galactose, mannose, xylose, fucose and arabinose in a molar ratio of 3.48:1.83:1.00:0.79:0.74:0.11 and its main component amino acids were phenylalanine (34.72%) and valine (27.84%). The neutral polysaccharides, AB-20-Ia and AB-20-IIa-2a showing lower activity than AB-20-IVa-2, consisted of xylose, glucose, mannose, fucose and arabinose in molar ratios of <0.05:<0.05:2.07:1.00:2.72 and 2.16:1.58:1.00:0.20:0.14, respectively. The molecular weights of AB-20-Ia, AB-20-IIa-2a and AB-20-IVa-2 were 840,000, 750,000 and 650,000 respectively. In the $^1H-\;and\;^{13}C-NMR$ spectra of AB-20-Ia and AB-20-IIa-2a, AB-20-Ia showed only ${\beta}-configuration\;(^1H:\;4.8\;ppm,\;^{13}C:\;107.0\;ppm)$ in the anomerization of the glycosidic linkages, while AB-20-IIa-2a had both ${\alpha}-anomer\;(^1H:\;5.4\;ppm,\;^{13}C:\;102.0\;ppm)\;and\;{\beta}-anomer$. Especially, AB-20-Ia and AB-20-IIa-2a showed acetyl signals $(^1H:\;2.5\;ppm,\;^{13}C:\;21.0\;ppm)$. In the methylation analysis of the three polysaccharides, high proportion of 1,6-linked glucofuranosyl residues were detected in AB-20-Ia, whereas 1,6-linked glucopyranosyl residues and branches linked at position 4 of those mainly contained in AB-20-IIa-2a. AB-20-IVa-2 consisted mainly of 1,2-linked xylofuranosyl residues and 1,6-linked glucopyranosyl residues and branches linked at position 3 of those.

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Analysis of 5-aza-2'-deoxycytidine-induced Gene Expression in Lung Cancer Cell Lines (폐암 세포주에서 5-aza-2'-deoxycytidine 처치에 의해 발현되는 암항원 유전자 분석)

  • 김창수;이해영;김종인;장희경;박종욱;조성래
    • Journal of Chest Surgery
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    • v.37 no.12
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    • pp.967-977
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    • 2004
  • Background: DNA methylation is one of the important gene expression mechanisms of the cell. When cytosine of CpG dinucleotide in promotor is hypomethylated, expression of some genes that is controlled by this promoter is altered. In this study, the author investigated the effect of DNA demethylating agent, 5-aza-2'-deoxycytidine (ADC), on the expressions of cancer antigen genes, MHC and B7 in 4 lung cancer cell lines, NCIH1703, NCIH522, MRC-5, and A549. Material and Method: After treatment of cell lines, NCIH1703, NCIH522, MRC-5 and A549 with ADC (1 uM) for 48 hours, RT-PCR was performed by using the primers of MAGE, GAGE, NY-ESO-1, PSMA, CEA, and SCC antigen gene. In order to find the optimal ADC treatment condition for induction of cancer antigen, we studied the effect of ADC treatment time and dose on the cancer antigen gene expression. To know the effect of ADC on the expression of MHC or B7 and cell growth, cells were treated with 1 uM of ADC for 72 hours for FACS analysis or cells were treated with 0.2, 1 or 5 uM of ADC for 96 hours for cell counting. Result: After treatment of ADC (1 uM) for 48 hours, the expressions of MAGE, GAGE, NY-ESO-1, and PSMA genes increased in some cell lines. Among 6 MAGE isotypes tested, and gene expression of MAGE-1, -2, -3, -4 and -6 could be induced by ADC treatment. However, CEA gene expression did not change and SCC gene expression was decreased by ADC treatment. Gene expression was generally induced 24 - 28 hours after ADC treatment and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC ADC teatment, and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC teatment in ADC-Free medium. Most gene expression could be induced at 0.2 uM of ADC, but gene expression increased dependently on ADC treatment dose. The expression of MHC and B7 was not increased by ADC treatment in all four cell lines, and the growth rate of 4 cell lines decreased significantly with the increase of ADC concentrations. Conclusion: Treatment of lung cancer cell lines with ADC increases the gene expression MAGE, GAGE and NY-ESO-1 that are capable of induction of cytotoxic T lymphocyte response. We suggest that treatment with 1 uM of ADC for 48 hours and then culturing in ADC-free medium is optimal condition for induction of cancer antigen. However, ADC has no effect on MHC and B7 induction, additional modification for increase of expression of MHC, B7 and cytokine will be needed for production of efficient cancer cell vaccine.

Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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