Derivatization procedure of estradiol with a combination of MPDNP-F and 4-dimethylaminopyridine to generate product ion containing estradiol-skeleton for reliable determination of its serum/plasma concentrations by LC/ESI-MS/MS.

The quantification of serum/plasma estradiol (E2) is useful for the diagnosis, pathological analysis, and monitoring of the therapeutic efficacy of estrogen-dependent diseases. In this study, an improved derivatization method using 1-(2,4-dinitro-5-fluorophenyl)-4,4-dimethylpiperazinium iodide (MPDNP-F) was developed and combined with liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the sensitive and specific quantification of the serum/plasma E2. In the new method, the reaction time was reduced to 15 min from 90 min (two-step reaction in the previous method) by the direct reaction of MPDNP-F with E2 at 60°C in the presence of 4-dimethylaminopyridine (DMAP). DMAP served as the organic catalyst and had a less negative effect on the LC/ESI-MS/MS instrument compared to the non-volatile inorganic salt (NaHCO3), which was used in the previous method. The collision-induced dissociation of the molecular cation ([M]+) of the resulting derivative provided a product ion containing the E2-skeleton ([M-NO2-H]+), which significantly enhanced the assay sensitivity and specificity; compared to the dansyl chloride derivatization, which is the currently most-used derivatization procedure for the LC/ESI-MS/MS assays of E2, the MPDNP-F derivatization had significantly fewer interfering peaks and a clear and flat baseline in the serum sample analysis. The MPDNP-F derivatization-LC/ESI-MS/MS method enabled the precise and accurate quantification of E2 even at a 5.0 pg/mL concentration (lower limit of quantification) with a small sample volume (100 µL of serum/plasma) and had a tolerance for the matrix effect. This method was also proven to serve as a more sensitive and specific alternative to the clinically used chemiluminescence enzyme immunoassay.

Malnutrition and inflammation status in nonobese patients with inflammatory bowel disease are associated with nonalcoholic fatty liver disease: a retrospective study.

BACKGROUND/AIMS: The frequency and details of nonalcoholic fatty liver disease (NAFLD) complications in patients with inflammatory bowel disease (IBD) remain unclear. This study aimed to clarify characteristics of NAFLD in patients with IBD. METHODS: We retrospectively identified and enrolled patients with IBD diagnosed with or without NAFLD by undergoing abdominal computed tomography (CT) at our institution between 2005 and 2020. The primary endpoint was the complication rate of NAFLD in patients with IBD. Secondary endpoints were the clinical characteristics of nonobese patients with IBD and comorbid NAFLD and their association with nutritional and inflammatory parameters. RESULTS: Twenty-one (21.9%) of 96 eligible patients with IBD also had NAFLD. In nonobese patients (defined as patients with a body mass index <25 kg/m2), C-reactive protein (CRP; P<0.001) and alanine aminotransferase (P=0.018) levels were higher and the albumin level (P=0.005) and prognostic nutritional index (PNI; P=0.002) values were lower in patients with NAFLD than in those without NAFLD. The PNI value was positively correlated (P<0.001) and the CRP level was negatively correlated (P=0.001) with the hepatosplenic ratio. However, in the NAFLD combined group, PNI (P<0.05) and CRP values (P<0.001) were improved over time after CT imaging by continuing IBD treatment. CONCLUSIONS: Worsening nutritional and inflammatory status in IBD patients is associated with complications of NAFLD. Diagnosis of NAFLD in IBD patients using CT imaging might be useful not only for early detection of NAFLD but also in assessing the need for therapeutic intervention for IBD.

Effects of Undernutrition on Swallowing Function and Activities of Daily Living in Hospitalized Patients: Data from the Japanese Sarcopenic Dysphagia Database.

This retrospective cohort study examined the effects of undernutrition on swallowing function and activities of daily living in hospitalized patients. Data from the Japanese Sarcopenic Dysphagia Database were used, and hospitalized patients aged ≥20 years with dysphagia were included in the analysis. Participants were assigned to the undernutrition or normal nutritional status group based on the Global Leadership Initiative on Malnutrition criteria. The primary outcome was the Food Intake Level Scale change, and the secondary outcome was the Barthel Index change. Among 440 residents, 281 (64%) were classified under the undernutrition group. The undernutrition group had a significantly higher Food Intake Level Scale score at baseline and Food Intake Level Scale change (p = 0.001) than the normal nutritional status group. Undernutrition was independently associated with the Food Intake Level Scale change (B = -0.633, 95% confidence interval = -1.099 to -0.167) and the Barthel Index change (B = -8.414, 95% confidence interval = -13.089 to -3.739). This was defined as the period from the date of admission to the hospital until discharge or 3 months later. Overall, our findings indicate that undernutrition is associated with reduced improvement in swallowing function and the ability to perform activities of daily living.

Resolution of Laryngeal Ulceration with Prednisolone Therapy in a Patient with Crohn's Disease.

A 23-year-old man diagnosed with Crohn's disease (CD) was treated with infliximab. He developed new-onset sore throat and dysphagia during admission, and nasopharyngoscopy revealed epiglottic ulceration. Laryngeal ulceration was considered as an extraintestinal manifestation of CD owing to treatment failure with antibiotics and hydrocortisone. This strongly suggested that laryngeal ulceration was a complication of CD because of the rapid improvement in the symptoms and lesions after prednisolone administration. Furthermore, this treatment process demonstrated the superior anti-inflammatory effect of prednisolone over that of hydrocortisone and supported the assumption of inflammation related to CD.

The recombinogenic history of turnip mosaic potyvirus reveals its introduction to Japan in the 19th century.

Characterizing the detailed spatial and temporal dynamics of plant pathogens can provide valuable information for crop protection strategies. However, the epidemiological characteristics and evolutionary trajectories of pathogens can differ markedly from one country to another. The most widespread and important virus of brassica vegetables, turnip mosaic virus (TuMV), causes serious plant diseases in Japan. We collected 317 isolates of TuMV from Raphanus and Brassica plants throughout Japan over nearly five decades. Genomic sequences from these isolates were combined with published sequences. We identified a total of eighty-eight independent recombination events in Japanese TuMV genomes and found eighty-two recombination-type patterns in Japan. We assessed the evolution of TuMV through space and time using whole and partial genome sequences of both nonrecombinants and recombinants. Our results suggest that TuMV was introduced into Japan after the country emerged from its isolationist policy (1639-1854) in the Edo period and then dispersed to other parts of Japan in the 20th century. The results of our analyses reveal the complex structure of the TuMV population in Japan and emphasize the importance of identifying recombination events in the genome. Our study also provides an example of surveying the epidemiology of a virus that is highly recombinogenic.

Observation of the drainage process of the residual lipoma after endoscopic unroofing technique during colonoscopic evaluation of post-procedural hematochezia.

A 57-year-old man was referred to our hospital for further management of a subepithelial lesion noted on colonoscopy. He underwent endoscopic treatment of unroofing technique, in which the protruding portion of the tumor was partially resected. Due to a small amount of hematochezia, colonoscopy was performed to re-evaluate the lesion post-treatment. This enabled the observation of the drainage process of the residual lipoma. Remission was achieved and confirmed 8 months after the treatment. Endoscopic unroofing technique has been reported as a safe and effective method of treating lipomas, particularly large ones. To the best of our knowledge, this is the first endoscopic unroofing case in which the drainage process of the residual lipoma was observed and the remission of the lesion was confirmed.

Epstein-Barr Virus-positive Intestinal Diffuse Large B-cell Lymphoma in a Japanese Patient with Celiac Disease: First Reported Case and a Literature Review.

A 60-year-old Japanese woman was diagnosed with celiac disease (CeD) and treated with a gluten-free diet. For five years, she had a good clinical course. However, she complained of inappetence and nausea. Colonoscopy revealed ulcerative tumors in the terminal ileum. A histological examination of biopsy specimens from the ulcerative tumor showed diffuse infiltration of large atypical lymphocytes. Immunohistologically, the atypical lymphoid cells were positive for cluster of differentiation (CD) 10 and CD20. Many Epstein-Barr virus-encoded small RNA (EBER)-positive atypical lymphocytes were detected by in situ hybridization. This represents the first reported case of Epstein-Barr virus-positive intestinal diffuse large B-cell lymphoma complicated with CeD.

Branched-chain amino acid synthesis and glucosinolate-myrosinase system during takuan-zuke processing of radish root.

The traditional Japanese fermented pickle, takuan-zuke, is produced via dehydration and salt-aging of the Japanese winter radish root (Raphanus sativus L.). It has been reported that γ-aminobutyric acid (GABA; antihypertensive factor) accumulates during this production. Herein, the results of the comprehensive study (metabolites, enzyme activity, and gene expression levels were investigated) on the mechanism of branched-chain amino acid (BCAA) synthesis and glucosinolate (GLS)-myrosinase system of daikon dehydration have been reported. BCAAs, GLS, and their precursors were temporarily upregulated, and the genes linked to BCAA, GLS, and isothiocyanate synthesis (BCAT4, MAM3, IPMDH1, RMB1, RMB2, and GRS1) were upregulated during daikon dehydration. BCAAs and GLS accumulated in daikon during dehydration owing to the upregulation of genes, encoding these synthases as a stress response. These results suggest that the biological response of daikon adds characteristic flavor and health functionality to takuan-zuke and helps optimize the processing parameters to produce pickles with improved health benefits. PRACTICAL APPLICATIONS: Takuan-zuke is a popular pickle in Japan, which is produced from a Japanese winter radish root (Raphanus sativus L.), commonly referred to as daikon in Japan. Takuan-zuke is produced by dehydrating daikon either by the process of salt-pressing (shio-oshi) or sun-drying (hoshi). The processes influence the variations in the nutritional value of the final product. The results reveal that both the daikon dehydration processes can be followed to generate increased amounts of health-promoting components (e.g., branched-chain amino acid and glucosinolate) in takuan-zuke.

Rare Anal Canal Cancer with Secondary Extramammary Paget's Disease (Pagetoid Spread) Complicated by Squamous Cell Carcinoma of the Skin.

A 91-year-old man had a node and erythema in the anal area resistant to treatment. A biopsy of the node in the anus showed atypical cells developing as Paget's disease, and staining revealed that the cells were CK7-positive, CK20-positive, and GCDFP15-negative. Therefore, tumor invasion with pagetoid spread (PS) from the anus to the skin was suspected, and the patient was referred to our department for a close examination and surgical treatment. Lower gastrointestinal endoscopy showed edematous, hemorrhagic mucosa in the anal canal, and he was diagnosed with adenocarcinoma via a biopsy. Additionally, redness and swelling with white moss were observed on the skin around the anus. Biopsy showed that Paget cells were diffusely present in the epithelium, and an image of squamous cell carcinoma directly under the epithelium was obtained. Taken together, the patient was diagnosed with the invasion of anal canal cancer with PS to the skin, and we performed laparoscopic abdominoperineal resection and skin carcinoma resection in the perineum. The histopathological analysis showed adenocarcinoma invading the external anal sphincter and subcutaneous adipose tissue in the vicinity of the pectinate line of the anal canal. Pagetoid spread of the adenocarcinoma was observed in the epidermis, and the open portion was slightly invaded up to the rectal mucosa. The anal skin region of the adenocarcinoma partially continued to the hair follicles, and it was complicated by squamous cell carcinoma invading the dermis. There are a few reports of anal canal cancer with PS, and the coexistence of adenocarcinoma and squamous cell carcinoma, as seen in the present case, is rare. We report our case together with relevant literature.

Suitability of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration versus Paired Transbronchial Biopsy Specimens for Evaluating Programmed Death Ligand-1 Expression in Stage III and IV Lung Cancer: A Comparative Retrospective Study.

Objectives: Cancer cells usually escape tumor-reactive T-cell responses using immune checkpoint proteins, such as programmed death protein-1 (PD-1) and its ligand, programmed death ligand-1 (PD-L1). These proteins can be blocked by immune checkpoint inhibitors (ICIs); the decision on ICI-based first-line treatment for advanced lung cancers depends on the PD-L1 levels in tumor specimens. Determining the PD-L1 expression conventionally requires histological specimens from resected tumors and core biopsy specimens. Non-small cell lung cancer (NSCLC) is usually diagnosed at stage III or IV; therefore, only small biopsy specimens, such as those obtained via endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are available. However, the suitability of EBUS-TBNA specimens determining the PD-L1 expression levels in advanced lung cancers remains unclear. Materials and Methods: Here, we investigated the concordance rate of PD-L1 expression between EBUS-TBNA and matched transbronchial biopsy (TBB) specimens. Using the 22C3 anti-PD-L1 antibody (immunohistochemistry), we determined the PD-L1 expression levels in paired specimens obtained from 69 patients (50 with advanced NSCLC and 19 with small cell lung cancer [SCLC]), as well as the efficacy of ICIs in these patients. Results: The concordance rate of PD-L1 expression between the EBUS-TBNA and TBB specimens was 78.3%. The κ values referent to the PD-L1-positive expression rate between EBUS-TBNA and TBB specimens were 0.707 and 0.676 at cutoff limits of ≥1% and ≥50%, respectively. Among the 19 SCLC patients, 16 (84.2%) exhibited no PD-L1 expression in both EBUS-TBNA and TBB specimens. Notably, the progression-free survival of patients with ≥50% PD-L1 expression in the paired specimens who received ICI treatment was 8.3 months. Conclusion: Collectively, our results validate the use of EBUS-TBNA specimens for the determination of the PD-L1 expression levels in the context of NSCLC and SCLC.

Nivolumab-Induced Polymyalgia Rheumatica in a Patient with Lung Adenocarcinoma.

A 79-year-old woman was diagnosed with stage IV (cT1aN1M1, OSS) lung adenocarcinoma with bone metastasis of the right femur. She received nivolumab as a third-line treatment. She developed pain in the right shoulder, left wrist, right knee, and waist as well as a low-grade fever and morning stiffness, after five courses of nivolumab. After closer examination, she was diagnosed with polymyalgia rheumatica (PMR) precipitated by an immune-related adverse event. Nivolumab was discontinued, and oral prednisolone was started. Her arthralgia improved. Caution should be exercised regarding the development of PMR when polyarthralgia occurs during nivolumab treatment in patients with lung cancer.

Metabolism of glutamic acid to alanine, proline, and γ-aminobutyric acid during takuan-zuke processing of radish root.

Takuan-zuke is a traditional Japanese fermented pickle, prepared by dehydration of radish root (daikon) by salt-pressing or sun-drying followed by aging with salt. We previously reported that alanine, proline, and γ-aminobutyric acid (GABA) accumulate during daikon dehydration, whereas the level of glutamic acid, their precursor, decreases. We have also reported that dehydration and salt-aging markedly influence the dynamics of free amino acids. In this study, we quantitatively analyzed free amino acid levels, enzyme activity, and gene expression to characterize takuan-zuke amino acid metabolism. Enzyme activities related to alanine, proline, GABA, and glutamic acid metabolism were sustained during dehydration. Moreover, genes encoding alanine, proline, and GABA synthases (ALT1, P5CS1, and GAD4) were significantly upregulated during dehydration. These effects may represent cellular stress responses to the dehydration process. The biological response of daikon contributes to the healthy functional aspects that characterize takuan-zuke. These findings could guide the selection of suitable vegetable varieties to produce pickled vegetables with health-promoting properties. PRACTICAL APPLICATION: The fermented pickle takuan-zuke, prepared by dehydration of radish root (daikon), accumulates amino acids, such as alanine, proline, and GABA, during preparation that provide taste and health benefits. In this study, the aforementioned amino acids were found to accumulate because of the stress response of daikon during the dehydration process and not because of the action of microorganisms during fermentation. Takuan-zuke processing is a method for improving the nutrition of daikon.

Intussusception caused by a small intestinal lipoma with ectopic gastric mucosa containing gastric cystica profunda component cells within the inverted Meckel's diverticulum: a case report.

BACKGROUND: Lipomas are the most common cause of intussusception in adults. To our knowledge, however, no cases of lipoma and ectopic gastric mucosa with gastritis cystica profunda (GCP) have been reported. We report a case of intussusception caused by a small intestinal lipoma with ectopic gastric mucosa containing GCP-component cells within the inverted Meckel's diverticulum. CASE PRESENTATION: A female in her 40s underwent computed tomography for postoperative follow-up of left breast cancer. A tumor, suspected to be a lipoma, was found in the ileum. Since there were no symptoms, the patient underwent regular follow-up. However, gradual enlargement was observed, and surgery was recommended due to the risk of intussusception. After reduction via the Hutchinson technique, laparoscopically assisted partial resection of the small intestine was performed due to suspicion that the tumor was causing intussusception starting from the ileum. Histopathologic examinations revealed proliferation of mature adipose tissue in the subserosal layer, which was diagnosed as lipoma. Furthermore, adipose tissue was found in the stem area and accordingly, we diagnosed lipoma associated with the inverted Meckel's diverticulum. Moreover, gastric mucosa-like crypt epithelium and proper glandular tissue were identified in the mucosal membrane at the area of onset, and signs of gastric pit dilatation over the submucosa and crypt epithelium hyperplasia were observed. Diagnosis was ectopic gastric mucosa containing GCP component tissue. CONCLUSIONS: Intussusception in the small intestine complicated with lipoma and ectopic gastric mucosa with GCP within the Meckel's diverticulum has not been reported, demonstrating the rarity of our case.

Low pH Enhances the Glucosinolate-Mediated Yellowing of Takuan-zuke under Low Salt Conditions.

This study was performed to clarify the enhancement of the 4-methylthio-3-butenyl isothiocyanate induced yellowing of salted radish root (takuan-zuke) by low pH during short-term salt-aging at low temperature and low salinity. We used two different methods to prepare the dehydrated daikon prior to salt-aging: air-drying outdoors (hoshi takuan-zuke) or salting with a stone press (shio-oshi takuan-zuke). Low salt-aging at low temperature was carried out under pH control with citrate-phosphate buffer. The yellowing of both types of takuan-zuke was accelerated below pH 5, and the color of air-dried takuan-zuke was deeper than that of salt-pressed takuan-zuke. To elucidate this phenomenon, several previously reported yellowing-related compounds were analyzed by high-performance liquid chromatography. The result showed that the production of the primary pigment, 2-[3-(2-thioxopyrrolidin-3-ylidene)methyl]-tryptophan, was low compared with that in previous reports. Therefore, we suggest that an unknown pigment was generated through a previously unreported pathway.

Successful Treatment of Advanced Thymic Carcinoma with Carboplatin plus nab-Paclitaxel and Maintenance Monotherapy with nab-Paclitaxel: Two Case Reports.

A standard chemotherapy regimen for advanced thymic carcinoma has not yet been established. We treated 2 cases of thymic carcinoma with carboplatin plus nanoparticle albumin-bound (nab)-paclitaxel, and nab-paclitaxel maintenance therapy. The first case was a 68-year-old female, admitted for dyspnea and left shoulder pain. Chest computed tomography (CT) showed a huge mass in the anterior mediastinum, pleural and pericardial effusions, and multiple lung metastases. Specimens obtained from the anterior mediastinal mass by CT-guided needle biopsy revealed squamous cell carcinoma of the thymus, which was in stage IVB. The patient was administered carboplatin plus nab-paclitaxel as first-line treatment. After 3 cycles of chemotherapy, a partial response was observed with marked shrinkage of the tumor. Following 6 cycles of chemotherapy, nab-paclitaxel maintenance therapy was initiated. Disease progression was seen 9.1 months after initiation of treatment. The patient experienced no serious adverse events. The second case was a 70-year-old male who had productive cough, dyspnea, and right-sided chest pain. Chest CT revealed a huge mass in the anterior mediastinum, pericardial effusion, and multiple lymphadenopathies. Specimens obtained from station 11s by endobronchial ultrasound-guided transbronchial needle aspiration revealed undifferentiated thymic carcinoma, which was in stage IVB. Six cycles of carboplatin plus nab-paclitaxel were administered, followed by 5 cycles of nab-paclitaxel for maintenance. A partial response was seen, which was sustained for more than 13 months. The patient experienced no serious adverse events. These cases show that chemotherapy with carboplatin plus nab-paclitaxel and nab-paclitaxel as maintenance therapy can be a useful regimen for advanced thymic carcinoma.

Potato virus Y; the Andean connection.

Potato virus Y (PVY) causes disease in potatoes and other solanaceous crops. The appearance of its necrogenic strains in the 1980s made it the most economically important virus of potatoes. We report the isolation and genomic sequences of 32 Peruvian isolates of PVY which, together with 428 published PVY genomic sequences, gave an alignment of 460 sequences. Of these 190 (41%) were non-recombinant, and 162 of these provided a dated phylogeny, that corresponds well with the likely history of PVY, and show that PVY originated in South America which is where potatoes were first domesticated. The most basal divergences of the PVY population produced the N and C: O phylogroups; the origin of the N phylogroup is clearly Andean, but that of the O and C phylogroups is unknown, although they may have been first to establish in European crops. The current PVY population originated around 156 CE. PVY was probably first taken from South America to Europe in the 16th century in tubers. Most of the present PVY diversity emerged in the second half of the 19th century, after the Phytophthora infestans epidemics of the mid-19th century destroyed the European crop and stimulated potato breeding. Imported breeding lines were shared, and there was no quarantine. The early O population was joined later by N phylogroup isolates and their recombinants generated the R1 and R2 populations of damaging necrogenic strains. Our dating study has confirmed that human activity has dominated the phylodynamics of PVY for the last two millennia.

The prevalence and clinical features of asthma-COPD overlap (ACO) definitively diagnosed according to the Japanese Respiratory Society Guidelines for the Management of ACO 2018.

Background Asthma-COPD overlap (ACO) is a disease that shares clinical features of both asthma and COPD. The purpose of this study is to investigate the prevalence and clinical features of ACO. Methods We retrospectively reviewed data for 170 patients with persistent airflow limitation and diagnosed them according to "The Japanese Respiratory Society Guidelines for the Management of ACO 2018". Results Of the 170 patients, 111 were diagnosed as follows : COPD (74 patients, 66.6%), ACO (34 patients, 30.6%), and asthma (3 patients, 2.8%). There was no significant difference in clinical features between ACO and COPD patients. The following pulmonary function tests were significantly lower in ACO than in COPD patients : forced expiratory volume in 1 second/forced vital capacity, peak expiratory flow, maximal mid-expiratory flow, and the maximum expiratory flow at 50%and75%. The following respiratory impedance parameters were significantly higher in ACO than in COPD patients : respiratory resistance (Rrs) at 5 Hz (R5), Rrsat 20 Hz (R20), R5-R20, and low-frequency reactance area. Conclusions About 30% of patients with persistent airflow limitation were diagnosed with ACO. ACO patients had lower lung function and higher respiratory impedance compared with COPD patients. J. Med. Invest. 66 : 157-164, February, 2019.

Hydrogen gas improves left ventricular hypertrophy in Dahl rat of salt-sensitive hypertension.

PURPOSE: Hypertension is an important risk factor for death resulting from stroke, myocardial infarction, and end-stage renal failure. Hydrogen (H2) gas protects against many diseases, including ischemia-reperfusion injury and stroke. The effects of H2 on hypertension and its related left ventricular (LV) function have not been fully elucidated. The purpose of this study was to investigate the effects of H2 gas on hypertension and LV hypertrophy using echocardiography. METHODS: Dahl salt-sensitive (DS) rats were randomly divided into three groups: those fed an 8% NaCl diet until 12 weeks of age (8% NaCl group), those additionally treated with 2% H2 gas (8% NaCl + 2% H2 group), and control rats maintained on a diet containing 0.3% NaCl until 12 weeks of age (0.3% NaCl group). H2 gas was supplied through a gas flowmeter and delivered by room air (2% hydrogenated room air, flow rate of 10 L/min) into a cage surrounded by an acrylic chamber. We evaluated interventricular septal wall thickness (IVST), LV posterior wall thickness (LVPWT), and LV mass using echocardiography. RESULTS: IVST, LVPWT, and LV mass were significantly higher in the 8% NaCl group than the 0.3% NaCl group at 12 weeks of age, whereas they were significantly lower in the 8% NaCl + 2% H2 group than the 8% NaCl group. There was no significant difference in systolic blood pressure between the two groups. CONCLUSION: Our findings suggest that chronic H2 gas inhalation may help prevent LV hypertrophy in hypertensive DS rats.

Protein components of water extracts from fruiting bodies of the reishi mushroom Ganoderma lucidum contribute to the production of functional molecules.

BACKGROUND: Mushrooms have been widely considered as health foods as their extracts have anti-hypertensive and anti-tumor activities. After a thorough literature survey, we hypothesized that enzymes in mushroom extracts play an important role in synthesizing functional molecules. Therefore, in this study, proteins extracted from reishi mushroom (Ganoderma lucidum), which is used in oriental medicine, were identified by the proteomic approach, and appropriate extraction methods for improving angiotensin-converting enzyme (ACE) inhibitory activities were investigated. RESULTS: Various glycoside hydrolases (GHs), such as ß-N-acetylhexosaminidase (GH family 20), α-1,2-mannosidase (GH family 47), endo-ß-1,3-glucanase (GH family 128), and ß-1,3-glucanase (GH152), that degrade glycans in the fruiting body were identified. The residual glucanase activities generated ß-oligosaccharides. Additionally, the glutamic acid protease of the peptidase G1 family was determined as the major protein in the extract, and the residual peptidase activity of the extracts was found to improve ACE inhibitory activities. Finally, it was observed that extraction at 50 °C is suitable for yielding functional molecules with high ACE inhibitory activities. CONCLUSION: Water extraction is generally believed to extract only functional macromolecules that exist in mushroom fruiting bodies. This study proposed a new concept that describes how functional molecules are produced by enzymes, including proteases and GHs, during extraction. © 2018 Society of Chemical Industry.