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Kodama N, Yamada M, Nanba H. Jpn J Pharmacol 2001;87:327-332. Maitake D-fraction, beta1,6-glucan having beta1,3-branches, has been reported to activate the immune system of the host. To elucidate whether the [...]

We investigated a situation of passive smoking and its damaging effects among high school students. Urine cotinine concentration was measured and quantified. Additionally, we evaluated the awareness of passive smoking and smoking regulations in high school students, and the educational effect on passive smoking using a questionnaire survey and educational videos produced by high school students. We conducted a self-reporting questionnaire survey with high school students before and after watching the video produced by the high school students. We gathered the scores of the Kano Social Nicotine Dependence Questionnaire (KTSND) and awareness of smoking restrictions. Consent was obtained through the questionnaire before watching the video and collecting urine samples. Urine cotinine concentrations from 54 samples were evaluated and indicated within the low value. The KTSND score significantly decreased for those who responded to both questionnaires, after watching the video. Furthermore, analysis of the KTSND questionnaire items showed a significant decrease in scores for lifestyle, stress, and smoking location. This suggests that the video produced in this study has a certain amount of educational effect on passive smoking and that the student-led educational method is effective. The survey using the KTSND revealed that there were some students who were not exposed to passive smoking, but instead had high smoking tolerance. Going forward, it will be necessary to promote education on passive smoking and smoking prevention by incorporating the video lecture and urine cotinine concentration was measured, as in this study, to encourage behavior that decreases passive smoking among high school students.

Purpose: Japan has been known as a super-aged society, and ageing is a well-known risk factor for blinding eye diseases. However, epidemiological studies in ophthalmology are still scarce in Japan, and the sizes of the cohorts are relatively small. "Ganka-Ekigaku Network" (GEN, an acronym for the epidemiological network in ophthalmology in Japanese) is established to develop a capacity to boost each epidemiological study and enrich a potential inter-study collaboration to identify risk factors of visual impairment in aged society., Methods: We reviewed cohort studies in Japan with the inclusion criteria as: (1) at least n = 1000 at baseline, (2) multiple modalities of ophthalmic data, and (3) diagnosis reviewed by ophthalmologist(s), and (4) ophthalmologists are involved in the investigators group. As of January 2020, GEN includes 4 individual Japanese epidemiological studies namely, Hisayama study, Yamagata Study (Funagata), Tsuruoka Metabolomics Cohort study, and the Nagahama Prospective Genome Cohort for Comprehensive Human Bioscience., Results: GEN includes approximately 25,000 Japanese participants. The baseline surveys started from 1998 to 2012, and since then the data has been prospectively collected approximately every 5 years. A variety of ophthalmic measurements and other factors have been collected in each study in GEN: ophthalmic measurements (fundus photography, optical coherence tomography, etc.), systemic conditions (laboratory data, etc.), and others (DNA, etc.)., Conclusion: GEN is an open platform for observational ophthalmic epidemiological studies to share standardized methodologies. While each study in GEN pursues specific and original research questions, standardization of the methods will enable us to conduct reliable meta-analysis/pooled data analyses.

A 47-year-old man with ulcerative colitis was transferred to our hospital due to progressive dyspnea. Electrocardiography on admission showed ST elevation in leads II, III, aVF, and V5-V6. Coronary angiography revealed no remarkable coronary stenosis, and left ventriculography showed a depressed left ventricular ejection fraction (EF) of 23%. Although the patient received percutaneous cardiopulmonary support, his EF progressively decreased (7-15%), and both ventricular tachycardia (VT) and high-degree atrial-ventricular block occurred. An endomyocardial biopsy showed eosinophilic infiltration in the myocardium. Steroid therapy improved the patient's EF. However, his severe inferior wall hypokinesis and non-sustained VT remained after the abovementioned treatment.

Recurrent corneal erosion (RCE) is a common disorder causing ocular pain, tearing, photophobia, and visual impairments. Various factors such as ocular trauma, ocular surgery, corneal dystrophy, contact lens wear, and diabetes mellitus (DM) can cause RCE. The purpose of this study was to determine the causative factors and clinical course of RCE.We retrospectively examined 21 eyes of 21 patients with RCE and investigated the patients' background, type of treatments, and clinical course after the treatments. All patients were treated with eye drops, ocular lubrication, or contact lens bandage for the RCE.Among the 21 patients with RCE, 9 were caused by trauma (Trauma group), 8 by DM (DM group), 1 by bacterial corneal ulcer, 1 by lagophthalmus and bacterial corneal ulcer, 1 by bandkeratopathy, and 1 by eyelid tumor (one eye). The mean age of the patients was 57.8 years with a range 34-91 years. The mean duration from the trauma to the onset of RCE was 5.2 ± 5.0 months (mean ± SD). The time required for a complete recovery of RCE was longer in the DM group (10.3 ± 3.1 weeks) than in the Trauma group (2.7 ± 1.1 weeks, P < .01). The presence of DM was significantly associated with the recovery duration of RCE (r = 0.72; P < .01). Multivariate analyses showed that the recovery duration of RCE was associated with the presence of DM (odds ratio = 139.8, P = .04). On the other hand, the type of treatments had no effect on the recovery duration of RCE.These findings suggest that trauma and DM are important causes of RCE. Wound recovery after RCE may be delayed in patients with DM.

Coenzyme Q (CoQ) is composed of a benzoquinone moiety and an isoprenoid side chain of varying lengths. The length of the side chain is controlled by polyprenyl diphosphate synthase. In this study, dps1 genes encoding decaprenyl diphosphate synthase were cloned from three fungi: Bulleromyces albus, Saitoella complicata, and Rhodotorula minuta. The predicted Dps1 proteins contained seven conserved domains found in typical polyprenyl diphosphate synthases and were 528, 440, and 537 amino acids in length in B. albus, S. complicata, and R. minuta, respectively. Escherichia coli expressing the fungal dps1 genes produced CoQ 10 in addition to endogenous CoQ 8 . Two of the three fungal dps1 genes (from S. complicata and R. minuta) were able to replace the function of ispB in an E. coli mutant strain. In vitro enzymatic activities were also detected in recombinant strains. The three dps1 genes were able to complement a Schizosaccharomyces pombe dps1, dlp1 double mutant. Recombinant S. pombe produced mainly CoQ 10 , indicating that the introduced genes were independently functional and did not require dlp1. The cloning of dps1 genes from various fungi has the potential to enhance production of CoQ 10 in other organisms.

The maturation of dendritic cells into more-immunostimulatory dendritic cells by stimulation with different combinations of immunologic agents is expected to provide efficient, adoptive immunotherapy against cancer. Soluble β-glucan maitake D-fraction, extracted from the maitake mushroom Grifola frondosa, acts as a potent immunotherapeutic agent, eliciting innate and adoptive immune responses, thereby contributing to its antitumor activity. Here, we evaluated the efficacy of maitake D-fraction, in combination with a Toll-like receptor agonist, to treat tumors in a murine model. Our results showed that maitake D-fraction, in combination with the Toll-like receptor 9 agonist, cytosine-phosphate-guanine oligodeoxynucleotide, synergistically increased the expression of dendritic cell maturation markers and interleukin-12 production in dendritic cells, but it did not increase interleukin-10 production, generating strong effector dendritic cells with an augmented capacity for efficiently priming an antigen-specific, T helper 1-type T cell response. Maitake D-fraction enhances cytosine-phosphate-guanine oligodeoxynucleotide-induced dendritic cell maturation and cytokine responses in a dectin-1-dependent pathway. We further showed that a combination therapy using cytosine-phosphate-guanine oligodeoxynucleotide and maitake D-fraction was highly effective, either as adjuvants for dendritic cell vaccination or by direct administration against murine tumor. Therapeutic responses to direct administration were associated with increased CD11c(+) dendritic cells in the tumor site and the induction of interferon-γ-producing CD4(+) and CD8(+) T cells. Our results indicate that maitake D-fraction and cytosine-phosphate-guanine oligodeoxynucleotide synergistically activated dendritic cells, resulting in tumor regression via an antitumor T helper cell 1-type response. Our findings provide the basis for a potent antitumor therapy using a novel combination of immunologic agents for future clinical immunotherapy studies in patients., (© Society for Leukocyte Biology.)

Coenzyme Q10 (CoQ10) is essential for energy production and has become a popular supplement in recent years. In this study, CoQ10 productivity was improved in the fission yeast Schizosaccharomyces pombe. Ten CoQ biosynthetic genes were cloned and overexpressed in S. pombe. Strains expressing individual CoQ biosynthetic genes did not produce higher than a 10% increase in CoQ10 production. In addition, simultaneous expression of all ten coq genes did not result in yield improvements. Genes responsible for the biosynthesis of p-hydroxybenzoate and decaprenyl diphosphate, both of which are CoQ biosynthesis precursors, were also overexpressed. CoQ10 production was increased by overexpression of Eco&#95;ubiC (encoding chorismate lyase), Eco&#95;aroF(FBR) (encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase), or Sce&#95;thmgr1 (encoding truncated HMG-CoA reductase). Furthermore, simultaneous expression of these precursor genes resulted in two fold increases in CoQ10 production.

A 33-year-old woman experienced near-syncope at a hospital. Electrocardiography revealed an intermittent ventricular rhythm. The echocardiogram at admission indicated mild hypokinesis and severe diffuse hypokinesis with reverse takotsubo cardiomyopathy on the following day. The patient experienced abdominal pain on the admission day, and computed tomography identified a large left adrenal mass. Her catecholamine levels increased remarkably on the third day. The wall motion improved on the twelfth day. The tumor was successfully resected and the patient was diagnosed with an ectopic pheochromocytoma. The ventricular rhythm with myocardial damage and hypotension induced by the reverse takotsubo cardiomyopathy masked the characteristic symptoms of pheochromocytoma.

Maitake D (MD)-Fraction is a highly purified soluble β-glucan derived from Grifola frondosa (an oriental edible mushroom). Intraperitoneal (i.p.) injection of MD-Fraction has been reported to inhibit tumor growth via enhancement of the host immune system. In this study, we demonstrated that oral administration of MD-Fraction as well as i.p. injection significantly inhibited tumor growth in murine tumor models. After oral administration, MD-Fraction was not transferred to the blood in its free form but was captured by antigen-presenting cells such as macrophages and dendritic cells (DCs) present in the Peyer's patch. The captured MD-Fraction was then transported to the spleen, thereby inducing the systemic immune response. Our study showed that MD-Fraction directly induced DC maturation via a C-type lectin receptor dectin-1 pathway. The therapeutic response of orally administered MD-Fraction was associated with (i) induced systemic tumor-antigen specific T cell response via dectin-1-dependent activation of DCs, (ii) increased infiltration of the activated T cells into the tumor and (iii) decreased number of tumor-caused immunosuppressive cells such as regulatory T cells and myeloid-derived suppressor cells. Our preclinical study suggests that MD-Fraction is a useful oral therapeutic agent in the management of patients with cancer., (Copyright © 2012 UICC.)

We operated rescue activities in Tohoku area after the earthquake of March 11th, 2011. From our hospital, a doctor-helicopter flew to the staging care unit at Hanamaki airport with two members of the disaster medical assistance team (DMAT), one of whom was an anesthesiologist. The helicopter carried ten patients by nine flight missions, who were the victims of tsunami after the earthquake. There were seven doctor-helicopters from all over Japan and did the missions based at Hanamaki airport. The missions was quite different from our usual job as an anesthesiologist, but we could transfer the patients safely by using some knowledge of stabilizing the unstable patients as flight doctors. We report the details of our activities by our doctor-helicopters in Tohoku area.

In Japan, the definition of unnatural death is not prescribed in law. However, a legal judgment recently defined unnatural death as all deaths, excluding natural deaths and deaths from diseases. Legally, unnatural deaths must be reported to the police. In the case of a reported death being considered as suspicious by the police, a forensic autopsy is required. The number of autopsies and the autopsy rate in Japan and Kyoto has increased over the last 10 years. Using data collected from 221 autopsy cases between 2008 and 2010 in Kyoto, Japan, the characteristics of locations where autopsy cases were discovered were analysed to identify reasons for the increase in autopsy numbers. It was found that factors including amount of human interaction and socioeconomic factors may help to explain the statistically significant correlations found.

MD-Fraction, a highly purified, soluble β-(1,3) (1,6)-glucan obtained from Grifola frondosa (an oriental edible mushroom), has been reported to inhibit tumor growth by modulating host immunity. β-Glucan, a major component of the fungal cell wall, is generally recognized by PRRs expressed on macrophages and DCs, such as Dectin-1, and the ability of β-glucans to modulate host immunity is influenced by their structure and purity. Most cellular studies have used particulate β-glucans, such as yeast zymosan (crude β-glucan) and curdlan (purified β-glucan). However, little is known about the cellular mechanism of soluble β-glucans, including MD-Fraction, despite significant therapeutic implications. In this study, we investigated the cellular mechanism of MD-Fraction in murine resident macrophages and compared it with two well-known β-glucan particles. MD-Fraction induced GM-CSF production rapidly through Dectin-1-independent ERK and p38 MAPK activation. Subsequently, MD-Fraction-induced GM-CSF enhanced proliferation and Dectin-1 expression, which permitted Dectin-1-mediated TNF-α induction through the Syk pathway. Curdlan induced not only the proliferation and activation of Dectin-1/Syk signaling in a manner similar to MD-Fraction but also the uncontrolled, proinflammatory cytokine response. Contrastingly, zymosan reduced proliferation and Dectin-1 expression significantly, indicating that the mechanism of macrophage activation by MD-Fraction differs from that of zymosan. This is the first study to demonstrate that purified β-glucans, such as MD-Fraction and curdlan, induce GM-CSF production directly, resulting in Dectin-1/Syk activation in resident macrophages. In conclusion, we demonstrated that MD-Fraction induces cell proliferation and cytokine production without excessive inflammation in resident macrophages, supporting its immunotherapeutic potential.

The purpose of this study was to examine the effects of exercise training on age-related impairment of immune parameters related to T-cell activation in elderly individuals. Twenty-four elderly subjects were assigned to an exercise training group (EXC: 3 men, 9 women; age 61-76 years) or a nonexercise control group (CON: 4 men, 8 women; age 62-79 years). Subjects in EXC participated in exercise sessions 2 d·wk(-1) for 12 weeks. The training session included stretching and endurance exercise (10 minutes), resistance training comprised leg extension, leg press, hip abduction, and hip adduction using exercise machine and each subject's body weight. Subjects in CON maintained their normal physical activity levels during the study period. Blood samples were collected before and after the training period. Samples were measured for the numbers of leukocytes, lymphocytes, and monocytes, and for CD3(+), CD4(+), CD8(+), CD28(+)CD4(+), CD28(+)CD8(+), TRL-4(+)CD14(+), and CD80(+)CD14(+) cells. The number of leukocytes, lymphocytes, monocytes, CD3(+), CD4(+), and CD8(+) cells did not change after 12 weeks in either EXC or CON. The number of CD28(+)CD8(+) cells increased significantly after training in EXC (p ≤ 0.05), although CON showed no significant change. In the EXC group, CD80(+)CD14(+) cell counts were significantly higher after training (p ≤ 0.05), but the TLR-4(+)CD14(+) cell counts were unchanged. In the CON group, no significant alteration existed in TLR-4(+)CD14(+) and CD80(+)CD14(+) cell numbers. In conclusion, exercise training in elderly people is associated with increased CD28-expressing Tc cells and CD80-expressing monocytes. Therefore, exercise training might upregulate monocyte and T-cell-mediated immunity in elderly people.

We previously isolated the novel heteropolysaccharide maitake Z-fraction (MZF) from the maitake mushroom (Grifola frondosa), and demonstrated that MZF significantly inhibited tumor growth by inducing cell-mediated immunity. In this study, we demonstrated that MZF upregulated the expression of CD80, CD86, CD83, and MHC II on bone marrow-derived dendritic cells (DCs) and significantly increased interleukin-12 (IL-12) and tumor necrosis factor-alpha production by DCs in a dose-dependent manner. MZF-treated DCs significantly stimulated both allogeneic and antigen-specific syngenic T cell responses and enhanced antigen-specific interferon-gamma (IFN-gamma) production by syngenic CD4(+) T cells; however, MZF-treated DCs did not affect IL-4 production. Furthermore, the enhancement of IFN-gamma production in CD4(+) T cells, which was induced by MZF-treated DCs, was completely inhibited by the addition of an anti-IL-12 antibody. These results indicate that MZF induced DC maturation and antigen-specific Th1 response by enhancing DC-produced IL-12. We also demonstrated that DCs pulsed with colon-26 tumor lysate in the presence of MZF induced both therapeutic and preventive effects on colon-26 tumor development in BALB/c mice. These results suggest that MZF could be a potential effective adjuvant to enhance immunotherapy using DC-based vaccination.

Cancer cachexia is reported to be a major cause of cancer-related death. Since the pathogenesis is not entirely understood, only few effective therapies have been established. Since myriad tumors produce parathyroid hormone-related protein (PTHrP), plasma concentrations of PTHrP are increased in cancer cachexia. We measured the food intake, gastric emptying, conditioned taste aversion (CTA), and gene expression of hypothalamic neuropeptides in mice after administering PTHrP intraperitoneally. We administered PTHrP intravenously in rats and examined the gastroduodenal motility and vagal nerve activities. We also examined whether chronic administration of PTHrP influenced the food intake and body weight. Peripherally administered PTHrP induced negative energy balance by decreasing the food intake and gastric emptying; however, it did not induce CTA. The mechanism involved the activation of hypothalamic urocortins 2 and 3 through vagal afferent pathways and the suppression of gastroduodenal motor activity. The continuous infusion of PTHrP reduced the food intake and body weight gain with a concomitant decrease in the fat and skeletal muscle. Our findings suggest that PTHrP influences the food intake and body weight; therefore, PTHrP can be considered as a therapeutic target for cancer cachexia., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A novel polysaccharide, MZF, with a molecular mass of 23 kDa was isolated from Grifola frondosa . Results from methylation and (1)H NMR led to the conclusion that MZF is a heteropolysaccharide consisting of -->6)-alpha-D-Galp-(1--> (36.2%), -->3)-alpha-L-Fucp-(1--> (14.5%), -->6)-alpha-D-Manp-(1--> (9.4%), -->3)-beta-D-Glcp-(1--> (10.1%), alpha-D-Manp-(1--> (23.2%), and -->3,6)-beta-D-Glcp-(1--> (6.5%). Although MZF did not affect the proliferation of colon-26 cells in vitro, it significantly inhibited tumor growth in BALB/cA mice inoculated with colon-26 cancer cells. Moreover, MZF significantly induced the proliferation of splenocytes and peritoneal macrophages. The mRNA expression of IL-12p40, IL-2 and IFN-gamma were increased significantly in MZF-treated spleen. Furthermore, MZF augmented the percentage of IFN-gamma-producing cells in both splenic CD4(+) and CD8(+) T cells and tumor infiltrating CD4(+) and CD8(+) T cells and enhanced the cytotoxic activity of NK cells and CTLs. These results indicate that MZF is a novel effective immunomodulator that has antitumor activity associated with induced cell-mediated immunity.

Previous studies have presented that Maitake beta-glucan (MD-Fraction) extracted from the fruit body of Grifola frondosa has an anti-tumor effect by activating the immune system. Recently, the stimulating effects of beta-glucans on hematopoiesis were identified as new characteristics of polysaccharides, possibly helping to relieve the immunosuppression which results from chemotherapies. We demonstrated that the production of granulocyte colony-stimulating factor (G-CSF) was significantly enhanced by MD-Fraction (8mg/kg, i.p.) in granulocytopenic model induced in mice using cyclophosphamide (200mg/kg, i.p.). In addition, MD-Fraction induced a biphasic increase in the number of granulocytes in the spleen. The mechanism for the increase in granulocytes on the early phase on day 1 might involve the increased mRNA expression of macrophage inflammatory protein-2 (MIP-2), in the splenic cells, thereby recruiting granulocytes into the spleen. Interestingly, a decline of myeloid progenitors in the bone marrow and an increase in granulocytes in the peripheral blood were observed on day 5, suggesting a mobilization of granulocytes and their progenitors from the bone marrow to the peripheral blood. We confirmed that a possible mechanism in which MD-Fraction promoted the mobilization of granulocytes and their progenitors from the bone marrow is down-regulating the expression of the chemokine receptor, CXCR4, and its ligand, stromal cell-derived factor 1 (SDF-1) in the bone marrow microenvironment. These results reveal a novel function of Maitake beta-glucan that enhances the granulopoiesis and mobilization of granulocytes and their progenitors by stimulating G-CSF production. This finding presents opportunities to develop new therapeutic strategies against the immunosuppression caused by chemotherapies in cancer patients.

Cisplatin is broadly used clinically as an anticancer drug. Despite its significant anticancer activity, cisplatin-induced nephrotoxicity and myelosuppression limit its use. MD-Fraction is glucan purified from maitake (Grifola frondosa), which has beta-1, 6-main chain with beta-1, 3-branches, has been reported to exhibit antitumor and antimetastatic activities by enhancing the immune system. In this study, we demonstrate that MD-Fraction in combination with cisplatin significantly enhanced antitumor and antimetastatic activity compared to cisplatin alone. MD-Fraction reduced decreases in body weight, spleen weight and the number of immunocompetent cells such as macrophages, DCs and NK cells in cisplatin-treated mice. MD-Fraction also induced IL-12p70 production by splenocytes, resulting in increased NK cell activity in cisplatin-treated mice. MD-Fraction significantly increased the mRNA expression of GM-CSF, G-CSF, M-CSF, IFN-gamma, IL-12 p40 in splenocytes and reduced the decrease in the number of CFU-GM colonies in cisplatin-treated bone marrow. These facts suggest that MD-Fraction can reduce cisplatin-induced myelosuppression. Moreover, treatment with MD-Fraction significantly reduced cisplatin-induced nephrotoxicity accompanied by increases in serum creatinine level, necrosis and apoptosis of renal tubular cells. These results suggest that MD-Fraction in combination with cisplatin cannot only enhance antitumor and antimentastatic acitivity, but also reduce cisplatin-induced myelotoxicity and nephrotoxicity.

We reported a case of an abdominal wall hematoma which caused by Seldinger method via the femoral artery. A 48-year-old female, suffered from direct carotid cavernous fistula, was treated by transfemoral transvenous embolization (TVE). The whole procedure was completed without difficulty except minor resistance of guide wire manipulation during left femoral artery catheterization. Four hours later, the patient became hypotensive and showed the sign of impending shock without definitive causes. Nine hours after the embolization a huge hematoma of the abdominal wall was found. It required the total 1200 m/ of blood transfusion before her blood pressure returned to normal. She recovered fully from this event and discharged uneventfully. There is a speculation that a deep circumflex iliac artery (DCIA) was injured with an angle-shaped guide wire and bled into the abdominal wall. And subsequent systemic heparinization prevented the coagulation process, resulting a large hematoma. Anatomically, an angle-shaped guide wire is easily able to migrate into DCIA. To prevent a vascular injury, it is very important to manipulate a guide wire under fluoroscopic control and to select a J-shaped guide wire instead of an angle-shaped one.

The anti-metastatic activity of MD-Fraction extracted from the maitake mushroom (Grifola frondosa) was examined in an experimental murine model of lung metastasis. Intraperitoneal administration of MD-Fraction 2 d before tumor implantation significantly inhibited lung metastasis of colon-26 carcinoma and B16/BL6 melanoma cells. In this model, MD-Fraction enhanced IL-12 production from antigen presenting cells (APCs). MD-Fraction treatment activated NK cells and increased cytotoxicity against YAC-1 and colon-26 carcinoma cells. Furthermore, the depletion of NK cells with anti-asialo GM1 abolished the inhibitory effect of MD-Fraction on lung metastasis of colon-26 cells. Ex vivo, B16/BL6 cell adhesion to LPS-activated murine lung vascular endothelial cells was inhibited by MD-Fraction and anti-intercellular adhesion molecule (ICAM)-1 antibody. These results suggest that MD-Fraction inhibits tumor metastasis by activating NK cells and APCs, and by suppressing of ICAM-1 leading to the inhibition of tumor cell adhesion to vascular endothelial cells.

Objective: D-Fraction, a polysaccharide extracted from maitake mushrooms (Grifola frondosa), has been reported to exhibit an antitumor effect through activation of immunocompetent cells, including macrophages and T cells, with modulation of the balance between T-helper 1 and 2 cells. We examined whether D-Fraction could decrease the effective dosage of the chemotherapeutic agent, mitomycin-C (MMC), necessary to control carcinoma in mice., Methods and Results: We determined that 0.25 mg.kg-1.d-1 was the optimal dosage of MMC because consecutive administration for 17 d resulted in antitumor effects and a survival ratio of 100% in mice bearing mammary cancer cells (MM-46). Although the dosage of MMC was lower than the effective level, spleen weight and total number of nuclear cells in the mouse spleen decreased, indicating that MMC showed immunosuppressive activity. In contrast, the combination of D-Fraction and MMC recovered the decreases in the dose response induced by MMC and inhibited tumor cell growth more than MMC alone. These effects were achieved through increased immunocompetent cell proliferation. We evaluated the expression of CD28 on splenic CD8+ T cells and the amount of interleukin-12 produced by whole spleen cells including macrophages after administering D-Fraction. The results showed enhancement of the T-helper 1 dominant response., Conclusion: These results suggest that D-Fraction can decrease the effective dosage in tumor-bearing mice by increasing the proliferation, differentiation, and activation of immunocompetent cells and thus provide a potential clinical benefit for patients with cancer.

In innate immunity, activated natural killer (NK) cells attack and damage pathogens such as bacteria and virus without restriction by the MHC antigen. NK cells activated by IL-12 have been reported to recognize and kill tumor cells in perforin-mediated apoptosis. We have reported that D-Fraction, a polysaccharide extracted from the maitake mushroom (Grifola frondosa), activates macrophages, dendritic cells, and T cells and inhibits the growth of tumor cells. However, the effects of D-Fraction on NK cell function in the innate immune response are not well known. In the present study, we administered D-Fraction to MM-46 mammary tumor-bearing C3H/HeJ mice intraperitoneally for 3 consecutive days and investigated its effects on the activation and cytotoxicity of NK cells. D-Fraction significantly enhanced the cytotoxicity against NK-sensitive YAC-1 cells and the expression of CD223 on NK cells. D-Fraction also increased the expression of CD86 on macrophages. In addition, the levels of IL-12 in the culture supernatant of whole spleen cells and in serum increased, compared with the control corresponding to an increase in expression of IL-12 receptor betaI on NK cells. These results suggest that D-Fraction enhances the cytotoxicity of NK cells through the production of IL-12 by macrophages activated by D-Fraction.

A wild type NADPH-dependent carbonyl reductase from Candida magnoliae (reductase S1) has been found not to utilize NADH as a coenzyme. A mutation to exchange the coenzyme specificity in reductase S1 has been designed by computer-aided methods, including three-dimensional structure modeling and in silico screening of enzyme mutants. Site-directed mutagenesis has been used to introduce systematic substitutions of seven or eight amino acid residues onto the adenosine-binding pocket of the enzyme according to rational computational design. The resulting S1 mutants show NADH-dependency and have lost their ability to utilize NADPH as a coenzyme, but retain those catalytic activities. Kinetic parameter V(max) and K(m) values of those mutants for NADH are 1/3- to 1/10-fold those of the wild type enzyme for NADPH. As a model system for industrial production of optically active alcohols, the S1 mutants can be applied to an asymmetric reduction of ketones, cooperating with a coenzyme-regeneration system that uses an NAD-dependent formate dehydrogenase.

We have reported that D-Fraction, a polysaccharide extracted from the edible maitake mushroom (Grifola frondosa), activates immunocompetent cells, thereby eliciting antitumor activity. To extend the application of D-Fraction as a nutritional supplement for healthy people as well as treatment for those with cancer, we investigated the effects of D-Fraction on the immune system in normal C3H/HeJ mice. Splenocytes from mice administered D-Fraction intraperitoneally for 17 consecutive days were cultured, and the culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-12 production by antigen-presenting cells (APCs), including macrophages and dendritic cells, and also for the T helper (Th)-1 cytokine interferon (IFN)-gamma and the Th-2 cytokines IL-4 and IL-10. The level of IL-10 as well as those of NO and IFN-gamma were increased by D-Fraction as compared with the control, in which the serum immunoglobulin E level was increased. The results suggest that D-Fraction induced a Th-2 dominant response through the activation of macrophages, resulting in the enhancement of humoral immunity rather than cell-mediated immunity. Furthermore, an increase in the percentage ratio of CD69 and CD89 expression on major histocompatibility complex II(+) cells revealed activation of APCs 4 h after D-Fraction administration. These results indicate that D-Fraction enhances both the innate and adaptive arms of the immune response in normal mice. Therefore, its administration may enhance host defense against foreign pathogens and protect healthy individuals from infectious diseases.