Search

Thiobacillus sp. strain KNK65MA, which produced an NAD-dependent formate dehydrogenase (FDH) highly resistant to alpha-haloketones, was newly isolated, i.e., the enzyme showed no loss of activity after a 5-h incubation with alpha-haloketones, such as ethyl 4-chloro-3-oxobutanoate. The enzyme was also resistant to SH reagents. The enzyme, purified to homogeneity, was a dimer composed of identical subunits. The specific activity was 7.6 u/mg, and the apparent Km values for formate and NAD+ were 1.6 and 0.048 mM, respectively. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 44,021; this gene was highly expressed in E. coli cells. The deduced amino acid sequence of this FDH had high identity to other bacterial FDHs.

Ancylobacter aquaticus strain KNK607M, which had high NAD-dependent formate dehydrogenase (FDH) activity, was newly isolated. The enzyme, purified to homogeneity, was a dimer composed of identical subunits with a molecular mass of 44 kDa. The specific activity was 9.5 u/mg, and the enzyme was optimum at pH 6.3 and 50 degrees C, most stable at pH 7.0, and stable at 50 degrees C or lower. The apparent Km values for formate and NAD+ were 2.4 and 0.057 mM, respectively. The enzyme was specific to formate and was inhibited by SH reagents and heavy metal ions. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 43,895; this gene was highly expressed in E. coli cells. The FDH had high identity to other FDHs, i.e., those of Pseudomonas, Mycobacterium, Moraxella, and Paracoccus, which were 91.3%, 90.8%, 84.2%, and 82.3%, respectively.

Dendritic cells (DCs) are known to not only induce the activation of T cells, but are also associated with the differentiation of T cells. The D-fraction, a beta-glucan extracted from maitake (Grifola frondosa) which expresses anti-tumor effects by establishing a helper (Th)-1 dominance in BALB/c mice, enhanced IL-12p70 production by DCs, when the ratio of CD8alpha(+) DCs to CD8alpha(-) DCs increased. In addition, examination of the tumor rejection effect of D-fraction-stimulated DCs loaded with tumor antigen revealed that tumor growth is inhibited completely by activating CD4(+) T cells and CD8(+) T cells.

Natural killer (NK) cells are directly cytotoxic for tumor cells and play a primary role in regulating immune responses. We monitored levels of NK cell cytotoxic activity in cancer patients receiving D-Fraction extracted from maitake mushrooms (Grifola frondosa). Elevated levels of cytotoxic activity were maintained for one year. To elucidate the mechanisms underlying long-term activation of NK cells during treatment with D-Fraction, we examined tumor volume and levels of IFN-gamma and TNF-alpha in MM46-bearing C3H/HeN mice to which D-Fraction was administered for 19 d. D-Fraction markedly suppressed tumor growth, corresponding with increases in TNF-alpha and IFN-gamma released from spleen cells and a significant increase in TNF-alpha expressed in NK cells. This suggests that the D-Fraction activates NK cells even on the 20th day after treatment. Furthermore, D-Fraction increased macrophage-derived interleukin (IL)-12, which serves to activate NK cells. These results suggest that NK cells are not only responsible for the early effects of D-Fraction on tumor growth, but also for the long-term tumor-suppressive effects of D-Fraction through increased IL-12 released from macrophages.

A polysaccharide, designated as the D-fraction, extracted from maitake (Grifola frondosa), was reported to have anti-tumor effects by activating macrophages and T cells in C3H/HeN mice in which a Th-1 dominant response was established. In this study using BALB/c mice in which a Th-2 response is genetically dominant, D-fraction reduced the expression of Th-2 cytokine interleukin (IL)-4 but markedly increased the expression of Th-1 cytokine interferon (IFN)-gamma in CD4(+) T cells and also increased IL-12p70 production as well as IFN-gamma production by antigen-presenting cells (APCs), suggesting that D-fraction promotes the differentiation into Th-1 cells of CD4(+) T cells through enhancement of IL-12p70 production by APCs.

Maitake mushroom (Grifola frondosa) MD-fraction containing beta-1,6 glucan with beta-1,3 branched chains has previously exhibited strong anticancer activity by increasing immune-competent cell activity.1,2 In this non-random case series, a combination of MD-fraction and whole maitake powder was investigated to determine its effectiveness for 22- to 57-year-old cancer patients in stages II-IV. Cancer regression or significant symptom improvement was observed in 58.3 percent of liver cancer patients, 68.8 percent of breast cancer patients, and 62.5 percent of lung cancer patients. The trial found a less than 10-20 percent improvement for leukemia, stomach cancer, and brain cancer patients. Furthermore, when maitake was taken in addition to chemotherapy, immune-competent cell activities were enhanced 1.2-1.4 times, compared with chemotherapy alone. Animal studies have supported the use of maitake MD-fraction for cancer.

We have already reported that the D-Fraction, a beta-glucan extracted from the fruiting body of the maitake mushroom (Grifola frondosa), activates cellular immunity and expresses anti-tumor effects. In this study we investigated the anti-tumor functions of D-Fraction in relation to its control of the balance between T lymphocyte subsets Th-1 and Th-2. D-Fraction decreased the activation of B cells and potentiated the activation of helper T cells, resulting in enhanced cellular immunity. It also induced the production of interferon (IFN)-gamma, interleukin (IL)-12 p70, and IL-18 by whole spleen cells and lymph node cells, but suppressed that of IL-4. These results suggest that D-Fraction establishes Th-1 dominance which induces cellular immunity in the population that was Th-2 dominant due to carcinoma.

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 D-fraction can reduce the clinical effective dosage of antibiotics in the treatment of opportunistic bacterial infection, we examined the effects of D-fraction on the treatment of Listeria monocytogenes-infected mice in combination with vancomycine (VCM), the only antibiotic used for methicillin-resistant Staphylococcus aureus (MRSA). Listeria-infection was introduced by its inoculation into the abdominal cavity of mice. Without treatment, all inoculated mice died within 3 days after the inoculation. In contrast, in the mice treated with combined therapy of D-faction (10 mg/kg per day) and VCM (10 mg/kg per day), the survival rate was maintained at 60% on the 10th day after the inoculation, which was superior to that of mice treated with VCM alone (10 mg/kg per day). To investigate the mechanism underlying the reinforcement of VCM treatment by the D-fraction, the activities of macrophages and splenic T cells of Listeria-infected mice were evaluated. In mice administered with both D-fraction and VCM, macrophages produced 2.7 times as much interleukin-1 as that of non-treated control mice. The bactericidal activity of splenic T cells was also enhanced by 2.6 times of that of non-treated control mice. These results indicate that D-fraction activates immuno-competent cells, induced cytokine production, and consequently enhanced the bactericidal activities of the splenic T cells against Listeria monocytogenes, suggesting the clinical benefit of D-fraction in the case of anti-bacterial treatment for patients with high risks.

We have reported that D-Fraction extracted from maitake (Grifola frondosa), activates immune competent cells, and indicates anti-tumor activities. The D-Fraction was observed to induce angiogenesis in vivo and to enhance the proliferation capability and migration capability of human vascular endothelial cell in vitro. The D-Fraction also increased plasma vascular endothelial growth factor (VEGF) concentration significantly. Also VEGF and TNF-alpha production by the activated peritoneal macrophages were enhanced. These results suggest that the anti-tumor activity of the D-Fraction is not only associated with the activation of the immuno-competent cells but also possibly related to the carcinoma angiogenesis induction.

We recently reported the anti-hepatitis effect of a polysaccharide, designated as the D-fraction, extracted from maitake. Its effect includes immuno-regulating activities. We investigated the effect of the glucan in collagen-induced arthritis (CIA). The D-fraction was administered to CIA mice for 30 consecutive days. Arthritis development was observed from the 4th day after the second immunization. The D-fraction did not have any influence on anti-type II collagen antibodies in blood serum or activated B cells. To determine how cellular immunity may be involved in the development of CIA, ratios of CD4+ T cells and their activated form in the axillary and inguinal lymph node T cells were detected by flow cytometry analysis. The ratios were not different between the D-fraction group and the control group. However, interleukin-1beta, granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha productions from splenic macrophages were significantly increased to 2.0, 4.7 and 1.9 times the control group level, respectively. The ratio of macrophages in the whole spleen cells was 2.3 times that of the control group, and their migrating ability was 1.9 times higher. Based on these results, we concluded that the arthritis development induced by D-fraction administration is attributable to the activation of splenic macrophages.

Background: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712., Results: The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases., Conclusions: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

For the improvement of N-carbamyl-D-amino acid amidohydrolase (DCase), which can be used for the industrial production of D-amino acids, the stability of DCase from Agrobacterium sp. KNK712 was improved through various combinations of thermostability-related mutations. The thermostable temperature (defined as the temperature on heat treatment for 10 min that caused a decrease in the DCase activity of 50%) of the enzyme which had three amino acids, H57Y, P203E, and V236A, replaced was increased by about 19 degrees C. The mutant DCase, designated as 455M, was purified and its enzymatic properties were studied. The enzyme had highly increased stability against not only temperature but also pH, the optimal temperature of the enzyme being about 75 degrees C. The substrate specificity of the enzyme for various N-carbamyl-D-amino acids was changed little in comparison with that of the native enzyme. Enzymochemical parameters were also measured.

A plasmid, pNT4553, was constructed for high level production of N-carbamyl-d-amino acid amidohydrolase (DCase), the thermostability of which has been improved by amino acid substitution. The DCase activity and the stability of the plasmid in the host cells were dependent on the Escherichia coli strains used. E. coli HB101 was the most suitable host strain among the 13 types of E. coli tested. E. coli HB101 exhibited the highest activity, i.e. 6.36 units/ml of culture broth in 2YT medium (1.6% tryptone, 1.0% yeast extract, and 0.5% NaCl, pH 7.0), and the plasmid was stably maintained by cultivation in 5 types of E. coli including HB101. Casamino acids, NZ-amine, peptone, and protein extract (a mixture of hydrolyzates of corn gluten, wheat gluten and soybean), were found to be suitable as natural nitrogen sources for both enzyme activity and growth. When cultivation was carried out in the presence of high concentrations of glycerol (6.5%) as the carbon source, and protein extract (3.0%) as the nitrogen source, in a small volume of the medium (20 ml of medium in a 500-ml shaking flask), in which the aeration level was estimated to be high, growth and activity reached OD550=63.8 (17.1 mg of dry cell weight/ml of culture broth) and 22.9 units/ml of culture broth, respectively. The economical hyperproduction of DCase using only inexpensive constituents for the medium was achieved.

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.

To improve the production of D-amino acids using an immobilized N-carbamyl-D-amino acid amidohydrolase, the enzyme gene of Agrobacterium sp. KNK712 was mutagenized randomly to increase its thermostability. The gene was inserted into M13mp19, mutagenized with hydroxylamine, ligated into pUC19 after restriction endonuclease digestion, and then used to transform Escherichia coli. The resultant transformants were screened by a newly developed colorimetric enzyme assay method, and the candidate colonies corresponding to red spots were separated from the master plates. Using cell-free extracts of these clones, the properties of the enzymes produced were investigated, it being proved that these enzymes had almost the same activity and improved thermostability by about 5 degrees C compared with those of the native enzyme. As found on enzyme gene analysis of these mutants, the 57th amino acid, histidine, of the enzyme was changed to tyrosine, or the 203rd amino acid, proline, to leucine or serine.

For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10 degrees C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.

For the production of D-amino acids, thermotolerant bacteria producing N-carbamyl-D-amino acid amidohydrolase were isolated from soil by enrichment culture at 45 degrees C with N-carbamyl-D-amino acids as the sole nitrogen source. The enzyme activities and substrate specificities of these strains were examined by the resting cells reaction. One of the enzymes, produced by Pseudomonas sp. strain KNK003A, was purified and characterized, and the amino acids of its N-terminal region were sequenced. A DNA fragment containing the gene for a thermostable N-carbamyl-D-amino acid amidohydrolase was then cloned into Escherichia coli. The gene encoded a peptide of 312 amino acids, with a calculated molecular weight of 35,000. The similarity of the deduced amino acid sequences of this enzyme and a related enzyme from a mesophile, Agrobacterium sp. strain KNK712, was 60%. A database was searched for similar sequences.

Agrobacterium sp. strain KNK712, which produced N-carbamyl-D-amino acid amidohydrolase (DCase) was isolated from soil. The bacterium had D-specific hydantoinase activity also. Both enzymes are suitable for use in the production of D-amino acids. The DCase gene from Agrobacterium sp. strain KNK712 was cloned into Escherichia coli. The cloned DNA fragment contained one open reading frame, predicted to encode a peptide of 304 amino acids, with a calculated molecular weight of 34,285. The DCase gene was overexpressed under the control of the lac promoter, and DCase accounted for 50% of the soluble protein in the cells. The enzyme was purified and some properties were investigated. Both the optimum pH and the pH that gave greatest stability were about pH 7.0. The optimum temperature was 65 degrees C, and the enzyme was stable at 55 degrees C. The enzyme had strict specificity toward N-carbamyl-D-amino acids, and was inhibited by thiol reagents, Cu2+, Hg2+, Ag+, and ammonia.

Experimental rat models (5-week-old Sprague-Dawley rats) with hyperlipemia were prepared by feeding high-cholesterol feed containing sodium cholate and casein as a protein source. Dried maitake (Grifola frondosa) powder was mixed with the basic high-cholesterol feed and the serum lipids were periodically measured. Values of cholesterol, triglyceride and phospholipid in serum of rats in the maitake-feed group were suppressed by 0.3-0.8 times those in animals fed the basic feed, the latter values being close to those in rats given normal feed. The value of high density lipoprotein (HDL)-cholesterol in serum which is generally reduced by the ingestion of high-cholesterol feed remained the level it was at the beginning of the experiment. Weights of extirpated liver and epididymal fat-pads were significantly less (0.6-0.7 times) than those in the basic feed group, indicating that maitake inhibits lipid accumulation in the body. Liver lipids were also measured and the values were found to be decreased by maitake administration as true of serum lipid, suggesting maitake has an anti-liver lipid activity. Measurement of the amount of total cholesterol and bile acid in feces showed, the ratio of cholesterol-excretion had increased 1.8 times and bile acid-excretion 3 fold by maitake treatment. From these results, it is believed that maitake helps to improve the lipid metabolism as it inhibits both liver lipid and serum lipid which are increased by the ingestion of high-fat feed.

Several 13-alkyl substituted analogs of berberine and palmatine were found to be highly active against two types of Staphylococcus aureus (S1 and S2) of different origin. The most active 13-hexylberberine was 8 times more active (against S1) and the same order active (against S2) as kanamycin sulfate. 13-Hexylpalmatine displayed an activity against S. aureus (S1) 4 times greater than that of kanamycin sulfate. The activities of 13-hexylberberine against two types of S. aureus were 64 and 128 times greater than those of the clinically used alkaloid berberine. Additionally two hexyl derivatives possessed antifungal activity.

Although stimulation of N-methyl-D-aspartate receptors or voltage-dependent calcium channels induces both the activation of c-fos and brain-derived neurotrophic factor (BDNF) genes, it is not certain how the activation of these genes is related. Using primary cultures of rat hippocampal neurons, we found that exposing the cells to cycloheximide allowed subsequent activation of BDNF mRNA expression, although activation of AP-1 DNA-binding activity resulting from the c-fos induction was abolished. Super-induction of BDNF gene was also caused by cycloheximide. The estimated half-life of BDNF mRNA was approximately 2.5 hrs, which was almost identical to that of c-fos mRNA. These results indicate that nascent AP-1 is not required for the activation of BDNF gene, leading to the notion that the BDNF gene can be activated by Ca2+ signals as an immediate early gene.

Objective: To determine the efficacy of maitake mushrooms in inhibiting the elevation of liver and serum lipids in rats., Design: Sprague-Dawley rats with hyperlipidemia were used to measure and compare the values of cholesterol, phospholipids, and triglycerides between cholesterol-fed rats and rats whose diets were fortified with 20% maitake mushroom dried powder., Results: The values in maitake-fed rats were consistently less than those in the basic cholesterol-fed rats. The value of high-density lipoprotein cholesterol, which usually is decreased by taking high-cholesterol feed, maintained the level that it had at the beginning of the experiment. Weights of extirpated liver and epididymal fat pads were significantly less than those in the basic feed group., Conclusion: Our data suggest that maitake mushrooms have the ability to alter lipid metabolism by inhibiting both the accumulation of liver lipids and the elevation of serum lipids. Further studies are needed to elucidate the mechanism of activity of maitake mushrooms and to establish whether their action in humans is similar to that in the animal model tested here.

Isoflurane often produces tachycardia during clinical anesthesia. We examined the effect of a stepwise increase of isoflurane concentration on hemodynamic parameters in the absence or presence of nitrous oxide (N2O). After induction with thiamylal (3 mg.kg-1), isoflurane in oxygen or in 66% N2O-oxygen was administered with mask ventilation. Inspired isoflurane concentration was increased in a stepwise fashion (1, 2, 3 and 4%) every 5 minutes and manual ventilation was performed to maintain ETCO2 within 35-40 mmHg. Blood pressure (BP), heart rate (HR) and cardiac output (CO) were measured before and at every minute after isoflurane administration until 20 minutes. Systolic BP decreased gradually with increasing isoflurane concentration, but was transiently elevated for 3%. HR increased in a dose-related manner. CO decreased significantly at 1-3%. N2O and this seemed to magnify the isoflurane-induced decrease in BP and increase in HR at 1% and 2%. CO increased from baseline at 4%. Isoflurane tended to increase HR in a dose-related manner and induced a hyperdynamic response during rapid increasing of isoflurane concentration. This response may have beed caused by the irritating effect of isoflurane on the airways. Addition of N2O attenuated this response because it increases the speed of induction and the depth of anesthesia.

The fruit body of Grifola frondosa (maitake), Basidiomycetes was confirmed to contain substances with anti-diabetic activity. When 1 g/d of powdered fruit body of maitake was given orally to a genetically diabetic mouse (KK-Ay), blood glucose reduction was observed, in contrast to the control group in which the blood glucose increased with ageing. Moreover, levels of insulin and triglyceride in plasma demonstrated a change similar to blood glucose with feeding of maitake. Ether-ethanol-soluble (ES) and hot water-soluble (WS) fractions were prepared from the fruit body and their hypoglycemic activity was examined. Blood glucose-lowering activity was found when ES-fraction or WS-50% ethanol float (X) fraction was administered orally, but other WS-fractions were inactive. These results suggest that the anti-diabetic activity was present not only in the ES-fraction consisting of lipid but also in the X-fraction of peptidoglycan (sugar:protein = 65:35).

Since Snyder reported first twelve cases of benign paroxysmal torticollis in infancy (BPT) in 1969, about 30 cases have been reported mainly in the North America and Europe, but not in Japan. The authors experienced such a case of one-year-old boy. The attack occurred at the age of 2 months for the first time and continued to repeat every 2 weeks periodically. The head tiltings were always noticed when he woke up in the morning and lasted for about 5 to 6 days, but they disappeared during sleep. He tilted his head to the lateral side more often to the left than to the right. There were associated symptoms and signs including irritability, vomiting, gait disturbance, and abnormal truncal posture during the attacks. He did not demonstrate any neurological abnormalities between the attacks. The study of cerebrospinal fluid, caloric test, computed tomography, electroencephalography and brainstem auditory evoked responses were also normal. The frequency of the attacks gradually decreased after the age of 2 years and disappeared at the age of 3 years, and he has not complained of severe headache or vertigo up to 6 years of age. This case is reported to emphasize that BPT has to be included in the differential diagnosis of acquired torticollis. And it is required to accumulate such cases to elucidate its etiological factors.

Sex hormones are closely related to the onset and progression of periodontal disease. The purpose of this study was to evaluate the effects of sex hormones on the metabolism of human periodontal ligament cells. Human periodontal ligament cells and gingival fibroblasts were prepared from 7 donors with normal gingiva. 17 beta-estradiol and progesterone were purchased from Sigma Chemical Company. The effects of sex hormones on cell morphology, alkaline phosphatase activity, cell proliferation, DNA synthesis, collagen synthesis and non-collagenous protein synthesis were investigated. The results were as follows: 1. Periodontal ligament cells showed osteoinductive-like cells in the cell morphology and alkaline phosphatase activity. 2. DNA synthesis was stimulated by 17 beta-estradiol and progesterone in the presence of 5% fetal calf serum. 3. Collagen synthesis was inhibited by 17 beta-estradiol and progesterone, while non-collagenous protein synthesis was inhibited by 17 beta-estradiol and low concentrations of progesterone. 4. The addition of serum into culture medium was necessary to induce the basic metabolism of human periodontal ligament cells. It was demonstrated that sex hormones are closely related to the metabolism of human periodontal ligament cells.