Expression and characterization of α-1,3-glucanase from Paenibacillus alginolyticus NBRC15375, which is classified into subgroup 2 (minor group) of GH family 87.

Bacterial α-1,3-glucanase, classified as glycoside hydrolase (GH) family 87, has been divided into 3 subgroups based on differences in gene sequences in the catalytic domain. The enzymatic properties of subgroups 1 and 3 of several bacteria have been previously investigated and reported; however, the chemical characterization of subgroup 2 enzymes has not been previously conducted. The α-1,3-glucanase gene from Paenibacillus alginolyticus NBRC15375 (PaAgl) belonging to subgroup 2 of GH family 87 was cloned and expressed in Escherichia coli. PgAgl-N1 (subgroup 3) and PgAgl-N2 (subgroup 1) from P. glycanilyticus NBRC16188 were expressed in E. coli, and their enzymatic characteristics were compared. The amino acid sequence of PaAgl demonstrated that the homology was significantly lower in other subgroups when only the catalytic domain was compared. The oligosaccharide products of the mutan-degrading reaction seemed to have different characteristics among subgroups 1, 2, and 3 in GH family 87.

Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

Dietary syringic acid reduces fat mass in an ovariectomy-induced mouse model of obesity.

OBJECTIVE: Postmenopausal women are at increased risk of metabolic diseases such as obesity and diabetes. Therefore, the chemoprevention of postmenopausal changes in health via dietary supplements is important. Syringic acid (SA) is a phenolic compound present in the fruit of the assai palm, Euterpe oleracea, and in the mycelium of the shiitake mushroom, Lentinula edodes. This compound shows no affinity for estrogen receptors and may exert disease-preventive effects. Reportedly, dietary SA ameliorates high-fat diet-induced obesity in mice; however, its effects on estrogen deficiency-induced obesity are still unclear. Therefore, in this study, we investigated whether and how dietary SA affects these factors in ovariectomized (OVX) mice. METHODS: Ten-week-old OVX mice were fed SA-containing diets (100 mg/kg body weight/d) for 12 weeks. Their body weights, food intake, and uterus weights as well as other parameters were measured and comparisons were made with mice in the control group. RESULTS: Dietary SA did not affect the body weight, food intake, or uterus weight of OVX mice over the study period; however, the SA-fed group showed lower fat mass (ie, visceral, subcutaneous, and total fat) than the OVX-control group (11.1 ±â€Š3.3 vs. 8.3 ±â€Š2.4, P < 0.05; 7.9 ±â€Š1.1 vs. 5.9 ±â€Š1.6, P < 0.05; 19.0 ±â€Š4.2 vs. 14.1 ±â€Š3.8, P < 0.05, respectively). Furthermore, blood analysis revealed that SA-treatment resulted in a dose-dependent decrease and increase in serum triglyceride (59.2 ±â€Š8.3 vs. 43.9 ±â€Š12.2 mg/dL P < 0.05) and adiponectin (7.7 ±â€Š0.3 vs. 9.5 ±â€Š0.6 µg/mL, P < 0.05) levels, respectively. CONCLUSIONS: These results suggest that the SA diet improves lipid metabolism without affecting the uterus in OVX mice. Therefore, dietary SA has potential applicability for the prevention of postmenopausal obesity and type 2 diabetes.

Purification and characterization of a glycoside hydrolase family 5 endoglucanase from Tricholoma matsutake grown on barley based solid-state medium.

An endoglucanase was isolated from solid-state culture of the ectomycorrhizal fungus Tricholoma matsutake (TmEgl5A) grown on rolled barley and vermiculite. The enzyme was purified by ammonium sulfate fractionation, ion-exchange, hydrophobic, and gel filtration. TmEgl5A showed a molecular mass of approximately 40 kDa as determined by SDS-PAGE. The single band of the protein was analyzed by peptide-mass-finger-printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the trypsin-digested peptide sequences were matched to a putative endoglucanase sequence (protein ID1465229) in the JGI T. matsutake 945 v3.0 genome database. Based on the sequence information, the gene encoding TmEgl was cloned and expressed in Pichia pastoris KM71H. The deduced amino acid sequence was similar to GH5 family endoglucanases from Basidiomycetes. The enzyme acts on barley ß-glucan, lichenan, and CMC-Na. The hydrolyzation products from these substrates were detected by thin-layer chromatography as oligosaccharides with minimal disaccharides. These results suggested that T. matsutake produces a typical endoglucanase in solid-state culture, and the fungus has the potential to degrade ß-linkage polysaccharides.

Anti-osteoporotic effects of syringic acid and vanilic acid in the extracts of waste beds after mushroom cultivation.

In recent years, the number of patients with osteoporosis has increased as population grows older. Therefore, the chemoprevention of osteoporosis by better nutrition is important. White-rot fungi degrades milled wood lignin for growth and development. This degradation results in the formation of phenolic compounds such as syringic acid (SA) and vanillic acid (VA). In the artificial culture of edible mushrooms using a mushroom bed, the disposal of waste beds after mushroom cultivation is an important issue. The present study investigated the presence and amount of both SA and VA in the discarded waste beds after mushroom cultivation. The extracts from waste beds after cultivation of shiitake mushrooms, Lentinula edodes; buna shimeji, Hypsizygus marmoreus; maitake, Grifola frondosa; king trumpet mushrooms, Pleurotus eryngii; and butterscotch mushrooms, Pholiota microspora were analyzed using high performance liquid chromatography. Although the content of SA and VA was considerably different among the mushrooms, SA and VA were present in extracts obtained from all the waste beds. We also demonstrated that SA and VA exert their anti-osteoporotic effect independently of the estrogen receptor-mediated pathway using murine monocytic RAW264.7 cells, ovariectomized mice, and human breast cancer MCF-7 cells. Thus, these results suggest that the extracts are effective sources of SA and VA, which are effective in preventing osteoporosis.

Gene cloning of the pink-colored protein from Pleurotus salmoneostramineus (PsPCP) and its species-specific chromoprotein are effective for colorization of the fruit body.

Pleurotus salmoneostramineus is a pink mushroom. This pink color is a protein and forms a complex with 3H-indol-3-one. The gene encoding the pink-colored protein from P. salmoneostramineus (PsPCP) was cloned, and its sequence was elucidated as a 681-bp. The ORF encodes 226 amino acid residues. The amino acid sequence of the protein did not show any significant homology in the DDBJ/EMBL/GenBank databases. Recombinant PsPCP was expressed as the soluble form in E. coli. The reaction mixture of purified recombinant PsPCP and 3H-indol-3-one showed a pink color as the native pigment. A real-time PCR analysis revealed the strong expression of PsPCP in the primordium formation stage of the life cycle of the fungus; however, its expression decreased with the maturation of the fruit body. A comparison of PsPCP gene expression profiles between two strains revealed high levels in the dark-colored strain.

Enzymatic characterization of an extracellular glucoamylase from Tricholoma matsutake and its cloning and secretory expression in Pichia pastoris.

A glucoamylase from the ectomycorrhizal fungus Tricholoma matsutake (TmGLA) was purified 33.2-fold to homogeneity as a single monomeric glycoprotein with a molecular mass of 63.9 kDa. Maximum activity was observed at 60°C and pH 5.0. The enzyme is active down to 50°C and in the pH range of 4.0-6.0, and its activity is strongly inhibited by Ag+. It degrades α-1,4- and α-1,6-glycosidic linkages in various polysaccharides. Its gene (TmGlu1) was cloned using information from the enzyme's internal amino acid sequences and the whole genome sequence of T. matsutake NBRC 30605. The deduced amino acid sequence showed clear homology with those of GH family 15 proteins. Pichia pastoris transformed with TmGlu1 secreted the active enzyme in a glycosylated form, and its characteristics were the same as the native enzyme.

Antiosteoporotic activity of a syringic acid diet in ovariectomized mice.

In recent years, the number of patients with osteoporosis has risen with the increase in average longevity. Therefore, the chemoprevention of osteoporosis using food materials or food components has become an increasingly important target. Syringic acid (SA) is a phenolic compound present in the fruit of the açaí palm Euterpe oleracea and the mycelium of the shiitake mushroom Lentinula edodes. This compound has no affinity for estrogen receptors and is potentially useful for disease prevention. However, little is known about the effects of a SA diet on bone metabolism, particularly bone resorption in vivo. Here, we demonstrated the effects of a SA diet on bone loss and uterine weight loss in ovariectomized (OVX) mice. Ten-week-old OVX mice were fed SA-containing diets (100 mg/kg body weight/day) for 10 weeks. After 10 weeks of dietary SA, the body weight, food intake, and uterine weight of the OVX mice were unaffected; however, femoral bone mineral density (cortical bone density, cancellous bone density, and total bone density) was higher in the SA-fed groups than in the OVX-control group. Furthermore, histomorphometric analysis revealed that the number of osteoclasts and osteoblasts was decreased and increased, respectively, in the SA-fed groups. These results suggest that a SA diet suppresses bone loss by downregulating bone resorption and upregulating bone formation without affecting the uterus in OVX mice. Although further studies are needed, SA may be a compound that can be used to prevent or retard osteoporosis.

Draft Genome Sequence of the Basidiomycetous Fungus Flammulina velutipes TR19.

Here, we report the draft genome sequence of Flammulina velutipes TR19, which was newly isolated from commercial strains in Japan. The genes related to fruiting body formation in the basidiomycete were identified by whole-genome analysis.

Antagonistic activity of endo-ß-1,3-glucanase from a novel isolate, Streptomyces sp. 9X166, against black rot in orchids.

A total of 123 actinomycetes was isolated from 12 varieties of wild orchids and screened for potential antagonistic activity against Phytophthora, which causes black rot disease in orchids. In vitro and in vivo experimental results revealed that Streptomyces sp. strain 9X166 showed the highest antagonistic activity; its ß-1,3-glucanase production ability was a key mechanism for growth inhibition of the pathogen. PCR amplification and DNA sequencing of the 16S ribosomal RNA gene allowed the identification of this strain, with high similarity (99.93%) to the novel species Streptomyces similaensis. The glucanase enzyme, purified to homogeneity by anion exchange and gel filtration chromatography, showed a specific activity of 58 U mg(-1) (a 3.9-fold increase) and yield of 6.4%. The molecular weight, as determined by SDS-PAGE and gel filtration, was approximately 99 and 80 kDa, respectively, suggesting that the enzyme was a monomer. The purified enzyme showed the highest substrate specificity to laminarin, indicating that it was ß-1,3-glucanase. The hydrolyzed products of cello-oligosaccharides suggested that this enzyme was endo-type ß-1,3-glucanase. Streptomyces sp. 9X166 culture filtrate, possessing ß-1,3-glucanase activity, could degrade both freeze-dried and living mycelium. This is the first report on a ß-1,3-glucanase-producing Streptomyces sp. that could be an effective biocontrol agent for black rot disease in orchids.

Inhibitory effects of whisky polyphenols on melanogenesis in mouse B16 melanoma cells.

Whisky exerts an inhibitory effect on melanogenesis in B16 cells, the anti-melanogenic activity being positively correlated with the aging period and anti-oxidative activity of whisky. We examined the correlation between the inhibition of melanogenesis and the concentration of each compound in various whiskies to evaluate the importance of 11 different whisky polyphenols, including ellagic acid, gallic acid and lyoniresinol, in the anti-melanogenic activity of whisky. The concentration of all the compounds was positively correlated with the anti-melanogenic activity of whisky. Ellagic acid, gallic acid and lyoniresinol were the predominant polyphenols in the whiskies measured by HPLC. These three compounds also significantly inhibited the melanogenesis and tyrosinase activity in B16 cells. Ellagic acid, gallic acid and lyoniresinol were confirmed as the major participants in the anti-melanogenic activity of whisky.

Profile of non-volatiles in whisky with regard to superoxide dismutase activity.

SOD (Superoxide dismutase)-like activities of 23 kinds of single malt whisky (Scotch and Japanese) were evaluated. There was a positive correlation between SOD-like activity and the maturation age of whisky that exceeded the difference resulting from the manufacturing region. The SOD-like activity of Yamazaki 18, a typical single malt whisky in Japan, was approximately 1333 U/ml and that of non-volatile components in the whisky was 388U/mg, indicating that single malt whisky generally has a very strong SOD-like activity. To elucidate their contribution to SOD-like activity, the non-volatile components of whisky (Yamazaki 18) were ultrafiltered and separated with a Diaion HP20/water-EtOH system. Elution of the fraction less than 5000 molecular weight (<5000 MW fraction) with 60% (v/v) EtOH contributed most to SOD-like activity of the whisky. As this elution contained a considerable amount of polyphenolics, the content and SOD-like specific activity of ellagic acid, gallic acid, and lyoniresinol--the main whisky polyphenolics--were evaluated. The contribution of these compounds to the SOD-like activity of whisky was approximately 15%. Polyphenolics in whisky were relatively distributed to a higher MW fraction compared to carbohydrates in whisky, and specific activity (SOD-like activity per weight) of the >10,000 MW fraction was greater than that of the <5000 MW fraction, although the content of this fraction was low. These results indicate that various polyphenolics with higher molecular weights also contribute to the SOD-like activity of whisky together with main whisky polyphenolics.

Food restriction improves glucose and lipid metabolism through Sirt1 expression: a study using a new rat model with obesity and severe hypertension.

AIMS: To determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr-fa), as a model of human metabolic syndrome. MAIN METHODS: Male, 10-week-old SHRSP/IDmcr-fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPARα, γ and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle. KEY FINDINGS: FR improved the general conditions as well as blood chemistry of SHRSP/IDmcr-fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPARα and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUT4 expression in skeletal muscle. SIGNIFICANCE: These results indicate that FR corrects adipokine dysfunction by activating PPARγ via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPARα in the liver.

Deciphering pactamycin biosynthesis and engineered production of new pactamycin analogues.

Pactamycin is an aminocyclopentitol-derived natural product that has potent antibacterial and antitumor activities. Sequence analysis of an 86 kb continuous region of the chromosome from Streptomyces pactum ATCC 27456 revealed a gene cluster involved in the biosynthesis of pactamycin. Gene inactivation of the Fe-S radical SAM oxidoreductase (ptmC) and the glycosyltransferase (ptmJ), individually abrogated pactamycin biosynthesis; this confirmed the involvement of the ptm gene cluster in pactamycin biosynthesis. The polyketide synthase gene (ptmQ) was found to support 6-methylsalicylic acid (6-MSA) synthesis in a heterologous host, S. lividans T7. In vivo inactivation of ptmQ in S. pactum impaired pactamycin and pactamycate production but led to production of two new pactamycin analogues, de-6-MSA-pactamycin and de-6-MSA-pactamycate. The new compounds showed equivalent cytotoxic and antibacterial activities with the corresponding parent molecules and shed more light on the structure-activity relationship of pactamycin.

Effects of arachidonic acid analogs on FcepsilonRI-mediated activation of mast cells.

Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) have been shown to modulate a number of inflammatory disorders. Mast cells play a critical role in the initiation and maintenance of inflammatory responses. However, the effects of PUFAs on mast cell functions have not been fully addressed. We here-in examined the effects of PUFAs on the high affinity IgE receptor (FcepsilonRI)-mediated mast cell activation using RBL-2H3 cells, a rat mast cell line, that were cultured in the medium containing palmitic acid (PA), AA, or the AA analogs mead acid (MA) and eicosapentaenoic acid (EPA). In AA-supplemented cells, the FcepsilonRI-mediated beta-hexosamidase and TNF-alpha release, calcium (Ca(2+)) influx, and some protein tyrosine phosphorylations including Syk and linker for activation of T cells (LAT) were enhanced, whereas, in MA- or PA-supplemented cells, they were not changed when compared with cells cultured in control medium. In EPA-supplemented cells, the enhancements of beta-hexosamidase release and protein tyrosine phosphorylations were observed. Furthermore, in AA- or EPA-supplemented cells, FcepsilonRI-mediated intracellular production of reactive oxygen species (ROS) that is required for the tyrosine phosphorylation of LAT and Ca(2+) influx were enhanced when compared with the other cells. Thus, preincubation of AA or EPA augmented FcepsilonRI-mediated degranulation in mast cells by affecting early events of FcepsilonRI signal transduction, which might be associated with the change of fatty acid composition of the cell membrane and enhanced production of ROS. The results suggest that some PUFAs can modulate FcepsilonRI-mediated mast cell activation and might affect FcepsilonRI/mast cell-mediated inflammation, such as allergic reaction.

Horseradish peroxidase degrades lipid hydroperoxides and suppresses lipid peroxidation of polyunsaturated fatty acids in the presence of phenolic antioxidants.

Linoleic acid hydroperoxide (LAOOH) was effectively degraded by horseradish peroxidase (HRP) in the presence of quercetin. Several natural phenolic antioxidants, such as quercetin, capsaicin, and alpha-tocopherol, acted as good hydrogen donors in the peroxidase reaction that occurs during lipid hydroperoxide degradation. However, glutathione, which is a non-phenolic antioxidant that acts as a hydrogen donor for glutathione peroxidase, could not suppress lipid peroxidation in the presence of HRP. Lipid hydroperoxides generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also degraded with HRP in the presence of quercetin, and oxidative decomposition of DHA was suppressed by this reaction.

The effects of maternal mild protein restriction on stroke incidence and blood pressure in stroke-prone spontaneously hypertensive rats (SHRSP).

The effect of maternal protein restriction during pregnancy on the offspring's blood pressure was assessed in stroke-prone spontaneously hypertensive rats (SHRSP) which are genetically predisposed to hypertension and stroke. After the confirmation of pregnancy, the control group was given a 20% casein diet, and the low-protein group was fed a 9% casein diet. After the confirmation of delivery, commercial feed was given to both of the groups. No differences were seen between the control and low-protein offspring in regard to body weight, blood pressure elevation, or life span. One percent saline solution was put in the control and low-protein groups after the age of 11 weeks. Blood pressure increased markedly in the low-protein group, on the blood pressure level in the low-protein group on week 2 after salt loading (242+/-6 mmHg) was significantly higher than that in the control group (223+/-9 mmHg; p<0.05). The survival duration was significantly shorter in the low-protein group (113+/-4 days) than in the control group (135+/-22 days; p<0.05). These results suggest that maternal protein malnutrition in SHRSP exerted a high salt sensitivity and a malignant influence on stroke incidence on offspring.

Microbial synthesis of trans isomer of eicosapentaenoic acid (EPA) from the chemically synthesized trans isomer of linolenic acid by a delta12 desaturase-defective mutant of Mortierella alpina 1S-4.

The mono trans geometrical isomer of eicosapentaenoic acid, 5c,8c,11c,14c,17t-eicosapentaenoic acid (20:5delta5c,8c,11c,14c,17t), was synthesized by fatty acid microbial conversion using a delta12-desaturase defective mutant of an arachidonic acid (AA)-producing fungus, Mortierella alpina 1S-4. The substrate for the bioconversion, a geometrical isomer of linolenic acid, was prepared by isomerization of linseed oil methyl ester by the nitrous acid method, followed by purification on a AgNO3-silica gel column. The structure and double bond geometry were identified after hydrazine reduction followed by permanganate oxidation to 20:5delta5c,8c,11c,14c,17t. The biosynthetic route from 18:3delta6c,9c,12t to 20:5delta5c,8c,11c,14c,17t was presumed to mimic the route from linoleic acid to arachidonic acid.