Effects of heat degradation of betanin in red beetroot (Beta vulgaris L.) on biological activity and antioxidant capacity.

Betanin is a red pigment of red beetroot (Beta vulgaris L.), providing the beneficial effects to maintain human health. Betanin is involved in the characteristic red color of red beetroot, and used as an edible dye. Betanin is known to be a highly unstable pigment, and water solutions of betanin are nearly fully degraded after heating at 99°C for 60 min in the experimental conditions of this study. The present study investigated the effects of red beetroot juice (RBJ) and betanin on immune cells, and found that stimulation with RBJ and betanin induces interleukin (IL)-1ß, IL-8, and IL-10 mRNA in a human monocyte derived cell line, THP-1 cells. This mRNA induction after stimulation with RBJ and betanin was not significantly changed after heat treatment when attempting to induce degradation of the betanin. Following these results, the effects of heat degradation of betanin on the inhibition of lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264 cells and the antioxidant capacity were investigated. The results showed that the inhibition activity of RBJ and betanin with the LPS induced NO production is not altered after heat degradation of betanin. In addition, the results of FRAP (ferric reducing antioxidant power) and DPPH (1,1-Diphenyl-2-picrylhydrazyl) assays indicate that a not inconsiderable degree of the antioxidant capacity of RBJ and betanin remained after heat degradation of betanin. These results suggest that it is important to consider the effects of degradation products of betanin in the evaluation of the beneficial effects of red beetroot on health.

Effects of chondroitin sulfate oligosaccharides on osteoclast differentiation of RAW264 cells, and myotube differentiation of C2C12 cells.

Chondroitin sulfate (CS) is a glycosaminoglycan, and CS derived from various animal species is used in drugs and food supplements to alleviate arthralgia. The CS is a high molecular weight compound, and hydrolysis of CS by intestinal microbiota is thought to be required for absorption in mammalians. Chondroitin sulfate oligosaccharides (Oligo-CS) are produced by hydrolysis with subcritical water from CS isolated from a species of skate, Raja pulchra for the improvement of bioavailability. The present study conducted in vitro experiments using murine cell lines, to compare the biological activities of Oligo-CS and high molecular weight CS composed with the similar disaccharide isomer units of D-glucuronic acid and N-acetyl-D-glucosamine (CS-C). The results show that Oligo-CS inhibits osteoclast differentiation of RAW264 cells significantly at lower concentrations than in CS. The cell viability of a myoblast cell line, C2C12 cells, was increased when the cells were grown in a differentiated medium for myotubes with Oligo-CS, where there were no effects on the cell viability in CS. These results suggest that in vitro Oligo-CS exhibits stronger bioactivity than high-molecular weight CS.

In vitro anti-inflammatory and anti-lipid accumulation properties of taxifolin-rich extract from the Japanese larch, Larix kaempferi.

The Japanese larch, (Larix kaempferi) is known to contain abundant taxifolin (dihydroquercetin) in its xylem. In this study, to assess the bioactivities of taxifolin rich methanol extract of L. kaempferi (LK-ME), anti-inflammatory effect, and the anti-lipid accumulation effect of LK-ME were investigated. The results showed that nitric oxide (NO) and reactive oxygen species (ROS) were reduced after treatment with LK-ME, and that lipid accumulation in adipocyte differentiated 3T3-L1 cells was inhibited after the cells were grown in medium containing LK-ME. Taxifolin, the major compound contained in LK-ME, and its related compounds, quercetin and luteolin also exhibited similar effects with LK-ME. The LK-ME exhibits relatively strong anti-inflammatory and anti-lipid accumulation activities compared with that of similar amounts of taxifolin contained in LK-ME, suggesting that other minor compounds contained in LK-ME is involved in the effects. These results indicate the potential of taxifolin-rich L. kaempferi extract for use as a supplement to prevent excess inflammation and obesity.

Cell cytotoxity and anti-glycation activity of taxifolin-rich extract from Japanese larch, Larix kaempferi.

The larches, the Larix genus of plants are known as a natural source of taxifolin (dihydroquercetin), and extracts of its taxifolin rich xylem are used in dietary supplements to maintain health. In the present study, to assess biological activities of a methanol extract of the Japanese larch, Larix kaempferi (LK-ME), the effects of LK-ME on cell viability, inflammatory cytokine expression, and glycation were investigated. The effects of taxifolin which is known to be a main compound of LK-ME, and its related flavonoids, quercetin and luteolin were also examined. The results show that taxifolin exhibits lower growth inhibition activity and lesser induction activity of inflammatory cytokines in a human monocyte derived cell line, THP-1 cells, while in vitro anti-glycation activities of taxifolin were inhibiting at comparable levels to those of quercetin and luteolin. The growth inhibition and the cytokine induction activities, and the anti-glycation effects of LK-ME are assumed to have properties similar to taxifolin. The results of high performance liquid chromatography (HPLC) analysis indicated that taxifolin was detected as the main peak of LK-ME at the absorbance of 280 nm, and the concentration of taxifolin was measured as 3.12 mg/ml. The actual concentration of taxifolin in LK-ME is lower than the concentration estimated from the IC50 values calculated by the results of glycation assays, suggesting that other compounds contained in LK-ME are involved in the anti-glycation activity.

The effects of gene disruption of Kre6-like proteins on the phenotype of ß-glucan-producing Aureobasidium pullulans.

Killer toxin resistant 6 (Kre6) and its paralog, suppressor of Kre null 1 (Skn1), are thought to be involved in the biosynthesis of cell wall ß-(1 → 6)-D-glucan in baker's yeast, Saccharomyces cerevisiae. The Δkre6Δskn1 mutant of S. cerevisiae and other fungi shows severe growth defects due to the failure to synthesize normal cell walls. In this study, two homologs of Kre6, namely, K6LP1 (Kre6-like protein 1) and K6LP2 (Kre6-like protein 2), were identified in Aureobasidium pullulans M-2 by draft genome analysis. The Δk6lp1, Δk6lp2, and Δk6lp1Δk6lp2 mutants were generated in order to confirm the functions of the Kre6-like proteins in A. pullulans M-2. The cell morphologies of Δk6lp1 and Δk6lp1Δk6lp2 appeared to be different from those of wild type and Δk6lp2 in both their yeast and hyphal forms. The productivity of the extracellular polysaccharides, mainly composed of ß-(1 → 3),(1 → 6)-D-glucan (ß-glucan), of the mutants was 5.1-17.3% less than that of wild type, and the degree of branching in the extracellular ß-glucan of mutants was 14.5-16.8% lower than that of wild type. This study showed that the gene disruption of Kre6-like proteins affected the cell morphology, the productivity of extracellular polysaccharides, and the structure of extracellular ß-glucan, but it did not have a definite effect on the cell viability even in Δk6lp1Δk6lp2, unlike in the Δkre6Δskn1 of S. cerevisiae.

Oral administration of the Aureobasidium pullulans-derived ß-glucan effectively prevents the development of high fat diet-induced fatty liver in mice.

Aureobasidium pullulans-derived ß-glucan (AP-PG) consisting of a ß-(1,3)-linked glucose main chain and ß-(1,6)-linked glucose branches is taken as a supplement to improve health. This study demonstrates that oral administration of AP-PG is effective to prevent the development of high-fat diet (HFD)-induced fatty liver in mice. Here, C57BL/6N mice were fed with a normal diet or HFD, and AP-PG diluted in drinking water was administered orally. After 16 weeks, the serological analysis showed that HFD-induced high blood cholesterol and triglyceride levels were reduced by the oral administration of AP-PG. Further, HFD induced-fatty liver was significantly reduced by the oral administration of AP-PG. The triglyceride accumulation in the liver was also significantly reduced in mice administered AP-PG. Liver injury as indicated by an increase in serum alanine aminotransferase (ALT) in the HFD-fed mice was significantly reduced in the mice administered AP-PG orally, and the gene expression of cholesterol 7 alpha-hydroxylase (CYP7A1) which is known to be involved in cholesterol degradation in the liver was significantly increased in the AP-PG administered mice. These results suggest the possibility that the oral administration of AP-PG is effective to prevent the development of non-alcoholic fatty liver disease (NAFLD).

Stimulation of macrophages with the ß-glucan produced by aureobasidium pullulans promotes the secretion of tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

A ß-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a ß-(1,3)-linked main chain with ß-(1,6)-linked glucose side residues. Various ß-glucans consisting of ß-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial ß-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.

Stimulation with the Aureobasidium pullulans-produced ß-glucan effectively induces interferon stimulated genes in macrophage-like cell lines.

A ß-(1,3),(1,6)-D-glucan produced by A. pullulans (AP-PG) is known to be an immune stimulating agent. In this study, we demonstrate that the stimulation with AP-PG effectively induces the interferon (IFN) stimulated genes (ISGs) in macrophage-like cell lines. The ISGs, Mx1, ISG15, and viperin mRNAs were significantly increased in RAW264.7 cells after stimulation with AP-PG. The stimulation with AP-PG transiently induced IFN-ß mRNA. However, the expression of viperin mRNA was also increased after stimulation with AP-PG even when new protein synthesis was completely blocked by treatment with cycloheximide. Further, in IFN-α receptor knockdown RAW264.7 cells, AP-PG stimulation more effectively induced viperin mRNA compared with that of IFN-α stimulation. The phosphorylation of Ser 727 in STAT1 involved in the enhancement of STAT1 activation was immediately increased after stimulation with AP-PG. In addition, viperin mRNA expression induced after stimulation with IFN-α was significantly increased by combined stimulation with AP-PG. These results suggest that stimulation with AP-PG effectively induces the ISGs through the induction of IFN and the enhancement of STAT1-mediated transcriptional activation.

A small scale study on the effects of oral administration of the ß-glucan produced by Aureobasidium pullulans on milk quality and cytokine expressions of Holstein cows, and on bacterial flora in the intestines of Japanese black calves.

BACKGROUND: The ß-(1 → 3),(1 → 6)-D-glucan extracellularly produced by Aureobasidium pullulans exhibits immunomodulatory activity, and is used for health supplements. To examine the effects of oral administration of the ß-(1 → 3),(1 → 6)-D-glucan to domestic animals, a small scale study was conducted using Holstein cows and newborn Japanese Black calves. FINDINGS: Holstein cows of which somatic cell count was less than 3 x 105/ml were orally administered with or without the ß-(1 → 3),(1 → 6)-D-glucan-enriched A. pullulans cultured fluid (AP-CF) for 3 months, and the properties of milk and serum cytokine expression were monitored. Somatic cell counts were not significantly changed by oral administration of AP-CF, whereas the concentration of solid non fat in the milk tended to increase in the AP-CF administered cows. The results of cytokine expression analysis in the serum using ELISA indicate that the expressions of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in all cows which were orally administered with AP-CF became slightly lower than that of control cows after the two-month treatment. On the other hand, IL-8 expression tended to indicate a moderately higher level in all treated cows after the three-month administration of AP-CF in comparison with that of the control cows. Peripartum Japanese Black beef cows and their newborn calves were orally administered with AP-CF, and bacterial flora in the intestines of the calves were analyzed by T-RFLP (terminal restriction fragment length polymorphism). The results suggest that bacterial flora are tendentiously changed by oral administration of AP-CF. CONCLUSIONS: Our data indicated the possibility that oral administration of the ß-(1 → 3),(1 → 6)-D- glucan produced by A. pullulans affects cytokine expressions in the serum of Holstein cows, and influences bacterial flora in the intestines of Japanese Black calves. The findings may be helpful for further study on the efficacies of oral administration of ß-(1 → 3),(1 → 6)-D-glucans on domestic animals.

Expression of Ca²âº-dependent activator protein for secretion 2 is increased in the brains of schizophrenic patients.

Ca(2+)-dependent activator protein for secretion 2 (CADPS2), a secretory granule associate protein, mediates monoamine transmission and the release of neurotrophins including brain-derived neurotrophic factor (BDNF) which have been implicated in psychiatric disorders. Furthermore, the expression of CADPS2deltaExon3, a defective splice variant of CADPS2, has been reported to be associated with autism. Based on these observations, we examined whether expression levels of CADPS2 and CADPS2deltaExon3 are altered in psychiatric disorders. Quantitative polymerase chain reaction analysis was performed for postmortem frontal cortex tissues (BA6) from 15 individuals with schizophrenia, 15 with bipolar disorder, 15 with major depression, and 15 controls (Stanley neuropathology consortium). The mean CADPS2 expression levels normalized to human glyceraldehyde-3phosphate dehydrogenase (GAPDH) or TATA-box binding protein levels was found to be significantly increased in the brains of the schizophrenia group, compared to the control group. On the other hand, the ratio of CADPS2deltaExon3 to total CADPS2 was similar in the 4 diagnostic groups. We then analyzed CADPS2 expression in blood samples from 121 patients with schizophrenia and 318 healthy controls; however, there was no significant difference between the two groups. Chronic risperidone treatment did not alter the expression of CADPS2 in frontal cortex of mice. The observed increase in the expression of CADPS2 may be related to the impaired synaptic function in schizophrenia.

Possible association of the semaphorin 3D gene (SEMA3D) with schizophrenia.

Semaphorins are ligands of plexins, and the plexin-semaphorin signaling system is widely involved in many neuronal events including axon guidance, cell migration, axon pruning, and synaptic plasticity. The plexin A2 gene (PLXNA2) has been reported to be associated with schizophrenia. This finding prompted us to examine the possible association between the semaphorin 3D gene (SEMA3D) and schizophrenia in a Japanese population. We genotyped 9 tagging single nucleotide polymorphisms (SNPs) of SEMA3D including a non-synonymous variation, Lys701Gln (rs7800072), in a sample of 506 patients with schizophrenia and 941 healthy control subjects. The Gln701 allele showed a significant protective effect against the development of schizophrenia (p = 0.0069, odds ratio = 0.76, 95% confidence interval 0.63 to 0.93). Furthermore, the haplotype-based analyses revealed a significant association. The four-marker analysis (rs2190208-rs1029564-rs17159614-rs12176601), in particular, not including the Lys701Gln, revealed a highly significant association (p = 0.00001, global permutation), suggesting that there may be other functional polymorphisms within SEMA3D. Our findings provide strong evidence that SEMA3D confers susceptibility to schizophrenia, which could contribute to the neurodevelopmental impairments in the disorder.

Identification of the 4-hydroxycinnamate decarboxylase (PAD) gene of Klebsiella oxytoca.

A 7.1-kbp DNA fragment isolated from a wild strain of Klebsiella oxytoca was sequenced, leading to the identification of 10 open-reading frames (ORFs), including a 504-bp Pad gene. The Pad gene of the Gram-negative bacterium was subsequently expressed in Escherichia coli as a chimeric Pad. The deduced amino acid (AA) sequence of the Pad gene from wild-type K. oxytoca showed approximately 50% homology to those of other bacterial PADs from Gram-positive bacilli plus a coccus. These data and a genomic library search of some gamma-proteobacteria, including E. coli and Vibrio sp., indicated that PAD of K. oxytoca is a member of the bacterial PAD family characteristic of Gram-negative bacteria. Using Pad-specific PCR primers designed from the Gram-negative bacterial Pad of K. oxytoca, Pad genes of two further strains of K. oxytoca, another wild isolate and JCM 1665 and two PAD-positive Enterobacter spp. were successfully amplified for specific Pad detection.