Polysaccharides from a Fermented Beverage Induce Nitric Oxide and Cytokines in Murine Macrophage Cell Line.

Super Ohtaka®, a fermented beverage of plant extracts, is prepared from approximately 50 kinds of fruits and vegetables. Natural fermentation is mainly performed by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp.). Four water-soluble polysaccharide fractions were obtained from Super Ohtaka® by dialysis, ion exchange chromatography, and gel filtration chromatography; these fractions were designated as OEP1-1, OEP1-2, OEP2, and OEP3. OEP1-1 is a polysaccharide composed solely of glucose. The other fractions contained polysaccharides composed of glucose, galactose, mannose, and a small amount of arabinose. OEP2 and OEP3 contained phosphorus, which was not detected in OEP1-1 and OEP1-2. Furthermore, the immunomodulatory activity of the polysaccharides was investigated in murine macrophage cell lines. OEP2 and OEP3 significantly induced nitric oxide (NO) secretion by macrophages in a dose-dependent manner (concentration range of 4 to 100 µg/mL). When the concentration of OEP3 was 100 µg/mL, NO production was almost identical to lipopolysaccharide (LPS; 10 ng/mL) used as a positive control. Notably, OEP3 induced NO secretion more strongly than OEP2. This trend was also observed for TNF-α, IL-1ß, IL-6, and IL-12 p40 secretion. Overall, our in vitro studies on polysaccharides isolated from Super Ohtaka® suggest that the fermented beverage stimulates macrophages and activates the immune system.

Regulation of MEK inhibitor selumetinib sensitivity by AKT phosphorylation in the novel BRAF L525R mutant.

BACKGROUND: Oncogenic mutations in BRAF genes are found in approximately 5-10% of colorectal cancers. The majority of BRAF mutations are located within exons 11-15 of the catalytic kinase domains, with BRAF V600E accounting for more than 80% of the observed BRAF mutations. Sensitivity to BRAF- and mitogen-activated protein kinase (MEK) inhibitors varies depending on BRAF mutations and tumor cell types. Previously, we newly identified, BRAF L525R-mutation, in the activation segment of the kinase in colorectal cancer patient. Here, we characterized the function of the BRAF L525R mutation. METHODS: HEK293 cells harboring a BRAF mutation (V600E or L525R) were first characterized and then treated with cetuximab, dabrafenib, and selumetinib. Cell viability was measured using WST-1 assay and the expression of proteins involved in the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways was evaluated using western blot analysis. RESULTS: The MEK inhibitor selumetinib effectively inhibited cell proliferation and ERK phosphorylation in BRAF L525R cells but not in BRAF V600E cells. Further studies revealed that AKT phosphorylation was reduced by selumetinib in BRAF L525R cells but not in BRAF V600E cells or selumetinib-resistant BRAF L525R cells. Moreover, the AKT inhibitor overcame the selumetinib resistance. CONCLUSIONS: We established a model system harboring BRAF L525R using HEK293 cells. BRAF L525R constitutively activated ERK. AKT phosphorylation caused sensitivity and resistance to selumetinib. Our results suggest that a comprehensive network analysis may provide insights to identify effective therapies.

Structural Analysis of Injection-Molded Polyoxymethylene Treated Below a Melting Point Using Field-Emission Scanning Electron Microscopy and Infrared Spectroscopy.

The heat treatment of an injection-molded polyoxymethylene slightly below the melting point and subsequent isothermal treatment at 130 °C were performed. The polyoxymethylene structure was examined using field-emission scanning electron microscopy and polarization infrared reflection measurements. After the heat treatment, a significant change in the surface morphology was observed, and the reflection spectrum derived from the polariton in the injection direction also changed dramatically. Since the reflection spectrum in the injection direction contains the reflection component of the perpendicular direction and vice versa, the polarization spectra of both directions can be calculated consistently. The mixing ratio of each crossed component and the pure relative permittivity both parallel and perpendicular to the main-chain direction were determined using the oscillator model. The heat treatment reduced the ratio of the perpendicular component and increased the order structure until just before melting. The structural changes characterized by the two techniques, along with Raman spectroscopy and differential scanning calorimetry, are discussed.

Analysis of Clinical Isolates of Extended-Spectrum ß-Lactamase-Producing Bacteria with Primer and Probe Sets Developed to Detect blaCTX-M, blaTEM, and blaSHV Using a Fully Automated Gene Detection System.

In this study, we evaluated extended-spectrum ß-lactamase (ESBL)-producing bacteria with the newly developed primer and probe sets to detect blaCTX-M, blaTEM, and blaSHV using BD MAXTM, a fully automated multiplex polymerase chain reaction assay system. In 36 isolates confirmed by whole-genome sequencing to have blaCTX-M, blaTEM, or blaSHV, the developed primer and probe sets accurately detected each gene without being influenced by the presence of other ß-lactamase genes. In nine control strains that do not harbor either blaCTX-M, blaTEM, or blaSHV no cross-reaction was observed. In 191 strains phenotypically determined to be ESBL-producers by conventional antimicrobial susceptibility tests, 189 strains were blaCTX-M-, blaTEM-, or blaSHV-positive as assessed by BD MAXTM using the developed primer and probe sets, and two strains were negative for these genes. Whole-genome sequencing revealed that these two strains were phenotypically false-positive ESBL-producers. The accuracy of the primer and probe sets seems to be satisfactory, and they may be applicable to detect CTX-M-type ESBL-producing bacteria.

A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

Structural Analysis of Novel Low-Digestible Sucrose Isomers Synthesized from D-Glucose and D-Fructose by Thermal Treatment.

The synthesis of the saccharide ß-D-fructopyranosyl-(2→6)-D-glucopyranose, which was isolated from Super Ohtaka®, has recently been reported. During the synthesis of this saccharide, the formation of two novel saccharides from D-glucose and D-fructose was observed. The present study aimed to confirm the structures of the two disaccharides synthesized from D-glucose and D-fructose by thermal treatment. Furthermore, various properties of the saccharides were investigated. Both saccharides were isolated from the reaction mixture by carbon-Celite column chromatography and an HPLC system and were determined to be novel sucrose-isomers, ß-D-fructopyranosyl-(2↔1)-ß-D-glucopyranoside (1) and ß-D-fructofuranosyl-(2↔1)-ß-D-glucopyranoside (2), by MALDI-TOF MS and NMR analyses. Both saccharides showed low digestibility in vitro, and the sweetness of saccharide 2 was 0.45 times that of sucrose.

Structural Analysis of a Novel Oligosaccharide Isolated from Fermented Beverage of Plant Extracts.

A fermented beverage of plant extracts (Super Ohtaka®) was prepared from about 50 kinds of fruits and vegetables. This natural fermentation was performed by yeast (Zygosaccharomyces spp. and Pichia spp.) and lactic acid bacteria (Leuconostoc spp.) and resulted in the production of a novel fructopyranose-containing saccharide, which was subsequently isolated using carbon-Celite column chromatography and preparative-HPLC. The structure of the saccharide was determined using MALDI-TOF MS and NMR, and the saccharide was identified as ß-D-fructopyranosyl-(2→6)-ß-D-fructofuranosyl-(2↔1)-α-D-glucopyranoside. This is the first description of this novel saccharide and its isolation from a natural source.

Cancer Cell Interaction with Adipose Tissue: Correlation with the Finding of Spiculation at Mammography.

PURPOSE: To investigate the relationship between spiculated masses at mammography and marginal adipose tissue invasion at histologic examination. MATERIALS AND METHODS: The institutional review board approved this retrospective study, and the requirement to obtain informed consent was waived. A total of 478 patients with invasive breast cancer who underwent surgery between 1999 and 2009 were included in this study. Clinical-pathologic findings from patients with spiculated masses on mammograms were compared with those from patients without spiculated masses by using logistic regression models, Cox proportional hazards regression models, and the Kaplan-Meier method. RESULTS: There were 136 spiculated tumors and 342 nonspiculated tumors. All 136 spiculated tumors (100%) were positive for adipose tissue invasion, whereas only 264 of the 342 nonspiculated tumors (77%) were positive for adipose tissue invasion (P < .001). Multivariate analysis revealed that adipose tissue invasion (P < .001), histologic grade (P < .001), dense breast (P = .002), and body mass index (P = .02) were independent factors associated with spiculation. With regard to survival, although many patients with spiculated tumors had a hormone-sensitive (estrogen receptor positive: P = .004; progesterone receptor positive: P = .001) or low-grade (P < .001) tumor, in contrast to the patients without spiculated masses, the prognosis in the two groups was similar (disease-free survival: P = .09; overall survival: P = .23). CONCLUSION: Cancer cell interaction with adipose tissue is crucial for the finding of spiculation at mammography.

Comparison of follicular fluid and serum anti-Mullerian hormone levels as predictors of the outcome of assisted reproductive treatment.

OBJECTIVE: To compare follicular fluid and serum concentrations of anti-Mullerian hormone (AMH) as predictors of the outcome of assisted reproduction. STUDY DESIGN: This observational study enrolled 58 women who were undergoing IVF or ICSI treatment with the long stimulation protocol. Patients diagnosed as having PCOS were excluded. Serum and follicular fluid AMH levels were assessed as predictors of clinical pregnancy. RESULTS: Both the serum and follicular fluid AMH levels were higher in the clinical pregnancy group than in the failed group. A significant correlation was found between the serum and follicular fluid AMH levels, but a discrepancy was observed in some patients with elevated AMH levels in the follicular fluid or serum alone. Assisted reproductive treatment resulted in clinical pregnancy in all of the patients with elevated AMH levels in the follicular fluid (>40pM) or in the serum (>10pM). The ROC-AUC for the combination of follicular and serum AMH was 0.772, which was relatively higher than that for either the serum AMH (AUC: 0.691) or follicular fluid AMH (AUC: 0.688) alone. CONCLUSION(S): Elevated AMH levels in either the serum or follicular fluid appeared to be predictive of clinical pregnancy, even if AMH levels in other fluids were low. This is a pilot study with preliminary data that need further confirmation.

Practical capability and cost effectiveness of a DNA pool-based genome-wide association study using BovineSNP50 array in a cattle population.

Genome-wide association mapping for complex traits in cattle populations is a powerful, but expensive, selection tool. The DNA pooling technique can potentially reduce the cost of genome-wide association studies. However, in DNA pooling design, the additional variance generated by pooling-specific errors must be taken into account. Therefore, this study aimed to investigate factors such as: (i) the accuracy of allele frequency estimation; (ii) the magnitude of errors in pooling construction and in the array; and (iii) the effect of the number of replicate arrays on P-values estimated by a genome-wide association study. Results showed that the Illumina correction method is the most effective method to correct the allele frequency estimation; pooling errors, especially array variance, should be taken into account in DNA pooling design; and the risk of a type I error can be reduced by using at least two replicate arrays. These results indicate the practical capability and cost-effectiveness of pool-based genome-wide association studies using the BovineSNP50 array in a cattle population.

Phenylbutenoid dimers isolated from Zingiber purpureum exert neurotrophic effects on cultured neurons and enhance hippocampal neurogenesis in olfactory bulbectomized mice.

Trans-3-(3'4'-dimethoxyphenyl)-4-[(E)-3",4"-dimethoxystyryl]cyclohex-1-ene (Comp.1) and cis-3-(3'4'-dimethoxyphenyl)-4-[(E)-3",4"-dimethoxystyryl]cyclohex-1-ene (Comp.2), phenylbutenoid dimers, have been isolated as neurotrophic molecules from an Indonesian medicinal plant, Zingiber purpureum. The aim of this study was to explore the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. Comp.1 (10-30 µM) or Comp.2 (30 µM) significantly induced neurite sprouting in PC12 cells. Comp.1 (0.03-3 µM) or Comp.2 (0.3-3 µM) significantly increased the neurite length and number of neurites in primary cultured rat cortical neurons. Comp.1 (30 µM) and Comp.2 (3-30 µM) also provided significant protection against cell death caused by deprivation of serum. The in vivo effects of both Comp.1 and Comp.2 were evaluated on hippocampal neurogenesis in olfactory bulbectomized (OBX) mice, an experimental depression and dementia animal model. Comp.1 (50mg/kg p.o.), Comp.2 (50mg/kg p.o.), or fluoxetine (10mg/kg i.p.), an antidepressant, were administrated once a day on days 15-28 after OBX. Neurogenesis was assessed by analysis of cells expressing NeuN, a neuronal marker, and 5-bromo-2'-deoxyuridine (BrdU) uptake. Immunohistochemical analysis showed that the number of BrdU/NeuN double-labeled cells in the dentate gyrus was significantly decreased 30 days after OBX. Chronic treatment with Comp.1, Comp.2 or fluoxetine significantly increased the number of BrdU/NeuN double-labeled cells. These results indicate that Comp.1 and Comp.2 have neurotrophic effects, and have the potential for disease modification in depression and dementia.

Isolation and structural confirmation of the oligosaccharides containing α-D-fructofuranoside linkages isolated from fermented beverage of plant extracts.

Fermented beverage of plant extracts was prepared from the extracts of approximately 50 types of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two oligosaccharides containing an α-fructofuranoside linkage were detected in this beverage and isolated using carbon-Celite column chromatography and preparative HPLC. The structural confirmation of the saccharides was determined by methylation analysis, MALDI-TOF-MS, and NMR measurements. These saccharides were identified as α-D-fructofuranosyl-(2→6)-D-glucopyranose, which was isolated from a natural source for the first time, and a novel saccharide ß-D-fructopyranosyl-(2→6)-α-D-fructofuranosyl-(2↔1)-α-D-glucopyranoside.

Synthesis of ß-D-fructopyranosyl-(2→6)-D-glucopyranose from D-glucose and D-fructose by a thermal treatment.

The synthesis is reported of ß-D-fructopyranosyl-(2→6)-D-glucopyranose that had previously been isolated from a fermented plant extract as a new saccharide. A disaccharide was predominately formed from an equal amount of D-glucose and D-fructose under melting conditions at 140 °C for 60 to 90 min. This saccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative HPLC, and was confirmed to be ß-D-fructopyranosyl-(2→6)-D-glucopyranose by TOF-MS and NMR analyses.

Novel fructopyranose oligosaccharides isolated from fermented beverage of plant extract.

Four oligosaccharides containing a fructopyranosyl residue have been found from fermented beverage of plant extract and isolated from the beverage using carbon-Celite column chromatography and preparative high performance liquid chromatography. Structure confirmation of the saccharides was provided by methylation analysis, MALDI-TOF-MS and NMR measurements. These saccharides were identified as oligosaccharides of fructopyranoside series; beta-D-fructopyranosyl-(2-->6)-D-fructofuranose (1), beta-D-fructopyranosyl-(2-->1)-D-fructopyranose (2), beta-D-fructopyranosyl-(2-->1)-beta-D-fructofuranosyl-(2<-->1)-alpha-D-glucopyranoside (3), and beta-D-fructopyranosyl-(2-->6)-alpha-D-glucopyranosyl-(1<-->2)-beta-D-fructofuranoside (4). Saccharides 3 and 4 among novel saccharides 1, 3, and 4 were named 'pyrano-1-kestose (pyrano-isokestose)' and 'pyrano-neokestose', respectively.

Magnolol and honokiol prevent learning and memory impairment and cholinergic deficit in SAMP8 mice.

The therapeutic use of neurotrophic factors to treat neurodegenerative disorders, including Alzheimer's disease, is considered feasible. Magnolol and honokiol, constituents of the Magnolia plant, are small organic compounds with neurotrophic activity. We investigated whether magnolol and honokiol can prevent age-related learning and memory impairment and cholinergic deficits in senescence-accelerated mice (SAM). Magnolol (1, 10 mg/kg) or honokiol (0.1, 1 mg/kg) were orally administered to SAMP8 mice once a day for 14 days in 2-month-old mice. Learning and memory performance were evaluated by passive avoidance tests and location and object novelty recognition tests. SAMP8 mice showed significant impairment of learning and memory at 4 and 6 months of age. This age-related learning and memory impairment was prevented by pretreatment with either magnolol (10 mg/kg) or honokiol (1 mg/kg). Cholinergic neuron densities in the medial septum and vertical limb of the diagonal band of the forebrain were evaluated by an immunohistochemical analysis of choline acetyltransferase (ChAT). SAMP8 mice showed a significant cholinergic deficit at 6 months of age. These age-related cholinergic deficits were prevented by treatment with either magnolol (10 mg/kg) or honokiol (1 mg/kg). Moreover, SAMP8 mice showed decreased activity of Akt, a member of the prosurvival pathway, in the forebrain at 2 months of age. A 14-day treatment with either magnolol (10 mg/kg) or honokiol (1 mg/kg) enhanced phosphorylation of Akt in the forebrain at 2 months of age. These results suggest that magnolol and honokiol prevent age-related learning and memory impairment by preserving cholinergic neurons in the forebrain. These compounds may have potential therapeutic applications to various neurodegenerative disorders.

Structural analysis of three novel trisaccharides isolated from the fermented beverage of plant extracts.

BACKGROUND: A fermented beverage of plant extracts was prepared from about fifty kinds of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). We have previously examined the preparation of novel four trisaccharides from the beverage: O-beta-D-fructopyranosyl-(2->6)-O-beta-D-glucopyranosyl-(1->3)-D-glucopyranose, O-beta-D-fructopyranosyl-(2->6)-O-[beta-D-glucopyranosyl-(1->3)]-D-glucopyranose, O-beta-D-glucopyranosyl-(1->1)-O-beta-D-fructofuranosyl-(2<->1)-alpha-D-glucopyranoside and O-beta-D-galactopyranosyl-(1->1)-O-beta-D-fructofuranosyl-(2<->1)- alpha-D-glucopyranoside. RESULTS: Three further novel oligosaccharides have been found from this beverage and isolated from the beverage using carbon-Celite column chromatography and preparative high performance liquid chromatography. Structural confirmation of the saccharides was provided by methylation analysis, MALDI-TOF-MS and NMR measurements. CONCLUSION: The following novel trisaccharides were identified: O-beta-D-fructofuranosyl-(2->1)-O-[beta-D-glucopyranosyl-(1->3)]-beta-D-glucopyranoside (named "3G-beta-D-glucopyranosyl beta, beta-isosucrose"), O-beta-D-glucopyranosyl-(1->2)-O-[beta-D-glucopyranosyl-(1->4)]-D-glucopyranose (4(1)-beta-D-glucopyranosyl sophorose) and O-beta-D-fructofuranosyl-(2->6)-O-beta-D-glucopyranosyl-(1->3)-D-glucopyranose (6(2)-beta-D-fructofuranosyl laminaribiose).

Nodular fasciitis with degeneration and regression.

Nodular fasciitis is a benign reactive proliferation that is frequently misdiagnosed as a sarcoma. This article describes a case of nodular fasciitis of 6-month duration located in the cheek, which degenerated and spontaneously regressed after biopsy. The nodule was fixed to the zygoma but was free from the overlying skin. The mass was 3.0 cm in diameter and demonstrated high signal intensity on T2-weighted magnetic resonance imaging. A small part of the lesion was biopsied. Pathological and immunohistochemical examinations identified the nodule as nodular fasciitis with myxoid histology. One month after the biopsy, the mass showed decreased signal intensity on T2-weighted images and measured 2.2 cm in size. The signal on T2-weighted images showed time-dependent decreases, and the mass continued to reduce in size throughout the follow-up period. The lesion presented as hypointense to the surrounding muscles on T2-weighted images and was 0.4 cm in size at 2 years of follow-up. This case demonstrates that nodular fasciitis with myxoid histology can change to that with fibrous appearance gradually with time, thus bringing about spontaneous regression. Degeneration may be involved in the spontaneous regression of nodular fasciitis with myxoid appearance. The mechanism of regression, unclarified at present, should be further studied.

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract.

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.

Structural analysis of a novel saccharide isolated from fermented beverage of plant extract.

Fermented beverage of plant extract was prepared from about 50 kinds of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Three kinds of saccharides have been found in this beverage and produced by fermentation. The saccharides isolated from the beverage using carbon-Celite column chromatography and preparative HPLC, were identified as a new saccharide, beta-d-fructopyranosyl-(2-->6)-d-glucopyranose, laminaribiose and maltose by examination of constituted sugars, GLC and GC-MS analyses of methyl derivatives and MALDI-TOF-MS and NMR measurements of the saccharides.

Identification and functional analysis of genes required for desulfurization of alkyl dibenzothiophenes of Mycobacterium sp. G3.

Mycobacterium sp. G3 was reported as a dibenzothiophene (DBT)-degrading microorganism and the first strain to have the ability to degrade high-molecular-weight alkyl DBTs, such as 4,6-dibutyl DBT and 4,6-dipentyl DBT, by the C-S bond cleavage pathway. Three genes (mdsA, mdsB, and mdsC) for desulfurization, which form a cluster, were cloned from Mycobacterium sp. G3. The expression of each gene in Escherichia coli JM109 showed that MdsC oxidized DBT to DBT sulfone, MdsA transformed DBT sulfone into 2-(2'-hydroxyphenyl)benzene sulfinate (HPBS), and MdsB desulfinated HPBS into 2-hydroxybiphenyl (HBP), indicating that the gene products of mdsABC are functional in the recombinant. MdsC oxidized 4,6-dimethyl DBT, 4,6-diethyl DBT, 4,6-dipropyl DBT and 4,6-dibutyl DBT to each sulfone form, suggesting that MdsC covers a broad specificity for alkyl DBTs.