Optimization of prediction methods for risk assessment of pathogenic germline variants in the Japanese population.

Predicting pathogenic germline variants (PGVs) in breast cancer patients is important for selecting optimal therapeutics and implementing risk reduction strategies. However, PGV risk factors and the performance of prediction methods in the Japanese population remain unclear. We investigated clinicopathological risk factors using the Tyrer-Cuzick (TC) breast cancer risk evaluation tool to predict BRCA PGVs in unselected Japanese breast cancer patients (n = 1,995). Eleven breast cancer susceptibility genes were analyzed using target-capture sequencing in a previous study; the PGV prevalence in BRCA1, BRCA2, and PALB2 was 0.75%, 3.1%, and 0.45%, respectively. Significant associations were found between the presence of BRCA PGVs and early disease onset, number of familial cancer cases (up to third-degree relatives), triple-negative breast cancer patients under the age of 60, and ovarian cancer history (all P < .0001). In total, 816 patients (40.9%) satisfied the National Comprehensive Cancer Network (NCCN) guidelines for recommending multigene testing. The sensitivity and specificity of the NCCN criteria for discriminating PGV carriers from noncarriers were 71.3% and 60.7%, respectively. The TC model showed good discrimination for predicting BRCA PGVs (area under the curve, 0.75; 95% confidence interval, 0.69-0.81). Furthermore, use of the TC model with an optimized cutoff of TC score ≥0.16% in addition to the NCCN guidelines improved the predictive efficiency for high-risk groups (sensitivity, 77.2%; specificity, 54.8%; about 11 genes). Given the influence of ethnic differences on prediction, we consider that further studies are warranted to elucidate the role of environmental and genetic factors for realizing precise prediction.

A Single-Batch Fermentation System to Simulate Human Colonic Microbiota for High-Throughput Evaluation of Prebiotics.

We devised a single-batch fermentation system to simulate human colonic microbiota from fecal samples, enabling the complex mixture of microorganisms to achieve densities of up to 1011 cells/mL in 24 h. 16S rRNA gene sequence analysis of bacteria grown in the system revealed that representatives of the major phyla, including Bacteroidetes, Firmicutes, and Actinobacteria, as well as overall species diversity, were consistent with those of the original feces. On the earlier stages of fermentation (up to 9 h), trace mixtures of acetate, lactate, and succinate were detectable; on the later stages (after 24 h), larger amounts of acetate accumulated along with some of propionate and butyrate. These patterns were similar to those observed in the original feces. Thus, this system could serve as a simple model to simulate the diversity as well as the metabolism of human colonic microbiota. Supplementation of the system with several prebiotic oligosaccharides (including fructo-, galacto-, isomalto-, and xylo-oligosaccharides; lactulose; and lactosucrose) resulted in an increased population in genus Bifidobacterium, concomitant with significant increases in acetate production. The results suggested that this fermentation system may be useful for in vitro, pre-clinical evaluation of the effects of prebiotics prior to testing in humans.

Catechins in tea suppress the activity of cytochrome P450 1A1 through the aryl hydrocarbon receptor activation pathway in rat livers.

Polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) develop various adverse effects through activation of an aryl hydrocarbon receptor (AhR). The suppressive effects of brewed green tea and black tea on 3-methylcholanthrene (MC)-induced AhR activation and its downstream events were examined in the liver of rats. Ad-libitum drinking of green tea and black tea suppressed MC-induced AhR activation and elevation of ethoxyresorufin O-deethylase activity in the liver, whereas the teas themselves did not induce them. Tea showed a suppressive fashion on the expression of cytochrome P450 1A1 (CYP1A1). Tea suppressed the AhR activation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ex vivo. A part of catechins and theaflavins was present in plasma and liver as conjugated and intact forms. The results of this study suggested that active component(s) of tea are incorporated in the liver and suppress the activity of CYP1As through the AhR activation pathway.

Antagonistic effect of the Ainu-selected traditional beneficial plants on the transformation of an aryl hydrocarbon receptor.

UNLABELLED: Transformation of an aryl hydrocarbon receptor (AhR) is the initial step to express the multiple toxicity of halogenated and polycyclic aromatic hydrocarbons (HAHs and PAHs) including dioxins. Therefore, it has been suggested that suppression of the transformation induced by HAHs and PAHs leads to reduce their toxicological effects. In this study, the antagonistic effect of 110 indigenous plants (192 plant parts) used as medicine and/or food by the Ainu on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation was investigated. Of these, a stalk of Aralia elata (Miq.) Seemann and a bark of Fraxinus mandshurica Rupr. var. japonica Maxim. exhibited the strong antagonistic effect in a dose-dependent manner. An antioxidative activity and polyphenol content were also measured, and the strong correlation (r= 0.96) between these two parameters could be confirmed. However, correlation coefficients of the antagonistic effect of 192 extracts compared to their antioxidative activity and polyphenol content were 0.17 and 0.20, respectively. These results suggest that the Ainu-selected traditional beneficial plants are useful source for findings of novel AhR antagonists, and the antagonistic activity of these plants may be independent on their antioxidative activity and polyphenol content. PRACTICAL APPLICATION: Our findings lead to discovery of the valuable plants used by the Ainu and the novel active compounds useful for human's life, and furthermore, may contribute to the development of new medicines and functional foods.

Green and black tea suppress hyperglycemia and insulin resistance by retaining the expression of glucose transporter 4 in muscle of high-fat diet-fed C57BL/6J mice.

To investigate the preventive effects of tea on hyperglycemia and insulin resistance, male C57BL/6J mice were given a high-fat diet containing 29% lard and also green or black tea ad libitum for 14 weeks. Both teas suppressed body weight gain and deposition of white adipose tissue caused by the diet. In addition, they improved hyperglycemia and glucose intolerance by stimulating glucose uptake activity accompanied by the translocation of glucose transporter (GLUT) 4 to the plasma membrane in muscle. Long-term consumption of the high-fat diet reduced levels of insulin receptor ß-subunit, GLUT4 and AMP-activated protein kinase α in muscle, and green and black tea suppressed these decreases. The results strongly suggest that green and black tea suppress high-fat diet-evoked hyperglycemia and insulin resistance by retaining the level of GLUT4 and increasing the level of GLUT4 on the plasma membrane in muscle.

Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes.

In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 µM catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. (-)-Epicatechin (EC) and (-)-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the dose- and time-dependent manner, whereas (-)-catechin 3-gallate (Cg) and (-)-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and time-dependent manner. When the cells were treated with 50 µM catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKCλ/ζ without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 3'-kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKCλ/ζ in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 cells.

Identification of epigallocatechin-3-gallate in green tea polyphenols as a potent inducer of p53-dependent apoptosis in the human lung cancer cell line A549.

The effects of green tea polyphenols on cultured cancer cells have been well characterized, especially the effects of epigallocatechin-3-gallate (EGCg), since EGCg suppresses oncogenic signaling pathways and induces cell cycle arrest or apoptosis by regulating cell cycle-associated proteins. In the present study, we attempted to identify signaling pathways or target molecules regulated by each of or a mixture of green tea polyphenols, including epicatechin (EC), epicatechin-3-gallate (ECg), epigallocatechin (EGC), and EGCg, in the human lung cancer cell line A549. ECg, EGC, and a catechin mixture, in addition to EGCg, significantly decreased cell viability. In contrast, caspase 3/7 activity, an apoptosis indicator, was specifically induced by EGCg. By conducting a series of luciferase-based reporter assays, we revealed that the catechin mixture only up-regulates the p53 reporter. EGCg was a more potent inducer of p53-dependent transcription, and this induction was further supported by the induced level of p53 protein. RNA interference (RNAi)-mediated p53 knockdown completely abolished EGCg-induced apoptosis. Finally, a proteome and western blot analysis using approximately 70 different antibodies failed to detect up-regulated proteins in catechin mixture-treated A549 cells. Taken together, these results indicate that EGCg, among several green tea polyphenols, is a potent apoptosis inducer that functions exclusively through a p53-dependent pathway in A549 cells.

Epigallocatechin gallate promotes GLUT4 translocation in skeletal muscle.

In this study, we investigated whether epigallocatechin gallate (EGCg) affects glucose uptake activity and the translocation of insulin-sensitive glucose transporter (GLUT) 4 in skeletal muscle. A single oral administration of EGCg at 75 mg/kg body weight promoted GLUT4 translocation in skeletal muscle of rats. EGCg significantly increased glucose uptake accompanying GLUT4 translocation in L6 myotubes at 1 nM. The translocation of GLUT4 was also observed both in skeletal muscle of mice and rats ex vivo and in insulin-resistant L6 myotubes. Wortmannin, an inhibitor of phosphatidylinositol 3'-kinase, inhibited both EGCg- and insulin-increased glucose uptakes, while genistein, an inhibitor of tyrosine kinase, failed to inhibit the EGCg-increased uptake. Therefore, EGCg may improve hyperglycemia by promoting GLUT4 translocation in skeletal muscle with partially different mechanism from insulin.

Cacao polyphenol extract suppresses transformation of an aryl hydrocarbon receptor in C57BL/6 mice.

Dioxins enter the body through the diet and cause various toxicological effects through transformation of an aryl hydrocarbon receptor (AhR). Plant extracts and phytochemicals including flavonoids are reported to suppress this transformation. This paper investigates the suppression by a cacao polyphenol extract (CPE) of AhR transformation in vivo. The CPE was administered orally to C57BL/6 mice at 100 mg/kg of body weight, followed 1 h later by 3-methylcholanthrene (MC), an AhR agonist, injected intraperitoneally at 10 mg/kg of body weight. CPE suppressed the MC-induced transformation to the control level by inhibiting the formation of a heterodimer between AhR and an aryl hydrocarbon receptor nuclear translocator in the liver at 3 h postadministration. It also suppressed MC-induced cytochrome P4501A1 expression and NAD(P)H:quinone-oxidoreductase activity, whereas it increased glutathione S-transferase activity at 25 h. CPE constituents and their metabolites might contribute, at least in part, to the suppression of AhR transformation. The results indicate that the intake of CPE suppressed the toxicological effects of dioxins in the body.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate.

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, substituted at the 2-position with piperidines bearing quaternary ammonium salt side chains, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Attachment of the charged entity using an N-ethylcarbamoyloxy linker led to the discovery of the highly soluble compound 22 (D13-9001), which maintained good potency in vitro and displayed excellent activity in vivo in a rat pneumonia model of P. aeruginosa.

Identification of FAZF as a novel BMP2-induced transcription factor during osteoblastic differentiation.

Bone morphogenetic protein 2 (BMP2) is a key factor in the regulation of osteoblastic differentiation; however, its downstream mediators are not fully understood. Previously, we identified and characterized transcription factor promyelocytic leukemia zinc finger protein (PLZF), composed of an N-terminal BTB/POZ and C-terminal zinc finger motifs, as an upstream factor of CBFA1 (Runx2/core-binding factor 1). PLZF was induced in an osteoblastic differentiation medium, but was not induced by BMP2. Here, we report the identification of transcription factor fanconi anemia zinc finger protein (FAZF), which is closely related to PLZF. FAZF was induced by BMP2 in human mesenchymal stem cells (hMSCs). In addition to the full-length FAZF, we also identified alternatively spliced mRNAs in which the C-terminal zinc finger motifs were deleted (designated BTB/POZ-only FAZF). Both the full-length and BTB/POZ-only FAZF mRNAs were equally expressed in BMP2-treated hMSCs. The full-length FAZF was exclusively detected in the nucleus, whereas the BTB/POZ-only FAZF protein was localized in the cytoplasm of the transfected cells. The full-length FAZF, but not the BTB/POZ-only FAZF, increased the expression of osteoblastic differentiation markers, including CBFA1, collagen 1A1, osteocalcin, and alkaline phosphatase in C2C12 cells. In conclusion, both FAZF and PLZF differentially participate in the regulation of osteoblastic differentiation via the BMP2 and CBFA1 signaling pathways, respectively.

Screening of indigenous plants from Japan for modulating effects on transformation of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with which halogenated and polycyclic aromatic hydrocarbons such as dioxins and benzo[a]pyrene interact as ligands. Since such compounds cause various toxicological effects, including cancer, through the transformation of AhR, it is important to determine influence of modulating factors. It has been reported that certain plant components such as flavonoids and indoles can affect AhR transformation. In this study, to obtain clues to novel ligands of AhR, 191 species of indigenous plants were collected in Japan, and their 50% methanolic extracts (total 368 plant parts) were tested for modulating effects on AhR transformation in a cell-free system using a rat hepatic cytosolic fraction. Among tested extracts at a concentration of 1 mg dry weight of plant/mL, 174 of 368 extracts suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation to 50% or less, while 9 extracts per se induced AhR transformation equivalent to more than 20% of that induced by 1 nM TCDD. Mallotus japonicus (Thunb.) Muell. (leaf) and Trichosanthes rostrata Kitamura (fruit and fruit skin) strongly suppressed 1 nM TCDD-induced AhR transformation, while Phellodendron amurense Ruprecht (seed) per se strongly induced AhR transformation. These results suggest that a large variety of plants in Japan contain various compounds modulating, mainly suppressing, AhR transformation.

Molokhia (Corchorus olitorius L.) extract suppresses transformation of the aryl hydrocarbon receptor induced by dioxins.

Dioxins enter the body mainly through diet and cause the various toxicological effects by binding to the cytosolic aryl hydrocarbon receptor (AhR) followed by its transformation. In recent reports, it has been shown that certain natural compounds suppress AhR transformation in vitro. In this study, we demonstrated that ethanolic extract from molokhia, known as Egyptian spinach, showed the strongest suppressive effect on AhR transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cell-free system using rat hepatic cytosol among 41 kinds of extracts from vegetables and fruits. The molokhia extract also suppressed TCDD-induced AhR transformation in mouse hepatoma Hepa-1c1c7 cells and in intestinal permeability system constructed with human colon adenocarcinoma Caco-2 cells and human hepatoma HepG2 cells. Moreover, oral administration of the molokhia extract (100mg/kg body weight) decreased 3-methylcholanthrene-induced AhR transformation to the control level by inhibiting translocation of the AhR from cytosol into the nucleus in the liver of rats. The molokhia extract-administered rat liver showed a tolerance to TCDD-induced AhR transformation by ex vivo experiment. These results indicate that molokhia is an attractive food for isolation and identification of a natural antagonist for the AhR.

Identification and characterization of a novel kelch-like gene KLHL15 in silico.

Kelch-like proteins are implicated in embryogenesis and carcinogenesis through cytoskeleton organization. KLHL (kelch homolog) genes, containing two evolutionary conserved domains--broad-complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domains, and kelch motif, are human homologs of Drosophila kelch gene. We identified the KLHL15 gene, a novel human homolog of Drosophila kelch, by using bioinformatics. KLHL15 gene, consisting of 4 exons, was located within human genome sequences RP11-47911 (AC079169.32) and RP11-793H5 (AC079376.26). Complete coding sequence of human KLHL15 cDNA was determined by assembling FLJ32736 cDNA (AK057298) and KIAA1677 cDNA (AB051464). The human, chicken, and zebra fish KLHL15 (604 aa) showed 85-93% total-amino acid identity. N-terminal BTB/POZ domain and C-terminal three KELCH motifs were identified within KLHL15 protein by using the Pfam program. Human KLHL15 mRNA was expressed ubiquitously in various tissues. This is the first report on identification and characterization of the KLHL15 gene.

Identification and characterization of human ZNF18 gene in silico.

Zinc finger protein genes are regulatory transcription factors that interact directly with DNA and/or RNA. Here, we identified the ZNF18 gene by using bioinformatics. Human ZNF18 gene, consisting of 9 exons, was located within human genome sequences RP11-1096G20 (AC005410.3). Complete coding sequence of human ZNF18 cDNA was determined by FLJ35337 cDNA (AK092656), which was identified by using partial coding sequence of human ZNF18 cDNA (X52342) as a query. Human, mouse, and rat ZNF18 showed 77-92% total-amino acid identity. N-terminal leucine rich region, krueppel-associated box, and C-terminal three C2H2 type zinc finger motifs were identified within ZNF18 protein. This is the first report on identification and characterization of the ZNF18 gene.

The promyelotic leukemia zinc finger promotes osteoblastic differentiation of human mesenchymal stem cells as an upstream regulator of CBFA1.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is the leading cause of myelopathy in Japan and is diagnosed by ectopic bone formation in the paravertebral ligament. OPLL is a systemic high bone mass disease with a strong genetic background. To detect genes relevant to the pathogenesis of OPLL, we performed a cDNA microarray analysis of systematic gene expression profiles during the osteoblastic differentiation of ligament cells from OPLL patients (OPLL cells), patients with a disorder called ossification of yellow ligament (OYL), and non-OPLL controls together with human mesenchymal stem cells (hMSCs) after stimulating them with osteogenic differentiation medium (OS). Twenty-four genes were up-regulated during osteoblastic differentiation in OPLL cells. Zinc finger protein 145 (promyelotic leukemia zinc finger or PLZF) was one of the highly expressed genes during osteoblastic differentiation in all the cells examined. We investigated the roles of PLZF in the regulation of osteoblastic differentiation of hMSCs and C2C12 cells. Small interfering RNA-mediated gene silencing of PLZF resulted in a reduction in the expression of osteoblast-specific genes such as the alkaline phosphatase, collagen 1A1 (Col1a1), Runx2/core-binding factor 1 (Cbfa1), and osteocalcin genes, even in the presence of OS in hMSCs. The expression of PLZF was unaffected by the addition of bone morphogenetic protein 2 (BMP-2), and the expression of BMP-2 was not affected by PLZF in hMSCs. In C2C12 cells, overexpression of PLZF increased the expression of Cbfa1 and Col1a1; on the other hand, the overexpression of CBFA1 did not affect the expression of Plzf. These findings indicate that PLZF plays important roles in early osteoblastic differentiation as an upstream regulator of CBFA1 and thereby might participate in promoting the ossification of spinal ligament cells in OPLL patients.