Fate of pesticides in a distilled spirit of barley shochu during the distillation process

Moromi (the fermented mash) of "mugi shochu" that had been artificially contaminated with pesticides was distilled to elucidate the fate of pesticides in the distillation process. The pesticides residing in the distillate were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of the analyzed pesticides (249 compounds), 89% were not detected in the distillate, showing that the distillation process minimized the risk of pesticide contamination.     

Characterization of newly established bovine intestinal epithelial cell line

Membranous epithelial cells (M cells) of the follicle-associated epithelium in Peyer’s patches have a high capacity for transcytosis of several viruses and microorganisms. Here, we report that we have successfully established a bovine intestinal epithelial cell line (BIE cells) and developed an in vitro M cell model. BIE cells have a cobblestone morphology and microvilli-like structures, and strongly express cell-to-cell junctional proteins and cytokeratin, which is a specific intermediate filament protein of epithelial cells. After co-culture with murine intestinal lymphocytes or treatment with supernatant from bovine PBMC cultured with IL-2, BIE cells acquired the ability of transcytosis. Therefore, BIE cells have typical characteristics of bovine intestinal epithelial cells and also have the ability to differentiate into an M cell like linage. In addition, our results indicate that contact between immune cells and epithelial cells may not be absolutely required for the differentiation of M cells. We think that BIE cells will be useful for studying the transport mechanisms of various pathogens and also the evaluation of drug delivery via M cells.     

Endothelin receptor type A expression defines a distinct cardiac subdomain within the heart field and is later implicated in chamber myocardium formation

The avian and mammalian heart originates from two distinct embryonic regions: an early differentiating first heart field and a dorsomedially located second heart field. It remains largely unknown when and how these subdivisions of the heart field divide into regions with different fates. Here, we identify in the mouse a subpopulation of the first (crescent-forming) field marked by endothelin receptor type A (Ednra) gene expression, which contributes to chamber myocardium through a unique type of cell behavior. Ednra-lacZ/EGFP-expressing cells arise in the ventrocaudal inflow region of the early linear heart tube, converge to the midline, move anteriorly along the outer curvature and give rise to chamber myocardium mainly of the left ventricle and both atria. This movement was confirmed by fluorescent dye-labeling and transplantation experiments. The Ednra-lacZ/EGFP-expressing subpopulation is characterized by the presence of Tbx5-expressing cells. Ednra-null embryonic hearts often demonstrate hypoplasia of the ventricular wall, low mitotic activity and decreased Tbx5 expression with reciprocal expansion of Tbx2 expression. Conversely, endothelin 1 stimulates ERK phosphorylation and Tbx5 expression in the early embryonic heart. These results indicate that early Ednra expression defines a subdomain of the first heart field contributing to chamber formation, in which endothelin 1/Ednra signaling is involved. The present finding provides an insight into how subpopulations within the crescent-forming (first) heart field contribute to the coordination of heart morphogenesis through spatiotemporally defined cell movements.     

Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts

1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse bone marrow cells and osteoblasts, 1,25(OH)₂D₃ induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than 10^?9 M of 1,25(OH)₂D₃. In the presence of bone morphogenetic protein 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)₂D₃ (greater than 10^?11 M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)₂D₃. In addition, the expressions of RANKL mRNA and proteins were induced by 1,25(OH)₂D₃ in osteoblasts, and further upregulated by BMP-2. In mouse bone marrow cell cultures without 1,25(OH)₂D₃, BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)₂D₃.     

Natural killer cell is a major producer of interferon γ that is critical for the IL-12-induced anti-tumor effect in mice

Although the anti-tumor effect of IL-12 is mediated mostly by IFNγ, which cell types most efficiently produce IFNγ and therefore initiate or promote the anti-tumor effect of IL-12 has not been clearly determined. In the present study, we demonstrated hydrodynamic injection of the IL-12 gene led to prolonged IFNγ production, NK-cell activation and complete inhibition of liver metastasis of CT-26 colon cancer cells in wild-type mice, but not in IFNγ knockout mice. NK cells expressed higher levels of STAT4 and upon IL-12 administration displayed stronger STAT4 phosphorylation and IFNγ production than non-NK cells. Adoptive transfer of wild-type NK cells into IFNγ knockout mice restored IL-12-induced IFNγ production, NK-cell activation and anti-tumor effect, whereas transfer of the same number of wild-type non-NK cells did not. In conclusion, NK cells are predominant producers of IFNγ that is critical for IL-12 anti-tumor therapy.     

Regulation of AMPA Receptor Trafficking by O-Glycosylation

The present study investigated the role of O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) in AMPA receptor trafficking. Alloxan, an inhibitor of O-GlcNAc transferase, potentiated responses of AMPA receptors composed of the GluR1 subunit expressed in Xenopus oocytes. No potentiating effect of alloxan was obtained with mutant GluR1 (S831A) receptor lacking CaMKII phosphorylation site. Alloxan facilitated basal synaptic transmission to approximately 120% of basal levels and enhanced Schaffer collateral-CA1 long-term potentiation (LTP) in rat hippocampal slices, especially in the late phase of the LTP. Alloxan stimulated translocation of the GluR1 and GluR2 subunit from the cytosol towards the plasma membrane in rat hippocampal slices with the LTP, although it had no effect on subcellular distribution of the NR1 subunit. Taken together, the results of the present study show that alloxan regulates AMPA receptor trafficking by inhibiting O-GlcNAcylation, to modulate hippocampal synaptic transmission and synaptic plasticity.     

Fatal exacerbation of type B chronic hepatitis triggered by changes in relaxed circular viral DNA synthesis and virion secretion

Virological features of fulminant liver disease-causing hepatitis B virus (HBV) have not been fully elucidated. We studied longitudinally the viruses obtained before and after fulminant liver disease in a patient with chronic HBV infection showing fatal exacerbation. HBV strains were obtained before and after exacerbation (designated as FEP1 and FEP2). Their virological features were investigated by in vitro transfection. FEP1 and FEP2 possessed higher activity of overall HBV DNA synthesis than the wild-type. FEP1 lacked competence for relaxed circular (RC) HBV DNA synthesis and RC HBV DNA-containing virion secretion, but FEP2 maintained it. Chimeric analysis revealed that the preS/S gene, where FEP1 had a considerable number of mutations and deletions but FEP2 did not, was responsible for impaired RC HBV DNA synthesis and virion secretion. Furthermore, incompetence of FEP1 strain was transcomplemented by the preS/S protein of wild-type strain. In conclusion, the viral strain after exacerbation showed resurgent RC HBV DNA synthesis and virion secretion, which was caused by conversion of the preS/S gene from a hypermutated to hypomutated state. This may have been responsible for disease deterioration in the patient. This is a novel type of HBV genomic variation associated with the development of fulminant liver disease.     

Connective tissue growth factor is a substrate of ADAM28

ADAM28, a member of the ADAM (a disintegrin and metalloproteinase) gene family, is over-expressed by carcinoma cells and the expression correlates with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, information about substrates of ADAM28 is limited. We screened interacting molecules of ADAM28 in human lung cDNA library by yeast two-hybrid system and identified connective tissue growth factor (CTGF). Binding of CTGF to proADAM28 was demonstrated by yeast two-hybrid assay and protein binding assay. ADAM28 cleaved CTGF in dose- and time-dependent manners at the Ala¹⁸¹-Tyr¹⁸² and Asp¹⁹¹-Pro¹⁹² bonds in the hinge region of the molecule. ADAM28 selectively digested CTGF in the complex of CTGF and vascular endothelial growth factor₁₆₅ (VEGF₁₆₅), releasing biologically active VEGF₁₆₅ from the complex. RT-PCR and immunohistochemical analyses demonstrated that ADAM28, CTGF and VEGF are commonly co-expressed in the breast carcinoma tissues. These data provide the first evidence that CTGF is a novel substrate of ADAM28 and suggest that ADAM28 may promote VEGF₁₆₅-induced angiogenesis in the breast carcinomas by the CTGF digestion in the CTGF/VEGF₁₆₅ complex.     

Functional role of acetylcholine and the expression of cholinergic receptors and components in osteoblasts

Recent studies have indicated that acetylcholine (ACh) plays a vital role in various tissues, while the role of ACh in bone metabolism remains unclear. Here we demonstrated that ACh induced cell proliferation and reduced alkaline phosphatase (ALP) activity via nicotinic (nAChRs) and muscarinic acetylcholine receptors (mAChRs) in osteoblasts. We detected mRNA expression of several nAChRs and mAChRs. Furthermore, we showed that cholinergic components were up-regulated and subunits/subtypes of acetylcholine receptors altered during osteoblast differentiation. To our knowledge, this is the first report demonstrating that osteoblasts express specific acetylcholine receptors and cholinergic components and that ACh plays a possible role in regulating the proliferation and differentiation of osteoblasts.     

Feeding rats a high fat/carbohydrate ratio diet reduces jejunal S/I activity ratio and unsialylated galactose on glycosylated chain of S–I complex

AimsWe examined whether decreasing jejunal sucrase/isomaltase (S/I) activity ratio by feeding rats a high fat/carbohydrate ratio diet is regulated by changing glycosylated chains on the S–I complex.Main methodsJejunal activities of sucrase, isomaltase and β-1,4-galactosyltransferase were examined in rats fed a high fat/carbohydrate or a low fat/carbohydrate ratio diet. The amount of galactose and mannose in the glycosylated chain on the S–I complex in rats fed both diets was determined using RCA₁₂₀ and Con A lectins, respectively. The effects of reducing unsialylated galactose from the glycosylated chain on the S–I complex were assessed by determining sucrase activity in purified S–I complex treated with β-galactosidase.Key findings and significanceFeeding rats a high fat/carbohydrate ratio diet reduced jejunal S/I activity ratio in mucosal homogenates and purified fractions. The level of unsialylated galactose in glycosylated chains on the S–I complex was reduced by feeding rats a high fat/carbohydrate ratio diet. The form with reduced levels of unsialylated galactose had lower sucrase activity than that with more unsialylated galactose. The reduction of galactose on the S–I complex by β-galactosidase in vitro reduced sucrase activity. Feeding rats a high fat/carbohydrate ratio diet also reduced jejunal β-1,4-galactosyltransferase activity. Taken together, decreasing the S/I activity ratio by feeding rats a high fat/carbohydrate diet is associated with the reduction of unsialylated galactose on the glycosylated chain of the S–I complex.