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Objective Adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) influences hepatic cholesterol transportation. Accumulation of hepatic cholesterol leads to fatty liver disease, which is improved by glucagon-like peptide 1 (GLP-1) in diabetes. Therefore, we analyzed the molecular mechanism in the regulation of hepatic ABCA1 by GLP-1 analogue exendin-4. Methods Hepatic ABCA1 expression and transcription were checked by western blotting, real-time polymerase chain reaction (PCR), and luciferase assay in HepG2 cells. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were employed to determine transcriptional regulation of the ABCA1 gene. Prolactin regulatory element-binding (PREB)-transgenic mice were generated to access the effect of exendin-4 on improving lipid accumulation caused by a high-fat diet (HFD). Results Exendin-4 stimulated hepatic ABCA1 expression and transcription via the Ca2+/calmodulin (CaM)-dependent protein kinase kinase/CaM-dependent protein kinase IV (CaMKK/CaMKIV) pathway, whereas GLP-1 receptor antagonist exendin9-39 cancelled this effect. Therefore, exendin-4 decreased hepatic lipid content. ChIP showed that PREB could directly bind to the ABCA1 promoter, which was enhanced by exendin-4. Moreover, PREB stimulated ABCA1 promoter activity, and mutation of PREB-binding site in ABCA1 promoter cancelled exendin-4-enhanced ABCA1 promoter activity. Silencing of PREB attenuated the effect of exendin-4 and induced hepatic cholesterol accumulation. Blockade of CaMKK by STO-609 or siRNA cancelled the upregulation of ABCA1 and PREB induced by exendin-4. In vivo, exendin-4 or overexpression of PREB increased hepatic ABCA1 expression and decreased hepatic lipid accumulation and high plasma cholesterol caused by a HFD. Conclusions Our data shows that exendin-4 stimulates hepatic ABCA1 expression and decreases lipid accumulation by the CaMKK/CaMKIV/PREB pathway, suggesting that ABCA1 and PREB might be the therapeutic targets in fatty liver disease., Graphical abstract Image 1, Highlights • The GLP-1R agonist exendin-4 suppressed lipid accumulation by upregulating ABCA1 expression in hepatocytes. • Exendin-4 regulated the expression and transcription of hepatic ABCA1 via the CaMKK/CaMKIV/PREB pathway. • Overexpression of PREB or exendin-4 protected mouse liver from fatty liver by upregulation of ABCA1.

Exendin-4 (Exenatide), a long-acting agonist of the glucagon-like peptide 1 receptor (GLP-1R), has been confirmed to protect the cardiovascular system from atherosclerosis in type 2 diabetes. However, the detailed mechanism of this process is not clear. Activation of endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO) plays an important role in endothelial homeostasis. Previous study shows that HDL activates eNOS via its receptor scavenger receptor type B class I (SR-BI). In this study, we checked the effect of exendin-4 on HDL/SR-BI-mediated eNOS activation in HUVECs. Firstly, we demonstrated the expression of GLP-1R in HUVECs and exendin-4 significantly induced HDL-mediated eNOS activation. Next, we found exendin-4 increased the expression and promoter activity of SR-BI, and then blocking of PKA or AMPK by its specific inhibitor cancelled this effect. Consistently, cAMP enhanced SR-BI promoter activity while domain negative AMPK inhibited the effect of exendin-4 on SR-BI promoter activity. Blocking of GLP-1R by exendin 9-39 cancelled exendin-4-induced SR-BI promoter activity and AMPK phosphorylation. As a downstream of AMPK, a transcription factor FoxO1 was confirmed to directly bind to the SR-BI promoter region by Chromatin Immunoprecipitation (ChIP) assay and overexpression of FoxO1 enhanced SR-BI promoter activity. Moreover, exendin-4 markedly increased the expression and nuclear recruitment of FoxO1 by decreasing its phosphorylation. As well as, mutation of FoxO1-binding site in the SR-BI promoter region or silence of FoxO1 cancelled exendin-4-induced SR-BI expression. In summary, our results show that exendin-4 upregulates the expression and transcription of SR-BI via PKA/AMPK/FoxO1 pathway, which contributes to HDL-mediated eNOS activation and NO generation in human endothelial cells. Disclosure J. Lyu: None. H. Imachi: None. K. Fukunaga: None. S. Sato: None. T. Dong: None. T. Ibata: None. T. Kobayashi: None. M. Matsumoto: None. K. Murao: None.

� Background and Aims Methyl CpG-binding proteins are considered to play critical roles in epigenetic control of gene expression by recognizing and interacting with 5-methylcytosine (m 5 C) in eukaryotes. However, among 13 corresponding genes in Arabidopsis thaliana, designated as featuring a methyl-binding domain (MBD), only four have so far been shown actually to bind to m 5 C. One example, AtMBD5, was selected here to screen for interacting proteins. � Methods Yeast two-hybrid assays were used for screening, and physical interaction was confirmed by pulldown and bimolecular fluorescence complementation (BiFC) assays. Cellular localization was analysed by fluorescence-tagged fusion proteins using tobacco (Nicotiana tabacum) cultured bright yellow 2 cells. � Key Results A gene finally identified was found to encode AtRAN3, a protein that belongs to the Ran GTPase family, which plays a critical role in nucleocytoplasmic transport and spindle bipolarization during cell division. AtMBD5 and AtRAN3 were clearly shown to interact in the nucleus by BiFC. On co-expression of AtMBD5-cyan fluorescence protein and yellow fluorescence protein-AtRAN3 in tobacco cells, both localized to the nucleus in the resting stage, migrating to the cytoplasm, primarily around chromatin, during mitosis, particularly at metaphase. � Conclusions These results suggest that AtMBD5 becomes localized to the vicinity of chromosomes with the aid of AtRAN3 during cell division, and may play an important role not only in maintenance of chromatin structures by binding to m 5 C, but also in progress through mitosis by detaching from m 5 C. The present findings also shed light on the physiological function of Ran GTPases, direct target proteins of which have not thus far been well defined, suggesting their key role in chromatin movements in plant cells.

Campylobacter jejuni causes foodborne disease associated with abdominal pain, gastroenteritis, and diarrhea. These symptoms are induced by bacterial adherence and invasion of host epithelial cells. C. jejuni infection can occur with a low infective dose, suggesting that C. jejuni may have evolved strategies to cope with the bacterial clearance system in the gastrointestinal tract. The mucosa layer is the first line of defense against bacteria. Mucus conditions are maintained by water and anion (especially Cl(-)) movement. Cystic fibrosis transmembrane conductance regulator (CFTR) is the main Cl(-) channel transporting Cl(-) to the lumen. Mutations in CFTR result in dehydrated secreted mucus and bacterial accumulation in the lungs, and recent studies suggest that closely related pathogenic bacteria also may survive in the intestine. However, the relationship between C. jejuni infection and CFTR has been little studied. Here, we used an (125)I(-) efflux assay and measurement of short-circuit current to measure Cl(-) secretion in C. jejuni-infected T-84 human intestinal epithelial cells. The basic state of Cl(-) secretion was unchanged by C. jejuni infection, but CFTR activator was observed to induce Cl(-) secretion suppressed in C. jejuni-infected T-84 cells. The suppression of activated Cl(-) secretion was bacterial dose-dependent and duration-dependent. A similar result was observed during infection with other C. jejuni strains. The mechanism of suppression may occur by affecting water movement or mucus condition in the intestinal tract. A failure of mucus barrier function may promote bacterial adhesion or invasion of host intestinal epithelial cells, thereby causing bacterial preservation in the host intestinal tract.

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).

Adherence of diarrhea-associated Escherichia coli was studied by scanning electron microscopy. Enteropathogenic E. coli (EPEC) adherence factor-positive (EAF+) E. coli of EPEC serotypes (class I EPEC) adhered to plastic and human jejunal and ileal mucosa, similar to case and HeLa cells. Localized adherence, elongation of cell microvilli, and "locking" of the bacterial aggregates by the elongated microvilli were evident after incubation for 20 min. EAF+ E. coli adhered strikingly to mucus but rarely to M cells in Peyer's patch-associated epithelium. Most enteroaggregative E. coli (EAggEC) strains adhered to plastic, similar to HeLa cells. Some diffuse-adhering E. coli (DAEC) strains displayed no adherence to plastic but formed "dimples" on HeLa cells. Both EAggEC and DAEC adhered at lower levels to human small intestines (except M cells) than did EAF+ E. coli. In all cases of EAF+ E. coli, EAggEC, and DAEC, strains were found with atypical characteristics. The data demonstrate the unique adherence characteristics of EAF+ E. coli, EAggEC, and DAEC.