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The rapid emergence of multi-drug-resistant bacteria has raised a serious public health concern. Therefore, new antibiotic developments have been highly desired. Here, we propose a new method to visualize antibiotic actions on translating ribosomes in the cell-free system under macromolecular crowding conditions by cryo-electron microscopy, designated as the DARC method: the Direct visualization of Antibiotic binding on Ribosomes in the Cell-free translation system. This new method allows for acquiring a more comprehensive understanding of the mode of action of antibiotics on the translation inhibition without ribosome purification. Furthermore, with the direct link to biochemical analysis at the same condition as cryo-EM observation, we revealed the evolution of 2-DOS aminoglycosides from dibekacin (DBK) to arbekacin (ABK) by acquiring the synthetic tailored anchoring motif to lead to stronger binding affinity to ribosomes. Our cryo-EM structures of DBK and ABK bound ribosomes in the cell-free environment clearly depicted a synthetic tailored γ-amino-α-hydroxybutyryl (HABA) motif formed additional interactions with the ribosome enhancing antibiotic bindings. This new approach would be valuable for understanding the function of antibiotics for more efficient drug development. [ABSTRACT FROM AUTHOR]

Melatonin plays physiological roles in various critical processes, including circadian rhythms, oxidative stress defenses, anti‐inflammation responses, and immunity; however, the current understanding of the role of melatonin in hepatic glucose metabolism is limited. In this study, we examined whether melatonin affects gene expression of the key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). We found that melatonin treatment increased PEPCK mRNA levels in rat highly differentiated hepatoma (H4IIE) cells and primary cultured hepatocytes. In addition, we found that melatonin induction was synergistically enhanced by dexamethasone, whereas it was dominantly inhibited by insulin. We also report that the effect of melatonin was blocked by inhibitors of mitogen‐activated protein kinase/extracellular signal‐regulated protein kinase (MAPK/ERK), RNA polymerase II, and protein synthesis. Furthermore, the phosphorylated (active) forms of ERK1 and ERK2 (ERK1/2) increased 15 min after melatonin treatment. We performed luciferase reporter assays to show that melatonin specifically stimulated promoter activity of the PEPCK gene. Additional reporter analysis using 5′‐deleted constructs revealed that the regulatory regions responsive to melatonin mapped to two nucleotide regions, one between −467 and −398 nucleotides and the other between −128 and +69 nucleotides, of the rat PEPCK gene. Thus, we conclude that melatonin induces PEPCK gene expression via the ERK1/2 pathway at the transcriptional level, and that induction requires de novo protein synthesis. [ABSTRACT FROM AUTHOR]

An abstract of the study "The effect of lycopene intake and treadmill running exercise training on bone mineral in growing male rats," by Satoshi Hattori, Kosuke Asano, and Naomi Omi, is presented.

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor. In this study, we examined the mechanism(s) involved in the regulation of the rat SHARP-2 gene expression by (-)-epigallocatechin-3-gallate (EGCG). The induction of SHARP-2 mRNA by EGCG was repressed by pretreatments with inhibitors for either phosphoinositide 3-kinase (PI3K) or RNA polymerase II. Then, we examined a biological relationship between EGCG and transcription factor NF-κB interfering with the insulin action. The protein levels of the NF-κB were rapidly decreased by an EGCG treatment. Finally, the mechanism(s) of transcriptional activation of the rat SHARP-2 gene by both NF-κB and EGCG was analyzed. While overexpression of the NF-κB p65 protein decreased the promoter activity of the SHARP-2 gene, EGCG did not affect it. Thus, we conclude that EGCG induces the expression of the rat SHARP-2 gene via both the PI3K pathway and degradation of the NF-κB p65 protein.

We previously reported that (-)-epigallocatechin-3-gallate (EGCG) increased the level of SHARP-1 mRNA via a phosphoinositide 3-kinase/atypical protein kinase C lambda signaling pathway in rat H4IIE hepatoma cells. In the present study, we investigated other signaling pathway(s). Treating with either compound-C, BAY11-7082, or both, partially blocked the up-regulation of the SHARP-1 gene by EGCG. This suggests that AMP-activated protein kinase (AMPK)- and nuclear factor-kappa B (NF-κB)-signaling pathways were additively involved in the induction mediated by EGCG. Indeed, an AMPK activator induced a level of SHARP-1 mRNA. Although actinomycin D partially blocked the EGCG-induction of that SHARP-1 mRNA level, the nucleotide sequence between -1501 and -1 in the rat SHARP-1 gene did not positively respond to EGCG and NF-κB, respectively. Thus, we conclude that EGCG stimulates multiple signaling pathways in the SHARP-1 gene expression at the transcriptional and post-transcriptional levels and that there is no regulatory region susceptible to EGCG and NF-κB in the examined region., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling insulin-independent diabetes mellitus. In this study, we investigated whether soybean isoflavones could induce the expression of SHARP-2, a downstream component of insulin-dependent signaling pathway, associated with the regulation of blood glucose. One such compound called genistein, rapidly and temporarily induced SHARP-2 mRNA levels in a dose-dependent manner in rat H4IIE hepatoma cells. This induction process was rapidly stimulated by a protein kinase C (PKC) activator and blocked by a PKC inhibitor, suggesting that SHARP-2 may be induced via PKC activation. Upon Western blot analysis, genistein showed a stimulation of PKC phosphorylation. Therefore, we concluded that genistein might transcriptionally induce SHARP-2 through the activation of PKC in H4IIE cells. Our results suggest that genistein might be a useful dietary supplement to control insulin-independent diabetes mellitus by inducing the SHARP-2 expression via a bypass of the insulin-dependent signaling pathway.

ZHX2 and ZHX3 are the members of the ZHX transcriptional repressor family. To investigate the regulatory role of the repressors in hepatocytes and their involvement in carcinogenesis, the expression levels of ZHX2 and ZHX3 mRNAs were examined. The dRLh-84 hepatoma cells considerably expressed cancer marker genes PKM and HK II that are expressed in developing fetal tissues and cancer cells but repressed in normal hepatocytes. In dRLh-84 cells, the expression levels of ZHX2 and ZHX3 were very low compared with rat hepatocytes. Upon the reporter gene analysis utilizing the promoter region of these genes, ZHX3 repressed the transcription of the reporter luciferase gene from both promoters while ZHX2 only repressed that from HK II promoter. The promoter activity of alpha-fetoprotein was also repressed by the expression of ZHX2 in HLE hepatoma cells in a dose-dependent manner. We concluded that ZHX2 and ZHX3 were involved in the transcriptional repression of the hepatocellular cacinoma markers in normal hepatocytes, suggesting that the failure of the ZHX2 and/or ZHX3 expression might be a critical factor in the hepatocellular carcinogenesis.

In the rat, estrogen receptor (ER) beta is preferentially expressed in the ovary and the prostate gland where it is transcriptionally regulated by testosterone. A single 5'-end of rERbeta cDNA was identified in these tissues by the 5'-RACE analysis in the present study. The transcription starting site was predicted at -335 from the translation starting signal (ATG), and a 640bp section of the 5'-flanking region of the gene was cloned. Luciferase reporter assays revealed this region to be responsible for cell-specific promoter activity and successive deletion analyses indicated that only 98bp were sufficient for basic promoter activity as well as for testosterone-dependent transcription. The sequence of the determined region found to demonstrate high homology with the mouse ERbeta promoter with more than 80% identity between positions -1 and -550. The rat region of -30/-110 also showed good homology with 69% identity to corresponding section of the human promoter. Putative cis-acting elements, USF/Arnt and AML1a, were found in common in the promoter regions of three species. The present study thus demonstrated the 5'-flanking region of the rERbeta gene to be a functional promoter.