"Rich arginine and strong positive charge" antimicrobial protein protamine: From its action on cell membranes to inhibition of bacterial vital functions.

Protamine, an antimicrobial protein derived from salmon sperm with a molecular weight of approximately 5 kDa, is composed of 60-70 % arginine and is a highly charged protein. Here, we investigated the mechanism of antimicrobial action of protamine against Cutibacterium acnes (C. acnes) focusing on its rich arginine content and strong positive charge. Especially, we focused on the attribution of dual mechanisms of antimicrobial protein, including membrane disruption or interaction with intracellular components. We first determined the dose-dependent antibacterial activity of protamine against C. acnes. In order to explore the interaction between bacterial membrane and protamine, we analyzed cell morphology, zeta potential, membrane permeability, and the composition of membrane fatty acid. In addition, the localization of protamine in bacteria was observed using fluorescent-labeled protamine. For investigation of the intracellular targets of protamine, bacterial translation was examined using a cell-free translation system. Based on our results, the mechanism of the antimicrobial action of protamine against C. acnes is as follows: 1) electrostatic interactions with the bacterial cell membrane; 2) self-internalization into the bacterial cell by changing the composition of the bacterial membrane; and 3) inhibition of bacterial growth by blocking translation inside the bacteria. However, owing to its strong electric charge, protamine can also interact with DNA, RNA, and other proteins inside the bacteria, and may inhibit various bacterial life processes beyond the translation process.

Direct visualization of ribosomes in the cell-free system revealed the functional evolution of aminoglycoside.

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.

Melatonin stimulates transcription of the rat phosphoenolpyruvate carboxykinase gene in hepatic cells.

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.

An insulin-inducible transcription factor, SHARP-1, represses transcription of the SIRT1 longevity gene.

The rat enhancer of split- and hairy-related protein (SHARP)-1 genes encode insulin-inducible transcriptional repressors. A longevity gene, sirtuin 1 (SIRT1) encodes protein deacetylase. These play an important role in regulating hepatic glucose metabolism. In this study, to evaluate a correlation with these gene expressions, we examined whether SIRT1 effects on expression of the SHARP-1 gene by a treatment with a SIRT1 inhibitor or activator in rat H4IIE hepatoma cells. Whereas the SIRT1 inhibitor increased the level of SHARP-1 mRNA, the SIRT1 activator decreased it. Next, whether SHARP-1 effect on the transcriptional activity of the human SIRT1 gene using luciferase reporter assays was determined. Promoter activity of the SIRT1 gene was specifically repressed by SHARP-1. Further reporter analysis using 5'- deleted or mutated constructs revealed that an E box sequence (5'-CACGTG-3') of the SIRT1 gene promoter was required for the inhibitory effect of SHARP-1. Thus, we conclude that expressions between the SHARP-1 and the SIRT1 genes show a negative correlation and that SHARP-1 represses transcription of the SIRT1 gene.

Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

5-Aminoimidazole-4-carboxyamide-1-ß-D-ribofranoside stimulates the rat enhancer of split- and hairy-related protein-2 gene via atypical protein kinase C lambda.

The 5'-AMP-activated protein kinase (AMPK) functions as a cellular energy sensor. 5-Aminoimidazole-4-carboxyamide-1-ß-D-ribofranoside (AICAR) is a chemical activator of AMPK. In the liver, AICAR suppresses expression of thephosphoenolpyruvate carboxykinase(PEPCK) gene. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcriptional repressor and its target is thePEPCKgene. In this study, we examined an issue of whether theSHARP-2gene expression is regulated by AICAR via the AMPK. AICAR increased the level of SHARP-2 mRNA in H4IIE cells. Whereas an AMPK inhibitor, compound-C, had no effects on the AICAR-induction, inhibitors for both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) completely diminished the effects of AICAR. Western blot analyses showed that AICAR rapidly activated atypical PKC lambda (aPKCλ). In addition, when a dominant negative form of aPKCλ was expressed, the induction of SHARP-2 mRNA level by AICAR was inhibited. Calcium ion is not required for the activation of aPKCλ. A calcium ion-chelating reagent had no effects on the AICAR-induction. Furthermore, the AICAR-induction was inhibited by treatment with an RNA polymerase inhibitor or a protein synthesis inhibitor. Thus, we conclude that the AICAR-induction of theSHARP-2gene is mediated at transcription level by a PI 3-K/aPKCλ pathway.

Fabrication and characterization of a deep ultraviolet wire grid polarizer with a chromium-oxide subwavelength grating.

A wire grid polarizer comprised of chromium oxide is designed for a micro-lithography system using an ArF excimer laser. Optical properties for some material candidates are calculated using a rigorous coupled-wave analysis. The chromium oxide wire grid polarizer with a 90 nm period is fabricated by a double-patterning technique using KrF lithography and dry etching. The extinction ratio of the grating is greater than 20 dB (100:1) at a wavelength of 193 nm. Differences between the calculated and experimental results are discussed.

(-)-Epigallocatechin-3-gallate stimulates both AMP-activated protein kinase and nuclear factor-kappa B signaling pathways.

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.

Analysis of induction mechanisms of an insulin-inducible transcription factor SHARP-2 gene by (-)-epigallocatechin-3-gallate.

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.

Analysis of regulatory mechanisms of an insulin-inducible SHARP-2 gene by (S)-Equol.

Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling type 2 diabetes mellitus. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor that decreases expression of the phosphoenolpyruvate carboxykinase gene, a gluconeogenic enzyme gene. In this study, we screened for soybean isoflavones that can induce the rat SHARP-2 gene expression and analyzed their mechanism(s). Genistein and (S)-Equol, a metabolite of daidzein, induced rat SHARP-2 gene expression in H4IIE rat hepatoma cells. The (S)-Equol induction was mediated by both the phosphoinositide 3-kinase- and protein kinase C (PKC)-pathways. When a dominant negative form of atypical PKC lambda (aPKCλ) was expressed, the induction of SHARP-2 mRNA level by (S)-Equol was inhibited. In addition, Western blot analyses showed that (S)-Equol rapidly activated both aPKCλ and classical PKC alpha. Furthermore, the (S)-Equol induction was inhibited by treatment with a RNA polymerase inhibitor or a protein synthesis inhibitor. Finally, a reporter gene assay revealed that the transcriptional stimulation by (S)-Equol was mediated by nucleotide sequences located between -4687 and -4133 of the rat SHARP-2 gene. Thus, we conclude that (S)-Equol is an useful dietary supplement to control type 2 diabetes mellitus.

(-)-Epigallocatechin-3-gallate enhances the expression of an insulin-inducible transcription factor gene via a phosphoinositide 3-kinase/atypical protein kinase C lambda pathway.

The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is an insulin-inducible transcriptional repressor. In this study, we examined issues of whether (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, regulates the expression of the rat SHARP-1 gene and which signaling pathway mediates the regulation. When H4IIE cells were treated with EGCG, SHARP-1 mRNA levels rapidly increased. Pretreatments with inhibitors for either phosphoinositide 3-kinase (PI 3-K) or protein kinase C partially blocked EGCG induction. Atypical protein kinase C lambda (aPKCλ) is known as a downstream target of PI 3-K in the liver. When a dominant-negative form of aPKCλ was expressed, the EGCG-induced SHARP-1 mRNAs was inhibited. Finally, Western blot analysis revealed that EGCG rapidly and temporarily stimulates aPKCλ phosphorylation. Thus, we conclude that EGCG induces SHARP-1 gene expression via a PI 3-K/aPKCλ signaling pathway.

Genistein stimulates the insulin-dependent signaling pathway.

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 repress cancer markers in normal hepatocytes.

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.

Transrectal high-intensity focused ultrasound for treatment for patients with biochemical failure after radical prostatectomy.

Four patients with biochemical prostate-specific antigen (PSA) failure with suspected local recurrence at the vesico-urethral anastomotic site after radical prostatectomy were treated using a high-intensity focused ultrasound (HIFU) device (Sonablate 500) under caudal or spinal anesthesia. The pretreatment PSA levels ranged from 0.318 to 0.898 ng/mL and their Gleason scores ranged between 5 and 7. HIFU treatment was carried out six times in four patients. The median time of operation and follow-up period were 30 min (range, 15-37) and 13 months (range, 7-18), respectively. In all patients, the median PSA levels decreased from 0.555 ng/mL (range, 0.318-0.898) to 0.137 ng/mL (range, 0.102-0.290). The median PSA nadir after each HIFU was 0.054 ng/mL (range, 0.008-0.097). No major complications were noted. HIFU may be useful for the therapy of vesicourethral anastomostic lesion in patients with PSA failure after prostatectomy.

Cloning and characterization of the 5'-flanking region of the rat estrogen receptor beta gene.

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.

Regulation of estrogen receptor alpha and beta expression by testosterone in the rat prostate gland.

Although ER beta is known to be expressed at high levels in the rat prostate gland, its regulation is not well understood. Here we examined ER mRNA expression and the effects of testosterone administration in male rats at 1, 4 and 9 weeks of age who were castrated and/or treated with testosterone for a week, and then sacrificed. ER alpha was the major type of ER expressed in 2 week-old animals while dominant expression of ER beta mRNA was apparent in older age groups. Interestingly while ER beta expression was diminished and ER alpha mRNA increased in the castrated group, testosterone administration reversed this effect. A time-course study indicated that induction of ER beta mRNA increased within 9 hr and ER alpha decreased in 2 days after an injection (i.p.) of testosterone. Our results suggested that 1) testosterone up-regulates ER beta mRNA expression while ER alpha is down-regulated; and that 2) great changes in ER alpha and beta expression in the prostate gland during development from the newborn to adult may be due to the influence of testosterone.