4-Acetylantroquinonol B ameliorates nonalcoholic steatohepatitis by suppression of ER stress and NLRP3 inflammasome activation.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is an inflammatory lipotoxic disorder with a prevalence of over 25% worldwide. However, safe and effective therapeutic agents for the management of NAFLD are still lacking. We aimed to investigate the hepatoprotective effect and molecular mechanism of 4-acetylantroquinonol B (4-AAQB), a natural ubiquinone derivative obtained from the mycelia of Antrodia cinnamomea. METHODS: RAW264.7 and J774A.1 cells were treated with 4-AAQB and then stimulated with LPS or tunicamycin (TM) for 24 h. Inflammatory responses, markers of endoplasmic reticulum (ER) stress, and NOD-like receptor protein 3 (NLRP3) inflammasome were analyzed in both cell lines. In the applied in vivo model, male C57BL/6J mice were fed with chow or a methionine/choline-deficient (MCD) diet along with vehicle or 4-AAQB (10 mg/kg, i.p. injected, once a day) for 10 consecutive days. Plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Liver tissues were analyzed using histological techniques; protein levels involved in ER stress, NLRP3 inflammasome, and inflammatory responses were measured. RESULTS: 4-AAQB significantly ameliorated the plasma levels of ALT and AST as well as the NAFLD activity score (NAS) in mice fed the MCD diet. In addition, 4-AAQB suppressed inflammatory responses, ER stress, and NLRP3 inflammasome activation, but increased the nuclear factor erythroid 2-related factor 2 (Nrf2) and Sirtuin 1 (SIRT1) signaling pathways in both in vitro and in vivo models. CONCLUSIONS: We suggest that 4-AAQB treatment might be a tangible therapeutic strategy in the management of NAFLD/NASH.

Evaluation of early resin luting cement damage induced by voids around a circular fiber post in a root canal treated premolar by integrating micro-CT, finite element analysis and fatigue testing.

OBJECTIVE: This study utilizes micro-CT image combined with finite element (FE) analysis and in vitro fatigue testing to investigate the mechanical behavior associating with early resin luting cement damage induced by voids around a circular fiber post in a root canal treated premolar. METHODS: Six similar mandibular first premolars with root canal treatment were scanned with high resolution micro-CT before and after fatigue testing. Micro-CT images of all teeth were processed to identify various materials (dentin, luting cement and void) to evaluate the volume/position of the void in each reconstructed tooth root canal model. Six corresponding mesh models from CT images were generated to perform FE simulations under receiving oblique concentrated loads (200N) to evaluate the luting cement layer mechanical behavior. All teeth were subjected to the fatigue test with 240,000 load cycles simulating chewing for one year to compare results with those in FE simulations. RESULTS: The result showed that most voids occurred adjacent to the apical third of the fiber post. Voids induced the fiber post to pull out, creating a stress concentration at the void boundary. Fatigue life in the experimental testing was found decreased with the stress value/micro-motion increasing in FE analysis. SIGNIFICANCE: This study establishes that micro-CT, FE simulation and fatigue testing can be integrated to understand the early de-bonding mechanism at the luting cement layer in a root canal treated premolar, suggesting that attention must be paid to resin luting cement dissolving/debonding easier when voids occur in the apical and peri-apical areas of fiber posts.

Regulation of ribonuclease E activity by the L4 ribosomal protein of Escherichia coli.

Whereas ribosomal proteins (r-proteins) are known primarily as components of the translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments.

Genomic analysis of mRNA decay in E. coli with DNA microarrays.

The decay of mRNA plays an important role in the regulation of gene expression. Although relatively ignored for many years and regarded as a simple ribonucleotide salvage pathway, mRNA decay has been established in recent years as a well-defined cellular process that plays an integral role in determining gene expression. The recent application of microarray methods to the study of diverse organisms will help us to better understand these gene regulatory circuits and the influence of transcript stability on gene expression. DNA microarray technology is the method of choice to study individual mRNA half-lives on a global scale. It is important to standardize these methods to generate reproducible and reliable results. In this chapter, we describe experimental designs for the analysis of mRNA decay on a genome-wide scale and provide detailed protocols for each experimental step. We also present an analysis of the decay of chromosomally encoded mRNAs in E. coli.

RhlB helicase rather than enolase is the beta-subunit of the Escherichia coli polynucleotide phosphorylase (PNPase)-exoribonucleolytic complex.

Escherichia coli polynucleotide phosphorylase (PNPase), a protein that has both ribonucleolytic and synthetic capabilities, binds, along with the 48-kDa glycolytic enzyme enolase, the 50-kDa DEAD-box protein RhlB helicase and other cellular proteins to the C-terminal "scaffold" region of RNase E to form a complex termed the RNA degradosome. PNPase itself has been reported to exist as a complex (alpha(3)beta(2)) containing trimers of a catalytic subunit (alpha) and dimers of another subunit (beta). The beta-subunit has been believed to be enolase; we report here that it is instead the RhlB helicase. Whereas interaction between PNPase-alpha and enolase was observed in bacteria that synthesize RNase E having a scaffold region, immunoprecipitates from cells expressing PNPase-alpha, RhlB, and enolase from single-copy chromosomal loci, plus a mutant RNase E protein lacking its C-terminal half, showed direct association of PNPase-alpha only with RhlB. Using affinity chromatography, we found that PNPase-alpha and RhlB form a ribonucleolytically active complex corresponding to the mass calculated previously for alpha(3)beta(2) (i.e., 377-380 kDa), whereas no association between PNPase-alpha and enolase was detected. Chromosomal deletion of the eno gene had no effect on the ability of PNPase to degrade either single- or double-stranded RNAs. Collectively, our findings show that direct interaction between PNPase-alpha and RhlB occurs physiologically in the absence of the RNase E C-terminal region, that enolase association with PNPase-alpha is a consequence of the interaction of both proteins with RNase E, and that, contrary to current notions, enolase is not the beta-subunit of E. coli PNPase complex.

Global analysis of Escherichia coli RNA degradosome function using DNA microarrays.

RNase E, an essential endoribonuclease of Escherichia coli, interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome. To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289 E. coli mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover. We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations. Our results, which argue that decay of some E. coli mRNAs in vivo depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs for decay by degradosomes.

Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production.

An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme.

Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays.

Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the approximately 4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although approximately 80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5' or 3' untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.