Comparison of the effects of pachymic acid, moronic acid and hydrocortisone on the polysome loading of RNAs in lipopolysaccharide-treated THP-1 macrophages.

We have proposed that analysis of ribosome-loaded mRNAs (i.e., the translatome) is useful for elucidation of pharmacological effects of phytocompounds in immune cells, regarding the involvement of post-transcriptional regulation mechanisms. In the present study, we compared the effects of pachymic acid from Poria cocos fungus and moronic acid from propolis with those of hydrocortisone on the translatomes of THP-1 macrophages exposed to bacterial lipopolysaccharide (LPS) to find clues to their biological effects. Polysome-associated RNAs collected from cells treated for 3 h with LPS plus each of the compounds were analyzed by DNA microarray followed by analyses of pathways/gene ontologies (GO). Upregulated mRNAs in enriched pathways that were found to contain AUUUA (AU)-rich motifs were checked by real-time PCR, and expression of candidate RNA-binding proteins stabilizing/destabilizing such AU-rich mRNAs was checked by Western blotting. The numbers of upregulated and downregulated genes (fold-changes ± 2.0 versus vehicle-control) were, respectively, 209 and 125 for moronic acid, 23 and 2 for pachymic acid, and 214 and 59 for hydrocortisone treatment. Overlapping with hydrocortisone treatment for upregulation were 158 genes in moronic acid and 17 in pachymic acid treatment; of these, 16 overlapped within all treatments (C-X-C motif chemokine ligands, interferon-induced protein with tetratricopeptide repeats, etc.). Pathway analyses showed GO enrichments such as 'immune response', 'receptor binding', 'extracellular space' etc. The pachymic acid-upregulated mRNAs (highly overlapped with the other 2 treatments) showed the presence of signal peptides and AU-rich motifs, suggesting regulation by AU-rich element (ARE)-binding proteins. The expression of ARE-binding protein HuR/ELAV-1 was increased by the 3 compounds, and AUF1/hnRNP D was decreased by pachymic acid. These results suggested that pachymic acid and moronic acid effects may involve as yet unknown post-transcriptional modulation via ARE-binding proteins resembling that of glucocorticoids.

Shikonin changes the lipopolysaccharide-induced expression of inflammation-related genes in macrophages.

We aimed to find candidate molecules possibly involved in the anti-inflammatory activity of shikonin (active compound of "Shikon") by analyzing its effects on gene expression of lipopolysaccharide (LPS)-treated THP-1 macrophages. Polysome-associated mRNAs (those expected to be under translation: translatome) from cells treated with LPS alone (LPS: 5 µg/mL), shikonin alone (S: 100 nM), or LPS plus shikonin (LPS&S) for 3 h were analyzed by DNA microarray followed by detection of enriched pathways/gene ontologies using the tools of the STRING database. Candidate genes in enriched pathways in the comparison of LPS&S cells vs. LPS cells were analyzed by reverse-transcription quantitative real-time PCR (RT-qPCR; 1, 2, and 3 h). DNA microarray showed shikonin significantly influences gene expression. Gene expression changes between LPS&S cells and LPS cells were compared to detect relevant proteins and/or mRNAs underlying its anti-inflammatory effects: shikonin downregulated pathways which were upregulated in LPS cells, for example, 'innate immune response'. Within changed pathways, three genes were selected for RT-qPCR analyses as key candidates influencing inflammatory responses: CYBA (component of the superoxide-generating Nox2 enzyme), GSK3B (controller of cell responses after toll-like receptor stimulation), and EIF4E (a key factor of the eukaryotic translation initiation factor 4F complex that regulates abundance of other proteins involved in immune functions). All three mRNAs were decreased at 2 h, and CYBA continued low at 3 h relative to LPS cells. Given that shikonin decreased the expression of CYBA gene of Nox2, in addition to the direct inhibition of the Nox2 activity that we have previously shown, it is suggested that one of its anti-inflammatory mechanisms could be attenuation of oxidative stress.

Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca(2+) fluxes.

Shikonin, an anti-inflammatory compound of "Shikon", inhibits the neutrophil superoxide (O2 (•-)) generation by NADPH oxidase 2 (Nox2); however, the mechanisms of how shikonin affects Nox2 activity remained unclear. We aimed to elucidate the relationship between the inhibition of Nox2 activity and influences on intracellular Ca(2+) concentration ([Ca(2+)]i) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca(2+)]i (by fluorescence) and O2 (•-) generation (by chemiluminescence) and evaluated the effects of shikonin on neutrophil-like HL-60 cells stimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Since fMLP activates Nox2 by elevation in [Ca(2+)]i via fluxes such as inositol 1,4,5-trisphosphate-induced Ca(2+) release (IICR) and store-operated Ca(2+) entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE. Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca(2+)]i and O2 (•-) generation (IC50 values of 1.45 and 1.12 µM, respectively) in a synchronized manner. Analyses of specific Ca(2+) fluxes showed that shikonin inhibits IICR and IICR-linked O2 (•-) generation (IC50 values: 0.28 and 0.31 µM for [Ca(2+)]i and O2 (•-), respectively), as well as SOCE and SOCE-linked O2 (•-) generation (IC50 values: 0.39 and 0.25 µM for [Ca(2+)]i and O2 (•-), respectively). These results suggested that shikonin inhibits the O2 (•-) generation by Nox2 in fMLP-stimulated neutrophils by targeting Ca(2+) fluxes such as IICR and SOCE.

Anti-inflammatory effects of alpinone 3-acetate from Alpinia japonica seeds.

We aimed to investigate the bioactive components of Alpinia japonica as anti-inflammatory compounds using searches of the Alpinia genus, and subsequently demonstrated that alpinone 3-acetate markedly inhibits 12-O-tetradecanoyiphorbol 13-acetate-induced inflammation in a mouse model of ear edema. To assess other bioactivities of alpinone 3-acetate, we performed translatome analyses and compared them with those of hydrocortisone. Polysome-associated mRNAs were prepared from alpinone 3-acetate- or hydrocortisone-treated and control cells from 12-O-tetradecanoyiphorbol 13-acetate-induced THP-1-derived macrophages cultured in the presence of Escherichia coli O-111 lipopolysaccharide. Subsequent microarray analysis revealed that alpinone 3-acetate and hydrocortisone upregulated and downregulated the same 155 and 41 genes, respectively. Moreover, direct comparisons of translationally regulated genes indicated 5 and 10 gene probes that were upregulated and downregulated by alpinone 3-acetate and hydrocortisone, respectively. In conclusion, assays of 12-O-tetradecanoyiphorbol 13-acetate-induced inflammation ear edema in mice and polysome profiling of alpinone 3-acetate bioactivities indicated similar medicinal possibilities to those of hydrocortisone.

Evaluation of radical scavenging properties of shikonin.

With the aim of developing effective anti-inflammatory drugs, we have been investigating the biochemical effects of shikonin of "Shikon" roots, which is a naphthoquinone with anti-inflammatory and antioxidative properties. Shikonin scavenged reactive oxygen species like hydroxyl radical, superoxide anion (O2 (•-)) and singlet oxygen in previous studies, but its reactivity with reactive oxygen species is not completely understood, and comparison with standard antioxidants is lacking. This study aimed elucidation of the reactivity of shikonin with nitric oxide radical and reactive oxygen species such as alkyl-oxy radical and O2 (•-). By using electron paramagnetic resonance spectrometry, shikonin was found unable of reacting with nitric oxide radical in a competition assay with oxyhemoglobin. However, shikonin scavenged alkyl-oxy radical from 2,2'-azobis(2-aminopropane) dihydrochloride with oxygen radical absorbance capacity, ORAC of 0.25 relative to Trolox, and showed a strong O2 (•-)-scavenging ability (42-fold of Trolox; estimated reaction rate constant: 1.7 × 10(5) M(-1)s(-1)) in electron paramagnetic resonance assays with CYPMPO as spin trap. Concerning another source of O2 (•-), the phagocyte NADPH oxidase (Nox2), shikonin inhibited the Nox2 activity by impairing catalysis when added before enzyme activation (IC50: 1.1 µM; NADPH oxidation assay). However, shikonin did not affect the preactivated Nox2 activity, although having potential to scavenge produced O2 (•-). In conclusion, shikonin scavenged O2 (•-) and alkyl-oxy radical, but not nitric oxide radical.

Diarylheptanoids from Alpinia officinarum Cause Distinct but Overlapping Effects on the Translatome of B Lymphoblastoid Cells.

Diarylheptanoids (AO-0001, AO-0002, and AO-0003) isolated from Alpinia officinarum inhibit proinflammatory mediators and exhibit cytotoxic and antiviral activity. However, the precise mechanisms of action of these diarylheptanoids are unknown as are their effects on expression of specific genes. Here, we used a translatome analysis to investigate the mechanisms and modes of action of these three diarylheptanoids. Polysome-associated messenger RNAs (mRNAs) were prepared from diarylheptanoids-treated and control cells from a human B lymphoblastoid cell line; these mRNA samples were then used for microarray analysis. Microarray Data Analysis Tool version 3.2 was used to analyze the microarray data analysis; this software uses pathway information of the WikiPathways for gene ontology analysis. Each of the diarylheptanoids caused upregulation or downregulation of the same 37 and 286 genes, respectively. Among the 37 upregulated genes, 16 were related to mRNA processing based on the WikiPathways analysis. Our findings provided new insights into the mode of action of diarylheptanoids from A. officinarum.

Thrombin-stimulated proliferation is mediated by endothelin-1 in cultured rat gingival fibroblasts.

Abstract Endothelin-1 (ET-1) appears to be involved in drug-induced proliferation of gingival fibroblasts. Thrombin induces proliferation of human gingival fibroblasts via protease-activated receptor 1 (PAR1). In this study, using cultured rat gingival fibroblasts, we investigated whether thrombin-induced proliferation of gingival fibroblasts is mediated by ET-1. Thrombin-induced proliferation (0.05-2.5 U/mL). Proliferation was also induced by a PAR1-specific agonist (TFLLR-NH(2,) 0.1-30 microm), but not by a PAR2-specific agonist (SLIGRL-NH(2)). Thrombin (2.5 U/mL) induced an increase in immunoreactive ET-1 expression, which was inhibited by cycloheximide (10 microg/mL), and an increase in preproET-1 mRNA expression, as assessed by reverse transcription polymerase chain reaction. TFLLR-NH(2) increased ET-1 release into the culture medium in both a concentration (0.01-10 microm)- and time (6-24 h)-dependent manner, as assessed by solid phase sandwich enzyme-linked immunosorbent assay. The thrombin (2.5 U/mL)-induced proliferation was inhibited by a PAR1-selective inhibitor, SCH79797 (0.1 microm) and an ET(A) antagonist, BQ-123 (1 microm), but not by an ET(B) antagonist, BQ-788 (1 microm). These findings suggest that thrombin, acting via PAR1, induced proliferation of cultured rat gingival fibroblasts that was mediated by ET-1 acting via ET(A).

Identification of AUF1 as a rapamycin-responsive binding protein to the 5'-terminal oligopyrimidine element of mRNAs.

In vertebrates, mRNAs containing a 5'-terminal oligopyrimidine (TOP) motif are coordinately post-transcriptionally regulated. Binding of specific proteins to this element has been proposed to downregulate expression of TOP mRNAs at the level of translational initiation. We previously reported that rapamycin induces binding activity to the TOP element of ribosomal protein (r-protein) L32 mRNA. In this study, we adapt DEAE-cellulose/oligo dT-cellulose tandem column chromatography to purify TOP element-binding proteins from bovine submaxillary lymph nodes (SLN). We also show by northwestern blot analysis that two proteins of molecular weight 47kDa (47BP) and 43kDa (43BP) specifically bind to a (32)P-labeled riboprobe containing TOP regulatory element of the r-protein L32. Microsequencing of the purified 47BP revealed an internal sequence of 15 amino acids identical to the consensus sequence of the 2x RBD-Gly family. Western blot analysis of the cytoplasm fractions using an AUF1 antibody revealed that these two proteins are p45 AUF1 and p42 AUF1. Increases of the four isoforms of AUF1 protein were observed in 100,000g supernatant fractions of rapamycin-administered rat SLN. Furthermore, decreases of p45 AUF1 and p42 and/or p40 AUF1 were observed in the polysomal fractions of BJAB cells in which translation of TOP mRNAs was selectively suppressed by rapamycin treatment. Taken together, these results suggest that AUF1 is a TOP mRNA-binding protein that may participate in the translational suppression of TOP mRNAs resulting from rapamycin treatment.

An IkappaB-beta COOH terminal region protein is essential for the proliferation of CHO cells under acidic stress.

CHO-K1 cells were able to proliferate and maintain pHi homeostasis at pH 6.3. A novel acidic sensitive mutant, AS-5B, which proliferated at pH 7.4 but failed to either proliferate or maintain pHi homeostasis at pH 6.3, was derived from CHO-K1 using a replica method. The acidic-sensitivity of AS-5B was not due to deficiencies in sodium proton exchangers, HCO3- (co)transporters or H+-ATPases. A cDNA clone encoding a COOH terminal region of IkappaB-beta conferred partial acidic-resistance on AS-5B, and the encoded protein was present in CHO-K1, but was nearly absent from AS-5B. Our data demonstrated that the expression of this small protein was essential for the proliferation of CHO cells under acidic stress.

Expression of chaA, a sodium ion extrusion system of Escherichia coli, is regulated by osmolarity and pH.

ChaA, one of the sodium ion extrusion systems of Escherichia coli, was found to function at high pH [Biochim. Biophys. Acta 1363 (1998) 231]. A chaA-lacZ transcriptional fusion gene was constructed using chaA of E. coli O157:H7 and its expression was observed in strains derived from E. coli K12. The fusion gene was expressed at high pH and was induced by the addition of NaCl, KCl or sucrose. The amount of chaA mRNA measured by reverse transcription-polymerase chain reaction (RT-PCR) was increased by the addition of sucrose to alkaline growth medium. These results suggested that chaA expression was regulated by medium osmolarity and pH.

Rapamycin induces binding activity to the terminal oligopyrimidine tract of ribosomal protein mRNA in rats.

The immunosuppressant rapamycin selectively suppresses the translation of mRNAs containing a terminal oligopyrimidine (TOP) tract adjacent to the cap structure. trans-Acting factors that bind to the 5'-untranslated region (5'-UTR) of TOP mRNAs may be involved in selective translational repression. Some of these factors are regulated by rapamycin-responsive signaling pathways. To identify candidates for the selective trans-acting factor, we examined whether administration of rapamycin alters the binding activity of proteins that bind to RNA containing the TOP element of mouse ribosomal protein (r-protein) L32 mRNA. Preadministration with Freund's complete adjuvant (FCA) prior to rapamycin treatment resulted in increased translational efficiency of r-protein L32 mRNA in submaxillary lymph node (SLN; 2.3-fold), thymus (1.5-fold), and parotid gland (PG; 1.6-fold). Translation of r-protein L32 or elongation factor 1A mRNAs in SLN and PG from FCA-pretreated rats were sensitive to rapamycin administration and the binding ability of p56 was generally increased in extracts from these tissues. On the other hand, in thymus, rapamycin had no effect on the translational efficiency of TOP mRNAs and no p56 binding was detected in the extracts from FCA-pretreated animals. Coadministration of FK506, another immunosuppressive macrolide, increased the p56 TOP-RNA-binding activity and induced selective translational repression of TOP mRNAs in a dose-dependent manner, even in thymus. These findings indicate that p56 is a plausible candidate for the trans-acting factor responsible for regulating the translation of TOP mRNA by a rapamycin-sensitive pathway and that TOP mRNA translational regulation may be responsible for the tissue specificity of rapamycin.

Different proteins are phosphorylated under acidic environments in Jurkat cells.

In order to test the ability of immune cells to grow under acidic conditions, Jurkat cells were cultured in medium at pH 6.3. At this acidic pH, the cells required approximately 2 days per generation, while the generation time was approximately 1 day for cells grown in near neutral medium. Western blot analysis using anti-phosphotyrosine antibody (4G10) showed that protein phosphorylation was affected by external pH. For example, the level of a 66-kDa phosphoprotein increased dramatically in acidic medium, whereas the amount of a 34-kDa phosphoprotein decreased. These results suggest that Jurkat cells use different signal pathways under acidic environments.

Bacterial strategies to inhabit acidic environments.

Bacteria can inhabit a wide range of environmental conditions, including extremes in pH ranging from 1 to 11. The primary strategy employed by bacteria in acidic environments is to maintain a constant cytoplasmic pH value. However, many data demonstrate that bacteria can grow under conditions in which pH values are out of the range in which cytoplasmic pH is kept constant. Based on these observations, a novel notion was proposed that bacteria have strategies to survive even if the cytoplasm is acidified by low external pH. Under these conditions, bacteria are obliged to use acid-resistant systems, implying that multiple systems having the same physiological role are operating at different cytoplasmic pH values. If this is true, it is quite likely that bacteria have genes that are induced by environmental stimuli under different pH conditions. In fact, acid-inducible genes often respond to another factor(s) besides pH. Furthermore, distinct genes might be required for growth or survival at acid pH under different environmental conditions because functions of many systems are dependent on external conditions. Systems operating at acid pH have been described to date, but numerous genes remain to be identified that function to protect bacteria from an acid challenge. Identification and analysis of these genes is critical, not only to elucidate bacterial physiology, but also to increase the understanding of bacterial pathogenesis.

Ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibits the growth of Escherichia coli at alkaline pH.

The growth of Escherichia coli was inhibited by ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in a medium of initial pH 8.8. The growth inhibition was reversed by the addition of CaCl(2). E. coli could grow in the presence of EGTA at pH values below 8. The concentration of free calcium ions increases with a decrease in medium pH because of a decrease in the calcium binding capacity of EGTA. So, although the results suggest that calcium ions are essential for the growth of E. coli, the minimum concentration required is very low.