m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress.

Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA binding protein whose mutations cause amyotrophic lateral sclerosis. TLS/FUS undergoes LLPS and forms membraneless particles with other proteins and nucleic acids. Interaction with RNA alters conformation of TLS/FUS, which affects binding with proteins, but the effect of m6A RNA modification on the TLS/FUS-RNA interaction remains elusive. Here, we investigated the binding specificity of TLS/FUS to m6A RNA fragments by RNA pull down assay, and elucidated that both wild type and ALS-related TLS/FUS mutants strongly bound to m6A modified RNAs. TLS/FUS formed cytoplasmic foci by treating hyperosmotic stress, but the cells transfected with m6A-modified RNAs had a smaller number of foci. Moreover, m6A-modified RNA transfection resulted in the cells obtaining higher resistance to the stress. In summary, we propose TLS/FUS as a novel candidate of m6A recognition protein, and m6A-modified RNA fragments diffuse cytoplasmic TLS/FUS foci and thereby enhance cell viability.

A novel challenge of nondestructive analysis on OGATA Koan's sealed medicine by muonic X-ray analysis.

OGATA Koan (1810-63) was a physician and the director of Tekijuku, and he contributed to Western medicine in the late Edo period. Osaka University preserves two of his medicine chests. One of the chests, which was used in his last years (the second chest) contained 22 glass bottles and 6 wooden cylinders. These bottles and cylinders contained formulated medicines; however, about half cannot be opened because of the long-term storage. It is necessary to comprehend the physical property of both the containers and their contents for investigation of this adequate preservation method; however, destructive analysis is not allowed. To analyze the medicines sealed in the glass bottles, we focused on muonic X-ray analysis, which has high transmittance. First, we certified the analytical methods using a historical medicinal specimen preserved in Osaka University. Thereafter, we applied the method on the bottles stored in the second chest. X-ray fluorescence identified the glass of those bottles to be lead potash glass. Among these bottles, we chose the bottle with the label "," which contains white powdered medication, for muonic X-ray analysis. We identified the contents of the medication in the glass to be Hg2Cl2. Through this study, we first applied muonic X-ray analysis on the medical inheritances and succeeded to detect the elements contained both in the container and in the contents of the sealed bottle. This would be a new method for nondestructive analysis of such cultural properties.

Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m6A modification.

pncRNA-D is an irradiation-induced 602-nt long noncoding RNA transcribed from the promoter region of the cyclin D1 (CCND1) gene. CCND1 expression is predicted to be inhibited through an interplay between pncRNA-D and RNA-binding protein TLS/FUS. Because the pncRNA-D-TLS interaction is essential for pncRNA-D-stimulated CCND1 inhibition, here we studied the possible role of RNA modification in this interaction in HeLa cells. We found that osmotic stress induces pncRNA-D by recruiting RNA polymerase II to its promoter. pncRNA-D was highly m6A-methylated in control cells, but osmotic stress reduced the methylation and also arginine methylation of TLS in the nucleus. Knockdown of the m6A modification enzyme methyltransferase-like 3 (METTL3) prolonged the half-life of pncRNA-D, and among the known m6A recognition proteins, YTH domain-containing 1 (YTHDC1) was responsible for binding m6A of pncRNA-D Knockdown of METTL3 or YTHDC1 also enhanced the interaction of pncRNA-D with TLS, and results from RNA pulldown assays implicated YTHDC1 in the inhibitory effect on the TLS-pncRNA-D interaction. CRISPR/Cas9-mediated deletion of candidate m6A site decreased the m6A level in pncRNA-D and altered its interaction with the RNA-binding proteins. Of note, a reduction in the m6A modification arrested the cell cycle at the G0/G1 phase, and pncRNA-D knockdown partially reversed this arrest. Moreover, pncRNA-D induction in HeLa cells significantly suppressed cell growth. Collectively, these findings suggest that m6A modification of the long noncoding RNA pncRNA-D plays a role in the regulation of CCND1 gene expression and cell cycle progression.

Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity.

Translocated in liposarcoma (TLS) is an RNA-binding protein and a transcription-regulatory sensor of DNA damage. TLS binds promoter-associated noncoding RNA (pncRNA) and inhibits histone acetyltransferase (HAT) activity of CREB-binding protein (CBP)/E1A-binding protein P300 (p300) on the cyclin D1 (CCND1) gene. Although post-translational modifications of TLS, such as arginine methylation, are known to regulate TLS's nucleocytoplasmic shuttling and assembly in stress granules, its interactions with RNAs remain poorly characterized. Herein, using various biochemical assays, we confirmed the earlier observations that TLS is methylated by protein arginine methyltransferase 1 (PRMT1) in vitro The arginine methylation of TLS disrupted binding to pncRNA and also prevented binding of TLS to and inhibition of CBP/p300. This result indicated that arginine methylation of TLS abrogates both binding to pncRNA and TLS-mediated inhibition of CBP/p300 HAT activities. We also report that an arginine residue within the Arg-Gly-Gly domain of TLS, Arg-476, serves as the major determinant for binding to pncRNA. Either methylation or mutation of Arg-476 of TLS significantly decreased pncRNA binding and thereby prevented a pncRNA-induced allosteric alteration in TLS that is required for its interaction with CBP/p300. Moreover, unlike WT TLS, an R476A TLS mutant did not inhibit CCND1 promoter activity in luciferase reporter assays. Taken together, we propose the hypothesis that arginine methylation of TLS regulates both TLS-nucleic acid and TLS-protein interactions and thereby participates in transcriptional regulation.

Internal evaluation of impregnation treatment of waterlogged wood; relation between concentration of internal materials and relaxation time using magnetic resonance imaging.

The purpose of this study is to clarify the degree of impregnation resulting from treatment of internal waterlogged wood samples using MRI. On a 1.5T MR scanner, T1 and T2 measurements were performed using inversion recovery and spin-echo sequences, respectively. The samples were cut waterlogged pieces of wood treated with various impregnation techniques which were divided into different concentrations of trehalose (C12H22O11) and polyethylene glycol (PEG; HO-(C2H4O)n-H) solutions. Then these samples underwent impregnation treatment every two weeks. From the results, we found that the slope of the T1-concentration curve using linear fitting showed the value of the internal area for PEG to be higher than the external area; internal, -2.73ms/wt% (R2=0.880); external, -1.50ms/wt% (R2=0.887). Furthermore, the slope of the T1-concentration curve using linear fitting showed the values for trehalose to have almost no difference when comparing the internal and the external areas; internal, -2.79ms/wt% (R2=0.759); external, -3.02ms/wt% (R2=0.795). However, the slope of the T2-concentration curve using linear fitting for PEG showed that there was only a slight change between the internal and the external areas; internal, 0.26ms/wt% (R2=0.642); external, 0.18ms/wt% (R2=0.920). The slope of the T2-concentration curve did not show a change in linear relationship between the internal and the external areas; internal, 0.06ms/wt% (R2=0.175); external, -0.14ms/wt% (R2=0.043). In conclusion, using visualization of relaxation time T1, it is possible to obtain more detail information noninvasively concerning the state of impregnation treatment of internal waterlogged wood.

Synthesis and properties of oxo-carboxylato- and dioxo-bridged diosmium complexes of tris(2-pyridylmethyl)amine.

A series of oxo-bridged diosmium complexes with tpa ligand (tpa = tris(2-pyridylmethyl)amine) are synthesized. The hydrolytic reaction of the mononuclear osmium complex [Os(III)Cl(2)(tpa)]PF(6) in aqueous solution containing a sodium carboxylate yields a µ-oxo-µ-carboxylato-diosmium(III) complex, [Os(III)(2)(µ-O)(µ-RCOO)(tpa)(2)](PF(6))(3) (R = C(3)H(7) (1), CH(3) (2), or C(6)H(5) (3)). One-electron oxidation of 1 with (NH(4))(2)Ce(IV)(NO(3))(6) gives a mixed-valent [Os(III)Os(IV)(µ-O)(µ-C(3)H(7)COO)(tpa)(2)](PF(6))(4) complex (4). A mixed-valent di-µ-oxo-diosmium complex, [Os(III)Os(IV)(µ-O)(2)(tpa)(2)](PF(6))(3) (5), is also synthesized from 1 in an aerobic alkaline solution (pH 13.5). All the complexes exhibit strong absorption bands in a visible-near-infrared region based on interactions of the osmium dπ and oxygen pπ orbitals of the Os-O-Os moiety. The X-ray crystallographic analysis of 1, 3, and 4 shows that the osmium centers take a pseudo-octahedral geometry in the µ-oxo-µ-carboxylato-diosmium core. The mixed-valent osmium(III)osmium(IV) complex 4 has a shorter osmium-oxo bond and a larger osmium-oxo-osmium angle as compared with those of the diosmium(III) complex 1 having the same bridging carboxylate. Crystal structure of 5 reveals that the two osmium ions are bridged by two oxo groups to give an Os(2)(µ-O)(2) core with the significantly short osmium-osmium distance (2.51784(7) Å), which is indicative of a direct osmium-osmium bond formation with the bond order of 1.5 (σ(2)π(2)δ(2)δ*(2)π*(1) configuration). In the electrochemical studies, the µ-oxo-µ-carboxylato-diosmium(III) complexes exhibit two reversible Os(III)Os(III)/Os(III)Os(IV) and Os(III)Os(IV)/Os(IV)Os(IV) oxidation couples and one irreversible redox wave for the Os(III)Os(III)/Os(II)Os(III) couple in CH(3)CN. The irreversible reductive process becomes reversible in CH(3)CN/H(2)O (1:1 Britton-Robinson buffer; pH 5-11), where the {1H(+)/2e(-)} transfer process is indicated by the plot of the redox potentials against the pH values of the solution of 1. Thus, the µ-oxo-µ-butyrato-diosmium(III) center undergoes proton-coupled electron transfer to yield a µ-hydroxo-µ-butyrato-diosmisum(II) species. The di(µ-oxo) complex 5 exhibits one reversible Os(III)Os(IV)/Os(IV)Os(IV) oxidation process and one reversible Os(III)Os(IV)/Os(III)Os(III) reduction process in CH(3)CN. The comproportionation constants K(com) of the Os(III)Os(IV) states for the present diosmium complexes are on the order of 10(19). The values are significantly larger when compared with those of similar oxo-bridged dimetal complexes of ruthenium and rhenium.

A bitter diterpenoid furanolactone columbin from Calumbae Radix inhibits azoxymethane-induced rat colon carcinogenesis.

The modifying effect of dietary administration of a diterpenoid furanolactone columbin isolated from the crude drug Calumbae Radix (the root of Jateorhiza columba MIERS, Menispermacea) on azoxymethane (AOM)-induced was investigated in male F344 rats. Animals were initiated with AOM (three weekly subcutaneous injections of 15 mg/kg body weight) to induce colonic neoplasms. They were fed the experimental diets mixed with columbin (4, 20, and 100 ppm) for 4 weeks, starting 1 week before the first dosing of AOM and thereafter maintained on the basal diet without columbin. Additional experimental groups included the AOM alone group, the columbin alone group (100 ppm in diet for 4 weeks), and the untreated control group. Dietary feeding of columbin (4, 20, and 100 ppm) during the initiation phase of AOM-induced colon carcinogenesis reduced the incidence and multiplicity of colonic adenocarcinoma and the inhibition by feeding of 20 ppm (incidence: 20%, P=0.0242 and multiplicity: 0.20+/-0.40, P<0.02) and 100 ppm (incidence: 10%, P=0.0029 and multiplicity: 0.10+/-0.30, P<0.002) columbin was significant when compared with the AOM alone group (incidence: 55% and multiplicity: 0.55+/-0.50). Also, columbin administration in diet lowered the number of argyrophilic nucleolar organizer regions protein per nucleus in non-lesional colonic crypts and the blood polyamine content, which are reflected in cell proliferation activity. These results indicate chemopreventive ability of dietary columbin against chemically induced colon tumorigenesis when fed during the initiation phase, providing a scientific basis for chemopreventive ability of columbin against human colon cancer.

Effects of extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver.

The effects of a standardized extract of Ginkgo biloba L. leaves (EGb) and its terpene constituents, bilobalide and ginkgolides, on the activities of detoxification enzymes, i.e., glutathione S-transferases (GSTs) and DT-diaphorase, and glutathione contents, were investigated in the mouse liver. Oral treatment with EGb (100-1,000 mg/kg) and bilobalide (10-30 mg/kg) once a day for 4 days caused a dose-dependent elevation in GST activity. Ginkgolide A (30 mg/kg, for 4 days) also significantly elevated GST activity, whereas ginkgolide B and ginkgolide C at the same dose had no effects. EGb significantly increased the protein level of GST pi, and bilobalide significantly increased those of GST alpha and GST mu Moreover, EGb-treatment and bilobalide-treatment caused significant elevations in DT-diaphorase activity and in hepatic glutathione contents.