SSA/Ro52 autoantigen interacts with Dcp2 to enhance its decapping activity

We identified human decapping enzyme 2 (hDCP2) as a binding protein with Ro52, being colocalized in processing bodies (p-bodies). We also showed that the N-terminus and C-terminus of Ro52 bound to hDCP2. Moreover, Ro52 enhanced decapping activity of hDCP2 in a dose-dependent manner. Our data support the novel notion of the association between Ro52 with hDCP2 protein in cytoplasmic p-bodies, playing a role in mRNA metabolism in response to cellular stimulation.     

Modulation of redox potential and alteration in reactivity via the peroxide shunt pathway by mutation of cytochrome P450 around the proximal heme ligand

In the thermophilic cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st), a phenylalanine residue at position 310 and an alanine residue at position 320 are located close to the heme thiolate ligand, Cys317. Single site-directed mutants F310A and A320Q and double mutant F310A/A320Q have been constructed. All mutant enzymes as well as wild-type (WT) P450st were expressed at high levels. The substitution of F310 with Ala and of A320 with Gln induced shifts in redox potential and blue shifts in Soret absorption of ferrous-CO forms, while spectral characterization showed that in the resting state, the mutants almost retained the structural integrity of the active site. The redox potential of the heme varied as follows: -481 mV (WT), -477 mV (A320Q), -453 mV (F310A), and -450 mV (F310A/A320Q). The trend in the Soret band of the ferrous-CO form was as follows: 450 nm (WT) < 449 nm (A320Q) < 446 nm (F310A) < 444 nm (F310A/A320Q). These results established that the reduction potential and electron density on the heme iron are modulated by the Phe310 and Ala320 residues in P450st. The electron density on the heme decreases in the following order: WT > A320Q > F310A > F310A/A320Q. The electron density on the heme iron infers an essential role in P450 activity. The decrease in electron density interferes with the formation of a high-valent oxo-ferryl species called Compound I. However, steady-state turnover rates of styrene epoxidation with H2O2 show the following trend: WT approximately equal to A320Q < F310A approximately equal to F310A/A320Q. The shunt pathway which can provide the two electrons and oxygen required for a P450 reaction instead of NAD(P)H and dioxygen can rule out the first and second heme reduction in the catalytic process. Because the electron density on the heme iron might be deeply involved in the k cat values in this system, the intermediate Compound 0 which is the precursor species of Compound I mainly appears to participate dominantly in epoxidation with H2O2.     

Antioxidant Activity of 3-Methylthiopropylamine Hydrochloride

Antioxidant activities of various amino acids and some of their related substances on the autoxidation of sodium lenolenate in aqueous system were examined by TBA method. As the results, 3-methylthiopropylamine hydrochloride (MTPA) was found to be most effective in antioxidant activity among the substances tested. Also, it was an unique characteristic of MTPA that it was quite soluble in organic solvents, fats and oils as well as in water. At a temperature of less than 100°C, MTPA was quite stable in aqueous solution over whole pH range. Acute toxicity test of MTPA by oral administration of mice has indicated that LD₅₀ of MTPA was 4.5 g per kg.     

IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^−/−) mice and RAG-2 and RORγt double deficient (RAG-2^−/− × RORγt^−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^−/− × RORγt^−/− mice that lacked T and NKT cells. Surprisingly, RAG-2^−/− × RORγt^−/− mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.     

Increased biological potency of hexafluorinated analogs of 1,25-dihydroxyvitamin D3 on bovine parathyroid cells

1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is known to be involved in regulating the proliferation of parathyroid cells and PTH synthesis through reactions involving its nuclear receptor. We evaluated the effects of 1,25-(OH)₂D₃ and its hexafluorinated analog, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D₃ (26,27-F₆-1,25-(OH)₂D₃), on parathyroid cells. The 1,25-(OH)₂D₃ and 26,27-F₆-1,25-(OH)₂D₃ each inhibited [³H]thymidine incorporation and ornithine decarboxylase (ODC) activity, which is important in cell proliferation, in primary cultured bovine parathyroid cells. The inhibitory effect of 26,27-F₆-1,25-(OH)₂D₃ on PTH secretion from parathyroid cells was significantly more potent than that of 1,25-(OH)₂D ₃ between 10⁻¹¹ M and 10⁻⁸ M. Study of 26,27-F₆-1,25-(OH)₂D₃ metabolism in parathyroid cells in vitro elucidated its slower degradation than that of 1,25-(OH)₂D₃. After 48 h of incubation with [1β-³H]26,27-F₆-1,25-(OH)₂D₃, two HPLC peaks, one for [1β-³H]26,27-F₆-1,25-(OH)₂D₃, and a second larger peak for [1β-³H]26,27-F₆-1,23(S),25-(OH)₃D₃, were detected. No metabolites were detected after the same period of incubation with 1,25-(OH)₂[26,27-³H]D₃. We observed that 26,27-F₆-1,23(S),25-(OH)₃D₃ was as potent as 1,25-(OH)₂D₃ in inhibiting the proliferation of parathyroid cells.Data suggest that the greater biological activity of 26,27-F₆-1,25-(OH)₂D₃ is explained by its slower metabolisms and by the retention of the biological potency of 26,27-F₆-1,25-(OH)₂D₃ even after 23(S)-hydroxylation.     

The role of ether-a-go-go-related gene K(+) channels in glucocorticoid inhibition of adrenocorticotropin release by rat pituitary cells

The present study investigated the role of K(+) channels in the inhibitory effect of glucocorticoid on adrenocorticotropin (ACTH) release induced by corticotropin-releasing hormone (CRH) using cultured rat anterior pituitary cells. Apamin and charybdotoxin (CTX) did not have a significant effect on ACTH release induced by CRH (1 nM). Tetraethylammonium (TEA), a broad spectrum K(+) channel blocker, increased the ACTH response to CRH only at the highest concentration (10 mM). The exposure to 100 nM corticosterone for 60 min inhibited the CRH-induced ACTH release. Neither TEA, apamin, nor CTX affected the inhibitory effect of corticosterone. In contrast, astemizole (Ast) and E-4031, ether-a-go-go-related gene (erg) K(+) channel blockers, abolished the inhibitory effect of corticosterone on CRH-induced ACTH release (1.25+/-0.10 vs. 1.45+/-0.11 ng/well at 10 microM Ast, p>0.05, 1.71+/-0.16 vs. 1.91+/-0.32 ng/well at 10 microM E-4031, p>0.05, vehicle vs. corticosterone). RT-PCR demonstrated all three subtypes of rat-erg mRNAs in the pituitary and corticosterone increased only erg1 mRNA up to 2.47+/-0.54 fold. In conclusion, erg K(+) channels were up-regulated by glucocorticoid, and have indispensable roles in delayed glucocorticoid inhibition of CRH-induced ACTH release by rat pituitary cells.