A novel method for determining vitamin B1 in a wide variety of foodstuffs with or without polyphenols.

Certain foodstuffs exhibit matrix interference effects on the vitamin B(1) analysis prescribed in the official methods of the European Union, AOAC International, and Japan. In this study, we demonstrated that one of the problematic polyphenols in green tea or cocoa was tannin. For these matrices, thiamine was found to interact with tannin in the extraction step and was thus removed by filtration as a residue. To resolve the matrix interference, we proposed lowering the pH of the extraction solution by adding hydrochloric acid after the enzyme reaction. High precision (2-3% RSDr) and good recovery (98.3-103%) were obtained with reference materials using the proposed method. We also confirmed the equivalence of the obtained data from the proposed method and the Japanese official method for nutritional labeling. From these results, the method was found to be effective for vitamin B(1) analysis regardless of the presence of interference matrices.

Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation.

Interferon regulatory factor (IRF)-3 is a critical transcription factor regulating innate immune responses against viral and bacterial infections. Signals activated by various pathogens are integrated by IRF-3 kinase, resulting in the specific phosphorylation of IRF-3 in the cytoplasm. This phosphorylation induces dimerization and association with the coactivators CREB-binding protein/p300, and the resultant complex activates the target genes in the nucleus. However, the phosphorylation sites that determine the active/inactive status of IRF-3 have been a source of controversy. In this study, we generated an antibody that specifically detects the phosphorylation of Ser-386 and used it as a probe. We found: 1) viral infection specifically induces phosphorylation of the Ser-386; 2) recently identified IRF-3 kinases (IKK-i/epsilon and TBK-1) phosphorylate Ser-386 and induce its dimerization; 3) phosphorylation of Ser-386 is exclusively observed with the dimer; 4) mutation at Ser-386 abolishes the dimerization potential; 5) a constitutively active 5D mutant designed to mimic phosphorylation of Ser/Thr residues other than Ser-385 and -386 is secondarily phosphorylated at Ser-386, presumably by an irrelevant kinase. These results strongly suggest that Ser-386 is the target of the IRF-3 kinase and critical determinant for the activation of IRF-3.

X-ray crystal structure of IRF-3 and its functional implications.

Transcription factor IRF-3 is post-translationally activated by Toll-like receptor (TLR) signaling and has critical roles in the regulation of innate immunity. Here we present the X-ray crystal structure of the C-terminal regulatory domain of IRF-3(175-427) (IRF-3 175C) at a resolution of 2.3 A. IRF-3 175C is structurally similar to the Mad homology domain 2 of the Smad family. Structural and functional analyses reveal phosphorylation-induced IRF-3 dimerization, which generates an extensive acidic pocket responsible for binding with p300/CBP. Although TLR and Smad signaling are evolutionarily independent, our results suggest that IRF-3 originates from Smad and acquires its function downstream of TLR.

Expression of FTZ-F1alpha in transgenic Xenopus embryos and oocytes.

Fushi tarazu transcription factor-1 (FTZ-F1) was originally found as a regulator of fushi tarazu gene expression in Drosophila. The frog homologue (FTZ-F1alpha) and the 3.5 kb 5'-flanking region of the FTZ-F1alpha gene have been cloned, and it has been shown by reverse transcription-polymerase chain reaction that FTZ-F1alpha expression begins in embryos at stage 11 and becomes stronger after that. By in situ hybridization analysis, the FTZ-F1alpha mRNA was also found in immature frog oocytes. In this study, immunohistology revealed that the product of FTZ-F1alpha was localized in the cytoplasm of the immature oocyte. To analyze the promoter activity of the Rana rugosa FTZ-F1alpha gene, transgenic Xenopus were produced carrying the fusion construct, consisting of truncated 5'-flanking regions (3.0, 1.8 and 0.3 kb) of the FTZ-F1alpha gene and the green fluorescent protein (GFP) open reading frame. The 0.3 kb 5'-flanking region could drive GFP expression in Xenopus embryos at stage 20 and in immature oocytes in the ovary 2 months after metamorphosis. Gel mobility shift assay was used to test whether proteins in extracts from Xenopus embryos and ovaries bound to the 0.3 kb DNA. The extract from embryos at stage 11 formed one retarded band. The extract from ovaries formed a different retarded band. The results, taken together, indicate that production of transgenic Xenopus is very useful for the analysis of the promoter activity of genes in amphibians. The results also suggest that at least two proteins (one in the embryo and the other in the ovary of 2-month-old postmetamorphosing Xenopus) bind the 0.3 kb 5'-flanking region of the FTZ-F1alpha gene. These proteins may be involved in the regulation of FTZ-F1alpha gene expression in amphibians.

Two novel genes expressed in Xenopus germ line: characteristic features of putative protein structures, their gene expression profiles and their possible roles in gametogenesis and embryogenesis.

We compared the secondary spermatogonia and the primary spermatocytes of Xenopus for the proteins in their microsomal fractions and identified a newly synthesized protein (94 kDa) and three other proteins (99, 85, and 72 kDa) which increased their amount after entering the meiotic phase. These four proteins were used as antigens to produce polyclonal antibody which was found to react with the four proteins as well as two other proteins (208 and 60 kDa). Immunoscreening of Xenopus testis cDNA library with this polyclonal antibody yielded two cDNA clones (Xmegs and Xtr) encoding novel proteins. Xmegs mRNA was specifically expressed in the spermatogenic cells from the mid-pachytene stage to completion of two meiotic divisions. The putative Xmegs protein contained 19 tandem repeats of 26 amino acid residues rich in proline as well as potential phosphorylation sites (i.e., serine and threonine residues). Around this repetitive area, we found five PEST sequences known as a proteolytic signal to target protein for degradation. The presence of PEST sequences was believed to allow protein levels to closely parallel mRNA abundance. These results suggested the possible role of this novel protein in the regulation of two meiotic divisions specific to the spermatogenesis in a phosphorylation- and/or dephosphorylation-dependent manner. On the other hand, Xtr mRNA was expressed in both spermatogenic and oogenic cells except for round spermatids and the later stage cells. This mRNA was also expressed in the early stage embryos and its amount was kept constant from the St. I oocyte to the gastrula stage and decreased thereafter. The putative Xtr protein contained four complete and one partial tudor-like domains that were discovered in Drosophila tudor protein which plays an important role in PGC differentiation and abdominal segmentation. The characteristic expression profile of Xtr and the protein structure similar to the Drosophila tudor protein suggested its possible role in the progression of meiosis and PGC differentiation.