[Inter-laboratory Study on the Modified Methods for Analyzing Bisphenol A Content for Migration Tests from Polycarbonate Food Apparatuses, Containers, and Packaging].

An inter-laboratory study involving 24 laboratories was conducted to validate the modified analytical method for the migration solution of heptane for the determination of bisphenol A migrating from polycarbonate food processing materials. In this study, two concentrations of samples were blindly coded. Each laboratory determined the analyte (bisphenol A, phenol and p-tert-butylphenol) concentration in each sample according to the established protocol. The obtained values were analyzed statistically using internationally accepted guidelines. Horwitz ratios were calculated based on the reproducibility relative standard deviation (RSDR), which was estimated from the inter-laboratory study, and predicted RSDR, which was calculated using the Horwitz/Thompson equation. Horwitz ratios of the two samples ranged from 0.15 to 0.37 for the three compounds, meeting the performance criteria of less than 2 set by the Codex Alimentarius for analytical method approval. These results showed that this modified analytical method shows good performance as an analytical method for the migration solution of heptane.

Structural basis of the transcription termination factor Rho engagement with transcribing RNA polymerase from Thermus thermophilus.

Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the adenosine triphosphate-dependent RNA translocase/helicase Rho, which causes RNA/DNA dissociation from the RNAP elongation complex (EC). However, the structural basis of the interplay between Rho and RNAP remains obscure. Here, we report the cryo-electron microscopy structure of the Thermus thermophilus RNAP EC engaged with Rho. The Rho hexamer binds RNAP through the carboxyl-terminal domains, which surround the RNA exit site of RNAP, directing the nascent RNA seamlessly from the RNA exit to its central channel. The ß-flap tip at the RNA exit is critical for the Rho-dependent RNA release, and its deletion causes an alternative Rho-RNAP binding mode, which is irrelevant to termination. The Rho binding site overlaps with the binding sites of other macromolecules, such as ribosomes, providing a general basis of gene regulation.

Adipose tissue-derived mesenchymal stem cells ameliorate bone marrow aplasia related with graft-versus-host disease in experimental murine models.

Graft-versus-host disease (GVHD) constitutes the most frequent complications after the allogeneic hematopoietic stem cell transplantation for a variety of hematological malignancies. In the present study, we explored the prophylactic potential of adipose tissue-derived mesenchymal stem cells (AD-MSCs) in controlling GVHD in murine models with a special focus on bone marrow aplasia related with acute GVHD. The CB6F1 mice were induced GVHD by the injection intravenously of C57BL/6 (B6-Ly-5.1) splenocytes without conditioning irradiation or chemotherapy. AD-MSCs from C3H mice were injected intravenously via tail veins. GVHD was assessed using flowcytometry analysis of peripheral blood cells and histopathologic analysis of target organs. Histopathological analyses revealed that AD-MSCs markedly suppressed the infiltration of lymphocytes into liver as well as the aplasia in bone marrow. This study is the first to clarify the effectiveness of AD-MSCs against bone marrow aplasia in GVHD, supporting a rationale of AD-MSCs for ameliorating bone marrow suppression and infectivity after allo-HSCT in human clinics.

A Thermus phage protein inhibits host RNA polymerase by preventing template DNA strand loading during open promoter complex formation.

RNA polymerase (RNAP) is a major target of gene regulation. Thermus thermophilus bacteriophage P23-45 encodes two RNAP binding proteins, gp39 and gp76, which shut off host gene transcription while allowing orderly transcription of phage genes. We previously reported the structure of the T. thermophilus RNAP•σA holoenzyme complexed with gp39. Here, we solved the structure of the RNAP•σA holoenzyme bound with both gp39 and gp76, which revealed an unprecedented inhibition mechanism by gp76. The acidic protein gp76 binds within the RNAP cleft and occupies the path of the template DNA strand at positions -11 to -4, relative to the transcription start site at +1. Thus, gp76 obstructs the formation of an open promoter complex and prevents transcription by T. thermophilus RNAP from most host promoters. gp76 is less inhibitory for phage transcription, as tighter RNAP interaction with the phage promoters allows the template DNA to compete with gp76 for the common binding site. gp76 also inhibits Escherichia coli RNAP highlighting the template-DNA binding site as a new target site for developing antibacterial agents.

Ratcheting of RNA polymerase toward structural principles of RNA polymerase operations.

RNA polymerase (RNAP) performs various tasks during transcription by changing its conformational states, which are gradually becoming clarified. A recent study focusing on the conformational transition of RNAP between the ratcheted and tight forms illuminated the structural principles underlying its functional operations.

The ratcheted and ratchetable structural states of RNA polymerase underlie multiple transcriptional functions.

DNA-dependent RNA polymerase (RNAP) accomplishes multiple tasks during transcription by assuming different structural forms. Reportedly, the "tight" form performs nucleotide addition to nascent RNA, while the "ratcheted" form is adopted for transcription inhibition. In this study, we performed Cys-pair crosslinking (CPX) analyses of various transcription complexes of a bacterial RNAP and crystallographic analyses of its backtracked and Gre-factor-bound states to clarify which of the two forms is adopted. The ratcheted form was revealed to support GreA-dependent transcript cleavage, long backtracking, hairpin-dependent pausing, and termination. In contrast, the tight form correlated with nucleotide addition, mismatch-dependent pausing, one-nucleotide backtracking, and factor-independent transcript cleavage. RNAP in the paused/backtracked state, but not the nucleotide-addition state, readily transitions to the ratcheted form ("ratchetable"), indicating that the tight form represents two distinct regulatory states. The 3' end and the hairpin structure of the nascent RNA promote the ratchetable nature by modulating the trigger-loop conformation.

Time-resolved Raman and polyacrylamide gel electrophoresis observations of nucleotide incorporation and misincorporation in RNA within a bacterial RNA polymerase crystal.

The bacterial RNA polymerase (RNAP) elongation complex (EC) is highly stable and is able to extend an RNA chain for thousands of nucleotides. Understanding the processive mechanism of nucleotide addition requires detailed structural and temporal data for the EC reaction. Here, a time-resolved Raman spectroscopic analysis is combined with polyacrylamide gel electrophoresis (PAGE) to monitor nucleotide addition in single crystals of the Thermus thermophilus EC (TthEC) RNAP. When the cognate base GTP, labeled with (13)C and (15)N (*GTP), is soaked into crystals of the TthEC, changes in the Raman spectra show evidence of nucleotide incorporation and product formation. The major change is the reduction of *GTP's triphosphate intensity. Nucleotide incorporation is confirmed by PAGE assays. Both Raman and PAGE methods have a time resolution of minutes. There is also Raman spectroscopic evidence of a second population of *GTP in the crystal that does not become covalently linked to the nascent RNA chain. When this population is removed by "soaking out" (placing the crystal in a solution that contains no NTP), there are no perturbations to the Raman difference spectra, indicating that conformational changes are not detected in the EC. In contrast, the misincorporation of the noncognate base, (13)C- and (15)N-labeled UTP (*UTP), gives rise to large spectroscopic changes. As in the GTP experiment, reduction of the triphosphate relative intensity in the Raman soak-in data shows that the incorporation reaction occurs during the first few minutes of our instrumental dead time. This is also confirmed by PAGE analysis. Whereas PAGE data show *GTP converts 100% of the nascent RNA 14mer to 15mer, the noncognate *UTP converts only ∼50%. During *UTP soak-in, there is a slow, reversible formation of an α-helical amide I band in the Raman difference spectra peaking at 40 min. Similar to *GTP soak-in, *UTP soak-in shows Raman spectoscopic evidence of a second noncovalently bound *UTP population in the crystal. Moreover, the second population has a marked effect on the complex's conformational states because removing it by "soaking-out" unreacted *UTP causes large changes in protein and nucleic acid Raman marker bands in the time range of 10-100 min. The conformational changes observed for noncognate *UTP may indicate that the enzyme is preparing for proofreading to excise the misincorporated base. This idea is supported by the PAGE results for *UTP soak-out that show endonuclease activity is occurring.

Crystallization and preliminary X-ray crystallographic analyses of Thermus thermophilus backtracked RNA polymerase.

DNA-dependent RNA polymerase (RNAP) synthesizes RNA complementary to the template DNA. During transcript elongation, RNAP often undergoes backward translocation ('backtracking') by dissociating the 3' end of the nascent RNA transcript from the template DNA. While the backtracked state of RNAP is inactive in RNA elongation, it actively hydrolyses the RNA 3' end to regenerate the active elongation complex. To study the structural basis of the backtracked state and its cleavage activity, two backtracked RNAP complexes were reconstituted by assembling Thermus thermophilus RNAP with designed nucleic acid scaffolds. The reconstituted backtracked complexes were active in the transcript-cleavage reaction. These complexes were crystallized and X-ray diffraction data sets were obtained at resolutions of 3.4 and 3.7 Å.

Isolation of human mAbs that directly modulate FMS-related tyrosine kinase 3 signaling.

FMS-related tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase that plays important roles in hematopoiesis, including early progenitors and dendritic cell development. FLT3 is expressed at high levels in 70-100% of cases of AML and in virtually all cases of B-lineage acute lymphoblastic leukemia. FLT3 is regarded as a molecular target in the development of novel therapies for acute leukemia patients. Currently, many small-molecule FLT3 inhibitors have been developed, but clinical trials have resulted in limited antileukemia effects because of off-target toxicities and drug resistance. The development of anti-FLT3 Abs might overcome these difficulties and enhance the antileukemia efficacy of FLT3 inhibitors. In the present study, we demonstrate the isolation of novel human mAbs against FLT3 with antagonistic or agonistic activities. An antagonistic Ab, designated A2, continuously inhibits FLT3 ligand (FL)-induced phosphorylation of FLT3 and MAPK. A2 cooperatively induces apoptosis with daunorubicin, even in the presence of FL. An agonistic Ab, designated 3E6, surprisingly induces the phosphorylation of FLT3 and MAPK, and supports the growth of a factor-dependent cell line independently of FL addition. In addition, A2 showed complement-dependent cytotoxicity activity, but was devoid of Ab-dependent cell mediated cytotoxicity. Finally, we evaluated Ab internalization in a cell line. Immunofluorescence and flow cytometry analyses revealed that A2 is efficiently internalized. Collectively, these data demonstrate that A2 is a potent human Ab that might be capable of delivering cytotoxic reagents and that has antagonistic effects on FLT3 signaling. In addition, 3E6 might be a potential scaffold for novel dendritic cell-based immunotherapies.

Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein.

The multi-subunit DNA-dependent RNA polymerase (RNAP) is the principal enzyme of transcription for gene expression. Transcription is regulated by various transcription factors. Gre factor homologue 1 (Gfh1), found in the Thermus genus, is a close homologue of the well-conserved bacterial transcription factor GreA, and inhibits transcription initiation and elongation by binding directly to RNAP. The structural basis of transcription inhibition by Gfh1 has remained elusive, although the crystal structures of RNAP and Gfh1 have been determined separately. Here we report the crystal structure of Thermus thermophilus RNAP complexed with Gfh1. The amino-terminal coiled-coil domain of Gfh1 fully occludes the channel formed between the two central modules of RNAP; this channel would normally be used for nucleotide triphosphate (NTP) entry into the catalytic site. Furthermore, the tip of the coiled-coil domain occupies the NTP ß-γ phosphate-binding site. The NTP-entry channel is expanded, because the central modules are 'ratcheted' relative to each other by ∼7°, as compared with the previously reported elongation complexes. This 'ratcheted state' is an alternative structural state, defined by a newly acquired contact between the central modules. Therefore, the shape of Gfh1 is appropriate to maintain RNAP in the ratcheted state. Simultaneously, the ratcheting expands the nucleic-acid-binding channel, and kinks the bridge helix, which connects the central modules. Taken together, the present results reveal that Gfh1 inhibits transcription by preventing NTP binding and freezing RNAP in the alternative structural state. The ratcheted state might also be associated with other aspects of transcription, such as RNAP translocation and transcription termination.

Formation of pyridine nucleotides under symbiotic and non-symbiotic conditions between soybean nodules and free-living rhizobia.

Enzymatic regulation of pyricline nucleotide formation, under symbiotic and non-symbiotic conditions, was analyzed using soybeans (Glycine max L. cv. 'Akisengoku') and rhizobia (Bradyrhizobia japonicum strain A1017), respectively. It was found that levels of pyridine nucleotides in bacteroids in root nodules were different from those in free-living cells of rhizobia. This difference was associated with differences in activities of enzymes involved in the pathway from L-tryptophan to NAD and NADP. That is, these activities were lower in bacteroids than in free-living bacteria and lower in the nodule cytosol than in root extracts. The optimum pH for NAD synthetase in bacteroids, was 9.0. Additionally, the optimum pH for ATP-nicotinamide mononucleotide (NMN) adenyltransferase, final step enzyme in NAD formation, was estimated to be 7.6. In the bacteroid fraction, the K(m) of NAD synthetase (22 microM) was approximately 1/22 of that of ATP-NMN adenyltransferase (482 microM). Vmax values were estimated to be almost in the same order for both NAD synthetase and ATP-NMN adenyltransferase. This is the first report on the formation of pyridine nucleotides originating from L-tryptophan in bacteroids in soybean nodules and free-living bacteria.