Background: Methyl-CpG binding domain 4 (MBD4) is a DNA glycosylase that excises mismatched bases generated in methylated CpG sequences. Results: We report the biochemical and structural properties of the methyl-CpG binding domain of MBD4 (MBD MBD4 ). Conclusion: MBD MBD4 recognizes a wide range of 5-methylcytosine modifications via an extensive hydration network. Significance: This study provides new insight into the structural mechanism of the broad base recognition that is unique to MBD MBD4 .

We studied the opening mechanism of Ca(2+)-permeable channels formed with mouse transient receptor potential type 5 (mTRP5) using Xenopus oocytes. After stimulation of coexpressed muscarinic M(1) receptors with acetylcholine (ACh) in a Ca(2+)-free solution, switching to 2 mM Ca(2+)-containing solution evoked a large Cl(-) current, which reflects the opening of endogenous Ca(2+)-dependent Cl(-) channels following Ca(2+) entry through the expressed channels. The ACh-evoked response was not affected by a depletion of Ca(2+) store with thapsigargin but was inhibited by preinjection of antisense oligodeoxynucleotides (ODNs) to G(q), G(11), or both. The mTRP5 channel response was also induced by a direct activation of G proteins with injection of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). The ACh- and GTP gamma S-evoked responses were inhibited by either pretreatment with a phospholipase C inhibitor, U73122, or an inositol-1,4,5-trisphosphate (IP(3)) receptor inhibitor, xestospongin C (XeC). An activation of IP(3) receptors with injection of adenophostin A (AdA) evoked the mTRP5 channel response in a dose-dependent manner. The AdA-evoked response was not suppressed by preinjection of antisense ODNs to G(q/11) or U73122 but was suppressed by either preinjection of XeC or a peptide mimicking the IP(3) binding domain of Xenopus IP(3) receptor. These findings suggest that the activation of IP(3) receptor is essential for the opening of mTRP5 channels, and that neither G proteins, phosphoinositide metabolism, nor depletion of the Ca(2+) store directly modifies the IP(3) receptor-linked opening of mTRP5 channels.

The picosecond transient infrared spectrum of the excited charge-transfer (CT) state of 4-(pyrrol-1-yl)benzonitrile (PBN) has been recorded in the frequency range of 1700−920 cm-1. The CT state of PBN gives several quite strong infrared bands. The band at 1219 cm-1 shifts toward the high-frequency side with increasing delay time between pump and probe pulses. This frequency shift is attributed to vibrational relaxation. Some of the transient bands (1429, ∼1290, 1219, and 964 cm-1) are very close in frequencies to those observed for the benzonitrile moiety of the excited CT state of 4-(dimethylamino)benzonitrile (DMABN). This finding implies that the benzonitrile groups of PBN and DMABN are decoupled from the electron-donating (pyrrole or dimethylamino) groups, and it can be explained by the twisted intramolecular charge-transfer model. To make this point clear, however, further discussion is necessary on the band assignments and on correlation between structure and spectral pattern.

Infrared spectra of the ground, charge transfer (CT), and locally excited (LE) states of isotope-labeled 4-(dimethylamino)benzonitriles (DMABNs) in the region between 1700 and 900 cm-1 are reported. The isotopomers measured are normal DMABN, NC–C6H4–15NMe2 (dimethylamino nitrogen labeled DMABN), and NC–C6H2D2–NMe2 (3,5-dideuterated DMABN). The infrared spectrum of the excited CT state of DMABN-d2 is consistent with the previous band assignments for normal DMABN and DMABNs isotopically labeled on dimethylamino group. For the LE state of normal DMABN, three bands are observed at 1481, 1415, and 1399 cm-1. This is in contrast with a previously reported transient infrared spectrum, where positions of bands due to the transient do not shift from the ground state ones. The band at ca. 1481 cm-1 is observed for normal and 15N labeled DMABN, but not for DMABN-d2. Except for this point, the band positions are almost identical for the three isotope-labeled species. The vibrational transitions observed at ca. 1415 and 1398 cm-1 are hence attributed to modes with atomic displacements localized on methyl groups and/or the part of the benzonitrile moiety adjacent to the cyano group or the cyano group itself. Quantum chemical calculations of the vibrational spectra for the CT and LE states of DMABN at present do not correctly reproduce the experimental spectra, which means that more accurate calculations are needed for a reliable analysis of these spectra.

Metabolic reprogramming of leukemia cells is important for survival, proliferation, and drug resistance under conditions of metabolic stress in the bone marrow. Deregulation of cellular metabolism, leading to development of leukemia, occurs through abnormally high expression of transcription factors such as MYC and Ecotropic Virus Integration site 1 protein homolog (EVI1). Overexpression of EVI1 in adults and children with mixed lineage leukemia-rearrangement acute myeloid leukemia (MLL-r AML) has a very poor prognosis. To identify a metabolic inhibitor for EVI1-induced metabolic reprogramming in MLL-r AML, we used an XFp extracellular flux analyzer to examine metabolic changes during leukemia development in mouse models of AML expressing MLL-AF9 and Evi1 (Evi1/MF9). Oxidative phosphorylation (OXPHOS) in Evi1/MF9 AML cells accelerated prior to activation of glycolysis, with a higher dependency on glutamine as an energy source. Furthermore, EVI1 played a role in glycolysis as well as driving production of metabolites in the tricarboxylic acid cycle. L-asparaginase (L-asp) exacerbated growth inhibition induced by glutamine starvation and suppressed OXPHOS and proliferation of Evi1/MF9 both in vitro and in vivo ; high sensitivity to L-asp was caused by low expression of asparagine synthetase (ASNS) and L-asp-induced suppression of glutamine metabolism. In addition, samples from patients with EVI1 + MF9 showed low ASNS expression, suggesting that it is a sensitive marker of L-asp treatment. Clarification of metabolic reprogramming in EVI1 + leukemia cells may aid development of treatments for EVI1 + MF9 refractory leukemia.

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The outcomes for relapsed and metastatic Ewing sarcoma (EWS) is extremely poor. Therefore, it is important to identify the tumor‐specific targets in these intractable diseases. High focal adhesion kinase (FAK) transcript expression levels in EWS cell lines are known. TAE226 is a dual inhibitor of FAK and insulin‐like growth factor‐I receptor (IGF‐IR), while PF‐562,271 is a dual inhibitor of FAK and proline‐rich tyrosine kinase 2. We compared the cytotoxicity of TAE226 and PF‐562,271 toward three EWS cell lines. TAE226 strongly inhibited proliferation of three cell lines when compared with PF‐562,271. Furthermore, we investigated the efficacy of TAE226 as well as its mechanism of action against EWS. A stable EWS cell line with FAK and IGF‐IR knocked down was established, and microarray analysis revealed dysregulated expression in various pathways. TAE226 treatment of EWS cell lines induced cell cycle arrest, apoptosis, AKT dephosphorylation, and inhibition of invasion. We demonstrated that TAE226 drastically inhibits the local growth of primary tumors and metastasis in EWS using mouse models. Furthermore, the combination of TAE226 and conventional chemotherapy proved to exert synergistic effects. TAE226 may be a candidate single agent or combined therapy drug to be developed for patients who have relapse and metastatic EWS tumors in future.

A new derivative of hexaaza[1]paracyclophane in which p-phenylenes are alternately replaced by 9,10-anthrylenes was prepared to investigate the impact on overall π-conjugation as well as conformational change of the macrocycle. The charge and spin distribution for one-electron and three-electron oxidation of the macrocycle was elucidated by means of electrochemical, spectroelectrochemical, EPR spectroscopic, and SQUID magnetometric methods. In particular, the triradical trication was successfully isolated as an air-stable salt, and moreover, its structure was disclosed by X-ray analysis. The triradical trication was characterized as a spin-frustrated three-spin system with the antiferromagnetic exchange interaction (J/k ≃ - 74 K).