Polypeptide N-acetylgalactosaminyltransferase-15 regulates adipogenesis in human SGBS cells.

Adipogenesis involves intricate molecular mechanisms regulated by various transcription factors and signaling pathways. In this study, we aimed to identify factors specifically induced during adipogenesis in the human preadipocyte cell line, SGBS, but not in the mouse preadipocyte cell line, 3T3-L1. Microarray analysis revealed distinct gene expression profiles, with 1460 genes induced in SGBS cells and 1297 genes induced in 3T3-L1 cells during adipogenesis, with only 297 genes commonly induced.

Genes for tRNA recycling are upregulated in response to infection with Theiler's mouse encephalitis virus.

The concept of tRNA recycling has recently emerged from the studies of ribosome-associated quality control. Therein tRNase Z removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase Z and TRNT1.

Perturbing the Normal Level of SIDT1 Suppresses the Naked ASO Effect.

Although antisense oligonucleotide (ASO) therapeutics can be taken up by living cells without carrier molecules, a large part of incorporated ASOs are trapped in the endosomes and do not exert therapeutic effects. To improve their therapeutic effects, it would be important to elucidate the mechanism of cellular uptake and intracellular trafficking of ASOs. In this study, we investigated how SIDT1 affects cellular uptake and intracellular trafficking of ASOs.

Transcription from the proximal promoter of ELAC1, a gene for tRNA repair, is upregulated by interferons.

tRNase Z (ELAC1) and TRNT1 function in tRNA recycling. Recently, we have shown that these genes are upregulated in the cells infected with Theiler's mouse encephalitis virus (TMEV), implying that tRNA recycling functions in response to viral infection. To address the molecular mechanism underlying the ELAC1 upregulation in the cells infected with TMEV, we performed luciferase assays using various plasmid constructs harboring the ELAC1 promoter region.

A naked antisense oligonucleotide with phosphorothioate linkages is taken up intracellularly more efficiently but functions less effectively.

We have been developing a gene silencing technology by harnessing a tRNA 3' processing endoribonuclease, tRNase Z, with antisense oligonucleotides. Here, to further improve this technology, we investigated how the length and the modifications of naked oligonucleotides affect the efficiency of their uptake by HeLa, HEK293, and HL60 cells by flow cytometry and fluorescence microscopy. 7-30-nt Alexa-Fluor-568-labeled DNAs with phosphorothioate linkages and 7-30-nt Alexa-Fluor-568-labeled, 2'-O-methylated RNAs without phosphorothioate linkages were examined, and, on the whole, longer oligonucleotides were shown to be intracellularly taken up more efficiently.

The heptamer sgRNA targeting the human OCT4 mRNA can upregulate the OCT4 expression.

TRUE gene silencing is one of the gene suppression technologies. This technology exploits the enzymatic property of the tRNA 3' processing endoribonuclease tRNase Z, which is that it can cleave a target RNA under the direction of a small guide RNA (sgRNA). We have been working on the development of therapeutic sgRNAs for hematological malignancies.

RNA-hydrolyzing activity of metallo-β-lactamase IMP-1.

Metallo-β-lactamases (MBLs) hydrolyze a wide range of β-lactam antibiotics. While all MBLs share a common αβ/βα-fold, there are many other proteins with the same folding pattern that exhibit different enzymatic activities. These enzymes, together with MBLs, form the MBL superfamily.

Heptamer-type small guide RNA that can shift macrophages toward the M1 state.

Multiple myeloma is a refractory cancer of plasma cells. Although treatment strategies for multiple myeloma are getting improved year by year, in most cases patients relapse due to the emergence of drug-resistant mutations in the myeloma cells. The interplay between myeloma cells and tumor-associated macrophages (TAM) is important for the pathology.

Mastication Affects Transcriptomes of Mouse Microglia.

Background/aim: We investigated whether mastication affects microglia, whose activity is thought to be associated with cognition and brain tumor progression.

Materials And Methods: We kept mice by feeding either a hard or soft diet for 2, 4 or 8 months. After each period, we removed the whole brains and isolated microglia.

The 31-nucleotide Y4-RNA fragment in plasma is a potential novel biomarker.

The 31- and 32-nt 5'-fragment of Y4-RNA (Y4RNAfr) exists abundantly in human peripheral blood plasma. Although physiological roles of the plasma Y4RNAfr are not well established, its potential utility as a diagnostic/prognostic marker for acute coronary syndrome was suggested. In this paper, to establish a normal range of the Y4RNAfr level in plasma, we measured plasma Y4RNAfr levels of 40 healthy persons using the method we have developed, and compared them with other blood test data.

Potential physiological roles of the 31/32-nucleotide Y4-RNA fragment in human plasma.

The 31- and 32-nt 5'-fragments of Y4-RNA (Y4RNAfr) exist abundantly in human plasma. The Y4RNAfr can function as 5'-half-tRNA-type sgRNA for tRNase Z, although we do not know yet what its physiological roles are and what cellular RNAs are its genuine targets. In this paper, we analyzed the effects of the Y4RNAfr on cell viability and transcriptomes using HL60, RPMI-8226, and HEK293 cells, and Y4RNAfr-binding RNAs in A549 cells.

Dual activity of PNGM-1 pinpoints the evolutionary origin of subclass B3 metallo--lactamases: a molecular and evolutionary study.

Resistance to -lactams is one of the most serious problems associated with Gram-negative infections. -Lactamases are able to hydrolyze -lactams such as cephalosporins and/or carbapenems. Evolutionary origin of metallo--lactamases (MBLs), conferring critical antibiotic resistance threats, remains unknown.

Evaluation of double heptamer-type sgRNA as a potential therapeutic agent against multiple myeloma.

Emergence of drug-resistant mutations in the course of myeloma cell evolution and subsequent relapse of myeloma appears to be currently inevitable in most patients. To remedy this situation, we are trying to develop therapeutic small guide RNAs (sgRNAs) based on tRNase Z-utilizing efficacious gene silencing (TRUE gene silencing), an RNA-mediated gene expression control technology. We designed two sets of double heptamer-type sgRNA, which target the human BCL2 mRNA.

The Y4-RNA fragment, a potential diagnostic marker, exists in saliva.

The 94-nt full-length Y4-RNA is thought to have roles in the initiation of DNA replication and RNA quality control. Although its 31/32-nt fragment also exists abundantly in plasma, little is known about its physiological role. Since the 31/32-nt Y4-RNA fragment in sera is reported to be more abundant in patients with coronary artery disease than healthy persons, the fragment may have a potential for a diagnostic and/or prognostic biomarker for some diseases regardless of its functionality.

Microbicidal Effects of Stored Aqueous Ozone Solution Generated by Nano-bubble Technology.

Background/aim: Clinically used disinfectants are often irritating and cause skin problems. Ozone water is unique among disinfectants. It does not damage skin cells and readily decomposes to oxygen without generating harmful residues.

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents.

TRUE gene silencing (termed after tRNase Z(L)-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3' processing endoribonuclease, tRNase Z(L), to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines.

IOP1 protein is an external component of the human cytosolic iron-sulfur cluster assembly (CIA) machinery and functions in the MMS19 protein-dependent CIA pathway.

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear.

Lesion bypass activity of DNA polymerase θ (POLQ) is an intrinsic property of the pol domain and depends on unique sequence inserts.

DNA polymerase θ (POLQ, polθ) is a large, multidomain DNA polymerase encoded in higher eukaryotic genomes. It is important for maintaining genetic stability in cells and helping protect cells from DNA damage caused by ionizing radiation. POLQ contains an N-terminal helicase-like domain, a large central domain of indeterminate function, and a C-terminal polymerase domain with sequence similarity to the A-family of DNA polymerases.

MMXD, a TFIIH-independent XPD-MMS19 protein complex involved in chromosome segregation.

Xeroderma pigmentosum group D (XPD) protein is one of the subunits of TFIIH that is required for nucleotide excision repair and transcription. We found a XPD protein complex containing MMS19 that was assumed to be a regulator of TFIIH. However, the MMS19-XPD complex did not contain any other subunits of TFIIH.

DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability.

"Replicative stress" is one of the main factors underlying neoplasia from its early stages. Genes involved in DNA synthesis may therefore represent an underexplored source of potential prognostic markers for cancer. To this aim, we generated gene expression profiles from two independent cohorts (France, n=206; United Kingdom, n=117) of patients with previously untreated primary breast cancers.

Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity.

POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases.

Lack of DNA polymerase theta (POLQ) radiosensitizes bone marrow stromal cells in vitro and increases reticulocyte micronuclei after total-body irradiation.

Abstract Mammalian POLQ (pol theta) is a specialized DNA polymerase with an unknown function in vivo. Roles have been proposed in chromosome stability, as a backup enzyme in DNA base excision repair, and in somatic hypermutation of immunoglobulin genes. The purified enzyme can bypass AP sites and thymine glycol.

Low-fidelity DNA synthesis by human DNA polymerase theta.

Human DNA polymerase theta (pol or POLQ) is a proofreading-deficient family A enzyme implicated in translesion synthesis (TLS) and perhaps in somatic hypermutation (SHM) of immunoglobulin genes. These proposed functions and kinetic studies imply that pol may synthesize DNA with low fidelity. Here, we show that when copying undamaged DNA, pol generates single base errors at rates 10- to more than 100-fold higher than for other family A members.

DNA polymerase theta (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct.

DNA polymerase theta (pol theta) is a nuclear A-family DNA polymerase encoded by the POLQ gene in vertebrate cells. The biochemical properties of pol theta and of Polq-defective mice have suggested that pol theta participates in DNA damage tolerance. For example, pol theta was previously found to be proficient not only in incorporation of a nucleotide opposite a thymine glycol or an abasic site, but also extends a polynucleotide chain efficiently from the base opposite the lesion.

DNA polymerases eta and theta function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes.

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ.