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The eukaryotic mRNAs produced by RNA polymerase II (Pol II) are capped with an inverted 7-methyl-guanosine (m7G) linked to the first residue of the mRNA (36). This event occurs by a series of three enzymatic reactions; The 5′ triphosphate end of the nascent RNA Pol II transcript is cleaved by 5′ RNA triphosphatase to produce the diphosphate end. RNA guanylyltransferase forms a covalent enzyme-GMP complex and subsequently caps the RNA substrate by adding a guanosine residue in a 5′-5′ triphosphate linkage. The cap is then methylated by RNA (guanine-7) methyltransferase (23, 39). In higher eukaryotes, a bifunctional monomeric polypeptide carries both the RNA triphosphatase and guanylyltransferase activities, while the capping enzyme from yeast is a complex of RNA triphosphatase and guanylyltransferase subunits (40). The polypeptides are encoded by the CET1 and CEG1 genes, respectively in Saccharomyces cerevisiae, and both are essential for the cell viability. Capping, the first mRNA modification, occurs by the time the transcript is only 25 to 30 nucleotides long in an early transcription phase. Such cotranscriptional capping is mediated by recruitment of capping enzyme machinery to the phosphorylated carboxy-terminal domain (CTD) of the largest subunit of Pol II (7, 15, 22, 51). The CTD of Pol II has an unusual structure with many heptapeptide repeats (YSPTSPS). The capping enzyme binds directly and specifically to the phosphorylated CTD of Pol II via the Ceg1 subunit (yeast) or the guanylyltransferase domain (metazoan). Furthermore, the guanylyltransferase activity of Ceg1 associated with phosphorylated CTD is allosterically regulated by both the Cet1 and phosphorylated CTD to ensure it has a coordinated capping activity (8). The mammalian capping enzyme is also allosterically regulated by an interaction with the phosphorylated CTD (16, 25). The CTD phosphorylated at serine 5 of the heptapeptide repeat appears to be important for the capping reaction because it stimulates the guanylyltransferase activity, although both serine 2 phosphorylation and serine 5 phosphorylation can mediate the protein interaction. An interesting extension to these findings was added by a chromatin immunoprecipitation assay, which provides in vivo evidence that the capping enzyme machinery interacts dynamically with Pol II during a transcription cycle (20, 35). The capping enzyme interacts with Pol II immediately after the serine 5 of CTD is phosphorylated. As serine 5 phosphorylation decreases in an early elongation phase, the capping enzyme dissociates from the transcription complex. Recently, data from several groups have raised the concept of “checkpoints” in transcription, especially in an early phase (summarized in reference 26). As operated during the cell cycle to ensure that each phase of the cycle is complete before the next one begins, checkpoint in early transcription is suggested to play a role in ensuring that only the properly modified RNA at the 5′ end is extended. The Pol II transcription is thus subjected to checkpoint control for the coordinated transcription with mRNA capping (6, 25, 26). Several transcription factors have been reported to play in this window. DSIF, a human homolog of the yeast transcription factor Spt4/Spt5, increases the pausing of Pol II and thus plays a role as a negative factor (42, 46, 49). Within this temporal and spatial interval, while Pol II with the hypophosphorylated CTD is paused at the promoter-proximal region, many factors are intended to target the capping enzymes to increase their recruitment or to enhance their catalytic activities. In addition to TFIIH, which creates a binding epitope for the capping enzyme on CTD by serine 5 phosphorylation, as described above, Spt5 itself interacts with the triphosphatase and guanylyltransferase components of the capping enzyme (21, 27, 44). In the case of human immunodeficiency virus (HIV), DSIF/Spt5-induced transcription arrest allows HIV-encoded Tat to interact with capping enzymes and to stimulate their catalytic activities (5, 6). Phosphorylation of Pol II CTD is critical for the transition to the elongation phase. At this step, the elongation factor P-TEFb, a DRB-sensitive protein kinase, phosphorylates the Pol II CTD and Spt5 (19, 29, 38). HIV Tat also interacts with P-TEFb in this step (29). In S. cerevisae, Ctk1 kinase complex and the Bur1 kinase complex facilitate the transcription elongation (18, 50). The phosphorylation of Pol II CTD is thus meant to lead to the formation of the processive transcription elongation complexes. According to recent reports, the capping enzyme in Schizosaccharomyces pombe interacts with Cdk9/Pch1, a yeast P-TEFb homolog (28). How does the capping enzyme fit into the complicated scheme to delineate it in the order of pausing, capping, and the reversion of pausing in the checkpoint model? If the elongation-competent transition does not occur until the RNA is capped and the capping is the major determinant to shift the Pol II status, it could be indicative that a capping enzyme plays a critical role in regulation of an early transcription in addition to its role in simple cap formation. To study whether the capping enzyme plays a key role in coordinating mRNA processing and transcription elongation, we used a well-characterized yeast system. Because Ceg1, a capping enzyme subunit, contributes to transcription via its typical cap formation activity, this study examined various ceg1 temperature-sensitive alleles to determine if there is any additional role in transcription elongation. Among them ceg1-63 displayed 6-azauracil (6AU) sensitivity and a defect in PUR5 induction by 6AU treatment. This study shows that transcription through the pause sites artificially inserted at the promoter-proximal region was severely inhibited in ceg1-63. We show that such an elongation defect was coupled to the reduced guanylyltransferase activity. However, it happened independently of the turnover of uncapped transcripts. These results indicate that the transcription ternary complexes are held at the promoter as long as their RNAs have not been properly capped. That is, capping enzyme plays a critical role in the promoter-proximal checkpoint window by reinforcing the checkpoint security circuit and probably by reversing the transcription arrest in time. This finding also strongly supports the transcription checkpoint model, in which an early transcription is tightly regulated.

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70(s6k) and extracellular signal-regulated kinase 1/2 (ERK1/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H(2)O(2), because the arsenite-induced increase in dichlorofluorescein (DCF) fluorescence was completely abolished by pretreatment with catalase but not with heat-inactivated catalase. Elimination of H(2)O(2) by catalase or N-acetyl-L-cysteine inhibited the arsenite-induced activation of p70(s6k) and ERK1/2, indicating the possible role of H(2)O(2) in the arsenite activation of the p70(s6k) and the ERK1/2 signaling pathways. A specific inhibitor of p70(s6k), rapamycin, and calcium chelators significantly blocked the activation of p70(s6k) induced by arsenite. While the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 completely abrogated arsenite activation of p70(s6k), ERK1/2 activation by arsenite was not affected by these inhibitors, indicating that H(2)O(2) might act as an upstream molecule of PI3K as well as ERK1/2. Consistent with these results, none of the inhibitors impaired H(2)O(2) production by arsenite. DNA binding activity of AP-1, downstream of ERK1/2, was also inhibited by catalase, N-acetyl-L-cysteine, and the MEK inhibitor PD98059, which significantly blocked arsenite activation of ERK1/2. Taken together, these studies provide insight into mechanisms of arsenite-induced tumor promotion and suggest that H(2)O(2) plays a critical role in tumor promotion by arsenite through activation of the ERK1/2 and p70(s6k) signaling pathways.

We previously reported that the activation of p21(WAF1/Cip1) transcription by histone deacetylase inhibitor apicidin was mediated through Sp1 sites and pointed to the possible participation of protein kinase C (PKC). In this study, we investigated the role and identity of the specific isoforms of PKC involved and identified phosphatidylinositol 3-kinase (PI 3-kinase) as an upstream effector in HeLa cells. Using an isoform-specific pharmacological inhibitor of PKC, a PKC epsilon dominant-negative mutant, and antisense oligonucleotide to inhibit PKC epsilon specifically, we found that among PKC isoforms, PKC epsilon was required for the p21(WAF1/Cip1) expression by apicidin. In addition to PKC epsilon, PI 3-kinase appeared to participate in the activation of p21(WAF1/Cip1) promoter by apicidin, since inactivation of PI 3-kinase either by transient expression of dominant-negative mutant of PI 3-kinase or its specific inhibitors, LY294002 and wortmannin, attenuated the activation of p21(WAF1/Cip1) promoter and p21(WAF1/Cip1) protein expression by apicidin. Furthermore, membrane translocation of PKC epsilon in response to apicidin was blocked by the PI 3-kinase inhibitor, indicating the role of PI 3-kinase as an upstream molecule of PKC epsilon in the p21(WAF1/Cip1) promoter activation by apicidin. However, the p21(WAF1/Cip1) expression by apicidin appeared to be independent of the histone hyperacetylation, since apicidin-induced histone hyperacetylation of p21(WAF1/Cip1) promoter region was not affected by inhibition of PI 3-kinase and PKC, suggesting that the chromatin remodeling through the histone hyperacetylation alone might not be sufficient for the expression of p21(WAF1/Cip1) by apicidin. Taken together, these results suggest that the PI 3-kinase-PKC epsilon signaling pathway plays a pivotal role in the expression of the p21(WAF1/Cip1) by apicidin.

We have previously reported on the identification of the endogenous transmethylation inhibitor oligosaccharide-linked acyl carrier protein (O-ACP). In this study, the role of the transmethylation reaction on cell cycle progression was evaluated using various transmethylase inhibitors, including O-ACP. O-ACP significantly inhibited the growth of various cancer cell lines, including NIH3T3, ras-transformed NIH3T3, MDA-MB-231, HT-1376, and AGS. In addition, exposure of ras-transformed NIH3T3 to O-ACP caused cell cycle arrest at the G0/G1 phase, which led to a decrease in cells at the S phase, as determined by flow cytometry. In contrast, transmethylase inhibitors did not affect the expression of p21(WAF1/Cip1), a well known inhibitor of cyclin dependent kinase, indicating that the cell cycle arrest by transmethylase inhibitors might be mediated by a p21(WAF1/Cip1)-independent mechanism. Therefore, O-ACP, a novel transmethylase inhibitor, could be a useful tool for elucidating the novel role of methylation in cell proliferation and cell cycle progression.

The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5′-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts Schizosaccharomyces pombe and Candida albicans . Surprisingly, the S. pombe capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The S. pombe RTPase gene ( pct1 + ) is essential for viability. Pct1 can replace the S. cerevisiae RTPase when GTase activity is supplied by the S. pombe or mouse enzymes but not by the S. cerevisiae GTase. The C. albicans capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.

The canonical Wnt pathway is critical for embryonic stem cell (ESC) pluripotency and aberrant control of β-catenin leads to failure of exit from pluripotency and lineage commitments. Hence, maintaining the appropriate level of β-catenin is important for the decision to commit to the appropriate lineage. However, how β-catenin links to core transcription factors in ESCs remains elusive. C-terminal-binding protein (CtBP) in Drosophila is essential for Wnt-mediated target gene expression. In addition, Ctbp acts as an antagonist of β-catenin/TCF activation in mammals. Recently, Ctbp2, a core Oct4-binding protein in ESCs, has been reported to play a key role in ESC pluripotency. However, the significance of the connection between Ctbp2 and β-catenin with regard to ESC pluripotency remains elusive. Here, we demonstrate that C-terminal-binding protein 2 (Ctbp2) associates with major components of the β-catenin destruction complex and limits the accessibility of β-catenin to core transcription factors in undifferentiated ESCs. Ctbp2 knockdown leads to stabilization of β-catenin, which then interacts with core pluripotency-maintaining factors that are occupied by Ctbp2, leading to incomplete exit from pluripotency. These findings suggest a suppressive function for Ctbp2 in reducing the protein level of β-catenin, along with priming its position on core pluripotency genes to hinder β-catenin deposition, which is central to commitment to the appropriate lineage.

일제강점기는 한국미술사의 출발점이라는 시각에서 기존 논문에서 다루지 않았던 당시의 요지(窯址) 조사와 고려청자 연구가 어떠한 배경속에서 시작했으며 시기별로 어떤 변화를 보이고 그 내용은 무엇이었는지에 대해 살펴보았다. 당시 요지조사와 고려청자의 연구는 일본 제국주의가 설정한 조선예술쇠망론의 식민사관과 식민지에 대한 인류학적 자료조사라는 큰 틀에서 시작되었다. 1910년대부터 1930년대까지 강진을 비롯하여 공주 학봉리, 광주 분원리, 부안 유천리·진서리, 고령사부동·기산동 등 중요한 요지 대부분이 조사되었으나, 현재 각 요지의 발굴보고서는 공주 학봉리를 제외하고는 확인할 수 없는 한계가 있다. 그러나 1914년 강진 요지 발굴자료도 다른 논문에서 단편적으로 확인되기 때문에 모든 발굴보고서는 작성되었던 것으로 보인다. 그렇기 때문에 각 요지의 발굴조사보고서 부재(不在)는 조선총독부박물관의 의도적인 자료 폐기 또는 누락를 추정하는 근거이기도 하다. 고려청자에 대한 인식과 연구는 요지조사 자료 축적과 함께 50여 년간 시기별로 변화를 보였다. 19세기부터 1900년대는 본격적인 요지조사가 실시되기 이전으로 고려청자는 고가의 골동품으로서 수집과 감상의 대상으로 여겨지던 시기이다. 전세품(傳世品)이나 개인적인 수집품만을 분석하여 한국 도자기의 역사를 관념적인 시각에서 계보(系譜)를 정리하고 종류를 구분하였다. 이후 1910-1920년대에는 일본의 대외확장에 따라 식민지에 대한 인류학적 조사가 본격적으로 진행된 시기이다. 도자기 연구는 고고학적 발굴을 통한 요지 현황파악과 조사를 바탕으로 한 것이었으나 여전히 감상도자(鑑賞陶磁)라는 관점도 유지되었다. 특히 도아회(匋雅會), 도자기연구회(陶磁器硏究會), 채호회(彩壺會) 등을 중심으로 감상, 품평활동과 도자사적인 연구를 병행했다는 것이 특징이다. 고려청자를 도자산업 진흥을 위한 중국 도자기 연구의 부수적인 연구대상으로 다루면서 비색청자를 생산할 수 있었던 기술적 요인과 문양소재의 원류를 찾는 논문이 발표되기도 하였다. 1928년과 1933년 각각 강진과 부안 청자요지가 발굴되면서 1930년대부터 1945년 까지는 그동안의 조사결과를 토대로 고려시대의 시기별 변화, 고려와 중국의 문헌사료 분석, 소비지 출토 편년(編年)유물, 간지(干支)명을 비롯한 명문(銘文)청자 연구 등으로 연구범위를 확대해갔다. 오늘날의 도자사 연구, 문헌사 연구, 고고학적 연구와 같이 분야가 세분화된 단행본들이 출간되었다. 그중에서 나카오 만죠(中尾万三)는 중국 도자기 역사에 대한 지식을 배경으로 중국과 고려의 영향관계, 원류 규명, 시기구분 등 미술사적 시각에서 연구한 반면, 노모리 켄은 고고학적 조사 결과를 세부항목으로 분류하고 정리, 비교하는 방식이었다. 서로 다른 연구 방식을 취했으나 두 사람의 연구 결과는 광복 이후 국내 도자사 연구의 토대를 형성했다.

The hyperinflammation that begins with sepsis is essential for eradicating infection but also causes hypoperfusion and organ failure. To understand the innate immune status of septic patients, the functional and phenotypic changes of neutrophils during sepsis and their clinical implication were studied.Seventy-four patients who were admitted to intensive care unit due to severe sepsis or septic shock were enrolled. Surface antigens of neutrophils (CD64, CD10, and CD16) were detected by flow cytometry. Respiratory burst activity (RBA) was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate and phorbol-12-myristate-13-acetate. The parameters were serially examined at Days 1 and 8 in septic shock patients.High CD64 and low CD10 and CD16 on Day 1 was associated with sepsis severity (P = 0.003, 0.017, and 0.007, respectively). On Day 1, RBA and CD64 were higher in survivors than in nonsurvivors of septic shock patients (P = 0.012 and 0.027, respectively), and on Day 8, CD10 and CD16 were higher in survivors than in nonsurvivors (P = 0.019 and 0.036, respectively). High RBA and high CD64 on Day 1 showed low 28-day mortality in univariate analysis (P = 0.018 and 0.034, respectively). In multivariate analysis, RBA maintained statistical significance (P = 0.042) but CD64 revealed only a tendency (P = 0.064).Neutrophil surface antigen (CD64, CD10, and CD16) could reflect sepsis severity. High CD64 expression and high RBA at early phase of sepsis might be associated with better prognosis, whereas high expression of CD10 and CD16 at late phase of sepsis might be associated with better prognosis. © 2015 International Clinical Cytometry Society.

For cells to exit from pluripotency and commit to a lineage, the circuitry of a core transcription factor (CTF) network must be extinguished in an orderly manner through epigenetic modifications. However, how this choreographed epigenetic remodeling at active embryonic stem cell (ESC) genes occurs during differentiation is poorly understood. In this study, we demonstrate that C-terminal binding protein 2 (Ctbp2) regulates nucleosome remodeling and deacetylation (NuRD)-mediated deacetylation of H3K27 and facilitates recruitment of polycomb repressive complex 2 (PRC2)-mediated H3K27me3 in active ESC genes for exit from pluripotency during differentiation. By genomewide analysis, we found that Ctbp2 resides in active ESC genes and co-occupies regions with ESC CTFs in undifferentiated ESCs. Furthermore, ablation of Ctbp2 effects inappropriate gene silencing in ESCs by sustaining high levels of H3K27ac and impeding H3K27me3 in active ESC genes, thereby sustaining ESC maintenance during differentiation. Thus, Ctbp2 preoccupies regions in active genes with the NuRD complex in undifferentiated ESCs that are directed toward H3K27me3 by PRC2 to induce stable silencing, which is pivotal for natural lineage commitment. Stem Cells 2015;33:2442–2455

Pluripotent stem cells (PSCs) have distinct metabolic properties that support their metabolic and energetic needs and affect their stemness. In particular, high glycolysis is critical for the generation and maintenance of PSCs. However, it is unknown how PSCs maintain and acquire this metabolic signature. In this study, we found that core pluripotency factors regulate glycolysis directly by controlling the expression of glycolytic enzymes. Specifically, Oct4 directly governs Hk2 and Pkm2, which are important glycolytic enzymes that determine the rate of glycolytic flux. The overexpression of Hk2 and Pkm2 sustains high levels of glycolysis during embryonic stem cell (ESC) differentiation. Moreover, the maintenance of high glycolysis levels by Hk2 and Pkm2 overexpression hampers differentiation and preserves the pluripotency of ESCs in the absence of leukemia inhibitory factor. Overall, our study identifies a direct molecular connection between core pluripotency factors and ESC metabolic signatures and demonstrates the significance of metabolism in cell fate determination. Stem Cells 2015;33:2699–2711

Intraperitoneal (IP) chemotherapy is a promising post-surgical therapy of solid carcinomas confined within the peritoneal cavity, with potential benefits in locoregional and systemic management of residual tumors. In this study, we intended to increase local retention of platinum in the peritoneal cavity over a prolonged period of time using a nanoparticle form of platinum and an in-situ crosslinkable hyaluronic acid gel. Hyaluronic acid was chosen as a carrier due to the biocompatibility and biodegradability. We confirmed a sustained release of platinum from the nanoparticles (PtNPs) and nanoparticle/gel hybrid (PtNP/gel), receptor-mediated endocytosis of PtNPs, and retention of the gel in the peritoneal cavity over 4 weeks--conditions desirable for a prolonged local delivery of platinum. However, PtNPs and PtNP/gel did not show a greater anti-tumor efficacy than CDDP solution administered at the same dose but rather caused a slight increase in tumor burdens at later time points, which suggests a potential involvement of empty carriers and degradation products in the growth of residual tumors. This study alerts that although several materials considered biocompatible and safe are used as drug carriers, they may have unwanted biological effects on the residual targets once the drug is exhausted; therefore, more attention should be paid to the selection of the drug carriers.

ATP citrate lyase (ACLY) is a key enzyme that is involved in de novo lipogenesis by catalyzing conversion of cytosolic citrate into acetyl CoA and oxaloacetate. Up-regulation of ACLY in various types of tumors enhances fatty acid synthesis and supplies excess acetyl CoA for histone acetylation. However, there is evidence that its enzymatic activity alone is insufficient to explain ACLY silencing-mediated growth arrest in tumor cells. In this study, we found that ACLY knockdown in primary human cells triggers cellular senescence and activation of tumor suppressor p53. Provision of acetyl CoA to ACLY knockdown cells did not alleviate ACLY silencing-induced p53 activation, suggesting an independent role for ACLY activity. Instead, ACLY physically interacted with the catalytic subunit of AMP-activated protein kinase (AMPK) and inhibited AMPK activity. The activation of AMPK under ACLY knockdown conditions may lead to p53 activation, ultimately leading to cellular senescence. In cancer cells, ACLY silencing-induced p53 activation facilitated DNA damage-induced cell death. Taken together, our results suggest a novel function of ACLY in cellular senescence and tumorigenesis.

The C-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit is hyperphosphorylated during transcription. Using an in vivo cross-linking/chromatin immunoprecipitation assay, we found previously that different phosphorylated forms of RNA Pol II predominate at different stages of transcription. At promoters, the Pol II CTD is phosphorylated at Ser 5 by the basal transcription factor TFIIH. However, in coding regions, the CTD is predominantly phosphorylated at Ser 2. Here we show that the elongation-associated phosphorylation of Ser 2 is dependent upon the Ctk1 kinase, a putative yeast homolog of Cdk9/P-TEFb. Furthermore, mutations in the Fcp1 CTD phosphatase lead to increased levels of Ser 2 phosphorylation. Both Ctk1 and Fcp1 cross-link to promoter and coding regions, suggesting that they associate with the elongating polymerase. Both Ctk1 and Fcp1 have been implicated in regulation of transcription elongation. Our results suggest that this regulation may occur by modulating levels of Ser 2 phosphorylation, which in turn, may regulate the association of elongation factors with the polymerase.

The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5′ end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle.

The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5′-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present. Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD.

Background/purpose: In prior studies, we induced the antimicrobial activity of deglycyrrhizinated licorice root extract (DG-LRE) by inhibiting the growth and biofilm formation of Streptococcus mutans UA159. Here, we used clinical strains of mutans streptococci (MS) collected from Koreans to determine the optimal concentration of DG-LRE for oral hygiene products to prevent dental caries. Materials and methods: Antimicrobial effects of DG-LRE against 14 clinical strains of MS were evaluated through the minimum inhibitory concentration, minimum bactericidal concentration, time-kill assay, and biofilm-forming assay. Results: Minimum inhibitory concentration and minimum bactericidal concentration values of DG-LRE against the clinical strains of MS ranged from 4 mu g/mL to 8 mu g/mL and from 8 mu g/mL to 16 mu g/mL, respectively. Time-kill assay demonstrated that the antimicrobial effects of DG-LRE primarily resulted from bactericidal activity. DG-LRE significantly decreased the biofilm formation of S. mutans ranging from 57.6% to 92.8% at 16 mu g/mL. Conclusion: These findings reveal that a DG-LRE concentration of 16 mu g/mL may be used to prevent dental caries in Koreans. Copyright (C) 2014, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved.

A 56-year-old man (height, 165 cm; weight, 75 kg) suspected of having herbal-related fulminant hepatitis was admitted for emergency orthotopic liver transplantation (LT). His medical history was unremarkable, except for hypertension. Upon admission to our hospital, the patient was alert with a Glasgow Coma Scale (GCS) score of 15 (4/5/6) that gradually decreased to 10 (3/2/5) by 28 h after admission. After transfer to the intensive care unit (ICU), intubation was performed and a propofol infusion was started at 4 mg/kg/h. The patient's vital signs were stable, and mechanical ventilatory support was performed with air in O2 (FiO2 0.5). Before surgery, the patient's Model for End-Stage Liver Disease score was 21, and the Child-Pugh score was class C. The patient's pupils were 3 mm/3 mm in diameter (left/right), and brain computed tomography (CT) and magnetic resonance imaging (MRI) revealed no remarkable findings. Upon arrival to the operating room, the right radial artery was cannulated and arterial blood pressure, electrocardiography, heart rate, pulse oxygen saturation, end-tidal CO2 concentration, and bispectral index (BIS) were monitored continuously. The preoperative BIS value was 43. Anesthesia was induced and maintained with a 4-6 vol% of desflurane and a 5-15 ng/ml of target effect site-controlled remifentanil infusion. Mechanical ventilatory support was maintained with air in O2 (FiO2 0.5). A Swan-Ganz catheter was inserted into the right internal jugular vein to monitor cardiac output and pulmonary arterial pressure. Prior to the skin incision, the patient's vital signs were stable, and his BIS value was 14. Approximately 1 h after the skin incision, the patient's BIS values decreased rapidly to zero. The suppression ratio was 100, and there was an absence of electric activity on electroencephalography. The patient's pupils remained 3 mm/3 mm (left/right) in diameter. His vital signs were stable, and his body temperature was 35.2℃. The BIS sensors were checked for proper placement. An arterial blood gas analysis was performed to evaluate for metabolic disturbances, but the results did not show any abnormal findings. During surgery, the patient's vital signs were generally stable (Table 1). We tried to maintain the mean arterial pressure at > 60 mmHg and body temperature at 34-35℃. Despite efforts to increase the BIS level, the patient's BIS values remained at zero until the end of the surgery for a total duration of 10 h 50 min. The surgical procedures were completed uneventfully with a total anesthetic time of 13 h 10 min. Table 1 Patient Characteristics according to Liver Transplantation Stage The patient was transferred to the ICU with the endotracheal tube still in place. The patient was sedated using a propofol infusion. After 24 h, the propofol infusion was discontinued, and the patient awoke with a GCS score of (4/E/5) and pupil size of 3 mm/3 mm (left/right). Afterwards, the sedation agent was switched to fentanyl. After being sedated for 52 h, the patient had awakened with an alert mental status of GCS score of 15 (4/5/6) and no neurologic deficits. Brain CT and MRI scans 3 days after surgery revealed no interval changes. Adequate spontaneous respiration was maintained, and the possibility of brain death was excluded. Extubation was performed on the fourth postoperative day after the patient could communicate. Liver function after transplantation recovered sequentially, and the patient was discharged without any complications. The presence of a BIS value of zero during surgery is a sign of a poor neurologic prognosis [1]. This case, however, indicates that a BIS value of zero for 10 h 50 min during surgery may not necessarily be correlated with a bad neurological outcome or brain death. The deterioration of consciousness is the most critical complication of fulminant hepatic failure. Therefore, an emergency LT should be performed before irreversible brain damage occurs. Despite active monitoring of brain function in patients undergoing urgent LT, some still suffer brain damage [2]. Therefore, it is important to evaluate a patient's exact brain function status, especially in those with fulminant hepatitis who are already in an unconscious state prior to emergent LT. Brain CT, direct measurement of intracranial pressure, transcranial Doppler imaging, and jugular venous oximetry, can help evaluate cerebral function before surgery [3]. In addition, electroencephalography is the standard method to determine brain function in unconscious patients. One study stated that BIS monitoring may be a useful measurement tool for grading and monitoring the status of hepatic encephalopathy in patients with overt hepatic encephalopathy [4]. Authors reported that mean BIS value decreased in awake patients with chronic liver disease who had more serious conditions (45.4 ± 5.0) than those with mild conditions (90.2 ± 2.5) [4]. A BIS value of zero may be due to inadequate anesthesia, hemodynamic instability, hypothermia, insufficient muscle relaxation, artifacts by other people or electric devices, and any major metabolic disturbance. Cerebral hypoperfusion can lead to an acute BIS reduction. However, in our case, hemodynamic variables, including arterial blood pressure and cardiac output, were stable. In addition, we repeatedly rechecked the BIS sensor positioning during the operation and kept the patient's body temperature at 34-35℃. Given the patient's condition, a relative anesthetic agent overdose cannot completely be excluded. Changes in drug sensitivity of the central nervous system, protein binding, volumes of distribution, drug metabolism, and drug elimination are more common in patients with liver disease [5]. Therefore, an overdose even with the ordinary use of anesthetics may occur in patients with severe liver disease. Fortunately, our patient recovered normally from anesthesia and showed no neurologic deficits after 52 h postoperatively. This case has a limitation. We did not monitor BIS during ICU care before and after a BIS value of zero was observed. If we had monitored BIS in the ICU, an in-depth discussion could lead us to a reasonable assumption. We suggest that a BIS value of zero during surgery is not always correlated with postoperative poor neurological outcome.

A 3-month-old (39 weeks postconceptual age) male infant weighing 2.9 kg was scheduled for laser photocoagulation with a diagnosis of retinopathy of prematurity. He was born at 27 weeks gestation with a birth weight of 970 g. He had been mechanically ventilated from birth for 20 days for respiratory insufficiency due to respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). A chest X-ray performed before the sub sequent ligation of the patent ductus arteriosus showed bilateral haziness in the entire lung field due to the RDS and BPD during tracheal intubation. The infant required ventilation with a high concentration of oxygen and received surfactant therapy. Twenty days after birth, patent ductus arteriosus (PDA) liga tion was done under general anesthesia in which induction was achieved with inhalation of sevoflurane and 1 mg of rocuroni um. The operation proceeded uneventfully and the extubation was performed five days after the surgery. After extubation, the infant was able to breathe spontaneously with an incubator oxygen supply, and the oxygen saturation was maintained above 90%. After PDA ligation, the infant was diagnosed with retinopa thy of prematurity, and he was then scheduled for laser photocoagulation. On the chest X-ray, improvement of haziness was observed from five days before the operation. After consultation with pediatrics, the decision was made to operate. Upon arrival in the operating room, electrocardiography, pulse oximetry, and noninvasive blood pressure were monitored, and the patient’s vital signs were stable. Induction of anesthesia was achieved with thiopental (15 mg), rocuronium (2 mg), and sevoflurane. The tracheal intubation was performed with an uncuffed 3.5 mm internal diameter endotracheal tube, but there was no capnogram trace after three breaths. At this time, the oxygen saturation rapidly dropped to below 80%. The endotra cheal tube was removed because the anesthesiologist suspected esophageal intubation and the patient was ventilated with 100% oxygen via face mask. Mask ventilation was not performed well, and peak inspiratory pressure was revealed to be above 25 mmHg. The anesthesiologist suspected stiff lungs, which suggested bronchospasm. Hydrocortisone sodium succinate (SoluCortef Ⓡ , Pfizer Inc., New York, NY, USA) 20 mg was intravenously injected. Five minutes later, oxygen saturation slowly increased up to 99% and reintubation was attempted. Although it was confirmed by direct laryngoscopy that the tube had passed between the vocal cords, proper ventilation of the lungs was not achieved. No expired carbon dioxide was noted on the capnograph, and chest auscultation was equivocal. The oxygen saturation was then in the low sixties. After sevoflurane was administered by inhalation, a slight chest movement was noted, and oxygen saturation increased up to 80%. During that time, a portable chest radiograph was taken which revealed severe consolidation with air bronchograms (Fig. 1A). Despite ventilatory support for 10 minutes, the oxygen saturation failed to increase beyond 80%. Hydrocortisone sodium succinate 20 mg was then intravenously injected once more. Afterwards, SaO 2 was maintained at 88-93%. The surgery was cancelled, and glycopyrrolate

The objective of the study was to investigate the antimicrobial effects of purified single compounds from ethanol-extracted licorice root on Streptococcus mutans. The crude licorice root extract (CLE) was obtained from Glycyrrhiza uralensis, which was subjected to column chromatography to separate compounds. Purified compounds were identified by mass spectrometry and nuclear magnetic resonance. Antimicrobial activities of purified compounds from CLE were evaluated by determining the minimum inhibitory concentration and by performing time-kill kinetics. The inhibitory effects of the compounds on biofilm development were evaluated using crystal violet assay and confocal microscopy. Cell toxicity of substances to normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. Chlorhexidine digluconate (CHX) was used in the control group. Three antimicrobial flavonoids, 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein, were isolated from the CLE. We found that the three flavonoids and CHX had bactericidal effects on S. mutans UA159 at the concentration of ≥4 and ≥1 µg/ml, respectively. The purified compounds completely inhibited biofilm development of S. mutans UA159 at concentrations over 4 μg/ml, which was equivalent to 2 μg/ml of CHX. Confocal analysis showed that biofilms were sparsely scattered in the presence of over 4 μg/ml of the purified compounds. However, the three compounds purified from CLE showed less cytotoxic effects on NHGF cells than CHX at these biofilm-inhibitory concentrations. Our results suggest that purified flavonoids from CLE can be useful in developing oral hygiene products, such as gargling solutions and dentifrices for preventing dental caries.

In order to prevent fatty acid deficiency and to supply enough energy, intravenous fat emulsion is necessary for parenteral nutrition in preterm neonates. However, parenteral administration of intravenous fat emulsion can induce lipid intolerance. The purpose of this study was to analyze risk factors for lipid intolerance in very low birth weight infants. This retrospective study included 80 preterm neonates whose birth weight was less than 1,500 g. Subjects were divided into 2 categories: those with a serum triglyceride level of ≥ 200 mg/dl (n = 33, 41%) and those with a serum triglyceride level of < 200 mg/dl (n = 47, 59%). We conducted logistic regression analysis using variables which were significant in univariate analysis. All statistical analyses were processed in SPSS version 19.0. Four risk factors for lipid intolerance were obtained through analysis of the electronic medical record. Lipid intolerance occurred more frequently in neonates with sepsis; those with a birth weight less than 1,000 g; those who was administered intravenous fat emulsion more than 2.6 g/kg/day; and those whose gestational age was less than 28 weeks. It is suggested that serum triglyceride levels should be closely monitored to prevent lipid intolerance in preterm neonates with the aforementioned characteristics.

Cancer cells rely mainly on glycolysis rather than mitochondrial respiration for energy production, which is called the Warburg effect. p53 mutations are observed in about half of cancer cases, and p53 controls the cell cycle and cell death in response to cellular stressors. p53 has been emphasized as a metabolic regulator involved in glucose, glutamine, and purine metabolism. Here, we demonstrated metabolic changes in cancer that occurred through p53. We found that p53-inducible microRNA-34a (miR-34a) repressed glycolytic enzymes (hexokinase 1, hexokinase 2, glucose-6-phosphate isomerase), and pyruvate dehydrogenase kinase 1. Treatment with an anti-miR-34a inhibitor relieved the decreased expression in these enzymes following DNA damage. miR-34a-mediated inhibition of these enzymes resulted in repressed glycolysis and enhanced mitochondrial respiration. The results suggest that p53 has a miR-34a-dependent integrated mechanism to regulate glucose metabolism.

The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.

Background. The lumen of novel threadlike structures (NTSs) is enclosed by a single layer of endothelial cells surrounded by extracellular matrix (ECM). We hypothesized that collagen may be a component of the ECM associated with lymphatic NTSs.Methods. Six female New Zealand white rabbits were anesthetized, and the NTS structures within lymphatic vessels were identified by contrast-enhanced stereomicroscopy or alcian blue staining. Isolated NTS specimens were stained with acridine orange, YOYO-1, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). The structural and molecular composition of the ECM was investigated using transmission electron microscopy (TEM), electrospray ionization-mass spectrometry, and proteomic analysis.Results. The lymph vessel wall was stained red by DiI, and rod-shaped nuclei were stained green by YOYO-1. The area surrounding the NTS was also stained red and contained green rod-shaped nuclei. TEM images showed that the NTS consisted of many ECM fibers and the ECM fibers appeared to be ~100 nm in diameter and had narrowly spaced striated bands. Proteomic analysis of the lymphatic NTS-associated ECM identified 4 proteins: keratin 10, cytokeratin 3, cytokeratin 12, and soluble adenylyl cyclase.Conclusion. The TEM study suggested that the lymphatic NTS-associated ECM did not contain collagen. This was confirmed by proteomic analysis, which showed that keratin was the major component of the ECM.

Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.

Arginase II catalyzes the conversion of arginine to urea and ornithine in many extrahepatic tissues. We investigated the protective role of arginase II on lipopolysaccharide-mediated apoptosis in the macrophage cells. Adenoviral gene transfer of full length of arginase II was performed in the murine macrophage cell line RAW264.7. The role of arginase II was investigated with cell viability, cytoplasmic histone-associated DNA fragmentation assay, arginase activity, nitric oxide production, and Western blot analysis. Arginase II is localized in mitochondria of macrophage cells, and the expression of arginase II was increased by lipopolysaccharide (LPS). LPS significantly increased cell death which was inhibited by AMT, a specific inducible nitric oxide synthase (iNOS) inhibitor. In contrast, LPS-induced cell death and nitric oxide production were increased by 2-boronoethyl-L-cysteine, a specific inhibitor of arginase. Adenoviral overexpression of arginase II significantly inhibited LPS-induced cell death and cytoplasmic histone-associated DNA fragmentation. LPS-induced iNOS expression and poly ADP-ribose polymerase cleavage were significantly suppressed by arginase II overexpression. Furthermore, arginase II overexpression resulted in a decrease in the Bax protein level and the reverse induction of Bcl-2 protein. Our data demonstrated that inhibition of NO production by arginase II may be due to arginine depletion as well as iNOS suppression though its reaction products. Moreover, arginase II plays a protective role of LPS-induced apoptosis in RAW264.7 cells.

Checkpoint kinase 2 (CHK2) kinase is a key mediator in many cellular responses to genotoxic stresses, including ionizing radiation (IR) and topoisomerase inhibitors. Upon IR, CHK2 is activated by ataxia telangiectasia mutated kinase and regulates the S-phase and G1-S checkpoints, apoptosis and DNA repair by phosphorylating downstream target proteins, such as p53 and Brca1. In addition, CHK2 is thought to be a multi-organ cancer susceptibility gene. In this study, we used a tandem affinity purification strategy to identify proteins that interact with CHK2 kinase. Cyclin-dependent kinase 11 (CDK11)(p110) kinase, implicated in pre-mRNA splicing and transcription, was identified as a CHK2-interacting protein. CHK2 kinase phosphorylated CDK11(p110) on serine 737 in vitro. Unexpectedly, CHK2 kinase constitutively phosphorylated CDK11(p110) in a DNA damage-independent manner. At a molecular level, CDK11(p110) phosphorylation was required for homodimerization without affecting its kinase activity. Overexpression of CHK2 promoted pre-mRNA splicing. Conversely, CHK2 depletion decreased endogenous splicing activity. Mutation of the phosphorylation site in CDK11(p110) to alanine abrogated its splicing-activating activity. These results provide the first evidence that CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).

Rothia mucilaginosa is a gram-positive coccus of the family Micrococcaceae. R. mucilaginosa is considered a part of the normal flora of the human oropharynx and upper respiratory tract and lower respiratory tract infections attributable to R. mucilaginosa are not frequent. We present a case of pneumonia, in which the R. mucilaginosa infection was diagnosed by quantitative cultures of a bronchoalveolar lavage (BAL) specimen. A 46-yr-old woman with B lymphoblastic lymphoma was admitted to the hospital for scheduled chemotherapy. Her chest computed tomography (CT) scan revealed bilateral multifocal nodular and patchy consolidation in both lungs. Investigation of the BAL specimen revealed that 7% of leukocytes had intracellular gram-positive cocci. The quantitative cultures of the BAL specimen grew mucoid, non-hemolytic, and grayish convex colonies on blood agar at a count of approximately 200,000 colony-forming units/mL. The colonies were identified as R. mucilaginosa. The patient was empirically treated with levofloxacin for 7 days, after which findings on the chest radiograph and CT scan improved. She was discharged with improvement on hospital day 46. To our knowledge, this is the first report of R. mucilaginosa pneumonia diagnosed in Korea. Quantitative culture of BAL specimen and examination of intracellular organisms are crucial for assessing the clinical significance of R. mucilaginosa recovered from the lower respiratory tract.

Recent reports suggest the presence of heat-shock protein (HSP)–reactive T cells with a regulatory phenotype in various inflammatory diseases. To test whether HSP exerts renoprotective effects through regulatory T cells (Tregs), ischemia/reperfusion injury was done with or without heat preconditioning in mice. Splenocytes from heat-preconditioned mice had Treg expansion and a reduced proliferative response upon mitogenic stimulus. T cells from heat-preconditioned mice failed to reconstitute postischemic injury when adoptively transferred to T cell–deficient nu/nu mice in contrast to those from control mice. Tregs were also increased in heat-preconditioned ischemic kidneys. Depleting Tregs before heat preconditioning abolished the renoprotective effect, while adoptive transfer of these cells back into Treg-depleted mice partially restored the beneficial effect of heat preconditioning. Inhibition of HSP70 by quercetin suppressed Treg expansion, as well as renoprotective effects. Transferring Tregs in quercetin-treated heat-preconditioned mice partially restored the beneficial effect of heat preconditioning. The specificity of immune cell HSP70 in renoprotection was confirmed by partial restoration of kidney injury when T cells from HSP70-deficient heat preconditioned mice were adoptively transferred to nu/nu mice. Thus, the renoprotective effect of HSP70 may be partially mediated by a direct immunomodulatory effect through Tregs. Better understanding of immunomodulatory mechanisms of various stress proteins might facilitate discovery of new preventive strategies in acute kidney injury.

Cellular differentiation is a process in which the cells gain a more specialized shape, metabolism, and function. These cellular changes are accompanied by dynamic changes in gene expression programs. In most cases, DNA methylation, histone modification, and variant histones drive the epigenetic transition that reprograms the gene expression. Histone chaperones, HIRA and Asf1a, have a role for cellular differentiation by deposition of one of variant histones, H3.3, during myogenesis of murine C2C12 cells. In this study, we accessed the roles of histone chaperones and histone H3.3 in osteoblastic conversion of C2C12 myoblasts and compared their roles with those for myogenic differentiation. The unbiased analysis of the expression pattern of histone chaperones and variant histones proposed their uncommon contribution to each pathway. HIRA and Asf1a decreased to ∼50% and further diminished during differentiation into osteoblasts, while they were maintained during differentiation into myotubes. HIRA, Asf1a, and H3.3 were indispensable for expression of cell type-specific genes during conversion into osteoblasts or myotubes. RNA interference analysis indicated that histone chaperones and H3.3 were required for early steps of osteoblastic differentiation. Our results suggest that histone chaperones and variant histones might be differentially required for the distinct phases of differentiation pathway.

BACKGROUND/AIMS Recent studies have reported the potentials of endoscopic papillary large balloon dilatation (EPLBD) with minor endoscopic sphincterotomy (EST) for the complete removal of common bile duct (CBD) stone in the high risk groups. However, there have been no reports about the recurrence of the CBD stone after EPLBD with minor EST. The aim of this study was to evlauate the recurrence of CBD stone after EPLBD with minor EST. METHODS A total of 1,036 patients who underwent endoscopic treatment due to CBD stones at Pusan University Hospital were enrolled. The patients were classified into two groups: those who underwent EPLBD with minor EST (group 1) and those who underwent EST treatment (group 2). We investigated clinical factors and recurrence rate between two groups. RESULTS The recurrence of CBD stone occurred in total of 74 patients (7%), and the recurrence rates of CBD stone were 21/321 (6.5%) in Group 1 and 53/715 (7.4%) in Group 2. There were no difference in the presence of diverticulum and the number and size of recurrent CBD stone between the two groups. In case of diverticulum existence, recurrence rates were 12/158 (7.6%) in Group 1 and 21/101 (20.8%) in Group 2. When compared to the case of no diverticulum existence (Group 1: 9/163 [5.5%], Group 2: 32/614 [5.2%]), the recurrence rate of CBD stone was significantly lower if treated after EPLBD with minor EST (p < 0.01). CONCLUSIONS CBD stone that recurs after going through EPLBD with minor EST can be successfully removed with an endoscopic treatment. The recurrence of CBD stone was especially lower in cases with periampullary diverticulum and treated with EPLBD with minor EST. Our results will be helpful in endoscopic retreatment and preventing the recurrence of CBD stone.

Aims Phosphorylation of the adaptor protein p66shc is essential for p66shc-mediated oxidative stress. We investigated the role of the reducing protein/DNA repair enzyme apurinic/apyrimidinic endonuclease1 (APE1) in modulating protein kinase CβII (PKCβII)-mediated p66shc phosphorylation in cultured endothelial cells and PKC-mediated vasoconstriction of arteries. Methods and results Oxidized low-density lipoprotein (oxLDL)induced p66shc phosphorylation at serine 36 residue and PKCβII phosphorylation in mouse endothelial cells. Adenoviral overexpression of APE1 resulted in reduction of oxLDL-induced p66shc and PKCβII phosphorylation. Phorbol 12-myristate 13-acetate (PMA), which stimulates PKCs, induced p66shc phosphorylation and this was inhibited by a selective PKCβII inhibitor. Adenoviral overexpression of PKCβII also increased p66shc phosphorylation. Overexpression of APE1 suppressed PMA-induced p66shc phosphorylation. Moreover, PMA-induced p66shc phosphorylation was augmented in cells in which APE1 was knocked down. PMA increased cytoplasmic APE1 expression, compared with the basal condition, suggesting the role of cytoplasmic APE1 against p66shc phosphorylation. Finally, vasoconstriction induced by phorbol-12,13, dibutylrate, another PKC agonist, was partially inhibited by transduction of Tat-APE1 into arteries. Conclusion APE1 suppresses oxLDL-induced p66shc activation in endothelial cells by inhibiting PKCβII-mediated serine phosphorylation of p66shc, and mitigates vasoconstriction induced by activation of PKC.

Histone chaperones function in histone transfer and regulate the nucleosome occupancy and the activity of genes. HIRA is a replication-independent (RI) histone chaperone that is linked to transcription and various developmental processes. Here, we show that HIRA interacts with Mef2 and contributes to the activation of Mef2-target genes during muscle differentiation. Asf1 cooperated with HIRA and was indispensable for Mef2-dependent transcription. The HIRA R460A mutant, which is defective in Asf1 binding, lost the transcriptional co-activation. In addition, the role of Cabin1, previously reported as a Mef2 repressor and as one of the components of the HIRA-containing complex, was delineated in Mef2/HIRA-mediated transcription. Cabin1 associated with the C-terminus of HIRA via its N-terminal domain and suppressed Mef2/HIRA-mediated transcription. Expression of Cabin1 was dramatically reduced upon myoblast differentiation, which may allow Mef2 and HIRA/Asf1 to resume their transcriptional activity. HIRA led to more permeable chromatin structure marked by active histone modifications around the myogenin promoter. Our results suggest that histone chaperone complex components contribute to the regulation of Mef2 target genes for muscle differentiation.

Aim To characterize the pharmacodynamic profile of DA-1229, a novel dipeptidyl peptidase (DPP) 4 inhibitor. Main methods Enzyme inhibition assays against DPP4, DPP8 and DPP9. Antidiabetic effects of DA-1229 in HF-DIO mice and young db/db mice. Key findings DA-1229 was shown to potently inhibit the DPP4 enzyme in human and murine soluble forms and the human membrane-bound form with IC 50 values of 0.98, 3.59 and 1.26 nM, respectively. As a reversible and competitive inhibitor, DA-1229 was more selective to human DPP4 (6000-fold) than to human DPP8 and DPP9. DA-1229 (0.1–3 mg/kg) dose-dependently inhibited plasma DPP4 activity, leading to increased levels of plasma GLP-1 and insulin, and thereby lowering blood glucose levels in mice. In high fat diet-fed (HF) mice, a single oral dose of 100 mg/kg of DA-1229 reduced plasma DPP4 activity by over 80% during a 24 h period. Long-term treatment with DA-1229 for 8 weeks revealed significant improvements in glucose intolerance and insulin resistance, accompanied by significant body weight reduction. However, it remains unclear whether there is a direct causal relationship between DPP4 inhibition and body weight reduction. In young db/db mice, the DA-1229 treatment significantly reduced blood glucose excursions for the first 2 weeks, resulting in significantly lower levels of HbA1c at the end of the study. Furthermore, the pancreatic insulin content of the treatment group was significantly higher than that of the db/db control. Significance DA-1229 as a novel and selective DPP4 inhibitor improves the insulin sensitivity in HF mice and delays the onset of diabetes in young db/db mice.

Since persistent infection with high-risk human papillomavirus (HPV) is a known cause of high-grade cervical intraepithelial neoplasia and cervical cancer, several HPV DNA detection methods have been developed during the last decade. The Hybrid Capture II (HCII) assay, which allows detection of 13 high-risk HPVs, has been well validated; however, it does not provide any genotype-specific information. The oncogenic activity of HPV is dependent on its genotype. The prophylactic effects of HPV vaccines are based on L1 virus-like particles and are limited mainly to infections corresponding to the HPV type used to develop the immunogen. Therefore, accurate detection and genotyping are important for treatment as well as screening. A newly developed HPV genotyping system using a liquid bead array was evaluated with 286 cervical samples and the results were compared to two commercially available methods, i.e. the HCII and HPV DNA chip assays, and sequencing. The sensitivity for detection of high-risk HPV was 85.3 % (HCII), 94.7 % (DNA chip) and 99.0 % (liquid bead array). The liquid bead array showed almost perfect agreement (κ=0.95) with genotype information confirmed by sequencing, while substantial agreement (κ=0.8) was observed between DNA chip and sequencing. Furthermore, the liquid bead array had superior detection of 26 HPVs (16 high-risk and 10 low-risk types) and has proven to be as accurate as sequencing in identifying individual HPV types, even in cases with multiple HPV infections.

Parafibromin, a component of the RNA polymerase II-associated PAF1 complex, is a tumor suppressor linked to hyperparathyroidism-jaw tumor syndrome and sporadic parathyroid carcinoma. Parafibromin induces cell cycle arrest by repressing cyclin D1 via an unknown mechanism. Here, we show that parafibromin interacts with the histone methyltransferase, SUV39H1, and functions as a transcriptional repressor. The central region (128-227 amino acids) of parafibromin is important for both the interaction with SUV39H1 and transcriptional repression. Parafibromin associated with the promoter and coding regions of cyclin D1 and was required for the recruitment of SUV39H1 and the induction of H3 K9 methylation but not H3 K4 methylation. RNA interference analysis showed that SUV39H1 was critical for cyclin D1 repression. These data suggest that parafibromin plays an unexpected role as a repressor in addition to its widely known activity associated with transcriptional activation. Parafibromin as a part of the PAF1 complex might downregulate cyclin D1 expression by integrating repressive H3 K9 methylation during transcription.

The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells.

Ferritin, an iron storage protein, plays an essential role in iron homeostasis and a wide range of physiologic processes. Ferritin alleviates oxidative stress by regulating cellular labile iron concentration. The tumor suppressor p53 is induced upon iron depletion, and controls reactive oxygen species level. Although some functional connections between ferritin and p53 were implied in several reports, the direct links between ferritin and p53 has not yet been investigated. Here we report that ferritin physically interacts with p53 upon oxidative stress. Ferritin increases p53 protein level and p53 transcriptional activity in ferroxidase activity independent manner. Ferritin knocked down cells show retarded induction of p53 target genes upon oxidative stress. These findings suggest that ferritin cooperates with p53 to cope with oxidative stress.

The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, acts as a binding platform for various mRNA processing and histone-modifying enzymes that act co-transcriptionally. These factors are targeted to specific phosphorylation states of the CTD that predominate at different stages of transcription. Within the repeating sequence YSPTSPS, serines 2 and 5 are major phosphorylation sites, but serine 7 phosphorylation was recently discovered in mammalian cells. Here we show that CTD serine 7 is also phosphorylated in yeast and that Ser-7(P) chromatin immunoprecipitation patterns resemble those of Ser-5(P). The basal factor TFIIH can phosphorylate Ser-7 in vitro and is necessary for Ser-7(P) in vivo. Interestingly, deletion of the CTD Ser-5(P) phosphatase Rtr1 leads to an increase in Ser-5(P) but not Ser-7(P).

The embryonic stem cell-like gene expression signature has been shown to be associated with poorly differentiated aggressive human tumors and has attracted great attention as a potential target for future cancer therapies. Here, we investigate the potential of the embryonic stem cell signature as molecular target for the therapy and the strategy to suppress the embryonic stem cell signature. The core stemness gene Nanog is abnormally overexpressed in human embryonic carcinoma NCCIT cells showing gene expression profiles similar to embryonic stem cells. Down-regulation of the gene by either small interfering RNAs targeting Nanog or histone deacetylase inhibitor apicidin causes reversion of expression pattern of embryonic stem cell signature including Oct4, Sox2, and their target genes, leading to cell cycle arrest, inhibition of colony formation in soft agar, and induction of differentiation into all three germ layers. These effects are antagonized by reintroduction of Nanog. Interestingly, embryonic carcinoma cells (NCCIT, NTERA2, and P19) exhibit a higher sensitivity to apicidin in down-regulation of Nanog compared with embryonic stem cells. Furthermore, the down-regulation of Nanog expression by apicidin is mediated by a coordinated change in recruitment of epigenetic modulators and transcription factors to the promoter region. These findings indicate that overexpression of stemness gene Nanog in NCCIT cells is associated with maintaining stem cell-like phenotype and suggest that targeting Nanog might be an approach for improved therapy of poorly differentiated tumors. [Cancer Res 2009;69(14):5716–25]