Publications by authors named "Shin Murai"

13 Publications

  • Page 1 of 1

Computed tomographic colonography versus double-contrast barium enema for the preoperative evaluation of rectal cancer.

Surg Today 2021 Nov 23. Epub 2021 Nov 23.

Department of Surgical Oncology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Purpose: We investigated whether or not computed tomographic colonography (CTC) is a viable alternative to double-contrast barium enema (BE) for a preoperative rectal cancer evaluation.

Methods: The size and distance from the anal canal to the lower or upper tumor borders were laterally measured in 147 patients who underwent CTC and BE. Measurements were grouped into early cancer, advanced, and after chemoradiation therapy (CRT).

Results: In the early and advanced cancer groups, all lesions were visualized by BE. In contrast, 3 (7.8%) early and 8 (7.3%) advanced cases, located at the anterior wall near the anal canal, were not visualized by CTC because of liquid level formation. In the CRT group, 16 (23.5%) and 4 (5.8%) cases were not visualized by CTC and BE, respectively. The BE and CTC size measurements were similar among cohorts. However, the distance from the anal canal's superior margin tended to be longer with BE, especially in early cancer. The differences in distance from the anal canal were significantly larger in the early cancer group than in the other two groups (p = 0.0024).

Conclusion: CTC may be a viable alternative imaging modality in some cases. However, BE should be employed in anterior wall cases near the anal canal and CRT cases.
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http://dx.doi.org/10.1007/s00595-021-02411-5DOI Listing
November 2021

Time-Lapse Imaging of Necroptosis and DAMP Release at Single-Cell Resolution.

Methods Mol Biol 2021 ;2274:353-363

Department of Biochemistry, Toho University School of Medicine, Ota-ku, Tokyo, Japan.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). Necroptotic cells release a variety of cellular and nuclear factors, referred to as danger-associated molecular patterns (DAMPs). We recently developed a förster resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation based on FRET). SMART comprises a fragment of MLKL, and it monitors necroptosis, but not apoptosis or necrosis. We performed live-cell imaging for secretion activity (LCI-S) to observe the release of high-mobility group box 1 (HMGB1) from necroptotic cells at single-cell resolution. Moreover, we combined SMART and LCI-S imaging techniques and found two different modes of HMGB1 release from necroptotic cells. Thus, SMART and LCI-S are valuable tools for investigating intimate cross talk between necroptosis and DAMP release at single-cell resolution.
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http://dx.doi.org/10.1007/978-1-0716-1258-3_29DOI Listing
June 2021

MIND bomb 2 prevents RIPK1 kinase activity-dependent and -independent apoptosis through ubiquitylation of cFLIP.

Commun Biol 2021 01 19;4(1):80. Epub 2021 Jan 19.

Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omori-Nishi, Ota-ku, Tokyo, 143-8540, Japan.

Mind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48- and K63-linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIP), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II. TNF-induced RIPK1 Ser phosphorylation, a hallmark of RIPK1 death-inducing activity, was enhanced in MIB2 knockout cells, as was RIPK1 kinase activity-dependent and -independent apoptosis. Moreover, RIPK1 kinase activity-independent apoptosis was induced in cells expressing cFLIP mutants lacking MIB2-dependent ubiquitylation. Together, these results suggest that MIB2 suppresses both RIPK1 kinase activity-dependent and -independent apoptosis, through suppression of RIPK1 kinase activity and ubiquitylation of cFLIP, respectively.
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http://dx.doi.org/10.1038/s42003-020-01603-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815719PMC
January 2021

Identification of the hallmarks of necroptosis and ferroptosis by transmission electron microscopy.

Biochem Biophys Res Commun 2020 06 16;527(3):839-844. Epub 2020 May 16.

Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omori-Nishi, Ota-ku, Tokyo, 143-8540, Japan. Electronic address:

Apoptosis is the prototype for a regulated form of cell death, but recent studies have revealed other types of regulated forms of cell death, including necroptosis and ferroptosis. The molecular mechanisms underlying the execution of these processes have been intensively investigated, yet the hallmarks of their morphology are not fully understood. Here, we report that electron lucent cytoplasm was a common feature of both necroptosis and ferroptosis, which was consistent with cytoplasmic vacuolization due to a defect in the cytoplasmic membrane integrity. Notably, the perinuclear space was dilated in necroptosis, but such dilation did not occur in ferroptosis. Cells undergoing ferroptosis, but not necroptosis, exhibited an electron lucent nucleus. We previously reported that one of the nuclear danger-associated molecular patterns (DAMPs), high mobility group box (HMGB)1, is rapidly released from the nucleus to the extracellular spaces of cells undergoing necroptosis through the ruptured nuclear and cytoplasmic membrane. Via time-lapse imaging of cells stably expressing HMGB1 fused to a fluorescence protein, we found that HMGB1 was also released from the nucleus to the cytosol, and then eventually released into the extracellular spaces in cells undergoing ferroptosis. Thus, nuclear membrane damage was induced prior to cytoplasmic membrane rupture in ferroptosis. Thus, dilation of the perinuclear space and an electron lucent nucleus may be the hallmarks of necroptosis and ferroptosis, respectively.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.127DOI Listing
June 2020

Development of novel methods that monitor necroptosis and the release of DAMPs at the single cell resolution.

Cell Stress 2019 Jan 22;3(2):66-69. Epub 2019 Jan 22.

Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omori-Nishi, Ota-ku, Tokyo 143-8540, Japan.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). While danger-associated molecular pattern (DAMP)s are released from dead cells and involved in various pathological conditions, the mechanisms underlying regulation of the release of DAMPs are not fully understood. Apoptosis and pyroptosis can be detected by several types of sensors such as Forster resonance energy transfer (FRET) biosensors, termed SCAT1 (a sensor for caspase 1 activation based on FRET) and SCAT3, respectively. These sensors have provided better understanding of pyroptosis and apoptosis and . However, there have been no biosensors to monitor necroptosis. Development of a FRET biosensor that monitors necroptosis and generation of transgenic mice expressing such FRET biosensor might be useful to understand the mechanisms underlying the execution of necroptosis and also the consequences of necroptosis . In our recent study (Nat Commun, 9(1):4457), we developed a FRET biosensor for necroptosis, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Moreover, we recently developed a platform called Live-Cell Imaging for Secretion activity (LCI-S) to monitor protein secretion at the single cell level. This platform has enabled us to monitor the release of HMGB1 (High Mobility Group Box 1), one of the DAMPs, at the single cell level and reveals two different modes of the release of HMGB1 from necroptotic cells.
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http://dx.doi.org/10.15698/cst2019.02.177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551708PMC
January 2019

Addendum: A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs.

Nat Commun 2019 Apr 25;10(1):1923. Epub 2019 Apr 25.

Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omori-Nishi, Ota-ku, Tokyo, 143-8540, Japan.

The cDNA sequence of human SMART described in this Article was misreported, as described in the accompanying Addendum. This error does not affect the results or any conclusion of the Article.
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http://dx.doi.org/10.1038/s41467-019-09536-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483979PMC
April 2019

A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs.

Nat Commun 2018 10 26;9(1):4457. Epub 2018 Oct 26.

Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omori-Nishi, Ota-ku, Tokyo, 143-8540, Japan.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like (MLKL). While danger-associated molecular pattern (DAMP)s are involved in various pathological conditions and released from dead cells, the underlying mechanisms are not fully understood. Here we develop a fluorescence resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Mechanistically, SMART monitors plasma membrane translocation of oligomerized MLKL, which is induced by RIPK3 or mutational activation. SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Thus, SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis.
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http://dx.doi.org/10.1038/s41467-018-06985-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203740PMC
October 2018

Laparoscopic treatment of a vesicointestinal fistula due to a Meckel's diverticulum: a case report and review of the literature.

Clin J Gastroenterol 2018 Dec 18;11(6):476-480. Epub 2018 Jun 18.

Department of Surgery, Hitachi General Hospital, 2-1-1 Jonan-cho, Hitachi, Ibaraki, 317-0077, Japan.

While there have been numerous reports about colovesical fistulas and ruptured intestinal diverticula, there have been far fewer reports about vesicointestinal fistulas caused by Meckel's diverticula. Most Meckel's diverticula are asymptomatic. Furthermore, they seldom cause vesicointestinal fistulas, and the associated complications are non-specific. Thus, their preoperative diagnosis is difficult. We experienced a case in which a vesicointestinal fistula was caused by a Meckel's diverticulum and was treated with laparoscopic surgery. A 46-year-old male was referred to our hospital after exhibiting hematuria. Cystoscopy revealed a fistula between the small intestine and bladder. Contrast-enhanced computed tomography and magnetic resonance imaging showed a diverticulum in the ileum and a fistula between the ileum and bladder, which passed through the diverticulum. A Meckel's diverticulum was suspected. We conducted a laparoscopic operation. We dissected the Meckel's diverticulum with an automatic suturing device and removed it together with part of the ileum. The patient's postoperative course was good. We experienced a case in which a vesicointestinal fistula was caused by a Meckel's diverticulum and was successfully treated with laparoscopic surgery. In selected cases of Meckel's diverticulum, the dissection of the diverticulum with an automatic suturing device is appropriate.
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http://dx.doi.org/10.1007/s12328-018-0878-9DOI Listing
December 2018

Novel method to rescue a lethal phenotype through integration of target gene onto the X-chromosome.

Sci Rep 2016 11 15;6:37200. Epub 2016 Nov 15.

Institute of Resource Development and Analysis, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan.

The loss-of-function mutations of serine protease inhibitor, Kazal type 1 (SPINK1) gene are associated with human chronic pancreatitis, but the underlying mechanisms remain unknown. We previously reported that mice lacking Spink3, the murine homologue of human SPINK1, die perinatally due to massive pancreatic acinar cell death, precluding investigation of the effects of SPINK1 deficiency. To circumvent perinatal lethality, we have developed a novel method to integrate human SPINK1 gene on the X chromosome using Cre-loxP technology and thus generated transgenic mice termed "X-SPINK1". Consistent with the fact that one of the two X chromosomes is randomly inactivated, X-SPINK1 mice exhibit mosaic pattern of SPINK1 expression. Crossing of X-SPINK1 mice with Spink3 mice rescued perinatal lethality, but the resulting Spink3;XX mice developed spontaneous pancreatitis characterized by chronic inflammation and fibrosis. The results show that mice lacking a gene essential for cell survival can be rescued by expressing this gene on the X chromosome. The Spink3;XX mice, in which this method has been applied to partially restore SPINK1 function, present a novel genetic model of chronic pancreatitis.
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http://dx.doi.org/10.1038/srep37200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5109027PMC
November 2016

Dual inhibition of Cdc2 protein kinase activation during apoptosis in Xenopus egg extracts.

FEBS J 2015 Apr 18;282(7):1256-70. Epub 2015 Feb 18.

Department of Biochemistry, Toho University School of Medicine, Ota-ku, Tokyo, Japan.

When intracellular damage accumulates in proliferating somatic cells, the cell cycle usually arrests in G1 or G2 in a checkpoint-dependent manner, either to repair the damage or to die by apoptosis. In contrast, early embryonic cells lack checkpoint-mediated cell-cycle arrest, and it is not clear whether apoptosis in early embryonic cells occurs at a specific cell cycle stage or at random points. Here, we examined the functional molecular link between the embryonic cell cycle and apoptosis using Xenopus egg extracts. When apoptosis was induced in egg extracts by addition of exogenous cytochrome c during cell-cycle progression, cyclin B accumulation was inhibited, Cdc2 was not activated, and the cell cycle arrested at interphase. However, addition of recombinant cyclin B failed to activate Cdc2 due to the strong inhibitory phosphorylation of Cdc2 Tyr15 in apoptotic egg extracts. We found that endogenous Cdc25C, which activates the Cdc2-cyclin B complex by dephosphorylating Cdc2 Tyr15, was inactivated by caspase-mediated cleavage at two sites in the N-terminal regulatory domain. When the hyperactive Cdc25A catalytic fragment was added together with recombinant cyclin B to artificially dephosphorylate Cdc2 Tyr15, M-phase induction was restored in apoptotic egg extracts, indicating that the blockage of cyclin B accumulation and the caspase-mediated inactivation of Cdc25C dually inhibited Cdc2 activation. Apoptosis induction in cytostatic factor-arrested metaphase egg extracts resulted in inactivation of Cdc2 without cyclin B degradation. These results suggest that apoptotic inactivation of Cdc25C plays an important role in arresting the embryonic cell cycle at interphase during apoptosis.
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http://dx.doi.org/10.1111/febs.13217DOI Listing
April 2015

Maturation-associated Dbf4 expression is essential for mouse zygotic DNA replication.

Dev Growth Differ 2014 Dec 27;56(9):625-39. Epub 2014 Oct 27.

Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omorinishi Otaku, 143-8540, Tokyo, Japan.

Cdc7 is an S-phase-promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one-cell embryos at zygotic S-phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one-cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi-mediated knockdown of maternal Dbf4 expression prevents the maturation-associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one-cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 3' untranslated region (3'UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication.
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http://dx.doi.org/10.1111/dgd.12180DOI Listing
December 2014

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos.

Dev Biol 2010 May 26;341(1):205-12. Epub 2010 Feb 26.

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3' UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.
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http://dx.doi.org/10.1016/j.ydbio.2010.02.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854205PMC
May 2010

Apoptosis-inhibiting activities of BIR family proteins in Xenopus egg extracts.

FEBS J 2005 May;272(9):2237-50

Department of Biochemistry, Toho University School of Medicine, Tokyo, Japan.

In many animal species including Xenopus, ovulated eggs possess an intrinsic apoptotic execution system. This program is inhibited for a limited time by some maternal apoptosis inhibitors, although their molecular properties remain uncharacterized. Baculovirus IAP repeat (BIR) family proteins contain evolutionarily conserved BIR domains and play important roles in apoptosis suppression, and are therefore good candidates as maternal apoptosis inhibitors. We identified four maternal BIR family proteins in Xenopus eggs and, using the biochemical advantages of egg extracts, examined their physiological functions. These molecules included two survivin-related proteins, xEIAP/XLX, and a possible ortholog of XIAP named xXIAP. The addition of recombinant xXIAP greatly delayed apoptotic execution, whereas the immunodepletion of endogenous xXIAP significantly accelerated the onset of apoptosis. In contrast, xEIAP/XLX was a poor apoptosis inhibitor, and neither of the survivin orthologs showed anti-apoptotic activity in our assay. Both xEIAP/XLX and xXIAP were degraded by activated caspases, and also by a novel proteolytic system that required the presence of C-terminal RING finger domain but was insensitive to proteasome inhibition. Our data suggest that the regulation of endogenous xXIAP concentration is important for the survival of Xenopus eggs.
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http://dx.doi.org/10.1111/j.1742-4658.2005.04648.xDOI Listing
May 2005
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