Publications by authors named "Takako Hishiki"

54 Publications

On-tissue polysulfide visualization by surface-enhanced Raman spectroscopy benefits patients with ovarian cancer to predict post-operative chemosensitivity.

Redox Biol 2021 May 2;41:101926. Epub 2021 Mar 2.

Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Japan.

Chemosensitivity to cisplatin derivatives varies among individual patients with intractable malignancies including ovarian cancer, while how to unlock the resistance remain unknown. Ovarian cancer tissues were collected the debulking surgery in discovery- (n = 135) and validation- (n = 47) cohorts, to be analyzed with high-throughput automated immunohistochemistry which identified cystathionine γ-lyase (CSE) as an independent marker distinguishing non-responders from responders to post-operative platinum-based chemotherapy. We aimed to identify CSE-derived metabolites responsible for chemoresistant mechanisms: gold-nanoparticle (AuN)-based surface-enhanced Raman spectroscopy (SERS) was used to enhance electromagnetic fields which enabled to visualize multiple sulfur-containing metabolites through detecting scattering light from Au-S vibration two-dimensionally. Clear cell carcinoma (CCC) who turned out less sensitive to cisplatin than serous adenocarcinoma was classified into two groups by the intensities of SERS intensities at 480 cm; patients with greater intensities displayed the shorter overall survival after the debulking surgery. The SERS signals were eliminated by topically applied monobromobimane that breaks sulfane-sulfur bonds of polysulfides to result in formation of sulfodibimane which was detected at 580 cm, manifesting the presence of polysulfides in cancer tissues. CCC-derived cancer cell lines in culture were resistant against cisplatin, but treatment with ambroxol, an expectorant degrading polysulfides, renders the cells CDDP-susceptible. Co-administration of ambroxol with cisplatin significantly suppressed growth of cancer xenografts in nude mice. Furthermore, polysulfides, but neither glutathione nor hypotaurine, attenuated cisplatin-induced disturbance of DNA supercoiling. Polysulfide detection by on-tissue SERS thus enables to predict prognosis of cisplatin-based chemotherapy. The current findings suggest polysulfide degradation as a stratagem unlocking cisplatin chemoresistance.
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http://dx.doi.org/10.1016/j.redox.2021.101926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010883PMC
May 2021

OGT Regulates Hematopoietic Stem Cell Maintenance via PINK1-Dependent Mitophagy.

Cell Rep 2021 Jan;34(1):108579

Department of Stem Cell and Immune Regulation, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. Electronic address:

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.
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http://dx.doi.org/10.1016/j.celrep.2020.108579DOI Listing
January 2021

Reduced fatty acid uptake aggravates cardiac contractile dysfunction in streptozotocin-induced diabetic cardiomyopathy.

Sci Rep 2020 11 30;10(1):20809. Epub 2020 Nov 30.

Department of Cardiovascular Medicine, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma, 371-8511, Japan.

Diabetes is an independent risk factor for the development of heart failure. Increased fatty acid (FA) uptake and deranged utilization leads to reduced cardiac efficiency and accumulation of cardiotoxic lipids, which is suggested to facilitate diabetic cardiomyopathy. We studied whether reduced FA uptake in the heart is protective against streptozotocin (STZ)-induced diabetic cardiomyopathy by using mice doubly deficient in fatty acid binding protein 4 (FABP4) and FABP5 (DKO mice). Cardiac contractile dysfunction was aggravated 8 weeks after STZ treatment in DKO mice. Although compensatory glucose uptake was not reduced in DKO-STZ hearts, total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. Tracer analysis with C-glucose revealed that accelerated glycolysis in DKO hearts was strongly suppressed by STZ treatment. Levels of ceramides, cardiotoxic lipids, were similarly elevated by STZ treatment. These findings suggest that a reduction in total energy supply by reduced FA uptake and suppressed glycolysis could account for exacerbated contractile dysfunction in DKO-STZ hearts. Thus, enhanced FA uptake in diabetic hearts seems to be a compensatory response to reduced energy supply from glucose, and therefore, limited FA use could be detrimental to cardiac contractile dysfunction due to energy insufficiency.
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http://dx.doi.org/10.1038/s41598-020-77895-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705707PMC
November 2020

Fatty Acid Synthesis Is Indispensable for Survival of Human Pluripotent Stem Cells.

iScience 2020 Sep 6;23(9):101535. Epub 2020 Sep 6.

Department of Cardiology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan.

The role of lipid metabolism in human pluripotent stem cells (hPSCs) is poorly understood. We have used large-scale targeted proteomics to demonstrate that undifferentiated hPSCs express different fatty acid (FA) biosynthesis-related enzymes, including ATP citrate lyase and FA synthase (FASN), than those expressed in hPSC-derived cardiomyocytes (hPSC-CMs). Detailed lipid profiling revealed that inhibition of FASN resulted in significant reduction of sphingolipids and phosphatidylcholine (PC); moreover, we found that PC was the key metabolite for cell survival in hPSCs. Inhibition of FASN induced cell death in undifferentiated hPSCs via mitochondria-mediated apoptosis; however, it did not affect cell survival in hPSC-CMs, neurons, or hepatocytes as there was no significant reduction of PC. Furthermore, we did not observe tumor formation following transplantation of FASN inhibitor-treated cells. Our findings demonstrate the importance of FA synthesis in the survival of undifferentiated hPSCs and suggest applications for FASN inhibition in regenerative medicine.
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http://dx.doi.org/10.1016/j.isci.2020.101535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509212PMC
September 2020

-Glycan-Altered Extracellular Vesicles: A Specific Serum Marker Elevated in Pancreatic Cancer.

Cancers (Basel) 2020 Aug 31;12(9). Epub 2020 Aug 31.

Department of Pathology, Keio University School of Medicine, Tokyo 160-8582, Japan.

Pancreatic cancer (PC) is among the most lethal malignancies due to an often delayed and difficult initial diagnosis. Therefore, the development of a novel, early stage, diagnostic PC marker in liquid biopsies is of great significance. In this study, we analyzed the differential glycomic profiling of extracellular vesicles (EVs) derived from serum (two cohorts including 117 PC patients and 98 normal controls) using lectin microarray. The glyco-candidates of PC-specific EVs were quantified using a high-sensitive exosome-counting system, ExoCounter. An absolute quantification system for altered glycan-containing EVs elevated in PC serum was established. EVs recognized by -glycan-binding lectins ABA or ACA were identified as candidate markers by lectin microarray. Quantitative analyses using ExoCounter revealed that the ABA- or ACA-positive EVs were significantly increased in the culture of PC cell lines or in the serum of PC patients including carbohydrate antigen 19-9 negative patients with high area under curve values. The elevated numbers of EVs in PC serum returned to normal levels after pancreatectomy. Histological examination confirmed that the tumors stained with ABA/ACA. These specific EVs with -glycans recognized by ABA/ACA are elevated in PC sera and can act as potential biomarkers in a liquid biopsy for PC patients screening.
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http://dx.doi.org/10.3390/cancers12092469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563872PMC
August 2020

2-Nitroimidazoles induce mitochondrial stress and ferroptosis in glioma stem cells residing in a hypoxic niche.

Commun Biol 2020 08 17;3(1):450. Epub 2020 Aug 17.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.

Under hypoxic conditions, nitroimidazoles can replace oxygen as electron acceptors, thereby enhancing the effects of radiation on malignant cells. These compounds also accumulate in hypoxic cells, where they can act as cytotoxins or imaging agents. However, whether these effects apply to cancer stem cells has not been sufficiently explored. Here we show that the 2-nitroimidazole doranidazole potentiates radiation-induced DNA damage in hypoxic glioma stem cells (GSCs) and confers a significant survival benefit in mice harboring GSC-derived tumors in radiotherapy settings. Furthermore, doranidazole and misonidazole, but not metronidazole, manifested radiation-independent cytotoxicity for hypoxic GSCs that was mediated by ferroptosis induced partially through blockade of mitochondrial complexes I and II and resultant metabolic alterations in oxidative stress responses. Doranidazole also limited the growth of GSC-derived subcutaneous tumors and that of tumors in orthotopic brain slices. Our results thus reveal the theranostic potential of 2-nitroimidazoles as ferroptosis inducers that enable targeting GSCs in their hypoxic niche.
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http://dx.doi.org/10.1038/s42003-020-01165-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431527PMC
August 2020

The NOTCH-FOXM1 Axis Plays a Key Role in Mitochondrial Biogenesis in the Induction of Human Stem Cell Memory-like CAR-T Cells.

Cancer Res 2020 02 25;80(3):471-483. Epub 2019 Nov 25.

Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

Recent studies have shown that stem cell memory T (T) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor-engineered-T (CAR-T) cells. We previously reported that both human and murine-activated T cells are converted into stem cell memory-like T (iT) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iT reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into T-like CAR-T, "CAR-iT" cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iT formation, which are essential for the properties of iT cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iT differentiation. Like NOTCH-induced CAR-iT cells, FOXM1-induced CAR-iT cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCH- or FOXM1-driven CAR-iT formation is an effective strategy for improving cancer immunotherapy. SIGNIFICANCE: Manipulation of signaling and metabolic pathways important for directing production of stem cell memory-like T cells may enable development of improved CAR-T cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1196DOI Listing
February 2020

Xanthine oxidase inhibitor ameliorates postischemic renal injury in mice by promoting resynthesis of adenine nucleotides.

JCI Insight 2019 11 14;4(22). Epub 2019 Nov 14.

Department of Biochemistry, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

Although oxidative stress plays central roles in postischemic renal injury, region-specific alterations in energy and redox metabolism caused by short-duration ischemia remain unknown. Imaging mass spectrometry enabled us to reveal spatial heterogeneity of energy and redox metabolites in the postischemic murine kidney. After 10-minute ischemia and 24-hour reperfusion (10mIR), in the cortex and outer stripes of the outer medulla, ATP substantially decreased, but not in the inner stripes of the outer medulla and inner medulla. 10mIR caused renal injury with elevation of fractional excretion of sodium, although histological damage by oxidative stress was limited. Ischemia-induced NADH elevation in the cortex indicated prolonged production of reactive oxygen species by xanthine oxidase (XOD). However, consumption of reduced glutathione after reperfusion suggested the amelioration of oxidative stress. An XOD inhibitor, febuxostat, which blocks the degradation pathway of adenine nucleotides, promoted ATP recovery and exerted renoprotective effects in the postischemic kidney. Because effects of febuxostat were canceled by silencing of the hypoxanthine phosphoribosyl transferase 1 gene in cultured tubular cells, mechanisms for the renoprotective effects appear to involve the purine salvage pathway, which uses hypoxanthine to resynthesize adenine nucleotides, including ATP. These findings suggest a novel therapeutic approach for acute ischemia/reperfusion renal injury with febuxostat through salvaging high-energy adenine nucleotides.
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http://dx.doi.org/10.1172/jci.insight.124816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948864PMC
November 2019

Biochemical, Metabolomic, and Genetic Analyses of Dephospho Coenzyme A Kinase Involved in Coenzyme A Biosynthesis in the Human Enteric Parasite .

Front Microbiol 2018 30;9:2902. Epub 2018 Nov 30.

Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Coenzyme A (CoA) is an essential cofactor for numerous cellular reactions in all living organisms. In the protozoan parasite , CoA is synthesized in a pathway consisting of four enzymes with dephospho-CoA kinase (DPCK) catalyzing the last step. However, the metabolic and physiological roles of DPCK remain elusive. In this study, we took biochemical, reverse genetic, and metabolomic approaches to elucidate role of DPCK in . The genome encodes two DPCK isotypes (DPCK1 and DPCK2). Epigenetic gene silencing of and caused significant reduction of DPCK activity, intracellular CoA concentrations, and also led to growth retardation , suggesting importance of DPCK for CoA synthesis and proliferation. Furthermore, metabolomic analysis showed that suppression of gene expression also caused decrease in the level of acetyl-CoA, and metabolites involved in amino acid, glycogen, hexosamine, nucleic acid metabolisms, chitin, and polyamine biosynthesis. The kinetic properties of and human DPCK showed remarkable differences, e.g., the Km values of and human DPCK were 58-114 and 5.2 μM toward dephospho-CoA and 15-20 and 192 μM for ATP, respectively. Phylogenetic analysis also supported the uniqueness of the amebic enzyme compared to the human counterpart. These biochemical, evolutionary features, and physiological importance of DPCKs indicate that DPCK represents the rational target for the development of anti-amebic agents.
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http://dx.doi.org/10.3389/fmicb.2018.02902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284149PMC
November 2018

Myocardial fatty acid uptake through CD36 is indispensable for sufficient bioenergetic metabolism to prevent progression of pressure overload-induced heart failure.

Sci Rep 2018 08 13;8(1):12035. Epub 2018 Aug 13.

Department of Cardiovascular Medicine, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan.

The energy metabolism of the failing heart is characterized by reduced fatty acid (FA) oxidation and an increase in glucose utilization. However, little is known about how energy metabolism-function relationship is relevant to pathophysiology of heart failure. Recent study showed that the genetic deletion of CD36 (CD36KO), which causes reduction in FA use with an increased reliance on glucose, accelerates the progression from compensated hypertrophy to heart failure. Here, we show the mechanisms by which CD36 deletion accelerates heart failure in response to pressure overload. CD36KO mice exhibited contractile dysfunction and death from heart failure with enhanced cardiac hypertrophy and interstitial fibrosis when they were subjected to transverse aortic constriction (TAC). The pool size in the TCA cycle and levels of high-energy phosphate were significantly reduced in CD36KO-TAC hearts despite an increase in glycolytic flux. De novo synthesis of non-essential amino acids was facilitated in CD36KO-TAC hearts, which could cause a further decline of the pool size. The ingestion of a diet enriched in medium-chain FA improved cardiac dysfunction in CD36KO-TAC hearts. These findings suggest that myocardial FA uptake through CD36 is indispensable for sufficient ATP production and for preventing an increased glycolytic flux-mediated structural remodeling during pressure overload-induced hypertrophy.
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http://dx.doi.org/10.1038/s41598-018-30616-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6089997PMC
August 2018

Development of a Highly Sensitive Device for Counting the Number of Disease-Specific Exosomes in Human Sera.

Clin Chem 2018 10 18;64(10):1463-1473. Epub 2018 Jul 18.

Department of Nanoparticle Translational Research, Tokyo Medical University, Tokyo, Japan.

Background: Although circulating exosomes in blood play crucial roles in cancer development and progression, difficulties in quantifying exosomes hamper their application for reliable clinical testing. By combining the properties of nanobeads with optical disc technology, we have developed a novel device named the ExoCounter to determine the exact number of exosomes in the sera of patients with various types of cancer.

Method: In this system, individual exosomes were captured in the groove of an optical disc coated with antibodies against exosome surface antigens. The captured exosomes were labeled with antibody-conjugated magnetic nanobeads, and the number of the labeled exosomes was counted with an optical disc drive.

Results: We showed that the ExoCounter could detect specific exosomes derived from cells or human serum without any enrichment procedures. The detection sensitivity and linearity with this system were higher than those with conventional detection methods such as ELISA or flow cytometry. In addition to the ubiquitous exosome markers CD9 and CD63, the cancer-related antigens CD147, carcinoembryonic antigen, and human epidermal growth factor receptor 2 (HER2) were also used to quantify cancer cell line-derived exosomes. Furthermore, analyses of a cross-sectional cohort of sera samples revealed that HER2-positive exosomes were significantly increased in patients with breast cancer or ovarian cancer compared with healthy individuals and those with noncancer diseases.

Conclusions: The ExoCounter system exhibits high performance in the direct detection of exosomes in cell culture and human sera. This method may enable reliable analysis of liquid biopsies.
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http://dx.doi.org/10.1373/clinchem.2018.291963DOI Listing
October 2018

Neuroprotective Role of Astroglia in Parkinson Disease by Reducing Oxidative Stress Through Dopamine-Induced Activation of Pentose-Phosphate Pathway.

ASN Neuro 2018 Jan-Dec;10:1759091418775562

1 Department of Neurology, Keio University School of Medicine, Tokyo, Japan.

Oxidative stress plays an important role in the onset and progression of Parkinson disease. Although released dopamine at the synaptic terminal is mostly reabsorbed by dopaminergic neurons, some dopamine is presumably taken up by astroglia. This study examined the dopamine-induced astroglial protective function through the activation of the pentose-phosphate pathway (PPP) to reduce reactive oxygen species (ROS). In vitro experiments were performed using striatal neurons and cortical or striatal astroglia prepared from Sprague-Dawley rats or C57BL/6 mice. The rates of glucose phosphorylation in astroglia were evaluated using the [C]deoxyglucose method. PPP activity was measured using [1-C]glucose and [6-C]glucose after acute (60 min) or chronic (15 hr) exposure to dopamine. ROS production was measured using 2',7'-dichlorodihydrofluorescein diacetate. The involvement of the Kelch-like ECH-associated protein 1 (Keap1) or nuclear factor-erythroid-2-related factor 2 (Nrf2) system was evaluated using Nrf2 gene knockout mice, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction analysis for heme oxygenase-1. Acute exposure to dopamine elicited increases in astroglial glucose consumption with lactate release. PPP activity in astroglia was robustly enhanced independently of Na-dependent monoamine transporters. In contrast, chronic exposure to dopamine induced moderate increases in PPP activity via the Keap1/Nrf2 system. ROS production from dopamine increased gradually over 12 hr. Dopamine induced neuronal cell damage that was prevented by coculturing with astroglia but not with Nrf2-deficient astroglia. Dopamine-enhanced astroglial PPP activity in both acute and chronic manners may possibly reduce neuronal oxidative stress.
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http://dx.doi.org/10.1177/1759091418775562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960859PMC
July 2019

Gold-nanofève surface-enhanced Raman spectroscopy visualizes hypotaurine as a robust anti-oxidant consumed in cancer survival.

Nat Commun 2018 04 19;9(1):1561. Epub 2018 Apr 19.

Department of Biochemistry, Keio University School of Medicine, Tokyo, 160-8582, Japan.

Gold deposition with diagonal angle towards boehmite-based nanostructure creates random arrays of horse-bean-shaped nanostructures named gold-nanofève (GNF). GNF generates many electromagnetic hotspots as surface-enhanced Raman spectroscopy (SERS) excitation sources, and enables large-area visualization of molecular vibration fingerprints of metabolites in human cancer xenografts in livers of immunodeficient mice with sufficient sensitivity and uniformity. Differential screening of GNF-SERS signals in tumours and those in parenchyma demarcated tumour boundaries in liver tissues. Furthermore, GNF-SERS combined with quantum chemical calculation identified cysteine-derived glutathione and hypotaurine (HT) as tumour-dominant and parenchyma-dominant metabolites, respectively. CD44 knockdown in cancer diminished glutathione, but not HT in tumours. Mechanisms whereby tumours sustained HT under CD44-knockdown conditions include upregulation of PHGDH, PSAT1 and PSPH that drove glycolysis-dependent activation of serine/glycine-cleavage systems to provide one-methyl group for HT synthesis. HT was rapidly converted into taurine in cancer cells, suggesting that HT is a robust anti-oxidant for their survival under glutathione-suppressed conditions.
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http://dx.doi.org/10.1038/s41467-018-03899-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908798PMC
April 2018

Glucose is preferentially utilized for biomass synthesis in pressure-overloaded hearts: evidence from fatty acid-binding protein-4 and -5 knockout mice.

Cardiovasc Res 2018 07;114(8):1132-1144

Department of Cardiovascular Medicine, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan.

Aims: The metabolism of the failing heart is characterized by an increase in glucose uptake with reduced fatty acid (FA) oxidation. We previously found that the genetic deletion of FA-binding protein-4 and -5 [double knockout (DKO)] induces an increased myocardial reliance on glucose with decreased FA uptake in mice. However, whether this fuel switch confers functional benefit during the hypertrophic response remains open to debate. To address this question, we investigated the contractile function and metabolic profile of DKO hearts subjected to pressure overload.

Methods And Results: Transverse aortic constriction (TAC) significantly reduced cardiac contraction in DKO mice (DKO-TAC), although an increase in cardiac mass and interstitial fibrosis was comparable with wild-type TAC (WT-TAC). DKO-TAC hearts exhibited enhanced glucose uptake by 8-fold compared with WT-TAC. Metabolic profiling and isotopomer analysis revealed that the pool size in the TCA cycle and the level of phosphocreatine were significantly reduced in DKO-TAC hearts, despite a marked increase in glycolytic flux. The ingestion of a diet enriched in medium-chain FAs restored cardiac contractile dysfunction in DKO-TAC hearts. The de novo synthesis of amino acids as well as FA from glycolytic flux was unlikely to be suppressed, despite a reduction in each precursor. The pentose phosphate pathway was also facilitated, which led to the increased production of a coenzyme for lipogenesis and a precursor for nucleotide synthesis. These findings suggest that reduced FA utilization is not sufficiently compensated by a robust increase in glucose uptake when the energy demand is elevated. Glucose utilization for sustained biomass synthesis further enhances diminishment of the pool size in the TCA cycle.

Conclusions: Our data suggest that glucose is preferentially utilized for biomass synthesis rather than ATP production during pressure-overload-induced cardiac hypertrophy and that the efficient supplementation of energy substrates may restore cardiac dysfunction caused by energy insufficiency.
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http://dx.doi.org/10.1093/cvr/cvy063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6014234PMC
July 2018

Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target.

Int J Parasitol Drugs Drug Resist 2018 04 1;8(1):125-136. Epub 2018 Mar 1.

Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Japan. Electronic address:

The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells.
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http://dx.doi.org/10.1016/j.ijpddr.2018.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114107PMC
April 2018

Robust suppression of cardiac energy catabolism with marked accumulation of energy substrates during lipopolysaccharide-induced cardiac dysfunction in mice.

Metabolism 2017 12 20;77:47-57. Epub 2017 Sep 20.

Department of Cardiovascular Medicine, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan; Education and Research Support Center, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Electronic address:

Background: Myocardial contractile dysfunction in sepsis has been attributed mainly to increased inflammatory cytokines, insulin resistance, and impaired oxidative phosphorylation of fatty acids (FAs). However, precise molecular mechanisms underlying the cardiac dysfunction in sepsis remain to be determined. We previously reported major shift in myocardial energy substrates from FAs to glucose, and increased hepatic ketogenesis in mice lacking fatty acid-binding protein 4 (FABP4) and FABP5 (DKO).

Purpose: We sought to determine whether a shift of energy substrates from FAs to glucose and increased availability of ketone bodies are beneficial or detrimental to cardiac function under the septic condition.

Methods: Lipopolysaccharide (LPS, 10mg/kg) was intraperitoneally injected into wild-type (WT) and DKO mice. Twelve hours after injection, cardiac function was assessed by echocardiography and serum and hearts were collected for further analyses.

Results: Cardiac contractile function was more deteriorated by LPS injection in DKO mice than WT mice despite comparable changes in pro-inflammatory cytokine production. LPS injection reduced myocardial uptake of FA tracer by 30% in both types of mice, while uptake of the glucose tracer did not significantly change in either group of mice in sepsis. Storage of glycogen and triacylglycerol in hearts was remarkably increased by LPS injection in both mice. Metabolome analysis revealed that LPS-induced suppression of pool size in the TCA cycle was more enhanced in DKO hearts. A tracing study with C-glucose further revealed that LPS injection substantially reduced glucose-derived metabolites in the TCA cycle and related amino acids in DKO hearts. Consistent with these findings, glucose oxidation in vitro was similarly and markedly reduced in both mice. Serum concentration of β-hydroxybutyrate and cardiac expression of genes associated with ketolysis were reduced in septic mice.

Conclusions: Our study demonstrated that LPS-induced cardiac contractile dysfunction is associated with the robust suppression of catabolism of energy substrates including FAs, glucose and ketone bodies and accumulation of glycogen and triacylglycerol in the heart. Thus, a fuel shift from FAs to glucose and/or ketone bodies may be detrimental rather than protective under septic conditions.
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http://dx.doi.org/10.1016/j.metabol.2017.09.003DOI Listing
December 2017

Targeting Oxygen-Sensing Prolyl Hydroxylase for Metformin-Associated Lactic Acidosis Treatment.

Mol Cell Biol 2017 Aug 28;37(16). Epub 2017 Jul 28.

Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan

Metformin is one of the most widely used therapeutics for type 2 diabetes mellitus and also has anticancer and antiaging properties. However, it is known to induce metformin-associated lactic acidosis (MALA), a severe medical condition with poor prognosis, especially in individuals with renal dysfunction. Inhibition of prolyl hydroxylase (PHD) is known to activate the transcription factor hypoxia-inducible factor (HIF) that increases lactate efflux as a result of enhanced glycolysis, but it also enhances gluconeogenesis from lactate in the liver that contributes to reducing circulating lactate levels. Here, we investigated the outcome of pharmaceutical inhibition of PHD in mice with MALA induced through the administration of metformin and an intraperitoneal injection of lactic acid. We found that the PHD inhibitors significantly increased the expression levels of genes involved in gluconeogenesis in the liver and the kidney and significantly improved the survival of mice with MALA. Furthermore, the PHD inhibitor also improved the rate of survival of MALA induced in mice with chronic kidney disease (CKD). Thus, PHD represents a new therapeutic target for MALA, which is a critical complication of metformin therapy.
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http://dx.doi.org/10.1128/MCB.00248-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533879PMC
August 2017

The histone 3 lysine 9 methyltransferase inhibitor chaetocin improves prognosis in a rat model of high salt diet-induced heart failure.

Sci Rep 2017 01 4;7:39752. Epub 2017 Jan 4.

Department of Cardiology, Keio University School of Medicine, Shinjuku-ku Tokyo, Japan.

Histone acetylation has been linked to cardiac hypertrophy and heart failure. However, the pathological implications of changes in histone methylation and the effects of interventions with histone methyltransferase inhibitors for heart failure have not been fully clarified. Here, we focused on H3K9me3 status in the heart and investigated the effects of the histone H3K9 methyltransferase inhibitor chaetocin on prognoses in Dahl salt-sensitive rats, an animal model of chronic heart failure. Chaetocin prolonged survival and restored mitochondrial dysfunction. ChIP-seq analysis demonstrated that chronic stress to the heart induced H3K9me3 elevation in thousands of repetitive elements, including intronic regions of mitochondria-related genes, such as the gene encoding peroxisome proliferator-activated receptor-gamma coactivator 1 alpha. Furthermore, chaetocin reversed this effect on these repetitive loci. These data suggested that excessive heterochromatinization of repetitive elements of mitochondrial genes in the failing heart may lead to the silencing of genes and impair heart function. Thus, chaetocin may be a potential therapeutic agent for chronic heart failure.
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http://dx.doi.org/10.1038/srep39752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209701PMC
January 2017

A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system.

J Neuroinflammation 2016 May 4;13(1):99. Epub 2016 May 4.

Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, 160-8582, Tokyo, Japan.

Background: Toll-like receptor 4 (TLR4) plays a pivotal role in the pathophysiology of stroke-induced inflammation. Both astroglia and microglia express TLR4, and endogenous ligands produced in the ischemic brain induce inflammatory responses. Reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines produced by TLR4 activation play harmful roles in neuronal damage after stroke. Although astroglia exhibit pro-inflammatory responses upon TLR4 stimulation by lipopolysaccharide (LPS), they may also play cytoprotective roles via the activation of the pentose phosphate pathway (PPP), reducing oxidative stress by glutathione peroxidase. We investigated the mechanisms by which astroglia reduce oxidative stress via the activation of PPP, using TLR4 stimulation and hypoxia in concert with microglia.

Methods: In vitro experiments were performed using cells prepared from Sprague-Dawley rats. Coexisting microglia in the astroglial culture were chemically eliminated using L-leucine methyl ester (LME). Cells were exposed to LPS (0.01 μg/mL) or hypoxia (1 % O2) for 12-15 h. PPP activity was measured using [1-(14)C]glucose and [6-(14)C]glucose. ROS and NO production were measured using 2',7'-dichlorodihydrofluorescein diacetate and diaminofluorescein-FM diacetate, respectively. The involvement of nuclear factor-erythroid-2-related factor 2 (Nrf2), a cardinal transcriptional factor under stress conditions that regulates glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of PPP, was evaluated using immunohistochemistry.

Results: Cultured astroglia exposed to LPS elicited 20 % increases in PPP flux, and these actions of astroglia appeared to involve Nrf2. However, the chemical depletion of coexisting microglia eliminated both increases in PPP and astroglial nuclear translocation of Nrf2. LPS induced ROS and NO production in the astroglial culture containing microglia but not in the microglia-depleted astroglial culture. LPS enhanced astroglial ROS production after glutathione depletion. U0126, an upstream inhibitor of mitogen-activated protein kinase, eliminated LPS-induced NO production, whereas ROS production was unaffected. U0126 also eliminated LPS-induced PPP activation in astroglial-microglial culture, indicating that microglia-derived NO mediated astroglial PPP activation. Hypoxia induced astroglial PPP activation independent of the microglia-NO pathway. Elimination of ROS and NO production by sulforaphane, a natural Nrf2 activator, confirmed the astroglial protective mechanism.

Conclusions: Astroglia in concert with microglia may play a cytoprotective role for countering oxidative stress in stroke.
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http://dx.doi.org/10.1186/s12974-016-0564-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855896PMC
May 2016

Glutamine Oxidation Is Indispensable for Survival of Human Pluripotent Stem Cells.

Cell Metab 2016 Apr 31;23(4):663-74. Epub 2016 Mar 31.

Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan. Electronic address:

Human pluripotent stem cells (hPSCs) are uniquely dependent on aerobic glycolysis to generate ATP. However, the importance of oxidative phosphorylation (OXPHOS) has not been elucidated. Detailed amino acid profiling has revealed that glutamine is indispensable for the survival of hPSCs. Under glucose- and glutamine-depleted conditions, hPSCs quickly died due to the loss of ATP. Metabolome analyses showed that hPSCs oxidized pyruvate poorly and that glutamine was the main energy source for OXPHOS. hPSCs were unable to utilize pyruvate-derived citrate due to negligible expression of aconitase 2 (ACO2) and isocitrate dehydrogenase 2/3 (IDH2/3) and high expression of ATP-citrate lyase. Cardiomyocytes with mature mitochondria were not able to survive without glucose and glutamine, although they were able to use lactate to synthesize pyruvate and glutamate. This distinguishing feature of hPSC metabolism allows preparation of clinical-grade cell sources free of undifferentiated hPSCs, which prevents tumor formation during stem cell therapy.
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http://dx.doi.org/10.1016/j.cmet.2016.03.001DOI Listing
April 2016

Inhibition of the oxygen sensor PHD2 in the liver improves survival in lactic acidosis by activating the Cori cycle.

Proc Natl Acad Sci U S A 2015 Sep 31;112(37):11642-7. Epub 2015 Aug 31.

Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan; Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Suematsu Gas Biology Project, Core Research for Evolutional Science and Technology, Tokyo 160-8582, Japan;

Loss of prolyl hydroxylase 2 (PHD2) activates the hypoxia-inducible factor-dependent hypoxic response, including anaerobic glycolysis, which causes large amounts of lactate to be released from cells into the circulation. We found that Phd2-null mouse embryonic fibroblasts (MEFs) produced more lactate than wild-type MEFs, as expected, whereas systemic inactivation of PHD2 in mice did not cause hyperlacticacidemia. This unexpected observation led us to hypothesize that the hypoxic response activated in the liver enhances the Cori cycle, a lactate-glucose carbon recycling system between muscle and liver, and thereby decreases circulating lactate. Consistent with this hypothesis, blood lactate levels measured after a treadmill or lactate tolerance test were significantly lower in Phd2-liver-specific knockout (Phd2-LKO) mice than in control mice. An in vivo (13)C-labeled lactate incorporation assay revealed that the livers of Phd2-LKO mice produce significantly more glucose derived from (13)C-labeled lactate than control mice, suggesting that blockade of PHD2 in the liver ameliorates lactic acidosis by activating gluconeogenesis from lactate. Phd2-LKO mice were resistant to lactic acidosis induced by injection of a lethal dose of lactate, displaying a significant elongation of survival. Moreover, oral administration of a PHD inhibitor improved survival in an endotoxin shock mice model. These data suggest that PHD2 is a potentially novel drug target for the treatment of lactic acidosis, which is a serious and often fatal complication observed in some critically ill patients.
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http://dx.doi.org/10.1073/pnas.1515872112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577207PMC
September 2015

Activation of pyruvate dehydrogenase by dichloroacetate has the potential to induce epigenetic remodeling in the heart.

J Mol Cell Cardiol 2015 May 2;82:116-24. Epub 2015 Mar 2.

Department of Cardiology, Keio University, School of Medicine, Tokyo, Japan; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Tokyo, Japan. Electronic address:

Dichloroacetate (DCA) promotes pyruvate entry into the Krebs cycle by inhibiting pyruvate dehydrogenase (PDH) kinase and thereby maintaining PDH in the active dephosphorylated state. DCA has recently gained attention as a potential metabolic-targeting therapy for heart failure but the molecular basis of the therapeutic effect of DCA in the heart remains a mystery. Once-daily oral administration of DCA alleviates pressure overload-induced left ventricular remodeling. We examined changes in the metabolic fate of pyruvate carbon (derived from glucose) entering the Krebs cycle by metabolic interventions of DCA. (13)C6-glucose pathway tracing analysis revealed that instead of being completely oxidized in the mitochondria for ATP production, DCA-mediated PDH dephosphorylation results in an increased acetyl-CoA pool both in control and pressure-overloaded hearts. DCA induces hyperacetylation of histone H3K9 and H4 in a dose-dependent manner in parallel to the dephosphorylation of PDH in cultured cardiomyocytes. DCA administration increases histone H3K9 acetylation in in vivo mouse heart. Interestingly, DCA-dependent histone acetylation was associated with an up-regulation of 2.3% of genes (545 out of 23,474 examined). Gene ontology analysis revealed that these genes are highly enriched in transcription-related categories. This evidence suggests that sustained activation of PDH by DCA results in an overproduction of acetyl-CoA, which exceeds oxidation in the Krebs cycle and results in histone acetylation. We propose that DCA-mediated PDH activation has the potential to induce epigenetic remodeling in the heart, which, at least in part, forms the molecular basis for the therapeutic effect of DCA in the heart.
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http://dx.doi.org/10.1016/j.yjmcc.2015.02.021DOI Listing
May 2015

Hyperhomocysteinemia abrogates fasting-induced cardioprotection against ischemia/reperfusion by limiting bioavailability of hydrogen sulfide anions.

J Mol Med (Berl) 2015 Aug 6;93(8):879-89. Epub 2015 Mar 6.

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

Unlabelled: Elevated plasma homocysteine levels are considered an independent risk factor for cardiovascular diseases. Experimental evidence has shown that hydrogen sulfide anion (HS(-)) protects the myocardium from ischemia/reperfusion (IR) injury. Both homocysteine levels and endogenous HS(-) production are mainly regulated by two transsulfuration enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). We hypothesized that the transsulfuration pathway plays essential roles in the development of cardiac adaptive responses against ischemia, and investigated the roles of homocysteine, HS(-), and transsulfuration enzymes in fasting-induced cardioprotection against IR injury utilizing hyperhomocysteinemic Cbs (-/-) and Cth (-/-) mice. Langendorff-perfused hearts were subjected to 25-min global ischemia, followed by 60-min reperfusion. Two-day fasting ameliorated left ventricular dysfunction after reperfusion via propargylglycine- and glibenclamide-sensitive pathways in wild-type mice but not in Cbs (-/-) or Cth (-/-) mice, although fasting induced cardiac expression of several Nrf2 target antioxidant genes in both wild-type and Cth (-/-) mice. Intraperitoneal administration of sodium hydrosulfide (a HS(-) donor) at 24 h prior to IR improved myocardial recovery in wild-type mice but not in Cth (-/-) or high-methionine-diet-fed (thus intermediately hyperhomocysteinemic) wild-type mice. Quantitative analysis of reactive sulfur species using monobromobimane derivatization methods revealed that homocysteine efficiently captures HS(-) to form homocysteine persulfide in the hearts as well as in the in vitro reactions. Here we propose a novel molecular and pathophysiological basis for hyperhomocysteinemia; excessive circulatory homocysteine interferes with HS(-)-related cardioprotection against IR injury by capturing endogenous HS(-) to form homocysteine persulfide.

Key Message: Two-day fasting of mice ameliorates ischemia/reperfusion injury in Langendorff hearts. H2S-producing enzymes, CBS and CTH, are essential in fasting-induced cardioprotection. Administration of a H2S donor (NaHS) confers cardioprotection against IR injury. NaHS effects are absent in Cth (-/-), Cbs (-/-), and dietary hyperhomocysteinemic mice. Homocysteine captures cardioprotective HS(-) to form homocysteine persulfide.
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http://dx.doi.org/10.1007/s00109-015-1271-5DOI Listing
August 2015

Therapeutic hypothermia achieves neuroprotection via a decrease in acetylcholine with a concurrent increase in carnitine in the neonatal hypoxia-ischemia.

J Cereb Blood Flow Metab 2015 May 14;35(5):794-805. Epub 2015 Jan 14.

1] Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan [2] JST Exploratory Research for Advanced Technology (ERATO) Suematsu Gas Biology Project, Tokyo, Japan.

Although therapeutic hypothermia is known to improve neurologic outcomes after perinatal cerebral hypoxia-ischemia, etiology remains unknown. To decipher the mechanisms whereby hypothermia regulates metabolic dynamics in different brain regions, we used a two-step approach: a metabolomics to target metabolic pathways responding to cooling, and a quantitative imaging mass spectrometry to reveal spatial alterations in targeted metabolites in the brain. Seven-day postnatal rats underwent the permanent ligation of the left common carotid artery followed by exposure to 8% O2 for 2.5 hours. The pups were returned to normoxic conditions at either 38 °C or 30 °C for 3 hours. The brain metabolic states were rapidly fixed using in situ freezing. The profiling of 107 metabolites showed that hypothermia diminishes the carbon biomass related to acetyl moieties, such as pyruvate and acetyl-CoA; conversely, it increases deacetylated metabolites, such as carnitine and choline. Quantitative imaging mass spectrometry demarcated that hypothermia diminishes the acetylcholine contents specifically in hippocampus and amygdala. Such decreases were associated with an inverse increase in carnitine in the same anatomic regions. These findings imply that hypothermia achieves its neuroprotective effects by mediating the cellular acetylation status through a coordinated suppression of acetyl-CoA, which resides in metabolic junctions of glycolysis, amino-acid catabolism, and ketolysis.
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http://dx.doi.org/10.1038/jcbfm.2014.253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420853PMC
May 2015

Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry.

Nitric Oxide 2015 Apr 11;46:102-13. Epub 2014 Nov 11.

Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan; Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Suematsu Gas Biology Project, Tokyo 160-8582, Japan. Electronic address:

CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue.
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http://dx.doi.org/10.1016/j.niox.2014.11.005DOI Listing
April 2015

Involvement of the yciW gene in l-cysteine and l-methionine metabolism in Escherichia coli.

J Biosci Bioeng 2015 Mar 29;119(3):310-3. Epub 2014 Sep 29.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan.

We here analyzed a sulfur index of Escherichia coli using LC-MS/MS combined with thiol-specific derivatization by monobromobimane. The obtained sulfur index was then applied to evaluate the L-cysteine producer. E. coli cells overexpressing the yciW gene, a novel Cys regulon, accumulated l-homocysteine, suggesting that YciW is involved in L-methionine biosynthesis.
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http://dx.doi.org/10.1016/j.jbiosc.2014.08.012DOI Listing
March 2015

Large-area surface-enhanced Raman spectroscopy imaging of brain ischemia by gold nanoparticles grown on random nanoarrays of transparent boehmite.

ACS Nano 2014 Jun 2;8(6):5622-32. Epub 2014 Jun 2.

Frontier Core-Technology Laboratories, R & D Management Headquarters, FUJIFILM Corporation , 577, Ushijima, Kaisei-machi, Ashigarakami-gun, Kanagawa 258-8577, Japan.

Although SERS spectroscopy, which is sensitive to molecular vibration states, offers label-free visualization of molecules, identification of molecules and their reliable large-area imaging remains to be developed. Limitation comes from difficulties in fabricating a SERS-active substrate with homogeneity over a large area. Here, we overcome this hurdle by utilizing a self-assembled nanostructure of boehmite that is easily achieved by a hydrothermal preparation of aluminum as a template for subsequent gold (Au) deposition. This approach brought about random arrays of Au-nanostructures with a diameter of ∼125 nm and a spacing of <10 nm, ideal for the hot-spots formation. The substrate, which we named "gold nanocoral" (GNC) after its coral reef-like shape, exhibited a small variability of signal intensities (coefficient value <11.2%) in detecting rhodamine 6G molecule when 121 spots were measured over an area of 10 × 10 mm(2), confirming high uniformity. The transparent nature of boehmite enabled us to conduct the measurement from the back-side of the substrate as efficiently as that from the front-side. We then conducted tissue imaging using the mouse ischemic brain adhered on the GNC substrate. Through nontargeted construction of two-dimensional-Raman-intensity map using differential bands from two metabolically distinct regions, that is, ischemic core and contralateral-control areas, we found that mapping using the adenine ring vibration band at 736 cm(-1) clearly demarcated ischemic core where high-energy adenine phosphonucleotides were degraded as judged by imaging mass spectrometry. Such a detection capability makes the GNC-based SERS technology especially promising for revealing acute energy derangement of tissues.
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http://dx.doi.org/10.1021/nn4065692DOI Listing
June 2014

Neutral aminoaciduria in cystathionine β-synthase-deficient mice; an animal model of homocystinuria.

Am J Physiol Renal Physiol 2014 Jun 23;306(12):F1462-76. Epub 2014 Apr 23.

Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, Gunma, Japan; Department of Biochemistry, Keio University Graduate School of Pharmaceutical Sciences, Tokyo, Japan

The kidney is one of the major loci for the expression of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). While CBS-deficient (Cbs(-/-)) mice display homocysteinemia/methioninemia and severe growth retardation, and rarely survive beyond the first 4 wk, CTH-deficient (Cth(-/-)) mice show homocysteinemia/cystathioninemia but develop with no apparent abnormality. This study examined renal amino acid reabsorption in those mice. Although both 2-wk-old Cbs(-/-) and Cth(-/-) mice had normal renal architecture, their serum/urinary amino acid profiles largely differed from wild-type mice. The most striking feature was marked accumulation of Met and cystathionine in serum/urine/kidney samples of Cbs(-/-) and Cth(-/-) mice, respectively. Levels of some neutral amino acids (Val, Leu, Ile, and Tyr) that were not elevated in Cbs(-/-) serum were highly elevated in Cbs(-/-) urine, and urinary excretion of other neutral amino acids (except Met) was much higher than expected from their serum levels, demonstrating neutral aminoaciduria in Cbs(-/-) (not Cth(-/-)) mice. Because the bulk of neutral amino acids is absorbed via a B(0)AT1 transporter and Met has the highest substrate affinity for B(0)AT1 than other neutral amino acids, hypermethioninemia may cause hyperexcretion of neutral amino acids.
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http://dx.doi.org/10.1152/ajprenal.00623.2013DOI Listing
June 2014

Reduced methylation of PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway.

Nat Commun 2014 Mar 17;5:3480. Epub 2014 Mar 17.

1] Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan [2] Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology (ERATO), Suematsu Gas Biology Project, Tokyo 160-8582, Japan.

Haem oxygenase (HO)-1/carbon monoxide (CO) protects cancer cells from oxidative stress, but the gas-responsive signalling mechanisms remain unknown. Here we show using metabolomics that CO-sensitive methylation of PFKFB3, an enzyme producing fructose 2,6-bisphosphate (F-2,6-BP), serves as a switch to activate phosphofructokinase-1, a rate-limiting glycolytic enzyme. In human leukaemia U937 cells, PFKFB3 is asymmetrically di-methylated at R131 and R134 through modification by protein arginine methyltransferase 1. HO-1 induction or CO results in reduced methylation of PFKFB3 in varied cancer cells to suppress F-2,6-BP, shifting glucose utilization from glycolysis toward the pentose phosphate pathway. Loss of PFKFB3 methylation depends on the inhibitory effects of CO on haem-containing cystathionine β-synthase (CBS). CBS modulates remethylation metabolism, and increases NADPH to supply reduced glutathione, protecting cells from oxidative stress and anti-cancer reagents. Once the methylation of PFKFB3 is reduced, the protein undergoes polyubiquitination and is degraded in the proteasome. These results suggest that the CO/CBS-dependent regulation of PFKFB3 methylation determines directional glucose utilization to ensure resistance against oxidative stress for cancer cell survival.
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http://dx.doi.org/10.1038/ncomms4480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3959213PMC
March 2014