Publications by authors named "Tsuyoshi Tahara"

31 Publications

Synthesis of L-[5- C]Leucine and L-α-[5- C]Methylleucine via Pd -mediated C-Methylation and Microfluidic Hydrogenation: Potentiality of Leucine PET Probes for Tumor Imaging.

ChemMedChem 2021 Jun 14. Epub 2021 Jun 14.

Laboratory for Labeling Chemistry, RIKEN Center for Biosystems Dynamics Research, 6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan.

The efficient synthesis of L-[5- C]leucine and L-α-[5- C]methylleucine has been investigated using a continuous two-step sequence of rapid reactions consisting of Pd -mediated C-methylation and microfluidic hydrogenation. The synthesis of L-[5- C]leucine and L-α-[5- C]methylleucine was accomplished within 40 min with a decay-corrected radiochemical yield of 15-38 % based on [ C]CH I, radiochemical purity of 95-99 %, and chemical purity of 95-99 %. The Pd impurities in the injectable solution measured using inductively coupled plasma mass spectrometry met the international criteria for human use. Positron emission tomography scanning after an intravenous injection of L-[5- C]leucine or L-α-[5- C]methyl leucine in A431 tumor-bearing mice was performed. As a result, L-α-[5- C]methylleucine was found to be a potentially useful probe for visualizing the tumor. Tissue distribution analysis showed that the accumulation value of L-α-[5- C]methylleucine in tumor tissue was high [12±3% injected dose/g tissue (%ID/g)].
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http://dx.doi.org/10.1002/cmdc.202100255DOI Listing
June 2021

Disrupting tumor onset and growth via selective cell tagging (SeCT) therapy.

Sci Adv 2021 Apr 23;7(17). Epub 2021 Apr 23.

Biofunctional Synthetic Chemistry Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

This study presents the early framework of selective cell tagging (SeCT) therapy, which is the concept of preferentially labeling specific cells in vivo with chemical moieties that can elicit a therapeutic response. Using glycosylated artificial metalloenzyme (GArM)-based protein labeling, this study reports two separate functional strategies. In one approach, early tumor onset can be suppressed by tagging cancer cells in living mice with an integrin-blocking cyclic-Arg-Gly-Asp (cRGD) moiety, thereby disrupting cell adhesion onto the extracellular matrix. In another approach, tumor growth in mice can be reduced by tagging with a cytotoxic doxorubicin moiety. Subsequent cell death occurs following internalization and drug release. Overall, experiments have shown that mouse populations receiving the mixture of SeCT labeling reagents exhibited a significant delay/reduction in tumor onset and growth compared with controls. Highlighting its adaptability, this work represents a foundational step for further development of SeCT therapy and its potential therapeutic applications.
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http://dx.doi.org/10.1126/sciadv.abg4038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064634PMC
April 2021

A Strategy for Tumor Targeting by Higher-Order Glycan Pattern Recognition: Synthesis and In Vitro and In Vivo Properties of Glycoalbumins Conjugated with Four Different N-Glycan Molecules.

Small 2020 11 20;16(46):e2004831. Epub 2020 Oct 20.

Biofunctional Synthetic Chemistry Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.

Natural glycoconjugates that form glycocalyx play important roles in various biological processes based on cell surface recognition through pattern recognition mechanisms. This work represents a new synthesis-based screening strategy to efficiently target the cancer cells by higher-order glycan pattern recognition in both cells and intact animals (mice). The use of the very fast, selective, and effective RIKEN click reaction (6π-azaelectrocyclization of unsaturated imines) allows to synthesize and screen various structurally well-defined glycoalbumins containing two and eventually four different N-glycan structures in a very short time. The importance of glycan pattern recognition is exemplified in both cell- and mouse-based experiments. The use of pattern recognition mechanisms for cell targeting represents a novel and promising strategy for the development of diagnostic, prophylactic, and therapeutic agents for various diseases including cancers.
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http://dx.doi.org/10.1002/smll.202004831DOI Listing
November 2020

A viable strategy for screening the effects of glycan heterogeneity on target organ adhesion and biodistribution in live mice.

Chem Commun (Camb) 2018 Aug;54(63):8693-8696

Biofunctional Synthetic Chemistry Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan.

This work represents the first broad study of testing diverse heterogenous glycoconjugates (7 different glycoalbumins) for their differential in vivo binding (11 different cancer cell types) in both cell- and animal-based studies. As a result, various changes in biodistribution, excretion, and even tumor adhesion were observed.
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http://dx.doi.org/10.1039/c8cc01544aDOI Listing
August 2018

Sequential Double "Clicks" toward Structurally Well-Defined Heterogeneous -Glycoclusters: The Importance of Cluster Heterogeneity on Pattern Recognition In Vivo.

Adv Sci (Weinh) 2017 02 28;4(2):1600394. Epub 2016 Nov 28.

Biofunctional Synthetic Chemistry Laboratory RIKEN, Hirosawa Wako-shi, Saitama 351-0198 Japan; Biofunctional Chemistry Laboratory A. Butlerov Institute of Chemistry Kazan Federal University 18 Kremlyovskaya street Kazan 420008 Russia; JST-PRESTO, Hirosawa Wako-shi, Saitama 351-0198 Japan.

are prepared on albumin via a double click procedure. The number of glycan molecules present, in addition to the spatial arrangement of glycans in the heterogeneous glycoclusters, plays an important role in the in vivo kinetics and organ-selective accumulation through glycan pattern recognition mechanisms.
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http://dx.doi.org/10.1002/advs.201600394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323863PMC
February 2017

In Vivo Gold Complex Catalysis within Live Mice.

Angew Chem Int Ed Engl 2017 03 15;56(13):3579-3584. Epub 2017 Feb 15.

Biofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan.

Metal complex catalysis within biological systems is largely limited to cell and bacterial systems. In this work, a glycoalbumin-Au complex was designed and developed that enables organ-specific, localized propargyl ester amidation with nearby proteins within live mice. The targeted reactivity can be imaged through the use of Cy7.5- and TAMRA-linked propargyl ester based fluorescent probes. This targeting system could enable the exploitation of other metal catalysis strategies for biomedical and clinical applications.
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http://dx.doi.org/10.1002/anie.201610273DOI Listing
March 2017

Glycan multivalency effects toward albumin enable N-glycan-dependent tumor targeting.

Bioorg Med Chem Lett 2016 May 14;26(9):2251-4. Epub 2016 Mar 14.

Biofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; Biofunctional Chemistry Laboratory, A. Butlerov Institute of Chemistry, Kazan Federal University, 18 Kremlyovskaya Street, Kazan 420008, Russia; Japan Science and Technology Agency-PRESTO, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Electronic address:

Multivalent interactions play an essential role in molecular recognition in living systems. These effects were employed to target tumor cells using albumin clusters bearing ∼10 molecules of asparagine-linked glycans (N-glycans). Noninvasive near-infrared fluorescence imaging clearly revealed A431 tumors implanted in BALB/cA-nu/nu mice after 1h in an N-glycan structure-dependent manner, thereby demonstrating the efficient use of glycan multivalency effects for tumor targeting in vivo.
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http://dx.doi.org/10.1016/j.bmcl.2016.03.046DOI Listing
May 2016

Estradiol Facilitates Functional Integration of iPSC-Derived Dopaminergic Neurons into Striatal Neuronal Circuits via Activation of Integrin α5β1.

Stem Cell Reports 2016 Apr 17;6(4):511-524. Epub 2016 Mar 17.

Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Electronic address:

For cell transplantation therapy for Parkinson's disease (PD) to be realized, the grafted neurons should be integrated into the host neuronal circuit to restore the lost neuronal function. Here, using wheat-germ agglutinin-based transsynaptic tracing, we show that integrin α5 is selectively expressed in striatal neurons that are innervated by midbrain dopaminergic (DA) neurons. In addition, we found that integrin α5β1 was activated by the administration of estradiol-2-benzoate (E2B) in striatal neurons of adult female rats. Importantly, we observed that the systemic administration of E2B into hemi-parkinsonian rat models facilitates the functional integration of grafted DA neurons derived from human induced pluripotent stem cells into the host striatal neuronal circuit via the activation of integrin α5β1. Finally, methamphetamine-induced abnormal rotation was recovered earlier in E2B-administered rats than in rats that received other regimens. Our results suggest that the simultaneous administration of E2B with stem cell-derived DA progenitors can enhance the efficacy of cell transplantation therapy for PD.
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http://dx.doi.org/10.1016/j.stemcr.2016.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834042PMC
April 2016

In vivo imaging of advanced glycation end products (AGEs) of albumin: first observations of significantly reduced clearance and liver deposition properties in mice.

Org Biomol Chem 2016 Jun;14(24):5755-60

Biofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. and Biofunctional Chemistry Laboratory, A. Butlerov Institute of Chemistry, Kazan Federal University, 18 Kremlyovskaya Street, Kazan 420008, Russia and Japan Science and Technology Agency-PRESTO, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

Advanced glycation end products (AGEs) are associated with various diseases, especially during aging and the development of diabetes and uremia. To better understand these biological processes, investigation of the in vivo kinetics of AGEs, i.e., analysis of trafficking and clearance properties, was carried out by molecular imaging. Following the preparation of Cy7.5-labeled AGE-albumin and intravenous injection in BALB/cA-nu/nu mice, noninvasive fluorescence kinetics analysis was performed. In vivo imaging and fluorescence microscopy analysis revealed that non-enzymatic AGEs were smoothly captured by scavenger cells in the liver, i.e., Kupffer and other sinusoidal cells, but were unable to be properly cleared from the body. Overall, these results highlight an important link between AGEs and various disorders associated with them, which may serve as a platform for future research to better understand the processes and mechanisms of these disorders.
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http://dx.doi.org/10.1039/c6ob00098cDOI Listing
June 2016

Visualizing Trimming Dependence of Biodistribution and Kinetics with Homo- and Heterogeneous N-Glycoclusters on Fluorescent Albumin.

Sci Rep 2016 Feb 23;6:21797. Epub 2016 Feb 23.

Biofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

A series of N-glycans, each sequentially trimmed from biantennary sialoglycans, were homo- or heterogeneously clustered efficiently on fluorescent albumin using a method that combined strain-promoted alkyne-azide cyclization and 6π-azaelectrocyclization. Noninvasive in vivo kinetics and dissection analysis revealed, for the first time, a glycan-dependent shift from urinary to gall bladder excretion mediated by sequential trimming of non-reducing end sialic acids. N-glycoalbumins that were trimmed further, in particular, GlcNAc- and hybrid biantennary-terminated congeners, were selectively taken up by sinusoidal endothelial and stellate cells in the liver, which are critical for diagnosis and treatment of liver fibrillation. Our glycocluster strategy can not only reveal the previously unexplored extracellular functions of N-glycan trimming, but will be classified as the newly emerging glycoprobes for diagnostic and therapeutic applications.
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http://dx.doi.org/10.1038/srep21797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763176PMC
February 2016

A novel (11)C-labeled thymidine analog, [(11)C]AZT, for tumor imaging by positron emission tomography.

EJNMMI Res 2015 Dec 4;5(1):124. Epub 2015 Sep 4.

Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies (CLST), 6-7-3 Minatojima, Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan,

Background: Nucleoside analogs labeled with positrons, such as (11)C and (18)F, are considered valuable in visualizing the proliferative activity of tumor cells in vivo using positron emission tomography (PET). We recently developed the (11)C-labeled thymidine analogs [(11)C]zidovudine ([(11)C]AZT) and [(11)C]stavudine ([(11)C]d4T) via the Pd(0)-Cu(I) co-mediated rapid C-C coupling reaction. In this study, to examine whether [(11)C]AZT and [(11)C]d4T might be useful for visualization of tumors in vivo, we performed PET imaging, tissue distribution studies, and metabolite analysis in tumor-bearing mice.

Methods: Mice bearing tumors (rat glioma C6 and human cervical adenocarcinoma HeLa cells) were injected with 50 MBq of [(11)C]AZT or [(11)C]d4T, and PET was performed immediately thereafter. After PET imaging, the radioactivity in several tissues, including tumor tissues, was measured using a γ-counter. In addition, radioactive metabolites in plasma, bile, intestinal contents, and tumor were analyzed using thin layer chromatography (TLC). Cellular uptake of [(11)C]AZT in C6 was measured in the presence or absence of non-labeled thymidine (0.1 mM).

Results: In PET studies, C6 and HeLa tumors in mice were clearly visualized using [(11)C]AZT. Time-activity curves using [(11)C]AZT showed that the accumulation of radioactivity in tumors plateaued at 10 min after injection and persisted for 60 min, while most of the radioactivity in other tissues was rapidly excreted into the urine. In various tissues of the body, tumor tissue showed the highest radioactivity at 80 min after injection (five to six times higher uptake than that of blood). Compared with tumor tissue, uptake was lower in other proliferative tissues such as the spleen, intestine, and bone marrow, resulting in a high tumor-to-bone marrow ratio. Cellular uptake of [(11)C]AZT in C6 cells was completely blocked by the application of thymidine, strongly indicating the specific involvement of nucleoside transporters. In contrast, the time-activity curve of [(11)C]d4T in the tumor showed transient and rapid excretion with almost no obvious tumor tissue accumulation.

Conclusions: Tumors can be detected by PET using [(11)C]AZT; therefore, [(11)C]AZT could be useful as a novel PET tracer for tumor imaging in vivo.
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http://dx.doi.org/10.1186/s13550-015-0124-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4597405PMC
December 2015

Na+/H+ exchanger inhibitor augments hyperosmolarity-induced vasoconstriction by enhancing actin polymerization.

Vascul Pharmacol 2013 Nov-Dec;59(5-6):120-6. Epub 2013 Jul 19.

Laboratory of Cardiovascular Pharmacology, Department of Biopharmaceutical Sciences, Kobe Gakuin University, Minatojima 1-1-3, Chuo-ku, Kobe 6508586, Japan.

Vascular smooth muscle cells (VSMCs) exhibit shrinkage-induced activation of Na(+)/H(+) exchanger isoform 1 (NHE-1) and Na(+), K(+), 2Cl(-) cotransporter (NKCC) under hyperosmotic conditions. To investigate the roles of these ion transporters in vascular smooth muscle force induced by hyperosmotic stress, we tested the effects of 5-(N, N-dimethyl)-amiloride (DMA; NHE inhibitor), cariporide (a selective NHE-1 inhibitor), and bumetanide (NKCC inhibitor) on the contractile response of rat aortic rings to hyperosmolar solutions. NHE inhibitors significantly augmented the maximum force response and contractile sensitivity to hyperosmolar sucrose, NaCl, and glucose in endothelium-denuded rings. Bumetanide elicited a comparatively modest increase in sensitivity. NHE inhibitors blocked the increase in intracellular pH and enhanced the cell volume decrease of cultured VSMCs after exposure to hyperosmolar sucrose. However, DMA had no effect on the increase in cytosolic free Ca(2+) concentration ([Ca(2+)]i) in rat VSMCs and on the increases in phosphorylation of myosin phosphatase target subunit 1 and myosin light chain (MLC) in aortic rings in response to hyperosmolar sucrose. Hyperosmolar sucrose-induced force was significantly attenuated by cytochalasin B in the presence or absence of DMA. Exposure to hyperosmolar sucrose increased the ratio of F- to G-actin; the ratio was further elevated by DMA. These results suggest that the potentiation of hyperosmotic shrinkage by NHE inhibition promotes actin polymerization in VSMCs and augments force production independent of changes in [Ca(2+)]i and MLC phosphorylation.
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http://dx.doi.org/10.1016/j.vph.2013.07.004DOI Listing
July 2014

A conformationally fixed analog of the peptide mimic Grb2-SH2 domain: synthesis and evaluation against the A431 cancer cell.

Mol Biosyst 2013 May;9(5):1019-25

Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka-shi, Osaka 560-0043, Japan.

A small peptide mimic of the Grb2-SH2 domain, which was previously prepared through the template-assisted click approach and exhibited selective A431 tumor growth inhibition both in vitro and in vivo, was further elaborated on to enhance the interaction with target phosphorylated proteins. A conformationally fixed analog was efficiently synthesized by solid-supported ring-closing metathesis and Cu(i)/His-mediated self-activating Huisgen [3+2] cycloadditon as the key steps, and exhibited a 10-fold enhanced affinity to a phosphorylated peptide, a truncated peptide analog of the Grb2-SH2-interacting phosphoproteins. A stronger interaction with the target phosphorylated proteins gave this cyclic analog cytotoxic activity in A431 cells.
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http://dx.doi.org/10.1039/c3mb25462cDOI Listing
May 2013

Reduction of [11C](+)3-MPB binding in brain of chronic fatigue syndrome with serum autoantibody against muscarinic cholinergic receptor.

PLoS One 2012 11;7(12):e51515. Epub 2012 Dec 11.

Department of Physiology, Osaka City University Graduate School of Medicine, Abeno-ku, Osaka, Japan.

Background: Numerous associations between brain-reactive antibodies and neurological or psychiatric symptoms have been proposed. Serum autoantibody against the muscarinic cholinergic receptor (mAChR) was increased in some patients with chronic fatigue syndrome (CFS) or psychiatric disease. We examined whether serum autoantibody against mAChR affected the central cholinergic system by measuring brain mAChR binding and acetylcholinesterase activity using positron emission tomography (PET) in CFS patients with positive [CFS(+)] and negative [CFS(-)] autoantibodies.

Methodology: Five CFS(+) and six CFS(-) patients, as well as 11 normal control subjects underwent a series of PET measurements with N-[(11)C]methyl-3-piperidyl benzilate [(11)C](+)3-MPB for the mAChR binding and N-[(11)C]methyl-4-piperidyl acetate [(11)C]MP4A for acetylcholinesterase activity. Cognitive function of all subjects was assessed by neuropsychological tests. Although the brain [(11)C](+)3-MPB binding in CFS(-) patients did not differ from normal controls, CFS(+) patients showed significantly lower [(11)C](+)3-MPB binding than CFS(-) patients and normal controls. In contrast, the [(11)C]MP4A index showed no significant differences among these three groups. Neuropsychological measures were similar among groups.

Conclusion: The present results demonstrate that serum autoantibody against the mAChR can affect the brain mAChR without altering acetylcholinesterase activity and cognitive functions in CFS patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051515PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3519853PMC
May 2013

Template-assisted and self-activating clicked peptide as a synthetic mimic of the SH2 domain.

ACS Chem Biol 2012 Apr 26;7(4):637-45. Epub 2012 Jan 26.

A new synthetic strategy for obtaining artificial receptors that selectively regulate and/or control specific protein/protein interactions was developed based on the template-assisted and the self-activating click reaction applied to a combinatorial library. Synthetic mimics of the Grb2-SH2 domain, examined as a model case, selectively bound to a target signaling protein to induce cytotoxicity and inhibit tumor growth in vivo.
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http://dx.doi.org/10.1021/cb2003175DOI Listing
April 2012

Electrocyclization-based labeling allows efficient in vivo imaging of cellular trafficking.

ChemMedChem 2010 Jun;5(6):841-5

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka-shi, Osaka 560-0043, Japan.

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http://dx.doi.org/10.1002/cmdc.201000027DOI Listing
June 2010

Thiamine tetrahydrofurfuryl disulfide improves energy metabolism and physical performance during physical-fatigue loading in rats.

Nutr Res 2009 Dec;29(12):867-72

Department of Physiology, Osaka City University Graduate School of Medicine, Osaka 545-8585, Japan.

Impaired energy metabolism is considered a possible cause of fatigue. The thiamine derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), is prescribed and is also an over-the-counter drug for the attenuation of fatigue. It is readily absorbed from the intestinal tract and converted into thiamine pyrophosphate (TPP), which plays an important role as a cofactor for enzymes of metabolic pathways involved in the production of adenosine triphosphate (ATP). We postulated that TTFD has an anti-fatigue effect by improving energy metabolism during physical-fatigue loading. Here, we initially used the forced swimming test to determine whether daily TTFD or thiamine for 5 days has anti-fatigue effects on weight-loaded rats. The swimming duration of TTFD-, but not of thiamine-treated rats, was significantly longer than that of control rats (P < .05). Based on these findings, we examined changes in the levels of thiamine and its phosphate esters in various organs and the effect of TTFD on ATP levels in skeletal muscle after forced swimming, to determine the cellular mechanisms of the anti-fatigue effect of TTFD. Daily TTFD resulted in a characteristic distribution of thiamine and its phosphate esters in rat skeletal muscle, liver, kidney, heart, brain, and plasma. Furthermore, daily TTFD attenuated the decrease in ATP content in the skeletal muscle caused by forced swimming with a weight load for a defined period (150 s). These results indicate that TTFD exerts anti-fatigue effects by improving energy metabolism during physical fatigue.
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http://dx.doi.org/10.1016/j.nutres.2009.10.007DOI Listing
December 2009

Daily oral administration of crocetin attenuates physical fatigue in human subjects.

Nutr Res 2009 Mar;29(3):145-50

Department of Physiology, Osaka City University Graduate School of Medicine, Osaka 545-8585, Japan.

This study compared the effects of placebo with a carotenoid compound, crocetin, as well as an antioxidant, ascorbic acid, on physical fatigue in humans. In this double-blind, placebo-controlled, 3-way crossover study, 14 Japanese healthy volunteers (7 men and 7 women) were randomized to oral administration of crocetin (15 mg), ascorbic acid (3,000 mg), or placebo for 8 days. Subjects performed workload tests on a bicycle ergometer at fixed workloads for 120 minutes at 2 times (a total of 240 minutes) as a fatigue-inducing physical task. During the physical task, subjects performed nonworkload tests at maximum velocity (MV) of 10 seconds at 30 minutes (30-minute test) after the start of the physical task and at 30 minutes before the end of the task (210-minute test). The change in MV from the 30- to the 210-minute test was significantly higher in men who received crocetin compared with men who received placebo (P < .05). This effect of crocetin was specific to males. Administration of ascorbic acid did not change in MV from the 30-minute to the 210-minute test on males or females. These results suggest that daily administration of crocetin may attenuate physical fatigue in men.
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http://dx.doi.org/10.1016/j.nutres.2009.02.003DOI Listing
March 2009

Changes in plasma and tissue amino acid levels in an animal model of complex fatigue.

Nutrition 2009 May 11;25(5):597-607. Epub 2009 Feb 11.

Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan.

Objective: Fatigue can be classified as physical or mental, depending on its cause. In physical fatigue, changes in the plasma levels of some amino acids have been reported. However, complex fatigue, which is experienced in daily life, is a combination of physical and mental fatigue. We aimed to identify changes in amino acid levels in the plasma, skeletal muscle, liver, and brain in an animal model of complex fatigue.

Methods: Rats were kept in a cage filled with water to a height of 2.2 cm for 5 d. Because rats showed a reduction of body weight when the model was developed, we also included a food-restricted group showing a similar profile in weight reduction as the water-immersed rats. A non-treated control group was also included.

Results: Results indicated that levels of branched-chain amino acids (valine, leucine, and isoleucine) were increased in plasma (valine, leucine, and isoleucine; P < 0.01), skeletal muscle (valine, leucine, and isoleucine; P < 0.01), the liver (valine; P < 0.05), and brain (isoleucine; P < 0.05), whereas a reduction in other amino acid levels (total amino acids and glutamine in the plasma, skeletal muscle, and liver; and phenylalanine, tyrosine, arginine, and threonine in the brain; P < 0.01) was seen in animals with complex fatigue.

Conclusion: Complex fatigue may bring about systemic changes in amino acid metabolism in multiple organs.
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http://dx.doi.org/10.1016/j.nut.2008.11.021DOI Listing
May 2009

Mental and physical fatigue-related biochemical alterations.

Nutrition 2009 Jan 2;25(1):51-7. Epub 2008 Oct 2.

Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan.

Objective: To confirm fatigue-related biochemical alterations, we measured various parameters just before and after relaxation and fatigue-inducing mental or physical sessions.

Methods: Fifty-four healthy volunteers were randomized to perform relaxation and fatigue-inducing mental and physical sessions for 4 h in a double-blind, three-crossover design. Before and after each session, subjects were asked to rate their subjective sensations of fatigue, and blood, saliva, and urine samples were taken.

Results: After the fatigue-inducing mental and physical sessions, subjective scores of fatigue were increased. After the fatigue-inducing mental session, the vanillylmandelic acid level in urine was higher and plasma valine level was lower than after the relaxation session. In contrast, after the fatigue-inducing physical session, serum citric acid, triacylglycerol, free fatty acid, ketone bodies, total carnitine, acylcarnitine, uric acid, creatine kinase, aspartate aminotransferase, lactate dehydrogenase, cortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, plasma branched-chain amino acids, transforming growth factor-beta1 and -beta2, white blood cell and neutrophil counts, saliva cortisol and amylase, and urine vanillylmandelic acid levels were higher and serum free carnitine and plasma total amino acids and alanine levels were lower than those after the relaxation session.

Conclusion: Some mental or physical fatigue-related biochemical changes were determined. Various biochemical alterations reflecting homeostatic perturbation and its responses might be shown. We believe that our results contribute to clarifying the mechanism of fatigue, developing evaluation methods, and establishing a basis for treatment.
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http://dx.doi.org/10.1016/j.nut.2008.07.010DOI Listing
January 2009

Antifatigue effects of coenzyme Q10 during physical fatigue.

Nutrition 2008 Apr 13;24(4):293-9. Epub 2008 Feb 13.

Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan.

Objective: This study examined the effects of coenzyme Q10 administration on physical fatigue.

Methods: In a double-blinded, placebo-controlled, three crossover design, 17 healthy volunteers were randomized to oral coenzyme Q10 (100 or 300 mg/d) or placebo administration for 8 d. As a fatigue-inducing physical task, subjects performed workload trials on a bicycle ergometer at fixed workloads twice for 2 h and then rested for 4 h. During the physical tasks, subjects performed non-workload trials with maximum velocity for 10 s at 30 min (30-min trial) after the start of physical tasks and 30 min before the end of the tasks (210-min trial).

Results: The change in maximum velocity from the 30- to the 210-min trial in the 300-mg coenzyme Q10-administered group was higher than that in the placebo group. In addition, subjective fatigue sensation measured on a visual analog scale in the 300-mg coenzyme Q10-administered group after the fatigue-inducing physical task and recovery period was alleviated when compared with that in the placebo group.

Conclusion: Oral administration of coenzyme Q10 improved subjective fatigue sensation and physical performance during fatigue-inducing workload trials and might prevent unfavorable conditions as a result of physical fatigue.
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http://dx.doi.org/10.1016/j.nut.2007.12.007DOI Listing
April 2008

Effects of oral administration of caffeine and D-ribose on mental fatigue.

Nutrition 2008 Mar;24(3):233-8

Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan.

Objective: We examined the effects of administering two different candidate antifatigue substances, caffeine and D-ribose, on mental fatigue.

Methods: In a double-blinded, placebo-controlled, three-way crossover design, 17 healthy volunteers were randomized to oral caffeine (200 mg/d), D-ribose (2000 mg/d), or placebo for 8 d. As fatigue-inducing mental tasks, subjects performed a 30-min Uchida-Kraepelin psychodiagnostic test and a 30-min advanced trail-making test on four occasions.

Results: During the tasks, the task performance of the caffeine group was better than that of the placebo group. However, after the fatigue-inducing tasks, although subjective perception of fatigue, motivation, or sleepiness was not significantly different, plasma branched-chain amino acid levels in the caffeine group were lower than those of the placebo group. Administration of D-ribose had no effect.

Conclusion: Because plasma branched-chain amino acid levels are decreased by mental fatigue, these results suggest that administration of caffeine improved task performance through the enhancement of central nervous system activity without increasing the sensation of fatigue. However, further decreases in branched-chain amino acid levels indicate that caffeine might promote deeper fatigue than placebo. Unfortunately, research subsequent to our study design has shown that D-ribose dosing higher than we used is needed to see a clinical effect and therefore no conclusions can be made from this study as to the efficacy of D-ribose.
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http://dx.doi.org/10.1016/j.nut.2007.12.002DOI Listing
March 2008

Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate.

PLoS One 2007 Aug 29;2(8):e798. Epub 2007 Aug 29.

Oncotest GmbH, Institute of Experimental Oncology, Freiburg, Germany.

The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000798PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949049PMC
August 2007

Effects of Applephenon and ascorbic acid on physical fatigue.

Nutrition 2007 May;23(5):419-23

Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan.

Objective: We examined the effects of Applephenon and ascorbic acid administration on physical fatigue.

Methods: In a double-blinded, placebo-controlled, three-way crossover design, 18 healthy volunteers were randomized to oral Applephenon (1200 mg/d), ascorbic acid (1000 mg/d), or placebo for 8 d. The fatigue-inducing physical task consisted of workload trials on a bicycle ergometer at fixed workloads for 2 h on two occasions. During the test, subjects performed non-workload trials with maximum velocity for 10 s at 30 min (30-min trial) after the start of the test and 30 min before the end of the test (210-min trial).

Results: The change in maximum velocity between the 30- and 210-min trials was higher in the group given Applephenon than in the group given placebo; ascorbic acid had no effect.

Conclusion: These results suggest that Applephenon attenuates physical fatigue, whereas ascorbic acid does not.
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http://dx.doi.org/10.1016/j.nut.2007.03.002DOI Listing
May 2007

Decrease of hepatic delta-aminolevulinate dehydratase activity in an animal model of fatigue.

Biochem Biophys Res Commun 2007 Feb 28;353(4):1068-73. Epub 2006 Dec 28.

Department of Physiology, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan.

Fatigue can be defined physiologically as inability to maintain the expected power output. At present, no standard of fatigue are yet available. In order to find biomarkers of fatigue, we investigated the level of delta-aminolevulinic acid (ALA), the first intermediate metabolite in the heme biosynthetic pathway, in the plasma and urine of an animal model of fatigue. To prepare fatigued animals, we kept rats for 5 days in a cage filled with water to a height of 1.5 cm. As a result, the plasma and urinary ALA levels were increased in the fatigued animals as compared with those in the control animals. One day after the rats had been returned to their normal cages, these increased levels were restored to the control ones. We also examined the activity of the enzyme ALA dehydratase (ALAD), which is the second enzyme in the heme biosynthetic pathway, and ALAD gene expression during the fatigue and its recovery sessions. The ALAD activity, as well as its gene expression, in the liver of the fatigued animals was decreased as compared with those of the control animals. Both activity and gene expression of ALAD were recovered to their respective control levels after the rats had been allowed to rest in their normal cages for 1 day. Furthermore, the activity of ALA synthase (ALAS), the rate-limiting enzyme in the heme biosynthesis, in the liver was increased after the fatigue session for 5 days. Although this level of increase in the plasma concentration of ALA may not induce fatigue, increase in plasma and urinary ALA levels can be biomarkers of fatigue.
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http://dx.doi.org/10.1016/j.bbrc.2006.12.139DOI Listing
February 2007

Dual gene defects involving delta-aminolaevulinate dehydratase and coproporphyrinogen oxidase in a porphyria patient.

Br J Haematol 2006 Jan;132(2):237-43

Okayama Prefectural University, Japan.

Summary A Caucasian male had symptoms of acute porphyria, with increases in urinary delta-aminolaevulinic acid (ALA), porphobilinogen (PBG) and coproporphyrin that were consistent with hereditary coproporphyria (HCP). However, a greater than expected increase in ALA, compared with PBG, and a substantial increase in erythrocyte zinc protoporphyrin, suggested additional ALA dehydratase (ALAD) deficiency. Nucleotide sequence analysis of coproporphyrinogen oxidase (CPO) cDNA of the patient, but not of the parents, revealed a novel nucleotide transition G835-->C, resulting in an amino acid change, G279R. The mutant CPO protein expressed in Escherichia coli was unstable, and produced about 5% of activity compared with the wild-type CPO. Erythrocyte ALAD activity was 32% of normal in the proband. Nucleotide sequence analysis of cloned ALAD cDNAs from the patient revealed a C36-->G base transition (F12L amino acid change). The F12L ALAD mutation, which was found in the mother and a brother, was previously described, and is known to lack any enzyme activity. This patient thus represents the first case of porphyria where both CPO and ALAD deficiencies were demonstrated at the molecular level.
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http://dx.doi.org/10.1111/j.1365-2141.2005.05852.xDOI Listing
January 2006

Heme-dependent up-regulation of the alpha-globin gene expression by transcriptional repressor Bach1 in erythroid cells.

Biochem Biophys Res Commun 2004 Nov;324(1):77-85

Department of Biotechnology, Kyoto Institute of Technology, Kyoto 606-8585, Japan.

The transcriptional factor Bach1 forms a heterodimer with small Maf family, and functions as a repressor of the Maf recognition element (MARE) in vivo. To investigate the involvement of Bach1 in the heme-dependent regulation of the expression of the alpha-globin gene, human erythroleukemia K562 cells were cultured with succinylacetone (SA), a heme biosynthetic inhibitor, and the level of alpha-globin mRNA was examined. A decrease of alpha-globin mRNA was observed in SA-treated cells, which was restored by the addition of hemin. The heme-dependent expression of alpha-globin occurred at the transcriptional level since the expression of human alpha-globin gene promoter-reporter gene containing hypersensitive site-40 (HS-40) was decreased when K562 cells were cultured with SA. Hemin treatment restored the decrease of the promoter activity by SA. The regulation of the HS-40 activity by heme was dependent on the NF-E2/AP-1 (NA) site, which is similar to MARE. The NA site-binding activity of Bach1 in K562 increased upon SA-treatment, and the increase was diminished by the addition of hemin. The transient expression of Bach1 and mutated Bach1 lacking CP motifs suppressed the HS-40 activity, and cancellation of the repressor activity by hemin was observed when wild-type Bach1 was expressed. The expression of NF-E2 strengthened the restoration of the Bach1-effect by hemin. Interestingly, nuclear localization of Bach1 increased when cells were treated with SA, while hemin induced the nuclear export of Bach1. These results indicated that heme plays an important role in the induction of alpha-globin gene expression through disrupting the interaction of Bach1 and the NA site in HS-40 enhancer in erythroid cells.
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http://dx.doi.org/10.1016/j.bbrc.2004.09.022DOI Listing
November 2004

Heme positively regulates the expression of beta-globin at the locus control region via the transcriptional factor Bach1 in erythroid cells.

J Biol Chem 2004 Feb 1;279(7):5480-7. Epub 2003 Dec 1.

Department of Biotechnology, Kyoto Institute of Technology, Kyoto 606-8585, Japan.

The transcription factor Bach1 heterodimerizes with small Maf proteins to repress Maf recognition element (MARE)-dependent gene expression. The repressor activity of Bach1 is inhibited by the direct binding of heme. To investigate the involvement of Bach1 in the heme-dependent regulation of the expression of the beta-globin gene, mouse erythroleukemia (MEL) cells were cultured with succinylacetone (SA), a specific inhibitor of heme biosynthesis, and the level of beta-globin mRNA was examined. A marked decrease of beta-globin mRNA in SA-treated cells was observed, and this decrease was reversed by the addition of hemin. An iron chelator, desferrioxamine, also lowered the level of beta-globin mRNA. The heme-dependent expression of beta-globin is a transcriptional event since the expression of the human beta-globin gene promoter-reporter gene containing the microlocus control region (microLCR) was inhibited when human erythroleukemia K562 cells and MEL cells were cultured with SA. Hemin treatment restored the decrease in promoter activity caused by SA. The control of the microLCR-beta-globin promoter reporter gene by heme was dependent on DNase I-hypersensitive site 2 (HS2), which contains MARE. The MARE binding activity of Bach1 in K562 and MEL cells increased upon SA treatment, and the increase was diminished by the treatment with hemin. Transient expression of Bach1 suppressed the microLCR activity, and this repressor activity was cancelled by treatment with hemin. The expression of a mutated Bach1 lacking heme-binding sites led to a loss in the heme responsiveness of the microLCR. Furthermore, chromatin immunoprecipitation experiments revealed that Bach1 bound to the MARE of HS2 increased by the treatment of MEL cells with SA, and this was cancelled by hemin. We propose that heme positively regulates the beta-globin gene expression by blocking the interaction of Bach1 with the MARE in the LCR.
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http://dx.doi.org/10.1074/jbc.M302733200DOI Listing
February 2004
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