Tsuyoshi Tahara

Heme Positively Regulates the Expression of β-Globin at the Locus Control Region via the Transcriptional Factor Bach1 in Erythroid Cells

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 β-globin gene, mouse erythroleukemia (MEL) cells were cultured with succinylacetone (SA), a specific inhibitor of heme biosynthesis, and the level of β-globin mRNA was examined. A marked decrease of β-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 β-globin mRNA. The heme-dependent expression of β-globin is a transcriptional event since the expression of the human β-globin gene promoter-reporter gene containing the microlocus control region (μLCR) 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 μLCR-β-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 μLCR 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 μLCR. 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 β-globin gene expression by blocking the interaction of Bach1 with the MARE in the LCR.

Antifatigue effects of coenzyme Q10 during physical fatigue.

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.

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

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, Oncotests 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.

Mental and physical fatigue-related biochemical alterations.

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.

A Combined 6π-Azaelectrocyclization/Staudinger Approach to Protein and Cell Engineering: Noninvasive Tumor Targeting by N-Glycan-Engineered Lymphocytes

Graphical Abstract Combined azaelectrocyclization and Staudinger ligation allowed proteins and living cells to be modified by small molecules (i.e., biotin or N-glycans). Chemically engineered lymphocytes modified by complex-type N-glycan targeted DLD-1 tissues implanted in nude mice at the whole-body level.

Daily oral administration of crocetin attenuates physical fatigue in human subjects

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.

In Vivo Gold Complex Catalysis within Live Mice

Metal complex catalysis within biological systems is largely limited to cell and bacterial systems. In this work, a glycoalbumin-AuIII 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.

Electrocyclization-Based Labeling Allows Efficient In Vivo Imaging of Cellular Trafficking

Covalent chemical labeling of living cells has garnered significant attention in the fields related to molecular imaging due to direct and easy operations, and broad and general applicability to both in vitro and in vivo studies, as well as the smaller size of the chemical labels. Undesirable modification of key functions on cell surfaces should be avoided in order to retain the native functions of the cell. Therefore, bioorthogonal approaches, which can be combined with biological techniques, have actively been investigated. Successful examples include Bertozzi’s Staudinger ligation and the strain-accelerated Huisgen 1,3-cycloaddition reaction. These methods have been applied to the labeling of cells expressing oligosaccharides with azido-containing sugar residues on the cell surfaces through biosynthetic pathways, which subsequently reacted with the methoxycarbonyl phenyldiphenylphosphine or cyclooctyne derivatives. In contrast, as a “non-bioorthogonal approach”, we recently developed a new lysine-based technique for the labeling of peptides and proteins (antibodies), which is based on a rapid 6p-azaelectrocyclization. 12] We have used this method to efficiently and selectively introduce both DOTA (1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid), as a metal chelating agent (either for magnetic resonance imaging (MRI), positron emission tomography (PET), or other radiopharmaceutical purposes), and fluorescent groups to lysine residues via a reaction with unsaturated aldehyde probes (such as probes 1 a and 1 b in Scheme 1) at very low concentrations (~10nm) within a short time (10–30 min) at room temperature. Furthermore, DOTA-labeled somatostatin and glycoproteins have subsequently been radiometalated with Ga, and their receptormediated accumulation successfully visualized by PET. Our new method precisely controls the introduction of DOTA or fluorescence labels onto lysine residues in target proteins by adjusting the reaction concentration so that the activity of the biomolecules is retained. The efficiency of our rapid azaelectrocyclization protocol depends on the steric accessibility of the primary amino groups. Reactions with internal lysine residues of the tertiary protein structures, as well as the N-terminal amine (amine with a substitution on the a-carbon), are very slow (>5 h at 24 8C), while lysines at protein surfaces react rapidly (10–30 min at 24 8C); therefore, labeling occurs preferentially at these positions. 12] Site-selective labeling of the target protein has also been achieved by directing reactive groups (unsaturated aldehydes) to a specific site using a smallmolecule ligand of the protein so that the decrease in activity is suppressed to a minimum when lysines, which are critical for receptor binding, are situated at the most accessible site of a target protein. Dihydropyridines as the electrocyclization products, which retain the cationic charges as those of the inherent lysines, might also contribute to the retention of the protein activity. Based on previous results, we speculated that our azaelectrocyclization chemistry could covalently and selectively label most of the accessible amino groups on cell constituents via a one-step procedure under mild and physiological conditions, i.e. , lysines of membrane proteins and/or ethanol amine derivatives with minimal interference with their native functions. Herein, we report direct labeling of a whole cell through azaelectrocyclization chemistry under extremely diluted concentrations. While the conventional chemical approach, such as the succinimidyl ester method, causes a significant internalization of the reagents leading to cytotoxicity, the present azaelectrocyclization protocol tightly anchors the fluorescence labels on the cell surface. An investigation of the in vivo imaging of the fluorescence-labeled lymphocytes was also performed as an application of the present protocol; the trafficking of the cells into the immune-related organs was clearly visualized with markedly high image contrast. Although we previously established a preparation method for unsaturated aldehyde probes, such as 1 a and 1 b (Scheme 1), via the oxidation of stable and therefore storable alcohol precursors 2 a, b on a 5–20 mg scale in a reaction flask, such a specialized procedure can only be performed in synthetic laboratories. Hence, for a broader and more general application of labeling reagents to biology-directed research, it is desirable to develop an easy-to-use oxidation (activation) method, such as a kit-like procedure, which enables the in situ generation of the unstable aldehydes that are otherwise difficult to handle, and that can be performed even on a microgram scale. Therefore, an Eppendorf tube protocol was established as shown in Scheme 1 (see the Experimental Section for details). Using a simplified Eppendorf tube procedure, probes such as 1 a and 1 b, as well as other unsaturated aldehyde probes previously developed in our laboratory, can be [a] Dr. K. Tanaka, K. Minami, Y. Fujii, Dr. E. R. O. Siwu, Prof. Dr. K. Fukase Department of Chemistry, Graduate School of Science, Osaka University 1-1 Machikaneyama-cho, Toyonaka-shi, Osaka 560-0043 (Japan) Fax: (+ 81) 6-6850-5419 E-mail : ktzenori@chem.sci.osaka-u.ac.jp koichi@chem.sci.osaka-u.ac.jp [b] Dr. T. Tahara, Dr. S. Nozaki, Dr. H. Onoe, Prof. Dr. Y. Watanabe RIKEN Center for Molecular Imaging Science 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe-shi, Hyogo 650-0047 (Japan) [c] S. Yokoi, K. Koyama Kishida Chemical Co. , Ltd. 14-10 Technopark, Sanda-shi, Hyogo 669-1339 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000027.

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

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.

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

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.