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Articles by H Hayashi
Total Records ( 18 ) for H Hayashi
  H Hayashi , H Nakagami , Y Takami , H Koriyama , M Mori , K Tamai , J Sun , K Nagao , R Morishita and Y. Kaneda

Objective— In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs).

Methods and Results— Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers.

Conclusions— FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

  M Asai , K Takeuchi , M Saotome , T Urushida , H Katoh , H Satoh , H Hayashi and H. Watanabe

Hypoxia, ischaemia, and exogenous chemicals can induce extracellular and intracellular acidosis, but it is not clear which of these types of acidosis affects endothelial cell function. The synthesis and release of endothelium-derived relaxing factors (EDRFs) are linked to an increase in cytosolic Ca2+ concentration, and we therefore examined the effects of extracellular and intracellular acidosis on Ca2+ responses and EDRF production in cultured porcine aortic endothelial cells.

Methods and results

Cytosolic pH (pHi) and Ca2+ were measured using fluorescent dyes, BCECM/AM (pH-indicator) and fura-2/AM (Ca2+-indicator), respectively. EDRFs, nitric oxide (NO) and prostaglandin I2 (PGI2) were assessed using DAF-FM/DA (NO-indicator dye) fluorometry and 6-keto PGF1 enzyme immunoassay, respectively. HEPES buffers titrated to pH 6.4, 6.9, and 7.4 were used to alter extracellular pH (pHo), and propionate (20 mmol/L) was applied to cause intracellular acidosis. Extracellular acidosis strongly suppressed bradykinin (BK, 10 nmol/L)- and thapsigargin (TG, 1 µmol/L)-induced Ca2+ responses by 30 and 23% at pHo 6.9, and by 80 and 97% at pHo 6.4, respectively. During the examinations, there were no significant differences in pHi among the three groups at pHo 7.4, 6.9, and 6.4. Extracellular acidosis also inhibited BK-stimulated PGI2 production by 55% at pHo 6.9 and by 77% at pHo 6.4, and NO production by 38% at pHo 6.9 and by 91% at pHo 6.4. The suppressive effects of extracellular acidosis on Ca2+ responses and NO production were reversible. Propionate changed pHi from 7.3 to 6.9, without altering pHo (7.4). Intracellular acidosis had no effect on BK- and TG-induced Ca2+ responses or NO production.


These results indicate that extracellular, but not intracellular, acidosis causes endothelial dysfunction by inhibiting store-operated Ca2+ entry, so helping to clarify the vascular pathophysiology of conditions such as ischaemia, hypoxia, acidosis, and ischaemia-reperfusion.

  H Itoh , T Sakaguchi , W. G Ding , E Watanabe , I Watanabe , Y Nishio , T Makiyama , S Ohno , M Akao , Y Higashi , N Zenda , T Kubota , C Mori , K Okajima , T Haruna , A Miyamoto , M Kawamura , K Ishida , I Nagaoka , Y Oka , Y Nakazawa , T Yao , H Jo , Y Sugimoto , T Ashihara , H Hayashi , M Ito , K Imoto , H Matsuura and M. Horie

Background— Drugs with IKr-blocking action cause secondary long-QT syndrome. Several cases have been associated with mutations of genes coding cardiac ion channels, but their frequency among patients affected by drug-induced long-QT syndrome (dLQTS) and the resultant molecular effects remain unknown.

Methods and Results— Genetic testing was carried out for long-QT syndrome–related genes in 20 subjects with dLQTS and 176 subjects with congenital long-QT syndrome (cLQTS); electrophysiological characteristics of dLQTS-associated mutations were analyzed using a heterologous expression system with Chinese hamster ovary cells together with a computer simulation model. The positive mutation rate in dLQTS was similar to cLQTS (dLQTS versus cLQTS, 8 of 20 [40%] versus 91 of 176 [52%] subjects, P=0.32). The incidence of mutations was higher in patients with torsades de pointes induced by nonantiarrhythmic drugs than by antiarrhythmic drugs (antiarrhythmic versus others, 3 of 14 [21%] versus 5 of 6 [83%] subjects, P<0.05). When reconstituted in Chinese hamster ovary cells, KCNQ1 and KCNH2 mutant channels showed complex gating defects without dominant negative effects or a relatively mild decreased current density. Drug sensitivity for mutant channels was similar to that of the wild-type channel. With the Luo-Rudy simulation model of action potentials, action potential durations of most mutant channels were between those of wild-type and cLQTS.

Conclusions— dLQTS had a similar positive mutation rate compared with cLQTS, whereas the functional changes of these mutations identified in dLQTS were mild. When IKr-blocking agents produce excessive QT prolongation (dLQTS), the underlying genetic background of the dLQTS subject should also be taken into consideration, as would be the case with cLQTS; dLQTS can be regarded as a latent form of long-QT syndrome.

  T Fujita , M Nakano , J Ohtani , T Kawata , M Kaku , M Motokawa , N Tsuka , H Hayashi and K. Tanne

The present study was designed to examine the expression of Sox 9 and type II and X collagens in regenerated condyle resulting from the use of a functional appliance. Ninety, 3-week-old, mice were divided equally into the following groups: two experimental groups (condylectomy group and condylectomy with functional appliance group) and the corresponding control group. In the condylectomy group, a unilateral condylectomy was performed on the right side. In the condylectomy with appliance group, the mandible was repositioned in a forward direction using a functional appliance after unilateral condylectomy. The expression of Sox 9 and type II and X collagens in the condyle was determined immunohistochemically 4 weeks after surgery.

In mice with a condylectomy, the expression was minimal. On the other hand, these factors were highly expressed in the condylectomized side with the appliance. It is thus speculated that cartilaginous regeneration is due to the expression of chondrogenic factors, such as Sox 9 and type II and X collagens. It is also suggested that condyle regeneration results from an optimal intra-articular environment with appropriate joint spaces achieved by condylar repositioning.

  T Taira , H Hayashi , Y Tajiri , S Onaga , G. i Uechi , H Iwasaki , T Ohnuma and T. Fukamizo

Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)3 from the substrate (GlcNAc)6 through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

  N Ohoka , S Kato , Y Takahashi , H Hayashi and R. Sato

The nuclear receptor-type transcription factor retinoic acid receptor-related orphan receptor (ROR) is a multifunctional molecule involved in tissue development and cellular function, such as inflammation, metabolism, and differentiation; however, the role of ROR during adipocyte differentiation has not yet been fully understood. Here we show that ROR inhibits the transcriptional activity of CCAAT/enhancer-binding protein β (C/EBPβ) without affecting its expression, thereby blocking the induction of both PPAR and C/EBP, resulting in the suppression of C/EBPβ-dependent adipogenesis. ROR interacted with C/EBPβ so as to repress both the C/EBPβ-p300 association and the C/EBPβ-dependent recruitment of p300 to chromatin. In addition to the inhibitory effect on C/EBPβ function, ROR also prevents the expression of the lipid droplet coating protein gene perilipin by peroxisome proliferators-activated receptor (PPAR), acting through the specific mechanism of its promoter. We identified a suppressive ROR-responsive element overlapping the PPAR-responsive element in the perilipin promoter and verified that ROR competitively antagonizes the binding of PPAR. ROR inhibits PPAR-dependent adipogenesis along with the repression of perilipin induction. These findings suggest that ROR is a novel negative regulator of adipocyte differentiation that acts through dual mechanisms.

  M Ohshima , K Inoue , H Hayashi , D Tsuji , M Mizugaki and K. Itoh

DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m5dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were contructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of light chains. The scFv library was selected against m5Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m5Cyd-BSA. Two scFvs with high reactivity for m5Cyd-BSA termed 1–2 and 1–12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1–2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1–2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1–2 was similar to scFv 1–2, and thus, AcGFP-scFv 1–2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.

  H Hayashi , K Suruga and Y. Yamashita

SLC26A3, a Cl/HCO3 exchanger, is highly expressed in intestinal epithelial cells, and its mutations cause congenital chloride diarrhea. This suggests that SLC26A3 plays a key role in NaCl absorption in the intestine. Electroneutral NaCl absorption in the intestine is mediated by functional coupling of the Na+/H+ exchanger and Cl/HCO3 exchanger. It is proposed that the coupling of these exchangers may occur as a result of indirect linkage by changes of intracellular pH (pHi). We therefore investigated whether SLC26A3 is regulated by pHi. We generated a hemagglutinin epitope-tagged human SLC26A3 construct and expressed it in Chinese hamster ovary cells. Transport activities were measured with a fluorescent chloride-sensitive dye dihydro-6-methoxy-N-ethylquinolinium iodide (diH-MEQ). pHi was clamped at a range of values from 6.0 to 7.4. We monitored the transport activity of SLC26A3 by reverse mode of Cl/HCO3 and Cl/NO3 exchange. None of these exchange modes induced membrane potential changes. At constant external pH 7.4, Cl/HCO3 exchange was steeply inhibited with pHi decrease between 7.3 and 6.8 as opposed to thermodynamic prediction. In contrast, however, Cl/NO3 exchange was essentially insensitive to pHi within physiological ranges. We also characterized the pHi dependency of COOH-terminal truncation mutants. Removal of the entire COOH-terminal resulted in decrease of the transport activity but did not noticeably affect pHi sensitivity. These results suggest that Cl/HCO3 exchange mode of human SLC26A3 is controlled by a pH-sensitive intracellular modifier site, which is likely in the transmembrane domain. These observations raise the possibility that SLC26A3 activity may be regulated via Na+/H+ exchanger 3 (NHE3) through the alteration of pHi under physiological conditions.

  Y Itoh , N Hatano , H Hayashi , K Onozaki , K Miyazawa and K. Muraki

The activation of a vanilloid type 4 transient receptor potential channel (TRPV4) has an obligatory role in regulation of intracellular Ca2+ (Ca2+i) in several types of cells including vascular and sensory organs. In this study, we provide evidence that TRPV4 is a functional regulator of Ca2+i in human synoviocytes. Although significant expression of TRPV4 in synoviocytes from patients with (RA) and without (CTR) rheumatoid arthritis was detected at mRNA and protein level, those in the human fibroblast-like synoviocyte line MH7A were rather lower. Consistently, the selective TRPV4 agonist 4-phorbol 12,13-didecanoate (4PDD) effectively elevated Ca2+i in the RA and CTR cells, which was abolished by the removal of external Ca2+. Moreover, the elevation was inhibited by ruthenium red, a blocker of TRPVs. In MH7A cells transfected with human TRPV4 (MH7A-V4), 4PDD elevated the Ca2+i in a similar manner to those in the RA and CTR cells. Electrophysiological analysis also revealed that 4PDD activated nonselective cationic currents in RA cells. Application of 227 mosM solution to the RA and MH7A-V4 cells elevated their Ca2+i, but this does not occur when it was applied to MH7A cells. Treatment of RA but not MH7A cells with 4PDD for 24 h reduced their production of IL-8. These results suggest that an environmental sensor, TRPV4, is a novel regulator of intracellular Ca2+ in human synoviocytes.

  F Ishikawa , T Akimoto , H Yamamoto , Y Araki , T Yoshie , K Mori , H Hayashi , K Nose and M. Shibanuma

Mitochondrial dysfunction, in particular, interference in the respiratory chain, is often responsible for the toxicogenic effects of xenobiotics. In this study, changes in gene expression resulting from pharmacological inhibition of the respiratory chain were studied by DNA microarray analysis using cells treated with rotenone or antimycin A, which inhibit complexes I and III of the electron transport system, respectively. Forty-eight genes were either up- or down-regulated more than 3-fold. These included stress- and/or metabolic-related effector genes and several transcriptional regulators represented by CHOP-10. Further study using siRNA showed that among the four genes studied, up-regulation of three was dependent on CHOP-10. C/EBPβ, a dimerizing partner of CHOP-10, was also involved in two of the three genes including Trib3, implying that CHOP-10, heterodimerizing with C/EBPβ or another partner played a key role in the expression of a set of genes under stress. Although CHOP-10 and Trib3 were both ER-stress response genes, signal inducing Trib3 during mitochondrial stress was distinct from that during ER stress. Cytotoxicity caused by inhibition of the respiratory chain was attenuated by treatment with siRNA for CHOP-10. This study demonstrated the importance of CHOP-10 in coordinating individual gene expression in response to the mitochondrial stress.

  M Ohshima , T Tadakuma , H Hayashi , K Inoue and K. Itoh

We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m5dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly4-Ser)3. The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m5dCyd and weakly with 5-methylcytidine (m5Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC50 0.054 µg/ml) was ~80 times higher than that of the parent MoAb (IC50 4.27 µg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m5dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained ~1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

  H Hayashi , S Tanase and T. Yagi

Esmond E. Snell (1914–2003) was a giant of B-vitamin and enzyme research. His early research in bacterial nutrition had lead to the discovery of vitamins such as lipoic acid and folic acid, and an anti-vitamin avidin. He developed microbiological assay methods for riboflavin and other vitamins and amino acids, which are still used today. He also investigated the metabolism of vitamins, discovered pyridoxal and pyridoxamine as the active forms of vitamin B6 and revealed the mechanism of transamination and other reactions catalysed by vitamin B6 enzymes. His research in later years on pyruvoyl-dependent histidine decarboxylase unveiled the biogenesis mechanism of this first built-in cofactor. Throughout his career, he was a great mentor of many people, all of whom are inspired by his philosophy of science.

  M Koivusalo , C Welch , H Hayashi , C. C Scott , M Kim , T Alexander , N Touret , K. M Hahn and S. Grinstein

Inhibitors of Na+/H+ exchange proteins block macropinocytosis by lowering the pH near the plasma membrane, which in turn inhibits actin remodeling by Rho family GTPases.

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