Biomedical subjects

Y Hayashi

Publications and source records attributed to Y Hayashi.

At least 109 records · Page 6Linked to original sources

PubMed2001

Characterization of three members of murine alpha1,2-fucosyltransferases: change in the expression of the Se gene in the intestine of mice after administration of microbes.

We cloned three members of a GDP-fucose:beta-galactoside alpha1,2-fucosyltransferase (alpha1,2-fucosyltransferase) family, MFUT-I, -II, and -III, from a cDNA of murine small intestine, and determined their enzymatic properties after transfection of the genes into COS-7 cells, and their expression in murine tissues by Northern blotting. MFUT-I, -II, and -III exhibited sequence homology with the human H (78.4%), Se (79.0%), and Sec1 (74.9%) gene products, respectively. COS-7 cells transfected with MFUT-I and -II exhibited alpha1,2-fucosyltransferase activity and the best acceptor substrate for both gene products was GA1 to yield a fucosyl GA1 structure, but no activity was detected in COS-7 cells with MFUT-III. MFUT-II yielded a 3.5-kb mRNA transcript in several tissues, whereas MFUT-I and -III were predominantly expressed in epididymis and testis, respectively. The administration of microbes into germ-free mice resulted in a rapid increase of the MFUT-II gene (Se gene) for the synthesis of fucosyl GA1 in the intestine.

Amino Acid Sequence↗

PubMed2001

Optimal time for predicting myocardial viability after successful primary angioplasty in acute myocardial infarction: a study using myocardial contrast echocardiography.

This study sought to elucidate serial changes in microvascular integrity during papaverine-induced hyperemia in the risk area for myocardial infarction. In addition, we attempted to determine the optimal time for predicting myocardial viability. Seventy-two patients who underwent serial myocardial contrast echocardiography (MCE) before and shortly after (day 1), 1 day (day 2), and 3 weeks (day 21) after recanalization were studied. In 18 of 72 patients, MCE was performed at baseline and during hyperemia using selective intracoronary infusion of papaverine. Both the peak grayscale ratio (PGSR) within the risk area, and the no- and low-reflow ratio (LR ratio) were analyzed in each stage. Left ventricular regional wall motion (RWM) was determined 6 months after recanalization. The correlation coefficient between PGSR with papaverine on day 1 and that on day 2 was 0.54 (p = 0.02); it was 0.50 (p = 0.04) between day 1 and day 21, and 0.82 (p = 0.001) between day 2 and day 21. On day 1, the correlation coefficient between the LR ratio with papaverine and RWM was 0.60 (p = 0.02), which changed to 0.72 (p = 0.003) on day 2 and 0.54 (p = 0.04) on day 21, respectively. The best time to predict viable myocardium was established on day 2 by receiver operating characteristics curves. ST-segment re-elevation, elapsed time from onset to recanalization, and antecedent angina pectoris were independent factors for PGSR on day 2 using stepwise and multiple linear regression analysis. This study suggests that the optimal time to estimate microvascular integrity for predicting myocardial viability might be 1 day after recanalization, which is neither shortly after recanalization nor during the convalescent stage.

Aged↗

PubMed2001

Activation of BMK1 via tyrosine 1062 in RET by GDNF and MEN2A mutation.

Big mitogen-activated protein kinase 1 (BMK1) is a new member of mitogen-activated protein kinase (MAPK) family. In the present study, we investigated whether glial cell line-derived neurotrophic factor (GDNF) can induce activation of BMK1 through RET tyrosine kinase. Its activation reached a maximal level at 30 min and continued at least for 120 min after GDNF stimulation. In addition, we detected BMK1 activation in NIH3T3 cells expressing RET with a multiple endocrine neoplasia (MEN) 2A mutation. The level of BMK1 activation markedly decreased by replacement of tyrosine 1062 with phenylalanine (designated Y1062F) in RET, indicating the importance of downstream signaling via tyrosine 1062. However, although both RAS/MAPK and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways are activated via tyrosine 1062, BMK1 activation by GDNF was not significantly impaired by treatment with an MEK1 inhibitor, PD98059, or two distinct PI3-K inhibitors, LY294002 and wortmannin, suggesting that the RAS and PI3-K signaling pathways are not crucial for BMK1 activation by GDNF. Moreover, luciferase reporter assays revealed that RET-MEN2A mutant proteins can activate the MEF2C transcription factor that is known to be a cellular target for BMK1, and that its activation is impaired by the Y1062F mutation or by expression of a dominant negative form of MEK5.

Animals↗

PubMed2001

Immunohistochemical investigation of caspase-1 and effect of caspase-1 inhibitor in delayed neuronal death after transient cerebral ischemia.

The localization of caspase-1 protein, interleukin-1beta (IL-1beta)-converting enzyme, was immunohistochemically examined in the hippocampal CA-1 subfield by a transient occlusion of bilateral common carotid arteries in Mongolian gerbils. Immunoreactivities for caspase-1 were found in microglias, astrocytes, endothelial cells of capillaries and some non-pyramidal neurons. Immunopositive microglias increased in number from 3 days until 7 days from the transient ischemia, and astrocytes also increased in number from 3 days until 28 days. At the electron microscopic level, caspase-1 immunoreaction endproducts were associated with Golgi apparatus in glial cells, endothelial cells of blood vessels and non-pyramidal neurons. The delayed neuronal death of CA-1 pyramidal cells was significantly protected by the treatment of specific caspase-1 inhibitor (Ac-WEHD-CHO) or broad caspase family inhibitor (z-VAD-FMK). Cell death was protected in a dose dependent manner by the former by 43-57%, and by the latter by 66-91% when injected at 1 and 10 microg, respectively. On the other hand, the protective effect of specific caspase-3 inhibitor (Ac-DMQD-CHO) was less significant at higher dose (10 microg) by 33% (P<0.05), and not detectable at lower dose (1 microg) by 13% (P=0.27). Furthermore, a significant decrease of microglias and astrocytes was found in the CA-1 as well as the reduction of IL-1beta and caspase-1 immunoreactivities by the treatment of Ac-WEHD-CHO. Extravasation of serum albumin was also extremely reduced by this treatment. These findings suggest that the inhibition of caspase-1 activity ameliorates the ischemic injury by inhibiting the activity of IL-1beta.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Activation of the subfornical organ enhances extracellular noradrenaline concentrations in the hypothalamic paraventricular nucleus in the rat.

Experiments were carried out to investigate whether angiotensinergic efferent pathways from the subfornical organ (SFO) regulate the noradrenergic system in the region of the hypothalamic paraventricular nucleus (PVN). Intracerebral microdialysis techniques were utilized to quantify the extracellular content of noradrenaline (NA) in the PVN area. In urethane-anaesthetized male rats, electrical stimulation (5-20 Hz, 600 microA) of the SFO significantly increased the NA concentration in the region of the PVN, and the increase was significantly prevented by pretreatment with the angiotensin II (ANG II) antagonist saralasin (Sar, 5 microg), into the third ventricle (3V). Injections of ANG II (5 microg) into the 3V significantly enhanced NA release in the PVN area. These results suggest that the angiotensinergic pathways from the SFO to the PVN may act to enhance NA release in the region of the PVN.

Angiotensin II↗

PubMed2001

Controlled drug release: new water-soluble prodrugs of an HIV protease inhibitor.

We designed and synthesized a series of highly water-soluble prodrugs of an HIV protease inhibitor, KNI-727 (1), containing tandem-linked two auxiliary units, a solubilizing moiety and a self-cleavable spacer. Prodrugs with an ionized amino group at the solubilizing moiety exhibited a remarkable increase of water-solubility (>10(4) fold) compared to the parent drug 1. These prodrugs released I not enzymatically, but chemically via an intramolecular cyclization-elimination reaction through an imide formation in physiological conditions. Diversified rates of parent drug release were observed when the chemical structure of both the solubilizing and the spacer moieties were modified. This new approach for water-soluble prodrugs will enable to control chemically the release of parent drug as well as to maintain high water-solubility.

Chromatography, High Pressure Liquid↗

PubMed2001

Circadian and photic regulation of MAP kinase by Ras- and protein phosphatase-dependent pathways in the chick pineal gland.

Chick pineal mitogen-activated protein kinase (MAPK) exhibits circadian activation and light-dependent deactivation at nighttime. Here we report that, in the chick pineal gland, levels of active forms of MAPK, MEK, Raf-1 and Ras exhibited synchronous circadian rhythms with peaks during the subjective night, suggesting a sequential activation of components in the classical Ras-MAPK pathway in a circadian manner. In contrast, the light-dependent deactivation of MAPK was not accompanied by any change of MEK activity, but it was attributed to the light-dependent activation of protein phosphatase dephosphorylating MAPK. These results indicate that the photic and clock signals regulate MAPK activity via independent pathways, and suggest a pivotal role of MAPK in photic entrainment and maintenance of the circadian oscillation.

Animals↗

PubMed2001

Sodium nitroprusside-induced seizures and adenosine release in rat hippocampus.

In the present study, we examined the effects of nitric oxide (NO)-related compounds, i.e. sodium nitroprusside (NO donor), diethyldithiocarbamate (NO trapper) and dithiothreitol (superoxide radical scavenger) on release of aspartate and adenosine from rat hippocampus using electrophysiological and microdialysis methods. Perfusion with 0.05 or 0.5 mM sodium nitroprusside significantly reduced high K(+)-evoked release of aspartate during high K(+) perfusion. Perfusion with 0.5 mM sodium nitroprusside always induced seizures and significantly increased release of aspartate and adenosine during washout of sodium nitroprusside. Diethyldithiocarbamate (5 mM) reversed the effects of sodium nitroprusside. Dithiothreitol (1 mM) significantly reduced the increase in adenosine release by sodium nitroprusside. These findings indicate that adenosine release is closely related to development of seizures, which are triggered by an increase in both NO itself and in part peroxynitrite, which results in reaction with superoxide radicals.

Adenosine↗

PubMed2001

Angiotensinergic and noradrenergic mechanisms in the hypothalamic paraventricular nucleus participate in the drinking response induced by activation of the subfornical organ in rats.

The present study was done to investigate the contribution of the hypothalamic paraventricular nucleus (PVN) to the drinking response caused by activation of the subfornical organ (SFO) following angiotensin II (ANG II) injections in the awake rat. Microinjection of ANG II into the SFO elicited the drinking response. Previous injections of either saralasin, an ANG II antagonist, or phentolamine, an alpha-adrenoceptor antagonist, bilaterally into the PVN resulted in the significant attenuation of the drinking response to ANG II. Similar injections of any of the beta-adrenoceptor antagonist timolol, the muscarinic antagonist atropine, or saline vehicle into the PVN had no significant effect on the drinking response. In an attempt to clarify the neural mechanisms in the PVN involved in the drinking response to ANG II injected into the SFO, the effect of microinjection of ANG II into the SFO on noradrenaline (NA) release in the PVN was examined using intracerebral microdialysis techniques. The injection of the ANG II, but not saline vehicle, significantly enhanced the NA release in the region of the PVN. These results indicate the involvement of both the angiotensinergic and alpha-adrenergic systems in the PVN in the drinking response caused by angiotensinergic activation of the SFO, and imply that the angiotensinergic projections from the SFO to the PVN may serve to increase NA release which results in mediating water intake.

Adrenergic alpha-Antagonists↗

PubMed2001

Downregulation of MLL-CBP fusion gene expression is associated with differentiation of SN-1 cells with t(11;16)(q23;p13).

The translocation t(11;16)(q23;p13) has only been documented in patients with acute leukemia or myelodysplasia secondary to therapy with drugs targeting DNA topoisomerase II. We have established a myeloid cell line (SN-1) with the MLL-CBP fusion gene from an acute leukemia patient with t(11;16)(q23;p13). Although SN-1 cells were not induced to differentiate by all-trans retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D(3) (VD3), retinoid X receptor (RXR) agonists, such as 9-cis retinoic acid and Ro48-2250, effectively induced differentiation of the cells. Downregulation of the expression of the MLL-CBP fusion gene occurred during the differentiation of SN-1 cells. When SN-1 cells were treated with MLL-CBP antisense oligonucleotide, the cells were induced to differentiate by ATRA or VD3, suggesting that the MLL-CBP fusion gene dominant-negatively suppresses ATRA- or VD3-induced differentiation. Moreover, suboptimal concentrations of sodium butyrate, a histone deacetylase inhibitor, had a cooperative effect with ATRA or VD3 in inducing the differentiation of SN-1 cells. The downregulation of the expression of MLL-CBP mRNA was accompanied by the induction of differentiation. These findings suggest that RXR agonists or a clinically applicable combination of ATRA and butyrate derivatives might be useful for differentiation therapy in leukemia patients with the MLL-CBP fusion gene.

Alitretinoin↗

PubMed2001

Inhibition of cytochrome c release in Fas-mediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins.

Persistent hepatitis C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Numerous viruses have been reported to escape from apoptotic mechanism to maintain persistent infection. In the present study, we characterized the effect of HCV proteins on the Fas signal using HCV transgenic mice, which expressed core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching system. The transgene expression of HCV transgenic mice caused resistance to Fas antibody stimulated lethality. Apoptotic cell death in the liver of HCV protein expressing mice was significantly reduced compared with nonexpressing mice. Histopathological analysis and DNA fragmentation analysis revealed that the HCV proteins suppressed Fas-mediated apoptotic cell death. To identify the target pathway of HCV proteins, we characterized caspase activity. The activation of caspase-9 and -3/7 but not caspase-8 was inhibited by HCV proteins. Cytochrome c release from mitochondria was inhibited in HCV protein expressing mice. These results indicated that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis and death in mice by repressing the release of cytochrome c from mitochondria, thereby suppressing caspase-9 and -3/7 activation. These results suggest that HCV may cause persistent infection, as a result of suppression of Fas-mediated cell death.

Animals↗

PubMed2001

Immunohistochemical distribution of the receptor for advanced glycation end products in neurons and astrocytes in Alzheimer's disease.

Advanced glycation end products (AGE) and the receptor for AGE (RAGE) have been implicated in the chronic complications of diabetes mellitus (DM), and have been reported to play an important role in the pathogenesis of Alzheimer's disease (AD). In this study, we established a polyclonal anti-RAGE antibody, and examined the immunohistochemical localization of amyloid beta protein (Abeta), AGE, and RAGE in neurons and astrocytes from patients with AD and DM. Our anti-RAGE antibody recognized full-length RAGE (50 kd) and N-terminal RAGE (35 kd) in human brain tissue. Abeta-, AGE-, and RAGE-positive granules were identified in the perikaryon of hippocampal neurons (especially from CA3 and CA4) in all subjects. The distribution and staining pattern of these immunopositive granules showed good concordance with each antibody. In AD, most astrocytes contained both AGE-and RAGE-positive granules and their distribution was almost the same. Abeta-positive granules were less common, but Abeta-, AGE-, and RAGE-positive granules were colocalized in one part of a single astrocyte. In DM patients and control cases, AGE-and RAGE-positive astrocytes were very rare. These finding support the hypothesis that glycated Abeta is taken up via RAGE and is degraded through the lysosomal pathway in astrocytes. In addition to the presence of AGE, the process of AGE degradation and receptor-mediated reactions may contribute to neuronal dysfunction and promote the progression of AD.

Aged↗

PubMed2001

GABAergic modulation of neurons in the nucleus of the solitary tract with ascending projections to the subfornical organ in the rat.

Twenty-five neurons in the region of the nucleus of the solitary tract (NTS) were antidromically activated by electrical stimulation of the subfornical organ (SFO) in male rats under urethane anesthesia. Microiontophoretically applied bicuculline, a gamma-aminobutyric acid (GABA)(A) antagonist, but not phaclofen, a GABA(B) antagonist, attenuated the post-antidromic inhibitory response evoked by SFO stimulation of approximately two-third (n=17) of identified neurons, indicating the existence of recurrent inhibitory systems through GABA(A) receptors. Iontophoretically applied GABA decreased the spontaneous activity of all identified neurons, and the GABA-induced inhibition was prevented by simultaneously applied bicuculline, but not by phaclofen. Activation of peripheral baroreceptors, achieved by rising arterial blood pressure with an intravenous infusions of phenylepherine, suppressed the activity of the majority (n=20) of identified neurons. The inhibitory response of identified neurons (n=7) to baroreceptor activation was partially antagonized by iontophoretically applied bicuculline, but not by phaclofen. These results imply that GABAergic mechanisms may modulate the baroreceptor reflex acting on GABA(A) receptors of NTS neurons with ascending projections to the SFO in the region of the NTS.

Animals↗

PubMed2001

Increased sensitivity to thyroid hormone in mice with complete deficiency of thyroid hormone receptor alpha.

Only three of the four thyroid hormone receptor (TR) isoforms, alpha1, beta1, and beta2, bind thyroid hormone (TH) and are considered to be true TRs. TRalpha2 binds to TH response elements on DNA, but its role in vivo is still unknown. We produced mice completely deficient in TRalpha (TRalpha(o/o)) that maintain normal serum thyroid-stimulating hormone (TSH) concentration despite low serum thyroxine (T(4)), suggesting increased sensitivity to TH. We therefore examined the effects of TH (L-3,3',5-triiodothyronine, L-T3) given to TH-deprived and to intact TRalpha(o/o) mice. Controls were wild-type (WT) mice of the same strain and mice resistant to TH due to deficiency in TRbeta (TRbeta(-/-)). In liver, T3 produced significantly greater responses in TRalpha(o/o) and smaller responses in TRbeta(-/-) as compared with WT mice. In contrast, cardiac responses to L-T3 were absent or reduced in TRalpha(o/o), whereas they were similar in WT and TRbeta(-/-) mice, supporting the notion that TRalpha1 is the dominant TH-dependent TR isoform in heart. 5-Triiodothyronine (L-T3) given to intact mice produced a greater suppression of serum T(4) in TRalpha(o/o) than it did in WT mice and reduced by a greater amount the TSH response to TSH-releasing hormone. This is an in vivo demonstration that a TR deficiency can enhance sensitivity to TH. This effect is likely due to the abrogation of the constitutive "silencing" effect of TRalpha2 in tissues expressing the TRbeta isoforms.

Animals↗

PubMed2001

Elution of bisphenol-A from hemodialyzers consisting of polycarbonate and polysulfone resins.

This study deals with bisphenol-A (BPA) analysis of the BPA-derived polymer pellets, polycarbonate (PC) and polysulfone (PS), and in the hemodialyzer casings made of PC, and the leaching of BPA from commercially available hemodialyzers into water and bovine serum, using HPLC, GC-MS, and LC-MS analyses, and NMR spectroscopy. Total contents of BPA in polymer pellets of each resin were 4.0 and 7.2 microg/g (PC) and 34.5 microg/g (PS). Amounts of BPA released from hemodialyzer PC casings lacking PS hollow-fiber were 11.7 and 13.7 ng/casing by water extraction, and 296 and 345 ng/casing by methanol extraction. On the other hand, BPA of 3.78 to 141.8 ng/module was recovered using water circulation of hemodialyzers, and 140.7 to 2,090 ng/module was detected when bovine serum was used as a circulation solvent. The elution profiles using various concentrations of ethanol/water mixtures indicated that a 17.2% (v/v) ethanol solution rather than bovine serum can be used as an extraction solvent, where a similar amount of BPA as with bovine serum circulation was eluted from the hemodialyzer. Thus, this solvent may be useful for evaluating BPA elution from hemodialyers under similar conditions to medical use.

Animals↗

PubMed2001

The CDCREL1 gene fused to MLL in de novo acute myeloid leukemia with t(11;22)(q23;q11.2) and its frequent expression in myeloid leukemia cell lines.

We report on an adult patient with de novo acute myeloid leukemia (AML) with a t(11;22)(q23;q11.2) involving CDCREL1 and MLL genes. Reverse transcriptase (RT)-polymerase chain reaction (PCR) followed by direct sequencing analysis revealed the MLL-CDCREL1 fusion transcript in his leukemic cells. Analysis of the fusion transcript showed that exon 6 of MLL was fused to exon 4 of CDCREL1, which contains an AT-hook domain of MLL and a GTP binding domain of CDCREL1. To investigate the roles of CDCREL1 further, we examined the expression of the CDCREL1 gene in various cell lines. Expression of CDCREL1 was detected in 11 (85%) of 13 AML cell lines and 3 (21%) of 14 acute lymphoblastic leukemia (ALL) cell lines, but none of 11 EB virus transformed B-cell lines by RT-PCR. The expression rate of CDCREL1 was significantly higher in AML cell lines than in ALL cell lines (P = 0.0035). Platelet glycoprotein 1B beta (GP1B beta), which is located downstream of CDCREL1 and is cotranscribed with CDCREL1 due to a nonconsensus polyadenylation sequence, was expressed in all these cell lines. The higher expression rate of CDCREL1 in AML cell lines than in ALL cell lines suggests that this gene may play some role in myeloid leukemogenesis.

Adult↗

PubMed2001

Coduplication of the MLL and FLT3 genes in patients with acute myeloid leukemia.

Tandem duplication (TD) of the MLL or FLT3 gene in acute myeloid leukemia (AML) has been reported. We examined whether TD of these two genes occurs simultaneously. We analyzed 13 AML and 2 myelodysplastic syndrome patients, including 6 adult patients with trisomy 11 and 9 pediatric patients with TD of the FLT3 gene, using RT-PCR followed by sequencing. Among these, TD of the MLL and FLT3 genes was found in 5 and 10 patients, respectively. Notably, TD of both the MLL and FLT3 genes (coduplication) was detected in two AML patients, who died 6 and 14 months after diagnosis. TD of these two genes in AML is rare; thus, coduplication of these genes in the same patient is predicted to be very rare. Although the mechanisms of TD of both genes are different, development of TD of both genes may be related to an unknown similar etiology in leukemia because the frequency of coduplication of these genes in a single patient is considered to be very low. Further studies of the coduplication of these genes in AML patients may lead to the clarification of its mechanism and clinical implications.

Acute Disease↗

PubMed2001

Homozygous deletion in a neuroblastoma cell line defined by a high-density STS map spanning human chromosome band 1p36.

Recent molecular studies have shown a relatively high rate of loss of heterozygosity (LOH) in neuroblastoma (NB) as well as other types of tumors in human chromosome band 1p36. To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in NB cell lines with PCR according to a high-density sequence tagged site (STS)-content map spanning 1p35-36. Among 25 NB cell lines examined, only one cell line, NB-1, showed no signal with 27 STSs in a 480 kb region in 1p36.2. The sequence analysis has revealed that the defective region included seven known genes (E4, KIF1B, SCYA5, PGD, Cortistatin, DFF45, and PEX14), nine expressed sequence tags (ESTs), and two microsatellite markers. These genes are related to apoptosis, an ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule, and components of a common translocation machinery. The region between the DFF45 and KIF1B genes was defined as homozygous deletion by Southern blotting. The search in LOH regions with high-density STSs may be useful for the isolation and identification of tumor suppressor genes in other tumors as well as NBs.

Child, Preschool↗