Biomedical subjects

Takeshi Hayashi

Publications and source records attributed to Takeshi Hayashi.

At least 19 recordsLinked to original sources

PubMed2007

Priming of centromere for CENP-A recruitment by human hMis18alpha, hMis18beta, and M18BP1.

The centromere is the chromosomal site that joins to microtubules during mitosis for proper segregation. Determining the location of a centromere-specific histone H3 called CENP-A at the centromere is vital for understanding centromere structure and function. Here, we report the identification of three human proteins essential for centromere/kinetochore structure and function, hMis18alpha, hMis18beta, and M18BP1, the complex of which is accumulated specifically at the telophase-G1 centromere. We provide evidence that such centromeric localization of hMis18 is essential for the subsequent recruitment of de novo-synthesized CENP-A. If any of the three is knocked down by RNAi, centromere recruitment of newly synthesized CENP-A is rapidly abolished, followed by defects such as misaligned chromosomes, anaphase missegregation, and interphase micronuclei. Tricostatin A, an inhibitor to histone deacetylase, suppresses the loss of CENP-A recruitment to centromeres in hMis18alpha RNAi cells. Telophase centromere chromatin may be primed or licensed by the hMis18 complex and RbAp46/48 to recruit CENP-A through regulating the acetylation status in the centromere.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Vascular endothelial growth factor promotes brain tissue regeneration with a novel biomaterial polydimethylsiloxane-tetraethoxysilane.

In the brain after infarction or trauma, the tissue eventually becomes pannecrotic and forms a cavity. In such situations, a scaffold is necessary for the implanted or migrated cells to produce new tissue. In this present study, therefore, we attempted to restore brain tissue using a novel biomaterial, polydimethylsiloxane-tetraethoxysilane (PDMS-TEOS) hybrid with or without vascular endothelial growth factor (VEGF), which is crucial for new vessel formation. When PDMS-TEOS scaffold was implanted into the artificial brain defect, it remained at the implanted site and kept the integrity of the brain shape. At 30 days after the implantation, the marginal territory of PDMS-TEOS scaffold became occupied by newly formed tissue. Immunohistochemical analysis revealed that the new tissue was constituted by astrocytes and endothelial cells. Addition of VEGF increased the newly produced tissue volume, and the immunohistochemical analysis showed that the numbers of astrocytes and endothelial cells were increased. Double staining with proliferation maker Ki67 demonstrated that VEGF significantly increased newly formed astrocytes and endothelial cells, indicating that addition of VEGF accelerated tissue restoration and angiogenesis. These findings show that implantation of PDMS-TEOS scaffold with VEGF might be effective for treating old brain infarction or trauma.

Animals↗

PubMed2006

Effect of modification of ovary preservation solution by adding glucose on the maturation and development of pig oocytes after prolonged storage.

Oocytes lose their developmental competence during prolonged storage of the ovary. In the present study, we supplemented the preservation solution for pig ovaries (phosphate buffered saline, PBS) with glucose and preserved the ovaries for 6 h at 25 C. Subsequently, we examined the glucose concentration of the follicular fluid (FF), pH of the FF, survival rate of the granulosa cells, and maturation and developmental competence of oocytes after storage. During storage, the glucose concentration of the FF (2.1 mM), pH of the FF (7.4), and survival rate of the granulosa cells (69.5%) rapidly decreased (glucose concentration: under 1.1 mM; pH: 6.8; and survival rate: 43%). On the other hand, when the preservation solution was supplemented with glucose (15 mM), the glucose concentration of the FF increased and the survival rate of the granulosa cells improved, although the pH of the FF decreased further (from 6.8 to 6.6). In addition, supplementation with glucose significantly improved the rates of oocytes at metaphase II (0 h: 65.0%; 6 h without glucose: 23.8%; and 6 h with glucose: 43.8%) and attenuated the decline in the rates of fertilization and development that resulted from prolonged storage, although there were no significant differences. In conclusion, modification of the preservation solution by the addition of glucose increased the glucose concentration of the FF and improved the rate of maturation of pig oocytes.

Animals↗

PubMed2006

Distribution of inducible nitric oxide synthase and cell proliferation in rat brain after transient middle cerebral artery occlusion.

Nitric oxide (NO) can be neuroprotective or neurotoxic during cerebral ischemia, depending on the NO synthase (NOS) isoform involved. In addition to neurotoxic effect in ischemic brain, inducible NOS (iNOS) also adversely affect ischemic outcome by blocking neurogenesis. In the present study, therefore, we studied the chronological and spatial change of the distribution of iNOS and cell proliferation in subventricular zone (SVZ) after transient focal cerebral ischemia. After 90 min of transient middle cerebral artery occlusion (tMCAO), iNOS-positive cells decreased in the ischemic core at 1 to 21 days, and increased in the ipsilateral periischemic area at 1 and 3 days. 5-Bromodeoxyuridine (BrdU)-positive cells appeared in the ischemic core at 3 to 21 days, appeared in the periischemic area at 3 and 7 days, and increased in the ipsilateral SVZ at 7 days. ED-1-positive cells appeared in the ischemic core at 3 to 21 days, and some of them were double positive with BrdU or iNOS, but the majority were BrdU-negative. The present study suggests that astrocytes are born within the periischemic area at early stage after tMCAO and migrate from SVZ into periischemic area at later stage, and that time-dependent and spatial changes of iNOS expression may be involved in the proliferation and differentiation of adult neurogenesis after focal cerebral ischemia.

Animals↗

PubMed2006

Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation.

The reiterated nature of histone genes has hampered genetic approach to dissect the role of histones in chromatin dynamics. We here report isolation of three temperature-sensitive (ts) Schizosaccharomyces pombe strains, containing amino-acid substitutions in the sole histone H2B gene (htb1+). The mutation sites reside in the highly conserved, non-helical residues of H2B, which are implicated in DNA-protein or protein-protein interactions in the nucleosome. In the allele of htb1-72, the substitution (G52D) occurs at the DNA binding loop L1, causing disruption of the gene silencing in heterochromatic regions and lagging chromosomes in anaphase. In another allele htb1-223 (P102L) locating in the junction between alpha3 and alphaC, the mutant residue is in contact with H2A and other histones, leading to structural aberrations in the central centromere chromatin and unequal chromosome segregation in anaphase. The third allele htb1-442 (E34K) near alpha1 displayed little defect. Evidence is provided that monoubiquitinated H2B is greatly unstable in P102L mutant, possibly owing to proteasome-independent destruction or enhanced deubiquitination. Histone H2B thus plays an important role in centromere/kinetochore formation.

Amino Acid Sequence↗

PubMed2006

Implantation of a new porous gelatin-siloxane hybrid into a brain lesion as a potential scaffold for tissue regeneration.

For brain tissue regeneration, any scaffold for migrated or transplanted stem cells with supportive angiogenesis is important once necrotic brain tissue has formed a cavity after injury such as cerebral ischemia. In this study, a new porous gelatin-siloxane hybrid derived from the integration of gelatin and 3-(glycidoxypropyl) trimethoxysilane was implanted as a three-dimensional scaffold into a defect of the cerebral cortex. The porous hybrid implanted into the lesion remained at the same site for 60 days, kept integrity of the brain shape, and attached well to the surrounding brain tissues. Marginal cavities of the scaffolds were occupied by newly formed tissue in the brain, where newly produced vascular endothelial, astroglial, and microglial cells were found with bromodeoxyuridine double positivity, and the numbers of those cells were dose-dependently increased with the addition of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Extension of dendrites was also found from the surrounding cerebral cortex to the newly formed tissue, especially with the addition of bFGF and EGF. The present study showed that a new porous gelatin-siloxane hybrid had biocompatibility after implantation into a lesion of the central nervous system, and thus provided a potential scaffold for cell migration, angiogenesis and dendrite elongation with dose-dependent effects of additive bFGF and EGF.

Animals↗

PubMed2006

Modulation of proline-rich akt substrate survival signaling pathways by oxidative stress in mouse brains after transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: A proline-rich Akt substrate (PRAS) contributes to the regulation of apoptosis after a variety of cell death stimuli, as well as in an in vivo transient focal cerebral ischemia (tFCI) model. We reported previously that overexpression of copper/zinc-superoxide dismutase (SOD1) reduces apoptotic cell death after tFCI. Our present study was designed to clarify the relationship between the PRAS signaling pathway and oxidative stress in the regulation of apoptosis after tFCI. METHODS: We used a tFCI model with SOD1 transgenic mice and wild-type littermates to examine the expression of phosphorylated PRAS (pPRAS) by Western blotting and immunohistochemistry and the interaction of pPRAS with phosphorylated Akt (pPRAS/pAkt) or the 14-3-3 protein (pPRAS/14-3-3) by coimmunoprecipitation. Direct oxidation of the carbonyl groups, an indication of oxidative injury to total and individual proteins caused by tFCI, was examined using a 2,4-dinitrophenylhydrazone reaction assay. RESULTS: Expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 decreased 2 hours after tFCI. Oxidized hydroethidine did not colocalize with expression of pPRAS. Individual oxidized carbonyls in pPRAS remarkably increased 2 hours after tFCI but were significantly reduced by SOD1 2 hours after tFCI. Expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 was promoted by SOD1 during the same time course. CONCLUSIONS: These results suggest that overexpression of SOD1 may affect the PRAS pathway after tFCI by reducing the direct oxidative reaction to pPRAS after reperfusion injury.

14-3-3 Proteins↗

PubMed2006

Regulatory role of heme oxygenase 1 in inflammation of rheumatoid arthritis.

OBJECTIVE: To examine the expression and pathogenetic roles of heme oxygenase 1 (HO-1), an inducible heme-degrading enzyme with antiinflammatory properties, in rheumatoid arthritis (RA). METHODS: HO-1 expression in synovial tissue from patients with RA, patients with osteoarthritis, and patients with noninflammatory joint diseases was determined by immunoblotting and immunohistochemistry. Effects of various agents, such as hemin (a chemical inducer of HO-1), small interfering RNA (siRNA) specific for HO-1, HO-1 expression vector, and antirheumatic agents, on HO-1 expression in RA synovial cell lines were analyzed by real-time reverse transcription-polymerase chain reaction (PCR) and immunoblotting. Cytokine synthesis was evaluated by real-time PCR and enzyme-linked immunosorbent assay. RESULTS: HO-1 was expressed more abundantly in the lesions of synovial tissue from patients with RA than in those from the other patient groups. Hemin, auranofin, and HO-1 expression vector induced HO-1 and reduced expression of tumor necrosis factor alpha (TNFalpha) messenger RNA, lipopolysaccharide (LPS)-induced secretion of interleukin-6 (IL-6) and IL-8, and expression of cyclooxygenase 2 in the synovial cell lines. Treatment with HO-1-specific siRNA augmented the synthesis of TNFalpha, IL-6, and IL-8 and canceled the suppressive effects of auranofin on TNFalpha secretion. When hemoglobin, as a scavenger of carbon monoxide, was added to auranofin-treated synovial cell lines, LPS-dependent production of IL-6 and IL-8 was increased. CONCLUSION: Our data demonstrate that HO-1 is expressed in RA synovial tissues and plays a regulatory role in the development of inflammation. The pharmacologic effects of auranofin depend, in part, on the levels of HO-1, suggesting that HO-1 induction is a novel therapeutic strategy for RA.

Arthritis, Rheumatoid↗

PubMed2006

Temporal profile of neural stem cell proliferation in the subventricular zone after ischemia/hypoxia in the neonatal rat brain.

OBJECTIVES: Ischemia/hypoxia (I/H) causes severe neonatal brain injury, such as periventricular leukomaracia and hypoxic/ischemic encephalopathy. Neural stem cell research could lead to a treatment for such disorders. In order to elucidate the dynamic changes in neural stem cells in the neonatal brain after I/H, we investigated the proliferation of new cells in the subventricular zone (SVZ). METHODS: Seven-day-old Wister rats were subjected to ligation of the left carotid artery followed by 2 hours of hypoxic stress (8% O(2) and 92% N(2), at 33 degrees C). In order to elucidate the dynamic change of neural stem cells in the SVZ, single bromodeoxyuridine (BrdU; 50 mg/kg) was administered 2 hours before death 1, 7, 14 and 21 days after I/H. Immunohistochemical and immunofluorescent studies for BrdU and doublecortin (DCX) were carried out. As a control, a group of rats was subjected to sham surgery (incision of skin, but no ligation of the carotid artery) and no I/H. RESULTS: The numbers of BrdU-labeled cells in the SVZ, for both the ipsilateral side and the contralateral side of the I/H brain, were twice the level of the control at 7 days after I/H, but the numbers for both sides returned to the control level at 21 days. In the ipsilateral side of the I/H brain, the number of BrdU-labeled cells in the SVZb (lining the upper wall of lateral ventricle) was 4-fold at 7 days and 15-fold at 21 days after I/H compared with the control level. This chronological pattern is very similar to the pattern for I/H results of the posterior periventricle (pPV). DCX appeared in most BrdU-labeled cells in the SVZb and pPV. DISCUSSION: These findings indicate that I/H enhances neural stem cell proliferation in the SVZ, and some newborn cells migrate as neural precursors to the SVZb and pPV after I/H in the neonatal rat brain.

Animals↗

PubMed2006

Cerebral ischemia and angiogenesis.

Angiogenesis occurs in a wide range of conditions. As ischemic tissue usually depends on collateral blood flow from newly produced vessels, acceleration of angiogenesis should be of therapeutic value to ischemic disorders. Indeed, therapeutic angiogenesis reduced tissue injury in myocardial or limb ischemia. In ischemic stroke, on the other hand, angiogenic factors often increase vascular permeability and thus may deteriorate tissue damage. In order to apply safely the therapeutic angiogenesis for ischemic stroke treatment, elucidating precise mechanism of brain angiogenesis is mandatory. In the present article, we review previous reports which investigated molecular mechanisms of angiogenesis. Endothelial cell mitogens, enzymes that degrade surrounding extracellular matrix, and molecules implicated in endothelial cells migration are induced rapidly in the ischemic brain. Their possible neuroprotective or injury exacerbating effects are discussed. Because therapeutic potential of angiogenic factors application had gained much attention, we here extensively reviewed relevant previous reports. In the future however, there is a need to consider angiogenesis in relation with regenerative medicine, as angiogenic factors sometimes possess neuron producing property.

Angiogenic Proteins↗

PubMed2006

Molecular mechanisms of ischemic neuronal cell death--with relevance to Alzheimer's disease.

There are many similarities in molecular mechanisms of neuronal cell death observed in ischemic stroke and Alzheimer's disease. From point of organelle damage, we introduced molecular events seen in ischemic stroke, and compared the findings with that observed in Alzheimer's disease. In the brain after ischemia, transmembrane potential and ion gradient are disturbed at very early stage. Several drugs are aimed to minimize this change, some of which were effective in experimental models. Calcium blocker and glutamate antagonist were also effective for Alzheimer's disease. As for mitochondrial and endoplasmic reticulum damage, both disorders share common pathological findings such as pro-apoptotic signals activation. However, there are some molecules which are neuroprotective in Alzheimer's disease but pro-apoptotic in ischemic neurons. We need to be so careful for judging the significance of a phenomenon obtained by an experiment. Lysosome, called as suicide bag, play important roles both in the brain of ischemic stroke and Alzheimer's disease. Leak of lysosomal enzymes influence, at least partially, the fate of neurons under pathological conditions in both disorders.

Alzheimer Disease↗

PubMed2006

Job strain, worksite support, and nutrient intake among employed Japanese men and women.

BACKGROUND: The association of job strain (as defined by the job demands/control model) and worksite support with nutrient intake is not clear. METHODS: A questionnaire survey was conducted of 25,104 workers employed in nine companies in Japan. Job strain and worksite support were assessed using the Job Content Questionnaire. Daily intake of 17 nutrients was measured using a dietary history questionnaire. Data from 15,295 men and 2,853 women were analyzed, controlling for age, education, marital status, occupation, and study site. RESULTS: Among men, job strain was positively associated with average daily intakes of fat, vitamin E, cholesterol, poly- and mono-unsaturated fatty acids (p for trend<0.05), and worksite support was positively associated with average daily intakes of total energy, crude fiber, retinol, carotene, vitamins A, C, and E, cholesterol, and saturated fatty acid (p for trend<0.05). Among women, worksite support was positively associated with average daily intakes of total energy, protein, vitamin E, and polyunsaturated fatty acid (p for trend<0.05). However these differences were generally small. CONCLUSIONS: The present study showed that job strain and worksite support were only weakly and inconsistently associated with nutritional intakes. It does not seem that changes in nutritional intakes explain the association between job strain or worksite support and coronary heart disease.

Adolescent↗

PubMed2006

[Mask anesthesia for cerebral angiography in childhood moyamoya disease].

Cerebral angiography is performed for diagnosis and management of moyamoya disease and in childhood moyamoya disease is usually carried out under general anesthesia after tracheal intubation. Mechanical irritation to trachea resulting in pain,cough,and increase in secretion after termination of the general anesthesia sometimes occurs and it sometimes causes hyperventilation resulting in hypocapnea. Continuous hypocapnea sometimes causes appearance of ischemic attacks in moyamoya disease. In the present study, we examine cerebral angiography conducted under general anesthesia using face mask ventilation in fourteen children with moyamoya disease. Sevoflurane was used as inhalation anesthetics. Face mask anesthesia was sixteen times in total in the 14 patients. Cerebral angiography terminated uneventfully in these patients except one patient who showed bronchospasm after induction of anesthesia and required tracheal intubation. However, the patient showed uneventful course after termination of the angiography. Tracheal irritation did not appear and all the patients were asleep just after termination of face mask anesthesia except for the patient who required tracheal intubation. In the latter case, the patient frequently coughed out phlegm after general anesthesia with tracheal intubation. In conclusion, general anesthesia with face mask ventilation was thought to be one of the suitable anesthetic methods introduced for cerebral angiography in childhood moyamoya disease.

Anesthesia, Inhalation↗

PubMed2005

Prevention of spinal motor neuron death by insulin-like growth factor-1 associating with the signal transduction systems in SODG93A transgenic mice.

The role of insulin-like growth factor-1 (IGF-1) in amyotrophic lateral sclerosis (ALS) and its mechanism of action are important from both pathogenic and therapeutic points of view. The present study investigated the changes of IGF-1Rbeta and the key intracellular downstream protein insulin receptor substrate-1 (IRS-1) by using SOD1(G93A) transgenic mice with continuous intrathecal IGF-1 treatment. The number of lumbar spinal motor neurons was preserved with IGF-1 treatment in a dose-dependent manner. The numbers of immunopositive motor neurons for IGF-1Rbeta and IRS-1 were not significantly different between wild-type and Tg mice with vehicle treatment, whereas treatment of Tg mice with IGF-1 decreased the numbers of immunopositive motor neurons in a dose-dependent manner. On the other hand, the ratio of immunopositive motor neurons per total living motor neurons in vehicle-treated mice was greatly increased in Tg mice with vehicle treatment compared with wild-type mice. With IGF-1 treatment, the ratio was dramatically decreased in a dose-dependent manner. These results suggest that IGF-1 treatment prevents motor neuron loss by affecting the signal transduction system through IGF-1R and the main downstream signal, IRS-1.

Analysis of Variance↗

PubMed2005

The protective effect of dantrolene on ischemic neuronal cell death is associated with reduced expression of endoplasmic reticulum stress markers.

The endoplasmic reticulum (ER) plays an important role in ischemic neuronal cell death. In order to determine the effect of dantrolene, a ryanodine receptor antagonist, on ER stress response and ischemic brain injury, we investigated changes in ER stress-related molecules, that is phosphorylated form of double-stranded RNA-activated protein kinase (PKR)-like ER kinase (p-PERK), phosphorylated form of eukaryotic initiation factor 2alpha (p-eIF2alpha), activating transcription factor-4 (ATF-4), and C/EBP-homologous protein (CHOP), as well as terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in the peri-ischemic area and ischemic core region of rat brain after transient middle cerebral artery occlusion (MCAO). In contrast to the cases treated with vehicle, the infarct volume and TUNEL-positive cells were significantly reduced at 24 h of reperfusion by treatment with dantrolene. The immunoreactivities for p-PERK, p-eIF2alpha, ATF-4, and CHOP were increased at the ischemic peripheral region after MCAO, which were partially inhibited by dantrolene treatment. The present results suggest that dantrolene significantly decreased infarct volume and provided neuroprotective effect on rats after transient MCAO by reducing ER stress-mediated apoptotic signal pathway activation in the ischemic area.

Activating Transcription Factor 4↗

PubMed2005

Up-regulation of low-density lipoprotein receptor expression in the ischemic core and the peri-ischemic area after transient MCA occlusion in rats.

Low-density lipoprotein (LDL) receptor is involved in cholesterol metabolism of CNS as a receptor of apolipoprotein E (ApoE), which plays an important role in regenerative process after brain ischemia. Temporal and spatial changes of LDL receptor were investigated after 90 min of transient middle cerebral artery occlusion (MCAO) in relation to those of microtubule-associated protein 2 (MAP2) and ApoE. In the ischemic core, LDL receptor became positive at 1 d after transient MCAO, which was not double positive for MAP2 or ApoE, and disappeared in 7 and 56 d. In the peri-ischemic area, LDL receptor became observed at 7 d, which peaked at 21 d, most of which were double positive for MAP2. The number of LDL receptor and ApoE double-positive cells increased at 7 d and decreased at 21 d with the shift of LDL receptor immunoreactivity from cytoplasm at 7 d to dendrites at 21 d in the peri-ischemic area. These results suggest that LDL receptor, interacting with ApoE, is profoundly involved in lipid transport of CNS for tissue repair in the peri-ischemic area after brain ischemia.

Animals↗

PubMed2005

Neural precursor cells division and migration in neonatal rat brain after ischemic/hypoxic injury.

Ischemia/hypoxia (I/H) causes severe perinatal brain disorders such as cerebral palsy. The neonatal brain possesses much plasticity, and to enhance new cell production would be an innovative means of therapy for such disorders. In order to elucidate the dynamic changes of neural progenitor cells in the neonatal brain after ischemia, we investigated new cells production in the subventricular zone and subsequent migration of these cells to the injured area. Newly produced cells were confirmed by incorporation of bromodeoxyuridine (BrdU), and attempt for differentiation was investigated by immunohistochemistry for molecular markers of each cellular lineage. In the sham-control brain, there were many BrdU-labeled cells which gradually decreased as the animal becomes older. Many of these cells were oligodendroglial progenitor or microglial cells. Although there were only few neuronal cells labeled for BrdU in the sham-control, they dramatically increased after I/H. They were located at just beneath the subventricular zone where the progenitor cells reside and to the injured area, indicating that newly produced cells migrated to the infarct region and differentiated into neuronal precursor cells in order to compensate the lost neural cells. We found that BrdU-labeled astroglial, oligodendroglial progenitor, and microglial cells were also increased after I/H, suggesting that they also play active roles in recovery. Progenitor cells may have potential for treating perinatal brain disorders.

Animals↗

PubMed2005

Expression of neurocan after transient middle cerebral artery occlusion in adult rat brain.

Neurocan is one of the major chondroitin sulfate proteoglycans in the nervous tissues. The expression and proteolytic cleavage of neurocan are developmentally regulated in the normal rat brain, and the full-length neurocan is detected in juvenile brains but not in normal adult brains. Recently, some studies showed that the full-length neurocan was detectable even in the adult brain when it was exposed to mechanical incision or epileptic stimulation. In the present study, we demonstrated by Western blot analysis that the full-length neurocan transiently appeared in the peri-ischemic region of transient middle cerebral artery occlusion (tMCAO) in adult rat with a peak level at 4 days after tMCAO. Immunohistochemical analysis showed that a clear positive signal of neurocan was observed 4 days after tMCAO in the peri-ischemic region of cerebral cortex and caudate, where cells strongly positive in GFAP expression were also distributed. These results indicate that accumulation of the full-length neurocan produced by reactive astrocytes may be one of the processes for tissue repair and reconstruction of neural networks after focal brain ischemia as well.

Animals↗