Biomedical subjects

Hiroyuki Nakayama

Publications and source records attributed to Hiroyuki Nakayama.

At least 19 recordsLinked to original sources

PubMed2006

Etoposide induces apoptosis and cell cycle arrest of neuroepithelial cells in a p53-related manner.

We clarified that etoposide (VP-16), a topoisomerase II inhibitor, induced apoptosis in the mouse fetal brain. Apoptotic mechanisms and cell cycle arrest in this system were investigated. Four mg/kg of VP-16 was injected into pregnant mice on day 12 of gestation (GD12). The cell cycle and expression of protein and mRNA of p53 and its transcriptional target genes were examined in the fetal brain. The number of p53- and p21-protein-positive cells peaked at 4 h after treatment (HAT). The expression of p21 mRNA was significantly increased at 4 HAT and 8 HAT. The expression of fas mRNA was significantly increased from 2 to 12 HAT. Significant expression of puma mRNA was observed from 1 HAT to 48 HAT. Flow cytometric analysis revealed that VP-16 induced S-phase accumulation and G2 arrest at 4 and 8 HAT, and VP-16-induced apoptosis was significantly increased from 4 to 24 HAT. In an experiment using BrdU treatment of pregnant mice, the migration of neuroepithelial cells in the fetuses was delayed as compared to the migration of controls, and BrdU-positive signals were observed in some pyknotic cells from 8 to 12 HAT. The present results suggest that VP-16 might induce cell cycle arrest at G2/M phase and apoptosis in a p53-related manner.

Animals↗

PubMed2006

Gene expression profiles of drug-metabolizing enzymes (DMEs) in rat liver during pregnancy and lactation.

A cDNA microarray analysis was conducted to examine hepatic gene expression profiles in pregnant and lactating F344 rats compared to a virgin control group using an Affymetrix GeneChip system. Of the approximately 16000 gene transcripts interrogated, more than 1000 were significantly modified in their expression when detected either in late pregnancy (19 days of gestation, GD 19, 513 genes upregulated and 579 downregulated) or on the day of delivery (postpartum 0 day, PPD 0, 497 upregulated and 733 downregulated). Particular interest was paid to the gene expression of drug-metabolizing enzymes (DMEs) and nuclear receptors (NRs). Though the expression of a few genes, those for CYP7A1, CYP51 and Sultx3, increased, the expression of a number of genes encoding DMEs (Phase I and Phase II) and NRs decreased during pregnancy and lactation. Changes in the expression of 9 genes encoding DMEs and NRs were confirmed by quantitative real-time PCR. For all 9 genes tested, overall, the results of the microarray and real-time PCR analyses were in agreement. This is the first application of a microarray analysis to the expression profiling of genes encoding DMEs and NRs in the liver of pregnant and lactating rats. When combined with other studies, the present study may provide a basis for investigating the mechanism of toxicity of environmental or other nonphysiologic chemicals to the fetus and mother and drug safety during pregnancy and lactation.

Animals↗

PubMed2006

Cell cycle progression is required for nuclear migration of neural progenitor cells.

In the developing brain, neural progenitor cells in the ventricular zone (VZ) show a typical migration pattern-interkinetic nuclear migration, in which nuclear position within the VZ is correlated with the cell cycle. However, the mechanisms underlying this regulation remain unclear. To clarify whether the cell cycle progression controls nuclear migration of neural progenitor cells, we determined whether chemically induced cell cycle arrest affected nuclear migration patterns in the VZ. Administration of 5-azacytidine (5AzC) or cyclophosphamide (CP) to pregnant mice induced cell cycle arrest in the fetal neural progenitor cells of the telencephalon: 5AzC induced G2/M-phase arrest, and CP induced S-phase arrest. We used 5-bromo-2'-deoxyuridine (BrdU) labeling to determine the position of the cell in the cell cycle and the nuclei within the VZ at the same time. Cells arrested in G2/M-phase stopped migrating in the inner area of the VZ. Cells arrested in S-phase stopped migrating in the outer area. These results indicate that nuclear position within the VZ was correlated with cell cycle phase, even when the cell cycle was disrupted, and that the nuclei of neural progenitor cells can migrate only when their cell cycle is going. Our results suggest that cell cycle regulators might control the machinery of migration through a common regulatory mechanism.

Animals↗

PubMed2006

Cell cycle and cell death regulation of neural progenitor cells in the 5-azacytidine (5AzC)-treated developing fetal brain.

In the developing brain, neural progenitor cells are susceptible to many extrinsic stresses, including DNA damage. We treated pregnant rats with 5-azacytidine (5AzC), a DNA demethylating and damaging agent, to investigate the cellular responses of the fetal brain, focusing on the regulation of proliferation and cell death. 5AzC first induced the accumulation of cells in abnormal mitosis, G2-phase accumulation, and then apoptosis of the neural progenitor cells. Most of the apoptotic cells were in G1 phase. Cell cycle transition studies suggested that G2/M progression was blocked, after which the cells moved to G1 phase or underwent apoptosis. p53, a key factor for response to DNA damage, and some of its target genes showed increased expression in Western blot and DNA microarray analyses. In 5AzC-treated fetal brains of p53-deficient mice, apoptosis did not occur, although G2/M accumulation was induced. These results suggest that, in the developing brain, apoptosis is p53-dependent but that another mechanism governs the G2/M checkpoint. The G2/M regulator, Cdc2, was activated by dephosphorylation through G2/M accumulation, suggesting accelerated entry into mitosis leading to accumulation of cells showing abnormal mitosis. Furthermore, some cells may have died due to mitotic catastrophe. Throughout brain development, various cell cycle and cell death regulation mechanisms provide neural progenitor cells with options for defense from DNA damage.

Animals↗

PubMed2006

Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart.

The manner in which Ca2+-sensitive signaling proteins are activated in contracting cardiomyocytes is an intriguing theoretical problem given that the cytoplasm is continually bathed with systolic Ca2+ concentrations that should maximally activate most Ca2+-sensitive signaling kinases and phosphatases. Store-operated Ca2+ entry, partially attributed to transient receptor potential (TRP) proteins, can mediate activation of the Ca2+-sensitive phosphatase calcineurin in nonexcitable cells. Here we investigated the gain-of-function phenotype associated with TRPC3 expression in the mouse heart using transgenesis to examine the potential role of store-operated Ca2+ entry in regulating cardiac calcineurin activation and ensuing hypertrophy/myopathy. Adult myocytes isolated from TRPC3 transgenic mice showed abundant store-operated Ca2+ entry that was inhibited with SKF96365 but not verapamil or KB-R7943. Associated with this induction in store-operated Ca2+ entry, TRPC3 transgenic mice showed increased calcineurin-nuclear factor of activated T cells (NFAT) activation in vivo, cardiomyopathy, and increased hypertrophy after neuroendocrine agonist or pressure overload stimulation. The cardiomyopathic phenotype and increased hypertrophy after pressure overload stimulation were blocked by targeted disruption of the calcineurin Abeta gene. Thus, enhanced store-operated Ca2+ entry in the heart can regulate calcineurin-NFAT signaling in vivo, which could secondarily impact the hypertrophic response and cardiomyopathy.

Animals↗

PubMed2006

Evidence of apoptosis in the subventricular zone and rostral migratory stream in the MPTP mouse model of Parkinson disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is commonly used to create animal models of Parkinson disease. There is conflicting evidence on the occurrence of apoptosis induced by MPTP in the mouse substantia nigra pars compacta. We demonstrated that a single acute injection of MPTP induced apoptosis in the subventricular zone (SVZ) and rostral migratory stream (RMS) in the adult C57BL/6 mouse brain. The number of TUNEL-positive cells peaked at 24 hours after injection and decreased thereafter, paralleling the change in the number of cleaved caspase-3-positive cells after MPTP injection. Results of immunohistochemistry and ultrastructural analyses indicated that the majority of apoptotic cells in the SVZ and RMS were migrating neuroblasts (type A cells), whereas a few were astrocytes (type B cells). No apoptosis occurred in transit-amplifying progenitors (type C cells). The decrease in A cell numbers was most marked on day 2 and lasted to day 8 after the administration. A rapid and transient phagocytosis of apoptotic cells by microglial cells was demonstrated to parallel the MPTP-induced apoptosis. The present findings provide new insight into the extensive neurotoxicity of MPTP and may be valuable in reevaluating the MPTP mouse model of Parkinson disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2006

Repair process of fetal brain after 5-azacytidine-induced damage.

The fetal brain is susceptible to many extrinsic stresses. Some of these stresses induce excessive cell death in the prenatal stage, leading to anomalies in the neonatal brain. However, it is unclear how the developing brain responds to and repairs the prenatal tissue damage. We treated pregnant rats on day 13 of gestation with 5-azacytidine, one of the compounds that induces excessive cell death and inhibits proliferation in neural progenitor cells, to damage the fetal brain, and investigated the repair process up to 60 h after treatment. Histological analysis showed that 5-azacytidine induced strong apoptosis of neural cells. By 60 h, apoptotic cells disappeared and the tissue was repaired, although the telencephalic wall remained thinner than in controls. Flow cytometry analysis showed that the cell cycle distribution also returned to control levels at 60 h, suggesting that the repair process was completed around 60 h. During the repair period, amoeboid microglia infiltrated the brain and ingested the apoptotic cells. These microglial cells were positive for the multiple microglial markers, and mRNAs for the microglia-related cytokines tumor necrosis factor alpha, interleukin 1beta and macrophage colony stimulating factor (M-CSF) were up-regulated. DNA microarray analysis showed the up-regulation of genes relevant to glial cells, inflammation, the extracellular matrix, glycolysis, proliferation and neural development. We show here that the developing brain has the capacity to respond to the damage induced by extrinsic chemical stresses, including changing the expression of numerous genes and the induction of microglia to aid the repair process.

Animals↗

PubMed2006

The activity of matrix metalloproteinases (MMPS) and tissue inhibitors of metalloproteinases (TIMPs) in mammary tumors of dogs and rats.

We conducted zymography for detecting the activity of matrix metalloproteinases (MMPs) and reverse zymography for the activity of tissue inhibitors of metalloproteinases (TIMPs) in canine spontaneous and rat 7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor tissues. The activities of MMPs of canine mammary tumors were quite higher than those of the rat chemically induced tumors. The activities of MMPs were significantly higher in malignant tissues than in benign ones of canine tumors, whereas the activity of only MMP-2 was higher in both benign and malignant rat tumors compared to normal tissues. There were no differences of MMPs activities between benign and malignant rat tumors. The results of reverse zymography indicated that the activities of TIMP-1, -2 and -3 were strikingly higher in rat tumors than in canine tumors. The activities were higher in malignant tissues than in benign ones of dogs, and higher in tumor tissues than in normal mammary tissues of rats. The results of film in situ zymography for tissue localization of gelatinolytic activity showed that the digested area was more extended in malignant tumors than in benign ones of dogs. However, the area was similarly extended in both benign and malignant rat tumors. These results may indicate that the canine spontaneous malignant mammary tumors possess more aggressive nature than the rat chemically induced counterpart, resulting from the high level of MMPs and low level of TIMPs activities of the tumor tissues.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2006

Histopathological characteristics of Ito cells and Kupffer cells in the feline liver.

The histopathological characteristics of Ito cells and Kupffer cells were investigated in the liver of 21 cats (age range: 6 months -18 years) autopsied in our laboratory during 2003. Immunohistochemical examinations were performed using antibodies against lysozyme, desmin and alpha-smooth muscle actin. No Kupffer cells reacted with the antibody against lysozyme. However, macrophages in the lung and spleen showed a positive reaction with the antibody. This finding suggests a possibility that the amount of lysozyme in the Kupffer cells of feline liver is comparatively small. On the other hand, large vacuole-laden cells were observed in the hepatic perisinusoid of some feline cases, and these cells showed a positive reaction with antibodies against desmin and alpha-smooth muscle actin. These cells could be Ito cells with large lipid vacuoles. This conclusion was supported by electron microscopic observation and oil red O staining. However, no such large vacuole-laden perisinusoidal cells were detected in the liver of young cats less than 2 years old. The present study revealed the histopathological features of Kupffer cells and Ito cells in the feline liver.

Actins↗

PubMed2006

Prognostic factors associated with survival in dogs with lymphocytic-plasmacytic enteritis.

Prognostic factors associated with survival in dogs with lymphocytic-plasmacytic enteritis (LPE) were investigated through a retrospective study. Using case records, 48 dogs diagnosed with LPE were classified as survivors (n = 32) or non-survivors (n = 16), and the clinical and clinicopathological parameters were reviewed between the 2 groups by using univariate and multivariate prognostic analysis. Compared to the hospital population, non-survivors had an overrepresentation of the Shiba breed. Results of univariate analysis indicated that anorexia, severe weight loss, packed cell volume, and total protein were significantly associated with survival for 6 months after diagnosis. In multivariate analysis, anorexia and hypoproteinemia were significantly associated with survival. Furthermore, initial response to treatment was strongly associated with poor prognosis. Based on these clinical and laboratory parameters such as anorexia, hypoproteinemia and initial response to treatment, it may be possible to predict poor prognosis in canine LPE.

Animals↗

PubMed2006

Changes in submaxillary gland gene expression in F344 rats by multiple dosing of theophylline.

Multiple exposure to theophylline, a phosphodiesterase (PDE) inhibitor, induces acinar hypertrophy in the salivary gland. This study examined the effect of theophylline on the gene expression of secretory proteins and phosphodiesterases in the submaxillary gland. Male F344 rats received saline or theophylline (50 mg/kg) intraperitoneally for 4 days. The gene expressions for the secretory protein, cystatin S (CysS), and PDE subfamilies 3A and 4D in the submaxillary gland were quantified using RT-PCR. Theophylline exposure resulted in a sustained increase in mRNA expression for CysS and PDE3A, but PDE4D gene expression was unchanged. Our results suggest that submaxillary hypertrophy is primarily caused by the enhanced transcription of CysS, and that the transcription of each PDE subfamily gene is regulated differently.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

The critical roles of serum/glucocorticoid-regulated kinase 3 (SGK3) in the hair follicle morphogenesis and homeostasis: the allelic difference provides novel insights into hair follicle biology.

Mutation in the serum/glucocorticoid regulated kinase 3 (Sgk3, also known as Sgkl or Cisk) gene causes both defective hair follicle development and altered hair cycle in mice. We examined Sgk3-mutant YPC mice (YPC-Sgk3(ypc)/Sgk3(ypc)) and found expression of SGK3 protein with altered function. In the hair follicles of YPC mice, the aberrant differentiation and poor proliferation of hair matrix keratinocytes during the period of postnatal hair follicle development resulted in a complete lack of hair medulla and weak hair. Surprisingly, the length of postnatal hair follicle development and anagen term was shown to be dramatically shortened. Also, phosphorylation of GSK3beta at Ser9 and the nuclear accumulation of beta-catenin were reduced in the developing YPC hair follicle, suggesting that phosphorylation of GSK3beta and WNT-beta-catenin pathway takes part in the SGK3-dependent regulation of hair follicle development. Moreover, the above-mentioned features, especially the hair-cycling pattern, differ from those in other Sgk3-null mutant strains, suggesting that the various patterns of dysfunction in the SGK3 protein may result in phenotypic variation. Our results indicate that SGK3 is a very important and characteristic molecule that plays a critical role in both hair follicle morphogenesis and hair cycling.

Alleles↗

PubMed2005

Changes in cytochrome P450 isozymes (CYPs) protein levels during lactation in rat liver.

The effects of pregnancy on CYPs protein level in the liver have been investigated in our previous study. Since pregnancy was associated with a decrease in CYPs protein level, the objective of this study was to investigate whether CYPs protein can revert to the virgin control level after delivery. Western blot analysis was performed to investigate the changes of total nine CYPs protein (CYP1A1, CYP2B1/CYP2B2, CYP2C6, CYP2C12, CYP2D1, CYP2D4, CYP2E1, CYP3A1 and CYP4A1) at three distinct phases: delivery (postpartum day 0, PPD 0), peak lactation (PPD 14) and on weaning (PPD 28). By PPD 0, CYP1A1, 2B1, 2B2, 2C6, 2E1 and CYP4A1 were markedly down-regulated when compared with virgin controls. By PPD 14, however, CYP1A1, 2B1, 2B2 and CYP2C6 returned to the virgin control level. All the decreased CYPs during lactation were at the virgin control level at PPD 28. The expression of CYP2C12, CYP2D1 and CYP 3A1 did not differ between lactating, post-lactation and virgin control rats. CYP2D4 was not detectable in microsomal proteins obtained from virgin control rats at a protein loading of 20 mug total protein per lane.

Animals↗

PubMed2005

Presenilin 2 regulates the systolic function of heart by modulating Ca2+ signaling.

Genetic studies of families with familial Alzheimer's disease have implicated presenilin 2 (PS2) in the pathogenesis of this disease. PS2 is ubiquitously expressed in various tissues including hearts. In this study, we examined cardiac phenotypes of PS2 knockout (PS2KO) mice to elucidate a role of PS2 in hearts. PS2KO mice developed normally with no evidence of cardiac hypertrophy and fibrosis. Invasive hemodynamic analysis revealed that cardiac contractility in PS2KO mice increased compared with that in their littermate controls. A study of isolated papillary muscle showed that peak amplitudes of Ca2+ transients and peak tension were significantly higher in PS2KO mice than those in their littermate controls. PS2KO mouse hearts exhibited no change in expression of calcium regulatory proteins. Since it has been demonstrated that PS2 in brain interacts with sorcin, which serves as a modulator of cardiac ryanodine receptor (RyR2), we tested whether PS2 also interacts with RyR2. Immmunoprecipitation analysis showed that PS2, sorcin, and RyR2 interact with each other in HEK-293 cells overexpressing these proteins or in mouse hearts. Immunohistochemistry of heart muscle indicated that PS2 colocalizes with RyR2 and sorcin at the Z-lines. Elevated Ca2+ attenuated the association of RyR2 with PS2, whereas the association of sorcin with PS2 was enhanced. The enhanced Ca2+ transients and contractility in PS2KO mice were observed at low extracellular [Ca2+] but not at high levels of [Ca2+]. Taken together, our results suggest that PS2 plays an important role in cardiac excitation-contraction coupling by interacting with RyR2.

Animals↗

PubMed2005

Encepalomyocarditis virus-induced apoptosis and ultrastructural changes in the lacrimal and parotid glands of mice.

Development of acinar cell apoptosis and ultrastructural changes in the exorbital lacrimal and parotid glands was examined in DBA/2 mice infected with 10(2) PFU/mouse of EMC-D virus. Pyknotic acinar cells, most of which were positive for TUNEL and cleaved caspase-3 and had ultrastructural characteristics of apoptotic cells, developed earlier and were more frequently observed in the parotid gland than in the exorbital lacrimal gland, while the total damage of acinar cells and interstitial infiltration of macrophages were more prominent in the latter than in the former. These findings indicate that EMC-D virus induces acinar cell apoptosis in these glands. In addition, corresponding to the results of the detection of viral RNA signals by in situ hybridization, small aggregates of virus-like particles having typical size and structure of EMC virus were frequently observed in both the cytoplasm and the nucleus of acinar cells in the exorbital lacrimal gland, while they were found only in the cytoplasm of a few acinar cells in the parotid gland. In conclusion, between the exorbital lacrimal and parotid glands, there was a reverse relationship observed between the development of acinar cell apoptosis and that of total damage of acinar cells.

Animals↗

PubMed2005

Hydroxyurea (HU)-induced apoptosis in the mouse fetal lung.

In this study, the cytotoxicologic effects of HU on the fetal lung were assessed by exposing pregnant mice to HU on day 13 of gestation. The number of TUNEL-positive cells, i.e., apoptotic cells, in the fetal lung began to increase at 3 h after treatment (h), peaked at 6 h, and decreased thereafter, and the sequence of the number of cleaved caspase 3-positive cells corresponded to that of TUNEL-positive cells. Such positive reactivity for TUNEL and cleaved caspase 3 was mainly seen in pulmonary mesenchymal cells. Prior to the induction of apoptosis, the number of p53-positive cells in the fetal lung prominently increased at 1 and 3 h, and decreased thereafter. Among p53 transcriptional target genes (p21, fas, bax, apaf1, cyclin G, mdm2, and gad 45) examined, the expression levels of p21, bax, and cyclin G mRNAs were significantly elevated. In addition, the expression of fas mRNA tended to show higher levels compared with controls until 24 h. In addition, the results of flow cytometric analysis suggested that cell cycle arrest might be induced in S phase at 3 h. The present results suggest that HU-induced apoptosis in the mouse fetal lung may be closely related with the induction of p53.

Animals↗

PubMed2005

Impaired proliferation of non-parenchymal cells participates in an impairment of liver regeneration in db/db mice.

In this study, we examined the possibility that impaired proliferation of non-parenchymal cells affects in an impairment of liver regeneration in db/db mice, which are congenitally deficient in receptors for leptin. Liver regeneration after a two thirds partial hepatectomy (2/3 PH) was impaired in 10-week-old female db/db mice. The proliferation of both hepatocytes and non-parenchymal cells estimated from a bromodeoxyuridine (BrdU) labeling index was suppressed, and the protein expression of vascular endothelial growth factor was blocked in db/db mice. Although the extent of fatty change and the level of epidermal growth factor receptor protein expression in the liver were improved in 5-week-old db/db mice, the regeneration of liver was impaired after 2/3 PH in both 5- and 10-week-old db/db mice. These results suggested that suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration in db/db mice. As leptin has also the angiogenic effect, the angiogenic inhibitor FR-118487 was administered to ICR mice to examine liver regeneration after 2/3 PH, and the rate of regeneration was affected. In conclusion, it is suggested that the suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration probably through a disrupted angiogenesis in db/db mice.

Angiogenesis Inhibitors↗

PubMed2005

Microarray analysis of genes in fetal central nervous system after ethylnitrosourea administration.

BACKGROUND: Ethylnitrosourea (ENU), a monofunctional alkylating agent, induces apoptosis and cell cycle arrest in neuroepithelial cells, neural stem cells in the fetal central nervous system (CNS). These effects occur immediately after the administration of ENU to pregnant animals resulting in fetal brain anomalies and long-term effects include brain tumors in the offspring. METHODS: Changes in gene expression were investigated in the fetal CNS after ENU administration to pregnant rats using microarray to identify the genes involved in the injury and recovery of the fetal CNS. RESULTS: The up-regulation of 21 genes in injury and 15 genes in recovery phases and down-regulation of 5 genes in injury and 3 genes in recovery phases were identified. The genes up-regulated in the injury phase contained p53-target genes that mediate apoptosis and cell cycle arrest, and those in the recovery phase contained cell proliferation-promoting genes. The genes down-regulated in the injury phase contained cholesterol biosynthesis-related genes. In addition, there were some genes that have not been identified to be involved in the CNS injury and recovery. CONCLUSIONS: The present study will provide a better understanding of the mechanisms of development, regeneration and carcinogenesis of the CNS as well as the mechanisms of ENU-induced fetal CNS injury and recovery.

Alkylating Agents↗