Biomedical subjects

Haruhiko Sakamoto

Publications and source records attributed to Haruhiko Sakamoto.

15 recordsLinked to original sources

PubMed2006

Effects of aging and HIF-1alpha deficiency on permeability of hippocampal vessels.

We examined age-related changes in the blood-brain barrier (BBB) of neural cell-specific hypoxia inducible factor-1alpha (HIF-1alpha) deficient mice, which showed hydrocephalus with neuronal cell loss, to investigate an effect of neural cell-specific HIF-1alpha deficiency or hydrocephalus on vascular function. Vascular permeability of horseradish peroxidase (HRP) and binding of cationized ferritin (CF) particles to the endothelial cell luminal surface, as a marker of glycocalyx, were investigated. The thickness of CF-labeled glycocalyx was significantly decreased in the cortex in mutant mice compared with that of control mice, although it was not paralleled by increased vascular permeability. In addition, strong staining for HRP was seen around vessels located along the hippocampal fissure in 24-month-old mutant mice. The reaction product of HRP appeared in an increasing number of the endothelial cell abluminal vesicles and within the thickened basal lamina of arterioles in the hippocampus, showing increased vascular permeability. There were no leaky vessels in 10-week-old mutant mice or 10-week-old and 24-month-old control mice. These findings suggest the necessity of two factors, aging and hydrocephalus, for BBB dysfunction in HIF-1alpha deficient mice.

Aging↗

PubMed2005

Two pathways of apoptosis are simultaneously induced in the embryonal brains of neural cell-specific HIF-1alpha-deficient mice.

The aim of this study was to clarify the mechanism of apoptosis seen in the cortex of neural cell-specific hypoxia inducible factor-1alpha (HIF-1alpha)-deficient embryos. A previous study showed that the neural cells in the cortical area of the mutant embryos underwent apoptosis coincident with vascular regression. Through histological, immunohistochemical, and electron microscopic technique, two kinds of apoptotic cells were detected in the mutant embryonal cortex. Apoptotic cells of one type were clustered in small round structures, 10-20 mum in diameter, whereas the others, present in large numbers, were distributed in a group at the cortical plate located more to the outer side than the round structures. The histochemical and electron microscopic findings indicate that the former represented the appearance of macrophages, in which cellular fragments including vascular cells underwent oxidative stress-related, TNF receptor-mediated, caspase-2-induced apoptosis, while the latter showed c-Myc-related, caspase-3-activated apoptosis of the neural cells. These results suggest that two pathways of apoptosis are induced in neuronal and vascular cells of the cortex in the neural cell-specific HIF-1alpha-deficient mouse.

Animals↗

PubMed2005

Regional distribution and age-dependent expression of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D in rat brain.

The endocannabinoid anandamide (N-arachidonoylethanolamine) and other bioactive long-chain N-acylethanolamines are thought to be formed from their corresponding N-acylphosphatidylethanolamines by a specific phospholipase D (NAPE-PLD) in the brain as well as other tissues. However, regional distribution of NAPE-PLD in the brain has not been examined. In the present study, we investigated the expression levels of NAPE-PLD in nine different regions of rat brain by enzyme assay, western blotting and real-time PCR. The NAPE-PLD activity was detected in all the tested brain regions with the highest activity in thalamus. Similar distribution patterns of NAPE-PLD were observed at protein and mRNA levels. We also found a remarkable increase in the expression levels of protein and mRNA of the brain NAPE-PLD with development, which was in good agreement with the increase in the activity. The age-dependent increase was also seen with several brain regions and other NAPE-PLD-enriched organs (heart and testis). p-Chloromercuribenzoic acid and cetyltrimethylammonium chloride, which inhibited recombinant NAPE-PLD dose-dependently, strongly inhibited the enzyme of all the brain regions. These results demonstrated wide distribution of NAPE-PLD in various brain regions and its age-dependent expression, suggesting the central role of this enzyme in the formation of anandamide and other N-acylethanolamines in the brain.

Age Factors↗

PubMed2004

Prepro-orexin mRNA expression in the rat brain is increased during pregnancy.

The purpose of this study was to investigate whether orexin expression in the rat brain was changed during pregnancy. Brain samples were obtained from 5 nonpregnant rats and 10 pregnant rats (5; day 10 of gestation, and 5; day 20 of gestation). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to investigate the expression of prepro-orexin mRNA and the housekeeping gene in the rat brain. The signals were quantified by the densitometric analysis. The distribution and expression of orexin-A and orexin-B were determined using immunohistochemistry. The ratio of the prepro-orexin mRNA expressions to the housekeeping gene expression in pregnant rat brain were significantly higher than that in nonpregnant control. There was no significant difference between prepro-orexin mRNA levels of day 10 and day 20 of gestation. Immunohistochemical staining for orexin-A and orexin-B was present in neurons within and around the lateral and posterior hypothalamic areas in both nonpregnant and pregnant rats. These results suggest that increased prepro-orexin mRNA levels at early gestational age in the maternal rat has a role on energy metabolism during pregnancy.

Actins↗

PubMed2004

Blood-brain barrier disruption in the hypothalamus of young adult spontaneously hypertensive rats.

Vascular permeability and endothelial glycocalyx were examined in young adult spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and Wistar Kyoto rats (WKY) as a control, in order to determine earlier changes in the blood-brain barrier (BBB) in the hypothalamus in chronic hypertension. These rats were injected with horseradish peroxidase (HRP) as an indicator of vascular permeability. Brain slices were developed with a chromogen and further examined with cationized ferritin, a marker for evaluating glycocalyx. Staining for HRP was seen around vessels in the hypothalamus of SHR and SHRSP, but was scarce in WKY. The reaction product of HRP appeared in the abluminal pits of endothelial cells and within the basal lamina of arterioles, showing increased vascular permeability in the hypothalamus of SHR and SHRSP, whereas there were no leaky vessels in the frontal cortex of SHR and SHRSP, or in both areas of WKY. The number of cationized ferritin particles binding to the capillary endothelial cells was decreased in the hypothalamus of SHR and SHRSP, while the number decreased in the frontal cortex of SHRSP, compared with those in WKY. Cationized ferritin binding was preserved in some leaky arterioles, while it was scarce or disappeared in other leaky vessels. These findings suggest that BBB disruption occurs in the hypothalamus of 3-month-old SHR and SHRSP, and that endothelial glycocalyx is markedly damaged there without a close relationship to the early changes in the BBB.

Animals↗

PubMed2004

Blood-brain barrier is impaired in the hippocampus of young adult spontaneously hypertensive rats.

A causative role of blood-brain barrier (BBB) impairment is suggested in the pathogenesis of vascular dementia with leakage of serum components from small vessels leading to neuronal and glial damage. We examined the BBB function of young adult spontaneously hypertensive rats (SHR) in order to determine earlier changes in the BBB in chronic hypertension. SHR and stroke-prone SHR (SHRSP) were injected with horseradish peroxidase (HRP) as an indicator of BBB function and compared with Wistar Kyoto rats (WKY). The brain tissues were further examined with cationized ferritin, a marker for evaluating glycocalyx. The staining for HRP was distributed around the vessels in the hippocampal fissure of SHR and SHRSP, but not in WKY. With electron microscopy, the extravasated reaction product of HRP appeared in abluminal pits of the endothelial cells of arterioles and within the basal lamina in the hippocampus, but not the cerebral cortex, of SHR and SHRSP. On the contrary, the reaction product of HRP was never seen in the abluminal pits of the endothelial cells or the basal lamina of vessels in WKY. The number of cationized ferritin particles binding to the endothelial cells of capillaries was decreased in the hippocampus of SHR and SHRSP, while the number decreased in the cerebral cortex of SHRSP compared with those in WKY. However, the cationized ferritin binding was preserved in the endothelial cells of the arterioles with an increased vascular permeability. These findings suggest that the chronic hypertensive state induces BBB dysfunction in the hippocampus at an early stage.

Animals↗

PubMed2004

Expression of Epstein-Barr virus in Langerhans' cell histiocytosis.

Langerhans' cell histiocytosis (LCH) is a proliferative histiocytic disorder of unknown etiology. We previously reported that Epstein-Barr virus (EBV) infects and proliferates in macrophages, and investigated the possibility that EBV exhibits etiologic effects in LCH. To detect EBV expression, paraffin sections from 17 LCH cases were examined by mRNA in situ hybridization for EBV BamHIW, Epstein-Barr virus nuclear antigen-2 (EBNA2), and Epstein-Barr virus-encoded small nonpolyadenylated RNA (EBER1) sequences, and by indirect immunofluorescence staining for EBNA2, latent membrane protein 1 (LMP1), and BamHIZ-coding leftward-reading frame 1 (BZLF1). To detect EBV DNA, polymerase chain reaction (PCR)-Southern blotting was used. All cases showed positive hybridization signals by BamHIW mRNA in situ hybridization. Also, 13 and 14 cases showed positive signals for EBNA2 and EBER1 RNA in situ hybridization, respectively. Furthermore, almost all cases exhibited fluorescence after immunofluorescence staining with monoclonal anti-EBNA2 and anti-BZLF1 antibodies, and 15 cases were positive after treatment with monoclonal anti-LMP1 antibody. PCR-Southern blotting detected an amplified EBER1 sequence in all 9 cases examined. EBV expression was confirmed in LCH using in situ hybridization and immunofluorescence. Furthermore, EBV DNA was also detected by PCR-Southern blotting. These positive results of BZLF1 suggest that EBV replicates in LCH tissues.

Adolescent↗

PubMed2004

Effects of platelet release products on neutrophilic phagocytosis and complement receptors.

INTRODUCTION: Platelets enhance leukocytic phagocytosis via the action of ATP and ADP in platelet release products (PRPr). The present study was designed to clarify the type of complements and complement receptors that are involved in phagocytosis activation by PRPr, ATP and ADP. MATERIALS AND METHODS: Human peripheral blood was used as the source of neutrophils and platelets. The supernatant of the platelet suspension after simulation was used as PRPr. The effects of PRPr, ATP, ADP, and other substances on neutrophilic phagocytosis, rosette formation and expression of several antigens were investigated. For the markers of neutrophilic phagocytosis and rosette formation, IgM-sensitized sheep red blood cells (SRBC) were treated with diluted human serum (EAC) or purified complements (C1, C4, C2 and C3) (EAC3b) followed by C3 inactivation (EAC3bi). The expressions of CD11b, CD11c, CD18, and CD35 were evaluated using a flow cytometer. RESULTS: Neutrophilic phagocytosis of EAC and EAC3bi was enhanced by PRPr, ATP, and ADP, whereas this phagocytosis activation was abolished by antibodies against CD11b and CD18. Neutrophil rosette formation with EAC3bi was increased by ATP and ADP. Flow cytometry revealed that the expressions of CD11b and CD35 on neutrophils were increased by PRPr, but not by ATP and ADP. The component in PRPr, responsible for the increase in expressions of these antigens, could not be identified. CONCLUSION: PRPr increases the neutrophilic phagocytosis of complement-coated particles through the action of ATP and ADP by increasing the binding avidity with iC3b, but not the number of Mac-1 (CD11b/CD18).

Adenosine Diphosphate↗

PubMed2004

Hypoxia-inducible factor-2alpha is involved in enhanced apoptosis in the placenta from pregnancies with fetal growth restriction.

The aim of the present study is to investigate whether hypoxia-inducible factors (HIF-1alpha, HIF-2alpha, HIF-1beta) are involved in enhanced apoptosis in the human placenta from pregnancies with fetal growth restriction (FGR). Placental samples were obtained from women with normal term pregnancy (n = 18) or from pregnancy with FGR (n = 12). Placenta apoptosis was assessed by the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) staining. The expressions of HIF-1alpha, HIF-2alpha, and HIF-1beta were examined by immunohistochemical analysis. Enhanced apoptosis was observed in the placenta from pregnancies with FGR compared with normal term placenta. The apoptosis index in FGR group (1.45 +/- 1.26%) was significantly higher than that in the normal control group (0.18 +/- 0.16%; P < 0.01). There were no significant differences in the intensity of the staining for HIF-1alpha and HIF-1beta expressions between two groups, while HIF-2alpha was overexpressed in the placenta from pregnancies with FGR group (P < 0.05). The upregulation of HIF-2alpha protein expression in the placenta from pregnancies with FGR may, at least in part, be involved in the increased placental apoptosis.

Adult↗

PubMed2003

Transferrin microheterogeneity in pregnancies with preeclampsia.

BACKGROUND: It has been reported that concentrations of serum transferrin (Tf) and its highly sialylated subfraction increase in normal pregnancy. This study investigated changes in the concentrations of serum transferrin and its subfractions in preeclampsia. METHODS: The serum concentration of transferrin was determined by a standard turbidimetric assay and microheterogeneous transferrin subgroups (low sialylated, 4-sialo and highly sialylated transferrins) were assessed by crossed immuno-isoelectric focusing. RESULTS: Compared to normal pregnancy, the concentrations of total, 4-sialo and highly sialylated transferrins decreased by 27%, 16% and 38%, respectively, in severe preeclampsia, while these values did not significantly decrease in mild preeclampsia. The concentration of low sialylated transferrin involving 2-sialo- and 3-sialo-transferrins significantly decreased both in mild and severe preeclampsia, the value in severe preeclampsia was even significantly lower than that in nonpregnant women. The serum concentrations of total and highly sialylated transferrins in preeclampsia were correlated positively with infant birth weights (r=0.587 and r=0.645, respectively). CONCLUSIONS: The serum concentrations of total and highly sialylated transferrins in severe preeclampsia decrease significantly. This might have a negative impact on intrauterine growth. Additionally, the serum low sialylated transferrin decreases more sensitively in preeclampsia, although the concentration is low even in normal pregnancy.

Birth Weight↗

PubMed2003

Defective brain development in mice lacking the Hif-1alpha gene in neural cells.

Hypoxia-inducible factor 1alpha (HIF-1alpha) is essential for vascular development during embryogenesis and pathogenesis. However, little is known about its role in brain development. To investigate the function of HIF-1alpha in the central nervous system, a conditional knockout mouse was made with the Cre/LoxP system with a nestin promoter-driven Cre. Neural cell-specific HIF-1alpha-deficient mice exhibit hydrocephalus accompanied by a reduction in neural cells and an impairment of spatial memory. Apoptosis of neural cells coincided with vascular regression in the telencephalon of mutant embryos, and these embryonic defects were successfully restored by in vivo gene delivery of HIF-1alpha to the embryos. These results showed that expression of HIF-1alpha in neural cells was essential for normal development of the brain and established a mouse model that would be useful for the evaluation of therapeutic strategies for ischemia, including hypoxia-mediated hydrocephalus.

Animals↗

PubMed2002

Blood-brain barrier disruption in white matter lesions in a rat model of chronic cerebral hypoperfusion.

Blood-brain barrier damage has been implicated in the pathogenesis of cerebrovascular white matter lesions. This type of lesion is responsible for cognitive impairment in the elderly and can be induced by permanent ligation of the bilateral common carotid arteries in the rat. Because it is unclear whether the blood-brain barrier is impaired, we examined whether vascular permeability to horseradish peroxidase is altered using this model. According to light microscopic results, the reaction product of horseradish peroxidase was most intensely localized to the paramedian part of the corpus callosum in the brain, occurring to a small degree at 3 hours, day 1, markedly on day 3, but reduced on days 7 and 14. By electron microscopic study of the same area, the reaction product of horseradish peroxidase was localized to the plasmalemmal vesicles in the endothelial cells 3 hours after ligation, but appeared in the cytoplasm on days 1 and 3, suggesting a diffuse leakage of horseradish peroxidase. In addition, the reaction product was dispersed into the cytoplasm of glial cells in the perivascular regions on day 3. The luminal surface of the endothelial cell cytoplasm appeared irregular on day 7, suggesting a conformational change of the endothelial cells. Collagen fibrils proliferated in the thickened basal lamina and mitochondria degenerated in the pericyte on days 7 and 14. Perivascular glial endfeet were swollen throughout the survival period. In sham-operated rats, the reaction product of horseradish peroxidase was not observed at any time interval, except in vesicular structures. These findings indicate that chronic cerebral hypoperfusion induces blood-brain barrier damage with subsequent morphologic changes of the vascular structures in the corpus callosum. An extravasation of macromolecules, such as proteases and immunoglobulins, may contribute to the pathogenesis of white matter lesions.

Animals↗

PubMed2002

Polymerase chain reaction-based clonality analysis in thyroid lymphoma.

A previous patho-epidemiological study indicated that thyroid lymphoma (TL) evolves among active lymphoid cells into chronic lymphocytic thyroiditis (CLTH), a thyroid-specific autoimmune disease. In this study, clonality of B-cells in the CLTH and TL lesions was analyzed using polymerase chain reaction (PCR)-based method on surgically resected samples from 10 cases of TL; 7 mucosa-associated lymphoid tissue (MALT) lymphoma and 3 diffuse large B-cell lymphoma (DLBCL). CLTH lesions coexisted in all the cases with MALT lymphoma, but not in the three DLBCL cases. In cases of MALT lymphoma, the lymphomatous and CLTH areas were separately microdissected from each section and analyzed for clonality. In the cases of DLBCL, the whole specimens were used for clonality analysis. CLTH lesions showed smear in 6 samples, two bands in one, and more than three (oligoclonal pattern) in 2. MALT lymphoma lesions showed single or two bands (monoclonal pattern) in 4, oligoclonal pattern in 4, and smear in one. DLBCL showed monoclonal pattern in two and oligoclonal pattern in one. One common band was present among two separate MALT lesions in one case, but no common bands were found in the remaining six cases. These findings suggested the clonal evolution of B-cell from polyclonal to monoclonal proliferation to take place in the continuum of lymphoproliferative lesions into autoimmune thyroiditis.

Aged↗

PubMed2002

Two types of lymphatic invasion in lymph node metastasis with special references to the morphology of gastric carcinomas and immunohistology of e-cadherin and -catenin.

Two types of lymphatic invasion were distinguishable in lymph nodes (LN) with metastasis of a gastric carcinoma (GC). One was an embolic (E) type of lymphatic invasion that was characterized with embolus of carcinoma cells in collecting lymphatics. Infiltration of carcinomas as single cells or as tiny clusters through lymphatic capillaries were features of the other infiltrative (I) type of lymphatic invasion and were found in the interstitial spaces of peri-lymphnodal fat tissues. The latter was an early event during metastasis, even when not yet apparent in LN. Both types of lymphatic invasion coexisted, not infrequently, in advanced stages, but pure I type occurred exclusively in diffuse type GC ( 2< 0.005) C regardless of the existence of metastasis in LN. E type was more frequently encountered solely or predominantly in intestinal type GC ( 2< 0.05). Of the destructive lesions of collecting lymphatics, fibrous occlusion or stenosis and large emboli were seen in intestinal type GC ( 2< 0.005). Early I type lesions were more seen in diffuse type GC ( 2< 0.005). The incidence of destruction of collecting lymphatics by carcinoma cells from outside and the existence of single cells in collecting lymphatics were seen more in diffuse type GC, but not significantly. Immunohistology revealed that E-cadherin (Ec) and -catenin( c) were expressed positively in cytoplasm of carcinoma cells of diffuse GC at 50.0% and 78.5%, respectively. However, c of carcinoma cells exhibiting I type of lymphatic invasion were expressed negatively or much less. The mechanism of I type of lymphatic invasion, its low expression of c, and the heterogeneity of GC were discussed. It is necessary to clarify the I type of lymphatic invasion to gain improvements in curability.

Cadherins↗

PubMed2002

Clonality analysis of follicular lymphoma using laser capture microdissection method.

Whether a common and a single clone present, or not, among follicles of follicular lymphoma (FL) was examined in 12 cases with FL. Histologic grade was I in 6 cases, II in 3, and III in 3. DNA was selectively extracted from the neoplastic follicles of paraffin-embedded samples with use of laser capture microdissection method, and used for PCR-based analysis of rearrangement of immunoglobulin heavy chain variable region gene. Three different follicles in each case of FL were microdissected. Semi-nested PCR was performed using two sets of primers (Fr2A and Fr3A). In PCR with Fr2A primers, nine of 12 cases showed a common band among neoplastic follicles. The remaining three cases showed no PCR products. With Fr3A primers, eight of 12 cases showed a common band among follicles of the same case. The other four cases showed oligoclonal bands, among them presence of a common band was difficult to assess. Oligoclonal bands were more frequently observed in PCR with Fr3A than that with Fr2A and in grade I or II than in grade III cases. In total, 11 of 12 cases showed a common band in PCR with either Fr2A or Fr3A primers. In two cases, DNA extracted from whole section was amplified with both Fr2A and Fr3A or only Fr3A primers, showing smear or oligoclonal bands. These results showed the presence of a single clone of cells in neoplastic follicles of FL and the usefulness of PCR-based rearrangement analysis of immunoglobulin heavy chain gene combined with microdissection methods for differential diagnosis of FL from follicular hyperplasia.

Adult↗