Biomedical subjects

N Harada

Publications and source records attributed to N Harada.

At least 73 records · Page 4Linked to original sources

PubMed2001

Age differences in muscarinic cholinergic receptors assayed with (+)N-[(11)C]methyl-3-piperidyl benzilate in the brains of conscious monkeys.

Age-related changes in muscarinic cholinergic receptors were evaluated with the novel ligand (+)N-[(11)C]methyl-3-piperidyl benzilate ((+)3-MPB) in the living brains of young (5.9 +/- 1.8 years old) and aged (19.0 +/- 3.3 years old) monkeys (Macaca mulatta) in the conscious state using high-resolution positron emission tomography (PET). For quantitative analysis of receptor binding in vivo, metabolite-corrected arterial plasma radioactivity curves were obtained as an input function into the brain, and kinetic analyses using the three-compartment model and graphical Logan plot analysis were applied. Kinetic analyses of [(11)C](+)3-MPB indicated a regionally specific decrease in the receptor binding in vivo determined as binding potential (BP) = k(3)/k(4) in aged animals compared with young animals. Thus, the frontal and temporal cortices as well as the striatum showed age-related reduction of muscarinic cholinergic receptors in vivo, reflecting the reduced receptor density (B(max)) determined by Scatchard plot analysis in vivo. In the hippocampus, although BP of [(11)C](+)3-MPB indicated no significant age-related changes, it showed an inverse correlation with individual cortisol levels in plasma. When the graphical Logan plot analysis was applied, all regions assayed showed significant age-related decrease of [(11)C](+)3-MPB binding. These results demonstrate the usefulness of kinetic three-compartment model analysis of [(11)C](+)3-MPB with metabolite-corrected arterial plasma input as an indicator for the aging process of the cortical muscarinic cholinergic receptors in vivo as measured by PET.

Aging↗

PubMed2001

Brain aromatase is neuroprotective.

The expression of aromatase, the enzyme that catalyzes the biosynthesis of estrogens from precursor androgens, is increased in the brain after injury, suggesting that aromatase may be involved in neuroprotection. In the present study, the effect of inactivating aromatase has been assessed in a model of neurodegeneration induced by the systemic administration of neurotoxins. Domoic acid, at a dose that is not neurotoxic in intact male mice, induced significant neuronal loss in the hilus of the hippocampal formation of mice with reduced levels of aromatase substrates as a result of gonadectomy. Furthermore, the aromatase substrate testosterone, as well as its metabolite estradiol, the product of aromatase, were able to protect hilar neurons from domoic acid. In contrast, dihydrotestosterone, the 5 alpha-reduced metabolite of testosterone and a nonaromatizable androgen, was not. These findings suggest that aromatization of testosterone to estradiol may be involved in the neuroprotective action of testosterone in this experimental model. In addition, aromatase knock-out mice showed significant neuronal loss after injection of a low dose of domoic acid, while control littermates did not, indicating that aromatase deficiency increases the vulnerability of hilar neurons to neurotoxic degeneration. The effect of aromatase on neuroprotection was also tested in male rats treated systemically with the specific aromatase inhibitor fadrozole and injected with kainic acid, a well characterized neurotoxin for hilar neurons in the rat. Fadrozole enhanced the neurodegenerative effect of kainic acid in intact male rats and this effect was counterbalanced by the administration of estradiol. Furthermore, the neuroprotective effect of testosterone against kainic acid in castrated male rats was blocked by fadrozole. These findings suggest that neuroprotection by aromatase is due to the formation of estradiol from its precursor testosterone. Finally, a role for local cerebral aromatase in neuroprotection is indicated by the fact that intracerebral administration of fadrozole enhanced kainic acid induced neurodegeneration in the hippocampus of intact male rats. These findings indicate that aromatase deficiency decreases the threshold for neurodegeneration and that local cerebral aromatase is neuroprotective. Brain aromatase may therefore represent a new target for therapeutic approaches to neurodegenerative diseases.

Animals↗

PubMed2001

Evaluation of PET ligands (+)N-[(11)C]ethyl-3-piperidyl benzilate and (+)N-[(11)C]propyl-3-piperidyl benzilate for muscarinic cholinergic receptors: a PET study with microdialysis in comparison with (+)N-[(11)C]methyl-3-piperidyl benzilate in the conscious monkey brain.

We developed PET ligands (+)N-[(11)C]ethyl-3-piperidyl benzilate ([(11)C](+)3-EPB) and (+)N-[(11)C]propyl-3-piperidyl benzilate ([(11)C](+)3-PPB) for cerebral muscarinic cholinergic receptors. The distribution and kinetics of the novel ligands were evaluated for comparison with the previously reported ligand (+)N-[(11)C]methyl-3-piperidyl benzilate ([(11)C](+)3-MPB) in the monkey brain (Macaca mulatta) in the conscious state using high-resolution positron emission tomography (PET). At 60-91 min postinjection, regional distribution patterns of these three ligands were almost identical, and were consistent with the muscarinic receptor density in the brain as previously reported in vitro. However, the time-activity curves of [(11)C](+)3-EPB and [(11)C](+)3-PPB showed earlier peak times of radioactivity and a faster clearance rate than [(11)C](+)3-MPB in cortical regions rich in the receptors. Kinetic analysis using the three-compartment model with time-activity curves of radioactivity in metabolite-corrected arterial plasma as input functions revealed that labeling with longer [(11)C]alkyl chain length induced lower binding potential (BP = k(3)/k(4)), consistent with the rank order of affinity of these ligands obtained by an in vitro assay using rat brain slices and [(3)H]QNB. The cholinesterase inhibitor Aricept administered at doses of 50 and 250 microg/kg increased acetylcholine level in extracellular fluid of the frontal cortex and the binding of [(11)C](+)3-PPB with the lowest affinity to the receptors was displaced by the endogenous acetylcholine induced by cholinesterase inhibition, while [(11)C](+)3-MPB with the highest affinity was not significantly affected. Taken together, these observations indicate that the increase in [(11)C]alkyl chain length could alter the kinetic properties of conventional receptor ligands for PET by reducing the affinity to receptors, which might make it possible to assess the interaction between endogenous neurotransmitters and ligand-receptor binding in vivo as measured by PET.

Animals↗

PubMed2001

Synthesis, absolute configuration, and enantiomeric enrichment of a cruciferous oxindole phytoalexin, (S)-(-)-spirobrassinin, and its oxazoline analog.

Stereochemical studies of a cruciferous oxindole phytoalexin, (S)-(-)-spirobrassinin [(-)-4], and its oxazoline analogue, spirooxazoline (11), were carried out. Racemic spirobrassinin [(+/-)-4] was synthesized by SOCl(2)- or MsCl-mediated cyclization of dioxibrassinin [(+/-)-8]. Treatment of (3-hydroxyoxindol-3-yl)methylammonium chloride [(+/-)-9] with CSCl(2) and subsequent methylation of the obtained spirooxazolidinethione (+/-)-10 afforded spirooxazoline [(+/-)-11]. Enantioresolution of (+/-)-4 and (+/-)-11 was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate (12), chromatographic separation of diastereomeric amides 13, 14 or 15, 16, and their cleavage with CH(3)ONa. Absolute configuration of the stereogenic center in natural (S)-(-)-4 was derived from the exciton, calculated via CD methods, and unequivocally confirmed by X-ray crystallographic analyses of 1-[1'S,4'R-(-)-camphanoyl] derivatives [(-)-19 and (-)-20] of (+)- and (-)-4. Novel enantiomeric enrichment phenomena of 4 and 11 were discovered during their chromatographic separations under achiral HPLC conditions. Screening of antifungal activity against the fungus Bipolaris leersiae revealed no significant dependence of this activity on absolute configuration.

Antifungal Agents↗

PubMed2001

Isochromosome consisting of terminal short arm and proximal long arm X in a girl with short stature.

A 16-year-old girl with short stature, short neck, shield chest, and cubitus valgus was studied. FISH analyses of her structurally altered X chromosome showed a der(X)- (wcpX+,TelXp/Yp++,SHOX++,STS++,KAL-, 37A12-,DXZ1+,XIST++,97L7++,300O13-,404F- 18-,417G15-,404F18-,140A-,TelXq/Yq-). These results, together with the high-resolution banding analysis, indicated her karyotype to be 46,X,der(X)(Xpter-->Xp22.3::Xq22.3--> cen-->Xq22.3::Xp22.3-->Xpter). The der(X) was an isochromosome, consisting of duplicated terminal short arms and duplicated proximal long arms. This in turn suggested that the chromosome was formed through pericentric inversion of an X chromosome, followed by isochromosome formation through sister chromatid exchange at Xp, close to the centromere. Replication R-banding analysis showed that the abnormal X chromosome was late replicating. Analysis of digestion patterns with a methylation-sensitive restriction endonuclease of the phosphoglycerate kinase 1 gene, located in Xq13.3, indicated that its inactivation patterns were completely skewed.

Adolescent↗

PubMed2001

Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen.

Serological expression cloning of antigens eliciting a humoral immune response to a syngeneic mouse sarcoma identified pem (mouse placenta and embryonic expression gene) as a new member of the cancer/testis family. To identify the human homologue of pem, mouse pem sequences and pem-related expressed sequence tags from human testis were used as PCR primers for amplification using human testis cDNA. However, rather than pem, another gene, designated OY-TES-1, was isolated and found to be the human homologue of proacrosin binding protein sp32 precursor originally identified in mouse, guinea pig, and pig. OY-TES-1 maps to chromosome 12p12-p13 and contains 10 exons. Southern blot analysis suggests the presence of two OY-TES-1-related genes in the human genome. In normal tissues, OY-TES-1 mRNA was expressed only in testis, whereas in malignant tissues, a variable proportion of a wide array of cancers, including bladder, breast, lung, liver, and colon cancers, expressed OY-TES-1. Serological survey of 362 cancer patients with a range of different cancers showed antibody to OY-TES-1 in 25 patients. No OY-TES-1 sera reactivity was found in 20 normal individuals. These findings indicate that OY-TES-1 is an additional member of the cancer/testis family of antigens and that OY-TES-1 is immunogenic in humans.

Amino Acid Sequence↗

PubMed2001

Development of novel telomerase inhibitors based on a bisindole unit.

Telomerase is the enzyme that elongates telomere repeat at the ends of a chromosome. As high telomerase activity is observed in most cancer cells, inhibitors of human telomerase have been expected as new chemotherapeutic agents for cancer. We describe here the discovery of novel inhibitors with IC50 values in the submicromolar range. The structure of the novel inhibitors will be useful as a scaffold for construction of the library in the search for telomerase inhibitors.

Enzyme Inhibitors↗

PubMed2001

Introduction of antisense CD44S CDNA down-regulates expression of overall CD44 isoforms and inhibits tumor growth and metastasis in highly metastatic colon carcinoma cells.

We created antisense CD44 transfectants using LS174T, a colon adenocarcinoma cell line and assessed the effects of overall CD44 down-regulation on colorectal tumor growth and metastasis. The expression of antisense CD44s (the standard form of CD44) cDNA markedly inhibited the overall expression of CD44 variants. In vitro studies showed a significantly reduced ability of the stable antisense transfectants (LS174TAS1 and LS174TAS2) to bind hyaluronate and osteopontin, ligands for CD44. These cells developed tumors more slowly than controls (parental LS174T and mock transfectants) when the cells were subcutaneously injected into SCID mice. However, in vitro proliferation assays demonstrated no significant difference between the antisense transfectants and the controls on a hyaluronate-coated surface, suggesting the participation of ligands other than hyaluronate in tumor growth in vivo. Intrasplenic injection of parental LS174T cells produced colonies in the liver in 10 of 11 mice, whereas mice injected with the antisense transfectants were completely free of metastasis. In peritoneal dissemination, the weight of nodules and amount of ascites were significantly reduced in LS174TAS1 and AS2 compared with the controls. In vitro adhesion assays between the transfectants or controls and human peritoneal mesothelial cells revealed that the binding of LS174T cells to mesothelial cells was partly mediated by CD44-hyaluronate interaction. These data suggest that CD44-hyaluronate interaction plays a crucial role in peritoneal dissemination in colorectal carcinoma. The results of our study demonstrate the possible application of antisense CD44s to the treatment of colorectal carcinoma.

Adenocarcinoma↗

PubMed2001

Phototrophic N2 fixation suppressed by activated sulfate reduction in anoxic rice soil slurries.

Interactions between sulfate reduction (SR) and phototrophic nitrogenase activities were investigated in rice soil slurries mixed with rice straw. Activation of SR by adding exogenous sulfate suppressed acetylene-reducing activity (ARA) of the slurries, which was associated with phototrophic purple bacteria (PB) enumerated to 108-109 MPN g-1 dry weight (dw) soil. Adding 5 mm sodium molybdate, an inhibitor of SR, markedly increased ARA. However, in the slurries receiving both molybdate and exogenous sulfate, the effects declined simultaneously with partial recovery of SR. These results indicate outcompetition of sulfate-reducing bacteria (SRB) with PB in rice soil, when sulfate concentrations are high enough to support SR. The increasing effects of molybdate on ARA continued during the incubation in the sulfate-depleted condition, probably because of absence of SR and toxicity of molybdate to methanogenesis. Accordingly, stopping activities of the competitive microorganisms may be efficient to increase N2 fixation in rice soil.

Enzyme Inhibitors↗

PubMed2001

Strict closed-system drainage for treating chronic subdural haematoma.

A comparative study chiefly of the recurrence rate of chronic subdural haematoma after two treatment modalities was conducted. Patients were divided into a burr hole strict closed-system drainage group (SCD group; n=56) and a burr hole closed-system drainage with irrigation group (CDI group; n=45). The burr hole strict closed-system drainage involved simply inserting a drainage tube into the haematoma cavity as quickly as possible after minimally incising the haematoma capsule. The introduction of air into the haematoma cavity was prevented, and irrigation was not performed. Symptoms in both groups disappeared soon after surgery, with no postoperative complications. Haematoma recurred in one patient (1.8%) of the SCD group compared with 5 (11.1%) of the CDI group. The rate of recurrence was significantly lower for the SCD than for the CDI group (p<0.05). In 4 of 5 recurrences in the CDI group, the volume of residual intracapsular air was sufficient after initial surgery. These results suggested that postoperative residual intracapsular air is a factor contributing to recurrence. Burr hole strict closed-system drainage is a simple, less invasive procedure with which to treat chronic subdural haematoma and the outcome is excellent. Furthermore, prevention of intracapsular air intrusion during surgery might help prevent recurrence.

Adult↗

PubMed2001

Direct inhibitory effect of adrenomedullin, calcitonin gene-related peptide, calcitonin, and amylin on cholecystokinin-induced contraction of guinea-pig isolated caecal circular smooth muscle cells.

We recently reported the direct inhibitory effect of adrenomedullin on caecal circular smooth muscle cells via cAMP system. This study was designed to determine whether the structurally related peptides to adrenomedullin (i.e.; calcitonin gene-related peptide (CGRP), calcitonin, and amylin) can inhibit the cholecystokinin octapeptide (CCK-8)-induced contractile response by exerting a direct action on guinea-pig caecal circular smooth muscle cells, and to compare the inhibitory potency of these peptides. In addition, to elucidate each intracellular mechanisms, the effects of an inhibitor of cAMP-dependent protein kinase, inhibitors of particulate or soluble guanylate cyclase on the each peptide-induced relaxation were investigated. Adrenomedullin, CGRP, calcitonin, and amylin inhibited the contractile response produced by CCK-8 in a dose-dependent manner, with IC50 values of 0.14 nM, 0.37 nM, 5.4 nM, and 160 nM, respectively. An inhibitor of cAMP-dependent protein kinase significantly inhibited the relaxation produced by all of these peptides. On the contrary, inhibitors of particulate or soluble guanylate cyclase did not have any significant effect on the relaxation produced by these peptides. In this study, we demonstrated the direct inhibitory effects of the structurally related peptides to adrenomedullin (i.e.; CGRP, calcitonin, and amylin) on the isolated caecal circular smooth muscle cells via cAMP system. The order of potency was as follows; adrenomedullin falling dots CGRP > calcitonin > amylin.

Adrenomedullin↗

PubMed2001

Age-specific prevalence of open-angle glaucoma and its relationship to refraction among more than 60,000 asymptomatic Japanese subjects.

To clarify the prevalence of open-angle glaucoma (OAG) and its relationship to refraction among a Japanese population with a broad range of ages including children and adolescents, an 1-year epidemiological survey was conducted. The subjects of this study were 64,394 asymptomatic individuals who had attended the glasses and contact lens center in Yokohama, Japan from February 15, 1999 to February 14, 2000 and had been subjected to several optical examinations. The results of this study showed that the overall prevalence of OAG was 1.19% (1.14% for men, and 0.98% for women). Prevalence for children aged 6 to 14 of both sexes was approximately 0.5%. There were significant positive associations between the strength (diopter) of myopic refraction and OAG prevalence among all the examined subjects.

Adolescent↗

PubMed2001

Aromatase in atherosclerotic lesions of human aorta.

It is well documented that estrogens have atheroprotective effects in humans. Peripheral aromatization of circulating androgens has been demonstrated to exert estrogenic actions in many human tissues, especially in men and post-menopausal women. Recently, production of estrogens mediated by aromatase was detected in cultured smooth muscle cells and aortic endothelial cells and it has been proposed that this in situ produced estrogen may influence the development of atherosclerosis. In this study, we first examined aromatase expression by immunohistochemistry in human aortic tissues obtained from 85 autopsy cases (50 males, 35 females, 49.6 +/- 2.9-year-old) and by mRNA in situ hybridization in 10 cases. We then semi-quantified the level of aromatase mRNA in aortic tissues of 12 men and 12 post-menopausal women by reverse transcriptase-polymerase chain reaction (RT-PCR) to examine whether or not and in which cell types aromatase was expressed. We also studied alternative use of multiple exon 1 of its gene and immunolocalization of 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD I), which converts estrone produced by aromatase to estradiol, a biologically active estrogen. Aromatase immunoreactivity and mRNA hybridization signals and 17beta-HSD I immunoreactivity were all detected in smooth muscle cell (SMC) of the media and thickened intima, especially in SMC adjacent to an atheromatous plaque. The levels of aromatase mRNA were significantly higher in female cases than in male cases (P<0.05). The amount of aromatase mRNA was significantly higher in the specimens with fibroatheroma (P<0.05) than other lesions, and was also significantly higher in the cases utilizing 1c (I.3) or 1d (PII) of exon 1, i.e. gonadal types than those utilizing 1b (I.4), i.e. fibroblasts type as the promoter (P<0.01). These results suggest that estrone and estradiol are produced in SMC of the human aortic wall and that their production is mediated by aromatase and 17beta-HSD I, respectively. Moreover, it was suggested that aromatase overexpression, possibly as a result of alternative splicing, may play some roles in the development of atherosclerosis.

Adolescent↗

PubMed2001

Characterization and purification of a protein binding to the cis-acting element for brain-specific exon 1 of the mouse aromatase gene.

The functional differences between male and female brains commit to the existence of androgen that the testis secretes during the perinatal period. Androgen exerts its action on the brain after conversion to estrogen by brain aromatase. The aromatase appears in some neural nuclei such as in the hypothalamus and amygdala, and has been indicated to be involved in the expression of sexuality by the results of neurobehavioral analyses involving aromatase-knockout mice. We analyzed the brain-specific promoter in order to clarify the control mechanism for the expression of brain aromatase, which is deeply concerned in the sexual differentiation of the brain. The 202bp upstream region of brain-specific exon 1 contains at least three kinds of cis-acting elements, Arom-Aalpha, -Abeta and -B. In particular, the binding activities as to the Abeta sequence show a tissue-specific pattern. Gel shift analysis revealed that the Abeta binding factor recognizes the TTGGCCCCT sequence. Abeta binding activity is detectable at the perinatal stage, but is undetectable at the adult stage in the brain. Furthermore, a protein which binds to the Abeta sequence was purified from the fetal mouse brain. The molecular mass of the Abeta binding protein was estimated to be 49kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Animals↗

PubMed2001

Analysis of transcriptional regulation of human breast aromatase by in vitro and in vivo studies.

Aromatase mRNA in normal breast tissues is mainly transcribed from exon 1b (I.4) of the gene. However, in breast cancer tissues, it is often transcribed from exon 1c/1d (I.3/PII). Such a switching from exon 1b to exon 1c/1d has often found concomitantly with elevated levels of aromatase mRNA. To elucidate the molecular mechanism of a switching of multiple exons 1 and enhanced expression of aromatase in the breast tissues, we identified essential elements responsible for transcription from exon 1b. Gel shift assays indicated that there are two essential elements for transcription from exon 1b between -300 and -500 bp in the promoter region. The two unique elements have homologous consensus DNA sequences, and competed for binding of the specific nuclear protein with each other. We next generated transgenic mouse expressing a reporter gene controlled by multiple promoters in the human aromatase gene. The mouse gave tissue-specific expression of the reporter gene and tissue-specific utilization of the alternative promoter regions. These results show that this transgenic mouse is a good model animal for the study of tissue-specific regulation of human aromatase gene.

Alternative Splicing↗

PubMed2001

Somatic activation of beta-catenin bypasses pre-TCR signaling and TCR selection in thymocyte development.

Mutation or ablation of T cell factor 1 and lymphocyte enhancer factor 1 indicated involvement of the Wnt pathway in thymocyte development. The central effector of the Wnt pathway is beta-catenin, which undergoes stabilization upon binding of Wnt ligands to frizzled receptors. We report here that conditional stabilization of beta-catenin in immature thymocytes resulted in the generation of single positive T cells that lacked the alpha beta TCR and developed in the absence of pre-TCR signaling and TCR selection. Although active beta-catenin induced differentiation in the absence of TCRs, its action was associated with reduced proliferation and survival when compared to developmental changes induced by the pre-TCR or the alpha beta TCR.

Animals↗

PubMed2001

Vitamin C inhibits corpus gastritis in Helicobacter pylori-infected patients during acid-suppressive therapy.

BACKGROUND: Previous studies have shown that gastric acid suppression worsens corpus gastritis in Helicobacter pylori (H. pylori)-positive patients. We evaluated the effect of acid-suppressive therapy and vitamin C on H. pylori-associated gastritis. METHODS: Forty patients with reflux esophagitis were divided into three groups by the status of H. pylori and therapy: group A (n=15), H. pylori (+) and omeprazole 20 mg; group B (n=15), H. pylori (+) and omeprazole 20 mg + vitamin C 1200 mg; and group C (n=10), H. pylori (-) and omeprazole 20 mg. In all three groups, the mucosal interleukin (IL)-8 contents, H. pylori colonization density, neutrophil infiltration in the corpus, and serum gastrin were evaluated at entry and 2 weeks after starting therapy; in group B, serum vitamin C levels were also measured. RESULTS: In group A, the IL-8 contents and the degree of neutrophil infiltration during therapy exceeded those at entry, whereas in groups B and C, these values did not change significantly with treatment. Helicobacter pylori colonization density during therapy was similar to that at entry in all three groups. The serum gastrin (in all groups) and vitamin C levels (in group B) during therapy exceeded those at entry. CONCLUSIONS: Potent acid suppression worsens H. pylori-associated corpus gastritis, although such worsening gastritis may be inhibited by vitamin C.

Acids↗

PubMed2001

Motoneuron pool excitability of hemiplegic patients: assessing recovery stages by using H-reflex and M response.

OBJECTIVES: To compare the excitability of the motoneuron pools of both the spastic and the unimpaired sides of patients with hemiplegia with a new method by using H-reflexes and M responses. The method determines the ratio of the developmental slope of the H-reflex (Hslp) to the slope of the M response (Mslp). We also examined the relation between the Brunnstrom stages and the Hslp/Mslp. DESIGN: Experiment. SETTING: Electrophysiologic experimental laboratory in a Japanese medical school. PATIENTS: Fifteen hemiplegic patients (9 men, 6 women; age range, 48-71yr; mean, 60yr) with spasticity caused by a stroke. INTERVENTIONS: The subject was instructed to relax while seated in a reclining chair with his foot fixed on an immobile pedal. After the soleus H-reflex and M responses on one side were recorded, the same experimental procedures were carried out on the other side. MAIN OUTCOME MEASURES: Hslp/Mslp Brunnstrom stages. RESULTS: Hslp/Mslp had better predictive value than conventional indicators of motoneuron pool excitability. Hslp/Mslp appeared to be a better match for the bell-shaped pattern of the Brunnstrom stages. CONCLUSION: Hslp/Mslp is the preferred index for evaluating the motoneuron pool excitability of the spastic side of hemiplegic patients.

Aged↗