Biomedical subjects

N Harada

Publications and source records attributed to N Harada.

At least 163 records · Page 9Linked to original sources

PubMed2000

Bond strengths of self-etching primer adhesives to in vitro-demineralized dentin following mineralizing treatment.

PURPOSE: The purpose of this study was to determine if treatment of experimentally demineralized dentin with a calcium phosphate precipitating (CPP) solution could restore the bond strength of self-etching adhesives to that of normal mineralized control values. MATERIALS AND METHODS: The occlusal surfaces of human teeth were exposed for bonding. The teeth were assigned to a mineralized control group or one of three demineralized (40% phosphoric acid for 60 s) experimental groups. One of the experimental groups remained untreated, while the other two were treated with the CPP solutions for either 1 or 10 min. All experimental groups were then chemically dehydrated with ascending acetone concentrations to prevent shrinkage prior to bonding with Clearfil Liner Bond 2 (LB2) or MacBond II (MBII). Composite buildups were made and vertically, serially sectioned into multiple slabs about 0.7 mm thick. Each slab was then trimmed for microtensile bond testing and SEM examination. RESULTS: Both self-etching adhesives produced high bond strengths to control mineralized dentin (ca 42 and 37 MPa for LB2 and MBII, respectively). Acid etching with 40% phosphoric acid for 60 s followed by chemical dehydration significantly lowered (p < 0.05) bond strength to 26 and 27 MPa for LB2 and MBII, respectively, and created hybrid layers between 7 and 8 microns thick. When the demineralized dentin was treated with the CPP solution for 1 min, the bond strengths increased (p < 0.05) to 39 for LB2 and 42 MPa for MBII, and the hybrid layers were 11 microns thick. Increasing the CPP treatment time to 10 min lowered (p < 0.05) bond strengths but increased the hybrid layer thickness to 16 to 20 microns. CONCLUSION: The use of calcium phosphate precipitating solutions may permit higher bond strengths of self-etching primers to demineralized (e.g., caries-affected) dentin.

Acid Etching, Dental↗

PubMed2000

An integrated analysis and database system for full-length cDNA.

Annotation and database system of full-length cDNA sequences was developed. As the components of the system, ORF annotation system, functional annotation system based on database search results, mapping annotation system, and integrated retrieval and display system were developed. In the ORF annotation system integrated analyses using conventional tools are performed and useful retrieval interface using motif list are introduced. In the functional annotation system based on database search results, a new method that characterizes a given unknown cDNA was developed by using a profile of similarity level over words appearing in sequence database entries. In the mapping annotation system, we linked by similarity searches full-length cDNA sequences with database DNA sequences that are already mapped on chromosomes. By using these links, full-length cDNAs can be retrieved by the retrieval condition of physical mapping information. Genetic disease information mapped on the physical mapping site can also be displayed by this system. Furthermore, we constructed an integrated database system for these analyzed data, and thus enabled annotation and selection of full-length cDNAs from points of both gene function and mapping information.

Chromosome Mapping↗

PubMed1999

Production of soluble granulocyte colony-stimulating factor receptors from myelomonocytic cells.

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b-CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b-CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.

Amino Acid Sequence↗

PubMed1999

Change in hepatic antioxidant defense system with liver injury development in rats with a single alpha-naphthylisothiocyanate intoxication.

The change in hepatic antioxidant defense system with the development of alpha-naphthylisothiocyanate (ANIT)-induced liver injury was examined in rats injected once with the toxicant (75 mg/kg body weight). Liver injury with cholestasis did not occur 12 h after ANIT injection, but appeared at 24 h, progressed at 48 h, and recovered at 72 h, judging from the serum levels of marker enzymes and components. Liver lipid peroxide content increased 12 h after ANIT injection and further increased 24 and 48 h, but this increase was attenuated at 72 h. Liver superoxide dismutase and catalase activities decreased 24 and 48 h, respectively, after ANIT injection, although the catalase activity increased at 12 h, but these decreases were attenuated at 72 h. Liver Se-glutathione peroxidase activity remained unchanged 24, 48, and 72 h after ANIT injection, although the activity increased at 12 h. Liver reduced glutathione content increased 24 h after ANIT injection, but the increase was reduced time dependently thereafter. Liver ascorbic acid content increased 12 h after ANIT injection and further increased at 24 h, but the increase was reduced time dependently thereafter. These results indicate that the change in hepatic antioxidant defense system occurs before and with the development of ANIT-induced liver injury in rats, and suggest that the reduction of hepatic antioxidant defense system mediated by SOD and catalase could contribute to the liver injury development through an enhancement of hepatic lipid peroxidation.

1-Naphthylisothiocyanate↗

PubMed1999

Isoflurane anesthesia enhances the inhibitory effects of cocaine and GBR12909 on dopamine transporter: PET studies in combination with microdialysis in the monkey brain.

The effects of the dopamine transporter (DAT) inhibitors cocaine and GBR12909 on DAT and dopamine D(2) receptors were evaluated in the brains under awake and isoflurane-anesthetized monkeys using high-resolution positron emission tomography (PET) in combination with microdialysis. The striatal DAT availability and dopamine D(2) receptor binding were assayed with [11C]beta-CFT (WIN35,428) and [11C]raclopride, respectively. Cocaine or GBR12909 at a dose of 2 mg/kg was administered intravenously 30 min prior to the injection of labeled compounds. In the awake state, the in vivo binding of [11C]beta-CFT to DAT was significantly decreased by administration of cocaine or GBR12909 at a dose of 2 mg/kg. In contrast, [11C]raclopride binding to dopamine D(2) receptors was decreased only by GBR12909. Under isoflurane anesthesia, dopamine concentration in the striatal extracellular fluid (ECF), as measured by microdialysis, was markedly increased by cocaine or GBR12909 compared to the awake state. Isoflurane anesthesia more markedly enhanced the binding of [11C]beta-CFT in the saline-injected animals, and the degrees of reduction by cocaine and GBR12909 were more marked than those observed in the awake state. Under isoflurane anesthesia, the binding of [11C]raclopride was reduced not only by GBR12909 but also by cocaine which did not affect the binding in the awake state. Taken together, these observations indicated that isoflurane anesthesia enhanced not only the direct inhibitory effects of cocaine and GBR12909 on DAT, but also their indirect effects on dopamine D(2) receptors.

Anesthesia, Inhalation↗

PubMed1999

Digynic triploid infant surviving for 46 days.

We report on a triploid infant who survived for 46 days. She had severe intrauterine growth retardation, relative macrocephaly, and a small, noncystic placenta, which are manifestations compatible with type II phenotype. Cultured amniotic fluid cells, skin fibroblasts, cord blood, and peripheral blood lymphocytes all showed a nonmosaic 69,XXX karyotype. Analysis of chromosomal heteromorphisms and microsatellite DNA polymorphisms in the infant and her parents indicated that the extra haploid set in the infant resulted from nondisjunction at maternal second meiosis. Postzygotic, mitotic nondisjunction was ruled out because of the presence of both homozygous and heterozygous markers of maternal origin. A search of the literature demonstrated five triploid infants, including the girl we described, who survived 4 weeks or more, and the parental origin of whose triploidy was studied: four were digynic and one was diandric. These findings support the notion that type II triploids are digynic in parental origin and that they survive longer than type I, diandric triploids.

Abnormalities, Multiple↗

PubMed1999

A GCM motif protein is involved in placenta-specific expression of human aromatase gene.

A new cis-element, trophoblast-specific element 2 (TSE2) is located in the placenta-specific enhancer of the human aromatase gene that dictates its tissue-specific expression. In the minimum enhancer region, an element similar to the trophoblast-specific element (TSE), originally described for the human chorionic gonadotropin alpha-subunit gene, also exists (Yamada, K., Harada, N., Honda, S., and Takagi, Y. (1995) J. Biol. Chem. 270, 25064-25069). The co-presence of TSE and TSE2 is required to direct trophoblast-specific expression driven by a heterologous thymidine kinase promoter. A 2562-base pair cDNA clone encoding a 436-amino acid protein that binds to TSE2 was isolated from a human placental cDNA library using a yeast one-hybrid system with the TSE2 as a reporter sequence. The protein was revealed to be identical to hGCMa, a mammalian homologue of the Drosophila GCM (glia cells missing) protein. Expression of hGCMa is restricted to the placenta. The protein also binds to PLE1 in the leptin promoter among other cis-elements reported to confer placenta-specific expression, suggesting that hGCMa is a placenta-specific transcription regulator, possibly involved in the expression of multiple placenta-specific genes.

Aromatase↗

PubMed1999

Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene.

Ectopic expression of certain Wnt genes in mouse mammary tissue is tumorigenic, and mutations that stabilize beta-catenin are found in various human cancers including colorectal cancer. To determine the role of stabilized beta-catenin in intestinal tumorigenesis in mice, we constructed by embryonic stem (ES) cell-mediated homologous recombination, a mutant beta-catenin allele whose exon 3 was sandwiched by loxP sequences. When the germline heterozygotes were crossed with mice expressing Cre recombinase in the intestines, the serines and threonine encoded by exon 3 and to be phosphorylated by glycogen synthase kinase 3beta (GSK3beta) were deleted in the offspring intestines, which caused adenomatous intestinal polyps resembling those in Apc(Delta716) knockout mice. Some nascent microadenomas were also found in the colon. These results present experimental genetic evidence that activation of the Wnt signaling pathway can cause intestinal and colonic tumors.

Animals↗

PubMed1999

Significance of Steroid Sulfatase Expression in Human Breast Cancer.

The sulfatase pathway has been thought to be a primary means of local production of estrone in human breast cancer tissue. We measured steroid sulfatase (STS) mRNA levels in 97 breast cancers and evaluated its association with disease-free survival. High levels of STS mRNA proved to be a significant predictor of reduced relapse-free survival, both as a continuous variable (log STS mRNA; P = 0.028) and as a dichotomous variable with an optimized cutoff point (P=0.002). In multivariate analysis a high level of STS mRNA was an independent factor for predicting relapse-free survival. These results suggest a putative role of STS in breast cancer growth and metastasis, and administration of sulfatase inhibitors to breast cancer patients with high levels of STS mRNA might be an additional treatment option.

Journal Article↗

PubMed1999

Is synaptic dopamine concentration the exclusive factor which alters the in vivo binding of [11C]raclopride?: PET studies combined with microdialysis in conscious monkeys.

The effects of dopamine release manipulated by drugs on the in vivo binding of [11C]raclopride in the striatum were evaluated in conscious monkeys combined with microdialysis. The in vivo binding of [11C]raclopride was evaluated by high resolution positron emission tomography (PET), and the dopamine concentrations in the striatal extracellular fluid (ECF) were measured by microdialysis in the same animals. The systemic administration of the direct dopamine enhancers, GBR12909 (a dopamine transporter (DAT) blocker, at 0.5, 2 and 5 mg/kg) or methamphetamine (a dopamine releaser, at 0.1, 0.3 and 1 mg/kg) dose-dependently increased the dopamine concentration in the striatal ECF, and decreased in vivo [11C]raclopride binding in the striatum. The administration of the indirect dopamine modulators benztropine (a muscarinic cholinergic antagonist, at 0.1, 0.3 and 1 mg/kg) or ketanserine (a 5-HT2 antagonist, at 0.3, 1 and 3 mg/kg) also increased dopamine level in the striatal ECF, and decreased [11C]raclopride binding in a dose-dependent manner. However, the plots of percentage change in dopamine concentration in striatal EFC against that in [11C] raclopride binding indicated different relationships between the effects of direct dopamine enhancers (GBR12909 and methamphetamine) and indirect dopamine modulators (benztropine and ketanserine). These results suggested that the alternation of [11C]raclopride binding in vivo as measured by PET was differently affected by different neuronal manipulations, and not simply by the synaptic concentration of dopamine.

Animals↗

PubMed1999

Light-driven monodirectional molecular rotor.

Attempts to fabricate mechanical devices on the molecular level have yielded analogues of rotors, gears, switches, shuttles, turnstiles and ratchets. Molecular motors, however, have not yet been made, even though they are common in biological systems. Rotary motion as such has been induced in interlocked systems and directly visualized for single molecules, but the controlled conversion of energy into unidirectional rotary motion has remained difficult to achieve. Here we report repetitive, monodirectional rotation around a central carbon-carbon double bond in a chiral, helical alkene, with each 360 degrees rotation involving four discrete isomerization steps activated by ultraviolet light or a change in the temperature of the system. We find that axial chirality and the presence of two chiral centres are essential for the observed monodirectional behaviour of the molecular motor. Two light-induced cis-trans isomerizations are each associated with a 180 degrees rotation around the carbon-carbon double bond and are each followed by thermally controlled helicity inversions, which effectively block reverse rotation and thus ensure that the four individual steps add up to one full rotation in one direction only. As the energy barriers of the helicity inversion steps can be adjusted by structural modifications, chiral alkenes based on our system may find use as basic components for 'molecular machinery' driven by light.

Alkenes↗

PubMed1999

Synthesis of taxoids 5. Synthesis and evaluation of novel water-soluble prodrugs of a 3'-desphenyl-3'-cyclopropyl analogue of docetaxel.

A novel 3'-desphenyl-3'-cyclopropyl analogue of docetaxel was synthesized from 10-deacetyl-baccatin III. The cytotoxicity of the new taxoid was evaluated against several human tumor cell lines, and it had ca. 20 times stronger activity against human colon cancer cell lines (WiDr and Colon 320) than that of docetaxel. This taxoid was converted to its water-soluble prodrugs that have 2'-substituted amino acid derivatives with spacer. The prodrugs had good solubility in saline and showed more potent antitumor activity against B 16 melanoma in mice than that of docetaxel.

Animals↗

PubMed1999

Localized expression of aromatase in human vascular tissues.

The atheroprotective effects of estrogen are well established and the presence of an estrogen receptor in vascular tissues has recently been reported. Therefore, we investigated the localization of the estrogen-producing enzyme aromatase in vascular tissues to assess the possible contribution of endocrine, paracrine, and autocrine modes of action. Aromatase was found in human vascular smooth muscle cells (SMCs) but not in endothelial cells on in situ hybridization. These observations were further supported by quantitative analysis of aromatase mRNA and the activity in 15 human vascular specimens. Only trace levels of expression were detected in the 3 infants examined, whereas 0.0088 to 0.0806 amol/ microg RNA of aromatase mRNA and 12.9 to 122.3 fmol. h-1. mg-1 protein of the activity were detected in 12 of the adult individuals. The switching of tissue-specific exon 1 of the human aromatase gene was also observed in some cases. Aromatase was found to be expressed only in cultured SMCs and not in cultured endothelial cells of human aorta and pulmonary artery and to be regulated through dexamethasone and the signaling pathways of protein kinase A and C. Study results revealed the localized expression of aromatase in vascular SMCs, which indicated a possible direct action of locally produced estrogen in an autocrine or paracrine manner, with possible cross talk between smooth muscle and endothelial cells.

Adolescent↗

PubMed1999

In vivo interaction of AF-6 with activated Ras and ZO-1.

AF-6 contains two putative Ras-associating domains (RA domains) which are seen in several Ras effectors such as RalGDS and RIN1. We previously showed that an AF-6 fragment containing the amino-terminal (N-terminal) RA domain directly binds to activated Ras and ZO-1 in vitro. In this study, we showed that a single amino acid mutation in the N-terminal RA domain of AF-6 abolished the interaction of AF-6 with activated Ras and that the sites of this critical amino acid residue were similar to those for Raf-1 and RalGDS. The overexpression of the N-terminal RA domain of AF-6 inhibited the Ras-dependent c-fos promoter/enhancer stimulation in NIH3T3 cells. Endogenous AF-6 was coimmunoprecipitated with activated Ras from Rat1 cells expressing activated Ras. Moreover, we showed that AF-6 was coimmunoprecipitated with ZO-1 from Rat1 cells. Taken together, these results indicate that the Ras-interacting region on AF-6 is structurally similar to that on Raf-1 and on RalGDS and that AF-6 interacts with activated Ras and ZO-1 in vivo.

3T3 Cells↗

PubMed1999

An association between lipid peroxidation and alpha-naphthylisothiocyanate-induced liver injury in rats.

An association between lipid peroxidation and alpha-naphthylisothiocyanate (ANIT)-induced liver injury was examined in rats injected once with the toxicant (75 mg/kg body weight). The severity of liver injury was estimated 12, 24, 48, and 72 h after ANIT injection. Liver injury appeared 24 h after ANIT injection, progressed at 48 h, and recovered at 72 h, judging from the serum levels of marker enzymes and components. Serum lipid peroxide (LPO) concentration increased 24 h after ANIT injection and further increased at 48 h, but this increase was attenuated at 72 h. In contrast, liver LPO content increased 12 h after ANIT injection and further increased 24 and 48 h, but this increase was attenuated at 72 h. Similarly, myeloperoxidase (MPO) activity, an index of neutrophil infiltration, in the liver tissue increased 12 h after ANIT injection and further increased at 24 and 48 h, but this increase was attenuated at 72 h. Either serum LPO concentration or liver LPO content was significantly correlated with liver MPO activity (r = 0.661 for serum LPO concentration; r = 0.585 for liver LPO content). These results suggest that lipid peroxidation might be associated with ANIT-induced liver injury in rats and that this lipid peroxidation might occur via oxygen radicals derived from neutrophils infiltrated into the liver tissue of ANIT-intoxicated rats.

1-Naphthylisothiocyanate↗

PubMed1999

Identification of cis-acting elements in the proximal promoter region for brain-specific exon 1 of the mouse aromatase gene.

Among multiple exons 1 of the mouse aromatase gene, brain-specific exon 1 is only utilized in the hypothalamus and amygdala regions. In this study, identification of the promoter region necessary for basal transcription of the aromatase gene in the brain was undertaken. Deletions of various lengths were introduced into the overall promoter region, which was fused to the chloramphenicol acetyltransferase gene. The resulting reporters were transfected into cultured neurons from the diencephala of fetal mouse brains on embryonic day 13 and then their CAT mRNA levels were determined. The reporter plasmid containing the promoter region 202 bp upstream from the transcriptional initiation site gave the greatest expression. Then binding of trans-acting factors in a nuclear extract of the diencephala to the -202 bp promoter region was investigated by DNase I footprint analysis, multiple protected areas, referred to as Arom-Aalpha, Abeta, Agamma, B and C, being found. Gel shift assays, performed with oligonucleotides corresponding to the protected areas, showed that nuclear DNA binding factors form specific complexes exhibiting different mobilities. Substitution in the Arom-Aalpha or -B sequence in the promoter region in the CAT reporters decreased the CAT mRNA expression levels to about one-fifth the wild type one. These results suggest that multiple nuclear factors bound to the core promoter region participate in the expression of the aromatase gene in mouse brain neurons.

Albumins↗

PubMed1999

Steroid sulfatase expression is an independent predictor of recurrence in human breast cancer.

Steroid sulfatase (STS) hydrolyzes several sulfated steroids such as estrone sulfate, dehydroepiandrosterone sulfate, and cholesterol sulfate. In the present study, we have measured STS mRNA levels in 97 breast cancers by reverse transcription-PCR using a fluorescent primer in the presence of an internal standard RNA and evaluated its association with disease-free and overall survival. The median value was 728.0 amol/ng RNA (range, 0-11,778 amol/ng RNA). Levels were significantly higher in tumors demonstrating lymph node metastasis than in those without nodal involvement (P = 0.033) and in patients who experienced a recurrence during the follow-up period (mean, 40.8 months; median, 39 months) compared with those with no evidence of further disease (mean, 49.2 months; median, 48 months; P = 0.029). No significant associations were found between STS mRNA expression and age, menopausal status, tumor size, histological grade, estrogen receptor status, or postoperative adjuvant therapy. High levels of STS mRNA proved to be a significant predictor of reduced relapse-free survival as a continuous variable (log STS mRNA; P = 0.028). As a dichotomous variable with an optimized cutoff point of 1,240 amol/ng RNA, expression was also associated with a significantly shorter relapse-free survival rate (P = 0.002), but no significant correlation was found between the STS mRNA level and overall survival. Expression was found to be an independent factor for predicting relapse-free survival on multivariate analysis. The results thus support a putative role of STS in breast cancer growth and metastasis.

Adult↗

PubMed1999

Trizygotic pregnancy consisting of two fetuses and a complete hydatidiform mole with dispermic androgenesis.

A triplet pregnancy in a 23-year-old woman was terminated at 15 weeks of gestation because of her severe hypertension, lung edema, and secondary hyperthyroidism. The pregnancy consisted of a hydatidiform mole with a 46,XY karyotype and two fetuses each with 46,XX and a 46,XY karyotype. To determine the zygosity and genetic origin of the mole and fetuses, PCR- and computer-assisted genotyping were performed at 27 CA-repeat marker loci that were distributed evenly over the genome. As a result, genotypes of the three pregnancy products were distinct from each other, indicating that the triplets were trizygotic. The mole lacked any maternal alleles but inherited both of the paternal alleles and/or one paternal allele in duplicate. This, along with the XY sex chromosome constitution, indicated that the mole resulted from dispermic androgenesis. The mother developed a persistent trophoblastic tumor thereafter.

Abortion, Therapeutic↗