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Y Nakahara

Publications and source records attributed to Y Nakahara.

At least 19 recordsLinked to original sources
PubMed2005

Detection of p16 promoter methylation in the serum of oral cancer patients.

P16 promoter methylation occurs frequently in oral squamous cell carcinoma (OSCC). For the early detection of tumour-related aberrant DNA, we examined p16 methylation using the methylation-specific polymerase chain reaction (MSP) in tumour and serum samples of 17 OSCC patients. Aberrant p16 methylation was detected in 11 (64.7%) cases of primary OSCC. Of these 11 patients, 6 (54.5%) showed the same alteration in their serum. No methylation was found in control groups. Interestingly, DNA was detected in the serum of 3 out of 4 patients with recurrence. These results suggest that the MSP may be a sensitive and useful method for detecting recurrent OSCC.

Adult↗
PubMed2004

Hormonal control of a metamorphosis-specific transcriptional factor Broad-Complex in silkworm.

Insect metamorphosis is induced by the steroid 20-hydroxyecdysone (20E) in the absence of sesquiterpenoid juvenile hormone (JH). In Drosophila melanogaster, the Broad-Complex (BR-C) transcriptional factor plays critical roles during metamorphosis. We isolated cDNAs encoding BR-C in the silkworm Bombyx mori and examined their mRNA expression. cDNAs for three BR-C isoforms with zinc finger pairs (Z1, Z2 and Z4) and four isoforms lacking them were cloned. Their mRNAs were expressed in multiple tissues at the larval-pupal metamorphosis. In the anterior silk gland, BR-C mRNAs were expressed at the end of the last larval instar but not expressed during the penultimate instar. 20E administration induced BR-C mRNA expression and JH suppressed 20E-induced BR-C expression in this tissue both in vivo and in vitro. Thus, BR-C mRNAs are inducible by 20E only in the absence of JH, a finding that explains their metamorphosis-specific expression.

Animals↗
PubMed2003

Effect of daily walking steps on ultrasound parameters of the calcaneus in elderly Japanese women.

In this cross-sectional study, we investigated the effect of daily walking steps on ultrasound parameters of the calcaneus in elderly Japanese women. The subjects were 143 community-dwelling elderly women aged 61-87 years (mean age 71.4+/-5.5 years). The speed of sound (SOS), broadband ultrasound attenuation (BUA) and the stiffness index (Stiffness) of the calcaneus were measured. Walking steps were recorded using a pedometer for 7 consecutive days as an outcome measure of physical activity. In univariate analyses, steps/day significantly decreased with aging. SOS, BUA and Stiffness showed negative correlations with age and positive correlations with weight. Linear relationships were not seen between any of the ultrasound parameters and daily walking steps. Then, the ultrasound parameters were adjusted for age and/or weight using multiple regression models, and the relationships between the adjusted ultrasound measurements and walking steps were examined using quadratic regression models. Walking activity up to approximately 12,000 steps/day was positively associated with the adjusted ultrasound measurements, above which additional walking steps had no positive effect. We conclude that daily walking steps may be suitable for evaluating the relationship between ultrasound parameters and physical activity in elderly women, but further research is needed to confirm the effect of daily walking steps on the rate of bone loss.

Age Factors↗
PubMed2001

Determination of triazolam involving its hydroxy metabolites in hair shaft and hair root by reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair analysis.

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry has been developed for simultaneous determination of triazolam and its hydroxy metabolites in hair. After the addition of deuterium-labeled 1-hydroxymethyltriazolam as an internal standard, the analytes in hair shaft and hair root samples were extracted with a basic medium, CH(2)Cl(2):MeOH:28% NH(4)OH (20:80:2) at room temperature overnight. The chromatographic separation of the analytes was achieved using a semimicro HPLC column (3-microm particle size; 100 x 2.0-mm i.d.) by gradient elution with acetonitrile in water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at quasi-molecular ions [M+H](+) of triazolam and its metabolites. The method has been applied to determine the incorporation of triazolam and its metabolites into the hair shafts and hair roots of Dark Agouti rats administered 3 or 6 mg/kg triazolam intraperitoneally twice a day for 5 days. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were incorporated into the hair shafts and the hair roots. The concentration of 4-hydroxytriazolam was the highest of all compounds detected. An unknown substance considered to be 1,4-dihydroxytriazolam also appeared in the hair samples. The structural elucidation was performed with online HPLC-MS after acetylation of the substance with acetic anhydride and pyridine. The time course studies of triazolam and the metabolites in both rat hair roots and plasma were carried out after single intraperitoneal administration of triazolam. The concentrations of triazolam and the metabolites in the hair roots reflected those in the plasma. The proposed method using selected-reaction monitoring was applied to the determination of triazolam and the metabolites in human hairs of a triazolam addict. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were identified in the black hair shafts, whereas only triazolam was detected in the hair roots and the white hair shafts. This is the first report on the detection of triazolam and its metabolites in human hairs.

Aged↗
PubMed2001

Molecular cloning of silkworm paralytic peptide and its developmental regulation.

The silkworm paralytic peptide (PP) is a member of the ENF peptide family that exerts multiple biological activities involved in defense reaction and growth regulation. We isolated its cDNA and examined mRNA expression profiles. cDNA encoded 131 amino acids from which the 23-residue PP sequence was found at the C-terminal portion. Immunoblot analysis and paralytic activity assay indicated that inactive pro-protein in larval hemolymph was processed into active peptide immediately after bleeding. In the last larval instar, 0.6-kb PP mRNA was expressed in various tissues, of which the fat body was predominant. Its expression in the fat body decreased during the feeding period and then increased during metamorphic process. Juvenile hormone and 20-hydroxyecdysone upregulated its expression. At the embryonic stage, 1.5-kb mRNA, in addition to 0.6-kb mRNA, was expressed from 1 day after oviposition to hatching. PP was thus expressed stage-specifically under hormonal control.

Amino Acid Sequence↗
PubMed2001

Salmonella enteritidis FliC (flagella filament protein) induces human beta-defensin-2 mRNA production by Caco-2 cells.

Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a approximately 2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-kappaB (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-kappaB activation and thus plays an important role in up-regulation of the innate immune response.

Amino Acid Sequence↗
PubMed2001

High frequency of homozygous deletion and methylation of p16(INK4A) gene in oral squamous cell carcinomas.

p16(INK4A) inactivation was analyzed in ten squamous cell carcinoma (SCC) cell lines and 32 primary SCCs, using the polymerase chain reaction (PCR), PCR-single-strand conformation polymorphism, methylation-specific PCR, and cycle sequencing. In the study of cell lines, we detected three deletions in exon 1alpha and exon 2, and detected two methylations. Among tumor samples, we detected the homozygous deletions (HDs) of 43.8% in exon 1alpha 34.4% in exon 2, and methylation was found in 50.0%. The lack of p16(INK4A) with immunohistochemistry was detected in 71.9% and matched the alteration of p16(INK4A) gene. These results suggest that p16(INK4A) inactivation is predominantly caused by HD and methylation, and immunohistochemical evaluation of p16(INK4A) is a useful method.

Carcinoma, Squamous Cell↗
PubMed2001

Association of IL-8 and MCP-1 with the development of reexpansion pulmonary edema in rabbits.

BACKGROUND: The aim of this study is to determine the relationships between the cytokines and the inflammatory response in reexpansion pulmonary edema (RPE). METHODS: We examined the cell population, epithelial permeability measured by Evans blue dye (EB), betaglucuronidase and cytokine concentrations in bronchoalveolar lavage fluid (BALF) and/or blood using a rabbit RPE model. RESULTS: We confirmed that RPE is characterized by recruitment of polymorphonuclear leukocytes (PMNs), the release of PMN granular contents into the air spaces, and increased vascular permeability. These findings were highly correlated with increased interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) concentrations in the BALF. Growth related oncogene (GRO) was detected in the BALF from only 2 of the 7 reexpanded lungs while TNFalpha was not detected in any rabbits. A similar but less severe inflammatory response to the reexpanded lung was found in the contralateral lung. CONCLUSIONS: IL-8 and MCP-1 may play important roles in the development of RPE; the inflammatory response is independent of TNFalpha and unilateral reexpansion of the lung induces an inflammatory response not only in the reexpanded lung but also in the contralateral lung.

Animals↗
PubMed2001

Inactivation of the p14(ARF), p15(INK4B) and p16(INK4A) genes is a frequent event in human oral squamous cell carcinomas.

The p14(ARF), p15(INK4B) and p16(INK4A) genes were localized to 9p21, where genetic alterations have been reported frequently in various human tumors. We performed a molecular analysis of the mechanism of inactivation in cell lines and 32 oral squamous cell carcinoma (OSCC), using deletion screening, PCR-SSCP, methylation-specific-PCR and cycle sequencing. We detected homozygous deletion of p14(ARF)-1Ebeta in 9 (26.5%), of p15(INK4B) in one (3.1%), and of p16(INK4A) in 22 (56.3%) tumor samples. Three mutations were detected in the p16(INK4A) genes. We detected aberrant methylation of the p14(ARF) genes in 14 (43.8%), of the p15(INK4B) gene in 9 (28.1%), and of the p16(INK4A) gene in 16 (50.0%) tumor samples. Altogether, 87.5% of the samples harbored at least one of the alterations in the p14(ARF), p15(INK4B), and p16(INK4A) genes, indicating that the frequent inactivation of these genes may be an important mechanism during OSCC development.

Carcinoma, Squamous Cell↗
PubMed2001

Simultaneous determination of morphine and its glucuronides in rat hair and rat plasma by reversed-phase liquid chromatography with electrospray ionization mass spectrometry.

The simultaneous determination of morphine and the glucuronide metabolites [morphine-3-beta-D-glucuronide (M3G) and morphine-6-beta-D-glucuronide (M6G)] in rat hair and rat plasma was carried out using reversed-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). The chromatographic separation of the analytes was achieved using a semi-micro-HPLC column (3 microm particle size; 100 x 2.0 mm id) by gradient elution with 50 mM ammonium acetate and acetonitrile as eluents. After separation, morphine and the glucuronides were determined by selected ion monitoring (SIM) of ESI-MS using the quasi-molecular ions [M + H]+ at m/z = 286 and 462, respectively. The calibration curves were linear between the concentration of the analytes and the deuterium-labelled morphine (M-d3) selected as internal standard. The method was applied for the determination of the incorporation of morphine and the glucuronides into the hair shafts and hair roots of Dark Agouti rats after single intraperitoneal administration of morphine hydrochloride. Plasma concentrations of morphine and glucuronides were simultaneously determined after administration. Morphine and M3G were detected in the hair shafts and the hair roots. The concentrations of M3G in the hair root were lower than those of morphine in all sampling periods. In contrast, M3G concentrations in plasma were relatively higher at each sampling time. Small quantities of M6G were also identified in the plasma up to 4 h after administration. The concentration difference between the hair root and plasma seems to be due to the incorporation ratio of morphine and glucuronide into hair. As M3G was also identified in the hair shaft 1 week after administration, the incorporation of glucuronide metabolites into hair is obvious. This is the first report of the identification of morphine glucuronide in hair samples without the use of acid hydrolysis or enzyme digestion.

Animals↗
PubMed2001

Helicobacter pylori-mediated transcriptional regulation of the human beta-defensin 2 gene requires NF-kappaB.

Human beta-defensin 2 (hBD-2) is an antimicrobial peptide involved in host defence against bacterial infection in epithelial tissues. Its levels are dramatically increased after bacterial infection. The involvement of NF-kappaB in Helicobacter pylori-mediated induction of hBD-2 promoter activity was examined. A luciferase reporter plasmid containing the hBD-2 promoter extending from -2110 base pairs to -1 was transiently expressed in MKN45 cells, and promoter activity was determined after incubation with H. pylori for 6 h. Deletion or mutation of the NF-kappaB site at -208 abolished activation of the hBD-2 promoter. Only H. pylori strains carrying a cag pathogenicity island (PAI) induced activation of the NF-kappaB site of the hBD-2 promoter gene. By gel retardation analyses, H. pylori increased NF-kappaB binding to hBD-2 promoter gene sequences. Supershift analysis demonstrated that whereas H. pylori activated NF-kappaB p65-p65 and p50-p50 homodimers, and the p65-p50 heterodimer of NF-kappaB, only the p65-p65 homodimer bound to the NF-kappaB site of the hBD-2 promoter. Thus, specific NF-kappaB proteins are important cis-elements for induction of hBD-2 gene transcription by H. pylori.

Cell Line↗
PubMed2001

Overexpression of CDK2 is a prognostic indicator of oral cancer progression.

Cyclins and cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation, a process of which dysregulation can lead to uncontrolled cell growth and hence to cancer. We have already reported the alteration of CDK4 and cyclin D1 expression in oral cancer. In this study, we examined by immunohistochemistry the expression of CDK2, and cyclins A and E in 20 normal oral mucosa, 42 dysplastic epithelia, and 103 oral squamous cell carcinomas (SCCs). The expressions of CDK2, and cyclins A and E were not detected in the normal epithelium and significantly altered from epithelial dysplasia to SCC. While there were no significant correlations between the expression of cyclins A, E and the patients' survival, CDK2 expression was significantly correlated with lymph node involvement (P = 0.025), tumor differentiation (P = 0.032), mode of tumor invasion (P = 0.017), and shorter survival period (P = 0.0173). These results suggest that the elevated expression of CDK2 is a critical factor in oral cancer progression and can be used as a negative predictive marker of the patients' prognosis.

Adult↗
PubMed2001

Expression of human beta-defensin 2 (hBD-2) in Helicobacter pylori induced gastritis: antibacterial effect of hBD-2 against Helicobacter pylori.

BACKGROUND: Human beta-defensin 2 (hBD-2) plays a role in the innate defence system at mucosal surfaces. Colonisation of Helicobacter pylori in the stomach is an important pathological factor in gastrointestinal illnesses, including gastritis, peptic ulcer, and gastric adenocarcinoma. AIMS: To evaluate the antibacterial role of hBD-2 against H pylori infection in the gastric mucosa. SUBJECTS: Biopsied gastric mucosa specimens from H pylori positive (n=6) and H pylori negative (n=6) individuals were used. H pylori was determined by the presence of urease activity and microscopic examination. METHODS: The specimens were examined for hBD-2 expression by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridisation. The antibacterial effect of hBD-2 against H pylori was evaluated by the number of colony forming units of H pylori after incubation with 0, 10(-9), 10(-8), 10(-7), 10(-6), or 10(-5) M of hBD-2 peptide. RESULTS: All six H pylori positive specimens expressed a high level of hBD-2 mRNA while hBD-2 mRNA was not detected in the H pylori negative specimens by RT-PCR. Immunohistochemistry using anti-hBD-2 antiserum revealed that hBD-2 was expressed in the surface epithelium of H pylori infected specimens. In gastric specimens obtained after H pylori eradication, hBD-2 immunoreactivity had dramatically decreased. In situ hybridisation confirmed that hBD-2 transcripts were localised in the epithelium of H pylori infected gastric specimens. Incubation with hBD-2 reduced the growth rate of cultured H pylori in a dose dependent manner, and incubation with 10(-5) M hBD-2 completely inhibited the proliferation of H pylori. CONCLUSIONS: H pylori infection induces hBD-2 expression in the human gastric epithelium. hBD-2 inhibited the growth of H pylori in vitro, suggesting that hBD-2 plays an antibacterial role in H pylori induced gastritis.

Aged↗
PubMed2001

Increased interleukin-5 levels in bronchoalveolar lavage fluid is a major factor for eosinophil accumulation in acute eosinophilic pneumonia.

BACKGROUND: Increased interleukin-5 (IL-5) levels have been reported in bronchoalveolar lavage fluid (BALF) from patients with acute eosinophilic pneumonia (AEP); however, it still remains to be determined whether IL-5 is responsible for the eosinophil accumulation in the lung. OBJECTIVE: We examined the effect of antibodies against cytokines on eosinophil chemotaxis induced by BALF from AEP patients to identify factors responsible for eosinophil accumulation. METHODS: We measured a series of specific cytokines, including IL-3, IL-4, IL-5, IL-6, IL-8, GM-CSF, RANTES, MCP-1, MIP-1alpha and eotaxin, in the BALF from 4 patients with AEP. BALF from 4 patients with chronic eosinophilic pneumonia (CEP) and 13 patients with non-eosinophilic interstitial lung diseases (ILD) were examined as controls. The eosinophil chemotactic activity in the BALF was examined using tissue culture insert furnished with a polycarbonate membrane. RESULTS: The total protein content in BALF from patients with AEP was extremely elevated. Even after standardization with protein concentration, IL-5 levels in AEP patients were significantly higher than those in CEP and ILD. IL-3 and chemokines were rather lower in the AEP group than in the CEP and ILD groups. In AEP BALF, anti-IL-5 neutralizing antibody significantly inhibited eosinophil chemotaxis. Antibodies against IL-3, GM-CSF, and IL-8 did not affect the eosinophil migration. CONCLUSION: These findings suggest that locally produced IL-5 plays an important role in eosinophil accumulation of AEP.

Acute Disease↗
PubMed2001

Hair analysis for pharmaceutical drugs. II. Effective extraction and determination of sildenafil (Viagra) and its N-desmethyl metabolite in rat and human hair by GC-MS.

In order to study the incorporation of sildenafil (SDF) and its N-demethylated metabolite (norSDF) into hair, animal model experiments were carried out. After shaving the back hair, SDF was dosed to two sets of three male dark-agouti pigmented rats (5 weeks old) per each group at 25 mg/kg once a day for 5 successive days with intraperitoneal (i.p.) (set1) and oral administration (set2). The regrown back hair was collected 14 d after the first administration. Three typical extraction methods, using methanol-5 M hydrochloric acid, methanol-trifluoroacetic acid and 1 M sodium hydroxide, were evaluated using the rat hair samples containing SDF and norSDF. Methanol-5 M hydrochloric acid was the best extraction method in terms of high efficiency and reproducibility. The extract was purified using Bond Elut Certify columns and was derivatized with trimethylsilylimidazole: N,O-bis(trimethylsilyltrifluoroacetamide): trimethylchlorosilane (3: 3: 2) at 90 degrees C for 30 min. The trimethylsilylated products were analyzed by GC-MS using selected ion monitoring. SDF and norSDF were simultaneously detected in the rat hair. The hair concentrations were 4.9-6.3 (av. 5.8) ng/mg and 15.6-20.3 (av. 17.6) ng/mg for SDF and norSDF, respectively, with i.p. administration, and 2.6-4.1 (av. 3.6) ng/mg and 8.1-10.4 (av. 9.1) ng/mg with oral administration. The hair concentrations of norSDF were about three times higher than those of SDF, and the ratios of both compounds showed no significant difference between i.p. and oral administrations. This method was applied to the scalp hair of two patients who orally took SDF at regular intervals for the treatment of penile erectile dysfunction. The hair concentrations of SDF and norSDF in the two patients were 19.8 and 55.9 ng/mg, and 1.7 and 5.6 ng/mg, respectively.

Administration, Oral↗
PubMed2001

Hair analysis for pharmaceutical drugs. I. Effective extraction and determination of phenobarbital, phenytoin and their major metabolites in rat and human hair.

In order to establish an analytical method for the determination of phenobarbital (PB), phenytoin (PPH) and their hydroxylated metabolites in hair, animal model experiments were performed. Five male dark-agouti pigmented rats, aged 5 weeks, were intraperitoneally and orally administered PB or PPH independently at 25 mg/kg once a day for 5 successive days. The growing back hair was collected 15d after the first administration. Four typical extraction methods, using NH4OH-methanol-acetone, TFA-methanol-acetone, 1M sodium hydroxide and proteinase K, were evaluated using the rat hair samples containing PB or PPH. Methanol-acetone-NH4OH (10: 10: 1) was the best extraction method from all aspects, such as high extraction efficiency and low noise. The analytes in the extract were methylated in acetonitrile with 20% tetramethylammonium hydroxide and methyliodide at 70 degrees C for 10 min. After purification with Bond Elut Certify, the methylated products were analyzed by GC-MS. From rat hair, PB, p-hydroxy PB, PPH and p-hydroxy PPH were detected at average concentrations of 26.9, trace, 4.2 and 0.4 ng/mg with an intraperitoneal (i.p.) injection, and at 30.9, trace, 4.0 and 0.4 ng/mg with oral administration, respectively. There was little difference in hair concentrations between i.p. injection and oral administration. This method was applied to the head hair of two patients who orally took toxic amounts of PB and two volunteers who orally took 100 mg of PPH daily for 5 d. The hair concentrations of PB in the two patients were 16.2 and 14.7 ng/mg, and those of PPH in the two volunteers were 3.3 and 0.1 ng/mg.

Adult↗
PubMed2001

Separation of sympathomimetic amines of abuse and related compounds by micellar electrokinetic chromatography.

Separation of twelve sympathomimetic amines and related compounds by micellar electrokinetic chromatography (MEKC) with UV absorbance detection is described. These amines were well separated within 25 min using 50 mM sodium tetraborate solution containing 15 mM sodium dodecylsulfate (SDS) of pH 9.3 as a running solution and detected at 210 nm. MEKC was performed with an applied voltage of 13 kV at 25 degrees C using a fused-silica capillary (50 cm x 75 mm i.d.) with effective length of 37.5 cm. The detection limits of these compounds were in the range from 4 to 97 fmol/injection at a signal-to-noise ratio (S/N) of 3. The reproducibility of the method expressed as relative standard deviation (RSD) for within-day (n=6) and between-day (n=5) assays was less than 4.8 and 8.8%, respectively. The proposed method could be applied to the determination of an anorectic drug, phentermine, in Chinese tea with a detection limit of 99 microg/g (105 fmol/injection, S/N=3).

Calibration↗