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Biomedical subjects

Y Ishida

Publications and source records attributed to Y Ishida.

At least 37 records · Page 2Linked to original sources
PubMed2003

A new generic column switching system for quantitation in cassette dosing using LC/MS/MS.

Cassette dosing is a method in which multiple drugs are administered to a single animal at the same time, and the plasma concentrations of the individual compounds are simultaneously determined. This method enables high-throughput rapid screening for pharmacokinetic assessment of new drug candidates. An available gradient method was modified for cassette dosing analysis to attain the advantages of high sensitivity and applicability to a wide range of compounds. However, two problems arose; (1). the time-consuming optimization of mobile phases for each compound group, which limited applicability and (2). the remarkable suppression of ionization by polyethyleneglycol, which is commonly used in intravenous administration. To resolve these problems, a new column switching method was established to attain wider applicability and avoid the ionization suppression. This column switching system is very simple because the trap column and the analytical column are specified and the mobile phase is selected from only two species. Method optimization requires only the selection of the mobile phase and takes only a few hours. About 200 compounds, which were administered as about 50 cassettes, were analyzed using this column switching system. Assay validation of one cassette was carried out, and good accuracy and precision were obtained. About 90% of the compounds could be determined within 20% bias. These results showed that this new column switching system for cassette dosing is accurate enough for the screening of drug candidates and offers wide applicability for various compounds. This system was shown to be very useful for the determination of cassette dosing samples, containing multiple compounds.

Animals↗
PubMed2003

Cryptic insertion and translocation or nondividing leukemic cells disclosed by FISH analysis in infant acute leukemia with discrepant molecular and cytogenetic findings.

Of 51 infants with acute leukemia, 13 (25%) had contradictory findings on 11q23/MLL rearrangements that were analyzed by cytogenetic and Southern blot methods: seven had rearranged MLL and normal karyotype, four had rearranged MLL and abnormal karyotype with no 11q23 translocation, and two had germline MLL and 11q23 translocations. Fluorescent in situ hybridization (FISH) analysis using an MLL probe that was performed to elucidate the discrepancy disclosed the presence of normal dividing cells and nondividing leukemic cells in the same bone marrow in five patients, and cryptic insertion or translocation in another five. Subsequent FISH and reverse transcription-polymerase chain reaction analysis identified the MLL-AF10, MLL-AF4, or MLL-AF1q fusions that were produced by the cryptic rearrangements in four of the five patients. In the remaining three patients, the breakpoint of 11q23 translocation was located distal to the MLL locus in one, and the discrepancy was unresolved in two. Thus, FISH should complement cytogenetic analysis when cytogenetic and molecular genetic findings are contradictory in infant leukemia, and when infant leukemia does not show 11q23 translocations or other specific translocations including t(7;12), t(1;22), etc that are recurrently found in infant leukemia.

Blotting, Southern↗
PubMed2003

Consensus primers for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell lymphomas.

AIMS: To demonstrate the usefulness of polymerase chain reaction (PCR) methodology with both the FR2A/LJH/VLJH and the FR1c/LJH/VLJH primer sets for detecting monoclonal immunoglobulin heavy chain (IgH) gene rearrangement in B cell non-Hodgkin lymphomas (B-NHLs). METHODS: Eighty three patients with B-NHL were enrolled in this study. DNA was isolated from paraffin wax embedded sections and amplified by PCR to determine the sequences of the rearranged IgH gene. Each PCR product was subcloned. Cycle sequences and sequence analyses were done to determine the clone specific IgH variable region (VH) sequences. RESULTS: Clonal IgH gene rearrangements were detected in 45 cases with FR2a/JH/VLJH and in 14 of the remaining cases with FR1c/JH/VLJH. Most of the cases detectable with FR2a/JH/VLJH were derived from VH3 and VH4 families. Five of six cases in the VH1 family and one in the VH7 family were amplified with the FR1c/JH/VLJH primer set only. CONCLUSION: The detection rate of IgH rearrangement in B-NHLs can be increased by using both FR2A/LJH/VLJH and FR1c/LJH/VLJH, and these two primer sets are suitable for routine PCR methodology. Moreover, each primer set appears to be closely related to VH family specificity.

Clone Cells↗
PubMed2003

Development of immunoglobulin variable heavy chain gene consensus probes with conjugated 3' minor groove binder groups for monitoring minimal residual disease in childhood acute lymphoblastic leukaemia.

AIMS: To develop immunoglobulin heavy chain variable (VH) gene probes that are shorter and more flexible in position for monitoring minimal residual disease (MRD) in childhood leukaemia (ALL), using minor groove binder (MGB) technology. METHODS: All VH germline sequences registered in the database were aligned and the consensus regions were determined. The reliability of the MGB probes was compared with non-MGB probes in all 24 cases of ALL. RESULTS: Ten MGB probes (16 to 18 mers) were designed that enabled all the germline sequences on the database to be analysed, whereas the conventional non-MGB probes (21 to 27 mers) did not allow the analysis of four of the VH1 and five of the VH3 germline sequences. The sequencing results in five of the 24 cases of ALL were not matched to the non-MGB probes. CONCLUSIONS: MGB technology allows shorter probes to be designed, enabling MRD to be detected in childhood ALL. This would provide a considerable reduction in cost for a large MRD study.

Child↗
PubMed2003

Increased cyclin D1 expression in cancer of the ampulla of Vater: relevance to nuclear beta catenin accumulation and k-ras gene mutation.

AIMS: Several studies have reported that dysregulation of beta catenin or k-ras mutation promotes cyclin D1 expression. This study investigated the relation between cyclin D1 expression and clinicopathological parameters in carcinoma of the ampulla of Vater (CAV), and also assessed the relation between increased cyclin D1 expression and beta catenin/k-ras status in this series. METHODS: Thirty CAVs were evaluated for cyclin D1 expression by immunohistochemistry in relation to patient clinicopathological features. Aberrant beta catenin expression and k-ras mutation were also investigated by immunostaining and direct sequencing, and related to cyclin D1 expression. RESULTS: Increased cyclin D1 expression was seen in 17 of 30 CAVs and was significantly correlated with tumour cell proliferation and disease free survival time (p = 0.018, p = 0.018, respectively). Nuclear accumulation of beta catenin was found in nine of 30 cases, including four cases with missense mutations in exon 3 of CTNNB-1, and was significantly correlated with increased cyclin D1 expression (p = 0.003). k-ras gene mutation was detected in 12 of 30 cases, and was also significantly correlated with increased cyclin D1 expression (p = 0.026). Overall, 14 of 17 CAVs with increased cyclin D1 expression showed nuclear accumulation of beta catenin and/or k-ras mutation. CONCLUSIONS: Increased cyclin D1 expression appears to be associated with tumour proliferation and poorer clinical outcome in CAV. It is also associated with both aberrant beta catenin expression and k-ras mutation. These results are consistent with the in vitro data that cyclin D1 can be transactivated by activated beta catenin-T cell factor/LEF and k-ras pathways.

Aged↗
PubMed2003

[Chondromatous hamartoma of the lung with a unique demonstration on diagnostic imaging; report of a case].

A case of pulmonary hamartoma is reported with clinical, roentgenographical and histopathologic findings. The patient was a 53-year-old woman who had undergone right hemithyroidectomy for thyroid cancer 4 years before. An abnormal shadow, which was a non-clearly demarcated tumor, 2 cm in diameter, in the left middle lung field, was noted on her routine X-ray in February 2001. Physical examination and laboratory data revealed no significant findings, but computed tomography(CT) scans of the chest showed a gathering of small-sized high-density lesions in the nodule. She underwent left S8 segmentectomy on March 21, 2001. The pathology report on the frozen section was pulmonary hamartoma. Histopathologically, the lesion was characterized by a composition of bronchial epithelium, fat tissue and cartilage, with being diagnosed as a chondromatous hamartoma of the lung. The patient's postoperative course was uneventful, and she was discharged with no supportive therapy 14 days after the operation. To date, 14 months after the operation, the patient has been in good condition, without evidence of recurrence or distant metastasis on diagnostic imaging. This case is particularly interesting because a gathering of small-sized pulmonary hamartoma was demonstrated by imaging.

Female↗
PubMed2002

Ubiquitin expression in skin wounds and its application to forensic wound age determination.

The time-dependent expression of ubiquitin (Ub) was examined in murine skin wounds and 55 human skin wounds with different wound ages (groups I: 0-12 h, II: 1-5 days, III: 7-14 days and IV: 17-21 days). In murine skin wound specimens, neutrophils, macrophages and fibroblasts showed intensive Ub-positive reactions in the nuclei. In the human wound specimens with wound ages between 4 h and 1 day, neutrophils with strong intranuclear positive reactions for Ub were observed. With increasing wound ages, the nuclei of macrophages and fibroblasts were more intensively stained with anti-Ub antibody. Morphometrically, the intranuclear Ub-positive ratios were very low in group I. The skin wound specimens in groups II and IV showed Ub-positive ratios of >10%, and all samples in group III had Ub-positive ratios of >20%. These results suggest that a ratio of >10% for Ub indicates a wound age of at least 1 day. In contrast, Ub-positive ratios of less than 10% indicate a wound age of <1 day. Moreover, there was a significant difference in the Ub-positive ratio between group III and the other three groups. Thus, Ub-positive ratios considerably exceeding 30%, possibly indicate a wound age of 7-14 days. From the viewpoint of a forensic pathology application, the present study showed that Ub is suitable as a marker of wound age determination.

Adolescent↗
PubMed2002

Expression of steroid-converting enzymes in osteoblasts derived from rat vertebrae.

Studies in vitro and in vivo have shown that glucocorticoids and sex steroids play an important role in bone physiology and pathophysiology. In this study we investigated glucocorticoid and sex steroid conversion in osteoblasts derived from lumbar vertebrae of adult male and female rats. Progesterone was converted to inactive 20alpha-OH-progesterone and the conversion at day 5 was 16-fold greater than that at day 13 in both sexes (male/ female, 2.7/1.7 and 0.16/0.10 nM/10(5)cells/24 h, respectively). The conversion of inactive androstenedione to active androgen testosterone in males and females was 1.2- and 2.4-fold greater at day 5 than at day 13, respectively (male/female, 0.40/0.70 and 0.34/0.30 nM/ 10 cells/24 h, respectively). These results suggest that osteoblasts possess 20alpha-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD and that their activities are dependent on the stage of cell differentiation. At day 5, dehydroepiandrosterone was converted to androstenedione (male/female, 0.25/0.098 nM/10(5)cells/24 h), to 7alpha-OH-dehydroepiandrosterone (male/female, 0.49/0.39 nM/10(5)cells/24 h) and to 5-androstene-3beta,17beta-diol (male/female, 0.18/0.37 nM/10(5)cells/24 h), indicating the presence of 3beta-HSD, 7alpha-hydroxylase and 17beta-HSD, respectively. Both 3beta-HSD and 7alpha-hydroxylase activities declined with cell differentiation. Hormonally inactive cortisone was converted to active cortisol (male/female, 0.34/0.29 microM/10(6)cells/6 h) while conversion of cortisol to cortisone was not detectable, suggesting the presence of oxoreductase activity of 11beta-HSD-1. These results show, for the first time, the presence of 7alpha-hydroxylase and 20alpha-HSD in osteoblasts, and provide further evidence that osteoblasts metabolize a variety of steroid hormones and can thus regulate tissue responsiveness to them.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2002

Pharmacologic doses of medroxyprogesterone may cause bone loss through glucocorticoid activity: an hypothesis.

A number of studies suggest that progestagens may have beneficial effects on bone metabolism. C(21) Progestin medroxyprogesterone acetate (MPA) is one of the most commonly prescribed progestins for hormone replacement therapy and in gynecologic practice. However, it appears that MPA with significant glucocorticoid (GC) activity may decrease bone density. In this review, we argue that bone loss associated with MPA administration is caused by decreased osteoblast differentiation as a result of MPA occupying the GC receptor, since increasing GC receptor occupancy beyond that reached at normal (= optimal) GC concentrations attenuates osteoblast differentiation. We propose that progestins with no GC activity may be a better choice for progestagen therapy to achieve more beneficial effects on bone metabolism.

Adolescent↗
PubMed2002

Monitoring C-reactive protein in beagle dogs experimentally inoculated with Ehrlichia canis.

The concentrations of C-reactive proteins (CRP) in the plasma of five beagle dogs experimentally inoculated with Ehrlichia canis increased markedly. The concentrations began to increase between 4 and 16 days and peaked between 15 and 42 days after inoculation of E. canis. The peak concentrations ranged from 217.8 to 788.8 microg/ml (452.6 +/- 228.1 SD). After the peak, the concentrations of CRP decreased rapidly. The PCR product of 16S rRNA of E. canis became detectable in the five dogs between 18 and 27 days after inoculation of E canis. Antibodies to E canis were detected in plasma from the dogs between 5 and 15 days after inoculation of E. canis. The timings of seroconversion and of the start of the increase in CRP were approximately similar and the high concentrations of CRP in the plasma of the dogs tended to become apparent when the PCR product of 16 S rRNA of E. canis became detectable.

Animals↗
PubMed2001

Quantitative analysis of detailed lignin monomer composition by pyrolysis-gas chromatography combined with preliminary acetylation of the samples.

Detailed quantitative analysis of lignin monomer composition comprising p-coumaryl, coniferyl, and sinapyl alcohol and p-coumaraldehyde, coniferaldehyde, and sinapaldehyde in plant has not been studied from every point mainly because of artifact formation during the lignin isolation procedure, partial loss of the lignin components inherent in the chemical degradative methods, and difficulty in the explanation of the complex spectra generally observed for the lignin components. Here we propose a new method to quantify lignin monomer composition in detail by pyrolysis-gas chromatography (Py-GC) using acetylated lignin samples. The lignin acetylation procedure would contribute to prevent secondary formation of cinnamaldehydes from the corresponding alcohol forms during pyrolysis, which are otherwise unavoidable in conventional Py-GC process to some extent. On the basis of the characteristic peaks on the pyrograms of the acetylated sample, lignin monomer compositions in various dehydrogenative polymers (DHP) as lignin model compounds were determined, taking even minor components such as cinnamaldehydes into consideration. The observed compositions by Py-GC were in good agreement with the supplied lignin monomer contents on DHP synthesis. The new Py-GC method combined with sample preacetylation allowed us an accurate quantitative analysis of detailed lignin monomer composition using a microgram order of extractive-free plant samples.

Acetylation↗
PubMed2001

Neurons in the lateral paragigantocellular nucleus projecting to the infralimbic cortex in rats: electrical activity and response to changes in blood pressure.

Neurons in the lateral paragigantocellular nucleus (LPGi) are known to project directly to the infralimbic cortex (IL). However, there have been no physiological studies on LPGi neurons projecting to the IL. In urethane-anesthetized rats, the single-unit activities of LPGi neurons were recorded extracellularly to investigate their electrophysiological properties and responses to changes in blood pressure (BP). The LPGi neurons antidromically driven from the IL were restricted to the ventral part of the LPGi and most spontaneously active neurons revealed suppression of activity following a fall in BP. These results suggest that the LPGi neurons in a very restricted location influence the IL in response to changes in BP.

Action Potentials↗
PubMed2001

Modification of Arg-13 of mu-conotoxin GIIIA with piperidinyl-Arg analogs and their relation to the inhibition of sodium channels.

mu-Conotoxin GIIIA, a peptide toxin isolated from the marine snail Conus geographus, preferentially blocks skeletal muscle sodium channels in vertebrates. In this study, analogs of mu-conotoxin GIIIA in which essential Arg-13 was replaced with arginine analogs consisting of a piperidyl framework to regulate length and direction of the side chain were synthesized. Synthesized analogs exhibited similar CD and NMR spectra to that of GIIIA, suggesting a three-dimensional structure identical to that of the native toxin. The biological activities of piperidyl analogs were decreased or lost despite the small change in the side chain of Arg-13. The investigated structure-activity relationships in inhibiting electrically stimulated muscle contraction suggest that the guanidinium group at amino acid position 13 interacts best when spaced with three to four carbons and placed in a vertical direction from the peptide loop. Thus, the position of the guanidinium group at Arg-13 of GIIIA must be located in a certain range for its strong interaction with the channel protein.

Amino Acid Sequence↗
PubMed2001

Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication.

To investigate the early events of JC virus (JCV) infection, including attachment, penetration, transport to the nuclei, and replication of the virus, we analyzed the susceptibility of 15 different cell lines to infection using a semiquantitative polymerase chain reaction (PCR) assay, in situ hybridization, laser scanning confocal microscopy, and a viral replication assay. The cell lines examined were human permissive and nonpermissive cells as well as cells of monkey and mouse origin. JCV entry into the nuclei of the all cell lines was observed within 10 min after inoculation, demonstrating that the virus receptor is widely distributed among mammalian cells. Inhibition of viral entry by an anti-JCV VP1 antibody and sialidase treatment to remove sialic acid residues, which are considered a candidate for the JCV receptor, suggested that VP1 may interact with the cellular surface sialic acid. In addition, chlorpromazine, a clathrin-dependent pathway inhibitor, significantly suppressed entry of JCV into nuclei. In spite of the broad spectrum of cells susceptible to JCV entry, replication of the virus occurred exclusively in human neuroblastoma cell lines. These results suggest that whereas JCV can enter a wide variety of cell types and localize to the nuclei, cell-specific intranuclear mechanisms are required for virus replication.

HeLa Cells↗
PubMed2001

Changes in cortical noradrenergic axon terminals of locus coeruleus neurons in aged F344 rats.

The noradrenergic innervations and noradrenaline contents of the frontal cortex in two age groups (9 and 25 months) of male F344 rats have been quantified by electrophysiological and biochemical methods. In the electrophysiological study, the percentage of locus coeruleus (LC) neurons activated antidromically from the frontal cortex decreased with age. In contrast, the percentage of LC neurons showing multiple antidromic latencies, which suggests axonal branching of individual LC neurons, increased markedly between 9 and 25 months in the frontal cortex. In the biochemical study, we found no significant difference in noradrenaline levels in the cortical terminal fields of LC neurons during aging. These results suggest that LC neurons give rise to axonal branches to retain noradrenaline levels in their target fields in the aged brain. Our findings show that LC neurons preserve a strong capability for remodeling their axon terminals even in the aged brain.

Aging↗
PubMed2001

Identification of coding single-nucleotide polymorphisms in human taste receptor genes involving bitter tasting.

T2Rs comprise a G-protein-coupled receptor superfamily that contains functionally defined bitter taste receptors. Here we report the tissue expressions and coding single-nucleotide polymorphisms (cSNPs) in human T2R genes (hT2R3, hT2R4, and hT2R5) on chromosome 7q31. We first demonstrated that hT2R3, hT2R4, and hT2R5 are actually expressed in the circumvallate papillae of the human tongue by reverse transcription-polymerase chain reaction (RT-PCR). We identified six cSNPs within the T2R receptor genes. The hT2R4 and hT2R5 contained four and one cSNPs that cause missense mutations, respectively, while hT2R3 included one silent nucleotide mutation. However, we could not find any nonsense mutations that resulted in a frameshift or a premature stop codon within the open reading frames. Genotype frequencies of each cSNP were in Hardy-Weinberg equilibrium. The identification of nucleotide diversity and amino acid polymorphisms in human T2R receptors could help clarify individual differences in the acceptability and sensitivity to bitter compounds.

Base Sequence↗
PubMed2001

Constitutive expression and delayed light response of casein kinase Iepsilon and Idelta mRNAs in the mouse suprachiasmatic nucleus.

Casein kinase Iepsilon (CKIepsilon) and casein kinase Idelta (CKIdelta) phosphorylate clock oscillating mPER proteins, and play a key role in the transcription (post)translation feedback loop that generates circadian rhythm. In the present study, the expression profiles of CKIepsilon and CKIdelta mRNAs were examined in the mice clock center, suprachiasmatic nucleus (SCN). Moderate levels of CKIepsilon and CKIdelta mRNAs were constantly expressed in the SCN in both light:dark and constant dark conditions. This finding supports the hypothesis that CKI may form a constant threshold to the nuclear entry of mPER proteins as in the Drosophila homologue, double-time. Further, we demonstrated that the light exposure at subjective night induced a delayed increase in CKIepsilon and CKIdelta mRNAs in the SCN. CKIepsilon and CKIdelta proteins may play a role on light-induced phase-shift.

Animals↗