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Biomedical subjects

K Ohyama

Publications and source records attributed to K Ohyama.

At least 37 records · Page 2Linked to original sources
PubMed2001

Identification of a novel frameshift mutation (383insT) in the RUNX2 (PEBP2 alpha/CBFA1/AML3) gene in a Japanese patient with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder due to mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer-binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1)/acute myeloid leukemia 3 (AML3). To investigate the RUNX2 mutations in a Japanese patient with classic CCD, we analyzed the RUNX2 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and PCR-restriction fragment length polymorphism. The patient had hypoplasia of the clavicles, patent fontanelles, short stature, supernumerary teeth, and retention of deciduous dentition. We identified a 1-bp insertion (383insT) at codon 128 of the RUNX2 gene. The 383T insertion affects the conserved residue in the runt domain and results in premature termination in the runt domain.

Adolescent↗
PubMed2001

Additional locus of rDNA sequence specific to the X chromosome of the liverwort, Marchantia polymorpha.

The molecular cytogenetic organization of 17S ribosomal RNA genes (17S rDNA), a part of the 45S rDNA repeat, was investigated on the chromosomes of the liverwort Marchantia polymorpha using fluorescence in-situ hybridization (FISH). The numbers of 17S rDNA loci visualized in female and male chromosomes were ten and nine. respectively. This heterogeneous localization was due to the presence of an additional 17S rDNA locus on the X chromosome and its absence on the Y chromosome. The signal on the X chromosome covered almost the entire region of its long arm. The other nine signals were observed on the same loci of respective autosomes in both sexes. Southern hybridization analysis revealed an additional band including 17S rDNA exclusively on EcoRI digested female genomic DNA supporting the existence of an additional 17S rDNA locus on the X chromosome.

Chromosomes↗
PubMed2001

Progressive apoptosis in chorion laeve trophoblast cells of human fetal membrane tissues during in vitro incubation is suppressed by antioxidative reagents.

Previously, we demonstrated apoptotic cell death in the chorion laeve trophoblast layer of human fetal membrane tissues during the late stages of pregnancy, the progression of apoptosis during incubation in vitro, and its suppression by a low concentration of glucocorticoid hormones. We now report examination of mRNA expression of inflammatory cytokines [interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha] and antioxidative enzyme genes [heme oxygenase 1, catalase, Mn-superoxide dismutase (SOD), Cu/Zn-SOD, glutathione S-transferase, glutathione reductase and glutathione peroxidase] and apoptosis-related genes during in vitro progression of apoptosis with or without glucocorticoid by a reverse transcription/PCR method. It was shown that the mRNA levels increased in chorion laeve tissue for each cytokine examined and for catalase, heme oxygenase 1 and Mn-SOD in direct correlation with the in vitro incubation period. By Western blotting the existence of Mn-SOD protein, and its slight increase with incubation time, was also shown. The investigation of the influence of antioxidative reagents [pyrrolidine dithiocarbamate (PDTC), N-acetyl-l-cysteine (NAC) and nordihydroguaiaretic acid (NDGA)] on DNA fragmentation showed that DNA fragmentation in chorion laeve tissues was inhibited by approximately 50% in the presence of 1 mm PDTC, 30 mm NAC and 1 mm NDGA. These results suggest that apoptotic cell death of the trophoblast layer of chorion tissues may be induced through intracellular oxidative stress at the stage of parturition.

Acetylcysteine↗
PubMed2001

Isolation of X and Y chromosome-specific DNA markers from a liverwort, Marchantia polymorpha, by representational difference analysis.

The liverwort Marchantia polymorpha has X and Y chromosomes in the respective female and male haploids. Here we report the successful exploitation of representational difference analyses to isolate DNA markers for the sex chromosomes. Two female-specific and six male-specific DNA fragments were genetically confirmed to originate from the X and Y chromosomes, respectively.

Chromosomes↗
PubMed2001

Common regulation of growth arrest and differentiation of osteoblasts by helix-loop-helix factors.

Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWIST and FGFR3 genes.

Alkaline Phosphatase↗
PubMed2001

Improvement of separation method of fragmented DNA from an apoptotic cell DNA sample for the quantitation using agarose gel electrophoresis.

In order to quantify fragmented DNA extracted from apoptotic cells, we devised a separation method which condenses fragmented DNA into a small band, separating it from larger-size DNA with agarose gel electrophoresis. Calf thymus DNA and standard fragmented DNA were loaded onto 1.0% gel for 0.5, 1.0, 1.5 and 2.0 cm length, and onto 0.7, 1.0, 1.5 and 2.0% of gels for 1 cm length. DNA was then extracted from gel slices with the UltraClean 15 DNA Purification Kit, and estimated by measuring fluorescence intensity using Hoechst No.33258 dye. DNA recovery from the gel showed constant values regardless of the amount of loaded DNA up to 1 microg/assay, and a plot of loaded DNA amounts vs. the DNA amount yielded resulted in a strait line in any gel concentration used. Our results show the best conditions to estimate DNA fragmentation rates in apoptotic cells in which fragmented DNA was separated from thymus DNA by loading on 1.0% gel for 1.0 cm length. We used our method to estimate fragmentation rates in DNA fractions extracted from apoptotic human cervical fibroblast, amnion epithelial and chorion laeve trophoblast cells by stimulation with actinomycin D. The results show that DNA fragmentation rates in these cells were consistent with the electrophoretic patterns of the DNA samples shown by their photographs.

Apoptosis↗
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↗
PubMed2001

Familial multiple trichoepithelioma associated with subclavian-pulmonary collateral vessels and cerebral aneurysm--case report.

A 63-year-old woman presented with cerebellar infarction caused by occlusion of the right posterior inferior cerebellar artery. She had papules on her face that were identified histologically as multiple trichoepithelioma. Angiography revealed right subclavian-pulmonary collateral vessels and a cerebral aneurysm arising from the bifurcation of the right middle cerebral artery. Her grandmother, mother, and uncle had had similar papules, and the deaths of her mother and uncle were due to subarachnoid hemorrhage.

Aged↗
PubMed2001

Presence of Vicia graminea or Vicia unijuga lectin-binding (Vgu) glycoproteins with and without Thomsen-Friedenreich (T) antigen in normal human seminal plasma.

Vgu glycoprotein (Vicia graminea lectin- or Vicia unijuga lectin-binding glycoprotein) has been reported as oncofetal antigen, which is found in many kind of tumor tissues, amniotic fluid and fetal membranes. In autoradiography with an 125I-labeled Vicia unijuga lectin (VUA) probe and an 125I-labeled Arachis hypogaea lectin (PNA, anti-T lectin) probe, seminal plasma samples of eight healthy men gave 2-7 Vgu glycoproteins without T antigen, 1-2 Vgu glycoproteins with Thomsen-Friedenreich antigen (T antigen) and 1-8 T-antigen glycoproteins, respectively. These results show that in addition to T-antigen glycoproteins, normal human seminal plasma contains Vgu glycoproteins and Vgu glycoproteins with T antigen as seminal plasma components as well as other tumor markers such as CA19-9, CA-125 and CEA.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2000

Generation of expressed sequence tags from low-CO2 and high-CO2 adapted cells of Chlamydomonas reinhardtii.

To characterize genes whose expression is induced in carbon-stress conditions, 12,969 and 13,450 5'-end expressed sequence tags (ESTs) were generated from cells grown in low-CO2 and high-CO2 conditions of the unicellular green alga, Chlamydomonas reinhardtii. These ESTs were clustered into 4436 and 3566 non-redundant EST groups, respectively. Comparison of their sequences with those of 3433 non-redundant ESTs previously generated from the cells under the standard growth condition indicated that 2665 and 1879 EST groups occurred only in the low-CO2 and high-CO2 populations, respectively. It was also noted that 96.2% and 96.0% of the cDNA species respectively obtained from the low-CO2 and high-CO2 conditions had no similar EST sequence deposited in the public databases. The EST species identified only in the low-CO2 treated cells included genes previously reported to be expressed specifically in low-CO2 acclimatized cells, suggesting that the ESTs generated in this study will be a useful source for analysis of genes related to carbon-stress acclimatization. The sequence information and search results of each clone will appear at the web site: http://www.kazusa.or.jp/en/plant/chlamy/EST/.

Animals↗
PubMed2000

Comparison of expressed sequence tags from male and female sexual organs of Marchantia polymorpha.

A total of 935 expressed sequence tags (ESTs) from male immature sexual organ were determined, of which 600 ESTs were assembled into 110 non-redundant groups, resulting in 445 unique EST sequences. Of these, 244 sequences shared significant similarities to known nucleotide or amino acid sequences in other organisms. The remaining 201 unique sequences showed no significant matches and thus are likely to be novel transcripts. ESTs from male and female immature sexual organs of a liverwort, Marchantia polymorpha, were compared to characterize gene expression patterns during sex differentiation. Ninety-nine male ESTs turned out to be common genes found also in the female library. Interestingly, one of the ESTs found only in male shows a significant similarity to the transformer-2 gene involved in sex determination in Drosophila. In female, several unique lectin ESTs were found that are not present in the male library.

Amino Acid Sequence↗
PubMed2000

Predictive factors for intrahepatic recurrence after percutaneous ethanol injection therapy for small hepatocellular carcinoma.

BACKGROUND: Percutaneous ethanol injection therapy has been used widely for small hepatocellular carcinoma. This study was undertaken to determine factors predictive of local recurrence or new nodular recurrence in patients with small hepatocellular carcinoma treated with percutaneous ethanol injection. METHODS: The authors studied 73 nodules treated with percutaneous ethanol injection in 49 patients with small hepatocellular carcinoma. The usefulness of predictive factors for recurrence was assessed with the Kaplan-Meier method. The clinicopathologic variables examined included age, gender, Child-Pugh classification, number of tumors (single vs. multiple), tumor size, degree of tumor differentiation, ultrasonographic findings such as peripheral hypoechoic band (so-called 'halo'), intratumoral echo pattern, tumor staining on enhanced computed tomography, combination therapy with transcatheter arterial embolization, and serum alpha-fetoprotein level. RESULTS: The local recurrence rates were 19%, 27%, 33%, 33%, and 33%, respectively, and the new nodular recurrence rates were 19%, 51%, 74%, 83%, and 83%, respectively, at 1, 2, 3, 4, and 5 years after percutaneous ethanol injection therapy. The frequency of local recurrence was associated with the histologic differentiation of more than moderately differentiated (P < 0.001), presence of a sonographic halo (P < 0. 005), an intratumoral heterogeneous echo pattern (P < 0.001), and positive tumor staining on enhanced computed tomography (P < 0.01). Multivariate analysis showed that the presence of a halo and an intratumoral heterogeneous echo pattern were the most important variables for predicting local recurrence. The frequency of new nodular recurrences was related to the presence of multiple tumors (P < 0.01) and a high serum alpha-fetoprotein level (P < 0.001). Multivariate analysis showed that a high serum alpha-fetoprotein level was a reliable predictor of new nodular recurrence. CONCLUSIONS: This study showed that the presence of a halo and an intratumoral echo pattern on ultrasonography were useful predictors for local recurrence after percutaneous ethanol injection therapy for small hepatocellular carcinoma, and that a high serum alpha-fetoprotein level was associated with a higher frequency of new nodular recurrences.

Adult↗
PubMed2000

Biosynthesis of sterols and ecdysteroids in Ajuga hairy roots.

Hairy roots of Ajuga reptans var. atropurpurea produce clerosterol, 22-dehydroclerosterol, and cholesterol as sterol constituents, and 20-hydroxyecdysone, cyasterone, isocyasterone, and 29-norcyasterone as ecdysteroid constituents. To better understand the biosynthesis of these steroidal compounds, we carried out feeding studies of variously 2H- and 13C-labeled sterol substrates with Ajuga hairy roots. In this article, we review our studies in this field. Feeding of labeled desmosterols, 24-methylenecholesterol, and 13C2-acetate established the mechanism of the biosynthesis of the two C29-sterols and a newly accumulated codisterol, including the metabolic correlation of C-26 and C-27 methyl groups. In Ajuga hairy roots, 3alpha-, 4alpha-, and 4beta-hydrogens of cholesterol were all retained at their original positions after conversion into 20-hydroxyecdysone, in contrast to the observations in a fern and an insect. Furthermore, the origin of 5beta-H of 20-hydroxyecdysone was found to be C-6 hydrogen of cholesterol exclusively, which is inconsistent with the results in the fern and the insect. These data strongly support the intermediacy of 7-dehydrocholesterol 5alpha,6alpha-epoxide. Moreover, 7-dehydrocholesterol, 3beta-hydroxy-5beta-cholest-7-en-6-one (5beta-ketol), and 3beta,14alpha-dihydroxy-5beta-cholest-7-en-6-one (5beta-ketodiol) were converted into 20-hydroxyecdysone. Thus, the pathway cholesterol-->7-dehydrocholesterol-->7-dehydrocholesterol 5alpha,6alpha-epoxide-->5beta-ketol-->5beta-k etodiol is proposed for the early stages of 20-hydroxyecdysone biosynthesis. 3beta-Hydroxy-5beta-cholestan-6-one was also incorporated into 20-hydroxyecdysone, suggesting that the introduction of a 7-ene function is not necessarily next to cholesterol. C-25 Hydroxylation during 20-hydroxyecdysone biosynthesis was found to proceed with ca. 70% retention and 30% inversion. Finally, clerosterol was shown to be a precursor of cyasterone and isocyasterone.

Carbon Isotopes↗
PubMed2000

Direct transformation and plant regeneration of the haploid liverwort Marchantia polymorpha L.

Thalli of the haploid liverwort Marchantial polymorpha were successfully used for direct particle bombardment with plasmid pMT, which carries a hygromycin phosphotransferase gene (hpt) controlled by the CaMV 35S promoter and the NOS polyadenylation region. Hygromycin-resistant cell masses arose from the thallus surface and developed directly into hygromycin-resistant thalli. Southern blot analyses indicated that these thalli carried at least 1-4 copies of the hpt gene, which were stably transmitted to their asexual thallus progenies via gemma propagation for three generations. This transformation and direct plant regeneration protocol is expected to be a valuable tool for the molecular analysis of this lower land plant.

Anti-Bacterial Agents↗
PubMed2000

Inhibitory effects of amiodarone and its N-deethylated metabolite on human cytochrome P450 activities: prediction of in vivo drug interactions.

AIMS: To predict the drug interactions of amiodarone and other drugs, the inhibitory effects and inactivation potential for human cytochrome P450 (CYP) enzymes by amiodarone and its N-dealkylated metabolite, desethylamiodarone were examined. METHODS: The inhibition or inactivation potency of amiodarone and desethylamiodarone for human CYP activities were investigated using microsomes from B-lymphoblastoid cell lines expressing CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. The in vivo drug interactions of amiodarone and desethylamiodarone were predicted in vitro using the 1+Iu/Ki values. RESULTS: Amiodarone weakly inhibited CYP2C9, CYP2D6, and CYP3A4-mediated activities with Ki values of 45.1-271.6 microm. Desethylamiodarone competitively inhibited the catalytic activities of CYP2D6 (Ki=4.5 microm ) and noncompetitively inhibited CYP2A6 (Ki=13.5 microm ), CYP2B6 (Ki=5.4 microm ), and CYP3A4 (Ki=12.1 microm ). The catalytic activities of CYP1A1 (Ki=1.5 microm, alpha=5.7), CYP1A2 (Ki=18.8 microm, alpha=2.6), CYP2C9 (Ki=2.3 microm, alpha=5.9), and CYP2C19 (Ki=15.7 microm, alpha=4.5) were inhibited by desethylamiodarone with mixed type. The 1+Iu/Ki values of desethylamiodarone were higher than those of amiodarone. Amiodarone inactivated CYP3A4, while desethylamiodarone inactivated CYP1A1, CYP1A2, CYP2B6, and CYP2D6. CONCLUSIONS: The interactions between amiodarone and other drugs might occur via the inhibition of CYP activities by its N-dealkylated metabolite, desethylamiodarone, rather than by amiodarone itself. In addition, the inactivation of CYPs by desethylamiodarone as well as by amiodarone would also contribute to the drug interactions.

Amiodarone↗
PubMed2000

Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha.

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.

Base Sequence↗