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Biomedical subjects

N Takagi

Publications and source records attributed to N Takagi.

At least 55 records · Page 3Linked to original sources
PubMed1999

Structure and localization of the mouse prolyl oligopeptidase gene.

We have cloned and characterized the genomic structure of the mouse gene for prolyl oligopeptidase that is mapped to chromosome 10B2-B3. The gene is about 92 kilobases in size and contains 15 exons. All exon-intron junction sequences conform to the GT/AG rule. Comparison with the presumed domain structures of the mouse prolyl oligopeptidase indicates that the propeller domain of the enzyme is encoded by exons 3-10, whereas the catalytic domain is encoded by exons 1-3 and 10-15. The catalytic triad residues are encoded by two exons (Ser(554) on exon 13 and His(680) and Asp(642) on exon 15). The 5'-flanking region of the mouse prolyl oligopeptidase gene has structural features found in housekeeping gene promoters, including a GC-rich segment and an absence of TATA and CAAT boxes. A primer extension assay showed the presence of multiple sites for the initiation of transcription. Transient transfection analysis demonstrated that the 5'-flanking region of the gene can direct efficient expression in COS1 cells. Deletion studies revealed that the downstream 125-base pair sequence of the region is required for promoter activity in the cells.

Animals↗
PubMed1999

Molecular cloning of antisense transcripts of the mouse Xist gene.

Prior to X-inactivation, Xist is transcribed in unstable form. The initiation of X-inactivation is associated with the appearance of stable Xist transcripts which coat the X chromosome to be inactivated. Using strand specific RT-PCR analysis of the 5' region of Xist, we have detected antisense transcripts (Xist AS) in undifferentiated embryonic stem (ES) cells, but not in female somatic cells. Screening of a female ES cell cDNA library allowed us to isolate one poly(A)-tailed cDNA clone corresponding to this RNA. 5' RACE analysis showed that XistAS and the P1 sense product of Xist overlap by at least 707 bp. Expression of XistAS was also detected in early mouse embryos before random X-inactivation in the epiblast lineage. Although XistAS is low in abundance, it may be involved in destabilizing Xist mRNA in undifferentiated ES cells.

Animals↗
PubMed1999

Microsphere embolism-induced elevation of nerve growth factor level and appearance of nerve growth factor immunoreactivity in activated T-lymphocytes in the rat brain.

Changes in nerve growth factor (NGF) level and type of cells producing NGF were investigated in the rat brain after sustained cerebral embolism. The NGF level was determined by a two-site enzyme immunoassay specific for NGF. The cerebral cortex, striatum, and hippocampus of the embolized hemisphere maximally contained 2.4-, 2.4-, and 1.7-times higher NGF levels than the corresponding regions of the nonembolized hemisphere. A significant increase was transiently observed for 1 week in the cerebral cortex and striatum, whereas the increase was longer lasting, at least of 4 weeks' duration, in the hippocampus. To examine the localization of NGF-like immunoreactivity (NGF-LI), we used a newly developed anti-NGF peptide antiserum that specifically recognized a 30-kDa molecule(s) in the hippocampal extracts or in NGF cDNA-transfected cells, suggesting that the antibody predominantly reacted with the putative NGF precursor protein(s). NGF-LI, which was localized in neurons of the normal or non-embolized hemisphere, was reduced, and on the embolized side new signals emerged in small non-neuronal cells having a round shape. These included cells with common leukocyte antigen CD45 and T-lymphocyte antigen CD3, which did not appear in the normal or non-embolized hemisphere. NGF-LI and CD3 were colocalized in a substantial number of the cells, suggesting that some activated T-lymphocytes produce NGF for neuronal regeneration after sustained cerebral embolism.

Animals↗
PubMed1999

Effect of CpG methylation on expression of the mouse imprinted gene Mest.

We previously reported isolation of the mouse gene, Mest (mesoderm-specific transcripts), which is mapped to the proximal part of chromosome 6 and predominantly expressed in the mesoderm and its derivatives during development. Peg1, a paternally expressed gene isolated by a systematic screening of imprinted genes, was recently demonstrated to be identical to Mest. We and others have shown that the human homolog (MEST) of Mest is also imprinted so as to be expressed from the paternal copy and maps to 7q32. To study transcriptional regulation of Mest/Peg1, we examined the effect of DNA methylation on its expression. In the embryonal carcinoma (EC) cell line, MC12, from which Mest was originally isolated, the 5'-region harboring presumptive promoter of the gene was undermethylated. On the other hand, C4XX, a subclone of MC12 which had lost expression of Mest, was characterized by extremely high levels of methylation in the 5'-region, demethylation of which resulted in activation of Mest. Furthermore, a methylated reporter construct with the luciferase gene under the control of the putative promoter region of Mest was not competent to produce luciferase activity in MC12 cells. These results suggest a suppressive role for DNA methylation in Mest expression. However, neither methylated nor unmethylated reporter constructs showed luciferase activity in a primary culture from the adult kidney, in which Mest is down-regulated despite apparent unmethylation of the paternal allele. Taken together, the data suggest that there are probably two modes of regulation for the Mest gene; one being a methylation-dependent mechanism that regulates imprinted expression of Mest during development, and the other being a methylation-independent mechanism that is involved in down-regulation of Mest in adult tissues.

Animals↗
PubMed1999

Compact chromatin packaging of inactive X chromosome involves the actively transcribed Xist gene.

The Xist gene responsible for X inactivation may take a unique chromatin structure because of exceptional expression from inactive X Chromosome, (Chr). We have examined differential chromatin packaging of the Xist gene region between active and inactive X Chr with a novel method consisting of the chromatin fractionation and allele-specific detection. Analysis of F1 heterozygous female mice from T(X;16)16H x MSM crosses and two cell clones derived from inter-subspecific F1 female mice demonstrated that the packaging level of the transcribed Xist region on inactive X Chr was as tight as that of the repressed Pgk-1 allele on the same chromosome. On the other hand, restriction endonuclease sensitivity assay of chromatin showed that the promoter region, but not transcribed regions, of the transcribed Xist allele retained accessibility to nucleases. These results may suggest a cis-element(s) in a regulatory region of the Xist gene to prevent the transcriptionally inhibitory effect of the chromatin packaging.

Animals↗
PubMed1999

Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin.

We have previously shown that replacing the P1-site residue (Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lys results in the acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively. However, the inhibitory activities thus induced are not strong. In the present study, we introduced additional amino acid replacements around the reactive site to try to make the P1-site mutants more effective inhibitors of chymotrypsin or trypsin. The amino acid replacement Asp-->Tyr at the P2' site of OMCHI3(P1Met) resulted in conversion to a 35000-fold more effective inhibitor of chymotrypsin with an inhibitor constant (K(i)) of 1. 17x10(-11) M. The K(i) value of OMCHI3(P1Met, P2'Ala) indicated that the effect on the interaction with chymotrypsin of removing a negative charge from the P2' site was greater than that of introducing an aromatic ring. Similarly, enhanced inhibition of trypsin was observed when the Asp-->Tyr replacement was introduced into the P2' site of OMCHI3(P1Lys). Two additional replacements, Asp-->Ala at the P4 site and Arg-->Ala at the P3' site, made the mutant a more effective inhibitor of trypsin with a K(i) value of 1. 44x10(-9) M. By contrast, Arg-->Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) resulted in a greatly reduced inhibition of chymotrypsin, and Asp-->Ala replacement at the P4 site produced only a small change when compared with a natural variant of OMCHI3. These results clearly indicate that not only the P1-site residue but also the characteristics, particularly the electrostatic properties, of the amino acid residues around the reactive site of the protease inhibitor determine the strength of its interactions with proteases. Furthermore, amino acids with different characteristics are required around the reactive site for strong inhibition of chymotrypsin and trypsin.

Amino Acid Substitution↗
PubMed1999

The effect of transient global ischemia on the interaction of Src and Fyn with the N-methyl-D-aspartate receptor and postsynaptic densities: possible involvement of Src homology 2 domains.

Transient ischemia increases tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B in the rat hippocampus. The authors investigated the effects of this increase on the ability of the receptor subunits to bind to the Src homology 2 (SH2) domains of Src and Fyn expressed as glutathione-S-transferase-SH2 fusion proteins. The NR2A and NR2B bound to each of the SH2 domains and binding was increased approximately twofold after ischemia and reperfusion. Binding was prevented by prior incubation of hippocampal homogenates with a protein tyrosine phosphatase or by a competing peptide for the Src SH2 domain. Ischemia induced a marked increase in the tyrosine phosphorylation of several proteins in the postsynaptic density (PSD), including NR2A and NR2B, but had no effect on the amounts of individual NMDA receptor subunits in the PSD. The level of Src and Fyn in PSDs, but not in other subcellular fractions, was increased after ischemia. The ischemia-induced increase in the interaction of NR2A and NR2B with the SH2 domains of Src and Fyn suggests a possible mechanism for the recruitment of signaling proteins to the PSD and may contribute to altered signal transduction in the postischemic hippocampus.

Amino Acid Sequence↗
PubMed1999

Renin-angiotensin system and fibronectin gene expression in Dahl Iwai salt-sensitive and salt-resistant rats.

OBJECTIVE: The tissue renin-angiotensin system and extracellular matrix are involved in the cardiovascular hypertrophy and remodeling induced by hypertension. In this study, we examined the gene expression of the tissue renin-angiotensin system and fibronectin in inbred Dahl Iwai salt-sensitive and salt-resistant rats. MATERIALS AND METHODS: Eight pairs of 6-week-old male Dahl Iwai salt-sensitive and salt-resistant rats were fed either a low- or high-salt diet (0.3% or 8% NaCl, respectively) for 4 weeks. Activities of the circulating renin-angiotensin system were measured by radioimmunoassay and the gene expression of tissue angiotensinogen, the angiotensin II type 1 receptor (AT1) and fibronectin were analyzed by Northern blot analysis. RESULTS: Salt loading significantly increased blood pressure and produced cardiovascular hypertrophy and nephrosclerosis in the salt-sensitive rats. Activities of the circulating renin-angiotensin system were lower in salt-sensitive rats than in salt-resistant rats fed the low-salt diet, and salt loading lowered these activities in salt-resistant rats but not in salt-sensitive rats. In salt-resistant rats, salt loading increased renal, cardiac and aortic angiotensinogen, AT1 and fibronectin messenger (m)RNA expression except for aortic fibronectin mRNA expression. In contrast, in the salt-sensitive rats, salt loading stimulated the expression of cardiac fibronectin and aortic angiotensinogen, AT1 and fibronectin mRNAs. Furthermore, the cardiac and aortic fibronectin mRNA levels in salt-sensitive rats were higher than those in salt-resistant rats when both strains were fed the high-salt diet. CONCLUSIONS: These results demonstrate that the expression of tissue angiotensinogen, AT1 and fibronectin mRNAs is regulated differently in Dahl Iwai salt-sensitive and salt-resistant rats, and indicate that salt-mediated hypertension activates the cardiac fibronectin gene independently of the tissue renin-angiotensin system and stimulates the aortic fibronectin gene with activation of the tissue renin-angiotensin system.

Angiotensin I↗
PubMed1999

Simultaneous improvement of minimal-change nephrotic syndrome and anemia with steroid therapy.

A 56-year-old man presented with transient anemia in minimal-change nephrotic syndrome. Following nephrotic syndrome, anemia suddenly appeared without renal dysfunction. The anemia might be attributable to hemodilution because of significant correlations between the values of hemoglobin concentration and serum total protein or blood urea nitrogen during the clinical course. A low serum level and a low urinary excretion of erythropoietin were found, and when nephrotic syndrome ameliorated with steroid therapy, urinary erythropoietin excretion and anemia disappeared. This case indicated disappearance of the exponential increase of endogenous erythropoietin in acute anemia in nephrotic syndrome probably due to urinary losses and altered biosynthesis of erythropoietin. We report a case of the simultaneous improvement of both nephrotic syndrome and anemia with steroid therapy.

Anemia↗
PubMed1999

Severe hyperparathyroidism with hypercalcemia associated with chronic renal failure at pre-dialysis stage.

We report a case of a 23-year-old Japanese woman who had severe hyperparathyroidism associated with chronic renal failure before the start of dialysis treatment. Her chief complaints were swelling and pain in both shoulders. Laboratory examination revealed renal failure (BUN 134 mg/dl, serum Cr 7.3 mg/dl), severe normocytic normochromic anemia (hemoglobin 4.3 g/dl), hypercalcemia (11.8 mg/dl), and hyperphosphatemia (9.7 mg/dl). Serum PTH levels were extremely increased (intact PTH >1,000 pg/ml: normal range 10-50 pg/ml). X-ray examination of the skull and shoulders showed a salt and pepper appearance, and cauliflower-like deformity of the distal end of both clavicles, respectively. Accelerated ectopic calcification was observed in the costal cartilages, internal carotid arteries, and splenic arteries. Ultrasonographic examination revealed enlargement of the four parathyroid glands. Thallium-technetium subtraction scintigraphy of the parathyroid glands showed increased uptake into the upper two. Renal needle biopsy revealed severe impairment of the interstitium and tubules with much milder changes in glomeruli. The etiology of the renal failure could not be identified. Hemodialysis, total parathyroidectomy and auto-transplantation into the forearm were immediately performed. The pathological diagnosis was chief cell hyperplasia of the parathyroid glands. Based on the presence of chronic renal failure, remarkable hyperphosphatemia with mild hypercalcemia, an unusually high level of serum PTH, and accelerated ectopic calcification, the patient was diagnosed to have severe secondary hyperparathyroidism caused by chronic renal failure with major impairment of the renal interstitium and tubules.

Adult↗
PubMed1998

Mechanism of angiotensin II-mediated regulation of fibronectin gene in rat vascular smooth muscle cells.

This study was performed to investigate a mechanism of angiotensin II (Ang II)-mediated activation of the fibronectin (FN) gene in rat vascular smooth muscle cells. Actinomycin D and CV11974 completely inhibited Ang II-mediated increase in FN mRNA levels. Inhibitors of protein kinase C (PKC), protein-tyrosine kinase (PTK), phosphatidylinositol-specific phospholipase C, Ras, phosphatidylinositol 3-kinase, p70 S6 kinase, and Ca2+/calmodulin kinase also decreased Ang II-induced activation of FN mRNA. In contrast, cycloheximide; PD123319; or inhibitors of Gi, protein kinase A, or mitogen-activated protein kinase kinase did not affect the induction. FN promoter contained a putative AP-1 binding site (rFN/AP-1; -463 to -437), and the results of a transient transfection and electrophoretic mobility shift assay showed that Ang II enhanced rFN/AP-1 activity. CV11974 and inhibitors of PKC or PTK suppressed Ang II-mediated increases in rFN/AP-1 activity, although neither PD123319 nor a protein kinase A inhibitor affected the induction. Furthermore, mutation of rFN/AP-1 that disrupted nuclear binding suppressed Ang II-induced transcription in the native FN promoter (-1908 to +136) context. Thus, Ang II activates transcription of the FN gene through the Ang II type 1 receptor in vascular smooth muscle cells, at least in part, via the activation of AP-1 by a signaling mechanism dependent on PKC and PTK.

Angiotensin II↗
PubMed1998

Reversed-phase ion-pair chromatographic analysis of tetracycline antibiotics. Application to discolored teeth.

A high-performance liquid chromatographic method with diode array detection was developed to simultaneously separate tetracycline antibiotics and applied to the analysis of discolored teeth. By a reversed-phase ion-pair chromatographic system using pentanesulfonate as a counter ion, minocycline, oxytetracycline, tetracycline and demeclocycline were eluted in this order, and they showed base-line separation within 9 min. When using oxytetracycline as an internal standard, the quantitative ranges were between 2.5 ng/ml and 7.5 microg/ml. Powdered dentine (10 mg) and enamel (40 mg) prepared from discolored primary teeth were sonicated in 0.25 ml of 10 mM HCl containing oxytetracycline (0.75 microg/ml) and 50 mM EDTA-2Na, thereafter the supernatants were chromatographed. Eluates from both discolored tooth samples were identified as minocycline based on diode array spectra of their peaks, while minocycline was not detected in any samples from nondiscolored normal teeth, indicating that discoloration of the tested teeth was due to minocycline incorporated into dentine and enamel. Replicate quantitative analyses of the identical tooth substances showed that intra- and inter-assay C.V.s were 2.63 and 4.95% for dentine, and 5.42 and 10.88% for enamel. Application of the developed method to nine discolored teeth revealed that the incorporated minocycline ranged from 20.13 to 84.62 ng/mg of dentine and 0.89 to 7.87 ng/mg of enamel.

Anti-Bacterial Agents↗
PubMed1998

Difference in chromatin packaging between active and inactive X chromosomes by fractionation and allele-specific detection.

Using a novel method consisting of chromatin fractionation and allele-specific detection, chromatin packaging is compared between active X (Xa) and inactive X (Xi) chromosomes for five tumor cell clones that were derived from inter-subspecific F1 female mice. Separation of heterochromatic (H) and euchromatic (E) fractions is monitored by hybridization with subtelomeric satellite DNA and ribosomal RNA gene and by PCR amplification of p53 gene/pseudogene with one primer set. The H fraction was enriched with satellite and p53 pseudogene probably existing in heterochromatic regions while the E fraction showed inverse, suggesting fair separation. Analysis with seven marker and three gene loci revealed concentration of alleles on Xi in the H fraction and those on Xa in the E fraction, though the concentration levels varied. This implies that the packaging level of Xi is higher than that of active or inactive euchromatin on Xa. Intriguingly, one cell line showed biallelic expression and chromatin relaxation of the Pgk-1 locus, suggesting that the relaxation occur regionally on X chromosome.

Alleles↗